The winemaker's bug: From ancient wisdom to opening new vistas with frontier yeast science.
ABSTRACT The past three decades have seen a global wine glut. So far, well-intended but wasteful and expensive market-intervention has failed to drag the wine industry out of a chronic annual oversupply of roughly 15%. Can yeast research succeed where these approaches have failed by providing a means of improving wine quality, thereby making wine more appealing to consumers? To molecular biologists Saccharomyces cerevisiae is as intriguing as it is tractable. A simple unicellular eukaryote, it is an ideal model organism, enabling scientists to shed new light on some of the biggest scientific challenges such as the biology of cancer and aging. It is amenable to almost any modification that modern biology can throw at a cell, making it an ideal host for genetic manipulation, whether by the application of traditional or modern genetic techniques. To the winemaker, this yeast is integral to crafting wonderful, complex wines from simple, sugar-rich grape juice. Thus any improvements that we can make to wine, yeast fermentation performance or the sensory properties it imparts to wine will benefit winemakers and consumers. With this in mind, the application of frontier technologies, particularly the burgeoning fields of systems and synthetic biology, have much to offer in their pursuit of "novel" yeast strains to produce high quality wine. This paper discusses the nexus between yeast research and winemaking. It also addresses how winemakers and scientists face up to the challenges of consumer perceptions and opinions regarding the intervention of science and technology; the greater this intervention, the stronger the criticism that wine is no longer "natural." How can wine researchers respond to the growing number of wine commentators and consumers who feel that scientific endeavors favor wine quantity over quality and "technical sophistication, fermentation reliability and product consistency" over "artisanal variation"? This paper seeks to present yeast research in a new light and a new context, and it raises important questions about the direction of yeast research, its contribution to science and the future of winemaking.
- SourceAvailable from: Cristian Varela[Show abstract] [Hide abstract]
ABSTRACT: Saccharomyces cerevisiae and grape juice are 'natural companions' and make a happy wine marriage. However, this relationship can be enriched by allowing 'wild' non-Saccharomyces yeast to participate in a sequential manner in the early phases of grape must fermentation. However, such a triangular relationship is complex and can only be taken to 'the next level' if there are no spoilage yeast present and if the 'wine yeast' - S. cerevisiae - is able to exert its dominance in time to successfully complete the alcoholic fermentation. Winemakers apply various 'matchmaking' strategies (e.g., cellar hygiene, pH, SO2 , temperature and nutrient management) to keep 'spoilers' (e.g., Dekkera bruxellensis) at bay, and allow 'compatible' wild yeast (e.g., Torulaspora delbrueckii, Pichia kluyveri, Lachancea thermotolerans and Candida/ Metschnikowia pulcherrima) to harmonize with potent S. cerevisiae wine yeast and bring the best out in wine. Mismatching can lead to a 'two is company, three is a crowd' scenario. More than 40 of the 1500 known yeast species have been isolated from grape must. In this article, we review the specific flavour-active characteristics of those non-Saccharomyces species that might play a positive role in both spontaneous and inoculated wine ferments. We seek to present 'single-species' and 'multi-species' ferments in a new light and a new context, and we raise important questions about the direction of mixed-fermentation research to address market trends regarding so-called 'natural' wines. This review also highlights that, despite the fact that most frontier research and technological developments are often focussed primarily on S. cerevisiae, non-Saccharomyces research can benefit from the techniques and knowledge developed by research on the former. This article is protected by copyright. All rights reserved.FEMS Yeast Research 10/2013; · 2.46 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: To investigate the assimilation and production of juice metabolites by Saccharomyces cerevisiae during winemaking, we compared the metabolite profiles of 63 Sauvignon Blanc (SB) grape juices collected over five harvesting seasons from different locations of New Zealand before and after fermentation by the commercial wine yeast strain EC1118 at 15 °C. Metabolite profiles were obtained using gas chromatography-mass spectrometry (GC-MS) and nuclear magnetic resonance (NMR) and the oenological parameters were determined by Fourier Transform Infrared Spectroscopy (FTIR). Our results revealed that the amino acids threonine and serine were the most consumed organic nitrogen sources, while proline and gamma-aminobutyric acid (GABA) were the least consumed amino acids during SB juice fermentation. S. cerevisiae metabolised some uncommon nitrogen sources (e.g. norleucine, norvaline and pyroglutamic acid) and several organic acids, including some fatty acids, most likely after fermenting the main juice sugars (glucose, fructose and mannose). However, consumption showed large variation between juices and in some cases between seasons. Our study clearly shows that preferred nitrogen and carbon sources were consumed by S. cerevisiae EC1118 independent of the juice fine composition, whilst the consumption of other nutrient sources mainly depended on the concentration of other juice metabolites, which explains the uniqueness of each barrel of wine.This article is protected by copyright. All rights reserved.FEMS Yeast Research 10/2014; · 2.46 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Different populations within a species represent a rich reservoir of allelic variants, corresponding to an evolutionary signature of withstood environmental constraints. Saccharomyces cerevisiae strains are widely utilised in the fermentation of different kinds of alcoholic beverages, such as, wine and sake, each of them derived from must with distinct nutrient composition. Importantly, adequate nitrogen levels in the medium are essential for the fermentation process, however, a comprehensive understanding of the genetic variants determining variation in nitrogen consumption is lacking. Here, we assessed the genetic factors underlying variation in nitrogen consumption in a segregating population derived from a cross between two main fermenter yeasts, a Wine/European and a Sake isolate. By linkage analysis we identified 18 main effect QTLs for ammonium and amino acids sources. Interestingly, majority of QTLs were involved in more than a single trait, grouped based on amino acid structure and indicating high levels of pleiotropy across nitrogen sources, in agreement with the observed patterns of phenotypic co-variation. Accordingly, we performed reciprocal hemizygosity analysis validating an effect for three genes, GLT1, ASI1 and AGP1. Furthermore, we detected a widespread pleiotropic effect on these genes, with AGP1 affecting seven amino acids and nine in the case of GLT1 and ASI1. Based on sequence and comparative analysis, candidate causative mutations within these genes were also predicted. Altogether, the identification of these variants demonstrate how Sake and Wine/European genetic backgrounds differentially consume nitrogen sources, in part explaining independently evolved preferences for nitrogen assimilation and representing a niche of genetic diversity for the implementation of practical approaches towards more efficient strains for nitrogen metabolism.PLoS ONE 01/2014; 9(1):e86533. · 3.53 Impact Factor
© 2012 Landes Bioscience.
Do not distribute.
complex wines from simple, sugar-rich grape juice. Thus any
improvements that we can make to wine, yeast fermentation
performance or the sensory properties it imparts to wine will
benefit winemakers and consumers. With this in mind, the
application of frontier technologies, particularly the burgeon-
ing fields of systems and synthetic biology, have much to offer
in their pursuit of “novel” yeast strains to produce high quality
wine. This paper discusses the nexus between yeast research
and winemaking. It also addresses how winemakers and
scientists face up to the challenges of consumer perceptions
and opinions regarding the intervention of science and
technology; the greater this intervention, the stronger the
criticism that wine is no longer “natural.” How can wine
researchers respond to the growing number of wine com-
mentators and consumers who feel that scientific endeavors
favor wine quantity over quality and “technical sophistication,
fermentation reliability and product consistency” over “artisa-
nal variation”? This paper seeks to present yeast research in a
new light and a new context, and it raises important questions
about the direction of yeast research, its contribution to
science and the future of winemaking.
The winemaker’s bug
From ancient wisdom to opening new vistas
with frontier yeast science
Isak S. Pretorius,1,* Christopher D. Curtin2and Paul J. Chambers2
1University of South Australia; Adelaide, Australia;2The Australian Wine Research Institute; Adelaide, Australia
Keywords: Saccharomyces cerevisiae, wine, yeast
The past three decades have seen a global wine glut. So far,
well-intended but wasteful and expensive market-intervention
has failed to drag the wine industry out of a chronic annual
oversupply of roughly 15%. Can yeast research succeed where
these approaches have failed by providing a means of
improving wine quality, thereby making wine more appealing
to consumers? To molecular biologists Saccharomyces cerevi-
siae is as intriguing as it is tractable. A simple unicellular
eukaryote, it is an ideal model organism, enabling scientists to
shed new light on some of the biggest scientific challenges
such as the biology of cancer and aging. It is amenable to
almost any modification that modern biology can throw at a
cell, making it an ideal host for genetic manipulation, whether
by the application of traditional or modern genetic techniques.
To the winemaker, this yeast is integral to crafting wonderful,
The winemaker’s bug, Saccharomyces cerevisiae, is so closely asso-
ciated with humans it is rarely found in environs removed from
human habitation.1In fact, its evolutionary success can probably
be explained by its relationship with humans, particularly in the
production of alcoholic beverages, an activity that has been with
us for at least 7,000 years.2Because of us, S. cerevisiae enjoys
phenomenal reproductive success with, for example, an estimated
600,000 tons of baker’s yeast being produced every year.3But
how did this close relationship get started?
It is likely that the first alcoholic fermentations were “happy
accidents”: harvested grapes were not eaten quickly enough and
began to rot, Saccharomyces spp “moved in” and took advantage
of the free sugary meal and the first wines were made (Fig.1).
These early wines presumably tasted good and had an interesting,
pleasing, psychotropic effect. One can only assume that early
farmers learned from this experience and repeated the “accidents”
of previous “vintages.” Winemaking was born and wine yeast had
a secure future in the hands of its human guardians.
To Intervene or Let Nature Take its Course
It has probably been known since the earliest of times that wine is
susceptible to spoilage. The discovery that it is largely microbes
that are responsible for this has led, in more recent times, to a
debate on whether wine should be “natural” or whether we should
protect it from undesirable microorganisms by “pasteurization” or
the addition of sulfur dioxide. And this debate has broadened in
the past decade to include questions on whether wine should be
the fermentation product of its “natural” microflora or of a
controlled inoculated wine yeast (Fig.1).
Some winemakers and commentators believe that the ambient
yeast population in the vineyard and winery constitute part of the
characteristics of a “natural” wine. They believe that the unique
contributions of diverse yeast species confer a complexity upon
wine not seen in inoculated ferments. This might be true,4but it
comes with the risk of spoilage. There’s also an increased risk that
the fermentation will become “stuck”1—i.e., the ferment will stop
and be difficult to restart. In addition, spontaneous—“natural,”
“wild” or “feral”—ferments also tend to take longer.
*Correspondence to: Isak S. Pretorius; Email: firstname.lastname@example.org
Submitted: 12/13/11; Revised: 02/09/12; Accepted: 02/13/12
Bioengineered Bugs 3:3, 147–156; May/June 2012;G2012 Landes Bioscience
© 2012 Landes Bioscience.
Do not distribute.
inocula at the start of fermentation. This is a very fertile research
field where advances in wine yeast strain development and
fundamental yeast science have leveraged from one another.
This new era in wine yeast research, embracing cross-
disciplinary expertise, is worthy of review. It started following
the revelation that genes are made of DNA; the stage was set
for an explosive growth of knowledge, driven by a convergence of
genetics, biochemistry, cell biology, microbiology and computing.
And work on yeast was often at the forefront of developments.
There were compelling reasons for molecular biologists from
all fields to look on this simple single-celled fungus as the ideal
“guinea pig” for fundamental research. Our close relationship
with S. cerevisiae in food and beverage production over millennia
tells us that it is safe to work with; for example, it is designated
“Generally Recognized as Safe” (GRAS) by the United States’
Food and Drug Administration.5In addition, it is inexpensive
and easy to grow and can be stored for long periods in sus-
pended animation. But, perhaps, its best asset is an accessible
genetic system that can be followed through asexual and sexual
cycles. The three basic cell types—a, a and a/a cells—can
undergo mitosis and reproduce through an asexual budding
process. The a and a haploid cells are also able to undergo mating,
a sexual process that culminates in nuclear fusion and creation of
a/a diploid cells, which can be induced to undergo meiosis to
produce asci carrying four haploid spores.
Since the mid 1970s, when recombinant DNA technologies
revolutionized the way research in biological sciences is con-
ducted, S. cerevisiae has been one of the most important model
The debate continues and sets the backdrop for wine yeast
research, but over the past two decades, active yeast strain
development programs have been launched the world over to
generate strains that can improve wine quality when used as
organisms in molecular biology and emerging fields. For exam-
ple, a haploid laboratory strain (S288c) was the first eukaryote
to have its genome sequenced, a feat achieved through a
collaborative international effort involving more than 600
scientists under the able leadership of André Goffeau and
Stephen Oliver.6This paved the way for the first chip-based
gene array experiments.7
S. cerevisiae was also the first organism to be used to build a
systematic collection of bar-coded gene deletion mutants enabling
high throughput functional genomics experiments.8. But the most
important resource available to the yeast scientific community is
the Saccharomyces Genome Database (SGD; www.yeastgenome.
org), which provides, free of charge, access or links to the most
comprehensive data sets (genomic, transcriptomic, proteomic,
metabolomics, etc.) available to a molecular biologist. All of this
has been achieved by international collaborations on a grand scale.
What does all of this mean for wine research? The above
international efforts have put the winemaker’s bug center stage
in thousands of laboratories worldwide. And our knowledge is no
longer limited to the S288c laboratory version of S. cerevisiae
whose genome sequence was announced in 1996. Recently, the
genomes of several industrial yeast strains were sequenced,
including two ale strains (Foster’s O and Foster’s B) and five
wine yeast, AWRI 696, QA23, VIN7, VIN13 and VL3,9,10
enabling us to conduct comparative genomic analyses. This means
we can better understand what makes wine yeasts “tick” and why
there is such variation in S. cerevisiae “winemaking phenotypes”.
Engineering Yeast to Make Better Wine
Wine research and wine yeast strain development are certainly
well placed to benefit from the privileged place that S. cerevisiae
Figure1. To the winemaker, yeast is integral to crafting wonderful, complex wines from simple, sugar-rich grape juice. Grape juice is converted into wine
by the action of wine yeast. Some wine components are wholly generated by yeast as part of metabolism while others are essentially created by
the grapevine. The large number of compounds synthesized or modified by wine yeast have a major impact on wine quality and style.
148Bioengineered Bugs Volume 3 Issue 3
© 2012 Landes Bioscience.
Do not distribute.
glycerol transporters, mostly leading to increased glycerol yields
and accompanying reduced ethanol production. Alternative
approaches have included expression of the Aspergillus niger
occupies in the life sciences. The following includes some
examples that demonstrate this.
Getting control of alcohol levels in wine. Without question
the greatest challenge faced by the wine industry is rapidly
mounting concerns over alcohol consumption; excess consump-
tion creates problems for society and human health. In addition,
too much alcohol in wine can overwhelm flavor and make the
wine “hot” on the palate. The technical challenges associated with
reducing the alcohol content of wine, however, are substantial.11
Several genetic modification (GM) based metabolic engineer-
ing strategies have been explored to generate wine yeasts that
partially divert carbon metabolism away from ethanol production,
with the aim of decreasing ethanol yields during vinification.
Two glycerol-3-phosphate dehydrogenase isozymes, GPD1 and
GPD2, which divert carbon from glycolysis to glycerol produc-
tion, have proven to be the best candidates to date (Fig.2).
Enhanced expression of either GPD paralog achieved the
desired outcome with regard to ethanol yields;12-14however,
increased glycerol production was accompanied by undesirable
increased concentrations of acetic acid. This was probably due
to a perturbation in redox balance in the engineered strain,
requiring the action of one or more of the five aldehyde
dehydrogenase (Ald) isozymes; these enzymes help maintain
redox balance by reducing coenzymes NAD+or NADP+, when
they oxidize acetaldehyde to acetic acid. The problem, however,
was alleviated quite simply by knocking out ALD6.15,16
Similar approaches11have targeted S. cerevisiae pyruvate
decarboxylase isozymes, alcohol dehydrogenase isozymes and
glucose oxidase encoding gene (GOX) in S. cerevisiae, which
redirects glucose to gluconic acid, and extensive modification of
S. cerevisiae hexose transporters, which forces the yeast to respire
rather than ferment, regardless of the concentration of glucose
and fructose it encounters. Arising from these research efforts
are several promising candidate “low-ethanol” wine yeast strains
awaiting widespread acceptance of their use in commercial
winemaking. These strains could immediately enable production
of wines that contain 12% alcohol instead of 15%, from optimally
Enhancing varietal wine flavor during fermentation. To casual
wine drinkers it may seem fanciful, even pretentious, when a wine
enthusiast states that Shiraz offers impressions of “black pepper;”
Pinot Noir displays overtones of “earthy strawberries;” or that
Sauvignon Blanc is characterized by traces of “asparagus” and
“passionfruit.” But these descriptors of wine flavors may become
clearer to casual wine drinkers if they are informed that, for
example, strawberry flavor is considered to be relatively complex
among fruits and contributed to by a large number of aroma
compounds;17and that wine flavor is really the sum of complex
interactions between more than a thousand volatile compounds,
many of which overlap with those found in strawberry, and some
impact compounds that are, in fact, found in black pepper
(e.g., rotundone18) and passionfruit (e.g., polyfunctional thiols19).
Therefore, perceiving the aromas of these aforementioned fruits,
vegetables and spices in wine is not surprising. The relative
amounts of each compound, and the resultant flavor profile,
ultimately define differences among the vast array of wines and
wine styles produced throughout the world.
Grape variety is the starting point for differentiation—many
volatile compounds provide varietal distinction in addition to
Figure2. Reducing alcohol levels in wine: several GM-based strategies have been explored to generate wine yeasts that partially divert sugar metabolism
away from ethanol production. (A) Two glycerol-3-phosphate dehydrogenase isozymes, GPD1 and GPD2, can be harnessed to divert carbon from
glycolysis to glycerol production. However, increased glycerol production was accompanied by undesirable increased concentrations of acetic acid.
This problem was alleviated by knocking out ALD6. (B) Wild-type yeast convert most of the sugar they consume into ethanol and CO2.
© 2012 Landes Bioscience.
Do not distribute.
By mining the S. cerevisiae genome for putative carbon-sulfur-
lyase encoding genes, we identified four candidates (BNA3, CYS3,
giving wine its basic structure. The concentrations of many
volatile compounds are, however, dependent upon an almost
infinite number of variations in production, whether in the
vineyard or the winery. It is known, for example, that commercial
yeast strains possess different abilities to form and modulate
volatile compounds during alcoholic fermentation (Fig.3) that
significantly affect the flavor and overall quality of wines.20
Therefore, while the proportion of wine volatiles modulated by
yeast may be relatively low,21the choice of yeast strain controlling
fermentation is an effective method for shaping wine aroma
according to the preferences of consumers in target markets.22
A case in point is the incidence of powerful synergies between
Sauvignon Blanc grapes and yeast strains in formation of the
compounds responsible for “box-hedge” and “tropical fruit”
flavors—the polyfunctional thiols 4-mercapto-4-methylpentan-2-
one (4MMP), 3-mercaptohexan-1-ol (3MH) and 3-mercaptohexyl
acetate (3MHA). Odorless cysteine and glutathione conjugates
of 3MH and 4MMP form in the grape berry and during crush-
ing and can be found at higher concentrations in Sauvignon
Blanc juice in comparison to other white varieties.23Due to the
potency of the free thiols, with perception thresholds in the ng/l
range, only a fraction of the available conjugated precursors need
to be released to impart strong “passionfruit,” “grapefruit,”
“gooseberry” and “guava” flavors to wine. Yeast carbon-sulfur-
lyase enzymes are responsible for the release of 4MMP and
3MH from their cysteine conjugates, while 3MHA is produced
by yeast metabolism through the esterification of 3MH during
GLO1 and IRC7) that when deleted decreased the ability of yeast
to release 4MMP.24Subsequent studies have narrowed this list to
one gene, the β-lyase encoding IRC7,25and in fact, a particular
allele of this gene26as the main determinant of 4MMP formation
during winemaking. This line of research has, therefore, provided
a clear quantitative trait locus (QTL) for molecular breeding of
wine yeast. 3MH release, on the other hand, is not mono-
genetically determined;25,26therefore, optimization of its release
through non-GM approaches remains an empirical exercise.
Early research into precursors for the polyfunctional thiols
utilized a column-immobilized Escherichia coli carbon-sulfur
lyase enzyme, apo-tryptophanase, in a method designed to
measure aromatic potential.27We engineered a wine yeast,
VIN13, to constitutively express the gene encoding this enzyme,
tnaA.28Wine made from warm-climate Sauvignon Blanc grapes
with this yeast exhibited intense tropical characters, while in
model ferments the VIN13-tnaA strain released up to 20-fold
more 3MH and 4MMP.
The same wine yeast, VIN13, engineered to overexpress
S. cerevisiae alcohol-acetyltransferase encoding genes (ATF1 and
ATF2), was able to produce high concentrations of acetate
esters.29In neutral-tasting grape varieties, such as Colombard,
this results in lifted “banana” characters. Overexpression of ATF1
in VIN13 increased conversion of 3MH into its acetate ester,30
3MHA, which, due to its lower perception threshold, is signi-
ficantly more potent. Taken together, it is therefore possible to
specifically manipulate 4MMP production by controlling the
expression of S. cerevisiae’s own IRC7; and to release high
concentrations of both 3MH and 4MMP through the expres-
sion of E. coli’s tnaA gene in yeast and increase the potency of
Figure3. Commercial yeast strains possess different abilities to form and modulate compounds that impact on wine sensory properties.
These compounds are produced as a result of yeast metabolic processes.
150Bioengineered Bugs Volume 3 Issue 3
© 2012 Landes Bioscience.
Do not distribute.
“tropical fruit” aromas by boosting the conversion of 3MH into
3MHA by overexpression of S. cerevisiae’s own ATF1 gene. While
significantly less powerful than the tnaA gene product, modest
enhancement of 3MH release is also possible through expression
of the S. cerevisiae cystathionine β-lyase encoding gene, STR3,31
thereby paving the way for a range of “self-cloned” thiol-
modulating wine yeast.
Polyfunctional thiols are not the only sulfur-containing aroma
compounds that contribute to wine style. One need not be a wine
expert to know that “reductive” aromas with descriptors such
as “rotten egg,” “burnt rubber” and “sewage” are not going to
appeal to wine consumers. While there are several chemical and
biological mechanisms that contribute to “reductive” aromas in
wine, “rotten egg” gas, also known as hydrogen sulfide (H2S), is
largely a by-product of yeast metabolism. Under certain ferment-
ation conditions, most wine strains of S. cerevisiae produce
H2S hile incorporating inorganic sulfur into the amino acids
methionine and cysteine—a process known as the sulfate
reduction sequence (SRS) pathway.
Several GM strategies in the laboratory have been successful in
limiting H2S production by S. cerevisiae and these are generally
based on overexpression or inactivation of one or more genes
involved in the SRS pathway.32,33One of the targets has been
sulfite reductase, which comprises two a- and two β-subunits
(a2β2) encoded by yeast’s MET10 and MET5 genes, respectively.
This knowledge informed a classical, non-GM, mutagenesis
approach to develop three “low-H2S” strains, derived from the
widely-used commercial wine yeast Maurivin PDM (Fig.5).34
These strains, commercialized under the names Maurivin
Advantage, Platinum and Distinction, provide winemakers with
new strategies to manage “reductive” aromas, especially in grape
musts low in assimilable nitrogen.
Making the first modest moves with GM yeast strains.
Australia, New Zealand and many European countries have
effectively banned the use of genetically-modified organisms
(GMOs) in commercial wine production. A multitude of inter-
connected agronomic, business, regulatory, cultural and social
factors have led to these bans, but consumer sentiment is clearly
one of the main drivers. While it is unlikely that the situation
will change in the near future, the first modest move to release
a commercialized GM yeast to market was made in 2005; a
transgenic wine yeast, ML01, was given the green light from
regulatory authorities in the USA, Canada and Moldova.
ML01 carries genes that enable it to perform malolactic
fermentation (MLF), in which grape-derived malic acid is
deacidified (decarboxylated) to lactic acid. MLF is performed
by lactic acid bacteria, particularly Oenococcus oeni, following
alcoholic fermentation. However, O. oeni is rather fastidious,
being inhibited by a range of conditions typical of fermented
grape juice (e.g., low pH, high alcohol content and poor nutrient
availability) and can become “stuck” or sluggish.35In addition,
some lactic acid bacteria produce biogenic amines that impose
Figure4. There are powerful synergies between Sauvignon Blanc grapes and yeast strains in formation of the compounds responsible for tropical fruit
flavors: 4-mercapto-4-methylpentan-2-one (4MMP), 3-mercaptohexan-1-ol (3MH) and 3-mercaptohexyl acetate (3MHA). Odorless cysteine and
glutathione conjugates are converted to aromatic thiols by carbon-sulfur-lyase enzymes. Alcohol acetyl transferase further modifies 3MH, converting it
to the more potent 3MHA.
www.landesbioscience.comBioengineered Bugs 151
© 2012 Landes Bioscience.
Do not distribute.
health risks. Clearly, a wine yeast that performs MLF should be of
great interest to both winemakers and consumers.
ML01 carries the Schizosaccharomyces pombe malate trans-
porter gene (mae1) and the O. oeni malolactic enzyme gene
(mleA);36both are chromosomally integrated and regulated by the
S. cerevisiae PGK1 promoter and terminator. This enables the
ML01 to perform MLF in parallel with alcoholic fermentation
(Fig.6). In fermentation trials it was shown that 5 g/l of malic
acid was decarboxylated to lactic acid within 5 days, without
negative impacts on the sensory aspects on wine. Further analyses
of the phenotype, genotype, transcriptome and proteome revealed
that ML01 is “substantially equivalent” to its parental industrial
An alternative GM approach to lowering malic acid levels in
wine has been to engineer a wine yeast that is able to conduct
malo-ethanolic fermentation. In this case, malate is decarboxy-
lated to pyruvate, which is then converted to ethanol. S. cerevisiae
requires two heterologous genes for this, a malate transporter gene
(mae1) and a malic enzyme gene (mae2), both of which come
from S. pombe. While this strategy appears to be successful,37there
has not been a commercially available version of a malo-ethanolic
wine yeast released to market.
A second commercially-available GM wine yeast, ECMo01,
received clearance from the American and Canadian regulatory
bodies in 2006. ECMo01 was engineered to reduce the risk of
ethyl carbamate production during fermentation. Ethyl car-
bamate, a potential carcinogen, is the product of urea reacting
with ethanol, but is typically produced at such low levels (if at all)
in winemaking that it is generally not a concern. Nonetheless, in
some fortified wines and in some wine-producing regions, it can
make an appearance.
ECMo01 has an extra copy of the S. cerevisiae DUR1,2 gene
(Fig.7) under the control of the yeast PGK1 regulatory
sequences.38DUR1,2 encodes urea amidolyase, which converts
urea into ammonia and carbon dioxide, thereby removing
substrate for ethyl carbamate production. The ammonia that is
produced is consumed as a preferred nitrogen source by yeast.
ECMo01 has been shown to reduce ethyl carbamate in
Chardonnay wine by almost 90%, and analyses of ECMo01’s
phenotype and transcriptome also revealed that the ECMo01
yeast is “substantially equivalent” to its parental strain.
Interestingly, this yeast is cis (or “self”) cloned; it carries no
foreign DNA and, therefore, is not transgenic. Nevertheless,
because it was generated using techniques that involve manipula-
tion of DNA in vitro, the regulations of many countries require it
to be classed as a GMO.
Because wine yeasts are classified as “processing aids” by
American and Canadian regulators, wines made with GM yeasts
are not required to be labeled as such. While no winemakers from
these two countries have admitted to using ML01 or ECMo01, it
is common knowledge that these GM yeasts have been used,
albeit on a very limited scale; for understandable reasons, in the
current anti-GMO climate, users prefer to keep this confidential.
For wine yeast researchers and many grapegrowers and
winemakers, it is frustrating that we cannot take full advantage
of the many beneficial outcomes arising from the application of
Figure5. Building on knowledge from work utilizing GM strategies, a classical, non-GM mutagenesis approach was used to develop three “low-H2S”
strains. These strains have impaired sulfite reductase activity due to mutations in their MET10 and MET5 genes.
152Bioengineered Bugs Volume 3 Issue 3
© 2012 Landes Bioscience.
Do not distribute.
GM technologies in the food and beverage sector. It is to be
hoped that, in the near future, consumers will see through the
misrepresentations and scaremongering of anti-GM lobby groups
and be more accepting of what GM science has to offer. To hasten
this, scientists who bioengineer bugs for industrial applications
must be prepared to communicate their views to the wider
Figure6. Therearetwooptionstogeneticallyengineerextraneousmalateutilizationinordertodeacidifywine.Oneapproachutilizesthe Schizosaccharomyces
pombe malate transporter gene (mae1) and the O. oeni malolactic enzyme gene (mleA), enabling yeast to perform malolactic fermentation in parallel with
alcoholic fermentation. Alternatively, Saccharomyces cerevisiae can be modified by the introduction of mae1 and the S. pombe malic enzyme gene (mae2),
thereby enabling the conversion of malate into ethanol.
Figure7. A wine yeast has been genetically engineered to reduce ethyl carbamate production during fermentation. Through increased expression
of DUR1/DUR2, this yeast breaks down urea to ammonia and CO2before it is able to react with ethanol.
© 2012 Landes Bioscience.
Do not distribute.
sequence that might be the result of horizontal gene transfer
from a different yeast, Zygosaccharomyces.40A limitation of the
whole-genome sequencing studies, however, was that haploid
representations of diploid, and often heterozygous, commercial
and environmental strains were used to expedite sequence
assembly. In more recent studies,9,10five wine strains (AWRI
796, QA23, VL3, VIN7 and VIN13) and two brewing strains
(Foster’s O and B ale strains) were sequenced in their industrially-
used forms. The genomes of these strains were compared with
one another and previously sequenced strains, S288c (the
reference laboratory strain), YJM789 (a clinical isolate), RM11–
1a (a strain derived from a vineyard isolate) and JAY291 (a biofuel
We found that these industrial yeasts displayed significant
genotypic heterogeneity both between strains but also between
alleles present within strains (i.e., heterozygosity). This variation
manifested as single nucleotide polymorphisms (SNPs), small
insertions and deletions, and as novel, strain and allele-specific
ORFs. None had been found previously in the S. cerevisiae
genome and may provide the basis for novel phenotypic
Interestingly, several strain-specific ORFs form a gene cluster,
which has been found in multiple copies and at a variety of
genomic loci in a strain-dependent manner, but which is entirely
lacking from the S288c laboratory strain. Furthermore, this
cluster of sequences appears to have integrated into genomic
locations by a novel circular intermediate, but without employing
classical transposition or homologous recombination,9,41which
community and ensure that the debate is not so one-sided. After
all, there is no intrinsic fear of the technologies; indeed the
pharmaceutical industry has been very successful in developing
GM therapeutics, which the vast majority of us have welcomed
because of their efficacy and safety.
Opening New Vistas with Frontier Yeast Omics
With the advent of omics approaches we are seeing great advances
in understanding wine yeast biology and what makes wine yeast
so different from other strains of S. cerevisiae. Comparing the
genomes of a wine yeast (AWRI1631), a laboratory strain (S288c)
and a clinical isolate (YJM789), we uncovered a 0.6% difference
in nucleotide sequence, but, perhaps more importantly, there
was 100 kb additional genome sequence—enough to carry at
least 27 genes.39Open reading frames (ORFs) in the additional
sequences do not resemble anything found in other species of
Saccharomyces but appear to be similar to genes found in distant
fungal relatives. Blast searches indicated that some of the wine
yeast-specific genes have similarities to genes encoding cell wall
proteins, perhaps contributing to the greater robustness of wine
yeast compared with laboratory strains. Others may encode
proteins associated with amino acid uptake, which is significant
in the context of wine sensory attributes; amino acid metabolism
is central to the production of many sensorially-important volatile
In a subsequent study, analysis of the genome sequence
of another related wine yeast, EC 1118, revealed an additional
represents the first time such an element has been characterized
in S. cerevisiae. Overall, this work suggests that, despite the
scrutiny that has been directed at the yeast genome, there remains
a significant reservoir of ORFs and novel modes of genetic
transmission that may have significant phenotypic impact in this
important model and industrial species.
There have also been numerous studies describing transcrip-
tomic and metabolomic analysis of wine yeast fermentations. This
work is beginning to provide insights into wine yeast fermenta-
tions, but it is still early days. Looking to the future, as the various
omics fields progress, it should be possible to build systems-based
mathematical models of metabolism that will facilitate the in silico
design of new wine yeast strains. In parallel with this, we see the
emergence of synthetic biology where, yet again, S. cerevisiae is a
key player. It should not be too long before there are synthetically-
customized S. cerevisiae genomic components (e.g., regulatory
elements to control expression of targeted genes; cassettes carrying
genes encoding metabolic pathways to shape wine relevant traits,
etc.) available “off the shelf” for designing, building and refining
metabolic processes in wine yeast. But the key question remains:
are consumers ready for this brave and exciting new world?
Looking to the Future
Truly great wines are born from great marriages between grape
variety, climate, soil and landscape, on the one hand, and
technology, innovation and craftsmanship on the other. Thus, the
art in the science of winemaking lies in the choices made
regarding which technological tools and innovations are selected
and how they are applied to craft the infinite diversity of wine
styles. Put differently, if we gave the same tools, i.e., paint,
brushes and canvasses, to different artists—Da Vinci, Monet,
Picasso—and all were asked to paint the same thing, they will
invariably come up with very different masterpieces.
There is a fear that technological innovation—including the
tailoring of wine yeast strains—could result in wine homogeneity
and uniformity. Such fear is unfounded. The reality is that
technology creates diversity by offering more options to grape-
growers and winemakers to respond to market needs and
consumer preferences. These are options that the wine industry
desperately needs as it faces so many challenges: an endemic
oversupply of wine globally; prohibitionist-like propaganda
campaigns from some anti-alcohol lobbyists; outbreaks of new
diseases and pests in vineyards; climate change and environmental
The story of yeast research raises some important questions,
therefore. It leads us to question public perception of the terms
“natural” and “unnatural”; “technological” and “traditional”.
Where is the dividing line between “natural” and “unnatural” in
the context of yeast and wine research, and indeed, science as a
The story of yeast also highlights the importance of cross-
disciplinary research. We may be wine scientists, microbiolo-
gists, molecular geneticists or researchers engaged in the new
omics technologies—genomics, transcriptomics, proteomics or
metabolomics—but we all share a common goal. We all seek
154Bioengineered BugsVolume 3 Issue 3
© 2012 Landes Bioscience.
Do not distribute.
Century. Food Technol Biotechnol 2003; 41:3-10.
Varela C, Siebert TE, Cozzolino D, Rose L, McLean H,
Henschke PA. Discovering a chemical basis for differ-
entiating wines made by fermentation with “wild”
indigenous and inoculated yeasts: role of yeast volatile
compounds. Aust J Grape Wine Res 2009; 15:238-48;
Verstrepen KJ, Chambers PJ, Pretorius IS. The deve-
lopment of superior yeast strains for the food and
beverage industries: Challenges, opportunities and
potential benefits. The Yeast Handbook: Yeasts in
Food and Beverages. 2006:399–444.
Goffeau A, Barrell BG, Bussey H, Davis RW, Dujon B,
Feldmann H, et al. Life with 6000 genes. Science 1996;
274:546-, 563-7; PMID:8849441; http://dx.doi.org/
Schena M, Shalon D, Davis RW, Brown PO. Quanti-
tative monitoring of gene expression patterns with a
complementary DNA microarray. Science 1995; 270:
467-70; PMID:7569999; http://dx.doi.org/10.1126/
Winzeler EA, Shoemaker DD, Astromoff A, Liang H,
Anderson K, Andre B, et al. Functional characterization
of the S. cerevisiae genome by gene deletion and parallel
analysis. Science 1999; 285:901-6; PMID:10436161;
Borneman AR, Desany BA, Riches D, Affourtit JP,
Forgan AH, Pretorius IS, et al. Whole-genome com-
parison reveals novel genetic elements that characterize
the genome of industrial strains of Saccharomyces
cerevisiae. PLoS Genet 2011; 7:e1001287; PMID:
greater understanding of yeast as a simple, model organism: a
microorganism that has the potential to shed new light on disease
as well as processes such as fermentation. Wine science has made a
significant contribution to understanding in this area of inquiry.
Finally, the story of yeast research raises questions about the
future of omics technologies and their perception by society. Does
yeast research bring into question, perhaps, the way that GMOs
and synthetic genomes are perceived? We imagine that anyone
reading this article holds his/her own views on this important
and highly controversial subject. And this is the story of the
journey of the winemaker’s bug—7,000 years and counting...
Research at the Australian Wine Research Institute (AWRI) is
financially supported by Australia’s grapegrowers and winemakers
through their investment body the Grape and Wine Research
and Development Corporation, with matching funds from the
Australian Government. Systems biology research at the AWRI
uses resources provided as part of the National Collaborative
Research Infrastructure Strategy (NCRIS), an initiative of the
Australian Government, in addition to funds from the South
Australian State Government. AWRI’s collaborating partners
within this NCRIS-funded initiative—which is overseen by
Bioplatforms Australia—are Genomics Australia, Proteomics
Australia, Metabolomics Australia (of which the Microbial
Metabolomics unit is housed at the AWRI) and Bioinformatics
Australia. Various results discussed in this paper stems from
research projects that were funded by yeast supplier companies,
Anchor Yeast, AB Mauri, Laffort and Lallemand. The AWRI is
part of the Wine Innovation Cluster in Adelaide. This paper
draws upon results from several AWRI research projects and
publications; therefore, we gratefully acknowledge the contribu-
tions from several past and present colleagues, particularly, Paul
Henschke, Anthony Borneman, Toni Cordente, Simon Schmidt,
Hentie Swiegers, Cristian Varela, Jenny Bellon and Robyn Kievit.
We also acknowledge the numerous yeast researchers, wine
scientists and wine commentators from around the world on
whose publications we have drawn to put this paper together.
1.Martini A. Origin and domestication of the wine yeast
Saccharomyces cerevisiae. J Wine Res 1993; 4:165-76;
McGovern PE, Zhang J, Tang J, Zhang Z, Hall GR,
Moreau RA, et al. Fermented beverages of pre- and
proto-historic China. Proc Natl Acad Sci U S A 2004;
101:17593-8; PMID:15590771; http://dx.doi.org/10.
Pretorius IS, Toit MD, van Rensburg P. Designer
yeasts for the fermentation industry of the 21st
10. Borneman AR, Desany BA, Riches D, Affourtit JP,
Forgan AH, Pretorius IS, et al. The genome sequence of
thewine yeast VIN7 reveals anallotriploid hybrid genome
kudriavzevii origins. FEMS Yeast Res 2012; 12:88-96;
11. Kutyna DR, Varela C, Henschke PA, Chambers PJ,
Stanley GA. Microbiological approaches to lowering
ethanol concentration in wine. Trends Food Sci
Technol 2010; 21:293-302;
12. Michnick S, Roustan J-L, Remize F, Barre P, Dequin S.
Modulation of glycerol and ethanol yields during
alcoholic fermentation in Saccharomyces cerevisiae strains
overexpressed or disrupted for GPD1 encoding glycerol
3-phosphate dehydrogenase. Yeast 1997; 13:783-93;
13. Remize F, Roustan JL, Sablayrolles JM, Barre P, Dequin
S. Glycerol overproduction by engineered saccharomyces
cerevisiae wine yeast strains leads to substantial changes in
By-product formation and to a stimulation of fermenta-
tion rate in stationary phase. Appl Environ Microbiol
1999; 65:143-9; PMID:9872772
14. De Barros Lopes MA. Ata-ur-Rehman, Gockowiak H,
Heinrich AJ, Langridge P, Henschke PA. Fermentation
properties of a wine yeast over-expressing the Saccharo-
myces cerevisiae glycerol 3-phosphate dehydrogenase
gene (GPD2). Aust J Grape Wine Res 2000; 6:208-
15. Eglinton JM, Heinrich AJ, Pollnitz AP, Langridge P,
Henschke PA, de Barros Lopes MA. Decreasing acetic
acid accumulation by a glycerol overproducing strain of
Saccharomyces cerevisiae by deleting the ALD6 aldehyde
dehydrogenase gene. Yeast 2002; 19:295-301; PMID:
16. Cambon B, Monteil V, Remize F, Camarasa C, Dequin
S. Effects of GPD1 overexpression in Saccharomyces
cerevisiae commercial wine yeast strains lacking ALD6
genes. Appl Environ Microbiol 2006; 72:4688-
17. Zabetakis I, Holden MA. Strawberry Flavor: Analysis
and Biosynthesis. J Sci Food Agric 1997; 74:421-34;
18. Wood C, Siebert TE, Parker M, Capone DL, Elsey GM,
Pollnitz AP, et al. From wine to pepper: rotundone, an
obscure sesquiterpene, is a potent spicy aroma com-
pound. J Agric Food Chem 2008; 56:3738-44; PMID:
19. Engel KH, Tressl R. Identification of new sulfur-
containing volatiles in yellow passionfruit (Passiflora
edulis f. flavicarpa). J Agric Food Chem 1991; 39:2249-
20. Swiegers JH, Kievit RL, Siebert T, Lattey KA, Bramley
BR, Francis IL, et al. The influence of yeast on the
aroma of Sauvignon Blanc wine. Food Microbiol 2009;
21. Robinson AL, Boss PK, Heymann H, Solomon PS,
Trengove RD. Influence of yeast strain, canopy manage-
ment, and site on the volatile composition and sensory
attributes of cabernet sauvignon wines from Western
Australia. J Agric Food Chem 2011; 59:3273-84; PMID:
22. King E, Osidacz P, Curtin C, Bastian S, Francis I.
Assessing desirable levels of sensory properties in
Sauvignon Blanc wines–consumer preferences and
contribution of key aroma compounds. Aust J Grape
Wine Res 2011; 17:169-80; http://dx.doi.org/10.1111/
23. Peña-Gallego A, Hernández-Orte P, Cacho J, Ferreira
V. S-cysteinylated and S-glutathionylated thiol precur-
sors in grapes. A review. Food Chem 2012; 131:1-13;
24. Howell KS, Klein M, Swiegers JH, Hayasaka Y, Elsey
GM, Fleet GH, et al. Genetic determinants of volatile-
thiol release by Saccharomyces cerevisiae during wine
fermentation. Appl Environ Microbiol 2005; 71:5420-
6; PMID:16151133; http://dx.doi.org/10.1128/AEM.
© 2012 Landes Bioscience.
Do not distribute.
Dubourdieu D, Tominaga T. Nitrogen catabolic
repression controls the release of volatile thiols by
Saccharomyces cerevisiae during wine fermentation.
FEMS Yeast Res 2008; 8:1076-86; PMID:18462383;
26. Roncoroni M, Santiago M, Hooks DO, Moroney S,
Harsch MJ, Lee SA, et al. The yeast IRC7 gene encodes
a β-lyase responsible for production of the varietal thiol
Microbiol 2011; 28:926-35; PMID:21569935; http://
27. Peyrot Des Gachons C, Tominaga T, Dubourdieu D.
Measuring the aromatic potential of Vitis vinifera L.
Cv. Sauvignon blanc grapes by assaying S-cysteine
conjugates, precursors of the volatile thiols responsible
for their varietal aroma. J Agric Food Chem 2000;
48:3387-91; PMID:10956121; http://dx.doi.org/10.
28. Swiegers JH, Capone DL, Pardon KH, Elsey GM,
Sefton MA, Francis IL, et al. Engineering volatile thiol
release in Saccharomyces cerevisiae for improved wine
aroma. Yeast 2007; 24:561-74; PMID:17492802;
29. Lilly M, Bauer FF, Lambrechts MG, Swiegers JH,
Cozzolino D, Pretorius IS. The effect of increased yeast
alcohol acetyltransferase and esterase activity on the
flavor profiles of wine and distillates. Yeast 2006; 23:
641-59; PMID:16845703; http://dx.doi.org/10.1002/
30. Swiegers JH, Pretorius IS. Modulation of volatile sulfur
compounds by wine yeast. Appl Microbiol Biotechnol
2007; 74:954-60; PMID:17262212; http://dx.doi.org/
C,Marullo P, Claisse O,Cullin C,
31. Holt S, Cordente AG, Williams SJ, Capone DL,
Jitjaroen W, Menz IR, et al. Engineering Saccharomyces
fermentation through overexpression of an S. cerevisiae
Gene, STR3, for improvement of wine aroma. Appl
32. Omura F, Shibano Y, Fukui N, Nakatani K. Reduction
of hydrogen sulfide production in brewing yeast by
constitutive expression of MET25 gene. J Am Soc Brew
Chem 1995; 53:58-62.
33. Tezuka H, Mori T, Okumura Y, Kitabatake K,
Tsumura Y. Cloning of a gene suppressing hydrogen
sulfide production by Saccharomyces cerevisiae and its
expression in a brewing yeast. J Am Soc Brew Chem
34. Cordente AG, Heinrich AJ, Pretorius IS, Swiegers JH.
Isolation of sulfite reductase variants of a commercial
wine yeast with significantly reduced hydrogen sulfide
production. FEMS Yeast Res 2009; 9:446-59; PMID:
35. Davis CR, Wibowo D, Eschenbruch R, Lee TH, Fleet
GH. Practical Implications of Malolactic Fermentation:
A Review. Am J Enol Vitic 1985; 36:290-301.
36. Husnik JI, Volschenk H, Bauer J, Colavizza D, Luo Z,
van Vuuren HJ. Metabolic engineering of malolactic
wine yeast. Metab Eng 2006; 8:315-23; PMID:
37. Volschenk H, Viljoen-Bloom M, Van Staden J, Husnik
J, Van Vuuren HJJ. Genetic engineering of an
industrial strain of Saccharomyces cerevisiae for L-malic
acid degradation via an efficient malo-ethanolic path-
way. S Afr J Enol Vitic 2004; 25:63-73.
38. Coulon J, Husnik JI, Inglis DL, van der Merwe GK,
Lonvaud A, Erasmus DJ, et al. Metabolic engineering
of Saccharomyces cerevisiae to minimize the production
of ethyl carbamate in wine. Am J Enol Vitic 2006;
39. Borneman AR, Forgan AH, Pretorius IS, Chambers PJ.
Comparative genome analysis of a Saccharomyces
cerevisiae wine strain. FEMS Yeast Res 2008; 8:1185-
40. Novo M, Bigey F, Beyne E, Galeote V, Gavory F,
Mallet S, et al. Eukaryote-to-eukaryote gene transfer
events revealed by the genome sequence of the wine
yeast Saccharomyces cerevisiae EC1118. Proc Natl Acad
Sci U S A 2009; 106:16333-8; PMID:19805302;
41. Galeote V, Bigey F, Beyne E, Novo M, Legras J-L,
Casaregola S, et al. Amplification of a Zygosaccharomyces
bailii DNA segment in wine yeast genomes by
extrachromosomal circular DNA formation. PLoS
One 2011; 6:e17872; PMID:21423766; http://dx.
156Bioengineered Bugs Volume 3 Issue 3