Biotechnology Advances xxx (xxxx) xxx–xxx

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Biotechnology Advances
journal homepage: www.elsevier.com/locate/biotechadv

Research review paper

Enhancing the performance of brewing yeasts

⁎
Marcel Karabína, Lukáš Jelíneka, Pavel Kotrbab, Rudolf Cejnara, Pavel Dostáleka,
a
Department of Biotechnology, University of Chemistry and Technology, Prague, Technická 5, 16628 Prague 6, Czech Republic
b
Department of Biochemistry and Microbiology, University of Chemistry and Technology, Prague, Technická 5, 16628 Prague 6, Czech Republic

A R T I C L E I N F O A B S T R A C T

Keywords: Beer production is one of the oldest known traditional biotechnological processes, but is nowadays facing in-
Beer creasing demands not only for enhanced product quality, but also for improved production economics. Targeted
Brewing yeasts genetic modification of a yeast strain is one way to increase beer quality and to improve the economics of beer
Saccharomyces cerevisiae production. In this review we will present current knowledge on traditional approaches for improving brewing
Saccharomyces pastorianus
strains and for rational metabolic engineering. These research efforts will, in the near future, lead to the de-
Strain improvement
velopment of a wider range of industrial strains that should increase the diversity of commercial beers.
Genetic engineering
Metabolic engineering

1. Introduction
Beer is one of the most popular beverages in the world and almost
2 × 109 hectoliters are produced per year (BARTH-HAAS Group,
2016). Brewing, along with bread making and winemaking are some of
the world's oldest biotechnologies. They are very closely related and
traditionally used similar microorganisms. The first conditions appro-
priate for these biotechnologies were created by the human-hunter-
gatherer gradually becoming a farmer and breeder. The most favour-
able conditions for agriculture have always been in the river deltas,
where nature has taken care of natural fertilization and irrigation.
These conditions led to the cultivation of cereals, especially emmer and
barley (Hornsey, 1999). The cradle of their cultivation was Mesopo-
tamia and later Egypt. Mesopotamia was also the first area known for
beer production. The first grain fermented beverage was a beverage
called kaš, brewed by the Sumerians over the period from 3000 to
2800 BCE. The fact that beer was a very important beverage was also
demonstrated in the collection of laws of the Babylonian king Ham-
murabi (1728–1686 BCE) (Kunze, 1999).
At this time, of course, it was not known that microorganisms were
responsible for fermentation. In the course of historical development,
hops were introduced into the production of beer as another raw ma-
terial (Karabin et al., 2016; Karabin et al., 2015) and the production of
malt from cereals was made more efficient. In fermentation, either a
similar sourdough was used as in bread making, and fermentation took
place at temperatures above 20 °C, or spontaneous fermentation was
used, even at similar temperatures. This meant that almost every
brewery had its specific composition of fermenting cultures. During the
early Middle Ages, beer production began to move to monasteries and
fermentation was carried out by monks at very low temperatures (5 to
14 °C). From the point of view of the regions, Bavaria was the main
region, where dark beer was produced in the monasteries. The most
popular and widespread beers in Europe and England were top fer-
mented beers, which we can divided into pale ales, semi-dark ales and
dark top fermented stouts. In Belgium at that time, beer was also pro-
duced by spontaneous fermentation. During the 19th century, however,
popularity and production of bottom fermented beers increased, mainly
due to their improved stability and mass expansion of machine cooling.
In 1842, a pale bottom fermented very bitter lager was brewed in the
Municipal Brewery in Pilsen, and this has rapidly expanded, today re-
presenting the type of beer with the largest production volume in the
world. The fact that living microorganisms (yeasts) are responsible for
fermentation was explained by Pasteur in 1861. The first pure yeast
culture was isolated and cultivated at the yeast propagation plant by
Emile Hansen of the Carlsberg Laboratories in 1883 (Hornsey, 1999).
His work was the first application of pure brewer's yeast cultures in
brewing. At the same time, about the end of the 19th century, collec-
tions of industrial brewer's yeast strains were produced. Breweries
could now maintain their own yeast, isolated from production, and very
soon, most brewer's yeast strains became available commercially.
1.1. Origin and classification of brewer's yeasts
Brewing yeast strains can traditionally be divided into two groups:
top strains used for the production of beer types such as ale, stout or
porter, and bottom strains now referred to as Saccharomyces pastorianus,

⁎
Corresponding author.
E-mail address: Pavel.Dostalek@vscht.cz (P. Dostálek).

https://doi.org/10.1016/j.biotechadv.2017.12.014
Received 30 August 2017; Received in revised form 23 November 2017; Accepted 20 December 2017
0734-9750/ © 2018 Elsevier Inc. All rights reserved.

Please cite this article as: Karabín, M., Biotechnology Advances (2018), https://doi.org/10.1016/j.biotechadv.2017.12.014
M. Karabín et al.
used for the production of lagers. Originally, these strains were classi-
fied based on their flocculation properties. Top yeast at the end of
fermentation tends to rise to the surface of the fermented wort, and its
name is derived from this fact. Conversely, bottom yeast at the end of
the fermentation settled to the bottom of the fermentation vessel. The
optimum growth temperature for genus Saccharomyces is between 25
and 30 °C, however, the growth and fermentation of bottom yeasts are
not restricted to the optimum temperature and fermentation with
bottom fermenting yeasts are performed between 4 and 12 °C. In the
case of top fermenting yeast 14 to 25 °C is used (Kunze, 1999). Until the
development of machine cooling in the mid-19th century, the produc-
tion of beer was exclusively associated with the use of top yeast (Kunze,
1999).
Many species of the genus Saccharomyces are top brewing yeasts,
most of which are closely related to Saccharomyces cerevisiae (Pedersen,
1986) and form a polyphyletic group that can be divided for brewing
yeast into two subgroups (Gallone et al., 2016; Goncalves et al., 2016).
The first subgroup is made up of German, British and wheat beer
strains. The second subgroup strains are related to sake, wine and bread
yeasts. Within the first and the second S. cerevisiae group, beer strains
are more diverse than are wine strains. However, many top yeast strains
are hybrids in nature. Molecular characterization of the top strains
found in Belgian Trappist beers has shown that 25% of tested strains are
likely to have been produced by the hybridization of S. cerevisiae and S.
kudriavzevii (Gonzalez et al., 2008). Some hybrids have the same origin
as yeast strains that were originally classified as S. cerevisiae (Querol
and Bond, 2009).
Even more complicated is the situation with bottom yeast strains. It
has long been known that bottom brewer's yeast strains of S. pastorianus
are hybrids, but it was assumed that they were hybrids of S. cerevisiae
and S. bayanus (Bond, 2009; Bond et al., 2004; Dunn and Sherlock,
2008; Nakao et al., 2009). However, S. bayanus had very low sequence
identity (92.5%) when compared with the non-S. cerevisiae genome of S.
pastorianus. Recently however, in 2011 in the Patagonia region of Ar-
gentina a S. eubayanus strain was isolated that had a high identity
(99.5%) with the non- S. cerevisiae part of the S. pastorianus genome
(Libkind et al., 2011). The yeast was then isolated in North America
(Peris et al., 2016; Peris et al., 2014), East Asia (Bing et al., 2014) and
New Zealand (Gayevskiy and Goddard, 2016). Thus, S. pastorianus
strains were clearly confirmed as hybrids of the species S. cerevisiae and
S. eubayanus (Borsting et al., 1997; Casaregola et al., 2001; Fernandez-
Espinar et al., 2003; Hansen et al., 1994; Hansen and Kiellandbrandt,
1994; Joubert et al., 2000; Kodama et al., 2001; Monerawela and Bond,
2017; Nakao et al., 2009; Tamai et al., 1998; Tamai et al., 2000;
Yamagishi and Ogata, 1999). Yeast S. eubayanus is known for its cold
tolerance, and the higher the proportion of the S. eubayanus genome the
more cold-tolerant was the strain (Gibson et al., 2013). Saccharomyces
eubayanus is also more sensitive to higher ethanol concentrations than
traditional ethanol producing strains. Based on these properties, the
lager yeast has been divided long time ago into two groups: I) Saaz and
II) Frohberg (Glendinning, 1899; Krogerus et al., 2017a). Saaz strains
are generally not capable of fermenting maltotriose and therefore have
slower growth and fermentation compared with the Frohberg strains
(Gibson et al., 2013; Magalhaes et al., 2016). It is currently assumed
that hybridization of S. cerevisiae (ale) with S. eubayanus was required
to produce S. pastorianus group I (Saaz); on the other hand, S. pastor-
ianus group II (Frohberg) was followed by further hybridization with a
different S. cerevisiae (stout). The second group thus required two hy-
bridizations and there are at least two independent origins of this
species (Dunn and Sherlock, 2008; Monerawela and Bond, 2017). The
genomes of the individual production strains of S. pastorianus are
therefore various combinations of genomes of the species S. cerevisiae
and S. eubayanus. It has even been found that some strains may also
contain portions of genomes from other yeasts belonging to the Sac-
charomyces evolutionary group (Rainieri et al., 2006). As a result, there
are differences between the properties and capabilities of individual
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Biotechnology Advances xxx (xxxx) xxx–xxx
production strains, with the particular genotype depending on the
origin of the strain. Specific strains and their properties can be assigned
to certain breweries or a locality, and each strain is defined by the
characteristic genomic arrangement, number of copies, ploidy and se-
quential polymorphisms (Saerens et al., 2010). All these findings were
then confirmed by sequencing the genomes of the lager brewing yeast
groups I and II (de Leon-Medina et al., 2016; Dostalek et al., 2013;
Hewitt et al., 2014; Nakao et al., 2009; Okuno et al., 2016; van den
Broek et al., 2015; Walther et al., 2014).
1.2. The importance of yeast in brewing technology
Beer production is a traditional and well-established process.
Modern times, however, put considerable demands on both the quality
of the product and the economics of production. Beer production re-
mains a batch process despite some attempts to make it continuous.
Brewhouse-produced wort, containing fermentable carbohydrates
(maltose, glucose, fructose, maltotriose) and non-fermentable dextrins,
amino acids (Kabelova et al., 2008), polypeptides, polyphenols
(Dvorakova et al., 2008), and bitter hop compounds (Karabin et al.,
2014) is cooled, aerated and pitched with brewing yeast. Primary fer-
mentation takes place in an open system, where ethanol, carbon dioxide
and by-products of fermentation are formed (Andres-Iglesias et al.,
2016). After the substrate is depleted, the yeasts flocculate (Soares,
2011) and, depending on their species, they are discharged to the
surface or sediment (Stewart et al., 2013). Then maturation takes place
where carbonyl compounds (Inoue, 2008) are reduced, the beer is
clarified and is carbonated at a lower temperature than the main fer-
mentation (Stewart et al., 2013). After primary fermentation and ma-
turation, filtration, or stabilisation filtration is carried out to remove
haze precursors (haze sensitive proteins and haze sensitive poly-
phenols) of colloidal turbidity (Jelinek et al., 2014; Kotlikova et al.,
2013). Beer is then pasteurized and packaged into appropriate con-
tainers (Kunze, 1999).
In terms of time, fermentation and maturation are the most time-
consuming part of the brewing process and fermentation tanks occupy
the largest area of the brewery. One method for enhancing the whole
process is High Gravity Brewing (HGB), using highly concentrated wort
that is fermented to beer and then diluted to produce a finished beer of
the desired ethanol concentration (Patkova et al., 2000; Puligundla
et al., 2011). Standard brewer's yeasts in classical technology are
usually not possible to use at such a high concentrations of extract
(> 20% by weight) and they are not produced > 6% (by volume) of
ethanol in regular final beers (substrate and product inhibition). This
problem may be resolved by using a hybrid yeast or special selected
yeast or using immobilised yeasts. If wort dextrins are to be fermented
(production of low-carbohydrate beers), yeast with glycoamylase ac-
tivity is used, which degrades the dextrins to glucose and then converts
this into ethanol. Conversely, in beers with reduced ethanol content or
non-alcoholic beer (< 0.5% ethanol by volume), fermentation is car-
ried out to produce glycerol, or maltose and maltotriose are not used, so
only minor monosaccharides are fermented (Kunze, 1999).
Fermentation performance is directly affected by the flocculation
process. Insufficient or premature flocculation leads to problems of
filtration or poor fermentation of the wort and results in a high residual
extract in the beer. Flocculation is controlled by FLO family genes
(FLO1, FLO5, FLO8, FLO9 and FLO10) that are responsible for ex-
pressing mannoproteins on the yeast walls and are important for cell-
cell aggregation (Kurec and Branyik, 2011; Soares, 2011). Another step
in beer production is filtration, which removes haze particles, mainly
yeasts. Filtration greatly depends on the state of flocculation and clar-
ification of beer, but sometimes a high concentration of beta-glucans
(derived from malt and not sufficiently degraded in the brew) may
interfere by increasing the brew viscosity and thus slowing the filtration
(Kunze, 1999).
For beer, in terms of flavour, fermentation is a very crucial step. For
M. Karabín et al. Biotechnology Advances xxx (xxxx) xxx–xxx

Fig. 1. Formation of diacetyl during the synthesis of valine and its suppression and de-
gradation.
each type of beer, a corresponding brewing yeast strain is used and the
final beer flavour is finished during maturation. For lagers, it is quite
important to remove diacetyl (buttery flavour) produced during the
metabolism of valine from α-acetolactate. α-Acetolactate is sponta-
neously decarboxylated to diacetyl outside of the cell. During matura-
tion, diacetyl is converted into acetoin (3-hydroxybutanone), which has
no buttery flavour (Fig. 1). However, at a low maturation temperature,
this conversion is very slow (Inoue, 2008). Various types of beers are
also typical of high, medium or low concentrations of aromatic esters
and higher alcohols (Branyik et al., 2008). Higher alcohols are formed
either by anabolism or catabolism (Ehrlich pathway) of amino acids.
Esters are formed by enzymatic condensation of organic acids and al-
cohols (Pires et al., 2015; Pires et al., 2014). A negative substance
produced during fermentation is dimethylsulfide (DMS), which has the
unpleasant aroma of cooked vegetables (Anness and Bamforth, 1982;
Herrmann et al., 2010). During fermentation, this substance is created
from dimethylsulfoxide (DMSO) by the action of yeast reductases
(Saerens et al., 2010; Stewart et al., 2013). Some beers (especially
wheat beers) also have a flavour characteristic of clove due to the 4-
vinylguaiacol content resulting from the transformation of ferulic acid
released from the malt (Langos et al., 2017; Langos and Granvogl,
2016). On the contrary, this phenolic flavour is undesirable in lagers, so
it is necessary to consider strain selection.
Colloidal and sensory stability of beer is very important. Because the
beer is a colloidal system, colloidal turbidity occurs during storage. Its
formation can be slowed by storage at low temperatures and without

Fig. 2. Overview of the most common targets for enhancing the performance of brewing yeast (↑ - increase, ↓ - decrease, ↑↓ - in equilibrium).

3
M. Karabín et al.
oxygen. Very often, colloidal stability is increased by the sorption re-
moval of protein and polyphenol precursors of colloidal haze (Kotlikova
et al., 2013). The sensory stability of beer is better the higher the
concentration of natural antioxidants. However, yeast cells also have a
natural formation of sulphur dioxide, which is a very powerful anti-
oxidant and thus increases the sensory stability of beer. Since the pro-
duction of SO2 is strictly under genetic control, it is possible to select a
yeast strain with higher SO2 production (Dvorak et al., 2006). For some
beers, the creation and stability of beer foam is very important. The
foam skeleton is made from thermostable albumin (Z protein) derived
from barley malt. This protein can then be decomposed by a yeast
proteinase A released during autolysis of the yeast during beer ma-
turation (Stewart et al., 2013).
Nowadays, attempts have been made to prepare different hybrids of
S. pastorianus from original parental strains by classical techniques
(Krogerus et al., 2017a; Krogerus et al., 2017b). It is also possible to
encounter attempts to isolate new strains for brewing isolated from
nature or related food industries (Mascia et al., 2015; Mascia et al.,
2016). Another alternative to meet the requirements of the brewing
industry is the targeted genetic modification of yeast strains. Genetic
modification of brewer's yeasts is very demanding. It is much easier to
genetically modify well-characterized haploid laboratory strains than
genetically complex industrial strains, which are typically polyploid,
aneuploid, and even alloploid (Dequin, 2001; Randez-Gil et al., 1999).
Evidence is that the strategy used to modify the laboratory strain does
not necessarily work (and often does not work) in the industrial strain
(Klein et al., 1996; Nevoigt et al., 2002).
Despite the difficulties in many laboratories around the world,
variously transformed brewer's yeasts have been prepared. Examples
include modifications to extend the flavour stability of beer, foam sta-
bility, reduce ethanol production, or shorten the fermentation time and
significantly improve production efficiency (Fig. 2). In addition, fer-
mentation is one of the most potent steps in beer production, and
modifications are already available to address at least some of the
problems. In particular, this is a modification that prevents a series of
sensory defects in beer (Dunn et al., 2017; Saerens et al., 2010;
Steensels et al., 2014b).
2. Public acceptance and regulation of genetically modified
microorganisms
Towards the end of the 20th century, when methods had been de-
veloped to describe and modify the genome of industrially used yeast
strains, including strains used in brewing, there was an assumption that
these newly engineered microorganisms would completely change
some sectors of the beverage industry. In the 1990s, genetically mod-
ified baker's and brewer's yeast strains were granted the British
Government's permission for commercial use, although they were never
actually used (Akada, 2002). Consequently, as a result of the rapid
development and testing of GM crops and the description of the real and
apparent risks that could arise from their commercial use, the public
acceptance of genetically modified organisms (GMO) significantly de-
creased and, in addition to these social and ethical barriers, legislative
barriers emerged. Although the issue of genetically modified micro-
organisms (GMM) is different for many reasons, the development of
novel brewer's yeasts is currently restricted to traditional hybridization
methods that do not fall under the restrictive legislation (Steensels
et al., 2014b) or to the development of new methods of genetic mod-
ification and their laboratory and pilot-scale testing. To the best of our
knowledge, none of these strains are used industrially, and no appli-
cation for authorization of GM yeast application in the brewing industry
has been submitted until 2016.
In general, acceptance of food prepared from GM feedstocks and/or
using GM microorganisms is not too high, and some studies suggest that
it has declined further over the last few years (Kellershohn and Russell,
2015). GM microorganisms are accepted more widely than plants and/
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Biotechnology Advances xxx (xxxx) xxx–xxx
or animals (Sorgo et al., 2012). However, public awareness is very low,
based mostly on information from the Internet, television and similar
sources (Wunderlich and Gatto, 2015). GMO-related risk perception by
the public in the United States is lower than that of the European Union
(Fischer et al., 2013; Frewer et al., 2013), preferences of Europeans for
natural food being among the reasons for these differences, whereas
Americans are more open to new, intensified technologies (Pollack and
Shaffer, 2009; Vogel, 2012). On top of that, compared to Americans,
Europeans attach more importance to the protection of the environment
(Peycheva et al., 2014; Vogel, 2012). The question of price and other
features of products is also interesting. In the Australian authors' study
on the customer acceptance of GM beer, it has been shown that the
negative approach of the consumer can be weakened by lowering the
price or by adding some positive (health-promoting) effect (Burton and
Pearse, 2002). In less developed countries, public attitudes are sig-
nificantly affected by the standard of living and availability of food
(Azadi and Ho, 2010).
In brewing, the situation is even more complicated by the fact that
beer is perceived as a natural or “organic” beverage, and the disruption
of this perception can directly affect the acceptability of the product by
the consumer (Caporale and Monteleone, 2004; Tenbült et al., 2008).
Additionally, brewing is a relatively conservative industry with a long-
time history, during which a number of standards, strictly defining the
use of raw materials and technologies have been established, starting
with the Reinheitsgebot (German Beer Purity Law) of the 16th century
(van Tongeren, 2011) and ending, for example, with the Protected
geographical indication “Czech Beer” (Anonymous, 2008; Olsovska
et al., 2014). Objections associated with the use of GMMs can be
summarized into several categories, incorporating ethical, ecological
and health risks. Addressing and resolving most of these issues are a
prerequisite for application of GMMs in brewing and other industries in
foreseeable future.
The first group includes risks, both real and supposed, in connection
with the possible impact on the environment, such as the danger of
obtaining a competitive advantage as a result of genetic manipulation
(Perez-Torrado et al., 2015). That could lead to a reduction in microbial
diversity when the modified industrial strain is disseminated into the
natural ecosystem. Another often mentioned risk is the horizontal
transfer of genes to other organisms, which could endanger human
health (Prakash et al., 2011). For brewer's yeast, the occurrence of these
phenomena is relatively unlikely, because the brewer's yeast industrial
strains are strongly adapted to the conditions (type of substrate, tem-
perature) in the given ecological niche (Gallone et al., 2016) and un-
controlled transfer to another environment is possible only as a result of
extensive changes in their properties through massive modification of
genome. However, genetic modifications of brewer's yeasts are usually
very mild because major modifications could lead to changes in the
production of secondary metabolites during fermentation (Steensels
et al., 2014a) and therefore to the undesirable deterioration in sensory
properties of beer (Pires et al., 2014).
Many concerns from both customers and experts are associated with
the introduction of foreign genetic material into the cell and conse-
quently into the consumer's organism. The risk of health damage re-
lated to antibiotic resistance, carcinogenicity or allergenicity is related
both to foreign DNA transfer and to unexpected effects of newly pro-
duced proteins (Aguilera et al., 2013). This concern is unnecessary, at
least in the case of filtered beer, from which virtually all yeast has been
removed (Briggs, 2004). In addition, the majority of modern methods
for genetic manipulation use only reduced amounts of transferred non-
yeast DNA, or integrate DNA from closely related non-pathogenic mi-
croorganisms or from GRAS (Generally Regarded As Safe) MOs used in
the food industry (Perez-Torrado et al., 2015). Some newly developed
methods did not even leave a trace of foreign DNA in the modified yeast
(Alexander et al., 2016). Also, isolation procedures using antibiotic-
resistance selection markers are increasingly being abandoned in favour
of methods using other types of selection markers that do not pose a risk
M. Karabín et al.
of spreading antibiotic resistance to pathogenic MO (Da Silva and
Srikrishnan, 2012).
The above-mentioned information demonstrates the public interest
in ensuring the safety of GMO technologies. The legislation governing
the definition of genetically modified organisms, their usage and GMO-
food labelling vary considerably from one country to another, which
can be best described by differences in the US and EU legislation (Bühl
et al., 2015). In the US, a product-based approach has traditionally been
applied, but this has been significantly modified during the last few
years (Marchant and Stevens, 2015). The FDA, under whose authority
GMO falls, classifies yeast as a processing aid, which is provided with
GRAS status and which is not required to be labelled on the packaging
(Fischer et al., 2013). On the other hand, in the EU, resolving GMO
issues is based on process-based principles and the corresponding leg-
islation strictly defines the techniques of genetic manipulation whose
use requires the microorganism to be labelled as genetically modified. It
is a paradox that this obligation does not apply to microorganisms
prepared by procedures causing extensive and difficult-to-control
changes of the genome such as mutagenesis or protoplast fusion
(Anonymous, 2009). On the contrary, it applies to methods that almost
fully control genetic modifications, do not leave problematic residues of
the vector system in the cell and virtually do not differ from natural
recombination events (Saerens et al., 2010). It is clear that the further
development of molecular biological methods will lead to further dis-
cussion on this legislation and its future reassessment.
3. Improvement of brewer's yeast by traditional methods
3.1. Principles of traditional methods used to modify brewer's yeast
Although some yeast strains have traditionally been used in a cer-
tain geographical area or even in a particular brewery, often for several
decades (Boulton, 2017; Briggs, 2004), efforts to breed new strains have
been carried out virtually since their isolation in the nineteenth century
(Gibson et al., 2017). Classical methods, based mostly on sexual and
asexual hybridization, play an important role in this process, especially
with regard to the legislative, marketing, sociological and ethical bar-
riers mentioned in the previous chapter, which often make methods
based on genetic engineering inapplicable.
Sexual crossing, used to obtain improved traits in plant and animal
species, is often not possible for industrial strains of microorganisms
(Steensels et al., 2014b). For yeast cells, mating is common, especially
when the cell is adapting to a new or inappropriate, harsh environment
(Gallone et al., 2016; Goddard and Greig, 2015). Brewers' yeast, how-
ever, grows virtually uninterruptedly in the environment in which they
are adapted and which is the source of sufficient nutrients, and only
limited osmotic, thermal or ethanol stress. Given that starvation is one
of the basic prerequisites for sporulation (Bilinski and Casey, 1989), the
viability of brewer's yeast spores is very low, and due to the homothalic
and aneuploid character of cells, most brewer's yeast strains have al-
most completely lost the capability of sexual reproduction via direct
cell-cell and/or spore-spore mating (Gallone et al., 2016; Steensels
et al., 2014b). To overcome this, a broad spectrum of approaches have
been proposed and applied, including rare mating, mass mating/
genome shuffling, or procedures based on asexual hybridization such as
protoplast fusion (Fig. 3).
Rare mating does not require a sporulation step and uses diploid
yeast cells, which, in exceptional cases, may lose heterozygosity
(Hiraoka et al., 2000), to produce a homozygous diploid cell that can be
used for further mating with a haploid cell of the opposite mating type
to produce triploid and subsequently higher-ploidy hybrids containing
genetic information of both parental strains (Krogerus et al., 2017a).
Mass mating is based on the random mating of a large number of
cells of different parent strains or within the heterogeneous population
of one strain, obtained, for example, by chemical or UV mutagenesis
(Wang and Hou, 2010). This leads to the production of a large number
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Biotechnology Advances xxx (xxxx) xxx–xxx
of genetically different cells, some of which may exhibit the desired
traits. Individual hybrids are isolated by growing under conditions
corresponding to the desired phenotype (Krogerus et al., 2017a), which
can be further enhanced by repeating the process (genome shuffling).
This process is more effective than other hybridization methods, but its
disadvantage lies in the fact that due to the large number of genotypic
changes, cells with the desired phenotype may fail in other technolo-
gical and sensorial aspects (Steensels et al., 2014a). This is un-
acceptable in brewing where, in addition to the parameters such as
speed and degree of fermentation, the ability to produce only the de-
sirable sensory active substances is essential for ensuring product
quality (Pires et al., 2014).
Protoplast (spheroplast) fusion is a procedure applicable for in-
traspecific, intrageneric (Choi et al., 2010; Perez-Traves et al., 2012)
and intergeneric (Guo et al., 2012) hybridization. It evades the re-
quirements of other types of hybridization by a procedure based on the
removal of the cell wall of the parental cells, merging of the cellular
material of both cells, the subsequent regeneration of the cell wall and
the fusion of nuclei (karyogamy). Generally, the resulting hybrid con-
tains the complete genome of one of the parents, enriched by a part of
the second parent's genome (Kavanagh and Whittaker, 1996). The
drawbacks of this procedure are the difficult predictability of the hybrid
phenotype, its mitotic instability and the legislative barriers associated
with the fact that these microorganisms are considered GMOs in some
regions (Steensels et al., 2014b).
3.2. Application of traditional methods
Over the last 50 years most attention has been focused on creating
brewer's yeast hybrids that could allow intensification of beer produc-
tion (Saerens et al., 2010), namely through HGB technology. For this
purpose, the possibility of increasing resistance against osmotic stress
and high concentrations of ethanol as well as improving fermentation
performance at non-optimal temperatures, for both lager and ale yeast,
was tested (Ekberg et al., 2013). In one of the first studies, interspecific
protoplasts fusion of S. cerevisiae and osmotolerant S. mellis was used in
the 1980s to produce osmotolerant hybrids (Legmann and Margalith,
1983). Hybrids obtained showed an improved ability to ferment in
high-glucose medium. Fusion of protoplasts stimulated by electric field
(electrofusion) was also used to modify the sporulation activity of
brewer's yeasts (Urano et al., 1993a; Urano et al., 1993b). In the 1980s,
some authors also succeeded in improving the sporulation of lager
yeasts by using a suitably selected medium with the addition of sodium
acetate at a suitable temperature, so these yeast cells were able to mate
directly (Bilinski and Casey, 1989; Gjermansen and Sigsgaard, 1981).
One of the hybrids did not lag behind in fermentation ability compared
to the parental strains and from the point of view of low production of
vicinal diketones, even surpassed them (Gjermansen and Sigsgaard,
1981). Protoplast fusion coupled with mass mating was later used for
the development of cryotolerant hybrids of top fermenting brewer's
yeast strain and wine yeast S. bayanus (Sato et al., 2002), interspecific
hybrids of top and bottom fermenting brewer's yeast strains with in-
creased resistance to stress, capable of fermenting high-gravity worts at
high temperatures (Sanchez et al., 2012) and hybrids of ale yeasts with
S. eubayanus, which were able to ferment at lower temperatures faster
than the parent yeast, flocculated well and retained the ability to fer-
ment maltotriose (Krogerus et al., 2015). In another work, hybrids of S.
cerevisiae and S. eubayanus with different ploidy (allodiploid to allote-
traploid) were obtained, which showed increased fermentation perfor-
mance in worts of up to 25°Plato in a ploidy-dependent manner
(Krogerus et al., 2016).
Usually, testing of fermentation performance of new hybrids under
various, often extreme, conditions is combined with studying their
ability to produce a modified content of sensory-active secondary me-
tabolites, especially volatile esters, higher alcohols and vicinal dike-
tones. In recent years, natural mutants or mutants prepared artificially,
M. Karabín et al.
mostly by UV radiation or chemical mutagenesis, often combined with
mass mating/genome shuffling, have been used for this purpose. The
genome of heat- and osmotolerant ethylmethane sulfonate (EMS)-in-
duced mutants exhibited extensive rearrangements, which could also
occur during fermentation under extreme conditions, probably due to
the plasticity of the genome of tested strains (James et al., 2008).
Natural 5,5,5-trifluoroleucine-resistant mutants capable of producing
higher amounts of alcohols and esters have been tested for suppression
of undesirable wort aroma in non-alcoholic beers (Strejc et al., 2013).
The natural 2-deoxyglucose-resistant mutants of top-fermenting bre-
wer's yeast showed the ability to produce higher amounts of ethanol
and lower amounts of acetic acid when fermenting high-gravity worts
(Mizuno et al., 2006).
The acceleration of fermentation of high-gravity worts, better re-
sistance to ethanol and a different profile of volatile substances was also
demonstrated for UV-induced mutants of lager (Blieck et al., 2007) as
well as ale yeast strains (Wang and Hou, 2010). Furthermore, UV mu-
tants of lager strains have been shown to have an increased ability to
ferment maltotriose (Liu et al., 2008) and were able to decrease the
production of acetaldehyde (Shen et al., 2014), whose grass/green
apple flavour is undesirable in lager beers (Wang et al., 2006). The
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Biotechnology Advances xxx (xxxx) xxx–xxx
Fig. 3. Principles of hybridization techniques applicable for
improvements in brewer's yeast a/α – opposite mating
types.
shortening of the fermentation was demonstrated for the osmotolerant
lager yeast mutant prepared by chemical mutagenesis (Ekberg et al.,
2013). However, accelerated fermentation led to an increase in the
content of some volatiles, including vicinal diketones, diacetyl and 2,3-
pentadione (buttery aroma), which can cause serious problems in the
production of lager beers characterized by clean, balanced aroma
without significant off-flavours (Krogerus and Gibson, 2013).
Efforts to obtain strains with considerably elevated production of
volatile flavour compounds thus relates in particular to top-fermenting
strains or lager strains used for production of special beers with char-
acteristic flavours. The fact that, in addition to the ability to ferment
high-gravity worts, the ability to produce volatile secondary metabo-
lites is also affected by the yeast's ploidy was confirmed in the study of
Krogerus et al. (2016). One way to increase ester production is inter-
specific hybridization with flavour-active species such as S. kudriavzevii
(Bizaj et al., 2012). Elevated production of these secondary metabolites,
especially isoamyl acetate, has also been demonstrated for hybrids
prepared by protoplast fusion or direct mating between Sake and top-
fermented brewer's yeast (Mukai et al., 2001; Steensels et al., 2014a).
Some of these hybrids contained also traits usable for the rapid fer-
mentation of high-gravity worts. In one of the latest studies (Krogerus
M. Karabín et al.
et al., 2017b) attention was paid to the mating of two strains of S.
cerevisiae and one of S. eubayanus, from which arose a hybrid containing
a part of the genome from all three strains and retained technologically
important traits whilst losing the undesirable ability to produce com-
pounds such as vinyl guaiacol, which can cause phenolic off-flavours,
undesirable especially in lagers (Vanbeneden et al., 2008).
In the 1980s, attempts were made to develop brewer's yeast with
antimicrobial properties that could be used to reduce the risk of mi-
crobial contamination and thus a deterioration in the sensory quality of
the product. For this purpose, fusion of protoplasts of three yeast strains
– bottom-fermenting brewer's yeast and two S. cerevisiae strains with
antibacterial and anti-wild yeast properties was used. Resulting hybrids
exhibited antibacterial effects against 11 species of common brewery
contaminants (Sasaki et al., 1984). At the same time, the potential of
UV mutants of both top and bottom-fermenting brewer's yeasts to
produce increased amounts of extracellular thiamine has also been
demonstrated (Silhankova, 1985). However, research in this field was
not continued, probably due to unacceptable sensory properties of beers
resulting from changes in sulphur amino acid metabolism and thiamine
degradation (Seifert et al., 1978).
4. Improvement of brewer's yeast by modern methods
4.1. Recent DNA technologies for rational site-specific yeast engineering
The intuitive natural selection of production cultures of domes-
ticated yeasts over thousands of years, followed by purpose-directed
mating and mutagenesis approaches in the modern era have selected for
phenotypes successful in various brewer's technologies. The principal
drawback of the traditional genetic approaches is the low frequency of
obtaining the winning combination of desired traits. An attractive al-
ternative approach is the rational modification of the genetic potential
through either putting appropriate genetic parts together into heritable
and expressible DNA molecules, site-directed modifications of in-
dogenous alleles or targeted knock-out of an undesired genetic de-
terminant(s). Such genetic modification generally consists of identifi-
cation of the target gene(s) of interest and genetic engineering to
construct a recombinant strain, mostly in a predetermined manner. It is
worth noting that the recombinant protein could be produced not only
as intracellular or free extracellular protein, but tools are also available
for arming the surface of production yeasts with desired protein activity
(Tanaka and Kondo, 2015a; Tanaka and Kondo, 2015b). The most
popular surface display method is the assembly of a translational fusion
of the target protein with the excretion signal and C-terminal domain of
yeast α-agglutinin, which contains the glycosylphosphatidyl anchor
attachment signal allowing for covalent binding of the fusion to cell
wall glucan via transglycosylation.
Studies in various –omics and systems biology teach us that essen-
tially any action (such as [over]expression of an enzyme activity) in
certain parts of a biological system could trigger (unpredicted) reac-
tions in another part. This may result in a recombinant strain gaining an
unfavourable trait in addition to a desired one. The construction of
recombinant production yeasts thus should consider optimization of the
expression rates of gene(s) of interest to avoid metabolic burdens on the
cell, such as exhaustion of precursor metabolites and the cofactor pool
or a misbalance in metabolic fluxes, particularly when introducing
multiple genes of a pathway - for examples see (Jin et al., 2003; Lu and
Jeffries, 2007). Tuning of gene expression rate could be achieved at the
transcriptional level by selection for the promoter and transcriptional
terminator of appropriate strengths. Studies also document that both
mRNA stability and translation rate of the same protein can be highly
influenced by codon usage (Brat et al., 2012; Harigaya and Parker,
2017; Kaishima et al., 2016) and thus alteration in the use of preferred
and non-preferred codons could provide a method of control over
translational efficiency.
The simplest genetic engineering approach, which achieved various
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Biotechnology Advances xxx (xxxx) xxx–xxx
successes in brewer's yeasts (see Section 4.2), involved the expression of
recombinant indogenous or heterologous genes of interest, selected
based on well-known properties of encoded proteins, in most cases the
enzymes for direct metabolic engineering. More complicated is when
the genetic determinant underlying a favourable phenotype observed in
certain strains or under specific conditions, and thus suitable for genetic
engineering in production yeast, remains unknown. To circumvent the
lack of information about phenotype-genotype relationships, inverse
metabolic engineering (Borodina and Nielsen, 2014; Kim et al., 2012)
can be used. In this approach, the identification of gene(s) of interest
relies upon genetic mapping or state-of-the-art high throughput as-
sessment of phenotype-related transcription, protein and metabolite
production rates and/or advanced genomic analysis tools, such as
quantitative trait loci (QTL) analyses (Bloom et al., 2013; Ehrenreich
et al., 2010; Parts, 2014; Sirr et al., 2015). Resulting information about
causative genetic factor(s) is then used to genetically modify the pro-
duction strain.
4.1.1. Engineering recombinant yeasts
There are two principal ways to introduce recombinant DNA in S.
cerevisiae and its allies: using an extrachromosomal replicable vector
(plasmid) or stable integration into the yeast genome (chromosomes in
particular) via homologous recombination. The identification of a re-
combinant strain may rely on phenotypic selections when the pheno-
type of the implemented gene/modified allele can be selected for, DNA
analyses or selectable marker inserted along with the target gene of
interest. When heterologous gene expression is considered and irre-
spective of the method, each coding sequence must be equipped with
promotor and terminator sequences that control the strength of ex-
pression. Suitable promotors, recruiting RNA-polymerase for tran-
scription, and transcriptional terminators differing in strength are
available (Da Silva and Srikrishnan, 2012; Hubmann et al., 2014).
Recently, a panel of 14 constitutive yeast promoters was sorted ac-
cording to their strength under varied glucose and oxygen conditions
(Sun et al., 2012). The inducible promoters proved useful in bio-
technology involving a modified glucose-repressed, galactose-induced
GAL induction system (Paddon et al., 2013) or ADH2 promoter, which
is induced upon glucose depletion and provides a higher level of tran-
scription than the GAL system (Shen et al., 2012; Tang et al., 2015;
Weinhandl et al., 2014). The fact that yeast promoters are hundreds of
base pairs (bp) long, prompted rational construction of minimal pro-
moters (with up to 80% reduction in length) with both constitutive and
regulated behaviour (Redden and Alper, 2015; Redden et al., 2015). As
documented, e.g., with a strong constitutive TEF1 promoter, fine tuning
of the gene expression for a specific application can be also achieved by
screening a library of mutant promoter sequences generated by using
error prone PCR, which for a strong constitutive TEF1 promoter,-gen-
erated variants with a 15-fold difference in expression level between
the weakest and strongest variant (Alper et al., 2005).
The selection of an appropriate efficacy of transcription terminator
sequence to be inserted downstream of the coding region may take
advantage of the work by Yamanishi et al. (2013) who scored > 5000
native S. cerevisiae terminators. In other work, detailed characterization
of 30 terminator sequences allowed identification of those enhancing
the expression in S. cerevisiae (Curran et al., 2013) and rationally de-
signed, fully functional minimal terminators (35 to 70 bp in size) syn-
thesized in vitro were also described (Curran et al., 2015).
Coding sequences of predicted rational targets themselves can be a
subject of optimization through the in vivo screening of coding libraries
generated in vitro using error prone PCR (for an example see
COMPACTER below). An alternative approach to generate artificial
diversity for directed evolution can be represented by DNA shuffling,
i.e. combinatorial in vitro DNA assembly from parts of the coding se-
quences of closely related genes with the aim of selecting for proteins
that show improved or novel properties (for review see Nair and Zhao,
2009).
M. Karabín et al.
Similar to genetic engineering approaches used to create libraries of
rational targets in vitro, other advancements in molecular methods
revealed possibilities to create site-directed artificial genetic diversity in
vivo, be it in target coding sequences or other genetic determinants
(e.g., promoters for tuning their activity). Improvements with certain
potential for modification of production yeasts include concepts such as
yeast oligo-mediated genome engineering (YOGE); (DiCarlo et al.,
2013a) and targeted glycosylases to embedded arrays for mutagenesis
(TaGTEAM); (Finney-Manchester and Maheshri, 2013). In YOGE, a pool
of single-stranded oligonucleotides that contain desired point mutations
is transformed into the cells for recombination into the target sequence.
While YOGE oligonucleotides target the chromosomes in a pre-
determined manner, TaGTEAM can introduce point mutations (transi-
tions, transversions, deletions) or initiate chromosomal rearrangement
(deletions spanning multiple kbp) within a specific region labelled in
the genome. To this end, Finney-Manchester and Maheshri (2013)
constructed a translational fusion of the mutator enzyme (3-methyla-
denine DNA glycosylase Mag1 capable of excising nucleotide bases)
with DNA-binding domains of the bacterial tetracycline resistance
regulator TetR that recognizes 19-bp tetO element for binding. When
expressed in yeasts, the mutator fusion increases “random” mutation
rates in the region by about 10 kbp upstream and downstream of an
array of 240 tetO sites previously introduced to the target region by
homologous recombination.
4.1.1.1. Gene expression plasmids. A number of replicable yeast
expression plasmids and toolkits exist, most of them pre-designed to
allow for constitutive or inducible expression of heterologous gene(s)
(recently reviewed by Gnugge and Rudolf, 2017). These plasmids are
equipped with various prototrophic selection markers and markers
conferring drug resistance (Siewers, 2014). Yeast expression plasmids
fall into two categories. The first is represented by high-copy episomal
plasmids (YEp; 10 to over 50 copies per cell), in which maintenance
relies upon the origin of replication derived from the endogenous 2 μ
plasmid. These plasmids ensure high gene dose but lack a partitioning
system. Stochastic segregation may result in substantial variations in
the copy-number between cells in the population or even loss of the
plasmids and thus cell-to-cell expression heterogeneity (Karim et al.,
2013; Lee et al., 2015; Zhang et al., 1996). The plasmids of the second
category, low-copy yeast centromeric plasmids (YCp; 1 or 2 copies per
cell), can be used to better ensure the segregation stability of
recombinant gene(s). The chromosome-derived elements (autonomous
replicating sequence and centromeric sequence) in YEps execute
control over correct doubling and segregational stability during
mitosis. Although recent studies indicate that in spite of the selection
pressure, plasmid loss and uneven distribution may also occur with
YEps, these plasmid still offer substantially more reliability for gene
expression than YEps (Gnugge et al., 2016; Lee et al., 2015).
4.1.1.2. Gene integration for genomic expression. The need for
(continuous) selection conditions during propagation, as well as
issues with segregation and copy number instability of the expression
plasmids, often make the integration of foreign DNA into the host
genome by homologous recombination the method of choice. It is
particularly suitable if the metabolic engineering of production yeasts
using multiple genes is considered. Homologous recombination occurs
in S. cerevisiae with high efficiency and fidelity as documented by the
successful one-step assembly of a circular 0.6 Mbp Mycoplasma
genitalium genome, which used 25 fragments, mostly flanked with
only 80 bp regions of overlapping homology (Gibson et al., 2008).
Although homologous targeting sequences of 30 to 45 bp can be
sufficient to achieve correct recombination and insertion of a
heterologous DNA cassette at a desired chromosomal location, the
increase in length of targeting sequences to several hundreds of bp
improves the reliability and efficiency of integration (Manivasakam
et al., 1995). Homologous recombination can be effectively used to
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Biotechnology Advances xxx (xxxx) xxx–xxx
produce knockout integrants in which the targeted cassette disrupts the
continuity and thus the function of the selected gene.
To increase gene dosage in S. cerevisiae and thus expression rates,
higher copy number can be achieved by the sequential targeting of
heterologous cassette(s) to different locations in the genome or in-
tegration using abundant genomic sequences. Noteworthy, the use of
abundant target sites also increases transformation efficiency, which is
particularly desirable when difficult-to-transform strains are engineered
(Steensels et al., 2014b). High-copy sites involve an rDNA locus with
100 to 200 repeats coding for ribosomal RNAs (Woolford and Baserga,
2013) or more popular delta elements. These occur dispersed on dif-
ferent chromosomes as a part of Ty1 yeast transposons, with a total
of > 300 copies per S. cerevisiae genome (Kim et al., 1998; Maury et al.,
2016). Since transcription rates vary between different chromosomal
regions, the insertion location may contribute to the modulation in gene
expression in addition to selected promoter and terminator. To this end,
various sites on S. cerevisiae chromosomes (including Ty1 loci) were
scored for their gene expression activity (Fang et al., 2011; Flagfeldt
et al., 2009).
There are generally two possible ways to construct recombinant
yeasts with genomic insertions: using yeast integrative plasmids (YIp)
available in a number of variants (Gnugge and Rudolf, 2017) or
transformation of the yeast with linear DNA cassettes produced by re-
striction digestion or PCR. Heterologous DNA cassettes integrated in
chromosomes via homologous recombination gain segregational stabi-
lity in the cell population during mitosis, being thus maintained
without the need for selection pressure; however, as with the expression
plasmids, the integration of a selection marker within the cassette is
necessary to select for integrants. Several strategies with many recent
improvements have been developed to excise/recycle marker gene by
using tailored repeat sequences flanking the cassette, which facilitate
marker looping-out through homologous recombination (with stimu-
lation by meganucleases in some techniques; see also below) or bac-
teriophage Cre recombinase/loxP site- or yeast FLP1 recombinase/FRT
site-mediated recombination (reviewed by David and Siewers, 2015).
The Cre/loxP method for efficient marker recycling uses the more re-
cently developed EasyCloneMulti vector system designed for multiple
integrations using delta elements (Maury et al., 2016).
4.1.2. DNA assembly technologies
The first successful construction of a recombinant plasmid using
restriction digestion with a type II endonuclease and ligation was re-
ported > 40 years ago (Cohen et al., 1973). This method remains
popular in molecular biology for inserting plasmids with one or a few
heterologous genes, but suffers from substantial limitations when con-
structs harbouring multiple genes are designed. There have been many
recent advances for in vitro and in vivo DNA assembly techniques that
circumvent this problem (detailed in review by Chao et al., 2015). Some
improvement in restriction digestion/ligation approach were achieved
with BioBrics™ and BglBrick designs, which are based on iterative cy-
cles of restriction digestion and ligation to assemble DNA parts into
large constructs (Anderson et al., 2010; Ellis et al., 2011; Sarrion-
Perdigones et al., 2011). Correct assembly is facilitated by annealing of
the complementary single-stranded overhangs produced by nucleases
on target sites engineered at the DNA ends. GoldenGate technology,
which also employs type II endonucleases, allows the combination of
DNA parts in a one pot-reaction (Engler et al., 2008; Engler and
Marillonnet, 2014; Lee et al., 2015). Another technique involves as-
sembling multiple DNA segments in one step, a Gibson assembly, which
utilizes exonuclease activity to generate complementary overhangs that
are covalently joined by the harmonized action of DNA polymerase and
ligase (Gibson, 2009).
Methods used to assemble complex DNA constructs in vivo largely
rely on the propensity of S. cerevisiae for homologous recombination. A
DNA Assembler can be used to construct complex metabolic pathways
from linear DNA parts with homologous arms between neighboring
M. Karabín et al.
DNA modules; these DNA fragments are directly transformed into the
yeasts in a single step and integrants harbouring correct assemblies are
selected (Eriksen et al., 2013; Shao and Zhao, 2013; Shao and Zhao,
2012). In the original work by Shao et al. (2009), DNA Assembler al-
lowed the construction of a functional xylose utilization plus zeax-
anthin biosynthetic pathways either on a plasmid (when a linearized
plasmid backbone was inserted along with pathway genes) or on the
yeast chromosome. Using the DNA Assembler scheme in a combina-
torial manner, the powerful pathway optimization dubbed COM-
PACTER (customized optimization of metabolic pathways by combi-
natorial transcriptional engineering) was developed (Yuan et al., 2013).
In the pioneering work, a library of promoters of different strength was
co-transformed into the yeasts along with heterologous promoterless
xylose- or cellobiose-utilization pathway genes (Du et al., 2012). High
throughput screening then identified the strains in which a high effi-
ciency xylose or cellulose utilization pathway was assembled. Further
improvement involves the use of orthologous genes coding for enzyme
variants from different species, and error prone PCR to generate mutant
promoters or coding sequences to optimize the pathway through di-
rected evolution (Eriksen et al., 2013; Kim et al., 2013; Yuan and Zhao,
2013). More recently, the RADOM method (rapid assembly of DNA
overlapping multi-fragments) was developed, which in principle,
combines the DNA Assembler approach with classical blue/white
screening in Escherichia coli (Lin et al., 2015). In this method, plasmids
harbouring DNA construct variants assembled in yeasts on an in-
troduced plasmid backbone are extracted from the transformed popu-
lation and the library transformed into and screened in E. coli for as-
sembly-generated disruption of lacZα. Plasmids with insertions are then
subjected to DNA sequencing. This enhanced screening strategy sub-
stantially reduces labour and time required for the identification of
correct assemblies.
Some chromosomal integration approaches revolve around the use
of meganucleases (nucleases targeting recognition sites larger than
14 bp) such as I-SceI plus HO (Ostrov et al., 2013; Wingler and Cornish,
2011) or I-SceI alone (Kuijpers et al., 2013; Solis-Escalante et al., 2014),
which introduce assembly-stimulating double stranded breaks into pre-
determined sites of the chromosome and the method facilitates marker
excision/recycling. In spite of these advancements, the development of
CRISPR (clustered regularly interspaced short palindromic repeats) and
CRISPR-associated systems (Cas) has opened a new era in the assembly
of heterologous, marker-less DNA in yeast chromosomes as well as in
other DNA editing options (Bortesi and Fischer, 2015; Mali et al., 2013;
Schwarzhans et al., 2017).
CRISPR/Cas9-based genome engineering in yeasts typically requires
i) Cas9 nuclease that is targeted to the nucleus to generate a double
stranded break that facilitates integration, ii) CRISPR RNA (crRNA) plus
trans-activation crRNA (tracrRNA), usually provided as a continuous,
chimeric guide RNA (gRNA) and iii) a donor DNA part with flanking
homology to the integration site (Mali et al., 2013; Walter et al., 2016).
Since the use of CRISPR/Cas9-based tools assures high integration ef-
ficiency, selectable markers are dispensable and only a few resulting
colonies must be screened to identify correct integrants. The system
stems from defense system in Streptococcus pyogenes in which the as-
sembly of the complex consisting of Cas9, crRNA and tracrRNA leads to
cleavage of invading foreign DNA. In engineering systems, the position
of cleavage by Cas9 can be guided by the extra spacer in gRNA, which is
complementary to the defined DNA sequence at the site of intended
engineering. Both gRNA and Cas9 can be delivered to the cells for
transient transcription from the non-integrating expression DNA cas-
sette on a plasmid or linear DNA, or as the corresponding in vitro
transcribed gRNA and Cas9 mRNA molecules, respectively. Alter-
natively, Cas9 can be delivered as purified recombinant protein. Donor
DNA can be either a short oligonucleotide (e.g., introducing nonsense
mutation for gene knockout) or larger DNA as part of an expression
cassette assembly.
The feasibility of CRISPR/Cas9 in yeast engineering was
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Biotechnology Advances xxx (xxxx) xxx–xxx
demonstrated by DiCarlo et al. (2013b) who documented that nearly
100% marker-less integration efficiency can be achieved when appro-
priate oligonucleotide donor DNA is used. More recent improvements in
CRISPR/Cas9 technology for multigene pathway assembly in yeasts,
dubbed CasEMBLR (Jakociunas et al., 2015) and CAM (Horwitz et al.,
2015), which differ in the strategies used to generate gRNAs and donor
DNA fragments, were described. CasEMBLR was used in i) integration
of 15 DNA segments (carotenoid pathway) into three separate loci, and
ii) 10 DNA segments (tyrosine pathway) into two loci with concurrent
deletion of undesired endogenous genes (Jakociunas et al., 2015). Using
CAM (CRISPR-assisted multiplexing), Horwitz et al. (2015) succeeded
in integrating six heterologous DNA segments (total of 24 kb for the
muconic acid pathway), targeted across three loci to knockout un-
desired genes.
4.2. Rational metabolic engineering of brewing yeast
It is true that the traditional approaches described above bring a
number of advantages, but their main drawback is the low probability
that the desired phenotype will not have side effects. An alternative to
these methods is rational metabolic engineering, which allows targeted
modification of genetic information and the corresponding phenotype.
4.2.1. Intensification of beer production
Fermentation is one of the most time consuming steps in beer pro-
duction. Its rate is primarily dependent on the utilization speed of C-
and N-sources, the type of C- and N-sources, and the flocculation ability
of the yeast strain. Additionally, beer fermentation and maturation
greatly influence the speed of the following technological steps such as
filtration, stabilization, etc. In the past, many attempts have been made
to intensify the brewing technology through rational metabolic en-
gineering. In 1991, Steyn and Pretorius (1991) incorporated STA2
genes (encoding glucoamylase) from S. diastaticus and AMY (α-amy-
lase) genes from Bacillus amyloliquefaciens into brewer's yeast. The
transformants were able to degrade up to 93% of water-soluble starch
onto glucose and maltose under laboratory conditions. This work, along
with the constantly increasing industrial demands for yeast strains
producing ethanol from starch, resulted in the development of so-called
amylolytic yeast strains (Eksteen et al., 2003; Kim and Kim, 1996; Lin
et al., 1998; Marin et al., 2001; Ulgen et al., 2002). Many of them were
constructed for brewing use to reduce the residual extract in green beer.
A typical example is work by Liu et al. (2004) where the DNA fragment
containing the AMY gene was used for construction of a recombinant
brewer's yeast strain that was able to use starch as a sole carbon source.
Fermentation experiments with these recombinants showed a 25% de-
crease in residual oligosaccharides. Another gene encoding α-amylase
introduced into brewer's yeast was ALP1 from Saccharomycopsis fibuli-
gera (Wang et al., 2010). The recombinant strain showed high amylo-
lytic activity and was able to use starch as the primary source of carbon.
Fermentation tests showed that wort fermented with the recombinant
yeasts contained about 76.8% less residual sugars compared to stan-
dardly fermented wort. Two years later, the same authors presented an
article where the LSD1 gene (dextrinase) was integrated into the 18S
rDNA. The transformant metabolized about twice as much starch as the
host strain and also exhibited a higher reproduction rate (Wang et al.,
2012). In another work an engineered yeast for production of low-
carbohydrate beer was introduced (Park et al., 2014). The authors in-
corporated the glucoamylase (GAM1) gene from Debaryomyces occi-
dentalis into top and bottom fermenting yeast strains. The recombinants
exhibited both α-1,4- and α-1,6-degradation activity. Some of the
GAM1 recombinants were able to hydrolyse 96% of 2% (w/v) dextrins
and 98% of 2% (w/v) isomaltose within 5 days of fermentation.
Another research was focused on the development a new yeast
strains that would be able to ferment faster and more efficiently stan-
dard as well as high gravity worts. Vidgren et al. (2009) found that
some lager yeast strains contain a defective AGT1 gene encoding
M. Karabín et al.
maltose and maltotriose cell transporters. Such yeasts cannot be used to
produce beers by HGB technology, since highly concentrated worts
would be fermented slowly and incompletely. The authors repaired one
or more copies of the defective AGT1 gene using a specific DNA se-
quence from a top fermenting yeast. The newly constructed re-
combinants fermented high gravity worts (24° Plato) faster and the
extract utilization was more complete than the original strain. In 2005,
a GM yeast strain that efficiently fermented residual maltotriose in the
medium, was introduced as part of research of genes encoding maltose
transporters. This transformant was constructed by introducing newly
identified MTT1 and MTT1alt genes encoding maltose/maltotriose
transporters (Dietvorst et al., 2005). Some scientific groups have also
dealt with improvements in brewer's yeast nitrogen metabolism. Omura
et al. (2005) have shown that brewer's yeasts have an inactive PUT4
gene encoding a transporter of proline (major amino acid in wort)
during fermentation. They constructed the yeast strain with improved
proline assimilation, which have a great potential for more efficient
fermentation of the wort.
In addition to the basic metabolism of C and N, the ability of the
yeast to properly flocculate significantly contributes to the intensifica-
tion of the fermentation process (Soares, 2011). Successful introduction
and expression of flocculation genes FLO1 and FLO5 into non-floccu-
lating yeast strains has already been carried out in the 1990s (Bidard
et al., 1994; Watari et al., 1994). Over the course of time, rational
engineering has contributed to optimizing the flocculation properties of
yeast, especially to suppress premature flocculation. In 2001,
Verstrepen et al. (2003) published a manuscript describing how the
FLO1 promoter of a common non-flocculating yeast S. cerevisiae was
successfully replaced with the new promoter HSP30. This process re-
sulted in a stable flocculation ability of the transformant at the end of
fermentation. An extensive experiment was performed by Govender
et al. (2008) who studied the expression of dominant flocculation genes
FLO1, FLO5 and FLO11 driven by ADH2 and HSP30 promoters. All
promoter-gene combinations resulted in a specific phenotype in terms
of flocculation timing and intensity. For brewing use, the combination
of FLO1-based constructs, characterized by induction of flocculation,
was considered very effective. In a later study, Van Mulders et al.
(2009) used the laboratory yeast strains in which FLO genes are tran-
scriptionally silenced due to a nonsense mutation in the transcriptional
activator FLO8, for incorporation of a constitutive TEF1 promoter. This
chromosomal change resulted in overexpression of five flocculation
genes and correspondingly improved flocculation ability of transfor-
mants. The latest work describes the unexpectedly strong flocculation
ability of brewer's yeast transformants constructed during the study of
endogenous lipid synthesis pathways. This yeast was constructed by
transcriptional modulation of the lipid desaturase gene OLE1 and was
characterized by increased expression of FLO1 under the low-oxygen
condition (Degreif et al., 2017).
Improvement of beer filterability can be achieved by the effective
degradation of malt β-glucans by engineered yeasts during beer fer-
mentation. To facilitate the filtration process, bacterial, fungal or plant
genes encoding β-glucanases were successfully cloned into brewer's
yeast strains (Saerens et al., 2010). One of the first attempts to clone the
gene encoding β-glucanase into brewer's yeast was performed by
Cantwell et al. (1985) who used the bacterial (Bacillus subtilis) gene,
however secretion of the desired enzyme into the medium was minimal.
Much better results were achieved by Lancashire and Wilde (1987) who
repeated the previously experiment, but the β-glucanase gene was ex-
pressed in a fusion with the α-factor signal sequence. The transformants
effectively secreted β-glucanase into the medium and the β-glucan
content in the wort was significantly reduced. In the same year, Enari
et al. (1987) and Penttila et al. (1987) cloned the Trichoderma reesei β-
glucanase gene into brewer's yeast. The β-glucans in wort were effec-
tively degraded and filtration of the beer ran much faster. Not only
microbial enzymes were cloned into yeast. The thermolabile β-gluca-
nase from barley was introduced into the brewer's yeast strain in a
10
Biotechnology Advances xxx (xxxx) xxx–xxx
fusion with the mouse α-amylase signal sequence (Jackson et al., 1986;
Olsen and Thomsen, 1989). These yeasts effectively reduced the levels
of β-glucans during fermentation.
4.2.2. Improvement of beer quality
Improving beer quality through engineered yeast is primarily fo-
cused on the production of sensory active compounds. In the course of
time, many papers dealing with this topic have been published. They
can be divided into three groups (i) modifying the metabolism of sen-
sory active compounds, (ii) improving foam stability, and (iii) im-
proving colloidal stability. The metabolism of sensory active com-
pounds was most frequently modified to reduce the diacetyl (buttery
flavour) concentration in beer. In early works, the main strategy for
suppression of diacetyl formation (see in Fig. 1) was to increase the
activity of α-acetolactate decarboxylase, the enzyme catalysing de-
gradation of α-acetolactate (diacetyl precursor) to 3-hydroxybutanone.
In 1988, Sone et al. (1988) inserted a DNA fragment containing the
gene encoding α-acetolactate decarboxylase from Enterobacter aero-
genes, under the control of the alcohol dehydrogenase promoter, into
brewer's yeast S. cerevisiae. Laboratory-scale fermentation tests revealed
that the transformants were able to lower the α-acetolactate con-
centration in the medium more intensively in comparison to parental
strains. A similar approach was than chosen by a number of research
groups. Their works differed only in the origin of genes encoding α-
acetolactate decarboxylase. For example, Blomqvist et al. (1991) used
the gene from Klebsiella terrigena and Yamano et al. (1994a, 1994b)
used the Acetobacter aceti var. xylinum gene. In the latest work, Cejnar
et al. (2016) presented the development of engineered brewery yeast
containing acetolactate decarboxylase from Acetobacter aceti var. xy-
linum anchored on the surface of the cell wall. Using these yeasts, the
highest diacetyl concentration observed during fermentation tests was
about 30% lower in comparison with control yeasts. However, the
tolerance of consumers to recombinant yeasts containing bacterial
genes is very low (Nevoigt, 2008; Wang et al., 2009), therefore new
strategies now focus on the engineering of the original yeast's metabolic
pathways e. g. enhancing the biosynthesis of valine from α-acetolactate.
A very elegant way to reduce diacetyl concentrations was presented in
2008 by Omura (2008). He constructed a yeast strain that was able to
metabolize α-acetolactate in the cytosol before it leaves the cell. This
transformant contained the mutant acetohydroxyacid reductoisomerase
(Ilv5pΔ46), which was expressed and localized in the cytosol. Over-
expression of acetohydroxyacid reductoisomerase (Ilv5) in brewer's
yeast was also described in the work by Lu et al. (2012). The scientific
group of Shi et al. (2016) studied three recombinant brewer yeast
strains with a disrupted acetolactate synthase (ILV2) gene, over-
expressed 2,3-butanediol dehydrogenase (BDH1) gene and a combina-
tion of both. The use of all these strains in fermentation tests resulted in
a decrease of diacetyl content in comparison with the host strain. The
same research group also presented a work where an engineered yeast
with overexpressed BDH2 and ILV5 genes and a disrupted ILV2 gene
were tested. Fermentation tests showed that each of these strategies
reduced the diacetyl content, but the best results have been achieved
using recombinant strains with a deletion of ILV2 and simultaneously
overexpressing ILV5 (Shi et al., 2017).
Methods of molecular biology can also be used for reducing the level
of dimethyl sulphide (flavour of cooked vegetables) in beer. Most of the
published manuscripts were dedicated to the prevention of dimethyl
sulfoxide reduction to dimethyl sulphide during beer fermentation.
Hansen (1999) reduced the dimethyl sulphide content in medium using
yeast with a disrupted MXR1 gene, encoding the enzyme catalysing the
conversion of dimethyl sulfoxide to DMS. Later, the key role of this gene
was confirmed when MXR1 was inserted under the control of non-na-
tive promoters in engineered brewer's yeasts. Various strengths of
overexpression of this gene positively correlated with dimethyl sul-
phide production (Hansen et al., 2002).
Volatile esters, produced by yeast during fermentation, play an
M. Karabín et al.
important role in the sensory profile of beer (especially top fermented).
Therefore, it is understandable that the metabolism of these substances
has attracted the interest of many scientists. Fujii et al. (1994) increased
the production of ethyl acetate (solvent flavour) and isoamyl acetate
(banana flavour) in brewer's yeast by overexpression of the acetyl-
transferase (ATF1) gene. On the other hand, the later published work
described the reduced production of these esters (compared to parental
strains) by the recombinant yeast containing a disrupted ATF1 (Fujii
et al., 1996). In the manuscript by Verstrepen et al. (2003), over-
expression/deletion of ATF1, Lg-ATF1 from S. bayanus and ATF2 genes
were studied. Fermentation tests with transformants have shown that
expression of ATF1 and ATF2 greatly affects the production of not only
ethyl acetate and isoamyl acetate but also a number of minor esters
such as propyl acetate, isobutyl acetate, pentyl acetate, hexyl acetate,
etc. In 2013 Zhang et al. (2013) introduced a new yeast transformant
with over-expression of ATF1 and the simultaneous deletion of the
BAT2 gene (playing a significant role in the production of sensory un-
desirable branched-chain alcohols).
Antioxidants play an important role in the sensory stability of beer,
as they prevent the formation of sensory undesirable products of oxi-
dation reactions (especially carbonyl compounds) (Bushnell et al.,
2003). The major beer antioxidant is sulphur dioxide produced by yeast
during fermentation. In the past, engineered yeast with disrupted
MET10 gene (encoding a key part of sulphite reductase) was used to
produce beers with improved sensory stability (Hansen and
KiellandBrandt, 1996; Lu et al., 2009). In the study by Donalies and
Stahl (2002), similar results were achieved. In addition, the authors
developed yeast transformants with overexpression of the adenosyl-
phosphosulfate kinase (MET14) and/or phosphoadenosylphosphate
reductase (MET16) genes. This resulted in a 2–3 fold higher production
of sulphites compared to the original, low sulphite producing strain.
The authors also introduced transformants with overexpression of the
putative sulphite pump (SSU1) along with MET14, which can increase
the production of sulphites up to 10-fold. Wang et al. (2014) focused on
the construction of a brewer's yeast overexpressing superoxide dis-
mutase (SOD1) and γ-glutamylcysteine synthase (GHS1). This yeast has
been characterized by increased glutathione production and the ability
to secrete superoxide dismutase to the medium. Analysis of beers pro-
duced using this strain shown a low concentration of carbonyl com-
pounds and excellent flavour stability.
Foam quality and stability is one of the most important sensory
parameters of beer, and it is no surprise that a number of research
groups have tried to improve it using engineered yeast. Liu et al. (2004)
disrupted the PEP4 gene encoding proteinase A, an enzyme capable of
degrading proteins involved in the formation of beer foam. However,
this procedure had a negative effect on metabolism in the yeast cell, and
therefore Chen et al. (2017) modified the Golgi → vacuole transport of
proteinase A through overexpression of the VPS10 gene encoding the
key sorting receptor of proteinase A transport. Deletion of the SEC5
gene, encoding an important protein participating in the secretion of
proteinase A was also tested, however this caused only a minor re-
duction of proteinase A activity in the medium. The manuscript of
Blasco et al. (2012) reported the identification of the CFG1gene en-
coding the Cfg1p protein involved in the stabilization of beer foam. The
foam-promoting effect of Cfg1p was further confirmed by expressing
CFG1 in a foamless S. cerevisiae strain.
Brightness is another sensory parameter of beer, which is very
strictly judged by the consumer. Recently, Cejnar et al. (2017) devel-
oped and constructed a yeast strain that had proline-rich peptides an-
chored on its outer cell wall. These proteins adsorbed the haze-active
polyphenols (proanthocyanidins) of beer onto the yeast cell surface.
Polyphenol uptake caused by the engineered yeast was 20% higher in
comparison with the parental strain.
4.2.3. Health promoting beer
Beer is the world's most popular alcoholic beverage, but its long-
11
Biotechnology Advances xxx (xxxx) xxx–xxx
term consumption, combined with poor lifestyle, increases the risk of
civilization diseases such as obesity, diabetes etc. It is of no surprise that
scientists, in collaboration with nutritionists and brewers, are trying to
develop a low alcohol and low energy beer that would retain its sensory
qualities. Reduction of the beer residual extract, composed mainly of
dextrins that are responsible for the high energetic value of beer is
described in chapter 4.2.1.
Numerous works have been published about reducing the ethanol
content of beer through engineered yeast. Disruption of alcohol dehy-
drogenase encoding enzymes cannot be use because (i) alcohol dehy-
drogenase is an isoenzyme, and deletion of the gene encoding one type
of alcohol dehydrogenase leads to upregulation of genes encoding other
forms of the isoenzyme (Ida et al., 2012); (ii) yeast responds to dis-
ruption of alcohol dehydrogenase by slowing metabolism and in-
creasing glycerol and acetate production (Kutyna et al., 2010).
Nevoigt et al. (2002) developed an engineered yeast strain with
overexpression of the GPD1 gene (glycerol-3-phosphate dehy-
drogenase). This strain produced about 18% less alcohol and 5.6 times
more glycerol compared to wild-type, however, the production of some
sensory-active by-products of fermentation such as acetoin, diacetyl
and acetaldehyde also significantly increased. A more effective strategy,
which has not yet been applied to brewer's yeast, was introduced by
Cordier et al. (2007). This strategy involves the overexpression of GDP1
and ALD3 (cytosolic NAD+-dependent aldehyde dehydrogenase) genes
and the simultaneous deletion of the ADH1 (NAD+-dependent alcohol
dehydrogenase) gene. Transformants showed increased production of
glycerol and acetate at the expense of ethanol.
5. Conclusions
Brewer's yeasts have historically evolved along with Saccharomyces
cerevisiae species, and have been used in the world's oldest bio-
technologies - beer, wine and bread production; they therefore re-
present the oldest domesticated microorganism. Over time, brewer's
yeast and yeasts for bread production have become more and more
distinct from wine ones, mainly as a result of repeated use and multiple
mutations. Brewing yeasts have also received DNA of other species from
the genus Saccharomyces (S. eubayanus, S. kudriavzevii, etc.) into their
genome and so are distinguishable from sourdough yeasts by their
genotypes. As a result, we now use aneuploidic and polyploidic hybrid
brewing yeast strains created by nature and these strains can be divided
into bottom brewing yeast strains, now known as S. pastorianus, and the
top brewing yeast strains known as S. cerevisiae. These strains contained
in the collections of industrial microorganisms do not differ much in
phenotype from each other, or in beers produced by them, especially
beer with uniform sensory characters produced in large breweries. In
addition, most of these strains at least partly meet the requirements for
intensive industrial production. Because the genomes of many in-
dustrial strains are known, original parental strains found in nature can
be used for hybridization. It is therefore possible to speed up evolution
and prepare strains with a range of industrially useful traits (e.g. tol-
erance to high ethanol content, no typical aroma profiles, better floc-
culation, etc.). At the same time, it is also possible to look for micro-
organisms in nature that can supplement current strains, thereby
increasing diversity and permitting the production of beers with dif-
ferent, mainly sensory, properties than those currently available on the
market. Another option is to create strains by procedures based pri-
marily on genetic modifications using DNA from other types of micro-
organism. This second possibility, however, has raised marketing bar-
riers due to the conservatism of customers who, at least for the time
being, have not accepted the concept of GMO brewing yeast, as well as
legislative barriers. This has limited the possibilities of genetically
modifying yeasts and their application. However, we believe that by
targeted education, gradually adopting different opinions on GMOs in
individual countries, and by introducing new techniques for the as-
sembly of heterologous DNA, marker-less DNA, and other DNA editing
M. Karabín et al.
options, GM brewer's yeast in the future will be introduced into in-
dustrial practice. In the future, therefore, a larger variety of brewer's
yeast strains with different technological traits can be expected to make
beer cheaper. At the same time, a wider choice of available strains will
make it possible to target the sensory characteristics of beer, offering
options for different or even totally new beer types.
Acknowledgments
The authors thank the Technological Agency of the Czech Republic
(project TE02000177) for its financial support.
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