Microbial acetyl-CoA

metabolism and metabolic

engineering
Anastasia Krivoruchko, Yiming
Zhang, Verena Siewers, Yun
Chen, Jens Nielsen

Acetyl-coA
Acetyl-CoA is a key molecule in microbial central carbon
metabolism
Acetyl-CoA is composed of coenzyme A and acetic acid
connected by a thioester bond.
Acetyl-coA is the substrate for TCA cycle that generates ATP
and precursor metabolites for amino acids and nucleotides.
Acetyl-CoA is also the building block for fatty acid synthesis
and the end product of fatty acid degradation.
Acetyl-CoA is the substrate for protein acetylation,
Acetyl-CoA is a precursor molecule to molecules including
Fattyacids,
1-butanol,
polyhydroxyalkanoates,
polyketides and
Isoprenoids

Overview of the acetyl-CoA metabolism in S.

cerevisiae

Acetyl coA transport

Carnitine/acetyl-carnitine shuttle,
C4 dicarboxylic acid synthesis from
acetyl-CoA via the glyoxylate shunt and
Acetyl-CoA re-generation from citrate
by cytosolic ATP citrate lyase.
The first two have been identified in S.
cerevisiae.

Glyoxalate pathway
When grown on substrates such as acetate that
will be degraded to form acetyl coA
The cell in turn activates the glyoxylate cycle
for producing 4C precursors and
gluconeogenesis pathways to provide sugar
phosphates and other metabolites for biomass
formation.
The glyoxylate cycle uses isocitrate lyase and
malate synthase to convert isocitrate to
malate, by bypassing two oxidative steps of the
TCA cycle.

Glyoxalate pathway

S.cerevesiae as a cell
factory

Advantages
Robust,
Grows to high densities
Grows at low pH.
Resistant to phage contaminations
Well known physiology and
Availability of genetic maniulation tools.
Disadvantages:
Acetyl-CoA metabolism in yeast is
compartmentalized
S.cerevesiae is a non oleaginous yeast.

PK pathway
When pentoses such as xylose was given as
substrate, Phoshoketolase pathway which is an
alternate of EmbdenMeyerhof Parnas pathway
takes place.
Phosphoketolase is an enzyme that generates acetyl
phosphate from xylulose 5-phosphate.
Acetate kinase (ACK) then catalyzes conversion of
acetyl-phosphate to acetate which in turn will be
converted to Acetyl-coA
Phosphotransacetylase (PTA), an enzyme that
catalyzes conversion of acetyl- phosphate to acetylCoA

Anaerobic routes to acetyl-CoA

production
Under anaerobic conditions, enzymes like
pyruvate
formate
lyase
(PFL),
pyruvate
ferrexdoxin oxidoreductase (PFOR) are utilized.
PFL catalyzes the conversion of pyruvate to
acetyl-CoA and formate.
The catalytic function of PFL is dependent on a
radical, and oxygen destruction of the radical
results in cleavage of PFL
PFL is believed to be involvede in production of
acetyl-CoA in organisms experiencing transient
low oxygen conditions

Increasing coA levels

Vadali et al., 2004a - overexpression
of the major rate-controlling enzyme
of CoA biosynthesis, pantothenate
kinase
When combined with pantothenate
increased CoA levels as well as acetylCoA levels.
Successful in Isoamylacetate
production in E.coli.

Construction of synthetic acetyl-CoA

pathways
Acetyl-CoA (C2) formation from pyruvate (C3) by
decarboxylation leads to carbon loss that limits the
theoretical yield of acetyl-CoA derived compounds
Mainguet et al. (2013) designed a reversed version of the
glyoxylate shunt to convert C4 TCA cycle intermediates
into acetyl-CoA without carbon loss.
A heterologous two-step pathway from malate to
glyoxylate via malate thiokinase and malyl-CoA lyase
(since the malate synthase reaction is irreversible), the
reversible isocitrate lyase and aconitase reactions as well
as heterologous ATP-citrate lyase
The pathway when introduced could produce 3 molecules
of acetyl-CoA per molecule of glucose

Progress in engineering acetyl co-A metabolism

PDH bypass

Shiba et al. (2007)- engineered the PDH bypass route to

enhance acetyl-CoA levels for production of amorphadiene, a
sesquiterpene precursor to the anti-malarial drug artemisinin.
This molecule is synthesized via the mevalonate pathway in
yeast, using acetyl-CoA as a starting molecule.
Overexpression
acetaldehyde dehydrogenase gene (ALD6),
mutant acetyl-CoA synthetase from S. enterica (ACS)
truncated form of HMG-CoA reductase (tHMGR),
Farnesyl pyrophosphate(FPP)-synthase,

Highest amorphadiene levels of about 120 mg/L was obtained

Cont..
Chen et al -produced of -santalene, a sesquiterpene that acts as a perfume
ingredient
Overexpression
Acetaldehyde dehydrogenase gene (ALD6),
Mutant acetyl-CoA synthetase (ACS) and
Alcohol dehydrogenase 2 (ADH2) gene
ERG10, native thiolase gene
Production of about 3.65 mg/L of -santalene, compared to 2.08mg/L
produced by the reference.
75% increase increase in -santalene production- Overexpression of all the
four genes
25% increase in -santalene production- Overexpression ALD6 and ACSSE
alone
Deletion
citrate synthase (CIT2) (4.98 mg/L)
cytosolic malate synthase (MLS1) (8.29 mg/L)

PK pathway engineering
Papini et al (2012) expressed the fungal PK pathway
in S. cerevisiae by expressing xylulose-5-phosphate
phosphoketolase (XpkA) and acetate kinase (Ack)
from Aspergillus nidulans.
Transhydrogenase enzyme was also expressed to
increase the drain of NADPH in order to increase flux
through the pathway.
13-C labeling was used to see the intracellular flux
distribution and determine if yeast used the
heterologous pathway.
Phospho trans acetylase gene from Bacillus subtilis
was also introduced but was not successful

Other heterologous pathway

Kozak et al. (2014a) - Acetylating acetaldehyde
dehydrogenase (A-ALD) and PFL are engineered in yeast.
A-ALD catalyzes the ATP-independent conversion of
acetaldehyde to acetyl-CoA. The five genes encoding
acetaldehyde dehydrogenase were deleted and A-ALD
genes from different sources were expressed.
Expression of A-ALD variants enabled fast growth of
ald2ald3ald4ald5ald6 S. cerevisiae on glucose
medium.
Deletion of ACS2 results in a strain unable to grow on
glucose due to glucose repression of ACS1.
Genes encoding PFL were expressed in this strain that
were able to grow on glucose.

Cont
Lian et al. (2014)- reduced the
metabolic flux towards ethanol.
Theydisrupted ethanol production by
deleting ADH1 and ADH4.
The flux was redirected towards
glycerol, hence they also disrupted
the glycerol pathway by deleting
GPD1 and GPD2.
Succeful in Butanol production

Cont
Kozak et al. (2014b) use bacterial PDH for
the generation of cytosolic acetyl- CoA in
yeast.
PDH complex from Enterococcus faecalis
was expressed in yeast.
This pathway replaced the ACS-dependent
pathway for production of cytosolic acetylCoA.
The use of ACL for acetyl-CoA generation
has also been reported.

Overview of different strategies employed for metabolic

engineering of acetyl-CoA metabolism in yeast.

Targets identified from this

study
Citrate synthase (Cit1, Cit2p & Cit3)
- Retrogade transcriptional factors
- Glutamate
Malate synthase(Mls1 & Mls2)
Acetyl coA carboxylase
Pyruvate dehydrogenase (PDH)
Acetyl co-A synthetase
-Positive Transcription factor Adr1p
-Negative Transcription factor UME6
-Transcriptional activator Abf1p
-Hst3p & Hst4P deacetylating proteins.
Citrate lyase
Cit and ACS1 undergoes catabolite repression by glucose.

Conclusion
Apart from structural genes,
regulatory genes could also be
potential targets for metabolic
engineering.

Thank you