Update TRENDS in Genetics Vol.22 No.
4 April 2006
Research Focus
How did Saccharomyces evolve to become
a good brewer?
Jure Piškur1, Elżbieta Rozpe˛dowska1, Silvia Polakova1, Annamaria Merico2
and Concetta Compagno2
1
Cell and Organism Biology, Lund University, Sölvegatan 35, 22362 Lund, Sweden
2
Biomolecular Science and Biotechnology, University of Milan, Italy
Brewing and wine production are among the oldest
technologies and their products are almost indispens-
able in our lives. The central biological agents of beer
and wine fermentation are yeasts belonging to the
genus Saccharomyces, which can accumulate ethanol.
Recent advances in comparative genomics and bioinfor-
matics have made it possible to elucidate when and why
yeasts produce ethanol in high concentrations, and how
this remarkable trait originated and developed during
their evolutionary history. Two research groups have
shed light on the origin of the genes encoding alcohol
dehydrogenase and the process of ethanol accumu-
lation in Saccharomyces cerevisiae.
Introduction
The fundamental physiological characteristic of beer- and
wine-brewing yeasts is their ability to degrade carbo-
hydrates, usually six-carbon (C6) molecules such as
glucose, to two-carbon (C2) components, in particular
ethanol, without completely oxidizing them to CO2, even
in the presence of oxygen, as many other yeasts do. Yeasts
such as Saccharomyces cerevisiae and Schizosaccharo-
myces pombe, which accumulate ethanol even in the
presence of oxygen are called Crabtree-positive yeasts,
whereas those that degrade sugars to CO2, such as
Kluyveromyces lactis and Candida albicans, are desig-
nated as Crabtree-negative yeasts [1]. During ethanol
production, the energy for growth is provided by the
glycolysis (see Glossary) and fermentation pathways
rather than by the oxidative respiration pathway
(Figure 1). In S. cerevisiae, the Crabtree effect relies, to
a large extent, on a glucose-repression circuit, in which
the presence of C6 carbohydrates, such as glucose,
represses respiration [2,3] (Figure 2a). However, in nature
it is not the most efficient strategy to degrade the
substrate to the C2 compounds. Indeed, after depletion of
glucose and accumulation of ethanol, the metabolism in
Crabtree-positive yeasts changes (Figure 2a,b). The
fermentation product – ethanol – becomes a substrate
and is degraded if oxygen is present. This change in
metabolism is known as the ‘diauxic shift’ [1]. Both the
initial fermentative metabolism and its subsequent shift
require several specialized elements, including transcrip-
tion factors (e.g. Mig1) and enzymes (e.g. Adh2), which
Corresponding author: Piškur, J. (Jure.Piskur@cob.lu.se).
Available online 24 February 2006
www.sciencedirect.com
have been studied during the past decades [3,4]. Com-
parative genomics approaches have shed light on these
metabolic processes and the origin and evolution of the
corresponding genes and enzymes. Two research groups
have recently identified the genetic changes that occurred
in ancient yeast progenitors to provide a foundation for
the development of basic brewing properties [5,6].
The ‘make-accumulate-consume’ strategy
During fermentation (aerobic or anaerobic), yeast recycles
NADH in the acetaldehyde-to-ethanol conversion. If
oxygen is subsequently available, the accumulated etha-
nol is converted back to acetaldehyde (Figure 1). The
acetaldehyde-to-ethanol conversion is catalyzed by alcohol
dehydrogenase (Adh), which can in principle catalyze the
reaction in both directions (i.e. acetaldehyde-to-ethanol
and ethanol-to-acetaldehyde), although with different
catalytic efficiencies. In S. cerevisiae, the cytoplasmic
Adh activity is encoded by two genes that arose by a gene-
duplication event, ADH1 is expressed constitutively,
whereas ADH2 is expressed only when the internal
sugar concentration drops. The Adh1 enzyme has an
elevated Km for ethanol, consistent with ethanol being
Glossary
Crabtree effect: alcoholic fermentation is a predominant pathway in the
degradation of hexose sugars in the presence of oxygen, because of insufficient
capacity or saturation or repression of the respiratory metabolism, leading to
pyruvate overflow.
Fermentation: conversion of pyruvate to ethanol, by oxidative decarboxyla-
tion, leading to generation of CO2 and re-oxidation of glycolytic NADH, where
one of the intermediates, acetaldehyde, serves as the ultimate electron
acceptor.
Glucose repression: a process whereby in the presence of glucose the gene
expression of a variety of enzymes (involved in using other carbon sources,
cytochromes, Krebs cycle and respiration) is shut down.
Glycolysis: a series of reactions leading from glucose (six-carbon compound)
to pyruvate (three-carbon compound), generating energy (ATP by substrate
level phosphorylation only) and NADH.
Krebs cycle (TCA cycle): a series of reactions starting with the condensation of
acetyl CoA (originating from pyruvate) and oxaloacetate into citric acid [tri
carboxylic acid (TCA)] and resulting in the main products: CO2, ATP, NADH and
precursors for anabolic reactions.
Oxidative phosphorylation: re-oxidation of NADH in the respiratory chain
generates a proton motive force, which provides energy for the phosphoryl-
ation of ADP to ATP.
Respiration: oxidation of pyruvate to CO2 by the TCA cycle, leading to the
production of NADH, which is re-oxidized while generating ATP by oxidative
phosphorylation, using oxygen as the terminal acceptor of electrons.
Whole genome duplication: the genome was duplicated w100 Mya in the
Saccharomyces cerevisiae lineage and was followed by gene loss and
rearrangements; but several duplicated gene pairs have been preserved.
184 Update TRENDS in Genetics Vol.22 No.4 April 2006

Glucose Glucose-6-P Pentose

phosphate
pathway

Fructose-1,6-bisP

NADH

Glycerol Dihydroxy- Glyceraldehyde-

acetone 3-phosphate
phosphate Ethanol

NADH
Ethanol

NADH Adh NADH

Pyruvate Acetaldehyde

CO2
NADH
Mitochondrion
Pyruvate Acetate

CO2

Acetyl-CoA Acetyl-CoA

TCA
cycle
CO2

TRENDS in Genetics

Figure 1. A scheme of the pathways involved in glucose and ethanol assimilation under aerobic conditions underlining the differences between a Crabtree-positive yeast,
Saccharomyces cerevisiae (red arrows), which can accumulate ethanol, and a Crabtree-negative yeast Kluyveromyces lactis (green arrows), which degrades hexoses directly
to CO2. The conversion between acetaldehyde and ethanol, catalyzed by alcohol dehydrogenase, Adh, is marked in gray. Abbreviation: TCA cycle, tri carboxylic acid cycle.

a product of the reaction, whereas the Km of Adh2 is ten
times lower, consistent with ethanol being its substrate.
Recently, Thomson et al. [5] reconstructed an ancient
Saccharomyces alcohol dehydrogenase progenitor gene
(encoded by ADHA), using a combination of comparative
sequence analysis and genetics. They generated ‘novel’
forms of ADH genes, which represented putative ancient
intermediates of the enzyme, and characterized their
kinetic and substrate specificity parameters. Eleven out
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of the 12 reconstructed enzymes had demonstrable
activity levels, and all 11 showed a preference to convert
acetaldehyde to ethanol, thus resembling the modern
Adh1 [5]. The authors interpreted these results to mean
that, pre-duplication, Saccharomyces alcohol dehydrogen-
ase was predominantly involved in the generation, and not
consumption, of ethanol. Thomson et al. used a molecular-
clock analysis to determine that the ADHA duplication
and specialization of the two novel enzymes took place
 Update TRENDS in Genetics Vol.22 No.4 April 2006 185

!80 million years ago (Mya) [5], after the separation of

(a) Saccharomyces cerevisiae
the K. lactis and S. cerevisiae lineages. This duplication of
30 100 ADHA also seems to have occurred after the whole-genome
duplication of S. cerevisiae, estimated at 100 Mya [7,8]

Biomass OD/ml
(Figure 2c). Further analysis of duplicated genes revealed
several gene pairs that were also involved in ethanol
Glu, EtOH (g/l)

20 10
metabolism, which had originated by duplication at the
same time [5]. These duplicated genes, along with the
ability of S. cerevisiae and its closest relatives to ferment
10 1
glucose and accumulate ethanol even in the presence of
oxygen [9], formed the basis for the ‘make-accumulate-
consume’ strategy of ethanol production, which provided
0 0.1 the ancestor of Saccharomyces yeast with an advantage
0 10 Ethanol 20 30 40
use over its competitors because ethanol is toxic to most other
Time (h) microbes. Saccharomyces kills its competitors by produ-
cing ethanol but can also consume the generated
(b) Kluyveromyces lactis ethanol later.
30 100

Rewiring the transcriptional network of yeast

Ihmels et al. [6] developed a different approach to under-
Glu, EtOH (g/l)

20 10
stand the origin of glucose repression and ethanol
Biomass OD/ml

accumulation in Saccharomyces yeasts [6]. During fer-

mentation, S. cerevisiae does not need fully active
10 1 mitochondria because sufficient energy is provided by
glycolysis and fermentation. Ihmels et al. found that in
S. cerevisiae the expression of genes encoding the
0 0.1 mitochondrial ribosomal proteins (MRPs), those encoding
0 10 20 30 40 cytoplasmic ribosomal proteins (CRPs) and those encoding
Time (h) the rRNA processing proteins were not coordinated;
instead MRP expression was correlated with the
Key: expression of stress genes induced during the slower
respiratory growth in non-fermentable carbon sources,
Ethanol Biomass (OD/ml) Glucose
such as glycerol and ethanol [6]. However, in C. albicans, an
aerobic and Crabtree-negative yeast [10], the expression of
genes encoding MRP, CRP and rRNA are all correlated [6].
(c) Saccharomyces cerevisiae The upstream regions of the genes encoding MRP, CRP and
rRNA were analyzed in several fully sequenced yeast
ADH duplication (80 Mya) species, considered to be intermediates in evolution
between C. albicans and S. cerevisiae. A common regulat-
Whole-genome duplication
(100 Mya) Kluyveromyces lactis ory motif, the rapid growth element (RGE), was found
upstream of the rRNA processing genes but not upstream
ADH duplication of the stress genes. However, differences were observed in
the MRP genes. In S. cerevisiae and its closest relatives,
Candida albicans which all accumulate ethanol, the MRP-encoding genes do
not contain the regulatory RGE motif (AATTTT). Pre-
sumably, a global rewiring of the transcriptional network
occurred after the whole-genome duplication in the S.
cerevisiae lineage, the ensuing loss of many of the
Schizosaccharomyces duplicated genes and the change in lifestyle (the ability to
pombe (200 Mya)
out-compete other microbes by using ethanol) [11]. The
promoter regions of genes that should not be expressed
when glucose is abundant (i.e. the MRP genes), all lost their
RGE regulatory elements [6].

TRENDS in Genetics
Figure 2. Glucose (Glu)-grown aerobic batch cultures of (a) a Crabtree-positive
yeast, Saccharomyces cerevisiae; and (b) a Crabtree-negative yeast, Kluyvero-
myces lactis. The consumption of glucose, and the appearance of biomass and
ethanol are shown as a function of time. The Crabtree-positive yeasts initially
accumulate ethanol, which is later, after depletion of glucose, metabolized
(for more details, see Ref. [1]). This shift in metabolism is indicated by a green
arrow. (c) A simple phylogenetic relationship between the four yeasts is shown. The
timing of the whole-genome duplication that occurred w100 Mya [8] is indicated by
a blue arrow. The S. cerevisiae and Schizosaccharomyces pombe lineages
separated O200 Mya, whereas S. cerevisiae and K. lactis lineages separated
O100 Mya. The two independent ADH gene duplications are shown (red arrows)
and the timing of the S. cerevisiae duplication w80 Mya is indicated.

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186 Update TRENDS in Genetics Vol.22 No.4 April 2006
Is ethanol consumption unique to S. cerevisiae?
The end of the Cretaceous age provided an excess of fruits
and therefore the increased amounts of fermentable
substrates became available for many microbial commu-
nities [12]. The ability of fast ethanol accumulation and
ethanol tolerance was first exploited by Saccharomyces
yeasts to inhibit the growth of competing organisms, and
then the accumulated ethanol could be ‘digested’ (the
‘make-accumulate-consume’ strategy). However, is this
strategy of ethanol consumption and the following proper-
ties unique to Saccharomyces yeasts?
Many different yeasts are capable of facultative
fermentation. S. pombe separated from the Saccharo-
myces–Kluyveromyces lineage at least 200 Mya (Figure 2c)
and is capable of aerobic alcoholic fermentation in the
presence of excess sugars, a property shared by
S. cerevisiae [9]. S. pombe can also consume ethanol but
not as its sole carbon source [13], owing to the absence of a
complete glyoxylate cycle rather than its inability to
convert ethanol to acetaldehyde. K. lactis is a poor
producer of ethanol but can use this compound efficiently
as its only carbon source. It has duplicated the ADH
gene(s) independently more than once in its evolutionary
history, resulting in four modern alcohol dehydrogenases.
The KlADH4 gene is induced by ethanol and not repressed
by glucose and the corresponding enzyme is involved in
consumption of ethanol originating from either the
external medium or previous intracellular production
[14,15].
The yeast lineages originated O200 Mya and probably
possessed the ability to ferment and thereby produce
ethanol in anaerobic conditions from then onwards [11].
Apparently, the ability to consume ethanol is also shared
by less related fungi: Aspergillus nidulans, for example,
has ethanol inducible alcohol dehydrogenase activities
[16]. Some yeast lineages, in particular the lineage that
leads to S. cerevisiae, might have developed the ability to
suppress the respiratory metabolism and thereby accu-
mulated ethanol. Alternatively, perhaps the ability to
suppress respiration was the original yeast property,
which was later lost in some lineages, such as K. lactis
and C. albicans. The elevated appearance of fermentable
sugars w50–100 Mya [12] has provided a selective
pressure for the development of more-specialized re-
usage of ethanol, and it seems that this happened
independently, in different ways and simultaneously in
several yeast lineages. But why are Saccharomyces yeasts
competitive in the production of ethanol, especially as
harnessed by humans?
Concluding remarks
Saccharomyces yeasts owe their competitiveness to a
combination of several properties including fast growth,
efficient glucose repression, good ability to produce and
www.sciencedirect.com
consume ethanol, and a tolerance for several environmen-
tal stresses, such as high ethanol concentration and low
oxygen levels [11]. These properties are unevenly dis-
tributed among different modern yeasts but are uniquely
combined, specialized to perfection, regulated and coordi-
nated through an efficient network [4,6] in S. cerevisiae
and its closest relatives. Perhaps ‘efficient regulation’ is
the most unique invention of the Saccharomyces yeasts,
providing a crucial competitive ‘advantage’ when pro-
duction strains are selected for industrial fermentations in
breweries and wineries [17].
Acknowledgements
We thank Morten Kielland-Brandt (The Carlsberg Laboratory) for
comments on an early version of this article.
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