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Evaluation of industrial Saccharomyces cerevisiae strains


for ethanol production from biomass

Ceyda Kasavi a,*, Ilaria Finore b, Licia Lama b, Barbara Nicolaus b, Stephen G. Oliver c,
Ebru Toksoy Oner d, Betul Kirdar a
a
Department of Chemical Engineering, Bog azici University, Bebek 34342, Istanbul, Turkey
b
Istituto di Chimica Biomolecolare (ICB), CNR, Napoli, Italy
c
Cambridge Systems Biology Centre, Department of Biochemistry, University of Cambridge, Sanger Building,
80 Tennis Court Road, Cambridge CB2 1GA, UK
d
Department of Bioengineering, Marmara University, Goztepe 34722, Istanbul, Turkey

article info abstract

Article history: Five industrial Saccharomyces cerevisiae strains were evaluated for their suitability for strain
Received 6 June 2011 improvement for future use in ethanol production processes. Principal components analysis
Received in revised form of growth-related and production-related fermentation parameters of the 5 strains grown
17 May 2012 on glucose demonstrated the superiority of the Y9 strain in terms of its rapid growth and
Accepted 7 June 2012 highest ethanol yields on both biomass and glucose. The growth and ethanol production
Available online 3 July 2012 performances of these strains on various agro-industrial wastes (including sugar beet pulp,
starch and sugar beet molasses) and biological residues (including carrot, tomato and
Keywords: potato peel) were also determined. Ethanol tolerance studies, using both solid and liquid
Ethanol cultures, revealed the remarkable abilities of the BC187 and Y9 strains to survive and grow
Biomass at high ethanol concentrations. Suspension cultures were found to be highly tolerant to
Industrial Saccharomyces cerevisiae 78.80 g L1 ethanol however their growth ability showed a distinct decrease with increasing
strains ethanol concentration such that only (1e2)% of the control growth was observed in media
Ethanol tolerance containing 118.20 g L1 ethanol. The importance of choosing the appropriate S. cerevisiae
Agro-industrial waste strain to be used in ethanol production was clearly established with this study. Fermen-
tation performances of the cultures under different cultivation conditions pointed to the
fact that the choice of strain will not only depend on the ethanol tolerance but also on the
preferential utilization of the carbon resources of biological residues.
2012 Elsevier Ltd. All rights reserved.

1. Introduction particular sugar, starch and oil crops. Biodiesel can be


produced from waste edible oils and fats by transesterification
Biomass appears to be the most feasible feedstock for current processes or cracking [3]; whereas biological residues,
routes to the production of biofuels since it is renewable, manure, and grasses can be suitable for biogas production [4].
economical, has a low sulfur content, involves no net release On the other hand, biological organic materials with consid-
of carbon dioxide, and so has a high potential to become erable amounts of starch or cellulose that can be converted
economically feasible in near future [1,2]. The main biomass into sugar can be used to produce bioethanol. Sugarcane,
sources are currently represented by dedicated crops, in sugar beetroot, and sugar sorghum are examples of raw

* Corresponding author. Tel.: 90 212 359 6876; fax: 90 212 287 2460.
E-mail address: ceyda.kasavi@boun.edu.tr (C. Kasavi).
0961-9534/$ e see front matter 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.biombioe.2012.06.013
b i o m a s s a n d b i o e n e r g y 4 5 ( 2 0 1 2 ) 2 3 0 e2 3 8 231

materials that contain sugar; whereas wheat, barley and corn Saccharomyces Genome Resequencing Project [15] strain
are starch crops. A significant part of the wood of trees and collection and used in this study in order to evaluate their
herbs is composed of cellulose, which can be converted into potential use in ethanol production processes. For this,
sugar by using available technologies. However, since important fermentation parameters of the strains were
conversion of cellulose into glucose is more complicated than investigated both on glucose-containing media and on sugar
the conversion of starch, ethanol production is currently beet pulp, starch and sugar beet molasses as well as biological
based on wheat and corn in the USA, sugarcane in Brazil, and residues like carrot, tomato and potato peel wastes. Moreover,
wheat, barley, and sugar beet in Europe [2,5e7]. the ethanol tolerances of the strains were determined by
The conventional ethanol production processes involve following their colony-forming ability on agar plates as well as
a pre-treatment step where the sugar in the raw material is their growth rates in liquid cultures in the presence of
separated and fed as substrate to the fermentor where it is different concentrations of ethanol.
converted to ethanol. These processes are based on traditional
brewing techniques and utilize Saccharomyces cerevisiae strains
because they give a high ethanol yield, a high productivity, 2. Materials and methods
and their ability to withstand high ethanol concentrations
keeps distillation costs low. In the conventional ethanol 2.1. Strains and media
production process when the raw materials employed are
grains, then only the germs of the corn and barley (i.e. the Five industrial strains of S. cerevisiae, namely BC187, L-1374, L-
starch-containing parts) are used. Since these parts represent 1528, K11 and Y9 were selected from the Saccharomyces species
only a small portion of the total mass of the plant, a significant collection of SGRP and used in this study. BC187 was isolated
amount of fiber residue is generated [5,6]. Although these from a wine barrel in California [16], L-1374 and L-1528 were
ethanol technologies are mature and play a significant role in isolated from Chile [15], Kyokai no. 11, an ethanol-tolerant
generating liquid fuels for the transportation sector, they rely sake yeast [17], was obtained from Japan [18], and Y9, a wine
on food-grade biomass resources. Recently, there have been yeast [19], was obtained from Indonesian ragi [18]. S. cerevisiae
growing concerns about the diversion of food and fodder- strains were kindly provided by Ed Louis (University of
grade feedstocks from the food chain to biofuel production Nottingham).
and therefore, much research has focused on technologies Precultures were inoculated with a single colony of cells
which use non-food crops, the non-food parts of edible crops, taken from yeast extractepeptoneeglucose (YPD) agar plates
or the residues from wood-based or food-based industries and incubated in YPD medium (20 g L1 D-glucose, 20 g L1
such as wood chips, and the skins and pulp from fruit pressing peptone, 10 g L1 yeast extract) at 30  C and 3 Hz.
[2,8e10].
These ethanol technologies, which provide sustainable 2.2. Cultivation conditions
energy without compromising food security or the environ-
ment, do not involve additional energy expenditures for S. cerevisiae strains were grown in YPD medium. A preculture
substrate production and collection. Depending on the at a volume fraction of 1% was used to inoculate the culture. In
biomass source and product employed, they include cellulose order to test the capability of bioconversion of different
hydrolysis followed by fermentation, pyrolysis, gasification, typologies of biomass substrates into ethanol, strains were
and anaerobic digestion, as well as their various combinations also grown in YP medium (20 g L1 peptone, 10 g L1 yeast
depending duct [6]. Such ethanol production processes are not extract) including biological residues as a carbon source.
yet in extensive commercial use; however, a number of pilot Sugar beet pulp, carrot, tomato and potato peel, starch and
and demonstration plants have been set up recently with sugar beet molasses at 1 g L1 total carbohydrate content were
considerable active research programs being carried out in added as carbon sources. The medium containing 20 g L1
North America, Europe, Brazil, China, India and Thailand [1]. glucose as carbon source was used to grow the cells as control.
In general, ethanol processes start with the hydrolysis of Cultures were kept with vigorous shaking at 30  C and 3 Hz.
the biomass followed by the yeast-based fermentation of the Optical densities were monitored by spectroscopic measure-
resulting sugars. Co-fermentation of C5 and C6 sugars has ments at 600 nm wavelength until the steady-state was
been quite challenging and, as with traditional ethanol reached. All experiments were carried out in duplicate.
production processes, recombinant S. cerevisiae strains are the Samples (1 mL) taken from the culture at regular intervals
preferred microorganisms for the fermentation step. In con- were centrifuged at 6800  g for 6 min (Eppendorf 5415C;
structing recombinant S. cerevisiae strains for ethanol Germany) to determine substrate utilization, extracellular
production processes utilizing biomass resources, the choice product formation, and metabolite concentrations.
of the host strain is crucial.
Accumulation of ethanol is one of the main environmental 2.3. Preparation of biomass substrates
changes that yeast cells are exposed to in industrial produc-
tion processes using yeast [11]. The increase in ethanol level From the different biomass resources used in this study,
acts as an inhibitor of microorganism growth and viability carrot, tomato and potatoes were obtained from a local
[12,13] and therefore, yeast cells that have high growth ability market. Sugar beet molasses (with average mass composition
under high ethanol concentrations, are desired substantially of 48e51% sucrose, 15e18% moisture, 11e13% ash, 8% betaine,
in ethanol production processes [14]. Considering these facts, 3.61% potassium, 0.9% chloride, 0.53% calcium, 0.45% sodium,
five industrial S. cerevisiae strains were selected from the 0.27% sulfate, 0.09% nitrate, 3% carbohydrate and 7.5% other
232 b i o m a s s a n d b i o e n e r g y 4 5 ( 2 0 1 2 ) 2 3 0 e2 3 8

organic compounds) and sugar beet pulp (with average mass the colony-forming ability of the cells, every 24 h a sample from
composition of 10.4% crude protein, 0.9% crude fat/ether the liquid culture was spotted onto a YPD agar plate by use of
extract, 18e30% dry matter and 4e9% total ash) were obtained a Singer Instruments Rotor HDA and cultured at 30  C for 48 h.
from Kutahya Sugar Factory (Kutahya, Turkey) as by-products After 72 h, optical densities were measured at 600 nm [24].
of the manufacture of sucrose from sugar beet .Starch
molasses containing a mass fraction of 50.6% total sugars and 2.9. Identification of nucleotide variations present in
20% glucose was a byproduct of dextrose manufacture from ethanol tolerances genes of industrial yeast strains
corn and it was obtained from Akmaya yeast factory (Avcilar,
Turkey). The difference in the observed ethanol tolerance of industrial
Whereas molasses and sugar beet pulp were used directly, S. cerevisiae strains was investigated using the known genome
peel from the carrots, tomatoes and potatoes were first sequences of these strains. 15 ethanol tolerance genes that are
washed with tap water and then with distilled water in order thought to be related with ethanol stress, including URA7,
to remove surface dust particles. After washing, a blanching LAP3, YOR139C, CYB5, SFL1, HSP26, RTC3, OLE1, EDE1, TPS1,
operation was carried out by immersing them into hot water ELO1, MSN2, DOG1, INO1, HAL1 [25e30] were selected and
at 75e80  C for 20 min, followed by a drying step in an oven at compared to reveal differences which might be associated
45  C [20]. The dried material was ground, then sterilized at with increased tolerance in industrial strains. The whole-
121  C for 20 min, and stored at 4  C before further use. genome sequences of the industrial S. cerevisiae strains were
obtained from the Saccharomyces Genome Resequencing
2.4. Determination of maximum specific growth rate Project at the Sanger Institute [15].
and dry cell weight The DNA sequence of each gene obtained from SGD
(Saccharomyces Genome Database) was used to define the
For maximum specific growth rate determination, optical precise chromosomal localization of ethanol tolerance genes
densities (OD600) of samples collected during the exponential in each strain by BLAST. Sequence variations in each gene
phase of growth were used. were determined by the pairwise comparison of each strain
Dry cell weights (DCW) of steady state cultures were using Matlab 7.4 (MathWorks Inc.). Protein sequences were
determined gravimetrically by first recovering cells from 1 ml identified from nucleotide sequences by ORF Finder tool [31],
culture samples through centrifugation at 6800  g for 6 min and compared by the multiple sequence alignment tool
(Eppendorf 5415C; Germany). Cells were washed with distilled ClustalW2 [32].
water three times and then dried at 65  C until constant
weight was achieved. Reported values are averages of five
biological replicates for each data point.
3. Results and discussion
2.5. Extracellular metabolite analysis
3.1. Fermentation parameters of S. cerevisiae strains
Extracellular glucose and ethanol concentrations were deter- on glucose
mined by using enzymatic analysis kits (Boehringer Mannheim).
The fermentation performances of the five industrial S. cer-
2.6. Sugar assays evisiae strains, grown on YPD medium, were investigated and
compared. Maximum biomass and ethanol concentrations
Reducing sugar concentration was assayed by DNS method reached by the cultures, as well as their glucose utilization
[21]. Starch was analyzed as reported earlier [22]. The sugars
composition of media was analyzed by thin-layer chromatog-
raphy (TLC) using butanol/acetic acid/water (60:20:20 by
Table 1 e Comparison of fermentation parameters of S.
volume) as solvent. Maltose, galactose, glucose, mannose,
cerevisiae strains.
trehalose, fructose, raffinose, sucrose (10 g L1) were used as
standards. Parameter Strains

BC187 L-1374 L-1528 K11 Y9


2.7. Principal components analysis (PCA) 1 a
mmax (h ) 0.52 0.53 0.56 0.54 0.58
Final DCWa 1.44 1.50 1.21 1.59 1.18
PCA was carried out with the PLS Toolbox of MATLAB 7.4 by Max. ethanol conc. (g L1)a 9.16 9.46 9.47 10.70 11.23
using the measured growth related and production related Glucose utilization 0.89 0.82 1.22 1.04 1.16
parameters as variables and the strains as the objects, as rate (g L1 h1)a
described by Wold et al. [23]. Vol. productivity (g L1 h1) 0.66 0.82 0.82 0.93 0.98
Sp. productivity 0.46 0.55 0.68 0.59 0.83
(g DCW1 h1)
2.8. Determination of ethanol tolerance
Yps (g ethanol g1 glucose) 0.46 0.48 0.49 0.50 0.52
Ypx (g ethanol g1 biomass) 6.37 6.31 7.83 6.73 9.52
5 ml of YPD medium containing (0, 23.64, 39.40, 47.28, 63.04,
78.80, 94.56 and 188.20) g L1 ethanol, was inoculated with a The results were analyzed by analysis of variance and highly
significant differences in these fermentation parameters were
a final optical density at 600 nm of 0.05 (OD600 0.05) and was
observed between the strains ( p < 0.0005).
incubated at 30  C under 3 Hz shaking for 72 h. In order to test
b i o m a s s a n d b i o e n e r g y 4 5 ( 2 0 1 2 ) 2 3 0 e2 3 8 233

rates and maximum specific growth rates (mmax) were


Table 2 e Comparison of fermentation parameters of S.
measured (see Table 1). In addition the volumetric and specific cerevisiae strains in different carbon sources.
ethanol productivities, ethanol yield on glucose (Yps) and
Waste source S. cerevisiae strain
biomass (Ypx) were also determined for each strain (Table 1).
PCA of growth-related and production-related parameters BC187 Y9 K11 L-1374 L-1528
resulted in two different plots. Growth patterns revealed two mmax (h )1 a

distinct groups of strains with similar growth profiles, namely, Starch molasses 0.33 0.39 0.38 0.33 0.35
BC187 and L-1374 strains and L-1528 and Y9 strains with K11 Sugar beet molasses 0.26 0.39 0.37 0.33 0.41
forming a separate group. Whereas glucose consumption Tomato peel waste 0.22 0.34 0.32 0.29 0.20
rates within the groups did not vary significantly, the 30% Carrot peel waste 0.25 0.27 0.35 0.22 0.34
Potato peel waste 0.06 0.08 0.07 0.03 0.08
lower utilization rate of the former group was also reflected in
Sugar beet pulp 0.31 0.38 0.32 0.42 0.47
their lower specific growth rates (Table 1).
Based on the production related parameters, L-1374 and L- OD600 at steady state
1528 strains and Y9 and K11 strains were mutually compa- Starch molasses 1.18 1.27 1.23 1.14 1.08
Sugar beet molasses 1.20 1.30 1.30 1.19 1.11
rable. Higher ethanol concentration values were reached by
Tomato peel waste 0.84 0.95 0.94 0.97 0.82
Y9 and K11 strains, with the volumetric and specific ethanol Carrot peel waste 1.06 1.14 1.20 1.13 0.90
productivities being highest in Y9 and lowest in the BC187 Potato peel waste 0.62 0.85 0.86 0.71 0.65
cultures. Hence generally, the Y9 strain was found to stand Sugar beet pulp 1.15 1.25 1.30 1.20 1.03
out with its rapid growth and the highest ethanol yields in
% reducing sugar utilizeda
terms of both cell mass of the producer and glucose Starch molasses 60.69 59.87 67.82 63.87 60.12
consumed. Sugar beet molasses 57.98 70.83 67.88 63.90 67.26
Tomato peel waste 54.66 48.24 54.75 42.31 45.36
3.2. Bioconversion of biological residues into ethanol Carrot peel waste 63.00 55.56 60.00 72.95 65.97
Potato peel waste 39.37 26.30 37.84 32.95 26.12
Sugar beet pulp 54.69 44.24 50.33 49.65 46.51
The ability of the five strains to produce ethanol from a variety
of agricultural residues, including carrot, tomato and potato Max ethanol conc. (g L1)a
peels and industrial wastes like sugar beet pulp, starch, and Starch molasses 0.22 0.29 0.37 0.35 0.35
Sugar beet molasses 0.32 0.49 0.43 0.40 0.45
sugar beet molasses, was evaluated. Growth, sugar composi-
Tomato peel waste 0.25 0.27 0.24 0.16 0.18
tion and ethanol production profiles of the batch cultures
Carrot peel waste 0.33 0.33 0.34 0.37 0.36
were followed and mmax, maximum ethanol concentrations Potato peel waste 0.12 0.17 0.16 0.12 0.13
reached, and the percentages of reducing sugars utilized are Sugar beet pulp 0.34 0.32 0.31 0.19 0.35
summarized in Table 2. Sugar analysis of culture supernatants
a These results were analyzed by analysis of variance and highly
at intervals during the fermentations showed that most of the
significant differences in fermentation parameters were observed
fermentable sugars were exhausted within the first 8 h of between the strains ( p < 0.0001).
incubation (data not shown). All the strains grown on sugar
beet and starch molasses were found to utilize (60e70)% of the
reducing sugar present in the fermentation media. The
maximum yeast biomass concentrations reached by the similar to those of sugar beet pulp with 0.71 g L1 initial sugar
strains as well as their mmax values on both types of molasses content (Table 2). Higher ethanol concentrations were reached
were found to be very similar. However, higher ethanol with sugar beet pulp rather than tomato peels and the L-1528
concentrations were reached with sugar beet molasses. The strain, with its high final ethanol concentrations and mmax,
Y9 strain was found to be superior to the others not only in its was found to outperform Y9 on this resource (Fig. 1). The
utilization of the reducing sugars present in sugar beet lowest initial reducing sugar contents were observed for
molasses, but also in the conversion of these sugars into media based on potato peels (0.43 g L1 on average) and less
ethanol (Table 2). After molasses, carrot peel was also found to than 30% of these sugars were utilized by the strains, and then
be efficiently utilized by all strains, reaching comparable at very low mmax values. These results suggested the presence
ethanol concentrations. of growth inhibitory substances that need to be removed prior
In fact, from all the biomass resources considered in this to fermentation. Though fermentable sugars were converted
study, the highest amount of reducing sugars was released to ethanol at high yields, ethanol concentrations and
from carrot peels into the fermentation medium (1.07 g L1); substrate uptake rates could be improved by an appropriate
TLC analysis revealed that glucose was the principal sugar pre-treatment step.
released (results not shown). For molasses, initial reducing This work was performed on vegetable substrates of
sugar contents were approximately 0.56 g L1 and 0.63 g L1 for unknown provenance, for which the chain of custody is not
starch molasses and sugar beet molasses, respectively. When known. The species and the cultivars cannot be specified.
these two types of molasses were compared in terms of their However, a single batch of each vegetable substrate has been
ethanol yield on total sugar (Yps), both molasses exhibited Yps used in all fermentation experiments in order to prevent
values almost twice as high as that on carrot peel, for all errors associated with batch-to-batch, geographical, or
strains (Fig. 1). Strains growing in fermentation media con- seasonal variations in the chemical composition of the
taining tomato peels with 0.78 g L1 average initial reducing substrates. Though the current study mainly demonstrates
sugar content utilized the fermentable sugars at amounts the differences in the fermentation performances of different
234 b i o m a s s a n d b i o e n e r g y 4 5 ( 2 0 1 2 ) 2 3 0 e2 3 8

Fig. 1 e Yield comparison of S. cerevisiae strains grown on different carbon sources.

yeast strains, there is a reasonable concern that there may be 118.2 g L1 ethanol. In contrast at 118.2 g L1 ethanol, the Y9
substrate factors that influence the results obtained. and BC187 strains showed significant colony-forming ability,
while K11 and L-1374 formed few colonies.
Among yeast species, S. cerevisiae is known to tolerate high
3.3. Ethanol tolerance of S. cerevisiae strains
ethanol concentrations [18] and, indeed, the industrial yeast
strains used in this study were all highly tolerant up to
The ethanol tolerances of the five S. cerevisiae strains were
78.8 g L1 ethanol for 72 h. The observed ethanol tolerance of
evaluated by growing the five industrial strains in the pres-
K11 is in agreement with earlier studies where K11, the
ence of varying amounts of ethanol and then comparing their
ethanol-tolerant mutant of yeast strain K7, was reported to
cell yields with those of the control cultures without ethanol
produce ethanol at high titers at the final stage of fermenta-
(Table 3). All strains showed 77% or higher yield of cell
tion [17].
biomass in media containing between 0 and 47.28 g L1
ethanol. Growth ability of strains showed a distinct decrease
with increasing ethanol concentration and strains showed 3.4. Identification of nucleotide variations present in
only (1e2)% growth in media containing 118.2 g L1 ethanol. ethanol tolerances genes of industrial yeast strains
The colony-forming ability of all strains in the presence of
varying amounts of ethanol ((23.6e118.2) g L1) was tested by There are several reports in literature on finding gene targets
culture on solid media as described in Materials and methods. for the construction of ethanol tolerant S. cerevisiae strains. By
Fig. 2 shows the colony-forming ability of S. cerevisiae Y9 strain screening the collection homozygous diploid deletants (rep-
grown in YPD media containing (0e118.2) g L1 ethanol. For all resenting 4741 non-essential genes), two null mutants (ura7D
strains, no significant changes in colony-forming patterns and gal6D) that grew faster than the wild type in medium
could be detected up to 78.8 g L1 ethanol for 72 h. The L-1528 containing 63.04 g L1 ethanol were identified [25]. By similar
strain displayed slow growth at 94.6 g L1 and no growth at means, strains carrying the cyb5D, sfl1D, and yor139cD

Table 3 e Ethanol tolerance of S. cerevisiae strains.


% volume EtOH (g L1) Growth % [OD600(x% EtOH)/OD600(0% EtOH)] * 100
fraction
of EtOH BC187 L-1374 L-1528 K11 Y9

0 0 100 100 100 100 100


3 23.64 99.55 86.53 85.44 98.79 87.33
5 39.40 98.63 84.16 80.35 95.04 78.31
6 47.28 98.22 81.48 78.15 85.38 77.26
8 63.04 95.40 77.99 71.10 76.87 73.29
10 78.80 67.70 57.53 59.56 53.44 65.36
12 94.56 4.66 1.94 1.72 2.07 2.07
15 118.20 2.03 1.26 1.11 1.29 1.82
b i o m a s s a n d b i o e n e r g y 4 5 ( 2 0 1 2 ) 2 3 0 e2 3 8 235

examined and improved tolerance in cells overexpressing


OLE1 was observed which, in turn, indicated the importance of
the total unsaturated fatty acid content (rather than the
degree of unsaturation) for ethanol tolerance [28]. Using
strain-specific differences in the transcriptional response to
ethanol by identifying gene expression differences between
strains with and without the ability to acquire increased
ethanol tolerance after ethanol pre-treatment, new genes
(EDE1, ELO1, MSN2, TPS1) were identified that increased
ethanol tolerance when overexpressed [29]. By an inverse
metabolic engineering approach, S. cerevisiae cells trans-
formed with a genomic library for higher ethanol tolerance
were screened and INO1, DOG1, HAL1 and MSN2 were identi-
fied as target genes whose overexpression resulted in
increased volumetric ethanol productivities and specific
growth rates of the cultures [30].
Within the scope of this study, genes that have been
reported to be associated with ethanol tolerance, including
Fig. 2 e Colony-forming ability of undiluted (C), 10 fold URA7, LAP3 (GAL6), YOR139C, CYB5, SFL1, HSP26, RTC3, OLE1,
(1/10) and 100 fold (1/100) diluted S. cerevisiae Y9 cultures EDE1, TPS1, ELO1, MSN2, DOG1, INO1, HAL1 were selected for
grown in YPD containing (0, 23.64, 39.40, 47.28, 63.04, genomic comparison of industrial yeast strains. Nucleotide
78.80, 94.56 and 188.20) g LL1 ethanol after 24 h, 48 h and variations in these genes were systematically analyzed
72 h. by pairwise comparisons to the most (Y9) and least (L-1528)
ethanol-tolerant strains characterized in this study.
The Saccharomyces Genome Resequencing Project at the
deletions were identified as conferring ethanol tolerance [26]. Sanger Institute reported the near-complete genome
The ethanol tolerance and fermentative performance of wine sequences of S. cerevisiae from different sources and locations
yeasts were improved through modifying the expression and identified single nucleotide polymorphisms and nucleo-
levels of the HSP26 and YHR087W (RTC3) genes [27]. In order to tide insertions and deletions in the S. cerevisiae nuclear
understand the effect of fatty acid composition on ethanol genome. The report pointed out the presence of five lineages
tolerance in S. cerevisiae, the ethanol tolerance of yeast cells exhibiting the same phylogenetic relationship across their
that contained various levels of unsaturated fatty acids were entire genome in the S. cerevisiae population [15]. Based on this

Fig. 3 e Pairwise nucleotide sequence comparison of ethanol-tolerance genes of two sets of strains. Whereas the former set
included two ethanol tolerant strains (Y9 and BC187), the latter one embraced a tolerant strain (Y9) and the least tolerant
strain (L-1528).
236 b i o m a s s a n d b i o e n e r g y 4 5 ( 2 0 1 2 ) 2 3 0 e2 3 8

Fig. 4 e Multiple ClustalW2 alignment of Dog1p amino-acid sequences in five industrial S. cerevisiae strains.

classification, Y9 is in the Sake lineage, whereas BC187 and L- unique domains/motifs, transmembrane domains, and signal
1528 are in the Wine/Europe lineage. peptides predicted for Dog1p, but it has 5 domains that it
In this study, significant nucleotide variations (quantified shares with known protein families: the Gene3D homologous
as per cent nucleotide differences) in ethanol tolerance genes superfamily: 3.40.50.1000, the phosphatase panther protein
between strains were identified (Fig. 3). Nucleotide variations class, the HAD-like superfamily, the haloacid dehalogenase-
in HSP26, TPS1, INO1, CYB5 and LAP3 genes appear to be same like hydrolases, and HAD-superfamily hydrolases [37].
between Y9 and BC187 strains and Y9 and L-1528 strains. In order to understand the possible effects of nucleotide
These differences could be explained by the different lineages sequence variations on phenotype, amino-acid sequences of
to which these strains belong. On the other hand, in the the DOG1 protein products were compared between all 5
remaining genes some nucleotide variations were found strains and 11 amino-acid differences were identified (Fig. 4).
between Y9 and L-1528 strains that are different from the Two of these differences were seen between the sake yeasts
variations between the Y9 and BC187 strains. These variations (Y9 and K11) and the European wine yeasts (BC187, L-1374 and
in genes previously associated with ethanol tolerance could be L-1528). Therefore, these differences could be explained by the
responsible for the observed ethanol tolerances of industrial different lineages to which these strains belong. On the other
yeast strains. Remarkably, 41 nucleotide variations were hand, the amino-acid sequence of the L-1528 Dog1p was
identified in the DOG1 gene between the Y9 and L-1528 strains, different from that of all other strains at 6 positions Thr51,
13 of these variations were also found between Y9 and BC187 Val54, Leu55, Arg57, Asp64, Ile77, and the amino-acid sequences
strains; the other 28 variations were not seen between Y9 and of L-1528 and L-1374 were different from all other strains at
BC187 strains. Therefore, these 28 variations in DOG1 gene positions His79 and Ser80. Since the ethanol tolerances of the
could be the reason for the decreased ethanol tolerance of L- L-1528 and L-1374 strains were less than those of Y9, BC187
1528 strain. and K11, these 8 amino-acid differences, which were located
Dog1p (2-deoxyglucose-6-phosphate phosphatase) cata- in the known domains of Dog1p, might be involved in the
lyzes the hydrolysis of 2-deoxy-D-glucose-6-phosphate to 2- decreased ethanol tolerances of L-1528 and L-1374.
deoxy-D-glucose and phosphate. It physically interacts with
two protein kinases (Kin2p and Rck1p), a protein related with
cell division (Cdc12p), a protein required for mRNA splicing 4. Conclusions
(Snp1p), and an essential gene involved in the assembly of
cytosolic and nuclear iron-sulfur proteins (Cia1p) [33,34], and This study clearly establishes the importance of choosing
has genetic interactions with 18 genes [35,36]. There are no the appropriate S. cerevisiae strain to be used in ethanol
b i o m a s s a n d b i o e n e r g y 4 5 ( 2 0 1 2 ) 2 3 0 e2 3 8 237

production from biological residues. The choice will not only [13] Fischer CR, Klein-Marcuschamer D, Stephanopoulos G.
depend on a strains ethanol tolerance but also on its ability to Selection and optimization of microbial hosts for biofuels
utilize carbon sources available in agri-food residues. In future production. Metab Eng 2008;10(6):295e304.
[14] Hirasawa T, Yoshikawa K, Nakakura Y, Nagahisa K,
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