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8 APPLIED MICROBIOLOGY
Volume 8
In this thesis a metabolic model of S. cerevisiae was enhanced with the biochemistry
of volatile metabolites that have been found by a literature investigation. Not only the
volatile metabolites in question have been added, but also substances and reactions that Christoph Halbfeld
connect them to metabolites already present in the model. In total, 225 metabolites and
What happens in yeast during

219 reactions were added to the model. Furthermore, 12 metabolic reactions could be
verified by physiological and enzyme assays of knockout mutants.
Additionally, volatile metabolite dynamics during the induction of the Crabtree effect in
a fully respiratory growing continuous culture of S. cerevisiae were explored with real-
the Crabtree effect?
time analyses of the fermentation off-gas. Secondary electrospray ionization-Orbitrap-
mass spectrometry was used for this endeavor. In these measurements, I detected about An investigation of S. cerevisiae’s
2,500 signals of which 16 showed a response to the perturbation of the metabolic state
prior to the detection of ethanol. volatile space
Furthermore, the possibility of online volatile metabolite monitoring due to multi capil-
lary column–ion mobility spectrometry (MCC-IMS) analysis of yeast fermentation off-gas
What happens in yeast during the Crabtree effect?

was established. The MCC-IMS used was developed for the detection of volatiles in
An investigation of S. cerevisiae’s volatile space

human breath, and several technical adaptations were applied to allow robust detection
of volatiles in the headspace of yeast fermentations.
The MCC-IMS in its optimized configuration was applied to monitor volatile metabolite
changes of a laboratory and an industrial yeast strain during the Crabtree effect. In ad-
dition, metabolic differences in this setting were examined on transcriptional level using
a cDNA microarray. The metabolic shift could be observed in the volatile space of both
strains and in all tested conditions. The transcriptome showed differences in the leucine
and isoleucine pathways, as well as in genes related to the TCA cycle and the respiratory
pathways. Interestingly, the expression data indicated that the industrial strain upregula-
ted its respiration during the shift, while it was downregulated in the laboratory strain.
Lastly, possible applications for the knowledge gained and methods developed in this
work are discussed. This thesis provides a blueprint for studies of the volatile space in
other organisms. For example, the knowledge gained about the changes in the vola-
tilome during metabolic transitions could be used to online determine and potentially
control the metabolic state of a yeast.
Christoph Halbfeld
“What happens in yeast during the Crabtree effect?
An investigation of S. cerevisiae's volatile space”
Von der Fakultät für Mathematik, Informatik und Naturwissenschaften der RWTH Aachen
University zur Erlangung des akademischen Grades eines
Doktors der Naturwissenschaften genehmigte Dissertation
vorgelegt von
M. Sc. Biologie Christoph Halbfeld
aus
Moers, Nordrhein-Westfalen, Deutschland
Berichter: Universitätsprofessor Dr.-Ing. Lars M. Blank
Universitätsprofessor Dr. rer. nat. Jörg Ingo Baumbach
Tag der mündlichen Prüfung: 12.04.2018
Diese Dissertation ist auf den Internetseiten der Universitätsbibliothek online verfügbar.
Bibliografische Information der Deutschen Nationalbibliothek
Die Deutsche Nationalbibliothek verzeichnet diese Publikation in der Deutschen
Nationalbibliografie; detaillierte bibliografische Daten sind im Internet
über https://portal.dnb.de abrufbar.
Christoph Halbfeld:
What happens in yeast during the Crabtree effect?

An investigation of S. cerevisiae’s volatile space
1. Auflage, 2018
Gedruckt auf holz- und säurefreiem Papier, 100% chlorfrei gebleicht.
Apprimus Verlag, Aachen, 2018

Wissenschaftsverlag des Instituts für Industriekommunikation und Fachmedien
an der RWTH Aachen
Steinbachstr. 25, 52074 Aachen
Internet: www.apprimus-verlag.de, E-Mail: info@apprimus-verlag.de
Printed in Germany
ISBN 978-3-86359-635-4
D 82 (Diss. RWTH Aachen University, 2018)

Danksagung
Zuerst möchte ich mich bei Prof. Lars M. Blank für die Erstkorrektur meiner Arbeit und all die
Unterstützung, die ich von ihm erhalten habe bedanken. Außerdem möchte ich mich für die
Möglichkeit meine Arbeit am iAMB durchführen zu dürfen bedanken.
Mein Dank gilt ebenfalls Prof. Jörg-Ingo Baumbach für die Zweitkorrektur meiner Arbeit. Außerdem
möchte ich mich dafür bedanken, dass ich in der B&S Analytik die Möglichkeit hatte erste
Erfahrungen mit dem IMS zu sammeln und jederzeit für weitere Messungen willkommen war.
Außerdem möchte ich mich für die vielen aufschlussreichen Gespräche über das IMS bedanken.
Ich möchte mich ebenfalls bei Prof. Alan Slusarenko für die Übernahme der Drittprüferschafft
bedanken.
Ich danke ebenfalls meiner Betreuerin Dr. Birgitta E. Ebert an die ich mich jederzeit wenden konnte.
Durch ihre Unterstützung war ich in der Lage die Art, wie ich Experimente plane und Texte schreibe
wesentlich zu verbessern.
Vielen Dank ebenfalls an alle Projektpartner für die gute Zusammenarbeit. Jessica Kuhlmann danke
ich für die gute Zusammenarbeit bei der Verbesserung des IMS und der IMS-Analytik. Ann-Kathrin
Sippel möchte ich für die Zusammenarbeit bei der IMS-Analytik und der Vermessung von GC-
Proben danken. Prof. Sven Rahmann und Elias Kuthe möchte ich für die Hilfe bei der Auswertung
der cDNA-Microarrays danken. Sven Wegerhoff und Prof. Sebastian Engell möchte ich für die
interessanten Diskussionen während den Projekttreffen danken. Dr. Michael Quantz und Dr. Erik
Pollmann möchte ich für die immerwährende Hilfe in der Erkundung der unendlich großen
Hefeliteratur, für die Hilfe bei Fermentationsproblemen sowie für die gute Zusammenarbeit und die
Unterbringung bei der VH-Berlin danken.
Mein Dank gilt ebenfalls Prof. Pablo Martinez-Lozano Sinues für die Möglichkeit Experimente an
der SESI-Orbitrap-MS in Zürich durchzuführen. Ebenfalls danke ich der Zenobi-Gruppe für die
freundliche Arbeitsatmosphäre.
Außerdem danke ich allen Studenten, die im Verlauf meiner Arbeit mit mir zusammengearbeitet
haben: Christina Redmers, Daniela Dey, Christiane Sonntag, Sandrine Nankia Tatepo, Kai Büchner,
Mathis Wolter, Niklas Kitschen und Birthe Halmschlag. Durch sie wurde meine Arbeit bereichert, da
mehr Arbeitspakete bearbeitet werden konnten, als ich alleine hätte schaffen können.
Ebenfalls danke ich allen, die während meiner Zeit am iAMB gearbeitet haben, durch alle wurde eine
einzigartige Arbeitsatmosphäre geschaffen, die die oftmals langen Tage teils sehr unterhaltsam
gemacht haben. Besonders möchte ich hierbei Dr. Thiemo Zambanini, Benedikt Wienands, Hamed
Tehrani, Mathias Lehnen, Vanessa Bayer und Birthe Halmschlag danken, durch sie wurde die Zeit
unvergesslich. Ebenfalls möchte ich mich bei Eik Czarnotta für die Starthilfe bei Hefefermentationen
bedanken.
Ein besonderer Dank gilt Dr. Martin Zimmermann, der mich schon während meines Studiums, aber
auch in der Phase der Promotion immer wieder mit motivierenden Gesprächen auf dem Weg begleitet
hat.
Ich möchte mich auch bei den Mitarbeitern der Mechanischen und Elektrotechnik Werkstatt
bedanken. Mit Hilfe des Teams war ich dazu in der Lage Ideen zu realisieren, die ich alleine nicht
hätte umsetzen können.
Ich möchte ebenfalls meiner Freundin Dr. Sandra Jumpertz danken, sie hat mich auch in schwierigen
Phasen der Promotion immer aufgebaut und mir die Stärke gegeben die Promotion erfolgreich
abzuschließen.
Zuletzt möchte ich meiner Familie danken, die es mir ermöglicht hat diesen Weg zu beschreiten und
mir immer Rückhalt gegeben haben.
Funding
The project was funded by the German Federal Ministry of Education and Research (BMBF).
Eidesstattliche Erklärung
„Hiermit erkläre ich, Christoph Halbfeld, an Eides statt, dass ich die vorliegende Dissertation
selbstständig verfasst und keine anderen als die angegebenen Quellen und Hilfsmittel benutzt habe.“
Table of Contents
SUMMARY ....................................................................................................................................... V
ZUSAMMENFASSUNG ............................................................................................................... VII
LIST OF ABBREVIATIONS ........................................................................................................ IX
LIST OF FIGURES ..................................................................................................................... XIII
LIST OF TABLES ......................................................................................................................... XV
GENERAL INTRODUCTION ............................................................................................... 5

SUMMARY .......................................................................................................................... 5
INTRODUCTION................................................................................................................... 5
VOLATILE ORGANIC COMPOUNDS ....................................................................................... 5
BREAD ............................................................................................................................... 6
WINE ................................................................................................................................. 6
Wine flavors............................................................................................................. 6
Off-flavors ............................................................................................................... 7
FUNGAL VOCS .................................................................................................................. 8
METABOLIC PATHWAYS OF YEAST ..................................................................................... 9
VOCs derived from carbohydrate catabolism and fermentation ............................. 9
VOCs produced by amino acid synthesis and degradation ................................... 10
VOCs from fatty acids biosynthesis and degradation............................................ 11
VOCs derived from terpene biosynthetic pathways .............................................. 12
ANALYTICAL METHODS FOR THE IDENTIFICATION OF VOCS ............................................ 14
ION MOBILITY SPECTROMETRY ......................................................................................... 14
SECONDARY ELECTROSPRAY IONIZATION MASS SPECTROMETRY ..................................... 17
PROTON-TRANSFER-REACTION TIME-OF-FLIGHT MASS SPECTROMETRY ........................... 17
GAS-CHROMATOGRAPHY BASED METHODS ...................................................................... 18
OFFLINE-SAMPLING .......................................................................................................... 19
PROS AND CONS OF METHODS FOR VOC DETECTION ........................................................ 21
METHODS FOR THE DELINEATION OF VOC METABOLIC PATHWAYS ................................. 22
BOTTOM-UP APPROACHES ................................................................................................ 23
VERIFICATION OF PREDICTED PATHWAYS......................................................................... 24
AIMS ............................................................................................................................... 25
RESULTS ................................................................................................................................ 33
EXPANDING THE CONSENSUS YEAST MODEL BY VOLATILE METABOLISM ......................... 33
SUMMARY ........................................................................................................................ 33
INTRODUCTION................................................................................................................. 34
MATERIAL AND METHODS ............................................................................................... 35
Software tools for pathway prediction ................................................................... 35
Strains, media and plasmids used .......................................................................... 35
I
Table of Contents
Enzymes and PCR components ............................................................................. 36

Commercial kits ..................................................................................................... 39
Creating multiple knockout mutants ...................................................................... 39
Biotransformation assay ........................................................................................ 40
Data evaluation ...................................................................................................... 40
RESULTS AND DISCUSSION ............................................................................................... 41
CONCLUSION AND OUTLOOK ........................................................................................... 50
YEAST VOLATILOME DYNAMICS DURING METABOLIC SHIFTS ........................................... 55
SUMMARY ........................................................................................................................ 55
INTRODUCTION................................................................................................................. 56
Dynamics of (volatile) compounds ........................................................................ 56
Yeast strains and growth conditions ...................................................................... 58
Online SESI-Orbitrap MS measurements .............................................................. 60
Data evaluation: ..................................................................................................... 61
Interpretation of the results .................................................................................... 62
MULTICAPILLARY COLUMN-ION MOBILITY SPECTROMETRY OF VOLATILE METABOLITES
EMITTED BY SACCHAROMYCES CEREVISIAE ........................................................................ 71
SUMMARY ........................................................................................................................ 71
INTRODUCTION................................................................................................................. 72
Analytics ................................................................................................................ 74
SPME GC-MS measurements for validation of volatile metabolites .................... 74
MCC-IMS measurements ...................................................................................... 74
Volatile metabolite identification .......................................................................... 75
Experimental setup ................................................................................................ 75
MCC-IMS monitoring of batch cultures................................................................ 76
MCC-IMS monitoring of glucose-limited chemostat fermentations ..................... 80
Identification of volatile metabolites ..................................................................... 84
THE CRABTREE EFFECT REVISITED ................................................................................... 91
A COMPARATIVE STUDY ON THE TRANSCRIPTIONAL CHANGES DURING THE CRABTREE EFFECT
USING LABORATORY AND INDUSTRIAL STRAINS AND CONDITIONS ................................... 91
SUMMARY ........................................................................................................................ 91
INTRODUCTION................................................................................................................. 92
MCC-IMS analysis ................................................................................................ 94
cDNA microarray analysis .................................................................................... 94
II
Table of Contents
MCC-IMS and microarray analysis of two yeast strains ....................................... 95

CONCLUSION AND OUTLOOK ......................................................................................... 100
GENERAL DISCUSSION ................................................................................................... 105

POSSIBLE IMPACT OF THIS THESIS ................................................................................... 105
IMPACT ON FUTURE STUDIES .......................................................................................... 105
STRAIN ENGINEERING & POSSIBLE USE OF VOLATILE PRODUCTS .................................... 106
THE USE OF DIFFERENT METABOLIC CONDITIONS ........................................................... 107
ONLINE FLUX ANALYSIS ................................................................................................. 107
CONCLUSION .................................................................................................................. 107
THE CONSENSUS YEAST METABOLIC MODEL................................................................... 107
SESI-ORBITRAP .............................................................................................................. 108
MCC-IMS ..................................................................................................................... 108
LABORATORY VS. INDUSTRIAL CONDITIONS ................................................................... 108
OUTLOOK............................................................................................................................ 113
APPENDIX ..................................................................................................................................... 115
REFERENCES ............................................................................................................................... 123
CURRICULUM VITAE ................................................................................................................ 145

PUBLICATIONS ............................................................................................................................ 147
ORAL PRESENTATIONS ............................................................................................................... 147
POSTER PRESENTATIONS ............................................................................................................ 148
III
Summary
Summary
The baker’s yeast Saccharomyces cerevisiae is one of the best investigated organisms and widely used in
science. This scientific interest is partially based on the yeast’s use in the industrial production of
pharmaceuticals, beverages and food. Even though the taste of food and beverages is clearly influenced by
volatiles, the yeast’s volatilome, i.e., the entirety of the volatile metabolites produced, is to date largely
uncharted, with ethanol and acetaldehyde being prominent exceptions.
In this thesis, in chapter 2.1, a metabolic model of S. cerevisiae was enhanced with the biochemistry of volatile
metabolites that have been found by a literature investigation. Not only the volatile metabolites in question
have been added, but also substances and reactions that connect them to metabolites already present in the
model. In total, 225 metabolites and 219 reactions were added to the model. Furthermore, 12 metabolic
reactions could be verified by physiological and enzyme assays of knockout mutants. These mutants were
created using the CRISPR-Cas9 method and contained only one gene coding for a protein with alcohol
dehydrogenase activity.
In chapter 2.2, volatile metabolite dynamics during the induction of the Crabtree effect in a fully respiratory
growing continuous culture of S. cerevisiae were explored with real-time analyses of the fermentation off-gas.
SESI (secondary electrospray ionization)-Orbitrap-mass spectrometry (MS) was used for this endeavor. In
these measurements, we detected about 2,500 signals of which 16 showed a response to the perturbation of the
metabolic state prior to the detection of ethanol. These observations not only revealed the extent of the yeast’s
volatilome, but also indicated that volatile metabolite dynamics correlate with the metabolic state of the cell
culture and hence might be a noninvasive and fast online analytical method to monitor and later control
fermentation processes.
In chapter 2.3, to evaluate the possibility of online volatile metabolite monitoring, multi capillary column–ion
mobility spectrometry (MCC-IMS) analysis of yeast fermentation off-gas was established. This analytical
device was chosen because it runs at ambient temperature and pressure resulting in lower investment and
operating costs compared to SESI-Orbitrap-MS. The MCC-IMS used was developed for the detection of
volatiles in human breath, and several technical adaptations were required to allow robust detection of volatiles
in the headspace of yeast fermentations.
In chapter 2.4, the MCC-IMS in its optimized configuration was applied to monitor volatile metabolite changes
of a laboratory and an industrial yeast strain during the transition from fully respiratory to respiro-fermentative
metabolism (Crabtree effect). In addition, metabolic differences in this setting were examined on
transcriptional level using a cDNA microarray. The metabolic shift could be observed in the volatile space of
both strains and in all tested conditions. The transcriptome showed differences in the leucine and isoleucine
pathways, as well as in genes related to the TCA cycle and the respiratory pathways. Interestingly, the
expression data indicated that the industrial strain upregulated its respiration during the shift, while it was
downregulated in the laboratory strain.
Lastly, possible applications for the knowledge gained and methods developed in this work are discussed.
This thesis provides a blueprint for studies of the volatile space in other organisms. The extended metabolic
model could be used to generate yeast strains with special flavors or for the production of fragrances, perfumes
or precursors of pharmaceuticals. Also, the knowledge gained about the changes in the volatilome during
metabolic transitions could be used to online determine and potentially control the metabolic state of a yeast.
Finally, the analytical methods developed in this work might be used for online flux analysis, if some more of
the detected volatiles can be identified.
V
Zusammenfassung
Zusammenfassung
Die Bäckerhefe Saccharomyces cerevisiae ist einer der am besten untersuchten Organismen und wird häufig in der
Wissenschaft genutzt. Dieses wissenschaftliche Interesse beruht teilweise auf der industriellen Nutzung von Hefe
bei der Produktion von Medikamenten, Getränken und Nahrungsmitteln. Obwohl der Geschmack von
Nahrungsmitteln und Getränken deutlich von flüchtigen Metaboliten beeinflusst wird, ist das Volatilom, also die
Gesamtheit der produzierten flüchtigen Metabolite, bei Hefen bisher noch weitgehend unerforscht, mit Ausnahme
der prominenten Ausnahmen Ethanol und Acetaldehyd.
In dieser Arbeit wurde in Kapitel 2.1 die Literatur nach flüchtigen Metaboliten durchsucht und ein metabolisches
Modell von S. cerevisiae durch deren Integration verbessert. Zusätzlich zu den gefundenen Metaboliten wurden
Substanzen und Reaktionen in das Modell eingepflegt, um die hinzugefügten volatilen Metaboliten mit dem bereits
vorhandenen Netzwerk zu verbinden. Insgesamt wurden 225 Metaboliten und 219 Reaktionen zu dem Modell
hinzugefügt. Zudem konnten 12 metabolische Reaktionen durch physiologische und enzymatische Versuche mit
knockout-Mutanten verifiziert werden. Die Mutanten wurden durch Anwendung des CRISPR-Cas9-Systems
erzeugt und enthalten jeweils nur ein Gen, welches für ein Protein mit Alkoholdehydrogenaseaktivität kodiert.
In Kapitel 2.2 wurden die flüchtigen Metabolite einer kontinuierlichen, respiratorischen Saccharomyces cerevisiae-
Kultur während der Induktion des Crabtree-Effekts untersucht. Dafür wurden die Abluft der Fermentation direkt in
ein SESI (Sekundäre Elektrospray-Ionisation)-Orbitrap Massenspektrometer (MS) geleitet, wo sie in Echtzeit
analysiert wurde. Pro Experiment wurden ca. 2.500 Signale aufgenommen, 16 von diesen zeigten nach der
Perturbation des metabolischen Zustandes, noch bevor Ethanol detektiert wurde, eine Änderung in der
Signalintensität. Diese Ergebnisse verdeutlichen nicht nur die Größe des Hefe-Volatiloms, sondern zeigen ebenfalls,
dass die Dynamiken der volatilen Metabolite im Gasraum über Hefefermentationen mit dem metabolischen Zustand
der Zellen korrelieren. Daher könnte eine nicht-invasive und schnelle online-Analysemethode für das Überwachen
von Hefekulturen dazu geeignet sein, Fermentationsprozesse zu kontrollieren.
Genau dies wurde in Kapitel 2.3 untersucht, dazu wurde die Analyse des Gasraums über einer Hefefermentation
mit einem Multikapillarsäulen-Ionen-Mobilitäts-Spektrometer (MCC-IMS) etabliert. Dieses analytische Gerät
wurde ausgewählt, da es bei Normaldruck und Raumtemperatur betrieben werden kann. Im Vergleich zur SESI-
Orbitrap-MS sind für das MCC-IMS geringere Wartungskosten aufzubringen und auch die Anschaffungskosten des
MCC-IMS liegen deutlich unter denen der SESI-Orbitrap-MS. Das hier verwendete MCC-IMS wurde für die
Detektion von Metaboliten in menschlicher Ausatemluft konzipiert, so dass einige technische Anpassungen
vorgenommen werden mussten, um eine robuste Detektion der volatilen Metaboliten von Hefefermentationen zu
ermöglichen.
In Kapitel 2.4 wurde das optimierte MCC-IMS verwendet, um Metabolitkonzentrations-Veränderungen im
Gasraum über Hefefermentationen während dem Übergang von ausschließlich respirativem hin zu
respirofermentativem Wachstum (Crabtree-Effekt) zu untersuchen. Unter diesen Bedingungen wurde auch mithilfe
eines cDNA-Microarrays die Änderungen auf transkriptioneller Ebene untersucht. Für diese Versuche wurde ein
Laborstamm und ein industriell genutzter Stamm verwendet. Die metabolischen Veränderungen spiegelten sich im
Gasraum der beiden unterschiedlichen Hefestämme deutlich wieder. Im Transkriptom konnten Unterscheide in der
Regulation der Gene, die mit dem Leucin- und Isoleucin-Metabolismus zusammenhängen, sowie bei den für die
Atmung und den Citratzyklus zuständigen Genen festgestellt werden. Überraschend war, dass die mit der Atmung
assoziierten Gene im Industriestamm mit Änderung des metabolischen Zustandes hochreguliert wurden, während
die gleichen Gene im Laborstamm herunterreguliert wurden.
Im letzten Teil der Arbeit werden mögliche Anwendungen für das in dieser Arbeit generierte Wissen diskutiert. So
könnte diese Dissertation als Vorlage für die Untersuchung von Volatilen anderer Organismen genutzt werden. Das
erweiterte metabolische Modell könnte dazu verwendet werden, Hefestämme mit besonderen Aromen für die
Produktion von Duftstoffen, Parfums oder als Vorläufer für Medikamente, zu entwerfen. Darüber hinaus könnte
das hier generierte Wissen über die Veränderung flüchtiger Metabolite während einer metabolischen
Zustandsänderung ebenfalls dazu verwendet werden, um eine Fermentation mittels online-Analytik zu
kontrollieren. Letztlich könnten die hier entwickelten analytischen Methoden zur online-Flussanalyse genutzt
werden, falls noch weitere der hier detektierten flüchtigen Metabolite identifiziert werden.
VII
List of Abbreviations
Abbreviations
% percent
(NH4)2HPO4 diammonium phosphate
(NH4)2SO4 ammonium sulfate
μF microfarad
μL microliter
μm micrometer
ACO aconitase
ADH alcohol dehydrogenase
AIMS aspiration IMS
BAT branched-chain amino acid transmaniase
BNICE biochemical network integrated computational explorer
CaCl2 2H2O calcium chloride diydrate
Ca-pantheonate calcium-pantheonate
Cas9 CRISPR associated protein 9
cDNA copy desoxyribonucleic acid
CDW cell dry weight
CFME continuous flow microextraction
CHA1 catabolic L-serine deaminase
CIT citrate synthase
cm centimeter
CO2 carbondioxide
CoA conenzyme A
COB cytochrome b
CoCl2 7H2O cobalt(II) chloride heptahydrate
COR core protein of QH2 cytochrome c reductase
COX cytochrom oxidase
CRISPR clustered regulatory interspaced short palindromic repeats
CuSO4 5H2O copper(II) sulfate pentahydrate
CYT1 cycochrome c1
D dimer
DI direct immersion
DMA differential mobility analyzer
DNA desoxyribonucleic acid
DO dissolved oxygen
DTIMS drift time IMS
DTT dithiothreitol
E. coli Escherichia coli
e.g. exempli gratia
EDTA ethylendiamintetraacetic acid
ESI electrospray ionization
et al. et alia
FAEE fatty acid ethyl esters
FAIMS high field asymmetric waveform IMS
IX
FBA flux balance analysis

FBA flux balance analysis
FDR false discovery rate
FeSO4 7H2O ferrous sulfate heptahydrate
FI flame ionization
FMM from metabolite to methabolite
g gram
GEM genome scale model
GENRE genome scale network reconstruction
GRE 3-methylbutanal reductase
h hour
H2O water
H3BO3 boric acid
HMG-CoA 3-hydroxy-3-methylglutaryl CoA
HPLC high-performance liquid chromatography
HS headspace
i.e. id est
IDH isocitrate dehydrogenase
ILV acetolactate synthase
IMS ion mobility spectrometry /spectrometer
K ion mobility
K0 reduced ion mobility
KEGG Kyoto encyclopedia of genes and genomes
KGD alpha-ketoglutarate dehydrogenase
KH2PO4 potassium phosphate
KI potassium iodide
kV kilovolt
L liter
LB lysogeny broth
LEU alpha-isopropylmalate synthase
LPD lipoamide dehydrogenase
LSC ligase of succinyl-CoA
M molar
M monomer
m/z mass to charge ratio
m/z mass per charge ratio
MALDI matrix assisted laser desorption ionization
MCC multi capillary column
MCCase 3-methylcrotonyl-CoA
MDH malate dehydrogenase
mg miligram
MgSO4 7H2O magnesium sulfate heptahydrate
min minutes
mL milliliter
mM millimolar
X
MnCl2 4H2O manganese(II) chloride tetrahydrate

MS mass spectrometry /spectrometer
N2 nitrogen
NAD nicotinamid adenin dinucleotide
NADP nicotinamid adenin dinucleotid phosphate
NaMoO4 2H2O sodium molybdate dihydrate
ND not detected
NDE NADH dehydrogenase, external
NDI NADH dehydrogenase, internal
ng nanogram
nL nanoliter
NMR nuclear magnetic resonance
OD optical density
OLIMS open loop IMS
OMS overtone mobility analyzer
ORF open reading frame
ppb parts per billion
ppm parts per million
ppt parts per trillion
PTM posttranslational modification
PTR proton transfer reaction
PYC pyruvate carboxylase
Q quadrupole
QCR ubiquinol-cytochom c oxidoreductase
RIP reaction ion peak
RIP rieske iron sulfur protein
RNA ribonucleic acid
rpm rounds per minute
s seconds
S. cerevisiae Saccharomyces cerevisiae
SBML systems biology markup language
SDH succinate dehydrogenase
SDME single drop microextraction
SESI secondary electrospray ionization
SFA bifunctional alcohol and formaldhyde dehydrogenase
SIM selected ion monitoring
SPME solid phase microextraction
TCA tricarboxylic acid
TDS thermal desorption spectroscopy
TFBA thermodynamics-based flux analysis
TIC total ion current
TIMS trapped IMS
TMIMS transversal modulation IMS
TNT 2,4,6-trinitrotoluene
TOF time of flight
XI
TWIMS traveling-wave IMS

USER uracil-specific excission reagent
VOC volatile organic compound
vvm gasvolume per fermentervolume per minute
w/w weight per weight
ZnSO4 7H2O zinc sulfate heptahydrate
Ω ohm
XII
List of Figures
List of Figures
Figure 1: Overview of volatile metabolites released by yeast. Depicted are few representatives and their origin from
central carbon metabolism [1]. ................................................................................................................ 9
Figure 2: The Ehrlich pathway. Biochemistry and main genes involved in amino acid degradation (A) and
intermediates and products derived from involved amino acids (B). Figure adapted from [2, 3]. Figure
previously published in [1]. ................................................................................................................... 11
Figure 3: Mitochondrial monoterpene biosynthesis via the leucine catabolic MCC pathway and possible relationships
with sterol formation in Saccharomyces cerevisiae as proposed by Carrau et al. [4]. Figure adapted from
[4]. Previously published in [1]. ............................................................................................................ 13
Figure 4: Working principle of a drift tube-IMS; adapted with permission of Journal of Physiology and Pharmacology
from [5], previously published in [1, 6]. ............................................................................................... 15
Figure 5: Schematic representation of a secondary electrospray ionization source. The here indicated DMA is
interchangeable with an Orbitrap. DMA= differential mobility analyzer. Reprinted with permission
from [300]. Copyright 2012 American Chemical Society..................................................................... 57
Figure 6: Influence of mass resolution on peak separation. Taken from [133]. ........................................................... 58
Figure 7: Schematic view (A) and picture (b) of the mini-bioreactor setup connected to the SESI-Orbitrap-MS
(Bioreactor constructed by Eik Czarnotta and Suresh Sudarsan, RWTH Aachen University). ............ 60
Figure 8: Data evaluation of SESI Orbitrap analyses of the off-gas of a continuous glucose-limited yeast culture at
steady state which was disrupted with a 27 mM glucose pulse. The filtered data is displayed as a
clustered heatmap in which the scaled intensity of single masses (m/z value) are plotted over time (A),
zoom-in excerpt of the heatmap shown in A (B). Smoothed data of interesting signal traces (blue line)
identified in the heatmap are plotted against time together with the signal of ethanol (black line). The
left black vertical line indicates the timepoint of the glucose pulse injection, while the right line indicates
the timepoint of the first detection of ethanol (C-F). ............................................................................. 64
Figure 9: Experimental set-up for the online MCC-IMS measurements of fermenter off-gas. Previously published in
[185]. ..................................................................................................................................................... 76
Figure 10: MCC-IMS topographic plot of sterile Verduyn medium. The reaction ion peak (1/K 0 = 0.5 Vs cm−2) was
compensated by the software VisualNow. ............................................................................................ 77
Figure 11: MCC-IMS topographic plot of S. cerevisiae during the early stationary phase of a batch fermentation.
Boxes indicate signals that showed the most significant changes during the growth (perturbation)
experiments. .......................................................................................................................................... 78
Figure 12: (A) Fermentation profile of S. cerevisiae during batch growth in glucose minimal medium, (B) trends in
intensity, (C) heat map of selected peaks detected by MCC-IMS analysis of the fermentation off-gas.
Areas in the heat map show the detected signal peak and the surrounding area; DO = dissolved oxygen.
............................................................................................................................................................... 79
Figure 13: (A) Fermentation profile of S. cerevisiae adh1Δ during batch growth in glucose minimal medium and (B)
trends in intensity and (C) heat map of selected peaks detected by MCC-IMS analysis of the
fermentation off-gas. The areas in the heat map show the detected signal peak and the surrounding area;
DO = dissolved oxygen. ........................................................................................................................ 80
Figure 14: MCC-IMS topographic plot of the off-gas of a glucose-limited continuous cultivation of S. cerevisiae.
Boxes indicate signals that showed the most significant changes during the growth (perturbation)
experiments. The reaction ion peak (1/K0 = 0.5 Vs cm−2) was compensated for by the VisualNow
software. ................................................................................................................................................ 81
Figure 15: (A) Fermentation profile of S. cerevisiae during growth in a glucose-limited chemostat and (B) trends in
intensity and (C) heat map of selected peaks detected by MCC-IMS measurements of the fermentation
off-gas after perturbation of the metabolic steady state with a pulse of 22 mmol glucose; DO = dissolved
oxygen. .................................................................................................................................................. 82
Figure 16: (A) Fermentation profile of S. cerevisiae during growth in a glucose-limited chemostat and (B) trends in
intensity and (C) heat map of selected peaks detected by MCC-IMS measurements of the fermentation
off-gas during transition to anaerobic conditions; DO = dissolved oxygen. ......................................... 83
Figure 17: Volatile signals of Saccharomyces cerevisiae CEN.PK 113-7D (A) and Saccharomyces cerevisiae DHW
(B) in a fed-batch that was slowly overfed to trigger the Crabtree effect. The grey background represents
XIII
List of Figures
the feed rate. The commercially available Alcoline sensor form Biotechnology Kempe was used for
ethanol quantification in the fermentation broth along with the MCC-IMS Breath Discovery from B&S
Analytik for the monitoring of volatile metabolites in the fermentation head space. The black dotted line
indicates the start of the overfeeding process, the red dotted lines indicate the division into early (left)
mid (middle) and late (right) phase. D and M behind the substance names describe the measured dimer
and monomer respectively. ................................................................................................................... 96
Figure 18: Transcriptional alterations of the most differing pathways based on expressional changes between
Saccharomyces cerevisiae CEN.PK 113-7D (left) and Saccharomyces cerevisiae DHW (right). The two
strains were run in fed-batch fermentations and overfeed gradually. The respiration pathway (A), the
TCA-cycle (B) and the branched-chain amino acid production (C) are depicted. The genes without color
coding are not displayed, since it had a false discovery rate of more than 5% in at least one of the tested
strains. ................................................................................................................................................... 99
XIV
List of Tables
List of Tables
Table 1. Features of different analytical devices for volatile compound measurement. Data on resolving power taken
from [181-184], all other data taken from product sheets of current devices; LOD, limit of detection.
Previously published in [1]. .................................................................................................................. 22
Table 2: Microbial strains ............................................................................................................................................ 36
Table 3: Primers (table continues over multiple pages) ............................................................................................... 37
Table 4: Oligonucleotides for homologous recombination .......................................................................................... 39
Table 5: Volatile organic compounds emitted from S. cerevisiae fermentations. Compounds that are included in the
latest yeast genome scale metabolic reconstruction (Yeast 7.11) and in the Saccharomyces Genome
Database SGD [185] are categorized. (Table continues over multiple pages.) ..................................... 41
Table 6: Volatile metabolites added to the metabolic yeast model. The column reference indicates, whether the
pathway was taken from KEGG, a scientific publication, that the pathway was inferred by pathway
prediction tools (blank space), or predicted by established yeast enzymes with reactions similar to those
needed to establish the pathway (also blank space). (Table continues over multiple pages.) ............... 44
Table 7: Enzymatic capacity of sADH mutants. The strains were tested for their ability to convert alcohols, ketones,
and aldehydes in enzymatic assays. The mutants showed either clear conversion (+), slight conversion
(+/-) or no conversion of the substrate (-). Green indicates accordance to literature data, red indicates
contrariety. ............................................................................................................................................ 49
Table 8: Mass to charge ratio (m/z) of the most interesting signals recorded during the metabolic transient state in a
continuous glucose-limited fermentation after setting a glucose pulse. + and # indicate masses whose
signal intensity linear correlated with each other and which probably originate from the same metabolite.
m/z 69.0701 and 89.0962 have been tentatively identified as 3-methyl-1-butanol and isoprene,
respectively. .......................................................................................................................................... 63
Table 9: Volatile organic compounds detected in fermentations of S. cerevisiae via GC-MS measurements growing in
glucose minimal salt medium. ND, not detected. .................................................................................. 85
Table 10: Profiles of fermentation parameters of the fed-batch fermentations. ........................................................... 94
XV
Chapter 1
General introduction
Exploration and Exploitation of the Yeast Volatilome
Partially published as:

Ebert, B.; Halbfeld, C.; Blank, L. Exploration and exploitation of the yeast volatilome. Current
Metabolomics 2017, 5, 102-118.
Contributions:
The introduction was written by Christoph Halbfeld. Birgitta E. Ebert contributed 1.2.4 to
1.3 and 1.4. Lars Blank critically read the text.
Chapter 1
Summary
Volatile organic compounds (VOCs) are small molecular mass substances, which exhibit high-vapor
pressures, low boiling points, and lipophilic character. VOCs are produced by all organisms including
eukaryotic microbes like yeast. Volatile metabolites are for centuries exploited, for examples as
flavors in bread, beer, and wine. Notably, while the applications of VOCs are many, the knowledge
on their biochemical synthesis is still limited.
This introduction reviews the current information of yeast volatile metabolites and techniques to
further explore the VOC landscape made possible by improvements of the analytical possibilities,
regarding sampling frequency, identification, and quantification and the development to
computationally interpret (high-throughput) data. Especially possibilities for online and even real-
time analysis should trigger new experimental approaches that elucidate the biochemistry as well as
the regulation of VOC synthesis. Baker’s yeast is here the organism of choice as the genetic inventory
can be linked to VOC formation and with this in hand improved applications can be envisaged. The
physical, chemical or biological properties make many VOCs interesting targets for different
industrial sectors, while their natural function as semiochemicals or in defense mechanisms can be
exploited to engineer synthetic microbial communities or to develop new antibiotics.
VOCs produced by microbes including yeast are a chemical diverse group of compounds with
highly different applications. The new analytical techniques briefly summarized here will enable the
use of VOCs in even broader applications including human health monitoring and bioprocess control.
We envisage a bright future for VOC research and for the resulting applications.
Introduction
Volatile organic compounds
Volatile organic compounds (VOCs) are small molecular mass substances (<300 Da), which exhibit
high-vapor pressures, low boiling points and lipophilic character. VOCs encompass various chemical
classes, e.g., low molecular weight fatty acids and their derivatives (hydrocarbons, alcohols,
aldehydes and ketones), terpenoids, aromatic compounds, nitrogen containing compounds, and
volatile sulphur compounds.
VOCs are ubiquitous in nature and play an important role in all domains of life, especially in the
intra- and interspecies communication and self-protection. Plants, for example, use VOCs as direct
or indirect defense mechanism to repel herbivores or attract carnivores that exterminate herbivore
populations [7]. Likewise, animals use pheromones as means of communication or behavior-altering
agents, while humans use VOCs formed by microbes as an indicator of spoiled food, another form of
interspecies communication [5][8]. Taste perception is also determined by volatiles since the papillae
in the human mouth can only distinguish between salty, sweet, bitter, sour and, umami and what we
experience as flavor is created through the binding of volatiles to sensory receptors [6]. It is for this
reason that volatiles play an important role in the food and beverage industry, where they are often
5
produced by microorganisms. A prominent example is the baker’s yeast Saccharomyces cerevisiae

which is used for everyday products like bread, wine, beer, and yeast extract and whose endogenous
metabolites contribute to the flavor of these products. Accordingly, aroma as well as unpleasant odor
components formed by yeast during food fermentation have been extensively investigated. The aroma
of food fermentation is explained in further detail in the following paragraphs.
Bread
Bread has been consumed by humankind for millennia as it is essential food and is well known for
its rich flavor. Its odor and flavor depend on a multitude of factors like dough-composition,
fermentation and baking parameters. While wheat flour contributes only little to the aroma of bread,
enzymes and yeast contribute a better part to it [9-12]. To investigate VOC pattern in dough
fermentation, Frasse et al. analyzed the headspace of first yeasted, secondly yeasted and fermented,
and thirdly non-yeasted doughs. The experiments showed that alcohols, esters, ketones, lactones and
sulfur compounds were produced during yeast fermentation, while aldehydes were consumed in the
process [12].
The main compounds contributing to the flavor prior to baking were mostly fusel alcohols (isoamyl
alcohol, 2-methyl-1-butanol, 2-Phenylethanol), but also aldehydes (diacetyl, methional) due to a high
odor threshold [12]. During the baking process the flavor changes, the caramelization reaction of the
sugars, and especially the Maillard reaction play an important role in changing the flavor profile [9,
13-15]. Depending on the composition of amino acids in the dough, the Maillard reaction will lead to
the production of acetaldehyde, phenylacetaldehyde, 2-methylebutanal and other carbonyl
compounds. Leucine, isoleucine and lysine lead to a pleasing aroma, while phenylalanine and
methionine lead to an unpleasant odor in a model Browning system, while phenylalanine led to a rose
oil like odor in baked bread. Especially lysine, arginine, histidine, and tryptophan lead to the browning
of the crumb [16, 17]. After the baking process, the aroma of fresh baked bread unfolds. This aroma
has been investigated in versatile studies, it is created mainly by a mixture of aldehydes (e.g., 3-
methylbutanal, 2-methylpropanal, (2E,4E)-deca-2,4-dienal, hexanal, 2-phenylacetaldehyde, 3-
methylsulfanylpropanal, 2-methylbutanal, non-2-enal), ketones (butane-2,3-dione, oct-1-en-3-one)
and acids (e.g., acetic acid, 2-methylbutanoic acid, 3-methylbutanoic acid) [18-22]. The aroma of the
baked bread will change over time and the desirable flavor will disappear, this is also caused by
volatiles that slowly fade (e.g., 2-acetyl-1-pyrroline, 3-methylbutanal), while other compounds
formed through lipid peroxidation are formed [20, 23].
Wine
Wine flavors
Many studies have been conducted in the field of volatile wine metabolites, since these compounds
are responsible for the rich wine aroma. Welke et al. investigated the volatile profile of Merlot wines
and tentatively identified a total of 334 compounds; the article also gives a good overview of volatile
metabolites that were identified in other studies [24]. The aroma is influenced by the different stages
of wine making: first the choice and processing of the grapes (pre-fermentative flavor), then the
6
Chapter 1
fermentative conditions, choice of the yeast and amount of inoculum (fermentative flavor) and finally
the post-fermentative flavor that comes from enzymatic or chemical reactions while storage in the
barrel and bottle [25-30]. The wine grapes differ in a multitude of aroma compounds; especially
important roles play hereby the monoterpenes. There are about 70 monoterpenes known at this time
and differences in their concentration can be used for varietal characterization. The most prominent
monoterpenes in grape aromas are linalool, geraniol, nerol, citronellol, 3,6-dimethyl-1,5-octadien-
1,7-diol, and α-terpineol [30].
In 2001, Mateo et al. showed that a different inoculum concentration of the same yeast strain can
influence the volatile composition of wine. They showed that the quantity of the yeast inoculum is
positively correlated to the amount of higher alcohols that can be found later in the wine, while higher
alcohols above 400 mg/L are regarded as negative factor in the final product [27, 31]. Other than
higher alcohols the amount of long chain esters is strain dependent which has to be considered because
the amount of higher alcohols and long chain esters relate to each other by the equation:
ͶͲͲ (1)[27]
ሾ݈‫ݏݎ݁ݐݏ݄݁݊݅ܽܿ݃݊݋‬ሿ‫ݔ‬
ሾ݄݄݅݃݁‫ݏ݈݋݄݋݈ܿܽݎ‬ሿ
The choice of the correct strain is also important because of different amount of alcohols, organic
acids and esters that are also important for the bouquet in the resulting wines [27].
During the aging in a bottle different reactions take place that have an influence on the aroma: the
ester content changes, the acetate concentration decreases, mono- and dicarboxylic acid ethyl esters
increase, carotenes and carbohydrates break down and monoterpenes react in the sour conditions.
Especially the acetates decrease over a period of about six years until an equilibrium between alcohol,
acetic acid and acetate is reached. All these factors have an influence to the taste difference between
young and aged wine, e.g., the acetates distribute the fresh and fruity note of young wine [30]. Also
a connection between D,L-piperitone [32] and 1,8-cineole [33] and the positive aging effects of red
wine could be associated.
Off-flavors
The following section will focus on the negative flavor compounds in wine. These compounds are
perceived as negative, if their concentration exceeds a certain sensory threshold. The first off-flavor
was detected in Europe, when European cultivars of Vitis vinifera were cross-bread with wild
American wine plants to increase the fungal resistance. This off flavor can be tracked back to 2,5-
dimethyl-4-hydroxy-2,3-dihydro-3-furanone (furaneol) [30, 34]. The sensorial detection limit for
furaneol lies in the range between 30 and 300 ppb [30, 35, 36]. Another undesirable off-flavor is 2-
iso-butyl-3-methoxy-pyrazine that has a low perception threshold of about 0.002 to 0.4 ppb. It smells
like herbs or potatoes and is enriched in ripening Sauvignon grapes [30, 37-39]. 2-
ethyltetrahydropyridine, 2-acetyl-tetrahydropyridine and 2-acetylpyroline are responsible for a
mousy (taste of mouse urine) like flavor in wine [40-46]. An amount of more than 800 μg/L of the
pure substances or a mixture of 4-vinylguajacol and 4-vinylphenol leads to a medicinal or Elastoplast
like off-flavor. This flavor is developed, especially if the grapes were exposed directly to sunlight in
7
warmer regions. A similar effect occurs if the sum of more than 400 μg/L of 4-ethylphenol and 4-
ethylguajacol are exceeded, these compounds however have leathery and respectively horse sweat
aroma [30, 47]. The corky off-note that some wines develop during the aging process are associated
with several components such as 2,3,6-trichloroanisole, 2,3,4-trichloroanisole, 2,3,5,6-
tetrachloroanisole, pentachloroanisole, 2,4,6-tribromoanisole, 2-methylisoborneol, ethylenguaiacol,
4-ethylenphenol and 2,4,6-trichloroanisole. The single compounds show earthy, mushroom and
cardboard-like aromas. The olfactory threshold for 2,4,6-trichlooanisole is about 4-10 ng/L and thus
way lower than that of 4-vinylguajacol, 4-vinylphenol, 4-ethylphenol and 4-ethylguajacol [48-52].
It is not possible to sum up the complete literature of wine flavors in this thesis. To learn more
about wine flavors, the research of Prof. Rapp and the book “Flavour and Fragrances” from 2007
provides a useful basis [30, 53, 54].
Fungal VOCs
On the contrary to the general capability of microorganisms, the production of volatiles has not been
thoroughly explored so far but is receiving increased attention because of a growing awareness of the
potential of volatile metabolites as antimicrobial agents, biofuels or plant growth promoting
compounds. Also, it has been shown that the volatile footprint is specific for different organisms,
allowing VOC analysis to be used for diagnostic purposes, the detection of food spoilage, and hidden
growth of molds in buildings [7, 55-60]. By the same token, VOC synthesis depends on growth
condition, carbon source and carbon source availability [61, 62], hence can be used in fermentation
process monitoring.
The synthesis of microbial VOCs has long been seen as a cellular detoxification or waste disposal
mechanism, but analogous to plant hormones or pheromones they have important ecological roles
and are released into the environment, to impact metabolism and growth of competing or symbiotic
organisms. Research on these microbial semio- or infochemicals is of interest in basic research but
also in applied sciences, e.g., for the design of synthetic microbial communities tailored for the
production of chemicals, degradation of pollutants, and wine manufacturing [55].
Still, the spectrum of fungal volatile metabolites, also referred to as volatilome [63], is today not
broadly and systematically explored. This becomes apparent when comparing known fungal VOCs
with the number identified from the plant and animal kingdom. While databases for the latter list up
to 8000 compounds [64, 65], the microbial specific VOC database, mVOC [66], contains 1174
metabolites, out of which 500 are of fungal origin (as of May 2016). The current knowledge about
the VOC biosynthesis pathways and their regulation is even scarcer. This might be attributed to
challenges in detection and identification of these mainly low abundant metabolites and their
chemical diversity which requires complimentary sampling and analytical techniques to capture the
complete VOC space. Furthermore, the synthesis of VOCs originating from secondary metabolism
might only be activated under special growth conditions different from those the cells encounter
during cultivation in laboratory settings. Advances in system-wide analytical techniques [67] and
methods for the identification and awakening of secondary biosynthetic pathways pave the way for
comprehensive elucidation of yeast’s metabolic potential for volatile metabolite synthesis [68-70].
8
Chapter 1
Metabolic pathways of yeast

Yeasts release a manifold of volatile metabolites, which contribute to their rich odor. The VOCs are
produced from primary and secondary metabolism and include hydrocarbons, heterocycles,
aldehydes, ketones, alcohols, phenols, thioalcohols, -esters, terpenes amongst others (Figure 1).
VOCs produced from primary metabolism pathways, including glycolysis, proteolysis, and lipolysis
are thought of as waste materials and their production as a means of detoxification, while VOCs
involved in defense or communication mechanisms are assigned to secondary metabolism [71, 72].
Figure 1: Overview of volatile metabolites released by yeast. Depicted are few representatives and
their origin from central carbon metabolism [1].
In the following we give a coarse overview about currently known volatile metabolites and the
biosynthetic pathways of yeast. Given the focus of prior volatile research the presented volatile
spectrum is biased for VOCs impacting on the flavor of fermented food as previously mentioned.
VOCs derived from carbohydrate catabolism and fermentation

Ethanol is the most well-known volatile metabolite of yeast. It is produced under anaerobic conditions
or aerobically at high glycolytic fluxes to re-oxidize NADH produced by glucose oxidation. Acetic
acid, on the other hand, is formed if alternative pathways do not satisfy the anabolic NADPH demand
and is, for example, observed during recombinant protein production [73, 74]. Acetaldehyde is the
direct metabolic precursor of ethanol and acetate and at low concentrations contributes a fruity flavor
to beer, wine, and other alcoholic beverages. At higher concentrations, however, it possesses a
pungent, irritating odor [75]. The physiological role of acetaldehyde secretion is not well understood
and it is argued to simply be a leakage product of ethanol fermentation [76]. However, it has also
been reported that low amounts of acetaldehyde in the fermentation medium support growth of some
yeast strains [77].
2,3-butanediol is another fermentative volatile byproduct of yeast along with its precursor
metabolite acetoin. Their synthesis might also be a means to adjust redox cofactor levels but it has
9
also been reported that acetoin, in a mix with other yeast VOCs, attracts the fruit fly Drosophila
melanogaster with which some yeasts undergo a symbiotic relationship [78].
VOCs produced by amino acid synthesis and degradation

The amino acids valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and tyrosine are
catabolized by the Ehrlich pathway [20] (Figure 2). The resulting aliphatic and aromatic alcohols and
corresponding acids are known as fusel alcohols and fusel acids [79]. As with acetaldehyde, at high
concentrations fusel alcohols impart off-flavors (thus the German term fusel, bad liquor), while low
concentrations of these compounds (and their esters) contribute to the flavor and aroma of fermented
foods and beverages [80]. During beer and wine fermentation, yeasts utilize the amino acids contained
in the wort or grape must as main nitrogen source. In the first step of their degradation the amino
acids are deaminated to α-keto acids (Figure 2). The ketoacids are further decarboxylated and then
either reduced or oxidized to the respective fusel acid or alcohol. [2]. While proposed already in 1907
[79], the genes encoding the enzymes of this pathway are still to be entirely explored [81]. Also, the
physiological role of fusel oil formation and its regulation is not well understood but it is argued that
tuning the alternative oxidation and reduction steps of this pathway helps to maintain the
NADH/NAD+ ratio and to balance redox cofactors [81, 82]. Moreover, the irreversible
decarboxylation of the keto acid acts as a thermodynamic pull as low levels of this intermediate allow
shifting the equilibrium of the reversible deamination to the right side, thereby fostering complete
degradation of the amino acids and maximal nitrogen supply.
The fusel alcohols 3-methyl-1-butanol (isoamyl alcohol) and 2-methyl-1-propanol (isobutanol),
the end products of the degradation of the branched amino acids leucine and valine bear potential as
biofuels [83], while the aromatic 2-phenylethanol, the catabolic end product of phenylalanine is used
as rose-like fragrance. Catabolism of the sulphur containing amino acids, methionine and cysteine
via the Ehrlich pathway releases volatile sulphur compounds, e.g., methional and dimethyldisulfide.
These VOCs are responsible for the flavor in potato-based snacks and are also formed by the heat-
induced Maillard reaction. To a lesser extent the higher alcohols are synthesized via an alternative
anabolic pathway from pyruvate [84].
The carboxylic acids are easily converted to acetate esters by condensation with acetyl coenzyme
A (CoA), with ethyl acetate (solvent-like aroma), isoamyl acetate (banana aroma), isobutyl acetate
(fruity aroma) and phenyl ethyl acetate (roses, honey) being important flavor active examples found
in fermented beverages [85]. The physiological role of these esters is not elucidated, yet. One
hypothesis is that ester formation serves the recycling of CoA under anaerobic conditions when
acetyl-CoA consumption in the TCA cycle is suppressed [86]. On the first sight, production of the
volatile esters might be costlier for the cell than acetate secretion, but it has the advantage of
preventing the accumulation of this toxic weak acid which at high concentrations entails more
detrimental growth defects. Studies, in which acetyl-CoA overproduction increased isoamyl acetate
synthesis, while increased acetyl-CoA consumption decreased acetate ester formation, support this
theory [87, 88]. Also, antimicrobial effects have been reported for some esters [55].
10
Chapter 1
Figure 2: The Ehrlich pathway. Biochemistry and main genes involved in amino acid degradation
(A) and intermediates and products derived from involved amino acids (B). Figure adapted from [2,
3] . Figure previously published in [1].
Diacetyl and 2,3-pentanediol, VOCs with butter or toffee-like flavor are undesired byproducts (off-
flavors) in beer fermentation and are formed when 2-acetolactate or 2-oxobutyrate, intermediates of
valine and isoleucine biosynthesis, accumulate. When these compounds diffuse into the medium, they
are non-enzymatically decarboxylated to diacetyl and 2,3-pentanediol [89]. As long maturation times
are required for diacetyl degradation, minimizing the synthesis of this byproduct is desired. This can
be achieved by adjusting the valine concentration in the wort as valine uptake results in feedback
inhibition of acetohydroxy acid synthase, responsible for the catalysis of the diacetyl and 2,3-
pentanediol precursors [90].
VOCs from fatty acids biosynthesis and degradation

Another source of volatile metabolites is fatty acid synthesis and degradation. The most abundant free
fatty acids released during yeast fermentations are hexanoic, octanoic, and decanoic acid [91].
Besides these medium chain fatty acids propanoic and butanoic acid have been detected in the
headspace of yeast cultures, which might, however be rather byproducts from fermentation [91]. Fatty
acids are decarboxylated to alkanes, 1-alkenes, and methyl-ketones or reduced to the respective
aldehydes and 1-alkanols [92]. Aldehydes can also be formed by autoxidation of fatty acids [93].
11
Lactones, e.g., γ-decalactone, γ-butyrolactone (peach flavor), 4-hydroxy cis-6-dodecenoic acid-γ-

lactone (buttery flavor) [94] are derived from fatty acids via hydroxylation, β-oxidation and a final
intramolecular esterification step that leads to molecule cyclization [95].
The acyl-CoA derivatives of volatile medium chain length fatty acids react with ethanol to form
fatty acid ethyl esters (FAEE). Compared to the biosynthetic pathways for the acetate esters described
above much less is known about the responsible enzymes and genes for these conversions and the
regulation of these pathways. This might be due to the comparable lower levels FAEEs and the
accordingly difficult measurements. These esters are, however, of similar industrial interest due to
their fruity or floral flavors [85]. FAEE synthesis is hypothesized to be a detoxification mechanism
under anaerobic conditions under which fatty acid synthesis is inhibited and medium chain length
fatty acids are prematurely released from the fatty acid synthase. Ester formation avoids accumulation
of these toxic acids and the diffusion of the volatile FAEE out of the cells further helps to shift the
equilibrium of the esterification reaction towards ester formation [96].
VOCs derived from terpene biosynthetic pathways

Terpenes are unsaturated hydrocarbons derived from isoprene and are mainly produced by plants.
Their oxygenated derivatives, the terpenoids, have a major impact on the sensory profile of many
foods and beverages [97].
It is known that yeasts transform terpenes present in the must into terpenoids thereby contributing
to the flavor profile of wine. One example is the transformation of the monoterpene α-pinene to
verbenol, a secondary allylic alcohol with camphor and mint-like flavor properties [98]. Also,
biotransformations of the terpenoids linalool, α-terpineol, nerol, and geraniol by S. cerevisiae and
other yeast have been reported [99] but the responsible enzymes for these conversions are ill-defined
as only one Saccharomyces cyclase, the lanosterol cyclase Erg7p, is currently known [100]. The
analysis of the biocatalytic potential of yeast or other microbes for the synthesis of terpenes and
terpenoids is also of interest for the pharmaceutical industry as many of these compounds show
promising biological activity [101].
Yeasts were assumed to be incapable of de novo synthesis of terpenes or terpenoids, with the
exception of farnesol and geraniol. Yet, Carrau et al. showed the production of a variety of terpenes
by wine yeasts of the genus Saccharomyces from simple carbon sources including linalool, α-
terpineol, citronellol, nerol, furan linalool oxides, pyran linalool oxides, amongst others [4]. Carrau
et al. further observed different responses in the synthesis of the terpenoids to varying supply of
nitrogen and oxygen compared to each other and to sterol synthesis and concluded that these
compounds are synthesized by two separate pathways. They hypothesized that the cytosolic
mevalonate pathway whose end product is drained for sterol synthesis in the endoplasmic reticulum
contributes to the synthesis of the sesquiterpenes farnesol and nerolidol, while the synthesis of the
monoterpene geraniol might take place in the mitochondrion. They proposed that its synthesis is
linked to the catabolism of leucine, which is converted to 3-hydroxy-3-methylglutaryl CoA (HMG-
CoA) via 3-methylcrotonyl-CoA carboxylase (MCCase) (Figure 3). This enzyme has not been
experimentally confirmed to be existent in Saccharomyces but strong genetic evidence exists for
12
Chapter 1
Aspergillus nidulans [102] and it is known to be present in the mitochondria of plants and mammals
[103, 104]. The further conversion of geraniol to linalool, nerol, and citronellol is proposed to take
place in the vacuole as the low pH might favor these reactions [105]. Again, no genes have been
identified yet [100]. The missing information in the VOC biochemistry is in our opinion also strongly
linked to the somewhat cumbersome experimental protocols consisting of sample preparation and
analysis. The increasing interest in VOCs develops parallel with the advancements of analytical
techniques, which are summarized briefly.
Figure 3: Mitochondrial monoterpene biosynthesis via the leucine catabolic MCC pathway and
possible relationships with sterol formation in Saccharomyces cerevisiae as proposed by Carrau et
al. [4]. Figure adapted from [4] . Previously published in [1].
13
Analytical methods for the identification of VOCs

The analysis of all VOCs an organism produces or even more the quantification of these VOCs faces
several challenges entailing the so far limited knowledge of this compound class. Samples taken from
the headspace of a culture are less complex compared to metabolome samples extracted from cells or
culture broth, principally easing separation and measurement, nevertheless the chemical diversity of
the analytes complicates the analysis as most techniques do not allow complete coverage. The volatile
nature and dilute concentrations of VOCs further aggravate their measurements – both in terms of
sampling and analytics – requiring efficient concentration techniques and / or sensitive analytics.
Also, the analytical devices suitable for the measurement of volatiles are partially very specific,
expensive or require expert knowledge for proper use impeding easy access to VOC measurements.
Compound identification by mass spectral analysis is a further challenge because existing databases
such as NIST or Wiley contain mostly VOCs of plants, animals and insects and do not cover exotic
microbial VOCs. Furthermore, some compounds are so far completely unknown and have to be
structurally elucidated, requiring mass spectrometry (MS) analysis of stable isotope labeled
metabolites or nuclear magnetic resonance (NMR) spectrometry. Sodorifen, a VOC emitted by the
rhizobacterium Serratia plymuthica, is one such novel compound, which has only been discovered
recently [106, 107].
However, new or advanced sample extraction and analytical techniques allow today to broadly
capture and survey volatile metabolites produced in microbial fermentations, some of which are
briefly reviewed in the following.
Ion mobility spectrometry

In ion mobility spectrometry (IMS), ionized gas phase molecules are separated based on different
mobilities that are determined by the applied electric field, their size, mass, and shape, the IMS has
been in use for decades [108]. Through the advancing computational development, ion mobility
spectrometry technology could be used out of the lab since the 1970s when it was still called plasma
chromatography. However, due to the increasing computation power that developed during the 1990s,
major changes occurred in IMS technology and the understanding of its fundamental principles [5].
IMS is used to detect volatile compounds and is already used in military-, medical-, and customs
control applications. In military, the IMS is used nowadays to detect chemical warfare agents and to
warn soldiers, before the concentrations of chemical warfare agents become harmful [109]. At
airports, the IMS is used to detect illegal drugs and explosives in a matter of seconds [110]. It is even
possible to detect explosives in concentrations as low as 80 ppt, which has been proven for the
explosive 2,4,6-trinitrotoluene (TNT) [108, 111]. In medicine, the detection of lung carcinoma can
be achieved within minutes using the IMS [112-114]. Even the possibility of process control has been
illuminated previously to this thesis [115]. There have also been other areas of application in the lab,
as for example the detection of lipids and characterization of proteins. For more information about
applications the reviews by Collins and Lee from 2002 and the reviews by Cumeras et al. from 2015
are recommended [108, 116, 117].
14
Chapter 1
Compared to gas chromatography the ion mobility spectrometry is a relatively unknown technique
in life sciences. There are eight main methods that can be used to measure the ion mobility: 1. Drift
time IMS (DTIMS), 2. Traveling–wave IMS (TWIMS), 3. High field asymmetric waveform IMS
(FAIMS), 4. Trapped IMS (TIMS), 5. Open loop IMS (OLIMS) also called Aspiration IMS (AIMS),
6. Differential mobility analyzers (DMA), 6. Transversal modulation IMS (TMIMS) and 8. Overtone
mobility spectrometers (OMS). Since this is not the focus of this thesis, only the basic DTIMS will
be illuminated in more detail. For further information concerning ion mobility spectrometry, the
review “Review on Ion Mobility Spectrometry” in two parts is recommended [108, 117].
Solid, liquid and gaseous samples can be analyzed by IMS. After injection of the sample, the
analytes are ionized in the ion source. Radioactive sources [118], corona dischargers [119],
photoionization sources [120, 121], matrix assisted laser desorption ionization (MALDI) sources
[122], and electrospray ionizers [123] are most commonly used for analyte ionization. The ionized
molecules are guided by an electric field and are usually held back by an ion shutter [124, 125]. The
shutter opens periodically and releases the bundled ions into the drift tube. In the classic DTIMS the
electric field constantly pulls the ions towards the detector, located at the end of the drift tube. The
detector is classically a faraday plate but MS detectors are becoming more common (Figure 4) [116].
Figure 4: Working principle of a drift tube-IMS; adapted with permission of Journal of Physiology
and Pharmacology from [5], previously published in [1, 6].
In detail, in the classic DTIMS, the drift tube is constantly flushed by drift gas, opposing the ions.
Based on the attributes of an ion more or less collisions occur and some ions travel faster through the
drift tube than others. This relationship is called ion mobility and defined as K. The ion mobility is
dependent on the electric field intensity E. The drift velocity (ߴ݀ሻ is directly proportional to the ion
mobility and the electric field intensity [126]:
ߴ݀ ൌ ‫ܧܭ‬ (2)
15
The ion mobility can also be calculated using the length of the drift tube (L), the voltage drop
across L, and the time it takes an ion to move through the drift tube (td):
‫ܮ‬ଶ (3)
‫ܭ‬ൌ
ܸ ଶ ‫ݐ‬ௗ
At the molecular level K can be defined as fundamental relationship between ion mobility and the
collisions that occur.
͵‫ݍ‬ ʹߨ ଵ ݉ ൅ ‫ ܯ‬ଵ ͳ (4)

‫ ܭ‬ൌ ‫ כ‬ሺ ሻଶ ‫ כ‬ሺ ሻଶ ‫כ‬
ͳ͸ܰ ݇ܶ ݉‫ܯ‬ π
With q as charge of ions, N as number density of the drift gas, k as Boltzmann’s constant, T as
absolute temperature, m as mass of the ion, M as mass of the drift gas, and Ω collision cross section
of the ion in the drift gas. Since the temperature and the pressure are often different in different
devices, respectively locations, the reduced ion mobility was introduced:
ܲଵ ܶ଴ (5)
‫ܭ‬଴ ൌ ‫ܭ‬
ܲ଴ ܶଵ
Here P0 is 101,325 kPa and T0 is 273 K [126-128].
In classical DTIMS the drift gas is also ionized and in combination with residual water, reaction
ion peaks (RIP) form. These peaks do not represent pure nitrogen or water, but ion clusters that are
dependent on the used drift gas. For nitrogen containing gasses e.g., air, this is how the measured
clusters are formed [129]:
N2 + e Æ N2+ + 2e (1)
N2+ + 2N2 Æ N4+ + N2 (2)
N4+ + H2O Æ H2O+ + 2N2 (3)
H2O+ + H2O Æ H3O+ + OH (4)
H3O+ + H2O + N2 Æ H+(H2O)2 + N2 (5)
H+(H2O)n-1 + H2O + N2 Æ H+(H2O)n + N2 (6)
The choice of drift gas and to some extent the water content can be influenced by the experimenter.
Higher water concentrations cause the formation of larger ion clusters that have a lower ion mobility,
and hence lead to longer drift times [130]. Thus, for IMS applications such as online process control
varying water content has to be avoided. This can be achieved using a chromatographic pre-separation
of the sample, for example using a multi capillary column (MCC), which separates water from other
analytes [131].
16
Chapter 1
MCCs consist of about 1000 parallel short columns of 20 cm to 1 m length each. Because of the
large cross-sectional area of these parallel columns, it is possible to work with high gas flow rates (up
to 250 mL/min) resulting in much shorter separation times of MCCs compared to classical GC
capillary columns. The water in the samples does not interact with the column material, it thus directly
passes the MCC and is flushed out of the system while other components have longer retention times
and are introduced into the IMS. This pre-separation step increases the overall separation performance
and is equally suited for online measurements as unhyphenated IMS as it can be used without prior
sample preparation [131].
Secondary electrospray ionization mass spectrometry

A new and highly efficient way of measuring volatile compounds online is the use of a secondary
electrospray ionization (SESI) source coupled to an Orbitrap mass analyzer. Compared to IMS, this
MS based system enables the identification of analytes by fragmentation of the compounds and
analyses of the resulting mass spectra [132-135].
To prevent condensation of metabolites with low boiling points, analytes are guided through a
heated transfer line into the ion source. Sample ionization via SESI proceeds in two steps. First a
liquid (usually 0.1% formic acid in water) is ionized by an electrospray in which the liquid is exposed
to a high voltage to form an aerosol of charged, minute droplets. Then, the sample gas flow is
introduced into this mist and the secondary ionization occurs through ion-molecule interaction. A
recent optimization of this system by Barrios-Collando et al. resulted in a 5-fold improvement in
sensitivity compared to standard SESI [136]. The ionized sample is guided into the Orbitrap [132],
where the ions oscillate along the z-axis of a spindle-like inner electrode. Based on their mass to
charge ratio (m/z) the analytes have different frequencies in traveling back and forth along the z-axis.
This frequency is detected by the outer barrel like electrode.
SESI-MS has successfully been used to discriminate between smokers and non-smokers [133] or
to screen the volatile space of plants. For the latter study, plants were cultivated in a closed glass
beaker flushed with a constant air flow, which was directly introduced into the SESI-MS device. In
these experiments, about 400 VOC species were identified which correlated with the diurnal cycle of
Begonia semperflorens and about 1200 VOCs were related to mechanical damages of the plant [134].
Proton-transfer-reaction time-of-flight mass spectrometry

Proton-transfer-reaction time-of-flight mass spectrometry (PTR-TOF-MS) is another online and
highly sensitive measurement technique for VOCs. The ionization of VOCs by H3O+ ions, applied
here, is similar to that in IMS except that PTR-MS generally does not use a radioactive, but a hollow
cathode ion source, which generates H3O+ ions from water through electric discharges. The gaseous
sample is introduced into the drift tube containing the ionized water, and analytes with a higher proton
affinity than water are protonated. The ionized molecules are periodically guided through a transfer
lens system and accelerated into the TOF-MS, where they are electrically deflected onto a specific
trajectory. Based on their mass to charge ratio the flight time of the ions differs. Thus, through every
pulse created by the lens system a complete mass spectrum can be measured [137]. PTR-TOF-MS
17
and PTR-MS (usually equipped with a quadrupole mass analyzer) devices have been used to detect
microorganisms, including human and plant pathogens [138-143]. Also lactic acid fermentation
during the production of yogurt and the storage processes of milk and meat were monitored [139,
144-147]. Moreover, VOC profiles of dough fermentations with various yeasts and flours were used
to determine product quality. For example, specific masses of three unidentified compounds were
suggested as biomarkers for the optimization of the aromatic quality of bread dough [139, 148-150].
Gas-chromatography based methods

The most common method for offline measurements of volatile metabolites is gas-chromatography
after appropriate sample extraction (see section 3.5) although in some cases direct sample injection
is also possible.
When liquid samples are to be measured, they are evaporated in the injector unit, while gaseous
samples can directly be injected onto the chromatographic column. The chromatographic separation
is based on analyte interaction with the stationary phase of a coated gas chromatographic column.
Thin capillary columns of 10 m to 100 m length, an inner diameter between 0.1 and 0.33 mm and a
film thickness between 0.1 and 0.33 μm are typically used, which limits the maximal gas flow rates
to 1-10 mL/min. The analytes are transported through the column by a carrier gas (e.g., helium or
hydrogen) and are separated depending on their interaction with the column material. The interaction
strength depends on the chemical properties of the analytes and the conditions in the column,
especially temperature and pressure, which in sum lead to different retention times and hence
separation of the analytes [151-153]. For increased analyte resolution, two-dimensional gas
chromatography can be employed, which connects the first column with a second, shorter column,
with a typical length of 1-2 m, in daisy-chain. The second column is usually coated with a different
stationary phase, consequently leading to different retention times of the analytes [154-156]. Two-
dimensional gas chromatography has already been used to investigate the volatiles in wine in great
detail and allowed to analyze hard to separate chemical compounds, such as ethyl carbamate [157].
Common GC detectors are flame ionization (FI) and mass spectrometer (MS). In VOC research,
olfactometers are also widely used in conjunction with specially trained persons to directly evaluate
the olfactory effect of single or complex mixtures of VOCs [158, 159]. This approach exploits the
human sense of smell and is commonly used to determine the sensory activity of VOCs and odor
threshold concentrations [160]. Also, product quality can be assessed which is difficult to achieve
without an olfactometer, since many substances smell good only in a limited range of concentration
and importantly in combination with other substances. GC-olfactometry has been used to analyze the
complex VOC mixtures of wine and to link VOCs to the organoleptic properties [161]. A plethora of
further GC-based applications for volatile metabolite measurements have been reported and are
reviewed, for example in [162].
18
Chapter 1
Offline-sampling
IMS, PTR-TOF-MS, SESI-MS and - in limited applications - GC-Q-MS are suited for online
measurements without any upstream sample preparation. While often advantageous, depending on
the scientific problem, online measurements are not always necessary and sample preparation can be
useful to concentrate low abundant analytes. Subject to the extraction method used, it is possible to
selectively concentrate compound classes. On the downside, this selective binding leads to loss of
analytes that do not interact with the solvent or fiber.
A common technique for sample preparation is thermal desorption spectroscopy (TDS) in which
a column packed with adsorbent is flushed with the gaseous sample. Depending on gas flow rate and
sampling time the amount of analyte adsorbed to the packing material can be adjusted. It is also
possible to directly adsorb metabolites during the fermentation process by using special stirring bars,
made of adsorbing material. This technique was previously used to measure off-flavor producing
components in wine and to profile the metabolites of different fungi [163, 164].
Analyte extraction via purge and trap is achieved by stripping the liquid sample with an inert gas,
which is then passed through a sorbent trap to bind the extracted metabolites. These traps are filled
with equivalent adsorbent material as used in TDS. The analytes are released by thermal desorption
and transferred onto the GC column. This approach has been used to measure volatiles in red wine or
to determine the impact of altered alcohol acetyltransferase expression on the synthesis of esters and
alcohols of different yeast strains [165, 166].
In solid phase microextraction (SPME), a fiber of the adsorbing material is loaded with volatile
metabolites by introducing it into the headspace of the liquid sample. SPME has been used to analyze
diverse biological samples including fungi and human samples for cancer marker detection [159, 167,
168]. After loading of the adsorbent, the TDS column or SPME fiber is connected to a gas flow
leading to a GC-MS system. For efficient desorption of the bound analytes, the adsorbent is heated.
The disadvantage of this sample preparation approach is that all analytes are desorbed and the sample
can only be measured once [167]. Common adsorbents are polyacrylate, CARBOWAXTM
(polyethylene glycols, methoxypolyethylene glycols), CarboxensTM (carbon molecular sieve),
polydimethylsiloxane, Tenax® TA (poly(2,6-diphenyl-p-phenylenoxid)), and silica gel [164, 168].
Direct immersion single drop microextraction (DI-SDME) is a miniaturization of classical liquid-
liquid extraction which greatly reduces the extractant to sample ratio and achieves higher analyte
concentrations [169-172]. A water-immiscible drop hanging at the tip of a needle is submersed into
the liquid sample. The drop is not mixed with the liquid but keeps attached to the needle. When the
extraction is at equilibrium the drop is withdrawn back into the needle and directly injected into the
GC-MS. The extraction efficiency can be enhanced by continuously pumping the fluid sample
alongside the drop as it is done in continuous flow microextraction (CFME) [169, 173]. A variant of
this technique for gaseous analytes is headspace single drop microextraction (HS-SDME) in which
the drop is placed in the headspace of the liquid sample and only volatile metabolites present in the
gas phase are extracted [169, 174]. SDME theoretically allows extraction of all volatile metabolites
as the solvents can be freely chosen. Only the following requirements apply: The viscosity of the
extraction solvent has to be high enough to form a drop that sticks to the needle. The solvent used in
19
DI-SDME and CFME has to be insoluble in the aqueous sample and the extractant for HS-SDME has
to have a sufficiently low vapor pressure to prevent evaporation at the applied temperature.
Commonly used solvents are toluene, n-octanol, decane and benzyl alcohol. Automated SDME
workflows have been reported, but the technique is still not routinely used in many laboratories [169,
175]. For more detailed information about SDME the review by Xu et al. is recommended [169].
20
Chapter 1
Pros and cons of methods for VOC detection

The analytical techniques presented here have pros and cons, and the user has to choose the most
appropriate method according to the application requirements but also available budget (Table 1).
Ion mobility spectrometers combine relatively low costs (acquisition, operation, maintenance)
with a low detection limit, and the operation at atmospheric pressure allows hand-held IMS devices
[109]. IMS can be used for online measurements as ionization is continuous and no derivatization is
required. If the IMS is used without a chromatographic pre-separation, 20 spectra per second can be
acquired. Combined with a MCC the acquisition is in the range of few minutes, still fast compared to
common GC analyses, which often take 30 minutes or longer. However, IMS alone does not allow
compound identification. This is possible with hyphenated IMS-MS devices which however, come
with the disadvantage of stationary use and higher price [176]. As GC-MS techniques have been used
for a long time and for a variety of analytical applications, comprehensive, commercial databases
such as NIST are available, while only comparably small databases exist for IMS applications [177].
Data analysis is further complicated by modifications of ionized analytes that occur by collisions with
other ions and uncharged drift gas during passage through the drift tube [178]. This process is
influenced by the ion source, the strength of the electric field and as well as the length of the drift
tube impeding the set-up of a consistent database. As a consequence reliable compound identification
currently requires the measurement of pure standard substances but endeavors exist to establish
workable IMS-MS databases [130]. A further disadvantage of IMS is the high gas requirement
(carrier gas, drift gas) compared to GC if no air-loops are used.
In GC-MS systems often quadrupole (Q) mass analyzers are the detectors of choice. GC-Q-MS
systems are usually more expensive than IMS but much cheaper than those with TOF or Orbitrap
detectors (Table 1). In GC-MS analyses compounds are identified based on their mass, fragmentation
pattern and retention index. It is possible to scan the full mass spectrum or to filter single masses in
selected ion monitoring (SIM) mode. The mass resolution of a quadrupole MS is limited to about half
a mass unit thus identification of non-fragmented analytes is rarely possible. Since the samples
usually need to be water-free before entering the GC column, online measurements are often not
possible. Also, the mass spectrometer is not mobile as it requires carrier gas and a turbo pump for the
high vacuum of about 1-100 mTorr.
TOF detectors are extremely fast and can measure mass spectra in the kHz region. In current
devices, however, the actual data acquisition time is limited by the sample inlet time and ionization.
Compared to Q-MS, TOF detectors have a higher mass range (up to 36 000 m/z) and a higher mass
resolution [179]. The mass resolution of the TOF is sufficient to estimate the elementary composition
of most analytes, which can be used to tentatively identify detected compounds. However, if a soft
ionization method such as PTR is used isomers cannot be distinguished as the metabolites rarely
decompose into informative fragments.
Orbitraps are the most expensive but also the most sensitive mass analyzers presented here. They
are also the newest detectors on the market, since the first publication describing an Orbitrap was
only published in the year 2000 [180]. Furthermore, Orbitraps have the highest mass accuracies, high
mass ranges and the highest mass resolutions of all the detectors introduced here. Through the high
21
mass resolution, it is possible to separate peaks that even in TOF detectors are measured as one peak.
The higher mass accuracy results in improved determination of the elemental composition, and
consequently more conclusive compound identification. Structural identification can further be
achieved by fragmentation of selected compounds. However, since there is usually no pre-separation
it is not useful to fragment all metabolites while measuring complex samples.
Table 1. Features of different analytical devices for volatile compound measurement. Data on
resolving power taken from [181-184], all other data taken from product sheets of current devices;
LOD, limit of detection. Previously published in [1].
SESI-
Device IMS GC-Q-MS PTR-TOF
Orbitrap
Price category $ $$ $$$ $$$$
Resolving power 60 2,8 up to 5,000 60,000 to
>100,000
Scanning speed 20 Hz 97 Hz 10 Hz (TOF 20 Hz
200 kHz)
Portability limited none none none
LOD ppt ppt ppt ppt
Application Environmental (Online) Real-time analyses
examples monitoring (e.g., VOC analyses of monitoring of of plant VOC
water plant or human microbial emission,
contamination), samples, pesticide contamination of breath analysis,
breath analysis screening [186- food, detection of [133, 134]
(biomarker 188] bacterial infections
detection), and drug level
detection of control via breath
chemical warfare analysis [138, 139,
agents [109, 113, 141, 144, 147,
131, 185] 189]
Methods for the delineation of VOC metabolic pathways

As outlined earlier in the introduction volatile metabolites are used in interspecies communication,
can signal metabolic shifts and are of industrial interest for use as fragrances, food flavor enhancer or
biofuels. We described tools for the investigation of the volatile spectrum of yeast, but to understand
the link between specific VOCs and the metabolic mode and to eventually exploit the metabolic
machinery of an organism for the production of valuable VOCs, it is crucial to also know the
metabolic and regulatory network involved in the biosynthesis of these compounds. The current
knowledge of microbial VOC biosynthesis and regulation is rather scarce and scattered although
recent endeavors exist to aggregate microbial VOC data including metabolic pathways in one
22
Chapter 1
database [66]. These knowledge gaps even exist for the industrial workhorse and eukaryotic model
organism S. cerevisiae. Having been investigated in numerous scientific studies and 78% of its 6,604
ORFs been assigned to an enzymatic, regulatory or further cellular function [190], its biochemistry
is generally well established, documented in several databases [191-193], and well-curated genome-
scale models exist [194-196]. Remarkably, however, biosynthetic pathways of volatile metabolites
are only moderately covered. This incompleteness of metabolic models and databases became
apparent when checking VOC coverage of metabolic pathway databases and models: Of 100 VOCs
identified in the literature to be synthesized by yeast only 20% were contained in yeast specific
metabolic databases and the S. cerevisiae latest genome-scale model [185].
A suite of computational and experimental methods is available to close this lack of knowledge,
i.e., to identify the metabolic network underlying the synthesis of these metabolites. For the in silico
identification two approaches exist: The bottom-up network reconstruction aims to predict possible
pathways from available metabolic reaction networks or databases, while the top-down approach
takes condition-specific omics data and extracts possible networks using statistical and bioinformatics
analyses. The latter does not rely on a priori knowledge of network component interactions and is
therefore well suited for the prediction of less studied biosynthetic pathways and regulatory networks
such as those of volatile metabolites. Also, powerful methods exist that combine both approaches
[197-199]. Metabolic networks predicted by either approach still have to be manually pruned and
experimentally verified.
After integration of the novel pathways into an existing metabolic model, the extended model can
be used for the computation of engineering strategies to improve or prohibit VOC synthesis or to
interrogate interactions between VOC synthesis and the overall metabolic behavior of the cell.
Metabolic models further allow to address questions, such as to why the organism releases volatile
metabolites. For example, the occurrence of the Crabtree effect in S. cerevisiae, that is aerobic ethanol
formation, was in silico reproduced by stoichiometric models taking into account the investment costs
for the synthesis of enzymes or mitochondria. These simulations disclosed a trade-off between these
anabolic costs and the metabolic yield of alternative fermentative pathways [200, 201] at high
glycolytic fluxes. In this way, experimentally testable hypotheses can be generated and engineering
strategies derived to improve the metabolic activity towards a defined target function. For a detailed
description of the use of metabolic models, we refer to [202, 203].
Bottom-up approaches
The basis of bottom-up approaches for the prediction of biosynthetic pathways is a (genome-scale)
metabolic model of the organism under study. Such a metabolic model is generated based on the
information provided by the annotated genome and experimental data, gathered from databases and
the scientific literature and rationalizes and structures the existing knowledge, scattered in these
resources. For metabolic pathway prediction, stoichiometric models, which do not capture kinetic
properties, are sufficient. These models store the stoichiometry of the enzymatic repertoire of the cell
and the gene-enzyme-reaction relationship and thereby allow to explore the metabolic capabilities of
an organism and the effect of environmental or genetic perturbations.
23
To comprehend metabolic models with enzymatic capabilities for the synthesis of additional
metabolites, a retrosynthetic approach is employed which parses species-unspecific metabolic
databases such as KEGG, MetaCyc or BiGG [204-206] for enzymatic transformations that allow
connecting the novel metabolite to the existing network. Several publicly available or web-based tools
exist for this bottom-up prediction of metabolic pathways [207-209].
The approach presented by Christian et al. [197] differs from these pathway prediction tools in
that it directly derives models that allow the synthesis of a novel compound from a defined carbon
and energy source instead of isolated pathways. The original network is first appended with all
reactions of a universal metabolic database and then stepwise reduced by eliminating previously
added but non-essential reactions to eventually derive at a minimally extended model capable of
target compound synthesis. As the order of reaction removal impacts the model extension, a huge
number of parallel runs is performed. In order to increase the number of biologically meaningful
models in this set and to reduce a posteriori pathway selection and verification, the algorithm
integrates pathway prioritization into the pathway prediction process. This is enabled by ranking all
foreign reactions according to the probability that the catalyzing enzyme is encoded in the organism’s
genome and a preferential extension with high-ranked reactions.
The Biochemical Network Integrated Computational Explorer, BNICE, [210] goes beyond
reconfiguring known enzymatic reactions and uses instead a set of generalized enzymatic reaction
rules to generate metabolic networks built of existing and novel, i.e., previously unobserved,
biochemical reactions and compounds. The use of reaction rules is organized according to the enzyme
classification system but reduced to the first three digits that define the reaction mechanism but do
not constrain the substrate. This approach eases the identification of enzyme candidates for de novo
biochemical reactions. Not relying on metabolic reaction databases, this approach can also predict
non-enzymatic reactions such as decarboxylations or oxgenations. The drawback of BNICE is the
extensive pathway extraction and validation procedure, which follows the network inference step.
Although mainly applied to predict novel pathways for the heterologous synthesis of industrially
interesting products, the general applicability of BNICE allows to equally apply it for the delineation
of uncharted metabolic pathways as recently shown on the example of lipid metabolism [211].
The integration of systems-wide omics data, mRNA, protein or metabolite levels into metabolic
modeling constrains the model to more physiological relevant solutions that is reaction rates more
likely reflecting the in vivo enzymatic activities for the specific experimental setup [212-217]
Embedding such constraints into the pathway computation procedure is desirable as this would
significantly reduce the number of predicted pathways and aid in filtering out meaningful solutions.
Verification of predicted pathways

Top-down and bottom-up network predictions often result in a plethora of pathway candidates, which
have to be pruned, ranked and experimentally verified.
A basic requirement for a functional pathway is a stoichiometric and thermodynamic feasible
synthesis of its product from substrates provided in the growth experiment in which its production
24
Chapter 1
has been observed. This can be verified by (thermodynamics-based) flux balance analysis [218, 219]
and models shown to be stoichiometrically or thermodynamically infeasible are to be rejected.
Experimental identification of pathway specific intermediates by targeted metabolite profiling
gives strong indications for pathway activity. This method is applicable for secondary metabolites,
produced by linear pathways, whose intermediates do not participate in other reactions but less
informative for pathways involving highly connected metabolites. A powerful alternative for the
discrimination of active pathways is an isotope labeling experiment. In the early days of biochemical
pathway analysis, the conversion of metabolites has been traced by tracking the incorporation of
heavy, radioactive isotopes. Today, stable isotopes are used and the resulting isotope isomers
(isotopomers) analyzed by MS or NMR techniques. The introduced tracer has to be carefully designed
so that the activity of alternative pathways results in distinct labeling pattern of pathway intermediates
or end products. Computational methods exist that aid in the design of informative tracer experiments
and data evaluation [220-223].
Top-down approaches integrating transcriptome and/or proteome data directly yield predictions
about genes and enzymes involved in VOC synthesis, while for many bottom-up prediction tools,
gene candidates have to be defined a posteriori. BridgIT is such a computational framework that
builds on BNICE results and suggests enzyme candidates for novel reactions identified in the pathway
prediction process [224]. Following this, BLAST searches are applied to identify gene candidates
encoding these enzymes. The proposed gene-enzyme-reaction relationships are to be validated
experimentally. For this purpose, gene deletion mutants are generated and subsequently evaluated for
VOC synthesis or screened for the accumulation of proposed pathway intermediates. While verifying
or disproving alternative one-step pathways catalyzed by single enzymes is straightforward, finding
gene knockout targets of intertwined and longer pathway alternatives is less intuitive. Computational
tools such as the forced coupling algorithm FOCAL support the identification of genetic and
environmental conditions for conclusive model discrimination [225].
Aims
The overall goal of this thesis was the exploration of the volatile metabolites of baker´s yeast.
Although well known to many of us from daily life, as outlined in the introduction, much is unknown
and only limited information about the interrelationship between phenotype and genotype exist.
To close the gap this thesis shall be listing the known volatile metabolites from the literature and
combine this information with the massive information on the biochemistry of known-volatiles. The
information gathered shall be used for the enhancement of the consensus metabolic yeast model. The
environmental driven production of volatiles shall be investigated, to be able to exploit this non-
invasive signal from yeast metabolism in industrial applications. Here, finding a reporter metabolite
that indicates the metabolic shift from respiratory to respiro-fermentative metabolism. This reporter
shall be used in the YeastScent project, to regulate the feed rate of a yeast fed-batch fermentation to
maximize the biomass yield while maximizing the growth rate, hence by operating the fed-batch
below the critical growth rate that triggers the Crabtree effect.
This transition point was investigated in great detail, but not in respect of volatile production. Here,
the transition from respiratory to respiro-fermentative growth (Crabtree effect) was investigation in
25
industrial-like conditions by volatile and transcriptome analyses. These aims are connected by the
YeastScent project that uses a process model based prediction for fed-batch pump control with a
volatile reporter metabolite as proxy for yeast metabolism.
26
Chapter 2
Results
Expanding the consensus yeast model by volatile metabolism
Contributions:
The study was designed by Christoph Halbfeld, Birgitta E. Ebert and Lars M. Blank. The experiments
were performed by Christoph Halbfeld, Birthe Halmschlag and Niklas Kitschen. The data was
evaluated by Christoph Halbfeld and Birgitta E. Ebert.
Chapter 2
Results
Expanding the consensus yeast model by volatile metabolism
Summary
The consensus metabolic yeast model has so far been lacking the representation of the volatile
metabolite space. To overcome this shortcoming, a literature investigation of all known volatile
metabolites produced by S. cerevisiae was performed and 92 volatile metabolites were found
of which 78 did not exist in the latest metabolic model. To connect the newly found metabolites
to the metabolic network, 219 reactions were added to the consensus yeast metabolic model
using computational tools as well as manual integration of pathways. Stoichiometric and
thermodynamic feasibility of steady state production of the amended volatile metabolites was
verified with flux balance analysis and thermodynamics-based flux analysis. 12 reactions were
further experimentally verified by enzymatic assays of knockout mutants, which contained only
one enzyme with alcohol dehydrogenase activity. The new version of the consensus yeast
model, Yeast8, covers the yeast volatile metabolite space and represents the necessary basis to
shed light on the metabolic constraints underlying volatile metabolite formation, offering new
possibilities in modeling for example to create yeasts with a special aroma.
33
Results
Introduction
Through computational advancements it became possible to simulate microbial behavior in
silico. However, performing these simulations requires comprehensive knowledge about the
biochemical reactions that occur inside a cell. For the well investigated baker’s yeast
Saccharomyces cerevisiae, such knowledge is already accessible and has been converted into a
metabolic model in a standardized informatic language, the systems biology markup language
(SBML). The first consensus yeast model was based on two older models the iMM904 [226]
and the iLL672 [196]. The new model was created using SBML and MIRIAM standards. The
new model introduced name conventions as well as identifiers and is based on S. cerevisiae
S288C [227]. The first major enhancements were made, with version 4 of the model, which
was appended with lipid metabolism [228]. In version 5 of the model, the sphingolipid
metabolism was added. Also, a script to test for anaerobic growth conditions was added. As
well in version 5, an apportionment in genome scale models (GEMs) and genome scale network
reconstructions (GENRES) was introduced. This enables the possibility to use models based on
a specific task. For modeling approaches GEMs are used, where assumptions are made that
enable modeling but have no experimental evidence. GENREs can be used if established
knowledge that has been verified experimentally is needed [229]. In version 6 of the model
general enhancements were included, such as the manual curation of the model [194]. In the
7th and still current model, the fatty acid, glycolipid, and glycerophospholipid metabolism of
the yeast model was curated in great detail [230].
Even though the model was constantly updated in the past, the representation of the volatile
space was not yet tackled. Except for the well investigated volatiles ethanol and acetaldehyde
only few volatile metabolites could be found in the model representation [185]. The lack of
volatiles in the model is most likely based on difficulties that come along with detecting volatile
organic compounds (VOCs). Recently better analytical techniques have become available and
thus more and more detailed information on the volatile space of S. cerevisiae will emerge by
time. To fully understand the metabolic processes inside of S. cerevisiae the inclusion of volatile
metabolites to the consensus metabolic yeast model is crucial, therefore here the model is
enhanced by the VOCs that were found by a literature investigation [185].
The addition of new pathways to a model is always challenging. The information gaps
between the added metabolites and the already existing network have to be closed by the most
probable reactions. To close the gaps several tools are available for example, FMM, Pathpred
and Metaroute [207-209]. These tools search for possible pathways using already known
reactions from a database listing data of multiple organisms, e.g., KEGG [231-233]. The
Biochemical Network Integrated Computational Explorer (BNICE) is a tool that suggests new
routes based on biochemical rules. It does not rely on known reactions and, hence, completely
novel pathways can be created [234]. In addition, manual integration of new reactions is an
option. Therefore, enzymes catalyzing similar reactions need to be identified as they could
potentially act on more than one substrate, thereby contributing to novel pathways. Ideally, new
pathways are also experimentally verified, e.g., by 13C-tracer experiments or biochemical
assays.
Here, we set out to comprehensively append the Yeast 7 model with biosynthetic pathway
for volatile metabolites based on extensive scientific literature and database searches as well as
computational analyses. Pathway predictions were checked for thermodynamic feasibility and
34
Chapter 2
partially verified by wet-lab experiments.[219, 235] Another way and more solid is
experimental verification of added reactions, was partially performed here. We focused on
reactions catalyzed by alcohol dehydrogenases (Adhps) as their biocatalytic spectrum of
volatile alcohols or aldehydes synthesis has already been investigated. Although industrial
alcohol production is a highly valuable yeast process; the single enzymes were biochemically
characterized for all tested alcohols.
Material and Methods

Software tools for pathway prediction
For pathway prediction, the web based tools FMM (http://fmm.mbc.nctu.edu.tw/) [207],
PathPred (http://www.genome.jp/tools/pathpred/) [209], and MetaRoute (http://abi.inf.uni-
tuebingen.de/Services/MetaRoute/) [208] were used. In addition, BNICE [234] analyses were
run at the laboratory of computational systems biology (LCSB, Prof. Vassily Hatzimanikatis).
Flux balance analysis (FBA) for the verification of the stoichiometrical feasibility of
metabolic pathways was performed using the Yeast 7.5 Cobra yeast model and the COBRA-
Toolbox for Matlab [230, 236]. Linear programming problems were solved with the
commercially available IBM CPLEX solver (IBM, North Castle, NY, USA). Maximization of
the production of the respective volatile pathway product was used as objective function, the
carbon source uptake rate was set to 1 mmol/(gCDW x h), in order to relate all other fluxes to the
carbon source uptake rate. The lower bound for the ATP production reaction was set to
1 mol/mol glucose uptake. To inhibit falsely added new reactions from disturbing the tests all
new reactions but the tested one were set to zero. Thermodynamics-based flux analysis (TFBA)
was performed at the Laboratory of Computational Systems Biotechnology (LCSB) in
Lausanne Switzerland [219]. A script for performing the TFBA is available there, however it is
not publicly accessible.
Strains, media and plasmids used

S. cerevisiae CEN.PK 113-17a (Euroscarf, Oberursel, Germany) was cultivated in Verduyn
minimal salt medium [237]. The medium contained 20 g/L glucose, 5 g/L (NH4)2SO4, 3 g/L
KH2PO4, 0,5 g/L MgSO47H2O, as well as 1 mL 1000x trace elements and 1 mL of 1000x
vitamin solution. The vitamin solution contained 0.05 g L−1 D-biotin, 1 g L−1 calcium D-
pantothenate, 1 g L-1 nicotinic acid, 25 g L−1 myo-inositol, 1 g L−1 thiamine hydrochloride, 1 g
L−1 pyridoxine hydrochloride and 0.2 g L−1 p-aminobenzoic acid. The trace element solution
consisted of 15 g L−1 EDTA, 4.5 g L−1 ZnSO4·7H2O, 1 g L−1 MnCl2·4 H2O, 0.3 g L−1 CoCl2·7
H2O, 0.3 g L−1 CuSO4·5 H2O, 0.4 g L−1 NaMoO4·2 H2O, 4.5 g L−1 CaCl2·2 H2O, 3 g L−1
FeSO4·7 H2O, 1 g L−1 H3BO3 and 0.1 g L−1 KI. The strains containing the Cas9 containing
plasmid pCfB2312 were cultivated in Verduyn medium containing 200 μg mL-1 geneticin
(G418). E. coli DH5α (Invitrogen, Carlsbad, CA, USA) was grown in LB media containing 10
g/L tryptone, 5 g/L yeast extract and 5 g/L NaCl. All strains used are listed in Table 2.
35
Results
Table 2: Microbial strains

strain relevant characteristics Origin
E. coli
supE44 ΔlacU169 (φ80 lacZΔM15) hsdR17 (rk-mk+)
DH5α Eurofins
recA1 endA1 gyrA96 thi-1 relA1
DH5α pCfB2312 DH5α bearing pCfB2312 [238]
DH5α pGRNA-
DH5α bearing pGRNA-ADH1 this work
ADH1
DH5α pGRNA-
DH5α bearing pGRNA-ADH2B this work
ADH2B
DH5α pGRNA-
ADH3
DH5α pGRNA-
DH5α bearing pGRNA-ADH4C this work
ADH4C
DH5α pGRNA-
ADH5
DH5α pGRNA-
ADH6
DH5α pGRNA-
DH5α bearing pGRNA-GRE2C this work
GRE2C
DH5α pGRNA-
DH5α bearing pGRNA-SFA1 this work
SFA1
DH5α pUSER-
DH5α bearing pUSER-ADH2B this work
ADH2B
DH5α pUSER-
DH5α bearing pUSER-GRE2C this work
GRE2C
DH5α pUSER-
DH5α bearing pUSER-NKO2 this work
NKO2
S. cerevisiae
MATα; ura3-52; leu2-3_112; TRP1; HIS3; MAL2-8C;
CEN.PK113-17a Eurofins
SUC2
CEN.PK113-17a
CEN.PK-113-17a bearing pCfB2312 this work
pCfB2312
sADH1 Δadh2 Δadh3 Δadh4 Δadh5 Δadh6 Δgre2 Δsfa1 this work
sSFA1 Δadh1 Δadh2 Δadh3 Δadh4 Δadh5 Δadh6 Δgre2 this work
sGRE2 Δadh1 Δadh2 Δadh3 Δadh4 Δadh5 Δadh6 Δsfa1 this work
Enzymes and PCR components

All enzymes used in this work were purchased from New England Biolabs (Frankfurt,
Germany), Agilent (Santa Clara, USA) and Fermentas (St. Leon-Rot, Germany). All
oligonucleotides (Table 3 and Table 4) were purchased from Eurofins MWG Operon
(Ebersberg, Germany). Sequencing was also carried out by Eurofins MWG Operon (Ebersberg,
36
Chapter 2
Germany).
Table 3: Primers (table continues over multiple pages)
Primer name Primer sequence Properties
amplification of adh1 for sequencing.
A1 seq fw CTACGAATCCCACGGTAAG
Annealing at: 48 °C
A1 seq rv CTGGCGAAGAAGTCCAAAGC
amplification of adh2 for knockout
A2B_seq fw CGTCTTCAGAGCTCATTG
confirmation. Annealing at: 46 °C
A2B_seq rv GGCATCCTTGACACATTC
amplification of adh3 for sequencing
A3 seq fw GCAATCCACAGCTGCAATC
A3 seq rv CGGTACCACATGGTCTAAC
A4B_seq fw CGTAGTGCGTTACAGTTC
A4B_seq rv ATATTGCGGCTGGTAAGG
A5 seq fw GCCTTCGCAAGTCATTCC
A5 seq rv CAGGACGACAGTACCATTG
A6 seq fw CAATCACACGAAGATTGG
Annealing at: 43 °Cv
A6 seq rv GGAACCTAAAGCACTGTAAG
amplification of gre2 for knockout
Gre2C_seq fw CAACAATTGGCCCTCACCTC
amplification of gre2 for knockout
Gre2C_seq rv TGTGGGGAGACGGGTAGAAG
amplification of sfa1 for sequencing.
S1 seq fw GCTGCTGTTGCGTATGATG
amplification of sfa1 for sequencing.
S1 seq rv CAGGCTTCCAAAGCATCTC
37
Results
gRNA ACTGACTTGCACGCTTGGCAGTTTTAGAGCTA specific 20bp + structural

A1 fw GAA part
ADH2 CTGGGGTCGTTGTTGCTATCGTTTTAGAGCTA specific 20bp + structural
B GAA part
gRNA GTCACACCGATTTACATGCTGTTTTAGAGCTA specific 20bp + structural
A3 fw GAA part
gRNA
TTCATAGGCTTTCTTGATAAGTTTTAGAGCTA specific 20bp + structural
AHD4
GAA part
C
gRNA GTCATAGTGACTTGCACGCGGTTTTAGAGCTA specific 20bp + structural
A5 fw GAA part
gRNA ATGAAGATGCCGCTAGTCGTGTTTTAGAGCTA specific 20bp + structural
A6 fw GAA part
GRE2 GACCCAGTTAACGCCTACTGGTTTTAGAGCTA specific 20bp + structural
C GAA part
gRNA TTATCAGGCTCTGATCCAGAGTTTTAGAGCTA specific 20bp + structural
S1 fw GAA part
gRNA sequencing of single
GGTCAAACGCTGTAGAAG
seq gRNA-plasmids
gRNA TJOS-20 reverse primer for
GATCATTTATCTTTCACTGCGGAGAAG
all rv all gRNAs [238]
amplification of USER
P1 fw CGTGCGAUAGGGAACAAAAGCTGGAGCT
insert
P1 rv CACGCGAUTAACTAATTACATGACTCGA
insert
P2 fw AGTGCAGGUAGGGAACAAAAGCTGGAGCT
insert
P2 rv ACCTGCACUTAACTAATTACATGACTCGA
insert
USER colony PCR to check
GTCAGTGAGCGAGGAAG
check 2 USER reaction
USER
colony PCR to check
check GTACCGGCCGCAAATTAAAG
USER reaction
fw
USER sequencing after USER
GTACCGGCCGCAAATTAAAG
seq reaction
USER sequencing after USER
CCGCTCACAATTCCACAC
seq fw reaction
USER
CTGGCACGACAGGTTTC sequencing of USER site
site seq
USERi TJOS-21, integration of
ATGTTGTGTGGAATTGTGAG
nt fw USER sequence [238]
USERi GAATGCGTGCGATCGCGTGCATTCATGAGTGA TJOS-68, integration of
nt rv GGTAACTCACAT USER sequence [238]
38
Chapter 2
Table 4: Oligonucleotides for homologous recombination

Gene
Enzyme Repair Oligonucleotide
Name
CGTTAAATACTCTGGTGTCTGTCACACTGACTTGCACGCTTGGC
ADH1 YOL086C ATAAGACTGGCCATTGCCAGTTAAGCTACCATTAGTCGGTGGTC
AC
ATATCAAGCTACAAAAAGCATACAATCAACTATCAACTATTAAC
ADH2 YMR303C TATATCGTAATACACAGCGGATCTCTTATGTCTTTACGATTTATA
GTTTTCATTATCAAGCATGCCTATATTAGTA
TCAACGTTAAATATTCTGGTGTATGTCACACCGATTTACATGCTT
ADH3 YMR083W AAACGGCGATTGGCCATTACCTGTTAAACTACCATTAGTAGGTG
G
TGCAAAACAACCATCAACAACAAGTTTACATTTGCAACAACTA
ADH4 YGL256W ATAGTCAAATAAGAAAAAAAAATCGAACGAACTCATAAACGTC
AATTATGCGTGTGCCTTATTTATTTAGTTGTGCG
CGTTAAATATTCTGGTGTTTGTCATAGTGACTTGCACGCGTAAA
ADH5 YBR145W CGGTGATTGGCCATTTCAATTGAAATTTCCATTAATCGGTGGTC
AC
CATTGTGCAGCTGGTCATTGGGGCAATATGAAGATGCCGCTAGT
ADH6 YMR318C CGTTAACATGAAATCGTTGGTAAAGTTGTCAAGCTAGGGCCCAA
GT
CGTTGATATAACGTGTACGATTTTCAAACAAACAGATAGCAGTA
GRE2 YOL151W TCACACGCCCGTAAATACTTTAAATGAAAATAGATAATATTTAT
ATATATTAACGTTATTACAATTATTTTTTATC
CTGTATGCCACACTGATGCGTACACTTTATCAGGCTCTGATCCA
SFA1 YDL168W GATAACTTTTCCCTTGCGTTCTGGGCCACGAAGGAGCCGGTATC
G
Commercial kits
DNA fragments were purified using the GenepHlow Gel/PCR Kit from Geneaid (New Taipei
City, Taiwan). Plasmid purification was carried out using the QiaPrep Spin Miniprep Kit from
Qiagen (Hilden, Germany). Plasmids were extracted using the QiaPrep Miniprep Kit (Qiagen,
Hilden, Germany).
Creating multiple knockout mutants

E. coli was used for plasmid amplification. The cells were made electrocompetent following
the protocol of the Barrick Lab [239]. For electroporation cells were thawed on ice. 1 μL of the
circular vector (~100 ng) was added to the cells and the mixture was added to a precooled
electroporation cuvette. The electroporation pulse (2.5 kV, 200 Ω, 25 μF) was applied and 1 mL
of LB medium was added quickly. The cells were regenerated at 37 °C for 1 h.
The gRNA was integrated into the vector pCFB2312 [238] using the USER technology (New
England BioLabs GmbH, Frankfurt am Main, Germany). To generate multiple gene knockout,
the CRISPR/Cas9 system was used. The Cas9 containing plasmid pCFB2312 [240] was
introduced to S. cerevisiae CEN.PK 113-17A via heat shock transformation [241]. In the next
step, a plasmid, which was constructed using the USER technology (New England BioLabs
GmbH, Frankfurt am Main, Germany) [242-244] and contained 3 gRNAs and 6 μg of 90 or 120
39
Results
bp ds oligonucleotides (see Table 4), was transformed into chemically competent S. cerevisiae
cells [241]. The oligonucleotides were used as template for homologous recombination. The 90
bp oligonucleotides were used for the first successful disruptions and lead to the introduction
of a stop codon into the gene sequences of ADH1,3,5,6 and SFA1. The 120 bp oligonucleotides
were used for complete deletion of ADH2, ADH4 and GRE2. The deletions were confirmed
using PCR (for the complete deletions) and subsequently by sequencing (see Table 3).
Biotransformation assay
The biotransformation of different aldehydes and alcohols was tested using the knockout
mutants that contained only one of the seven known Adhp encoding genes. The mutants were
cultivated in shake flasks using YEP medium and fed again with a 50 % (w/w) glucose solution,
14 h prior to harvest. Equal amounts of biomass of 0.04 g CDW were harvested from the culture
by centrifugation of appropriate volumes of the culture at a timepoint when excess glucose was
still present in the medium. The pellet was washed twice with 0.9% NaCl. The cells were
disrupted by incubating the cells for 1 h with Zymolyase 20T (5 mg in 500 μL, WAK-Chemie
Medical GmbH, Steinbach (Taunus), Germany) at 37 °C in lysis buffer (1 M sorbit, 10 mM
DTT, 10 μL protease inhibitor (Protease Inhibitor Cocktail, Merck, Darmstadt, Germany) and
subsequent treatment with 0.5 mm glass beads in the VXR basic Vibrax (IKA Wilmington,
USA) at 4 °C. After cell disruption, the suspension was filtered through a 4 μm filter and the
filtrate was kept on ice until further use. For the enzymatic assay, 10 μL of the extracted proteins
were mixed with 170 μL potassium phosphate buffer (pH 8 for Adh3p and Adh4p, pH 7 for all
other enzymes) and 10 μL of 5 mM NAD+/NADP+ for alcohols or NADH/NADPH for
aldehydes. The assays were performed in 96-well microtiter plates and the consumption or
production of the redox cofactors monitored by reading the absorbance change at 340 nm using
the well plate reader Synergy MX (BioTek, Friedrichshall, Germany). The final volume of the
assay added up to 200 μL in each well. The assay was started when the absorbance stabilized
by adding 10 μL of 1 M substrate and stopped when the absorbance stabilized.
All assay substrates and the redox cofactors were purchased from Carl Roth (Karlsruhe,
Germany), Merck (Kenilworth, NJ, USA), VWR (Radnor, PA, USA) or Alpha Aesar
(Haverhill, MA, USA) and were of < 97% purity (for biochemistry).
Data evaluation
The absorbance data was corrected with the data of the control experiment, which included
everything but the substrate. The control was included on each 96-well plate and for each time
point. The absorbance values obtained after the addition of substrate were subtracted from the
last value recorded prior to the addition of the substrate. The resulting data was evaluated for
values above zero indicating a change in NAD+/NADP+ concentrations higher than that of the
control. For values close to zero, the plot of raw absorbance data over time was visually
inspected to check if the slope of the assay data was larger than that of the control.
40
Chapter 2
Results and Discussion

Mathematical models help to structure the knowledge of an organism’s metabolism. Models
can be analyzed to estimate its biochemical capabilities or to predict genetic engineering targets
to improve production of a target chemical thereby rationalizing and enhancing metabolic
engineering of productions strains [245]. Many volatile metabolites are of industrial interest as
they can be used as fragrances or flavor compounds in the cosmetic and food industry.
Interestingly, although yeast is known to produce a variety of volatile, aromatic compounds,
the current yeast consensus metabolic model, yeast 7.11, is almost completely lacking those
volatile metabolites. Here, we are closing this gap by appending the model biochemical
pathways for the synthesis of 92 yeast volatile metabolites that were extracted from the
scientific literature (Table 5).
Table 5: Volatile organic compounds emitted from S. cerevisiae fermentations. Compounds that
are included in the latest yeast genome scale metabolic reconstruction (Yeast 7.11) and in the
Saccharomyces Genome Database SGD [185] are categorized. (Table continues over multiple
pages.)
included in
Compound PubChem
Compound Yeast Reference #
class ID SGD
7.11
(2-phenylcyclopropyl)
alcohols 317540 no no [246]
methanol
1,2-benzene dicarboxylic
acids 1017 no no [66, 247]
acid
1,3-butanediol alcohols 6440 no no [246]
1-butanol alcohols 263 no no [246]
1-heptanol alcohols 8129 no no [246]
1-hexanol alcohols 8103 no no [248-251]
1-propanol alcohols 1031 no no [248-251]
2,3-butanediol alcohols 262 yes yes [250, 251]
2,5-dimethylpyrazine * pyrazines 31252 no no [66, 247]
2-ethyl-1-hexanol alcohols 7720 no no [66, 247, 248]
2-furfuraldehyde aldehydes 7362 no no [249]
2-hexanol alcohols 12297 no no [246]
2-methyl-2-butanol * alcohols 6405 no no [248-250]
2-methylbutanal * aldehydes 7284 yes no [66, 247]
2-methylbutanoic acid acids 8314 no no [66, 247]
2-methylbutanol alcohols 8723 yes yes [248-250]
2-pentanone ketones 7895 no no [66, 247]
[66, 247, 249-
2-phenylethanol * benzenoids 6054 yes yes
252]
2-phenylethyl acetate esters 7654 no no [249, 252]
2-propane alkanes 6334 no no [248]
2-propanol alcohols 3776 no no [66, 247, 248]
2-xylene benzenoids 7237 no no [66, 247]
3-methylbutanal * aldehydes 11552 yes yes [248]
[66, 247, 249,
3-methylbutanoic acid * acids 10430 no no
250]
41
Results
3-methylheptyl acetate esters 537686 no no [248]

5-methyl-2-furfuraldehyde aldehydes 12097 no no [249]
acetaldehyde aldehydes 177 yes yes [248-250, 253]
acetaldehyde diethylacetal ethers 7765 no no [249]
acetic acid acids 176 yes yes [66, 247, 251]
2-propenyl acetate esters 11584 no no [248]
ethenyl acetate esters 7904 no no [66, 247]
acetoin ketones 179 yes yes [249]
acetone ketones 180 no no [66, 247]
acetophenone ketones 7410 no no [249]
benzaldehyde aldehydes 240 no no [249]
benzyl acetate alcohols 8455 no no [249]
benzyl alcohol * alcohols 244 no yes [249]
butanal aldehydes 261 no no [248]
butanone * ketones 6569 no no [66, 247]
butyric acid carboxylic acids 264 no no [250, 252]
cis-3-hexen-1-ol alcohols 5281167 no no [249, 250]
decanoic acid carboxylic acids 2969 no no [249-252]
diacetyl * ketones 650 no yes [248, 249]
diethyl succinate esters 31249 no no [249-251]
dimethyl disulfide * sulfides 12232 no no [66, 247]
dodecanoic acid * carboxylic acids 3893 no no [250, 252]
ethanol alcohols 702 yes yes [66, 247]
ethyl 2-methylbutyrate esters 24020 no no [249]
[66, 247-250,
ethyl acetate esters 8857 yes no
253]
ethyl benzoate esters 7165 no no [249]
ethyl butyrate esters 7762 no no [249, 251, 252]
ethyl caproate esters 31265 no no [248-252]
ethyl caprylate esters 7799 no no [248-252]
ethyl decanoate esters 8048 no no [249-252]
ethyl furoate esters 11980 no no [249]
ethyl heptanoate esters 7797 no no [249]
ethyl isobutyrate esters 7342 no no [248, 249]
ethyl isovalerate esters 7945 no no [248, 249]
ethyl lactate esters 7344 no no [249, 250]
ethyl phenylacetate esters 7590 no no [249]
ethyl propanoate esters 7749 no no [248, 249]
ethyl pyruvate esters 12041 no no [250]
ethyl valerate esters 10882 no no [249]
ethyl-2-hydroxy propionate esters 545098 no no [251]
furfuryl alcohol * alcohols 7360 no no [249]
guaiacol * alcohols 460 no yes [249]
heptanal * aldehydes 8130 no no [248]
heptanoic acid carboxylic acids 8094 no no [249]
hexanal aldehydes 6184 no no [249]
hexanoic acid * carboxylic acids 8892 no no [249-252]
hexyl acetate esters 8908 no no [249, 250, 252]
isoamyl acetate esters 31276 yes no [248]
isoamyl alcohol alcohols 31260 yes yes [248-252]
isobutanal * aldehydes 6561 no yes [248]
42
Chapter 2
[66, 247-251,
isobutanol alcohols 6560 yes yes
253]
isobutyl acetate esters 8038 yes no [248, 249]
limonene terpenes 22311 no no [66, 247]
linalyl propionate esters 61098 no no [251]
methanol * alcohols 887 no no [249, 250]
methyl acetate esters 76214 no no [249]
methylpropanoic acid * acids 6590 no yes [66, 247, 250]
monoethyl succinate esters 70610 no no [251]
n-butyl acetate esters 31272 no no [248, 249]
nonanal aldehydes 31289 no no [248]
nonanoic acid carboxylic acids 8158 no no [249]
n-propyl acetate esters 7997 no no [248, 249]
octanoic acid * carboxylic acids 379 no no [249-252]
pentanal aldehydes 8063 no no [248]
propionic acid carboxylic acids 1032 no no [250]
pyrazine pyrazines 9261 no no [66, 247]
undecane alkanes 14257 no no [66, 247]
α-terpineol terpenes 17100 no no [249]
β-phenylethyl formate esters 7711 no no [248]
* These compounds were also detected in yeast extract [254]. In reference [247],
yeast was cultivated in malt extract and tryptone soya, in reference [248] yeast
extract, plus bactopeptone and glucose was used. All other references reported
volatiles from wine fermentations.
To find the pathways that connect each of the 92 metabolites to the reactions already existing
in the consensus model, the computational tools FMM, PathPred and MetaRoute were used. In
addition, a manual search for metabolic pathways in the literature and the manual addition of
possible reactions was performed (Table 6) [207-209]. Apart from seven metabolites
(acetaldehyde diethyl acetal, acetic acid 2-propenyl ester, acetic acid ethenyl ester, undecane,
(2-phenylcyclopropyl)methanol, 2-ethyl-1-hexanol and 2-xylene), all metabolites could be
connected to the metabolic pathways using these tools. For the pathway prediction of two of
the missing seven metabolites, 2-ethyl-hexanol and 2-xylene, BNICE [234] was used. BNICE
predicts possible pathways using generic enzyme reaction rules and here was confined to the
use of compounds present in the KEGG database [231, 232, 255]. To make sure that
S. cerevisiae is capable of catalyzing the added reactions, gene sequences coding for enzymes
known to catalyze those reactions were blasted against the yeast genome of S. cerevisiae S288C.
Flux Balance Analysis (FBA) and thermodynamics-based flux analysis (TFBA) [219, 235, 256]
were performed on 36 of the newly introduced pathways to further verify stoichiometric and
thermodynamic feasibility of the biosynthetic pathways of the volatile metabolites. All 36 tested
pathways yielded feasible reactions for both FBA and TFBA. Using TFBA it was possible to
correct pathways that would have not been thermodynamically feasible. An example for this is
the production of methanol. Methanol was first proposed to be produced during the reduction
of formaldehyde, this reaction however, turned out not to be thermodynamically feasible.
Instead the decarboxylation of glycolate was added to the model. Stoichiometric and
thermodynamic feasibility was confirmed by FBA and TFBA.
43
Results
Table 6: Volatile metabolites added to the metabolic yeast model. The column reference
indicates, whether the pathway was taken from KEGG, a scientific publication, that the pathway
was inferred by pathway prediction tools (blank space), or predicted by established yeast
enzymes with reactions similar to those needed to establish the pathway (also blank space).
(Table continues over multiple pages.)
PubChe Pathway based
Substance KEGG Literature
m on
1,2-benzenedicarboxylic
1017 C01606
acid another organism KEGG
1,3-butanediol 6440 C20335 another organism KEGG
1-butanol 263 C06142 literature data [257]
pathway
1-heptanol 8129
prediction
pathway
1-hexanol 8103
prediction
1-propanol 1031 C05979 literature data [257]
2-furfuraldehyde 7362 C14279 another organism KEGG
2-methylbutanoic acid 8314 C18319 literature data [258]
2-pentanone 7895 C01949 another organism KEGG
2-phenylethyl acetate 7654 C12303 literature data [259, 260]
2-propane 6334 C20783 another organism KEGG
pathway
2-propanol 3776 C01845
prediction
2-xylene 7237 C07212 another organism KEGG
3-methylbutanoic acid 10430 C08262 literature data [258]
3-methylheptyl acetate 537686 literature data [259, 260]
5-methyl-2-
12097 C11115
furfuraldehyde another organism KEGG
pathway
acetone 180 C00207
prediction
acetophenone 7410 C07113 another organism KEGG
pathway
benzaldehyde 240 C00261
prediction
benzyl acetate 8785 C15513 literature data [259, 260]
pathway
benzyl alcohol 244 C00556
prediction
butanal 261 C01412 literature data [257]
butanone 6569 C02845 another organism KEGG
butyric acid 264 C00246 literature data [257]
pathway
cis-3-hexen-1-ol 5281167 C08492
prediction
pathway
diacetyl 650 C00741
prediction
pathway
diethyl succinate Knoll 1994
31249 prediction
dimethyl disulfide 12232 C08371 literature data [261]
C02679 pathway
dodecanoic acid 3893
prediction
pathway
ethyl 2-methylbutyrate Knoll 1994
7945 prediction
44
Chapter 2
pathway
ethyl benzoate Knoll 1994
7165 C01839 prediction
pathway
ethyl butyrate Knoll 1994
7762 prediction
pathway
ethyl caproate Knoll 1994
31265 prediction
pathway
ethyl caprylate Knoll 1994
pathway
ethyl decanoate Knoll 1994
7799 prediction
pathway
ethyl furoate Knoll 1994
11980 prediction
pathway
ethyl heptanoate Knoll 1994
7797 prediction
pathway
ethyl isobutyrate Knoll 1994
7342 prediction
pathway
ethyl isovalerate Knoll 1994
pathway
ethyl lactate Knoll 1994
7344 prediction
pathway
ethyl phenylacetate Knoll 1994
7590 prediction
pathway
ethyl propanoate Knoll 1994
7749 prediction
pathway
ethyl pyruvate Knoll 1994
12041 prediction
pathway
ethyl valerate Knoll 1994
10882 prediction
ethyl-2-hydroxy pathway
propionate 45258 C01013 prediction
furfuryl alcohol 7360 C20441 another organism KEGG
guaiacol 460 C01502 another organism KEGG
pathway
heptanal 8130 C14390
prediction
pathway
heptanoic acid 8094 C17714
prediction
pathway
hexanal 6184
prediction
pathway
hexanoic acid 8892 C01585
prediction
hexyl acetate 8908 literature data [259, 260]
Isobutanal 6561 literature data [258]
limonene 22311 C00521 another organism KEGG
pathway
linalyl propionate
61098 prediction
pathway
methanol 887 C00132
prediction
methyl acetate 6584 literature data [259, 260]
methylpropanoic acid
6590 C02632 [258]
(isobutyrate) literature data
45
Results
pathway
monoethyl succinate 70610 [262]
prediction
n-butyl acetate 31272 C12304 literature data [259, 260]
n-propyl acetate 7997 C14928 literature data [259, 260]
propionic acid 1032 C00163 literature data [257]
α-terpineol 17100 C16772 another organism KEGG
pathway
β-phenylethyl formate
7711 prediction
While the positive BLAST and FBA results increased reliability of the introduced pathways,
confirmation requires experimental verification. Here, we aimed for biochemical confirmation
of those new reactions that are catalyzed by alcohol dehydrogenases but which could not be
assigned to specific genes, yet. The Yeast 7.11 model already contained 8 genes encoding
alcohol dehydrogenases and 7 Adhp catalyzed reactions. This set of reactions was extended to
17 in this study.
Because the ADHs are well investigated, the probability of unknown isoenzymes that need
to be knocked out additionally was extremely low. Another reason for choosing the ADHs was
their importance, as fermentative growth on hexoses is not possible for yeast lacking Adhs. To
do both, verify new reactions and to assign enzymes and genes, we generated S. cerevisiae
mutants deficient in all but one of the known Adhp encoding genes. As we used S. cerevisiae
CEN.PK113-17a in this study, which in contrast to S. cerevisiae S288c does not possess ADH7,
in total 9 mutants were created (Table 2). The nomenclature of these mutants is s, for single,
plus the name of the remaining Adhp gene, e.g., sADH1 refers to the mutant possessing only
the ADH1 gene; the S. cerevisiae strain void of all 8 Adhp enzymes was named sKO8. The wild
type strain and the sKO8 strain were tested twice for their biocatalytic activity. Three alcohols,
seven aldehydes, and 3 ketones were tested (Table 7) and depending on the cofactor specificity
either NAD(H) or NADP(H) were used in the assay.
The wild type S. cerevisiae strain showed activity for all substrates but isovaleraldehyde in
the assays complemented with NAD(+/H). Like the wild type, sADH1 also showed a broad
substrate specificity and converted all tested substrates except of acetone. However, Adh1p is
known mostly for the conversion of acetaldehyde to ethanol [263-265]. Interestingly, sADH1
showed activity in the assay with isovaleraldehyde, while no activity could be detected for the
wildtype, although both strains were cultivated under identical conditions and harvested during
exponential growth in the presence of glucose excess. sADH2 only showed activity with half
of the here tested substrates (Table 7). In accordance with literature, Adh2p oxidized
acetaldehyde and propanol [263]. Another mutant with an interesting substrate spectrum was
sADH3 which accepted acetaldehyde, 2,3-butanedione, 2-butanone, propanol, and
formaldehyde. The discrepancy to Adh1p, Adh2p and Adh5p is surprising as we expected a
similar substrate spectrum for these enzymes given Adh3p shares 80% of amino acids with
Adh2p and Adh1p, a paralog of Adh5p. The role of these enzymes is reported to be similar,
while a big discrepancy in the biocatalytic activity has been shown here. Adh5p is not well
investigated and no substrate spectrum has been recorded so far. From the here tested
substances only acetaldehyde has been tested before and contrary to the investigation here, it
was converted by a mutant lacking Adh1p-Adh4p, however all other enzymes with Adh
function were still active. [263, 266-268]. Dickinson et al. tested in 2002 the possibility of
46
Chapter 2
knockout mutants to produce isoamyl alcohol and 2-phenylethanol, however they failed to
create knockout mutants carrying only 1 single gene with ADH activity [258]. However, these
findings are not in any way contradicting the here created results, since mutants with more than
one ADH gene still active were tested. Of the 13 tested compounds, sADH4, solely showed
activity with 1,3-butanediol. In the literature ADH4 is described to possess formaldehyde
dehydrogenase activity, as well as being able to convert butanol [263, 265], however this could
not be confirmed here.
sSFA1 and sADH5 showed no activity on either of the tested substrates, while a slight
conversion of 2-hexanone was found in both strains, a slightly stronger activity was found in
the sKO8 mutant. The latter observation indicates that there were still other enzymes active,
which could convert 2-hexanone. In contrast to literature [265, 269], sADH5 was not able to
catalyze the reaction from acetaldehyde to ethanol and sSFA1 was not able to convert
formaldehyde. However, the published data was collected using knockout strains which still
contained other enzymes with ADHAdh6p and Gre2p use NADP(H) instead of NAD(H).
Therefore, experiments with NADP(H) were performed with sADH6 and sGRE2 as well as the
wild type and the sKO8 mutant (Table 7). With this cofactor, the wild type was able to catalyze
the conversion of every tested substrate. Surprisingly, the sADH6 mutant showed a strong
conversion of 2,3-butanedione, while in literature it is only reported that Adh6p is involved in
fusel alcohol production [270, 271]. This could be explained by upregulation of another enzyme
(possibly Bdh1p) that converts 2,3-butanedione since a slightly lower activity is also found in
sGRE2 and even in the sKO8. However, the activity with sADH6 and sGRE2 was higher than
in sKO8, indicating that these enzymes contribute to the conversion of 2,3-butanedione but are
not concentrated high enough in the wild type to make an impact on activity. The sADH6 mutant
was also able to convert hexanal, 2-butanone, 1-heptanal, acetone and isobutyraldehyde. The
sGRE2 mutant showed lower conversion rates of hexanal and 2-butanone in comparison to the
sADH6 mutant and even lower rates than seen with the sKO8 mutant. Therefore, it can be
assumed that these two metabolites were converted by other enzymes and not by Gre2p. sGRE2
was also able to convert isovaleraldehyde in accordance with Hauser et al. [272].
Here it was demonstrated that sADH1,2,3,4,6 and sGRE2 showed activity for some of the
tested substances. However, the substances are not protein specific but mostly are converted by
multiple enzymes. Adh5p and Sfa1p do not seem to be involved in the here tested fusel
alcohol/aldehyde metabolism at all. One explanation for this discrepancy could be that in
literature other S. cerevisiae strains (S288C, YPH499) have been used whose enzymatic make-
up might differ from CEN.PK 113-7D. Another possible explanation is moonlighting or
promiscuity of Adhps, the ability of enzymes to harbor separate functions or convert diverse
substrates [273, 274]. A moonlighting function could be the inhibition or activation of another
enzyme and therefore have a regulatory effect (e.g., S. cerevisiae’s arginase inhibits the
ornithine transcarbamylase, when arginine and ornithine are both present [275, 276]). Here it is
possible that one of the Adhps is only expressed or active if one of the other ADH genes is being
expressed. Since all other ADH genes are knocked out this could explain the results shown for
sADH5 and sGRE2. Since Adh5p is an Adh1p paralog it would have been expected to have
similar functions to the ones of Adh1p [277].
It is important to notice that all here discussed results have been evaluated for yeast in the
exponential growth phase in a shake flask. Therefore, the cells were oxygen limited with excess
47
Results
glucose. It is possible that yeasts in a stationary growth phase, or yeasts that have excess oxygen
and that are glucose limited, show different results. However, since the here tested conditions
lead to yeast fermentation, it is unlikely that alcohol dehydrogenases are expressed higher under
aerobic conditions.
It was surprising that the main activity not only in the conversion of acetaldehyde to ethanol,
but also in the conversion of most of the tested substrates could be detected in S. cerevisiae´s
main ADH. Not only Adh1p, but most of the tested enzymes (all but Adh4p, Adh5p and Sfa1p)
showed catalytic activity with a number of the tested substrates. Single enzymes are not
responsible for the production of specific substances. The conversion of the 13 investigated
substrates by alcohol dehydrogenases or enzymes with alcohol dehydrogenase activity,
respectively, was confirmed.
48
Table 7: Enzymatic capacity of sADH mutants. The strains were tested for their ability to convert alcohols, ketones, and aldehydes in
enzymatic assays. The mutants showed either clear conversion (+), slight conversion (+/-) or no conversion of the substrate (-). Green
indicates accordance to literature data, red indicates contrariety.
cofactor NAD(H) NADP(H)
substrate WT sADH1 sADH2 sADH3 sADH4 sADH5 sSFA1 sKO8 WT sADH6 sGRE2 sKO8
acetaldehyde + + + + - - - - + - - -
hexanal + + - - - - - - + + - -
2,3-butanedione - + + + - - - - + - + +/-
2-butanone + + + + - - - - + + - -
1-heptanal + + - - - - - - + + - -
1,3-butanediol + + + - + - - - + - + +/-
propanol + + +/- + - - - - + - - +/-
2-hexanone + + + - - - - +/- + - - +/-
acetone + - - - - - - - + + + -
isobutyraldehyde + + - - - - - - + + + +/-
isovaleraldehyde - + - - - - - - + - + +/-
formaldehyde + + + + - - - - + - - -
2-methylbutanal + + +/- - - - - - + - + +/-
49
Chapter 2
Results
Computational models enable us to structure the vast field of knowledge related to an organism,
the enhancement of a model helps to close gaps in this accessible knowledge. All here made additions
to the model are non-essential for growth of the organism. The majority of amendments made here
are based on computational predictions and were only substantiated by BLAST searches and (T)FBA
analyses. To increase the reliability of the here expanded model, experimental verification of the new
pathways is required. To this end, analyses of pathway intermediates by, e.g., LC or GC combined
with mass spectrometry [278, 279], besides enzymatic assays of know-out mutants, as done here, can
be applied. The here added reactions could be especially interesting, when a yeast shall be enhanced
for the production of volatiles that may alter the taste of the food produced. It would be possible to
construct yeasts that have a fruity taste by enhancing the production of some esters. For example, the
upregulation of genes producing 2-phenylacetate would lead to a rose like odor. These aroma
properties could even be transferred from yeast to the final product, e.g., a bread that smells like apple
or banana, or a wine that has a rose-like bouquet. Also, it would be possible to create yeasts that
produce industrially relevant products, such as fuels or precursors for fuel production, such as butanol
(biofuels) [280] or 2,3-butanediol (bioplastic) [281].
Conclusion and Outlook

The consensus yeast metabolic model was lacking the volatile metabolite space nearly completely in
earlier versions. Here 225 metabolites were added to cover this part of the yeast metabolism. To
connect the metabolites to the rest of the existing network, 219 reactions were added to the model. It
represents a structured representation about the current knowledge of volatile metabolite synthesis in
yeast. Although stoichiometric and thermodynamic coherent, most of the introduced pathway lack
experimental evidence. In further work those pathways are to be substantiated by wet-lab
examinations. The resulting, curated model not only represent a knowledgebase of VOC biosynthesis
in yeast but can be computationally analyzed to guide rational engineering of, e.g., flavor-
overproducing strains or odorless yeast, void of VOC production. It would be also possible to create
yeast strains that do not produce a product that is hard or impossible to purify. To monitor the volatile
space this thesis concentrated on online measurement methods. In the next chapters a SESI-Orbitrap,
as well as an MCC-IMS are illuminated in greater detail.
50
Yeast volatilome dynamics during metabolic shifts
Contributions:
The study was designed by Christoph Halbfeld, Birgitta E. Ebert, Lars M. Blank and Pablo Martinez-
Lozano Sinues. The experiments were performed by Christoph Halbfeld and Pablo Martinez-Lozano
Sinues. The data was evaluated by Christoph Halbfeld, Henrik Cordes and Birgitta E. Ebert.
Results
Yeast volatilome dynamics during metabolic shifts

Summary
S. cerevisiae is an industrially important organism, with applications as different as baking and

bioethanol production for which the different growth modi of yeast, aerobic respiration and
fermentation are exploited. Notably, while intensely studied, the metabolic transition from aerobic
respiration to fermentation is still not fully understood. Also S. cerevisiae’s volatile space, while again
important in many applications like wine and beer making, was widely neglected in the past. For
example, no study reported the volatilome in dependence of the metabolic state of yeast. Here, we
analyzed the off-gas of a continuous glucose-limited cultivation of S. cerevisiae using a SESI-
Orbitrap that enabled online measurements in near real-time with high mass accuracy. This resulted
in a large amount of data (2,500 signals/sample) that was evaluated using a self-written MATLAB
script. The data evaluation revealed 16 signals that changed during the shift from fully respiratory to
respiro-fermentative metabolism prior to the detection of ethanol. Both, masses that showed an
increase and signals that showed a decrease of signal intensity upon the shift were detected. Two of
these masses were tentatively identified as isoprene and 3-methyl-1-butanol. The other volatiles
remain yet to be determined and might give valuable insights into the yeast’s volatile metabolism.
However, even without the exact knowledge about the volatile metabolites and their biochemical
synthesis, it can be concluded that the presented fast and sensitive SESI-Orbitrap off-gas analysis
allows to clearly determine the metabolic state (respirative- or fermentative growth) of a
microbiological culture in near real time.
55
Chapter 2
Introduction
The baker’s yeast Saccharomyces cerevisiae has been in the focus of research and industrial
production ever since industrialization has started. It has been used to produce beverages like beer
and wine since ancient times [282-286] but has also been in the focus of scientific research and was
the first eukaryotic organism for which the complete genome has been sequenced [287, 288]. A
specific trait of baker’s yeast is the Crabtree effect, which represents a metabolic shift from respiration
to aerobic fermentation when high sugar concentrations are supplied in the growth medium [289].
This shift has significant, economic relevance in baking yeast production since fermentation with
high initial sugar concentration or excess glucose feeding mainly leads to the production of ethanol
instead of biomass and worsens baking and storage ability of the product (Dr. Quantz, VH-Berlin,
personal communication). The Crabtree effect can easily be induced by overfeeding of glucose and
has been studied in nearly every aspect. However, the impact of the Crabtree effect on the formation
of volatile metabolites has not been investigated so far. While there are plenty of studies covering
volatile flavor compounds of yeast products, e.g., bread or wine [9, 290-293], almost no studies
exploring volatile metabolite formation during yeast growth in minimal salt medium exist. This trend
is changing lately with scientific investigations putting the focus on Saccharomyces’ volatile space
[1, 91, 185].
Changing the metabolic state is required for an organism to adapt to altered environmental
conditions for example by enzymatic adaptation to new nutrition sources or the production of
metabolites that fend off enemies. Here we used the induction of the Crabtree effect, i.e., the transition
from respiration to aerobic fermentation, as an example for a metabolic shift. Induction of the
Crabtree effect results in an increased flux through glycolysis and reduction of the relative
(normalized to the glucose uptake rate) pentose phosphate pathway flux. The major change, however,
is a downregulation of the citric acid cycle, the respiratory pathway, and the strong increase in
cytosolic pyruvate transformation to ethanol [294]. These changes in the central carbon metabolism
are well investigated but not necessarily the only changes that occur in the cell and additional changes
of the fluxes through the pathways responsible for the synthesis of amino acids, fatty acid synthesis
and degradation as well as terpene biosynthesis involving also volatile metabolites might occur.
Dynamics of (volatile) compounds

Until recently it was complicated to measure volatile compounds and nearly impossible to investigate
the dynamics of their production or consumption. However, new ionization techniques and improved
analytical devices allow the measurement of volatile compounds without sample preparation and in
high-throughput enabling to capture fast dynamics [1, 295].
For online measurements of volatile dynamics, the analytical device must have a high mass
resolution, short measurement times and be suited for direct sampling. These attributes are combined
in secondary electrospray ionization Orbitrap mass spectrometry (SESI-Orbitrap-MS). The SESI ion
source allows a continuous ionization of volatile compounds which are then directly injected into the
Orbitrap system [296, 297]. The ionization is fast and efficient also for low abundant analytes. The
principle can be briefly described as follows: First, electrospray ionization (ESI) is performed with a
dilute aqueous formic acid solution (0.1%) in the absence of analyte ions. The formic acid solution is
lead through a capillary in which an electric field between the tip of the capillary and a counter
56
Results
electrode is applied. The ions in the formic acid solution are pulled towards the negative charge in
the electric field and thus form a cone, where all ions are collected at the tip of the cone. When the
charge is high enough at the tip of the cone, droplets are formed that dissociate from the rest of the
water. The droplets are multiply charged and become smaller over time since the water evaporates
until the electrostatic repulsion in the drops becomes larger than the surface tension. The droplets thus
undergo Coulomb fission and smaller droplets are formed. This continues until one or no charge is
left per droplet [298]. The sample is introduced into the charged droplets. Through collisions of the
uncharged volatiles with the charged droplets, charges are transferred and the volatile molecules are
ionized. Since this happens instantaneously, a constant flow of ionized molecules can directly be
transferred to the Orbitrap [299]. The ionized gas is bundled by an electric field (focusing electrode)
and led through the impaction slit. Here the ionized gas collides with a counter flow of clean gas and
the charged ions are dragged further along the electric field while uncharged particles are flowing out
of the system (Figure 5). Fast and continuous ionization makes SESI-Orbitrap-MS especially
interesting for fermentation off-gas analysis as samples can be withdrawn and measured without any
sample preparation.
Figure 5: Schematic representation of a secondary electrospray ionization source. The here indicated
DMA is interchangeable with an Orbitrap. DMA= differential mobility analyzer. Reprinted with
permission from [300]. Copyright 2012 American Chemical Society.
The Orbitrap system analyzes the samples with a frequency of up to 20 Hz. This enables the
detection of real time changes and to resolve even very fast dynamics. Because of the high resolving
power of the device (100,000 to 1,000,000 depending on the device and m/z) (see Figure 6), no
chromatographic pre-separation is necessary [301]. The high mass resolution gives the possibility to
distinguish isobaric ions and the fast measurements allow to record a complete mass spectrum in each
measurement. Depending on the concentration and stability of the metabolite, single interesting
masses can be further fragmented if the machine is equipped with a collision chamber and thus be
identified based on their fragment spectrum.
57
Chapter 2
Figure 6: Influence of mass resolution on peak separation. Taken from [133].
SESI-MS has been used to investigate the dynamics of volatile compounds excreted by Begonia
semperflorens [297]. These measurements allowed to elucidate diurnal changes of 400 volatile
metabolites released by these plants. As consequence of mechanical damage 1200 volatile metabolites
were detected that showed changes compared to non-damaged plants. Some of the changes could not
have been detected with a lower sampling frequency, hence the direct ionization and high sampling
frequency were needed [297]. Similar studies have been conducted for the human exhalome by Sinues
et al. who reported that 40% of the 111 detected signals showed significant circadian modulation
[302, 303]. In yet another study Gaugg et al. showed that smokers and non-smokers could be
distinguished based on breath analysis [133].
In this study, we focused on off-gas analysis of fermentations of baker’s yeast. Here we set out to
exploit the SESI-Orbitrap-MS technology to investigate rapid changes in the volatilome of a
continuous culture after targeted perturbations of the metabolic steady-state and induction of the
Crabtree effect. We show that it is possible to distinguish metabolic states using the SESI-Orbitrap
and to monitor the transition from one metabolic state to another.
Identification of volatile metabolites correlating with metabolic shifts and their biosynthetic
pathways will shed new light on the regulatory mechanisms associated with the Crabtree effect.

Yeast strains and growth conditions
In this study, two yeast strains were used: S. cerevisiae CEN.PK 113-7D (Euroscarf, Oberursel,
Germany), which was recommended for yeast physiology and S. cerevisiae VH 2200
(Versuchsanstalt der Hefeindustrie e.V., Berlin, Germany), used for industrial baking yeast
production. The strains were grown in Verduyn minimal salt medium [237] containing 3 g L−1
glucose, 3 g L−1 KH2PO4, 0.5 g L−1 MgSO4·7 H2O, 20.4 g L−1 potassium hydrogen phthalate as well
as 1 mL of vitamin solution and 1 mL of trace elements. The vitamin solution contained 0.05 g L−1
D-biotin, 1 g L−1 calcium D-pantothenate, 1 g L-1 nicotinic acid, 25 g L−1 myo-inositol, 1 g L−1
thiamine hydrochloride, 1 g L−1 pyridoxine hydrochloride and 0.2 g L−1 p-aminobenzoic acid. The
trace element solution consisted of 15 g L−1 EDTA, 4.5 g L−1 ZnSO4·7 H2O, 1 g L−1 MnCl2·4 H2O,
0.3 g L−1 CoCl2·7 H2O, 0.3 g L−1 CuSO4·5 H2O, 0.4 g L−1 NaMoO4·2 H2O, 4.5 g L−1 CaCl2·2 H2O,
3 g L−1 FeSO4·7 H2O, 1 g L−1 H3BO3 and 0.1 g L−1 KI. The medium was buffered by addition of 100
58
Results
mM potassium hydrogen phthalate and the pH was adjusted to 5 using KOH prior to autoclavation,
while the vitamin solution and the trace elements were sterile filtered. The glucose was separately
autoclaved.
The pre-culture was incubated at room temperature and stirred with a stirrer bar at low rpm. The
continuous cultivation was performed with 4 parallel self-made 10 mL bioreactors (Seele
Glasapparatebau & Laborservice, Swisttal-Straßfeld, Germany). Mixing of each reactor was achieved
by a stirrer bar (10x3 mm, Karl Roth, Karlsruhe Germany) powered by a second magnet below the
glass vessel, which itself was driven by a motor. The fermentations were run at 30 °C; the temperature
was controlled by a water jacket connected to a Thermomix 1420 (B. Braun, Bethlehem, PA, USA).
The pH was not actively controlled but kept at pH 5 due to buffer and the incoming fresh medium.
The dilution rate and thus the specific growth rate, was set to 0.15 h-1 (below the critical dilution
rate of both strains in Verduyn minimal medium). For the medium feed an Ismatec Reglo ICC (Cole-
Parmer GmbH, Wertheim, Germany) and Ismatec Pharmed tubing with 0.25 mm ID (Cole-Parmer
GmbH, Wertheim, Germany) was used. The fermentation volume was kept constant at 10 mL by
adjusting the efflux tubing to a predefined height. For the efflux an Ismatec Reglo ICC pump (Cole-
Parmer GmbH, Wertheim, Germany) and Ismatec Pharmed tubing with 1.02 mm ID (Cole-Parmer
GmbH, Wertheim, Germany) were used. The reactor was aerated with 500 mL/min (50 vvm) of
compressed air via a Sho-Rate Series rotameter (Books Instruments, PA, USA). The air was filtered
and humidified prior to its introduction into the reactor (Figure 7). Safeflow connectors (B. Braun,
Melsungen Germany) were used for sampling. The gas outlet was coupled to a SESI-Orbitrap via a
transfer line that was heated to 130 °C.
The off-gas of the cultivation was continuously monitored by the SESI-Orbitrap. The
measurements were started when the fully respiratory, continuous culture had reached a metabolic
steady state. To capture potentially fast dynamics of volatile metabolites, the SESI-Orbitrap
measurement frequency was set to one full spectrum every 5 seconds. The S. cerevisiae culture was
perturbed by injecting a pulse of a sterile glucose solution (50 mg ‫ ؙ‬27.7 mM) and measurements
were taken until the ethanol signal reached its maximum. After the pulse, the system was left
undisturbed for 48 hours to re-establish a metabolic steady state.
59
Chapter 2
Figure 7: Schematic view (A) and picture (b) of the mini-bioreactor setup connected to the SESI-
Orbitrap-MS (Bioreactor constructed by Eik Czarnotta and Suresh Sudarsan, RWTH Aachen
University).
Online SESI-Orbitrap MS measurements

The off-gas of the mini bioreactor was transferred via a heated (130 °C) transfer tube (short Rotilabo
silicon tubing (Carl Roth GmbH, Karlsruhe, Germany) directly into the SESI ion source without prior
treatment. The SESI ion source was developed at the ETH Zurich in the group of Pablo Martinez-
60
Results
Lozano Sinues and is commercially available (SEADM, Boecillo, Spain). The Thermo Finnigan LTQ
Orbitrap (Thermo Fisher Scientific, Waltham, MA, USA) was used as detector.
The SESI and Orbitrap settings were similar to those used by Rioseras et al. 2017 [295]. The SESI
used a 0.1% formic acid solution that was infused at ~100 nL/min through a 20 μm ID silica capillary.
The voltage of the electrospray was set to 5.4 kV.
The mass range was 50-500 m/z, with an acquisition rate of 0.20 spectra/s. The pre-set resolution
of the device was 30,000, while the mass accuracies were typically within 2 ppm. After connecting
the bioreactor off-gas to the SESI-Orbitrap, a quick decline of the total ion current (TIC) could be
observed. Measurements were only started when the TIC had stabilized.
Data evaluation:
Data was acquired in Thermo Scientific’s .raw format and converted to mzXML files using the
ProteoWizards msconvert tool [304]. The data was imported into MATLAB R2012b (Mathworks,
Natick, MA, USA) and each dataset was interpolated linearly (106 points in the range of 50-500 Da);
interpolated profile mass spectra were then centroided (intensity threshold of 50 a.u.) as described by
Rioseras et al. [295]. The mass spectral data was evaluated using a self-written MATLAB script (see
Appendix). The data was first processed using the medfilt1 (equation 1) and sgolayfilt filter for
smoothing. Those processed datasets were used to detect signals whose concentration changed in the
period between glucose pulse injection and the increase of the smoothed ethanol signal above a
defined threshold. For visual inspection, two clustergrams were created (see Figure 8 A and B). The
first one shows all signals which displayed a maximum intensity between the glucose pulse and the
appearance of ethanol. The second one shows those signals having a maximum later than the
appearance of ethanol but that changed after the injection of the glucose pulse. In addition, the
normalized data was plotted against time, one plot per signal, and overlaid with the normalized
ethanol signal (see Figure 8 C).
‫ ݅ݔ‬െ ‫݊݅݉ݔ‬ Equation 1
ܺൌ
‫ ݔܽ݉ݔ‬െ ‫݊݅݉ݔ‬
The signals left after filtering, were further evaluated manually by scanning the clustergram for
changes in the timeframe between the glucose pulse and ethanol formation. In case the masses showed
interesting dynamics in more than 5 of the eight experiments they were considered to correlate with
the metabolic changes induced by the glucose pulses. One exception was the mass most likely
representing isoamyl alcohol (3-methyl-1-butanol), which was included in the list of relevant masses
although it only showed a rapid change in 5 of the 8 performed experiments.

To elucidate potential changes in the volatilome during the metabolic shift from full respiratory
growth to aerobic fermentation, S. cerevisiae was cultivated in an aerated glucose-limited chemostat.
The dilution rate of 0.15 h-1 was set to be sufficiently below the critical growth rate of >0.2 h-1 of
both strains, thereby guaranteeing full respiratory metabolism. The off-gas of the cultivation was
monitored to detect volatile metabolites using a SESI-Orbitrap. First, the metabolic steady state was
investigated, later the steady state was disturbed by injecting a concentrated glucose solution. The
61
Chapter 2
volatiles that were either consumed or produced in this transient state prior to the production of
ethanol were in the focus of this study. Per experiment about 2,500 signals were recorded (see Figure
8 A and B) that show clearly individual trends during the course of the experiment. Due to the large
amount of signals, the data had to be software filtered for interesting signals since manual evaluation
of the complete datasets would be cumbersome. The data recorded during the steady state clearly
differentiated from the data recorded after the glucose solution was injected, therefore the two
metabolic states could be clearly distinguished using the SESI-Orbitrap. After the computational
evaluation about 200 to 300 signals of each dataset were left that were subsequently evaluated
manually by plotting these signals against the ethanol signal. Most of the here found signals were
increasing in intensity after the glucose pulse, while only few were decreasing. Therefore, it can be
assumed that the flux rates during aerobic fermentation are higher than during respiratory growth. All
interesting signals were added to a list and a score, based on the number of experiments, the signal
resulted to be interesting, was generated. The most 16 interesting signals were evaluated further
(Table 8).
Interpretation of the results

Some of the 16 signals probably represent isotopologues (molecules with a different number of
neutrons) of the same metabolite. Most of the identified signals were not unambiguously identified
during this thesis (Table 8). The response of these volatile molecules to the glucose pulse indicate
that the metabolic changes of yeast are manifold and not just restricted to central carbon metabolism.
This conclusion is based on the knowledge that most of the metabolites in central carbon metabolism
are not volatile, while the here used detection method only reported signals of volatile compounds.
The interesting signals can be distinguished into signals that increased and signals that decreased
after the glucose pulse was applied to the system (see Figure 8 C-F). Most of the signals increased
and are possibly related to the production of metabolites that are required for the fermentative
metabolism but not used during fully respiratory metabolism. Some signals rose quickly and
disappeared again in a matter of minutes (see Figure 8 E). These signals are potentially linked to
intermediates that shortly accumulate during the transient metabolic state, e.g., metabolites involved
in the production of amino acids for protein synthesis or intermediates of metabolic pathways
activated by the glucose pulse. Because of the very fast dynamics in the seconds range, it can be
concluded that the catalyzing enzymes were already present in the cells before the metabolic shift.
We exclude that these quickly changing signals are contaminants introduced with the pulsed glucose
solution as these signals could not be reproduced by a glucose injection into sterile medium and did
not respond immediately with the pulse but with a delay of about 3 minutes. As an artefact of data-
smoothing some signals showed changes already prior to the glucose pulse (Figure 8 F). However,
signal changes close after the pulse region can reliably be attributed to the metabolic perturbation.
For 16 signals, intensity changes were detected prior to an increase of the ethanol peak (Figure 8 C,
D, F). Those metabolites might be precursors of ethanol or other metabolites that accumulate because
of an imbalance during the metabolic shift or either arise or deplete due to intracellular flux
rearrangements. Increasing signals might also be degradation products of storage compounds whose
catabolism is activated during the metabolic shift.
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Results
Table 8: Mass to charge ratio (m/z) of the most interesting signals recorded during the metabolic
transient state in a continuous glucose-limited fermentation after setting a glucose pulse. + and #
indicate masses whose signal intensity linear correlated with each other and which probably
originate from the same metabolite. m/z 69.0701 and 89.0962 have been tentatively identified as 3-
methyl-1-butanol and isoprene, respectively.
percentage of experiments in which the
m/z signal was detected (%) comment
57.0700 87.5
58.0735 87.5
69.0701 100 (+) isoprene
70.0732 100 (+) isoprene (protonated)
71.0857 100 cyclopentane
72.0887 100
73.0649 87.5 ethoxy ethene
74.0680 87.5
89.0962 75 3-methyl-1-butanol
118.0962 75
119.0885 100
127.1117 75 1-octen-3-one
128.1152 75
166.9860 100 (#)
177.0763 100
196.1123 100 (#)
Most signals have not been identified yet, but we speculate that they have not even been observed
and be linked to the Crabtree effect so far as detection of these low abundant molecules and
monitoring of the fast dynamics is only possible today with highly sensitive and fast analytics such
as SESI-Orbitrap-MS.
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Figure 8: Data evaluation of SESI Orbitrap analyses of the off-gas of a continuous glucose-limited
yeast culture at steady state which was disrupted with a 27 mM glucose pulse. The filtered data is
displayed as a clustered heatmap in which the scaled intensity of single masses (m/z value) are plotted
over time (A), zoom-in excerpt of the heatmap shown in A (B). Smoothed data of interesting signal
traces (blue line) identified in the heatmap are plotted against time together with the signal of ethanol
(black line). The left black vertical line indicates the timepoint of the glucose pulse injection, while
the right line indicates the timepoint of the first detection of ethanol (C-F).
Two compounds were tentatively identified as isoprene and 3-methyl-1-butanol (isoamyl alcohol).
Isoamyl alcohol is produced during the degradation of leucine via the Ehrlich pathway. Possibly
during the metabolic transition from aerobic respiration to aerobic fermentation, amino acids are
required in large quantities because of an increased reproduction and production of biomass.
Therefore, leucine might have exceeded a threshold that triggers leucine degradation resulting in
isoamyl alcohol production. However, the definite role of isoamyl alcohol remains unclear.
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Results
Isoprene is not known to be naturally produced by S. cerevisiae, however precursors of it are native
to S. cerevisiae’s metabolism and metabolic engineering has been performed to create isoprene
production strains [305]. It would be more likely that the measured volatile is not isoprene but another
volatile with the same molecular weight. However, since the volatile space in yeast fermentations has
been neglected thus far widely, it is possible that isoprene has not been detected before. The direct
precursor of isoprene, dimethylallyl pyrophosphate, is a part of S. cerevisiae’s mevalonate pathway
[306]. The possibility of spontaneous conversion in low quantities is given. While there were studies
that have investigated the volatile space of yeast in the recent past, they have not detected the presence
of isoprene [91, 307]. This could be due to the used indirect measurement methods that included
adsorption prior to the measurement, as isoprene might not be adsorbed due to its high volatility.
Nevertheless, as mentioned before it is more likely that the tentatively identified isoprene signal is
another compound.

With fast, high-resolution, accurate mass SESI-Orbitrap-MS measurements, we could finely resolve
the dynamics of S. cerevisiae’s volatilome. Those analyses showed not only the expected changes in
the ethanol concentration upon induction of the Crabtree effect but revealed changes of several other
signals that either correlated positively or negatively with ethanol or showed a short peak and then
dropped back to the initial value. This is an interesting observation as so far metabolic changes
initiated by the Crabtree effect were primarily associated to central carbon metabolism, i.e.,
upregulation of the pyruvate decarboxylase pathway and downregulation of the TCA cycle and less
to peripheral metabolic pathways that produce volatile metabolites [294].
The information content of the SESI-Orbitrap MS data generated in this study has not been
exploited to its full extent. Further research on the identity of these compounds, their biosynthetic
pathways and regulatory programs is necessary to understand their dynamics and the relation to the
Crabtree effect. To achieve a deeper insight in the metabolism during the shift from respiratory to
respiro-fermentative metabolism, besides (volatile) metabolite profiling transcriptome and / or
proteome analyses are required ideally complemented with the computation of intracellular reaction
rates.
We propose that the highly time and mass resolved time series can be used to infer enzymatic
reaction and enzyme kinetics. For example, the occurrence of linearly correlating traces differing by
18 Da indicates a dehydration or condensation reaction. Also, the identification of the measured
masses can be achieved by further analysis, e.g., fragmentation of interesting masses and could further
increase the insight into yeast’s metabolism. Moreover, SESI-Orbitrap analysis might be used for 13C
metabolic flux analysis and could allow near real-time determination of intracellular fluxes [308].
Through the high mass resolving power, the high measurement frequency and the low detection
limit, it was possible to detect changes in the yeast volatile space in near real time. The only limitation
to this work is that solely excreted metabolites can be measured using a SESI-Orbitrap. Here it would
be interesting to try out an approach, where whole cells are directly measured using a flow injection
system that takes samples out of a stirred bioreactor, comparably to what Link et al. reported [309].
This procedure could give even greater insights into the yeasts dynamic metabolome. However, up
to now the number of detected metabolites is small compared to what can be measured using the
SESI-Orbitrap. The SESI-Orbitrap is an ideal device for research, but it is not too robust due to a
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Chapter 2
capillary that feeds the SESI and high vacuum that is needed for measurements. A less costly and
more robust system is given with the multicapillary column ion-mobility spectrometer as explained
in detail in the next chapter.
66
Multicapillary column-ion mobility spectrometry of volatile metabolites
emitted by Saccharomyces cerevisiae
Partially published as:

Halbfeld, C.; Ebert, B.; Blank, L. Multi-capillary column-ion mobility spectrometry of volatile
metabolites emitted by Saccharomyces cerevisiae. Metabolites 2014, 4, 751-774.
Contributions:
Christoph Halbfeld performed the experiments. Christoph Halbfeld and Birgitta E. Ebert
analyzed the results. Christoph Halbfeld and Birgitta E. Ebert wrote the manuscript with the
help of Lars M. Blank. We thank Marco Fraatz (Justus-Liebig-University Gießen, Germany)
for GC-MS measurements and help with data evaluation and Jörg I. Baumbach (Reutlingen
University, Germany) for fruitful discussions. We also thank Peter Kaiser (B&S Analytik,
Dortmund, Germany) for technical support and Mathis Wolter (RWTH Aachen, Germany)
for assistance with MCC-IMS-measurements and implementation of the experimental set-up.
Results
Multicapillary column-ion mobility spectrometry of volatile metabolites

emitted by Saccharomyces cerevisiae
Summary
This chapter deals with the analysis of the headspace of Saccharomyces cerevisiae cultures using
multicapillary column-ion mobility spectrometry (MCC-IMS). The high sensitivity and fast data
acquisition of the MCC-IMS enabled online analysis of the fermentation off-gas. Of the 19 specific
signals, four were identified as ethanol, 2-pentanone, isobutyric acid, and 2,3-hexanedione by MCC-
IMS measurements of pure standards and cross validation with thermal desorption–gas
chromatography-mass spectrometry measurements. As shown in the previous chapter, monitoring of
VOCs produced during microbial fermentations can give valuable insight into the metabolic state of
the organism. Here, it is shown that the MCC-IMS technology is suitable for fast, robust, and
noninvasive online measurements. The low maintenance requirements of the MCC-IMS could enable
the device for easy to use monitoring of industrial fermentations.
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Chapter 2
Introduction
Yeasts are key model organisms in eukaryotic research and play a significant role in many
biotechnological processes. The use of yeast for biotechnological processes dates back to 7000–5000
BC, where it has been used for wine fermentation and in food processing [284, 310-312]. Today,
yeast strains are used in the chemical, food, and pharmaceutical industry for the synthesis of a broad
range of products from bakery products to bioethanol and pharmaceuticals [313-316]. With 4 million
tons of yeast biomass produced for baking worldwide in 2009 and an estimated yearly increase of 7%
[317], the yeast biomass market is in the top 10 of biotechnology products. The so far most often used
yeast cell factory is the baker’s yeast Saccharomyces cerevisiae. This yeast is not only the most
widely used yeast in biotechnology, but is additionally the model system for eukaryotic organisms.
Accordingly, much research has been conducted with S. cerevisiae and many of the today state-of-
the-art analytical techniques and molecular biology methods have been first developed for and with
this yeast. The simple and rapid cultivation, genetic accessibility, and industrial importance were and
still are drivers to maintain its lead in the yeast community in many disciplines of science.
As mentioned above, the volatile metabolites produced by yeast have been mainly neglected in
yeast research with the most prominent exceptions of acetaldehyde and ethanol. This can be attributed
to the limited knowledge of the biochemistry due to the complex analysis of volatiles. In addition,
genetics involved in the formation of volatile metabolites hinder the general incorporation in systems
biology studies. By far, most reports of volatile metabolites from yeast originate from researchers
interested in high quality wine making [249-252, 291, 318, 319], as the scent of wine impacts its
organoleptic properties [291]. The main classes of volatile metabolites observed are alcohols,
aldehydes, and esters (Table 5); while depending on grape and yeast strain used, many others can be
found. Notably, most studies were carried out on media that have wine-like compositions. Thus, the
media are most often complex, with alternative carbon and nitrogen sources present. The observed
volatile metabolites can therefore originate from glucose catabolism or are products from
biotransformation, i.e., do not originate from sugar (the carbon and energy source), but rather from
grape metabolites that were only modified by yeast enzymes.
One of the key bottlenecks in VOC research, and one of the reasons why these have not been
studied broadly so far, is that sample preparation of gaseous chemicals requires additional care and
that the analysis of volatiles is challenging. Most often, VOCs are extracted and enriched using head-
space/solid phase microextraction (HS/SPME) methods and analyzed with gas chromatography
coupled to advanced mass spectrometers [5, 159]. In the previous chapter, the use of a SESI-Orbitrap
was reported, but because of the high invest cost, this device is unlikely to be used in industrial
applications. As described in the next chapter, industrial applicability of the analytical system is of
interest, therefore an alternative to the SESI-Orbitrap had to be investigated to monitor the yeast’s
volatile space. For this purpose, the ion mobility spectrometry that was already explained broadly in
the general introduction was used here. The MCC-IMS’s characteristics, high time resolution, low
cost, and low maintenance, together with the high sensitivity perfectly suit this analytical technique
for online measurements of dilute volatile metabolites in the headspace of microbial fermentations.
GC-MS devices are suited for the identification of metabolites, but come with the disadvantage of
longer sampling times, although rapid GC techniques exist. Furthermore, GC-MS instruments require
special gases such as helium and high vacuum, hence come with relatively high operating costs and
technical expenditure. In contrast, MCC-IMS can be operated with nitrogen or air (not necessarily
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Results
synthetic air), at ambient temperature and pressure. In addition, handheld, mobile devices are
available underlining that MCC-IMS can be used flexible as they can be robust.
The potential of MCC-IMS analyses for fermentation monitoring has already been shown for
measurements of mVOCs produced during batch cultivation of Escherichia coli and Pseudomonas
aeruginosa in shake flasks [320, 321]. Also, MCC-IMS measurements of yeast fermentation have
been reported in which online measurements of the off-gas of yeast fermentations were performed.
While Kotiaho [322] focused on the quantification of one single metabolite, ethanol, Kohlemainen et
al. measured patterns of off-gas metabolites without any analyte identification [323]. The potential
of IMS analyses for quality control during beer fermentation was shown by Vautz et al. [324] by
monitoring the ripening indicators diacetyl and 2,3-pentanedione.
In this contribution, we evaluated the capabilities of MCC-IMS for online monitoring of mVOCs
produced by S. cerevisiae. Different growth conditions were tested and special emphasis was put on
the dynamics of the VOC profiles during transient metabolic conditions, i.e., the shift from respiratory
to fermentative metabolism.

The yeast strains used in this study were the reference strain S. cerevisiae CEN.PK 113-7D [325] and
the isogenic ADH1 knockout mutant S. cerevisiae CEN.PK 117 YOL086c::kanMX4 [326] devoid of
the main alcohol dehydrogenase Adh1p. The yeast strains were grown in Verduyn minimal salt medium
[237] containing 20 g L−1 glucose, 3 g L−1 KH2PO4, 0.5 g L−1 MgSO4·7 H2O 20.4 g L−1 potassium
hydrogen phthalate as well as 1 mL of vitamin solution and 1 mL of trace elements. The nitrogen
source (NH4)2SO4 was replaced by 2 g L−1 urea, as at higher concentrations of ammonia, the water
chemistry of the IMS ionization could shift to ammonia chemistry [327]. The vitamin solution
contained 0.05 g L−1 D-biotin, 1 g L−1 calcium D-pantothenate, 1 g L-1 nicotinic acid, 25 g L−1 myo-
inositol, 1 g L−1 thiamine hydrochloride, 1 g L−1 pyridoxine hydrochloride and 0.2 g L−1 p-
aminobenzoic acid. The trace element solution consisted of 15 g L−1 EDTA, 4.5 g L−1 ZnSO4·7 H2O,
1 g L−1 MnCl2·4 H2O, 0.3 g L−1 CoCl2·7 H2O, 0.3 g L−1 CuSO4·5 H2O, 0.4 g L−1 NaMoO4·2 H2O,
4.5 g L−1 CaCl2·2 H2O, 3 g L−1 FeSO4·7 H2O, 1 g L−1 H3BO3 and 0.1 g L−1 KI. All precultures were
performed in 500 mL shake flasks filled with 10% medium at 30 °C and 250 rpm. The bioreactor
experiments were run in a Sartorius Biostat A plus bioreactor (Göttingen, Germany) with a working
volume of 1 L at 30 °C. The fermentation parameters were controlled by an external computer and
the software PC Panel μDCU. The pH was monitored with a Mettler Toledo pH electrode and
controlled at pH 5 using 10 M potassium hydroxide and 4 M hydrochloric acid. In aerobic
fermentations, the fermenter was aerated with pressurized air with a flow rate of 2.8 L min−1. To
reduce impurities, the air was filtered by a Sartorius Midisart 2000 sterile filter (0.2 μm pore size)
and passed through water, resulting in water-saturated gas. Dissolved oxygen was monitored with a
Hamilton Oxyferm dissolved oxygen electrode. If not mentioned otherwise, the dissolved oxygen
tension was maintained at 90% by adjusting the stirrer speed. Bioreactor cultivations were started by
inoculating to an OD600 of 0.1. During batch cultivation, sample for OD and HPLC measurements
were taken regularly in the first 12 h. Continuous, glucose-limited cultivations were run at a dilution
rate of 0.13 h−1 by feeding fresh medium at a flow rate of 2.5 mL min−1. A constant volume of culture
broth was maintained by positioning a tube at a predetermined height that corresponded to 1120 mL
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Chapter 2
volume and connecting it to a separate pump that removed excess fluid. Both batch and continuous
cultivations were single experiments.
To check for possible contamination during fermentations, samples were examined daily under
the microscope (Leica DM750) with a 10X ocular and a 100X oil immersion objective.
Analytics
The optical density was determined with an Ultrospec 10 photometer (Amersham Bioscience,
Amersham, Switzerland) with a fixed wavelength of 600 nm. When necessary, the samples were
diluted using demineralized water.
For the determination of glucose and fermentative byproducts, samples were taken directly out of
the fermenter using a syringe and a steel pipe. Samples were harvested by centrifugation (Heraeus
Megafuge 16R, Thermo Fisher Scientific, Marietta, Ohio, USA) at 5,000 rpm for 5 min at 4 °C. The
supernatant was stored at −20 °C until further analysis. Analytes were separated using an organic acid
resin column (C-S Chromatography, Langerwehe, Germany) at 50 °C. 5 mM H 2SO4 was used as
eluent at a flow rate of 0.8 mL min−1 (System Gold 125 Solvent Module). Analytes were detected
with a UV detector (166 Detector, (Beckman Coulter, Krefeld, Germany) at a wavelength of 210 nm
and a RI detector (Melz Differential Refractometer LDC 201) operated at 50 °C. Standard solutions
of the analytes were measured in concentrations of 0.1, 0.5, 1, 5, 10, 20, 40 and 50 g L−1.
SPME GC-MS measurements for validation of volatile metabolites

To cross-check the identification of volatile compounds, 10 mL of the supernatant were sampled from
the bioreactor and transferred into a headspace vial (20 mL). Metabolites were extracted from the
headspace of culture supernatants via solid phase microextraction (CAR/PDMS fibers, Supelco,
Steinheim, Germany) and analyzed with GC-MS as described in [159]. Briefly, the samples were
incubated at 50 °C for 15 min and after that agitated for 10 s at 250 rpm. Afterwards, the analytes
were desorbed from the SPME liner of the GC at 250 °C. GC-MS analyses were carried out with an
Agilent 7890A gas chromatograph equipped with an Agilent 7000B triple-quadrupole mass
spectrometer (Agilent Technologies, Waldbronn, Germany). The split was set to a ratio of 5:1. The
column temperature was increased from 40 to 250 °C with a rate of 12 °C min−1. All other parameters
were identical as those described in [159]. For mVOC identification, retention indices and mass
spectra were compared with the NIST mass spectral library (Version: 2.0) and data published in the
FlavorNet database (flavornet.org, [64]).
MCC-IMS measurements
The MCC-IMS used was a BreathDiscovery (B&S Analytics, Dortmund, Germany) with an upstream
multi capillary column type OV-5 (Multichrom Ltd. Novosibirsk, Russia) of 17 cm length consisting
of approx. 1000 capillaries. The capillaries have an inner diameter of 40 μm and are coated with OV-
5 stationary phase with a film thickness of 0.2 μm. The column temperature was set to 40 °C. Samples
were ionized with a 550 MBq 63Ni ion source. The ionized analytes were introduced into the drift
column (length, 120 mm) through a shutter that had a pulse frequency of 50 ms and an opening time
of 30 μs. Separation in the drift chamber was carried out in a negative coaxial electric field with an
intensity of 300 Vcm−1 (positive measurement mode). The MCC-IMS was operated at ambient
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Results
temperature and pressure (i.e., laboratory conditions). Raw data of mVOCs was acquired using
VOCan (B&S Analytik) with a frequency of 10 Hz, 5 consecutive single spectra were averaged. A
single round of data acquisition required 0.5 s. The program was used to control all parameters of the
MCC-IMS like gas flow and temperature, and to program measurement sequences.
Nitrogen 5.0 (Westfalen, Münster, Germany) was used both as drift gas in the MCC-IMS and as
carrier gas in the MCC. The drift gas flow rate was set to 100 mL min−1; the carrier gas flow rate was
set to 150 mL min−1 during batch and 50 mL min−1 during continuous fermentation. The fermenter
off-gas was introduced into the system through a 10 mL stainless steel sampling loop coupled to a
six-port valve. Between single measurements the MCC-IMS and the sampling line was purged with
a nitrogen flow of 100 mL min−1.
The MCC-IMS topographic plots were evaluated using the software VisualNow (B&S Analytik).
Reduced inverse ion mobilities, 1/K0 (Vs cm−2), were calculated by normalizing the measured drift
velocities (drift time per drift distance) to the electric field, temperature, and pressure. This reduced
ion mobility is characteristic for the ion and independent of the experimental conditions. The program
allowed the definition of peak regions and comparison of the peak intensities in different datasets.
The intensities of detected peak regions, the reduced ion mobility and retention time were exported
to Excel for further data evaluation [328].
The MCC-IMS was connected to the fermenter off-gas with a Teflon tube (ID, 1.58 mm; length,
1000 mm). To prevent overloading of the MCC-IMS, the off-gas was diluted with sterile, moisturized
air or nitrogen at a flow rate of 2.4 mL min−1. The air was filtered with a 0.2 μm sterile filter. Mixing
was achieved by introducing both gas streams into a 500 mL Schott bottle.
Volatile metabolite identification

For mVOC identification, pure standard substance measurements were performed for which the
MCC-IMS was operated with identical parameters as during the continuous cultivations. One mL of
aqueous standard solutions of a concentration of 0.01 g L−1 was filled in 100 mL Erlenmeyer flasks,
which were closed with an aluminum cap. The sampling tube was introduced to the flask and the
measurement was started.

Experimental setup
To enable off-gas measurements, the MCC-IMS had to be connected to the bioreactor in a way that
guaranteed that all analytes are captured. To achieve this, the off-gas of the bioreactor was connected
to a mixing chamber where it was diluted with filtered, compressed air. To ensure proper mixing, the
gas inlets were positioned at the bottom, while the gas outlet to the MCC-IMS was positioned in the
upper section of the chamber. A second outlet leading through a filter into the environment was
positioned at the top, to ensure that no pressure build-up is possible (Figure 9).
Between measurements, the MCC-IMS and sampling line were flushed with nitrogen at a flow
rate of 100 mL min−1 (Figure 4). To avoid microbial contaminations and the introduction of volatile
impurities from the compressed air, the gas used for aeration of the bioreactor was passed through a
0.2 μm filter and a water bath. The overall setup allowed controlled sampling without disturbances
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Chapter 2
from the environment as no impurities were observed in the MCC-IMS during abiotic operation of
the bioreactor.
Figure 9: Experimental set-up for the online MCC-IMS measurements of fermenter off-gas.
Previously published in [185].
MCC-IMS monitoring of batch cultures

So far, most studies of yeast VOCs focused on the determination of volatiles produced during wine
fermentation and their impact on wine aroma. These studies did not discriminate whether the VOCs
originated from yeast fermentation or the products of biotransformations of the grape metabolites
[249-252]. Here, to explore the mVOCs de novo synthesized by baker’s yeast, minimal salt medium
with glucose as the sole carbon source was used. For a first evaluation of the MCC-IMS set-up for real-
time fermentation monitoring, S. cerevisiae was grown in batch culture. Before inoculation of the
fermenter, signals occurring from the sterile medium were measured under process conditions (stirrer
speed, temperature and pH control, aeration). During a period of 12 h, the MCC-IMS measurements
(Figure 10) showed four major signals of invariant intensity (data not shown).
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Results
Figure 10: MCC-IMS topographic plot of sterile Verduyn medium. The reaction ion peak
(1/K0 = 0.5 Vs cm−2) was compensated by the software VisualNow.
After inoculation to a starting OD600 of 0.1, MCC-IMS analyses were performed throughout the
growth experiment in one hour intervals. In addition to the signals detected in the sterile medium, 19
peaks emerged during the batch growth experiment, at retention times between 1 and 190 s (Figure 11).
The time course of the six most distinctive peaks is shown in Figure 12 B, C. To correlate the MCC-
IMS patterns of the volatile metabolites to the yeast physiology, the optical cell density, carbon source
consumption, and byproduct formation (ethanol, glycerol) were quantified in parallel to the MCC-
IMS measurements (Figure 12 A).
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Chapter 2
Figure 11: MCC-IMS topographic plot of S. cerevisiae during the early stationary phase of a batch
fermentation. Boxes indicate signals that showed the most significant changes during the growth
(perturbation) experiments.
In the single fermentation experiment, the profile of signal C3-5 correlated with the growth rate
since its signal increased after the lag phase and reached its maximum during the exponential growth
phase. In the stationary phase, the peak intensity of C3-5 decreased again. Peak C3-9 was detected at
the same time as ethanol, measured in the fermentation broth, and its signal diminished
simultaneously with complete ethanol consumption. The signal of C3-8 showed an increasing trend
in the late exponential to early stationary phase, when ethanol was nearly consumed. Signal C3-15
showed a rather peculiar profile. The signal intensity first increased, but rapidly decreased after 5 h
and maintained a constant level for about 11 h. After this period, the intensity of C3-15 abruptly
increased and reached its prior maximal value followed by a steady decrease at the end of the batch.
This behavior might be explained by incomplete ionization of the analyte molecules. The intensity of
the two signals decreased when the intensity of peak C3-5 increased. This signal might have a higher
proton affinity and therefore be preferably ionized to substance C3-15. This hypothesis is
substantiated by the low reaction ion peak (RIP) in the MCC-IMS chromatograms for the period
between 5 h–21 h (data not shown). The RIP consists of reaction ion molecules originating from the
drift gas, here nitrogen. In the ionization chamber, water molecules react with positively charged
nitrogen ions to a cluster of the type (H2O)nH+. These ions form the RIP and transfer the charge to
molecules with a higher proton affinity. Hence, with increasing analyte concentration the RIP
diminishes. For more detailed information about the charge transfer reactions, the reader is referred
to [128]. Note that the data presented here originates from single experiments and are thus not based
on statistics. The intention of this work was the development of a set-up for online MCC-IMS
measurements of volatile metabolites in the off-gas of yeast fermentations, with which we will
generate more comprehensive datasets in future experiments.
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Results
Figure 12: (A) Fermentation profile of S. cerevisiae during batch growth in glucose minimal medium,
(B) trends in intensity, (C) heat map of selected peaks detected by MCC-IMS analysis of the
fermentation off-gas. Areas in the heat map show the detected signal peak and the surrounding area;
DO = dissolved oxygen.
To elucidate the potential of MCC-IMS analyses for the differentiation of different S. cerevisiae
strains or mutants we compared the MCC-IMS chromatograms of S. cerevisiae CEN.PK 117
YOL086c::kanMX4, deficient in the major alcohol dehydrogenase Adh1p, with its isogenic reference
strain. Again, we want to stress that in this proof-of-principle work, only single experiments were
performed to show the general applicability of MCC-IMS for online measurements of fermentation off-
gas.
The ADH1 deletion mutant had a reduced growth rate and biomass yield, about 56% and 42% of
the reference values. The ethanol formation was clearly reduced (maximal accumulation of 0.8 g L−1
vs. 6 g L−1 for the reference strain), while glycerol production was increased (Figure 13 A). These
differences in the strain physiology were also reflected in the MCC-IMS pattern (Figure 13 B,C).
While no new peaks compared to the reference strain chromatograms were detected, the profiles of
several peaks differed. Peak C3-15 increased much slower compared to the reference strain
fermentation. The signal of C3-5 rose in the beginning, stagnated within the 5th–20th h after
inoculation and increased afterwards. While the absolute intensity of peak C3-6 was lower in the
measurements of the mutant strain compared to the reference strain, the time profile was similar for
both cultivations. The profile of peak C3-20 was similar to that in the reference strain cultivation but
showed a shallower increase at the start of the fermentation, while the intensity of C3-8 rose faster.
Although replicate experiments are required for a statistically valid statement, we hypothesize that
these distinct mVOC derived MCC-IMS signals allow differentiation of the two yeast strains.
Similarly, species specific volatile footprints or markers have are already used to detect cancer via
breath analysis of patients [113] or fungal contaminants in buildings [329].
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Chapter 2
Figure 13: (A) Fermentation profile of S. cerevisiae adh1Δ during batch growth in glucose minimal
medium and (B) trends in intensity and (C) heat map of selected peaks detected by MCC-IMS analysis
of the fermentation off-gas. The areas in the heat map show the detected signal peak and the
surrounding area; DO = dissolved oxygen.
These measurements gave first valuable information about the volatile metabolite patterns
produced by yeast and their dynamics during growth on glucose minimal medium. However, because
of the high signal of volatiles, most likely ethanol and acetaldehyde, in the off-gas and probable
incomplete ionization of metabolites, data interpretation and thorough metabolite detection under
these conditions was limited. To make batch observation possible, other column materials or longer
columns would be needed, however, longer columns would increase the analysis time by far [324].
MCC-IMS monitoring of glucose-limited chemostat fermentations

High glucose concentrations as in batch fermentations trigger glucose repression. One consequence
is the induction of respiro-fermentative metabolism that is aerobic ethanol formation, known as
Crabtree effect [289, 330]. This switch of the metabolic mode is undesirable for example during
production of yeast biomass or protein production, as ethanol formation reduces product yield and
quality [331-334]. Such processes are therefore run as glucose-limited fermentations in which this
regulatory mechanism is repressed. While industrial processes are most often run as fed-batch
fermentations, in academic research glucose-limited chemostats are favored as these continuous
cultivations can be performed under defined, controlled, and constant conditions allowing very
reproducible experiments. Moreover, this fermentation mode allows varying one single fermentation
parameter, making it ideal for studying the impact of specific perturbations on growth physiology or
metabolism. To elucidate possible changes in the volatile metabolites during the transition from
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Results
respiratory to fermentative metabolism, we cultivated S. cerevisiae in a glucose-limited chemostat at

a dilution rate of 0.13 h−1. Under these fully oxidative growth conditions, S. cerevisiae produced no
ethanol. At metabolic steady state of the single experiment, the MCC-IMS chromatogram showed 13
peaks, which were not detected in the sterile medium, while two peaks observed in the sterile medium
were not or with considerably less intensity detected in the chemostat culture (Figure 14). Because of
the overloaded chromatogram during batch cultivation, it is difficult to state, which of these are
specific for the glucose-limited respiratory growth conditions and which appear generally during
growth of S. cerevisiae. The most distinct peaks were identical for both growth conditions.
Figure 14: MCC-IMS topographic plot of the off-gas of a glucose-limited continuous cultivation of
S. cerevisiae. Boxes indicate signals that showed the most significant changes during the growth
(perturbation) experiments. The reaction ion peak (1/K0 = 0.5 Vs cm−2) was compensated for by the
VisualNow software.
To induce a shift from respiratory to fermentative metabolism, the steady state culture was
perturbed once with a pulse of 22 mmol glucose, which was rapidly injected into the bioreactor.
Immediately after the glucose pulse, ethanol accumulated in the fermentation broth (Figure 15 A).
Acetate (data not shown) and glycerol were detected as well and showed a similar profile as ethanol.
The surplus glucose was consumed within 75 min after which ethanol was catabolized and diminished
about 3 h after the pulse. The optical density (OD600) increased from 31–39. The intensity of several
signals detected with the MCC-IMS increased after the pulse and decreased again after about 2 h
(Figure 15 B,C). The most prominent changes were observed for the signals marked in Figure 14,
these are the same peaks as in the batch cultivation of the wild type yeast and the ADH1 mutant. Peak
C3-15 showed a strong correlation with the ethanol concentration determined in the fermentation
broth. However, other peaks, like C3-6 and C3-8, did not resume the intensities prior to the
perturbation, but maintained a higher level within the 7 h of MCC-IMS monitoring, hence, correlated
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Chapter 2
with the increase in biomass concentration. C3-5 was the only peak whose intensity decreased upon
glucose addition. About 3 h after the glucose pulse, its intensity increased again and regained its
original value in the next 3 h. C3-20 showed a rapid decrease directly after the pulse, remained at a
constant level and showed a decreasing trend after about 2 h and 30 min.
Figure 15: (A) Fermentation profile of S. cerevisiae during growth in a glucose-limited

chemostat and (B) trends in intensity and (C) heat map of selected peaks detected by
MCC-IMS measurements of the fermentation off-gas after perturbation of the metabolic
steady state with a pulse of 22 mmol glucose; DO = dissolved oxygen.
The physiological response of S. cerevisiae to limited oxygen availability is very similar to that of
the Crabtree effect. In both cases, the fluxes through glycolysis are upregulated, while the fluxes
through the TCA are downregulated and as a consequence ethanol is produced [294, 335]. To
elucidate possible differences in the volatile metabolite patterns of yeast cultures responding to a
glucose pulse and anaerobiosis, respectively, in a second perturbation experiment gassing was
switched from air to nitrogen. In this single perturbation experiment, the dissolved oxygen (DO)
concentration dropped to zero within 10 min. Simultaneously with this drop, ethanol and glycerol
accumulated in the fermentation broth, while only little acetate which accumulated only after 1 h of
anaerobic growth (Figure 16 A). The biomass concentration decreased slowly during anaerobiosis.
Note that the data of this perturbation experiment cannot be directly compared to the glucose pulse
as the air supply was permanently replaced by the same flow rate of nitrogen.
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Results
Figure 16: (A) Fermentation profile of S. cerevisiae during growth in a glucose-limited chemostat
and (B) trends in intensity and (C) heat map of selected peaks detected by MCC-IMS measurements
of the fermentation off-gas during transition to anaerobic conditions; DO = dissolved oxygen.
As in the glucose pulse perturbation experiment, peak C3-15 increased as soon as ethanol was
produced (Figure 16 B, C). The increase flattened after about 60 min and decreased after about 2 h.
Again, incomplete ionization and possible incomplete evaluation of the peak area might be
responsible for this trend. The intensities of peak C3-6 and C3-8 increased rapidly in the first 30 min
after the shift to nitrogen gassing, considerably faster than in the glucose pulse experiment. C3-9
showed a slight increase, while peak C3-5 stayed constant over the period of 3 h. Peak C3-20 showed
a similar trend as in the glucose pulse perturbation, a rapid decrease after the shift to nitrogen gassing
followed by a steady intensity during the 3 h of monitoring. The dynamics of the MCC-IMS signals
were very similar to that observed during the imposed Crabtree effect and no new peaks were
detected. However, S. cerevisiae responded faster to anaerobic than to glucose excess conditions.
Changes in the peak intensities of, for example, C3-15 were already observed 7 min after the shift to
nitrogen gassing, i.e., before complete anaerobiosis, while the first changes in the MCC-IMS peak
intensities after the rapid glucose pulse were observed only after 17 min.
In both perturbation experiments, samples were measured in intervals of 215 s over a period of
about 30 min. These rapid measurements show the potential of MCC-IMS analysis for online
monitoring and control of bioprocesses.
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Chapter 2
Identification of volatile metabolites

A limitation of MCC-IMS is its poor ability for analyte identification by either retention time or ion
mobility, or combination of both. While databases of reference molecules are developed, the
resolving power is low. To get first hints of the mVOC spectrum in the off-gas, we performed
HS/SPME GC-MS analyses. Volatile metabolites were extracted from 10 mL the headspace of culture
supernatant by solid phase microextraction. Peaks detected in the GC-MS analyses were identified
by comparing retention indices and spectra with data from the NIST library (Version 2.0) and the
FlavorNet database [64]. Although this identification procedure often results in ambiguous results, it
narrowed down the standard measurements conducted with the MCC-IMS for peak identification.
GC-MS samples were taken from the glucose-limited chemostat cultivation of S. cerevisiae
CEN.PK during steady state conditions, after perturbation of the continuous cultivation by a glucose
pulse and shift to anaerobic conditions (15 min and 50 min after pulse injection and shift to anaerobic
conditions, respectively). Samples were also taken of the batch cultivation of the isogenic ADH1
knock-out mutant during exponential growth and stationary phase. Several peaks in the GC-MS
analyses were found in all samples, while some peaks were specific for the different growth
conditions or strain used. In total, metabolites could be assigned to 10 peaks by database searches
(Table 9). Compounds found in the samples of all culture conditions and the S. cerevisiae adh1∆
mutant were ethanol (in different amounts), 2-pentanone, 2-phenylethanol, 2,3-hexanedione, butyric
acid, and isobutyric acid. Benzaldehyde was found in all samples except those from anaerobic cultures.
Acetoin and 2,3-butanediol were only detected in the samples of the S. cerevisiae adh1∆ strain.
Acetoin is an intermediate of the pathway to 2,3-butanediol and can be derived from pyruvate and
acetaldehyde via three different pathways [336-341]. With the reduced flux of acetaldehyde to ethanol
in this mutant, conversion of acetaldehyde to acetoin and further to 2,3-butanediol might be induced
as has already been reported for a ADH1, ADH3 and ADH5 triple knockout mutant of S. cerevisiae
BY4742 [342]. By contrast, isovaleric acid was detected in all samples except in those from the ADH1
knock-out mutant. Besides the 10 metabolites identified in the GC-MS analyses, standards of 1-
butanol and acetaldehyde were measured by MCC-IMS. mVOCs were assigned to unknown peaks
MCC-IMS signals by comparing the retention time and reduced inverse ion mobility to data of the
pure standard substance measurements. As already assumed from the correlation with the ethanol
concentration in the fermentation broth, peak C3-15 was identified as ethanol. Compounds found in
the GC-MS, which are part of the Ehrlich pathway, were isobutyric acid, isovaleric acid, and 2-
phenylethanol and were also measured via MCC-IMS. The Ehrlich pathway is a catabolic pathway
which degrades amino acid into aroma compounds such as higher alcohols or volatile fatty acids
[246]. The fusel acids (and corresponding alcohols) isobutyric acid, isovaleric acid, and 2-
methylbutanoic acid are, for example, derived from the branched chain amino acids valine, leucine,
and isoleucine, respectively, by the activity of aldehyde dehydrogenases, encoded by ALD2 to ALD6
[253]. Only isobutyric acid could be assigned to one of the peaks (C3-8) detected in the off-gas analyses
of yeast fermentations. Unambiguous identification of 2-phenylethanol was not possible due to
overlapping peak regions, which is due to co-elution and similar drift times, with 2,3-butanediol.
The pure standard MCC-IMS measurements identified peak C3-5 as 2-pentanone. In Penicillium
roqueforti, 2-pentanone is derived from β-oxidation of fatty acids and might be synthesized in S.
cerevisiae via the same pathway [343]. However, although 2-pentanone has been found in several S.
cerevisiae fermentations [66, 247], a biochemical confirmation of the synthesis via β-oxidation is so
far not described. Peak C3-7 (not shown in Figure 10 and Figure 14) was identified as 2,3-
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Results
hexanedione, which is reported as a metabolite of brewer’s yeast with a cheesy aroma [193] and has
recently been detected in the headspace of agar plates cultures of Corynebacterium glutamicum [344].
Table 9: Volatile organic compounds detected in fermentations of S. cerevisiae via GC-

MS measurements growing in glucose minimal salt medium. ND, not detected.
reduced inverse MCC-IMS GC-MS
SESI- MCC-
Compound peak ion mobility retention retention
Orbitrap IMS
1/K0 (Vs cm−2) time (s) time (min)
2,3-butanediol x x 0.575 4.5 26.517
2,3-hexanedione ND x C3-7 0.57 19.4 9.633
2-pentanone ND x C3-5 0.554 6.4 5.029
acetoin x x 0.532 8.5 16.932
benzaldehyde ND x 0.566 37.5 25.366
butyric acid ND x 0.63 24.4 30.127
ethanol x x C3-15 0.516 4 4.193
isobutyric acid ND x C3-8 0.618 10.4 27.827
isovaleric acid ND ND − − 31.594
2-phenylethanol ND x 0.578 4.5 39.67
The low recovery rate of metabolites identified by GC-MS might partially be explained by the
different sampling procedures. While for the MCC-IMS measurements 10 mL of the off-gas were
directly measured, for GC-MS analyses, VOCs were extracted from the culture broth at 50 °C and
were preconcentrated. Furthermore, GC-MS peaks were only tentatively identified by comparison
with databases and require validation by pure standard analyte measurements. However, 2,3-
butanediol and acetoin have also been found in the SESI-Orbitrap measurements and therefore
support the designation as identified (Table 9) [295]. To broaden the spectrum of analyte detection in
the MCC-IMS, measurements with both negative and positive ion mode are useful. Our future
experiments will focus on the identification of unknown MCC-IMS signals, including thorough
verification by GC-MS measurements of pure standard substances. Ideally, feeding experiments with
labelled precursors should be performed to conclusively prove whether the identified compounds are
actually produced by S. cerevisiae, this verification needs to be carried out using a suitable MS.

Application of MCC-IMS in positive ion mode for online monitoring of fermentation off-gas
detected 19 signals produced by S. cerevisiae during growth on glucose minimal salt medium.
Although the here presented first analyses probably captured only a minute fraction of S. cerevisiae’s
volatile metabolite spectrum, they were sufficient to differentiate S. cerevisiae strains and to reveal
the impact of different growth conditions on the production of mVOCs. Also, the capability of the
MCC-IMS for online measurements was shown. Four compounds were identified by complementary
GC-MS measurements of fermentation broth extracts and pure standard substance measurements.
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However, limitations of the device were apparent, when small amounts of ethanol lead to an
overloading of the device with problems in overall detection. Therefore, the setup cannot be used in
batch fermentations. Another problem is that all signals accumulate at low 1/k0 values and low
retention times. To increase the resolvability and to prevent overloading the MCC-IMS configuration
has to be optimized by, e.g., installation of a sample loop of lower volume or a more polar MCC.
In the following chapter, the investigation of VOCs is taken to another level, by adding a
transcriptome analysis to the MCC-IMS measurements. Also, the focus was shifted from solely
laboratory to near industrial conditions and a comparison of these two conditions.
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The Crabtree effect revisited
A comparative study on the transcriptional changes during the Crabtree
effect using laboratory and industrial strains and conditions
Contributions:
This study was designed by Christoph Halbfeld, Birgitta E. Ebert, Sven Rahman, Erik Pollmann and
Lars M. Blank. The experiments were performed by Christoph Halbfeld, Erik Pollmann, Ann-Kathrin
Sippel and Michael Quantz. The data was evaluated by Christoph Halbfeld, Ann-Kathrin Sippel, Sven
Rahmann and Birgitta E. Ebert. Here the microarray-data was statistically evaluated by Sven
Rahmann and checked for biological relevance by Christoph Halbfeld and Birgitta E. Ebert. The
MCC-IMS data analysis was performed by Ann-Kathrin Sippel, Christoph Halbfeld and Birgitta E.
Ebert evaluated the data. The chapter was written by Christoph Halbfeld.
Results
The Crabtree effect revisited

A comparative study on the transcriptional changes during the Crabtree effect
using laboratory and industrial strains and conditions
Summary
S. cerevisiae is broadly used in research and in industrial processes. The yeast strains used in the
laboratory and by the industry are related but differ in metabolic activity. However, the two groups
and growth conditions have up to now not been compared in relation to pathway usage before and
after a metabolic shift. Here the laboratory yeast strain S. cerevisiae CEN.PK 113-7D in minimal salt
medium was compared with the industrial strain S. cerevisiae DHW cultivated in industrial molasses
medium, for both strains and conditions the Crabtree effect was induced by overfeeding. The strains
were investigated using volatile metabolite as well as microarray analyses. No differences could be
detected between the two strains’ volatilome, however, differences in the transcription of genes
involved in leucine biosynthesis as well as oxidative phosphorylation were observed. This could be
especially interesting for the industrial yeast production since the lack of respiratory inhibition in the
industrial strain bears the potential of a higher biomass yield compared to the laboratory strain.
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Chapter 2
Introduction
The Crabtree effect describes alcoholic fermentation by Crabtree positive yeasts under aerobic
conditions with high sugar concentrations [289]. This metabolic shift causes a rerouting of the relative
central carbon metabolism fluxes from the pentose phosphate pathway to the glycolysis [294]. In
addition, high glucose concentrations repress parts of the electron transport chain and therefore
respiration, an effect called glucose catabolite repression. Beyond respiration, also the non-glucose
sugar metabolism as well as the hexose metabolism are specifically up- or downregulated on
transcriptional level, depending on the amount of available sugar [345, 346]. However, metabolic
effects outside the central carbon metabolism, especially on volatile compound synthesis, have not
yet been investigated in detail.
To gain insight into pathways that are differentially activated prior to and past a metabolic shift, it
is useful to investigate the transcriptome. For this purpose, microarray analysis is a powerful method
to generate a broad overview of transcriptional differences. In microarrays, probes consisting of
specific DNA sequences, representing the genotype of the specific strain, are spotted on a glass slide.
For the microarray, DNA is required, however, mRNA describes the expression state of the cell. To
convert mRNA into cDNA a reverse transcriptase is used. In the same step, the cDNA is also marked
with a fluorescent dye and subsequently applied to the microarray. The fluorescence intensity of the
single spots on the microarray correlates with the amount of bound cDNA, and thus the expression
level of the gene [347-349].
To cope with changing environmental conditions, the cells need to quickly adapt their repertoire
of enzymes. Here it is possible that the required enzymes are either needed to be produced or are
already dormant inside the cell and have to be activated by posttranslational modification (PTM).
Phosphorylation is the most used PTM in S. cerevisiae [350, 351]. If the enzymes need to be produced
de novo, a large amount of mRNA will be existent in the cell, and even if an enzyme is constitutively
present in the cells there is still some mRNA since the cell’s proteome is not static. Therefore, by
using DNA microarrays it is possible to get an in-depth insight into the transcriptional state of the cell
and based on that an estimation of the metabolic state.
The transcriptional changes of yeast during the Crabtree effect have been investigated before [352]
as well as the shift from aerobic to anaerobic conditions [353]. Kresnowati et al. found out that
transcripts of the TCA cycle genes are decaying one order of magnitude quicker than previously
expected leading to a quick metabolic transition [352]. However, all studies on S. cerevisiae’s
metabolic shift, the Crabtree effect, previous to this thesis have been based on chemostat experiments,
Regenber et al. reported that the growth rate in chemostats has an influence on the transcriptome
[354]. Even though a multitude of studies investigated the yeast transcriptome [352, 354, 355] there
has not been any study that investigated the Crabtree effect during fed-batch experiments and that
compares laboratory and industrial strains, however other comparisons between industrial and
laboratory strains have been performed [356].
Because of S. cerevisiae’s importance in both, scientific studies as well as industrial processes, it
is important to investigate and compare the yeast metabolism under both conditions. Many scientific
studies have been carried out with focus only on laboratory conditions, however in applied
microbiology the industrial aspect and therefore industry-near conditions should also be considered
because the behavior of the microorganism might change. Therefore, a laboratory strain in minimal
medium was compared to an industrial strain in molasses medium in this study.
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To find out more about the regulation of the volatile metabolism at the onset of the Crabtree effect,
custom microarrays based on the here measured samples were run. Samples were taken during
carbon-limited fed-batch fermentations while the feed rate was gradually increased to induce a
metabolic shift. This experiment was performed with a laboratory strain on minimal salt medium as
well as with an industrial strain on industrial molasses medium.

In this study two types of yeast strains were used, the laboratory strain S. cerevisiae CEN.PK 113-7D
(Euroscarf, Oberursel, Germany), from here on referred to as laboratory strain, and the industrial
strain S. cerevisiae DHW (Deutsche Hefewerke GmbH, Nürnberg, Germany, from here on referred
to as industrial strain. The laboratory strain was cultivated in Verduyn minimal salt medium (see
chapter 2.2 for the medium composition and preparation), while the industrial strain was cultivated
in industrial molasses medium. The strains were first cultivated in shake flasks at 30 °C in a New
Brunswick Controlled Environment Incubator shaker (Eppendorf AG, Hamburg, Germany) shaker at
180 rpm. Hereby the medium was complemented with 20 g/L glucose for the minimal medium and 6
g/L mixed sugars for the industrial molasses medium. To increase the biomass for inoculation of the
main culture, the strains were further cultivated in the same media (molasses medium with 7 g/L
mixed sugars and Verduyn medium with 20 g/L glucose, respectively) in 18 L batch cultivations at
30 °C pH 5 overnight. The biomass was harvested by centrifugation in a Hettich Rotanta 460
centrifuge (Rotor: Typ 5624, Andreas Hettich GmbH & Co.KG, Tuttlingen, Germany) for 5 minutes
at 4000 rpm. The cells were washed once with tap water and finally stored at 4 °C until further use.
In the next step, the fed-batch cultivations were carried out. 7,5 L of salt solution (34 g (NH4)2HPO4,
10 g MgSO4x7H2O, 0.6 g ZnSO4x7H2O, 30 mg biotine [2%], 13 mg thiamine, 21 mg pyridoxine, 65
mg nicotinamide, 18 mg Ca-pantheonate and 35g (NH4)SO4 were put into the vessel. Then 88 gCDW
of cells were diluted in 0.5 L tap water and filled into the reactor, finally the feed was started. The
feed consisted of 320 g/L glucose in ten times concentrated Verduyn minimal salt medium for the
laboratory strain, respectively, 320 g/L of fermentable sugar equivalents (based on CO2 formation)
the in molasses medium for the industrial strain.
The feed of the fed-batch was controlled based on the signal of the alcohol sensor (Alcoline,
Biotechnologie Kempe, Kleinmachnow, Germany), hereby ethanol was tolerated in the first 3 hours
of cultivation, after that the feed was adapted automatically to avoid ethanol formation. Later on, the
feed was controlled manually to slightly overfeed the system. The other fermentation parameters were
set as given in Table 10. The pH was controlled using 25% phosphoric acid and 20% ammonium
hydroxide solution.
The fed-batch was run carbon-limited with a growth rate of about 0.13 h-1 and about 0.19 h-1 for
the laboratory and the industrial strain, respectively. To induce the Crabtree effect, the feed rate was
slowly increased until ethanol formation was detected. After the detection of ethanol, the feed rate
was reduced to reestablish carbon-limited conditions. At each sampling timepoint (frequency of 1
sample per minute), three samples were taken. The three samples of 100 μL each were stored in an
ice-salt water bath, which had a temperature below 0 °C. When 24 samples were taken, the samples
were centrifuged in a Hettich Universal 32 (Rotor: Typ 1624, Andreas Hettich GmbH & Co.KG,
Tuttlingen, Germany) at 4000 rpm for 30 seconds. The supernatant was discarded and the pellet
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Chapter 2
subsequently frozen in liquid nitrogen. These samples were used later for the microarray analysis.
When ethanol was detected in the IMS signals, samples were taken every other minute. Based on the
ethanol sensor and IMS data, it was decided which samples were to be used for microarray
experiments. In total, 40 samples were sent to Oaklabs (Hennigsdorf, Germany), where transcriptome
analyses were performed with custom microarrays.
Table 10: Profiles of fermentation parameters of the fed-batch fermentations.

time [h] air [L/min] pH temp. [°C]
0 5 4.5 30
1 7 4.7 30
2 9 4.9 31
3 11 5.1 32
4 13 5.3 32
5 15 5.5 32
6 15 5.7 32
7 15 5.8 32
8 15 5.9 32
9 15 6.0 33
10 15 6.0 34
11 15 6.1 35
12 15 6.2 36
13 15 6.3 36
>14 15 6.4 36
MCC-IMS analysis
The ion mobility spectrometer Breath Discovery (B&S Analytik, Dortmund Germany) was used for
the detection of volatile metabolites in the fermentation off-gas. The MCC-IMS device was enhanced
by B&S analytic especially for the measurements in a yeast fermentation headspace. In comparison
to the device used in chapter 2.3, the multicapillary column was replaced by the MCC OV-1701 with
a film thickness of 0.6 μm. Also, the sample loop was shortened from 10 mL to 50 μL. The MCC-
IMS analysis measurements were performed as described in chapter 2.3 with the exception that the
gas mixing flask was not used and the device was directly coupled with the bioreactor with a
polytetrafluoroethylene tubing connected to the head plate of the bioreactor. Measurements were
taken every 5 minutes.
cDNA microarray analysis

The initial analysis of the microarray samples was carried out by Prof. Sven Rahman (Chair of
Genome Informatics at the Faculty of Medicine at Duisburg-Essen University, Ruhr Professor for
Bioinformatics at TU Dortmund). The samples were grouped into early (before a change of the IMS
signals), mid (after changes of IMS signals were observed but prior to a change of the ethanol sensor
signal) and late (after a change of the ethanol sensor signal) groups. Also, a false discovery rate value
(FDR) was calculated based on the method described by Benjamini and Hochberg indicating the
significance of the transcriptional change was [357]. Here only data with an FDR value of less than
or equal to 5% were considered for further analysis. Fold change rates of genes with an FDR below
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Results
5% in both strains were analyzed using BioCyc’s omics viewer [358]. For identified interesting
pathways the fold changes were also considered, even if the FDR was above 5%. However, these
results were evaluated cautiously and are not shown in the figures but descried in the text. Since the
FDR rates are partially very high, some trends are indicated, but these data cannot be taken for a fact.
All of the here indicated FDR values are listed in Supplementary table 1.

MCC-IMS and microarray analysis of two yeast strains
Carbon-limited fed-batch fermentations at pilot-scale (7.5-12 L) were run with a laboratory strain
using Verduyn minimal salt medium and an industrial strain using molasses medium. The Crabtree
effect was induced in both experiments by gradually increasing the feed rate. In each experiment, an
ethanol sensor and an MCC-IMS were used for online monitoring of aqueous ethanol and volatile
metabolites, respectively. Additionally, samples for microarray analyses were taken once per minute.
Because of the high cost of the microarray analyses, only 40 samples were analyzed. 19 samples
each were measured from the experiments of the laboratory strain on minimal medium and the
industrial strain on the molasses medium, and two samples from an experiment with the laboratory
strain on industrial molasses medium. From Figure 17, it is observable that in both experiments the
MCC-IMS detected changes in the ethanol concentration minutes before the commercially available
ethanol sensor. The MCC-IMS is not only capable of detecting ethanol but can also be used to detect
other signals in the same run. Here, besides ethanol, isoamyl alcohol was detected. This has the
advantage that detection of the Crabtree effect becomes more robust since it is not only relying on
one single substance but two. Because of the early detection of the signals in the MCC-IMS, the
microarray samples were divided in three groups. The first group (early) spans samples taken prior
to any observable changes of the IMS signals when compared to the period before the feed was
enhanced. This group also contained samples taken before the feed rate was enhanced. The early
group encompassed 7 samples of the lab strain and 3 samples of the industrial strain. The second
group (mid) included samples taken in the time period, when MCC-IMS signals changed, but the
ethanol detector signal stayed constant. The mid group contained 8 samples in the lab strain and 11
samples in the industrial strain. Finally, the third group (late) consisted of samples taken after
increased ethanol levels were detected with the ethanol sensor. The late group contained 4 samples
of the lab strain and 5 samples of the industrial strain.
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Chapter 2
Figure 17: Volatile signals of Saccharomyces cerevisiae CEN.PK 113-7D (A) and Saccharomyces
cerevisiae DHW (B) in a fed-batch that was slowly overfed to trigger the Crabtree effect. The grey
background represents the feed rate. The commercially available Alcoline sensor form Biotechnology
Kempe was used for ethanol quantification in the fermentation broth along with the MCC-IMS Breath
Discovery from B&S Analytik for the monitoring of volatile metabolites in the fermentation head
space. The black dotted line indicates the start of the overfeeding process, the red dotted lines indicate
the division into early (left) mid (middle) and late (right) phase. D and M behind the substance names
describe the measured dimer and monomer respectively.
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Results
The data was investigated for differences between the laboratory and the industrial strain. To
identify changes that might be based on the medium rather than on the strain, transcriptional changes
of the laboratory strain on molasses medium were evaluated as well. So far, this comparison has not
been performed in any other study that we know of. The strongest differences in single pathways
were detected in respiration, the TCA cycle and the metabolism of leucine and isoleucine. Some
changes could be observed between the early and the mid samples, however the strongest changes
were observed between the early and late samples. In the laboratory strain, the aerobic respiratory
pathway was downregulated alongside with the tricarboxylic acid pathway (Figure 18). This is
conform with the Crabtree effect induced changes described in the yeast literature [289, 359].
However, the industrial strain showed opposing result: In this strain, the genes of the TCA cycle and
the respiratory chain, indicated in Figure 18 A and B by an orange color, were upregulated. Such a
behavior has up to now not been described. In the aerobic respiratory pathway, the indicated genes
that did not show a strong transcriptional change (SDH3, SDH2, QCR10, QCR9, QCR8, QCR7,
QCR6, RIP1, COX11, COX19, COX16, COX20 and COX5B) had a FDR above 5%. However, the
trends followed the indicated ones for all genes but COX19 in the laboratory conditions and COX20
and COX5B in the industrial conditions. This means that these genes were downregulated in the
laboratory strain and upregulated in the industrial strain, while the exceptions showed the opposite
behavior. It also should be noted that the FDR values for COX20 and COX5B were above 50%,
indicating these values, as all values not indicated in Figure 18, only display tendencies and not hard
facts. In summary, respiration was downregulated in the laboratory strain, while it was upregulated
in the industrial yeast strain.
The TCA cycle genes showed a similar gene expression pattern as those of the respiratory chain,
i.e., downregulation of all genes of the lab strain and 4 genes of the industrial strain and upregulation
of zero genes of the lab strain and 3 genes of the industrial strain. In addition to the expression changes
indicated in Figure 18 B, the genes PYC1, PYC2, CIT1, LPD1, KGD1, KGD2, MDH1 also showed a
similar regulation as the in Figure 18 indicated ones for each strain, i.e., downregulation in the lab
strain and upregulation in the industrial strain, they were however not plotted, since their FDR was
above 5%. ACO2, IDH1 and IDH2 showed a different expression profile. ACO2 was upregulated in
the laboratory strain and downregulated in the industrial strain and therefore opposing the regulation
pattern of the other genes in the TCA cycle, IDH1 and IDH2 were upregulated in the lab strain.
However, the expression data of the IDH genes showed a 30% FDR value and might therefore not be
as reliable as the other trends indicated in Figure 18.
In experiments with the laboratory strain on industrial molasses medium, it was observed that the
laboratory strain had a much lower critical growth rate (about 0.13 h-1) in comparison to the industrial
strain (about 0.19 h-1) in 1 L fed-batch experiments (data not shown). These results in combination
with the microarray data evaluation (Figure 18 A and B) indicate that the industrial strain might not
slow down its respiration as the laboratory strain does, since the industrial strain did not seem to be
affected by the glucose repression as the laboratory strain was. Thus, in the industrial strain, the
biomass yield is theoretically higher, since more of the glucose could be channeled through the
respiratory pathway instead of the fermentative pathway compared to the laboratory strain. This
makes the industrial strain theoretically much more suitable for biomass production compared the
laboratory strains. However, as ethanol was still produced, it has to be shown if this negatively affects
the baking ability. This result also gives hints about the importance of the Crabtree effect in industrial
yeast production. Since the Crabtree effect seems not to be as distinctive in the industrial conditions
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Chapter 2
as anticipated from the laboratory results [294], its impact on the industrial yeast production might
not be as big as could be anticipated from experiments with laboratory strains. However, higher yields
could be achieved, if the Crabtree effect is avoided, since fermentative growth yields less biomass
compared to respiratory growth. Since the transcriptome could also vary from the proteome and
fluxome, further experiments would be necessary to proof this theory.
The leucine biosynthesis was upregulated in the laboratory strain but was downregulated in the
industrial strain. This is an interesting finding, since in both strains the production of the leucine
degradation product 3-methyl-1-butanol increases during aerobic fermentation. The downregulation
in the industrial strain however, might be attributed to the medium because the laboratory strain on
industrial molasses medium indicated also a downregulation of the leucine metabolism (data not
shown). Not depicted in Figure 18 C are the values for the genes ILV5, LEU9, LEU2 and BAT1
because their calculated false discovery rate was in at least one of the data sets above the threshold
of 5%. However, the data still indicates a vague change of expression. All genes not indicated in
Figure 18 C are upregulated in the laboratory strain and downregulated in the industrial strain. The
only exception is LEU5, which was upregulated in both strains, however, the FDR of the industrial
strain data was very high (55%) and might give a wrong indication.
LEU4 codes for the major isoenzyme of α-isopropylmalate synthase as described by Casalone et
al. in 2000 [360]. Therefore, it would be expected that the major gene coding for the enzyme
catalyzing 3-methyl-2-oxobutanoate to 2-isopropylmalate would show a similar expression pattern
compared to the other genes in the pathway, but this was not the case here. One possible explanation
is that in the here tested laboratory strain, LEU9 codes for the major isoenzyme, another possibility
is that based on the growth conditions either LEU9 or LEU4 is expressed as known for BAT1 and
BAT2 [361].
BAT2 codes for the major branched-chain amino acid aminotransferase isoenzyme in the stationary
phase, while the BAT1 encoded enzyme dominates in the exponential growth phase [361]. Here in
both strains BAT2 was downregulated, while BAT1 was upregulated in the laboratory strain. This
reflects the activity of the rest of the pathway, where genes were upregulated in the laboratory strain.
The transaminases encoded by BAT1 and BAT2 are both, involved in the leucine biosynthesis and
degradation. This and an indication based on the microarray data of higher THI3 (ketoisocaproate
decarboxylase) expression, both enzymes involved in the degradation of leucine, can explain the
higher levels of 3-methyl-1-butanol in the samples during the Crabtree effect at least for the laboratory
strain.
98
Results
Figure 18: Transcriptional alterations of the most differing pathways based on expressional changes
between Saccharomyces cerevisiae CEN.PK 113-7D (left) and Saccharomyces cerevisiae DHW
(right). The two strains were run in fed-batch fermentations and overfeed gradually. The respiration
pathway (A), the TCA-cycle (B) and the branched-chain amino acid production (C) are depicted. The
genes without color coding are not displayed, since it had a false discovery rate of more than 5% in
at least one of the tested strains.
99
Chapter 2

In this chapter, two different yeast strains, a laboratory and an industrial yeast strain were compared.
Since in industrial yeast production, undefined molasses medium, and in laboratory applications
defined medium is used, we replicated these conditions here in pilot scale. Here off-gas analysis and
microarrays have been used to compare the two strains and media. The laboratory and the industrial
strain showed no differences in the production of ethanol and isoamylalcohol in response of the
Crabtree effect. Therefore, it can be assumed that all tests described in chapter 2.3 can be transferred
to the here tested industrial strain under industrial conditions. While the metabolites detected in the
off-gas showed similar behavior in both strains, differences in the expression of the genes of the
leucine, valine biosynthesis pathway and the genes in the respiratory pathway including the TCA
cycle could be observed in the microarray experiments.
Due to upregulation of the respiratory pathway, it could be concluded that the Crabtree effect in
the industrial strain is not as distinct as in the laboratory strain. However, since transcriptome data
can only indicate hints and are not necessarily connected to the proteome and fluxome, especially in
such short time intervals, further experiments are necessary to verify this assumption. In further
experiments the physiology could be investigated. However, this might be challenging especially in
industrial molasses medium. This diverse medium might make it difficult to use standard analytics
(e.g., HPLC, OD-measurements). Therefore, special methods have to be developed to investigate the
physiology and prove if the here seen changes in the transcriptome can be extrapolated to the yeasts
physiology.
100
Chapter 3
General discussion
General discussion and outlook
General discussion
Possible impact of this thesis
The insights into yeast metabolism gained in this thesis could be used in many applications. The here
introduced methods could have a major influence on future studies and the production of volatile
compounds. These methods could be used as a blueprint for future studies in other organisms. Instead
of implementing completely new pathways, the here generated knowledge could be used to simplify
the production of volatiles. It would also be possible to use online metabolic flux analysis with the
methods developed here. Lastly, the differentiation of metabolic states through the headspace could
be used in process control along with product quality analysis and the possibility to detect possible
contaminations.
Impact on future studies

This thesis could be used as a blueprint for the investigation of volatile compounds in other organisms.
For example, the investigation of soil bacteria could be interesting, since some of them use VOCs to
communicate with each other in sparsely populated terrains [92]. The here used methods are not
specific for S. cerevisiae but can also be applied for other microorganisms including prokaryotic
organisms. Through minor adaptations to the methods used in this thesis, the VOCs even of multi-
cellular organisms can be analyzed. For example volatile measurements of Begonia semperflorens
have been conducted by Barrios-Collado et al. [297]. The identification of the measured volatiles is
a challenge and can be pursuit using fragmentation of the metabolites in a mass spectrometer and
subsequent comparison of the fragments with a database like NIST [362]. As many VOCs are rather
small, isomers not easily identified by differences in mass occur, and hence it is always a good idea
to implement a second source of verification. For that purpose, it is possible to run a standard of the
expected metabolite.
The yeasts metabolic network has been investigated in great detail, however, prior to this thesis
little was known about the volatile metabolism [64, 194, 227, 229, 230]. The lack of information
about the biochemistry of VOCs can be compared to the novel annotation of not well investigated
organisms: basic information, like the genome sequence, can be compared by computational tools to
existing databases. Here, with the aim to investigate possible pathways connecting new metabolites
to the already known biochemical network. As described in this thesis, in a next step, the enzymatic
capabilities can be investigated using databases, to make sure that the organism is capable of
enzymatically catalyzing the reaction. However, if no enzyme with the required activity is identified
or the enzymatic capabilities of an organism are not well investigated, there is still a chance of the
presence of undiscovered enzymes or enzymes with moonlighting activity. Moonlighting describes
enzymes that have the capability of catalyzing two independent reactions [276]. A good starting point
for connecting new metabolites to an already existing metabolic network is the use of biological
databases (e.g., KEGG) and tools that search for the connection (here fmm, pathpred and metaroute
have been used) [207-209, 231, 232]. For the production of compounds that have not been described
yet, the tool BNICE can be used, this program predicts new metabolic pathways based on a set of
enzymatic reactions [234]. Using these tools is very convenient, since not all pathways have to be
investigated manually. However, it has to be noted that the metabolic pathways need to be verified,
105
Chapter 3
before they can be assumed correct. FBA and TFBA can be used to increase the plausibility of added
reactions as described in chapter 2.1 [219, 235].
It was also attempted to calculate which metabolites are volatile at room temperature based on
their molecular weight. However, structural complexity and other factors like functional groups have
a big influence on volatility, making a priori estimation of volatility difficult [363-365], although
highly interesting for basic research and applications.
Strain engineering & possible use of volatile products

With all the new knowledge gained in this thesis, where can it be applied? The here gained
information could be used to knock out genes that would weaken the Crabtree effect and therefore, a
nearly Crabtree negative strain that still holds the advantages like taste and texture of the industrial
strain could be produced. The MCC-IMS could be used to detect changes in the 3D-topographic plot
of the wildtype and knockout strain, to ensure that the flavor of the yeast strain is not affected by
these changes. This could be tested in a laboratory strain and later the same deletions could be
introduced into the different industrial strains that are used in the yeast production plants. This is
possible because of the great similarities that were revealed in the microarray experiments between
the laboratory and the industrial strain and conditions. Since the industrial strains are mostly not
haploid, the CRISPR-Cas9 system is predestined to be used in these strains. It was shown before that
polyploid strains can be manipulated easily [366-368].
Also new yeast strains could first be developed in the dry lab through application of the newly
generated metabolic model and in the next step the created strains could be constructed in the wet
lab. For example, by overexpressing pathways investigated in this thesis, yeasts could be designed
that would produce a higher amount of selected VOCs and therefore have a special flavor. There are
several compounds in yeasts that can create pleasant flavors if concentrated correctly. For example,
acetate esters have a fruity aroma, while 2-phenylethanol smells like roses, also creamy or caramel
like flavors can be found in yeasts repertoire [100]. If treated correctly, the designed flavor would not
only enhance the yeasts flavor, but still appear in products created with this yeast. This way it would
be possible to create special beers with strong taste of banana or a wine with a rose like bouquet.
Since the initial research on volatile compounds comes mostly from the investigation of foodstuffs
and beverages like wine, previous studies could be used to create new products with just the right
amount of volatile compounds [9, 291, 369-371]. The balance of different volatiles is important
because too high concentrations are also recognized as unpleasant [75].
These metabolites are not restricted to foodstuffs and beverages but could also be used in
cosmetics, fragrances and perfumes. For some of the metabolites investigated in this thesis
biotechnological processes are already in development [1, 372-375].
Another application would be the production of valuable metabolites that can be used for the
production of bioplastics or biofuels. Furans and terpenes might be the biofuels of the future. Also,
mycodiesels are close in composition to conventional diesel fuel and could take their place in the
coming years [376, 377]. Terpenes are not only interesting, regarding biofuels but also for the
production of drugs. Based on isoprene, medical relevant drugs could be produced directly in the
yeasts [378].
106
The use of different metabolic conditions

An advantage of yeast is the possibility to easily control its metabolic shift from aerobe respiration to
fermentation also known as the Crabtree effect [289]. Using this effect, it would be possible to let the
strain accumulate biomass in a low sugar and highly aerated fermenter. Once enough biomass is
present, the fermentation could be overfed to induce the production of special volatiles. Therefore,
the strain needs to be engineered to only produce the product in question while fermenting. Through
this thesis this engineering should be a lot easier than before because of the vast additions to the yeast
metabolic model and the microarray experiments performed in both, a laboratory and an industrial
strain.
Online flux analysis

The here introduced methods are highly sensitive and online measurements are possible. When the
detected peaks and masses can be assigned to metabolites, together with the enhanced yeast model it
would be possible to take flux balance analysis to the next level and calculate live fluxes. Online flux
analysis has been performed before, however through the noninvasive measurement of analytes and
the large range, this method could still be enhanced [379]. This would increase the yield of industrial
products, since the process parameters could be adjusted according to the microbial current metabolic
state. If correctly applied, the yield of the desired product could be increased, especially in difficult
production processes, e.g., where overfeeding might be problematic.
Conclusion
In this thesis, the volatile space of S. cerevisiae has been investigated. Therefore, computational
methods, novel analysis techniques and finally transcriptome analyses have been performed. Since
volatile metabolites are of high value and are important in industrial processes (aroma, taste), not only
laboratory, but also near industrial conditions have been examined in this thesis. Finally, we were
able to enhance the yeasts metabolic network, to increase the number of tools available for the
investigation of volatile compounds in microorganisms and to use the newly gained knowledge to
compare laboratory with industrial conditions.
The consensus yeast metabolic model

The consensus yeast metabolic network model (Yeast 7.5), is considerably lacking the volatile space
[230, 380]. In this work, these shortcomings were addressed by investigating the yeast literature for
known volatile metabolites and expanding the consensus model. A list of known volatile compounds
has been created and possible pathways have been investigated. Therefore, the theoretical known
enzymatic repertoire of yeast has been used, as well as computational tools and new pathways have
been created. The newly created pathways have been verified through a thermodynamic flux analysis.
In addition, 13 pathways have been verified by investigating 8 ADH knockout mutants that contain
only one enzyme with ADH activity each and one mutant that contains no enzyme with any ADH
activity. In total 219 reactions and 225 metabolites have been added to the metabolic model, while it
was not practically possible to experimentally verify all of the reactions added; this might be
addressed in future studies. It is possible that not all enzymes that have been added to the model are
required for its function because some proteins might possess moonlighting activity [273, 276].
107
Chapter 3
Moonlighting describes enzymes with more than one catalytic activity, making it possible to have a
smaller number of enzymes carrying out more reactions than anticipated up to this day [273, 276]. In
conclusion, the metabolic yeast model covers now volatile compounds, and includes reactions that
connect the volatiles with the know biochemistry. Novel experimental evidence can be easily used to
curate the model in the future.
Sesi-Orbitrap
To examine the volatile space of S. cerevisiae, new analytical tools have been used. The SESI-
Orbitrap combines high time resolution with high mass resolving power and high mass accuracy,
making online investigation of volatile dynamics in near real time possible. The Crabtree effect was
examined as distinct metabolic shift with industrial importance in food production. Using the SESI-
Orbitrap it was possible to investigate the Crabtree effect in a detail that it has never been investigated
in before. It was possible to investigate sub-minute changes of metabolism for the first time and to
observe metabolic network operation in near real time. Due to the new technology and the high
redundancy of the measurements, the system delivers robust data. Up to now there are no comparable
measurements with such high time resolution and mass accuracy. However, due to the high cost and
high maintenance of an Orbitrap, its use outside of the lab is limited. For industrial applications
another device, the MCC-IMS, was considered for the evaluation of quick changes in the volatile
yeast space.
MCC-IMS
The MCC-IMS was used for measurements of yeast fermentations, since it can operate non-invasive,
online, is a low-cost device and requires a relatively low maintenance [109, 381, 382]. Because of
these properties, the IMS is already used in industrial applications, such as air quality control, food
quality analysis, medical diagnostics, scanning for drugs, chemical warfare agents and explosives in
the military and for airport safety controls [109, 115, 117, 383-388]. Here it was shown that the two
metabolic conditions of aerobic respiration and aerobic fermentation could be distinguished in yeast
using an MCC-IMS. In the beginning, there were problems with too high ethanol concentrations in
the MCC-IMS, these problems could be solved by introducing a shorter sample loop and changing
the MCC in cooperation with the B&S Analytik (Dortmund Germany). In its final state, the MCC-
IMS could successfully distinguish different metabolic conditions in yeast. This has been reported
before, but never with an MCC-IMS [61, 62, 185]. Through its high sensitivity the device was even
able to detect metabolic changes prior to an industrial ethanol sensor. Additionally, to its fast
detection, the measurement is also more robust since two reporter metabolites that are produced early
during the metabolic change, can be used to predict the alteration in metabolism. As shown by Vautz
and Baumbach in 2008, the IMS is also suitable for process control [389].
Laboratory vs. industrial conditions

Laboratory and industrial conditions are hardly comparable, yet studies are mostly performed under
laboratory conditions. Here, both were investigated and compared. The metabolic shift could be
detected in both conditions. While the laboratory strain S. cerevisiae CEN.PK 113-7D was tested in
108
minimal salt medium as well as in industrial molasses medium, the industrial strain S. cerevisiae
DHW was solely tested under industrial conditions. All tests were carried out in pilot scale with a
volume for the fed batch of 8-11 L. Distinguishing between the metabolic states aerobic respiration
and aerobic fermentation was possible in all conditions. This is based on the robust measurements
that did not only yield one, but two distinct metabolic reporters at an early stage. The advantage of
two reporters is that the system becomes more stable, if for example the drift gas flask is dirty and
one reporter cannot be detected anymore, it is likely that the other reporter will still show up.
In addition to the volatile space, the transcriptome of the cells was also investigated to compare
the two conditions. Here it turned out that the gene regulation of the two strains was essentially similar
with exceptions in leucine and isoleucine metabolism, the TCA cycle and the respiratory pathway.
This is especially interesting for industrial yeast production, since it indicates that the Crabtree effect
might not be as significant in industrial yeast fermentations as it is in the laboratory.
109
Chapter 4
Outlook
Outlook
Outlook
The results generated in this thesis might contribute to future studies. First of all, the expansion of the
metabolic model will be helpful for studies concerning the metabolic network of yeast. The yeast
community will benefit from these new pathways especially if solutions need to be found to produce
large amounts of volatile organic compounds. However, for a rich aroma it is important to take into
consideration that not only one compound is responsible. Therefore, further investigations need to be
performed to find out what combination of metabolites will produce a pleasant aroma. Once the aroma
is constructed, metabolically engineered yeasts could be used to produce the flavor and positively
alter the aroma of a finished product. The final strains will have to be adjusted repeatedly, to ensure
that the intended flavor is achieved in the final product.
The data generated using the SESI-Orbitrap is very rich. In this thesis, the data has been evaluated
for effects during the Crabtree effect, however due to the high time resolution and excellent mass
resolution of the single time points, it is possible to gain even more insight without the need of further
experiments. For example, it should be possible to detect enzymatic activity based on mass transfers
and the accompanying increase respectively decrease of masses involved in the enzymatic reaction.
In the best case, it is even possible to determine reaction rates based on this dataset.
The here identified “reporter” volatile compounds could be interesting in industrial processes that
need to monitor yeast fermentations, since the reporters can indicate the change of the metabolic state.
The system could be checked online for example using the MCC-IMS described in chapter 2.3 and
based on the desired product the feed could be adjusted to gain biomass or the production of ethanol.
However, the use of the MCC-IMS is not only limited to yeast fermentations, using this thesis as
blueprint, it would also be possible to adjust the systems to other industrial processes. In addition,
MCC-IMS data would enable yeast producers to detect contaminations in very low concentrations
and early in an industrial process and contribute to avoid unnecessary costs.
113
Appendix
Appendix
Matlab Scripts for the evaluation of SESI-Orbitrap data
Driver:
%% Script for loading the data and running the analysis automatically for the file that equals n
% Order as indicated in Exps
%Creates two clustergrams, the first shows all masses that are changing between the glucose pulse
%and the rise of the ethanol signal, the second shows all masses that rise after the increase of the
%ethanol signal
%%
cd('C:\Measurements');
Data = load('Filename');
Exps = {'id20160513_Mini6TestR1'; 'id20160513_Mini6TestR4'; 'id20160513_Mini6TestR3';
'id20160517_WachstumCENR1Puls1'; 'id20160518_WachstumCCENR2Puls110muL';
'id20160518_WachstumVHR4Puls1_100muL'; 'id20160519_PulsVHR3mit100mgGlc_160518172415';
'id20160519_PulsCENR1mit100mgGlc_160518172415'; 'id20160520_PulsVHR4_100mgGLc_160518172415';
'id20160520_PulsCENR2_100mgGLc_160518172415'};
PulsTimes = [23.4; 13.5; 20.1; 21.1; 50.4; 58; 40.5; 50; 44.5; 45]/60;
% Conversion of minutes to hours for each timepoint after that the glucose pulse was applied
ExpNames = fieldnames(Data.mz);n=4
[ClustergramMaxInInterval, ClustergramMaxPastInterval,...
resi, mz, tt, tPuls, tEtOHSig] = Analyse_Glc_Puls(Data,...
Exps(n), PulsTimes(n));
PosEtOH = find(roundn(Data.mz.(Exps[96]), -3) == 93.0910);
PosPuls = min(find(roundn(Data.tiempo.(Exps[96]), -3) > roundn(PulsTimes(n),-3)));
time = transpose(Data.tiempo.(Exps{n}))*60;
Analysis:
function [ClustergramMaxInInterval, ClustergramMaxPastInterval, resi, mz, tt, tPuls, tEtOHSig] =
Analyse_Glc_Puls(Data, Exps, PulsTimes)
%% Script for data analysis of high resolution SESI-Orbitrap data
% we are searching for a timeseries the slope of which rises or falls sooner over the threshold than
that of EtOH
% rawdata time in h
%%
Res = struct();
for e = 1: length(Exps)
PosEtOH = find(roundn(Data.mz.(Exps[79]), -3) == 93.0910);
tPuls = min(find(roundn(Data.tiempo.(Exps{e}), -3) > roundn(PulsTimes(e),-3)));
% deltas between 2 data points
RawData = Data.tt.(Exps{e});
%% smoothing of the data using “moving average” and “sgolay”
MyNormedData = medfilt1(RawData(:,:),27,[],2);
DeltaSig = diff(MyNormedData,1,2);
tEtOHSig = min(find(DeltaSig(PosEtOH,:) > max(DeltaSig(PosEtOH,1:tPuls))));
minEtOHSig = MyNormedData(PosEtOH, tEtOHSig+1); % detections threshold
MyNormedData(abs(MyNormedData) < minEtOHSig) = 0;
%% finds signals that have larger changes during the measurements than the minimal EtOH
% signal
MinMaxSignalPrePuls = iqr(MyNormedData(:,1:tPuls),2);
MinMaxSignalPostPuls = iqr(MyNormedData(:,PosEtOH:end),2);
RelevantSignals = abs(MinMaxSignalPrePuls - MinMaxSignalPostPuls) > minEtOHSig;
%create variable mz for plotting
mz=Data.mz.(Exps{e});
tt=Data.tt.(Exps{e});
res = zeros(size(MyNormedData));
for i = 1: size(res,1)
res(i,:) = sgolayfilt(MyNormedData(i,:),1,35);
end
MyNormedData=res;
resi = zeros(size(MyNormedData));
for i = 1: size(MyNormedData,1)
xmin = min(MyNormedData(i,:),[],2);
xmax = max(MyNormedData(i,:),[],2);
xdiff2 = xmax - xmin;
for o= 1: size(MyNormedData,2)
xi = MyNormedData(i,o);
xdiff1 = xi-xmin;
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Appendix
xdiff = xdiff1 / xdiff2;
resi(i,o) = xdiff ;
end
end
%% find relevant signals that max between tpulse and t EtOH
MaxInInerval = max(resi(:,tPuls:tEtOHSig),[],2) > max(resi(:,tEtOHSig:end),[],2);
MinInInerval = min(resi(:,tPuls:tEtOHSig),[],2) < min(resi(:,tEtOHSig:end),[],2);
x = find(RelevantSignals & MaxInInerval);
lables = cellstr(num2str(Data.mz.(Exps{e})));
lables = cellstr(num2str([1:length(Data.mz.(Exps{e}))]'));
ClustergramMaxInInterval.(Exps{e}) = clustergram(resi(x,
tPuls:tEtOHSig),'RowLabels',lables(x),'Cluster', 'Column','LogTrans','false');
%% Find signal with max after t EtOH signal but have a signal before t EtOH
MaxPostEtOH = max(resi(:,tEtOHSig:end),[],2) > max(resi(:,tPuls:tEtOHSig),[],2);
x = find(RelevantSignals & MaxPostEtOH);
lables = cellstr(num2str(Data.mz.(Exps{e})));
lables = cellstr(num2str([1:length(Data.mz.(Exps{e}))]'));
ClustergramMaxPastInterval.(Exps{e}) = clustergram(resi(x,
tPuls:tEtOHSig),'RowLabels',lables(x),'Cluster', 'Column','LogTrans','false');
end
end
Plot:
%x = vector of masses that will be plotted against ethanol
X=[57.0700 58.0735 69.0701 70.0732 71.0857 72.0887 73.0649 74.0680 118.0652 119.0885 127.1117
128.1152 166.9860 177.0763 196.1123]';
for i = 1: size(X,1)
figure;plot((time),resi(PosEtOH,:),'k','LineWidth', l)
hold on
%PlotX
a = find(roundn(mz,-4) == X(i,:));
tPulsi=time(tPuls) %transforms time value from column number to value
tEtOHSigi=time(tEtOHSig) %transforms time value from column number to value
b=mz(a,:);
plot((time),resi(a,:),'LineWidth', l)
plot([tPulsi;tPulsi],[0,1],'k','LineWidth', l)
plot([tEtOHSigi;tEtOHSigi],[0,1],'k','LineWidth', l)
legend('Ethanol',num2str(b))
xlabel('time [min]','fontsize', 15)
ylabel('normalized data','fontsize', 15)
set(gca,'fontsize',15)
%xlim([40 90]); %sets x axis to the wanted time
figure;plot(tt(a,:),'LineWidth', l)
Correlation:
%% a and b are the m/z fragments that are to compare
a = find(roundn(Data.mz.(Exps{n}),-4) == 89.0962);
b = find(roundn(Data.mz.(Exps{n}),-4) == 71.0857);
%normalization of the data
xmina = min(Data.tt.(Exps{n})(a,:),[],2);
xmaxa = max(Data.tt.(Exps{n})(a,:),[],2);
xdiff2 = xmaxa - xmina;
for o= 1: size(Data.tt.(Exps{n}),2)
xi = Data.tt.(Exps{n})(a,o);
xdiff1 = xi-xmina;
xdiffa = xdiff1 / xdiff2;
resia(1,o) = xdiffa ;
end
xminb = min(Data.tt.(Exps{n})(b,:),[],2);
xmaxb = max(Data.tt.(Exps{n})(b,:),[],2);
xdiff2 = xmaxb - xminb;
for o= 1: size(Data.tt.(Exps{n}),2)
xi = Data.tt.(Exps{n})(b,o);
xdiff1 = xi-xminb;
xdiffb = xdiff1 / xdiff2;
resib(1,o) = xdiffb ;
end
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Appendix
%plot the data

figure;plot(resi(PosEtOH,:),'k')
hold on
plot (resia, 'g')
plot (resib, 'r')
plot([tPuls;tPuls],[0,1],'k')
plot([tEtOHSig;tEtOHSig],[0,1],'k')
cx = corr2(resi(a,:),resi(b,:))
cxy = num2str(cx)
%axis labeling and legend
c=mz(a,:);
d=mz(b,:);
legend('Ethanol',num2str(c), num2str(d),'corr coeff: ', cxy)
xlabel('time [min]')
ylabel('normalized data')
119
Appendix
Supplementary data
Supplementary table 1: FDR Values of transcript level changes between early and late group. FDR-
LS describes the FDR values in the laboratory strain, while FDR-IS describes the FDR value in the
industrial strain.
Name FDR-LS [%] FDR-IS [%]
SDH4 0,12 0,04
SDH3 0,08 10,40
SDH2 3,65 34,23
SDH1 0,36 0,61
YJL045W 0,02 1,49
NDE2 0,02 0,10
NDI1 10,80 58,28
NDE1 93,21 10,13
QCR10 3,73 15,58
QCR9 0,70 10,40
QCR8 1,84 24,29
QCR7 6,75 28,01
QCR6 5,36 15,56
QCR2 0,92 0,93
COR1 0,08 1,79
CYT1 0,34 1,15
RIP1 0,20 5,43
COB 100,00 88,52
COX11 10,45 12,38
COX19 7,51 37,47
COX16 0,01 60,03
COX23 0,50 3,09
COX18 83,57 45,83
COX20 0,02 76,23
COX5B 0,09 53,37
PYC1 0,06 9,93
PYC2 11,27 15,10
CIT3 0,01 0,03
CIT1 0,05 48,23
ACO1 1,02 3,11
ACO2 0,09 45,97
IDH1 29,01 5,04
IDH2 30,80 12,36
LPD1 2,83 60,74
KGD1 0,02 14,72
KGD2 0,09 35,94
LSC1 0,03 1,42
LSC2 0,00 11,41
MDH1 13,22 2,45
ILV2 0,51 2,26
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Appendix
ILV6 10,92 3,71

ILV5 17,92 55,02
ILV3 0,32 0,56
LEU9 0,01 87,15
LEU4 0,01 0,20
LEU1 0,00 1,71
LEU2 16,98 24,35
BAT1 0,05 19,27
BAT2 0,18 0,51
CHA1 89,04 65,61
ILV1 0,00 51,96
121
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Curriculum vitae
Curriculum vitae
Personal Data
Name: Christoph Halbfeld
Born: February 7th, 1988 in Moers, Germany
Nationality: German
Mobile: 0049 1520 4453659
Email: christoph.halbfeld@rwth-aachen.de
Education
2012-2017 Research assistant and doctoral candidate at the Institute of Applied
Microbiology of the RWTH Aachen University, Aachen, Germany
2012 Braunwald course: Biosystems Engineering Summer School Braunwald,
Switzerland
2010-2012 Master of Science in Biology at the RWTH Aachen University, Aachen,
Germany
2008-2011 Bachelor of Science in Biology at the RWTH Aachen University, Aachen,
Germany
2007 Abitur at the Anne-Frank-Gesamtschule Rheinkamp, Moers, Germany
Work Experience
2016 Research stay in the Zenobi Group at the Laboratory of Organic Chemistry at
the ETH Zürich, Switzerland
2013-2017 Research assistant and doctoral candidate at the Institute of Applied
Microbiology of the RWTH Aachen University, Aachen, Germany
2013 Research stay at B&S Analytik-GmbH Dortmund, Germany
Publications
Halbfeld, C.; Ebert, B.; Blank, L. Multi-capillary column-ion mobility spectrometry of volatile
metabolites emitted by Saccharomyces cerevisiae. Metabolites 2014, 4, 751-774.
Ebert, B.; Halbfeld, C.; Blank, L. Exploration and exploitation of the yeast volatilome. Current
Metabolomics 2017, 5, 102-118.
Oral Presentations
Halbfeld, C.; Ebert, E.; Blank, L. (14.04.2015) YeastScent – Volatile metabolites as quantitative
proxies for metabolic network operation of Saccharomyces cerevisiae. 28th VH-Yeast
Conference. Berlin, Germany
proxies for metabolic network operation of Saccharomyces cerevisiae. 24th Annual
ISIMS Conference. Cordoba, Spain
proxies for metabolic network operation of Saccharomyces cerevisiae. 6. Symposium
Metaboliten in Prozessabluft und Ausatemluft. Reutlingen, Germany
147
Curriculum vitae
Halbfeld, C.; Sippel, A.K.; Pollmann, E.; Ebert, E.; Quantz, M.; Zierow, J.; Baumbach, J.I.;
Blank, L. (25.04.2017) Investigation of the Crabtree effect using off-gas analysis. 30th
VH-Yeast Conference. Berlin, Germany
Poster presentations
Halbfeld, C.; Ebert, E.; Blank, L. (22.02.2016) YeastScent – Volatile metabolite measurements
for yeast fermentation control. Bioprocessing days 2016. Recklinghausen, Germany
(Poster award)
Halbfeld, C.; Ebert, B.; Martinez-Lozano Sinues, P.; Blank, L. (20.02.2017) Discovering
dynamics of volatile metabolites in yeast fermentations using SESI-Orbitrap-MS.
Bioprocessing days 2017. Recklinghausen, Germany
148

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8 APPLIED MICROBIOLOGY

What happens in yeast during

What happens in yeast during the Crabtree effect?

An investigation of S. cerevisiae’s volatile space

M. Sc. Biologie Christoph Halbfeld

Moers, Nordrhein-Westfalen, Deutschland

Berichter: Universitätsprofessor Dr.-Ing. Lars M. Blank

Universitätsprofessor Dr. rer. nat. Jörg Ingo Baumbach

Tag der mündlichen Prüfung: 12.04.2018

What happens in yeast during the Crabtree effect?

Gedruckt auf holz- und säurefreiem Papier, 100% chlorfrei gebleicht.

Apprimus Verlag, Aachen, 2018

D 82 (Diss. RWTH Aachen University, 2018)

ZUSAMMENFASSUNG ............................................................................................................... VII

LIST OF ABBREVIATIONS ........................................................................................................ IX

LIST OF FIGURES ..................................................................................................................... XIII

LIST OF TABLES ......................................................................................................................... XV

GENERAL INTRODUCTION ............................................................................................... 5

Enzymes and PCR components ............................................................................. 36

MCC-IMS and microarray analysis of two yeast strains ....................................... 95

GENERAL DISCUSSION ................................................................................................... 105

APPENDIX ..................................................................................................................................... 115

REFERENCES ............................................................................................................................... 123

CURRICULUM VITAE ................................................................................................................ 145

FBA flux balance analysis

MnCl2 4H2O manganese(II) chloride tetrahydrate

TWIMS traveling-wave IMS

Partially published as:

produced by microorganisms. A prominent example is the baker’s yeast Saccharomyces cerevisiae

Metabolic pathways of yeast

VOCs derived from carbohydrate catabolism and fermentation

VOCs produced by amino acid synthesis and degradation

VOCs from fatty acids biosynthesis and degradation

Lactones, e.g., γ-decalactone, γ-butyrolactone (peach flavor), 4-hydroxy cis-6-dodecenoic acid-γ-

VOCs derived from terpene biosynthetic pathways

Analytical methods for the identification of VOCs

Ion mobility spectrometry

͵‫ݍ‬ ʹߨ ଵ ݉ ൅ ‫ ܯ‬ଵ ͳ (4)

Here P0 is 101,325 kPa and T0 is 273 K [126-128].

Secondary electrospray ionization mass spectrometry

Proton-transfer-reaction time-of-flight mass spectrometry

Gas-chromatography based methods

Pros and cons of methods for VOC detection

Methods for the delineation of VOC metabolic pathways

Verification of predicted pathways

Material and Methods

Strains, media and plasmids used

Table 2: Microbial strains

Enzymes and PCR components

gRNA ACTGACTTGCACGCTTGGCAGTTTTAGAGCTA specific 20bp + structural

Table 4: Oligonucleotides for homologous recombination

Creating multiple knockout mutants

Results and Discussion

3-methylheptyl acetate esters 537686 no no [248]

Conclusion and Outlook

Yeast volatilome dynamics during metabolic shifts

S. cerevisiae is an industrially important organism, with applications as different as baking and

Dynamics of (volatile) compounds

Figure 6: Influence of mass resolution on peak separation. Taken from [133].

Material and Methods

Online SESI-Orbitrap MS measurements

Results and Discussion

Interpretation of the results