AEM Accepted Manuscript Posted Online 29 January 2016 Appl. Environ. Microbiol. doi:10.1128/AEM.03718-15 Copyright © 2016, American Society for Microbiology. All Rights Reserved.
1
Improved acetic acid resistance in Saccharomyces cerevisiae by overexpression
2
of the WHI2 gene identified through inverse metabolic engineering
3 4 5
Yingying Chena, Lisa Stabrylab, Na Weia,b # a
Department of Civil and Environmental Engineering and Earth Sciences, University of Notre
6
Dame, USA, b Department of Civil and Environmental Engineering, University of Pittsburgh,
7
USA
8 9
Running Title: Improved acetic acid resistance by overexpression of WHI2
10 11
#Address correspondence to Na Wei: [email protected]
12
Address: 156 Fitzpatrick Hall, University of Notre Dame, Indiana, 46556, United States.
13
1
14 15
Abstract Developing acetic acid resistant Saccharomyces cerevisiae is important for economically
16
viable production of biofuels from lignocellulosic biomass, but the goal remains a critical
17
challenge due to limited information on effective genetic perturbation targets for improving
18
acetic acid resistance in the yeast. This study employed a genomic library based inverse
19
metabolic engineering approach to successfully identify a novel gene target WHI2 (encoding a
20
cytoplasmatic globular scaffold protein) which elicited improved acetic acid resistance in S.
21
cerevisiae. Overexpression of WHI2 significantly improved glucose and/or xylose fermentation
22
under acetic acid stress in engineered yeast. The WHI2 overexpressing strain had five times
23
higher specific ethanol productivity than the control in glucose fermentation with acetic acid.
24
Analysis of expression of WHI2 gene products (including protein and transcript) determined that
25
acetic acid induced endogenous expression of Whi2 in S. cerevisiae. Meanwhile, the whi2Δ
26
mutant strain had substantially higher susceptibility to acetic acid than the wild type, suggesting
27
the important role of Whi2 in acetic acid response in S. cerevisiae. Additionally, overexpressing
28
WHI2 and a cognate phosphatase gene PSR1 had a synergistic effect in improving acetic acid
29
resistance, suggesting that Whi2 could function in combination with Psr1 to elicit acetic acid
30
resistance mechanism. These results improve our understanding of yeast response to acetic acid
31
stress and provide a new strategy to breed acetic acid resistant yeast strains for renewable biofuel
32
production.
2
33 34
Introduction Lignocellulosic biomass from nonfood stocks such as agricultural and forestry residues
35
has been identified as the prime source to produce renewable biofuels for substituting
36
conventional fossil fuels in the face of growing demand for energy and rising concerns of
37
greenhouse gas emissions (1-4). Bioconversion of plant cell wall materials by microbial
38
fermentation is typically preceded by harsh (physico)chemical hydrolysis designed to release
39
sugars; this hydrolysis treatment also generates byproducts that are toxic to fermenting
40
microorganisms (5, 6). Since hemicellulose and lignin in the plant cell wall is ubiquitously
41
acetylated (7, 8), typical acidic pretreatment of lignocellulosic biomass generates substantial
42
amounts of acetic acid (with the concentration ranging from 1 g/L to 15 g/L) in the resulting
43
hydrolyzates (9, 10). Acetic acid severely inhibits cell growth and fermentation activity in
44
Saccharomyces cerevisiae (5, 6, 11-13), the predominant microorganism used in industrial
45
fermentation (14, 15). Therefore, improving S. cerevisiae resistance to acetic acid is highly
46
desired and critical for achieving efficient and economically viable bioconversion of cellulosic
47
sugars to biofuels.
48
The toxicity effects of acetic acid in S. cerevisiae have been intensively characterized and
49
toxicity mechanisms have been proposed (10, 11, 16-20). When external pH is lower than the
50
pKa of acetic acid (4.7), the undissociated form of acetic acid prevails and can enter the cells
51
simply by diffusion through plasma membrane. Dissociation of acetic acid at neutral cytosolic
52
pH can lead to intracellular acidification (6). As a result, the intracellular pH needs to be
53
recovered by pumping out protons at the expense of ATP hydrolysis, which may induce cell
54
growth arrest and reduced fermentation performance (21). In addition, intracellular anion
3
55
accumulation can reach high level and decrease the activity of some key enzymes for glycolysis
56
(16).
57
However, the genetic basis of yeast stress response to acetic acid remains unclear,
58
making it difficult to improve acetic acid resistance in S. cerevisiae. It is known that the yeast
59
response to acetic acid stress involves genome-wide transcriptional changes (22-25). For
60
example, upregulation of various genes involved in glycolysis, the Krebs cycle and ATP
61
synthesis was identified in yeast cells cultivated in the presence of acetic acid, indicating
62
substantial alteration of carbohydrate and energy metabolism in acetic acid-stressed cells (26).
63
Genome-scale transcriptional analyses suggested that the transcriptional activators Haa1 and
64
Msn2 could be involved in regulating yeast adaptation to acetic acid (21, 26, 27). A large scale
65
chemical genomics study identified 648 genes whose deletion increased susceptibility of yeast to
66
acetic acid, and these gene determinants were found to be involved in carbohydrate metabolism,
67
cell wall assembly, amino acid metabolism, internal pH homeostasis, biogenesis of mitochondria,
68
and signaling and uptake of various nutrients (26). Recent studies reported cell-to-cell
69
heterogeneity in acetic acid tolerance (28, 29). It was found that only fraction of the cells within
70
an isogenic S. cerevisiae population resumed growth under acetic acid stress (28) and that
71
variations in the cytosolic pH of individual cells could contribute to the differences between cells
72
(29). The findings suggested that genes related to cell-to-cell heterogeneity could be potential
73
pool for the search of genetic targets for improving acetic acid. These prior results illustrate that
74
acetic acid stress response mechanism in S. cerevisiae is rather complex and involves
75
coordinated regulations of multiple genes. Due to the currently incomplete understanding of
76
relevant genetic and biomolecular networks for acetic acid stress response, it is challenging to
77
develop acetic acid resistant yeast strains through knowledge-based rational metabolic
4
78
engineering (30). Particularly, information regarding genetic targets and perturbation strategy for
79
effectively engineering acetic acid resistant yeast strains is needed.
80
This study employed a genomic library based inverse metabolic engineering approach to
81
develop S. cerevisiae strains for improved fermentation of cellulosic sugars under acetic acid
82
stress and identify genetic perturbation targets for enhancing acetic acid resistance in the yeast.
83
Specifically, we first introduced a genome-wide library into a parent S. cerevisiae strain
84
containing an optimized xylose fermentation pathway, so that the transformants could be
85
evaluated in fermentation of glucose and/or xylose (the two most abundant sugars from
86
lignocellulosic biomass) under acetic acid stress. We then characterized the screened
87
transformants to identify the target of gene perturbation. Here we report on a novel gene target
88
WHI2. Overexpression of WHI2 substantially enhanced acetic acid resistance of S. cerevisiae.
89
We show that acetic acid induced endogenous expression of Whi2 and that deletion of WHI2
90
gene resulted in hypersensitivity to acetic acid. We further determined the function of Whi2 and
91
its binding partner Psr1 in eliciting acetic acid resistance mechanisms in S. cerevisiae. Lastly, we
92
characterized improved performance of an engineered strain for glucose and/or xylose
93
fermentation in the presence of toxic levels of acetic acid under industrially relevant conditions.
94
The results improve our understanding of stress response to acetic acid in S. cerevisiae and
95
provide a new strategy to breed acetic acid resistant yeast strains for renewable biofuel
96
production from lignocellulosic biomass.
97
Materials and Methods
98 99 100
Strains and plasmids. All the strains and plasmids used in this study were summarized in Table 1. The recombinant xylose-fermenting S. cerevisiae strain SR8 (31) was used in this study for glucose and xylose fermentation. The strain was constructed previously (31) through i) 5
101
heterologous expression of XYL1 (coding for xylose reductase, XR), XYL2 (coding for xylitol
102
dehydrogenase, XDH), and XYL3 (coding for xylulokinase, XK) from Scheffersomyces stipitis in
103
S. cerevisiae D452-2 (MATα leu2 ura3 his3, and can1), and optimization of expression levels of
104
XR, XDH, and XK, laboratory evolution on xylose, and deletion of ALD6 coding for
105
acetaldehyde dehydrogenase. The auxotrophic marker genes in the strains SR8-trp and SR8-4
106
(Table 1) were recovered by the CRISPR-cas9 method (32, 33). These strains were kindly
107
provided by Dr. Yong-Su Jin’s lab. The Escherichia coli TOP10 strain was used for gene cloning
108
and manipulation. The whi2 knockout mutant derived from the laboratory strain BY4741 (GE
109
Healthcare Dharmacon) was used for evaluating the susceptibility of the null mutant versus the
110
wild type. The msn2 knockout mutant derived from the strain SR8-trp was used for testing the
111
hypothesis regarding the interaction of Whi2 and Msn2 in eliciting acetic acid resistance.
112
Enzymes, primers and chemicals. Restriction enzymes, DNA-modifying enzymes and
113
other molecular reagents were obtained from New England Biolabs (Beverly, MA). The reaction
114
conditions were set up following the manufacturer’s instructions. All general chemicals and
115
media components were purchased from Sigma-Aldrich (St. Louis, MO) or Fisher Scientific
116
(Pittsburgh, PA). Primers for both PCR and sequencing were synthesized by Integrated DNA
117
Technologies (Coralville, IA) and listed in Table S1.
118
Media and culture conditions. Yeast strains were routinely cultivated at 30 °C in YP
119
medium (10 g/L of yeast extract and 20 g/L of peptone) or synthetic complete (SC) medium (6.7
120
g/L of yeast nitrogen base and appropriate amino acids or nucleotides) containing 20 g/L of D-
121
glucose (SCD). SC media containing 20 g/L agar and glucose without tryptophan and/or leucine
122
amendment was used to select transformants using TRP1 and/or LEU2 as auxotrophic markers. E.
6
123
coli strains were grown in Luria-Bertani medium at 37 °C and 100 μg/mL of ampicillin was
124
added to the medium when required.
125
Construction of S. cerevisiae genomic library and yeast transformation. The genomic
126
DNA of the S. cerevisiae S288C strain was used to construct the genomic library as previously
127
described (34). Briefly, genomic DNA fragments (2-5 kb) were generated by sonication and
128
ligated into a multicopy plasmid pRS424 (a yeast episomal plasmid) with TRP1 as an
129
auxotrophic selection marker (35, 36). Plasmid extraction was performed using the QIAprep
130
Spin Miniprep Kit (Germantown, MD). The genomic library was transformed to the S. cerevisiae
131
strain SR8-trp using LiAc/PEG methods (37).
132
Selection of transformants with acetic acid resistance. Yeast genomic library
133
transformants were inoculated on SC medium agar plates (15 mm diameter), which contained
134
glucose (20 g/L) or xylose (20 g/L) and different concentrations of acetic acid (2.0 g/L, 2.5 g/L,
135
3.0 g/L and 3.5 g/L). The pH of the agar plates was adjusted to be 4.0 so that acetic acid was
136
predominantly undissociated. The strain SR8-trp harboring pRS424GPD plasmid (i.e. the strain
137
S-C1 in Table 1) was used as the control at all conditions. Transformants were isolated from the
138
plates with acetic acid concentrations at which the corresponding plates inoculated with the
139
control strain had no colony grown. Then, the cell growth and sugar consumption rates of the
140
isolated transformants were evaluated in liquid SC medium containing toxic level of acetic acid
141
and 20 g/L glucose or 20 g/L xylose. The initial cell biomass was adjusted to an optical density
142
at 600 nm (OD600) of 0.05. Plasmids from the selected transformants were isolated by Zyppy
143
Plasmid Miniprep Kit (Zymo Research) and amplified by E. coli transformation. The isolated
144
plasmids were re-transformed to the strain SR8-trp to verify the effects of the plasmids on acetic
145
acid resistance.
7
146
Insert identification, plasmid and strain construction. The verified plasmids showing
147
effects in improving acetic acid resistance were sequenced using T3 and T7 primers (Table S1)
148
to determine the nucleotide sequences of the inserted genomic DNA fragments. Sequences were
149
compared to the S. cerevisiae genome sequence to identify the insert sizes and open reading
150
frames. To construct overexpression plasmids, the complete open reading frames of the target
151
genes (WHI2, PSR1, HAA1 or MSN2) were amplified by PCR with the primers listed in Table
152
S1. The PCR products were subsequently digested and ligated to appropriate multiple cloning
153
sites of the plasmid pRS424GPD or pSR425GPD. To construct the msn2 mutant strain from
154
SR8-trp, the KanMX marker gene flanked by about 200 bp homologous to upstream and
155
downstream of MSN2 gene was PCR amplified from the genomic DNA of a msn2 knockout
156
strain derived from the laboratory strain BY4741 (GE Healthcare Dharmacon) with primers
157
msn2-F and msn2-R listed in Table S1. Transformation of PCR product into SR8-trp was
158
performed using Yeast EZ-transformation Kit (BIO 101). Positive transformants were selected
159
on YP medium containing 20 g/L glucose and 300 µg/mL Geneticin G418. Diagnostic PCR was
160
performed to confirm successful deletion, yielding the strain S-msn2Δ. The overexpression
161
plasmids were transformed respectively to the strain SR8-trp, SR8-4 or S-msn2Δ using Yeast
162
EZ-transformation Kit (BIO 101).
163
Protein expression experiments and data analysis. A yeast GFP (Green Fluorescent
164
Protein) clone collection of S. cerevisiae BY4742 strains (Life Technology, no. 95702),
165
developed by oligonucleotide directed homologous recombination to tag each open reading
166
frame (ORF) with Aequorea victoria GFP (S65T) in its chromosomal location at the 3’ end (38),
167
were used in protein expression analysis in response to acetic acid stress. Pre-cultured yeast cells
168
with GFP fusion protein Whi2 were grown in SC+glucose (SCD) media to early exponential
8
169
phase, and then were inoculated to 200 µL SCD medium (OD600 = 0.2) in clear bottom black 96-
170
well plates (Costar, Bethesda, MD) containing different concentrations of acetic acid (0 g/L, 2
171
g/L and 3 g/L, pH=4.0). The plates were then incubated in a Microplate Reader (SynergyTM HT
172
Multi-Mode, Biotech, Winooski, VT) at 30°C with fast shaking, and OD600 and GFP signal
173
(fluorescence readings, excitation at 485nm and emission at 528 nm) were simultaneously
174
measured every one hour for six hours. All experiments were performed in triplicate.
175
The protein expression levels based on GFP signals were calculated using the method
176
previously described (39, 40). The raw data from the OD600 and GFP measurements were
177
corrected by considering the background signals in control with medium only, and the protein
178
expression per cell unit for each measurement was calculated by normalizing GFP data to cell
179
number (OD600) as P=GFP/OD600. The expression of PGK1 gene product was used as the
180
reference, and the P values for each protein of interest were normalized based on mean P level of
181
PGK1 corresponding to the same condition on the same plate. The relative expression (RE) for
182
the protein of interest at each time point under each acetic acid exposure condition versus the
183
control condition without acetic acid was calculated as RE = Paa/Pc, where Paa referred to
184
normalized P in the experiment condition with acetic acid and Pc represented corrected and
185
normalized P in the control condition without acetic acid.
186
Reverse transcription quantitative PCR (RT-qPCR). The wild type strain in early
187
exponential phase was grown in SC medium containing glucose (20 g/L) and acetic acid (2 g/L)
188
or in medium without acetic acid (control condition). After 6 hour incubation, cells from
189
triplicate experiments were harvested by centrifugation for 30 s at 1000 rpm under 4ºC and RNA
190
was immediately extracted by using PureLink® RNA Mini Kit (Life Technology, NY) according
191
to the manufacturer’s instructions. RNA was reverse transcribed into complementary DNA
9
192
(cDNA) using the iScriptTM cDNA synthesis kit (Bio-Rad Laboratories, CA). The cDNA was
193
then used for qPCR analysis using the primers in Table S1 on a CFX Connect Real-time System
194
(Bio-Rad Laboratories, CA) and by using iQ™ SYBR® Green Supermix (Bio-Rad Laboratories,
195
CA). To confirm amplification specificity, PCR products were subjected to melting curve
196
analysis and gel electrophoresis. All the measurements were performed in triplicate for each
197
biological triplicate (n=3x3). Gene expression was calculated by quantification method (41) as (1
198
+ E)Cq / (1 + Eref)Cq,ref, E stands for application efficiency and Cq stands for quantification cycle
199
value.
200
Fermentation experiments. Batch fermentation experiments under oxygen-limited
201
conditions were performed in 125mL Erlenmeyer flasks containing 20 mL medium, and
202
anaerobic batch fermentation experiments were performed in 160 mL serum bottles containing
203
20 mL medium sealed with butyl rubber stoppers, both at 30°C and 100 rpm.The anaerobic
204
fermentation media were prepared by flushing with nitrogen which had passed through a heated,
205
reduced copper column to remove trace oxygen. Pre-cultured yeast cells in SCD medium were
206
centrifuged and washed with sterilized water, and then inoculated into fermentation media
207
containing glucose/xylose and acetic acid. The initial cell densities were adjusted to OD600=1.
208
The initial pH of the media was adjusted to 4.0. For anaerobic fermentation experiments,
209
ergosterol and Tween 80 were added to final concentrations of 0.01 g/L and 0.42 g/L,
210
respectively (42). Culture samples were taken from fermentation experiments to measure the
211
OD600 and concentrations of metabolites. For anaerobic fermentation experiments, samples were
212
taken by sterile syringe and 26G BD needles. Yeast cell dry weight was determined using a
213
microwave method as described previously (43). All of the fermentation experiments were set up
214
in duplicate.
10
215
As for fermentation with cellulosic hydrolysates, the corn stover hydrolysate was
216
prepared by NREL (http://www.nrel.gov/biomass/pdfs/47764.pdf) through dilute acid
217
pretreatment. The hydrolysate liquid fraction contained 10.9 g/L acetic acid, 115 g/L xylose and
218
17 g/L glucose. The hydrolysate was mixed with YP medium at 50% v/v ratio to result in a
219
hydrolysate mixture containing around 3.5 g/L acetic acid, 40 g/L xylose, and an adjusted
220
glucose concentration of 20 g/L. The pH was adjusted to 4.0. The fermentation experiments were
221
set up in flasks under oxygen limited conditions as described above.
222
Analytical methods. Cell growth was monitored by measuring OD600 using a UV-visible
223
spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA). Glucose, xylose, xylitol,
224
glycerol, acetic acid, and ethanol were quantified by high performance liquid chromatography
225
(Agilent Technologies 1200 series) equipped with a refractive index detector and a Rezex ROA-
226
Organic Acid H+ (8%) column (Phenomenex Inc., CA). The column was eluted with 0.005 N
227
H2SO4 as the mobile phase under the flow rate of 0.6 mL/min at 50 °C.
228
Results
229
Screening of transformants exhibiting improved acetic acid resistance. With the goal
230
to obtain transformants that grew faster under acetic acid stress in both glucose and xylose
231
consuming conditions, a method was developed that combined selection on agar plates and
232
screening in liquid medium containing toxic levels of acetic acid. The plates inoculated with the
233
genomic-library transformants had colonies grown on glucose or xylose containing agar plates
234
with up to 3 g/L acetic acid (pH 4.0) within 3-5 days. In contrast, plates inoculated with the
235
control strain S-C1 (Table 1) did not have any colony grown at acetic acid concentrations above
236
2 g/L through two week incubation. Then 130 fast-growing transformants were isolated from
11
237
plates containing 2.5 g/L or 3 g/L acetic acid (65 from glucose-containing plates and 65 from
238
xylose-containing plates).
239
We further screened the isolated transformants in liquid SC medium to identify strains
240
with superior resistance to acetic acid under both glucose-consuming and xylose-consuming
241
conditions. Among the 65 transformants isolated from xylose-containing plates, ten
242
transformants (#7, #32, #34, #35, #36, #38, #41, #49, #52 and #59) grew much faster than the
243
control strain S-C1 in liquid medium containing glucose+acetic acid (see Fig. S1A in the
244
supplemental material), and five out of these ten (#34, #35, #38, #52 and #59) also exhibited
245
significant improvement in liquid medium containing xylose+acetic acid (see Fig. S1B in the
246
supplemental material). As for the 65 transformants isolated from glucose-containing plates,
247
while some transformants showed improved cell growth in medium containing glucose+acetic
248
acid, no significant improvement was observed when these transformants were grown in medium
249
containing xylose+acetic acid (data not shown). A possible reason might be that the yeast was
250
more sensitive to acetic acid when grown on xylose than on glucose; similar observation was
251
also reported in a previous study (44).
252
Plasmids from each of the five selected transformants were isolated and re-transformed
253
into the parental strain respectively to confirm the effects. Four plasmids (#34, #38, #52 and #59)
254
showed positive effects in the re-transformed strains comparable to that in the original
255
transformants in terms of cell growth and sugar consumption under acetic acid stress. The #35
256
plasmid did not result in improved acetic acid resistance in the re-transformed strain, indicating
257
that the increased resistance in the original transformant might be due to genome mutations.
258
Sequence analysis of the four confirmed plasmids revealed that three (#34, #38 and #59) had
259
similar genome coordinates of the identified targets (chrXV: 409,259-412,369 for #34 and #38,
12
260
chrXV: 410,675-412,722 for #59), while the plasmid #52 did not harbor any intact open reading
261
frame (chrIX: 4597-5728).
262
All the three inserts harbored a complete sequence of the gene WHI2 (chrXV: 410,870-
263
412,330). The WHI2 gene product in S. cerevisiae is a 55 kDa cytoplasmatic globular scaffold
264
protein (38). Whi2 is involved in coordinating cell growth and proliferation and plays an
265
important role in nutrient-dependent cell cycle arrest (45-48). The protein has also been reported
266
to be required for activation of general stress response in the yeast (49-51).
267
Enhanced acetic acid resistance by overexpression of WHI2 gene. The effect of
268
overexpressing WHI2 on acetic acid resistance in S. cerevisiae has not been reported before, and
269
the role of Whi2 in acetic acid stress response is unclear. To determine the effect of WHI2 as a
270
gene perturbation target for improving acetic acid resistance, we introduced a multicopy plasmid
271
overexpressing WHI2 under the control of the constitutive GPD (TDH3) promoter and CYC1
272
terminator (Table 1) into S. cerevisiae strain SR8-trp or BY4742 (i.e. WHI2 overexpression in
273
different strain backgrounds), yielding strains S-WHI2 and BY2-WHI2 (Table 1), and
274
corresponding control strains S-C1 and BY2-C1 harbored the plasmid without WHI2 insert. The
275
strains S-WHI2 had noticeably higher cell growth under acetic acid stress than the control strain
276
S-C1, according to the results of yeast spotting assays on SC agar plates containing glucose (20
277
g/L) and different concentrations of acetic acid (Fig. 1A). When grown on plates with xylose as
278
the substrate, the strain S-WHI2 also had substantially higher acetic acid resistance than the
279
control strain (Fig. 1B). Overexpression of WHI2 in another strain background (BY-WHI2) also
280
showed improvement (see Fig. S2 in the supplemental material).
281 282
Batch experiments were conducted to characterize the glucose/xylose fermentation performances of the strain S-WHI2 versus the control strain S-C1under acetic acid stress. Fig. 2
13
283
shows ethanol production and sugar consumption profiles of the two strains. In glucose
284
fermentation under oxygen limited conditions, glucose (20 g/L) was completely consumed
285
within 39 hours by S-WHI2 with the presence of 2.5 g/L acetic acid, while the control strain S-
286
C1 consumed only 3.2 g/L glucose (Fig. 2A). The specific sugar consumption rate and specific
287
ethanol productivity of the strain S-WHI2 were both over five times higher than those of the
288
control (see Table S2 in the supplemental material). The pH of the fermentation medium was
289
4.0 initially and decreased to 3.3 at the end of S-WHI2 fermentation; a toxic level of protonated
290
form of acetic acid prevailed during the fermentation process. It was noted that the fermentation
291
profiles of S-WHI2 and S-C1 had no significant difference under the condition without acetic
292
acid (with pH below 4.0 during fermentation) (Fig. 2B), suggesting that the improvement
293
brought by WHI2 overexpression is associated with cellular response to acetic acid stress. It was
294
noted that the ethanol yields in glucose fermentation with acetic acid were higher than the yields
295
without acetic acid for both S-WHI2 and S-C1 (see Table S2 in supplemental material),
296
probably due to stimulating effect on glucose fermentation by low concentrations of weak acids
297
as reported in previous studies (52, 53). Similar level of improvement in the fermentation by the
298
strain S-WHI2 versus S-C1 was also observed under strict anoxic conditions (see Table S2 in
299
the supplemental material).
300
Additionally, xylose fermentation under acetic acid stress also had significant
301
improvement by overexpression of WHI2. The strain S-WHI2 consumed xylose with a specific
302
rate of 0.245±0.004 g/g dry cell wt/hr under oxygen limited condition, while the control strain
303
did not consume xylose in experimental time frame (Fig. 2C). Xylose fermentation by the two
304
strains in the absence of acetic acid had similar profiles (Fig. 2D), suggesting that WHI2
305
overexpression did not have significant impact on xylose fermentation pathway itself. Xylose
14
306
fermentation under anaerobic conditions proceeded slowly due to intrinsic limitation of the fungi
307
xylose assimilating pathway. No cell growth was observed with 2.5 g/L acetic acid, and thus
308
lower level of acetic acid (1.5 g/L) was used in the experiment. There were significant
309
improvements (t-test, P
1
Improved acetic acid resistance in Saccharomyces cerevisiae by overexpression
2
of the WHI2 gene identified through inverse metabolic engineering
3 4 5
Yingying Chena, Lisa Stabrylab, Na Weia,b # a
Department of Civil and Environmental Engineering and Earth Sciences, University of Notre
6
Dame, USA, b Department of Civil and Environmental Engineering, University of Pittsburgh,
7
USA
8 9
Running Title: Improved acetic acid resistance by overexpression of WHI2
10 11
#Address correspondence to Na Wei: [email protected]
12
Address: 156 Fitzpatrick Hall, University of Notre Dame, Indiana, 46556, United States.
13
1
14 15
Abstract Developing acetic acid resistant Saccharomyces cerevisiae is important for economically
16
viable production of biofuels from lignocellulosic biomass, but the goal remains a critical
17
challenge due to limited information on effective genetic perturbation targets for improving
18
acetic acid resistance in the yeast. This study employed a genomic library based inverse
19
metabolic engineering approach to successfully identify a novel gene target WHI2 (encoding a
20
cytoplasmatic globular scaffold protein) which elicited improved acetic acid resistance in S.
21
cerevisiae. Overexpression of WHI2 significantly improved glucose and/or xylose fermentation
22
under acetic acid stress in engineered yeast. The WHI2 overexpressing strain had five times
23
higher specific ethanol productivity than the control in glucose fermentation with acetic acid.
24
Analysis of expression of WHI2 gene products (including protein and transcript) determined that
25
acetic acid induced endogenous expression of Whi2 in S. cerevisiae. Meanwhile, the whi2Δ
26
mutant strain had substantially higher susceptibility to acetic acid than the wild type, suggesting
27
the important role of Whi2 in acetic acid response in S. cerevisiae. Additionally, overexpressing
28
WHI2 and a cognate phosphatase gene PSR1 had a synergistic effect in improving acetic acid
29
resistance, suggesting that Whi2 could function in combination with Psr1 to elicit acetic acid
30
resistance mechanism. These results improve our understanding of yeast response to acetic acid
31
stress and provide a new strategy to breed acetic acid resistant yeast strains for renewable biofuel
32
production.
2
33 34
Introduction Lignocellulosic biomass from nonfood stocks such as agricultural and forestry residues
35
has been identified as the prime source to produce renewable biofuels for substituting
36
conventional fossil fuels in the face of growing demand for energy and rising concerns of
37
greenhouse gas emissions (1-4). Bioconversion of plant cell wall materials by microbial
38
fermentation is typically preceded by harsh (physico)chemical hydrolysis designed to release
39
sugars; this hydrolysis treatment also generates byproducts that are toxic to fermenting
40
microorganisms (5, 6). Since hemicellulose and lignin in the plant cell wall is ubiquitously
41
acetylated (7, 8), typical acidic pretreatment of lignocellulosic biomass generates substantial
42
amounts of acetic acid (with the concentration ranging from 1 g/L to 15 g/L) in the resulting
43
hydrolyzates (9, 10). Acetic acid severely inhibits cell growth and fermentation activity in
44
Saccharomyces cerevisiae (5, 6, 11-13), the predominant microorganism used in industrial
45
fermentation (14, 15). Therefore, improving S. cerevisiae resistance to acetic acid is highly
46
desired and critical for achieving efficient and economically viable bioconversion of cellulosic
47
sugars to biofuels.
48
The toxicity effects of acetic acid in S. cerevisiae have been intensively characterized and
49
toxicity mechanisms have been proposed (10, 11, 16-20). When external pH is lower than the
50
pKa of acetic acid (4.7), the undissociated form of acetic acid prevails and can enter the cells
51
simply by diffusion through plasma membrane. Dissociation of acetic acid at neutral cytosolic
52
pH can lead to intracellular acidification (6). As a result, the intracellular pH needs to be
53
recovered by pumping out protons at the expense of ATP hydrolysis, which may induce cell
54
growth arrest and reduced fermentation performance (21). In addition, intracellular anion
3
55
accumulation can reach high level and decrease the activity of some key enzymes for glycolysis
56
(16).
57
However, the genetic basis of yeast stress response to acetic acid remains unclear,
58
making it difficult to improve acetic acid resistance in S. cerevisiae. It is known that the yeast
59
response to acetic acid stress involves genome-wide transcriptional changes (22-25). For
60
example, upregulation of various genes involved in glycolysis, the Krebs cycle and ATP
61
synthesis was identified in yeast cells cultivated in the presence of acetic acid, indicating
62
substantial alteration of carbohydrate and energy metabolism in acetic acid-stressed cells (26).
63
Genome-scale transcriptional analyses suggested that the transcriptional activators Haa1 and
64
Msn2 could be involved in regulating yeast adaptation to acetic acid (21, 26, 27). A large scale
65
chemical genomics study identified 648 genes whose deletion increased susceptibility of yeast to
66
acetic acid, and these gene determinants were found to be involved in carbohydrate metabolism,
67
cell wall assembly, amino acid metabolism, internal pH homeostasis, biogenesis of mitochondria,
68
and signaling and uptake of various nutrients (26). Recent studies reported cell-to-cell
69
heterogeneity in acetic acid tolerance (28, 29). It was found that only fraction of the cells within
70
an isogenic S. cerevisiae population resumed growth under acetic acid stress (28) and that
71
variations in the cytosolic pH of individual cells could contribute to the differences between cells
72
(29). The findings suggested that genes related to cell-to-cell heterogeneity could be potential
73
pool for the search of genetic targets for improving acetic acid. These prior results illustrate that
74
acetic acid stress response mechanism in S. cerevisiae is rather complex and involves
75
coordinated regulations of multiple genes. Due to the currently incomplete understanding of
76
relevant genetic and biomolecular networks for acetic acid stress response, it is challenging to
77
develop acetic acid resistant yeast strains through knowledge-based rational metabolic
4
78
engineering (30). Particularly, information regarding genetic targets and perturbation strategy for
79
effectively engineering acetic acid resistant yeast strains is needed.
80
This study employed a genomic library based inverse metabolic engineering approach to
81
develop S. cerevisiae strains for improved fermentation of cellulosic sugars under acetic acid
82
stress and identify genetic perturbation targets for enhancing acetic acid resistance in the yeast.
83
Specifically, we first introduced a genome-wide library into a parent S. cerevisiae strain
84
containing an optimized xylose fermentation pathway, so that the transformants could be
85
evaluated in fermentation of glucose and/or xylose (the two most abundant sugars from
86
lignocellulosic biomass) under acetic acid stress. We then characterized the screened
87
transformants to identify the target of gene perturbation. Here we report on a novel gene target
88
WHI2. Overexpression of WHI2 substantially enhanced acetic acid resistance of S. cerevisiae.
89
We show that acetic acid induced endogenous expression of Whi2 and that deletion of WHI2
90
gene resulted in hypersensitivity to acetic acid. We further determined the function of Whi2 and
91
its binding partner Psr1 in eliciting acetic acid resistance mechanisms in S. cerevisiae. Lastly, we
92
characterized improved performance of an engineered strain for glucose and/or xylose
93
fermentation in the presence of toxic levels of acetic acid under industrially relevant conditions.
94
The results improve our understanding of stress response to acetic acid in S. cerevisiae and
95
provide a new strategy to breed acetic acid resistant yeast strains for renewable biofuel
96
production from lignocellulosic biomass.
97
Materials and Methods
98 99 100
Strains and plasmids. All the strains and plasmids used in this study were summarized in Table 1. The recombinant xylose-fermenting S. cerevisiae strain SR8 (31) was used in this study for glucose and xylose fermentation. The strain was constructed previously (31) through i) 5
101
heterologous expression of XYL1 (coding for xylose reductase, XR), XYL2 (coding for xylitol
102
dehydrogenase, XDH), and XYL3 (coding for xylulokinase, XK) from Scheffersomyces stipitis in
103
S. cerevisiae D452-2 (MATα leu2 ura3 his3, and can1), and optimization of expression levels of
104
XR, XDH, and XK, laboratory evolution on xylose, and deletion of ALD6 coding for
105
acetaldehyde dehydrogenase. The auxotrophic marker genes in the strains SR8-trp and SR8-4
106
(Table 1) were recovered by the CRISPR-cas9 method (32, 33). These strains were kindly
107
provided by Dr. Yong-Su Jin’s lab. The Escherichia coli TOP10 strain was used for gene cloning
108
and manipulation. The whi2 knockout mutant derived from the laboratory strain BY4741 (GE
109
Healthcare Dharmacon) was used for evaluating the susceptibility of the null mutant versus the
110
wild type. The msn2 knockout mutant derived from the strain SR8-trp was used for testing the
111
hypothesis regarding the interaction of Whi2 and Msn2 in eliciting acetic acid resistance.
112
Enzymes, primers and chemicals. Restriction enzymes, DNA-modifying enzymes and
113
other molecular reagents were obtained from New England Biolabs (Beverly, MA). The reaction
114
conditions were set up following the manufacturer’s instructions. All general chemicals and
115
media components were purchased from Sigma-Aldrich (St. Louis, MO) or Fisher Scientific
116
(Pittsburgh, PA). Primers for both PCR and sequencing were synthesized by Integrated DNA
117
Technologies (Coralville, IA) and listed in Table S1.
118
Media and culture conditions. Yeast strains were routinely cultivated at 30 °C in YP
119
medium (10 g/L of yeast extract and 20 g/L of peptone) or synthetic complete (SC) medium (6.7
120
g/L of yeast nitrogen base and appropriate amino acids or nucleotides) containing 20 g/L of D-
121
glucose (SCD). SC media containing 20 g/L agar and glucose without tryptophan and/or leucine
122
amendment was used to select transformants using TRP1 and/or LEU2 as auxotrophic markers. E.
6
123
coli strains were grown in Luria-Bertani medium at 37 °C and 100 μg/mL of ampicillin was
124
added to the medium when required.
125
Construction of S. cerevisiae genomic library and yeast transformation. The genomic
126
DNA of the S. cerevisiae S288C strain was used to construct the genomic library as previously
127
described (34). Briefly, genomic DNA fragments (2-5 kb) were generated by sonication and
128
ligated into a multicopy plasmid pRS424 (a yeast episomal plasmid) with TRP1 as an
129
auxotrophic selection marker (35, 36). Plasmid extraction was performed using the QIAprep
130
Spin Miniprep Kit (Germantown, MD). The genomic library was transformed to the S. cerevisiae
131
strain SR8-trp using LiAc/PEG methods (37).
132
Selection of transformants with acetic acid resistance. Yeast genomic library
133
transformants were inoculated on SC medium agar plates (15 mm diameter), which contained
134
glucose (20 g/L) or xylose (20 g/L) and different concentrations of acetic acid (2.0 g/L, 2.5 g/L,
135
3.0 g/L and 3.5 g/L). The pH of the agar plates was adjusted to be 4.0 so that acetic acid was
136
predominantly undissociated. The strain SR8-trp harboring pRS424GPD plasmid (i.e. the strain
137
S-C1 in Table 1) was used as the control at all conditions. Transformants were isolated from the
138
plates with acetic acid concentrations at which the corresponding plates inoculated with the
139
control strain had no colony grown. Then, the cell growth and sugar consumption rates of the
140
isolated transformants were evaluated in liquid SC medium containing toxic level of acetic acid
141
and 20 g/L glucose or 20 g/L xylose. The initial cell biomass was adjusted to an optical density
142
at 600 nm (OD600) of 0.05. Plasmids from the selected transformants were isolated by Zyppy
143
Plasmid Miniprep Kit (Zymo Research) and amplified by E. coli transformation. The isolated
144
plasmids were re-transformed to the strain SR8-trp to verify the effects of the plasmids on acetic
145
acid resistance.
7
146
Insert identification, plasmid and strain construction. The verified plasmids showing
147
effects in improving acetic acid resistance were sequenced using T3 and T7 primers (Table S1)
148
to determine the nucleotide sequences of the inserted genomic DNA fragments. Sequences were
149
compared to the S. cerevisiae genome sequence to identify the insert sizes and open reading
150
frames. To construct overexpression plasmids, the complete open reading frames of the target
151
genes (WHI2, PSR1, HAA1 or MSN2) were amplified by PCR with the primers listed in Table
152
S1. The PCR products were subsequently digested and ligated to appropriate multiple cloning
153
sites of the plasmid pRS424GPD or pSR425GPD. To construct the msn2 mutant strain from
154
SR8-trp, the KanMX marker gene flanked by about 200 bp homologous to upstream and
155
downstream of MSN2 gene was PCR amplified from the genomic DNA of a msn2 knockout
156
strain derived from the laboratory strain BY4741 (GE Healthcare Dharmacon) with primers
157
msn2-F and msn2-R listed in Table S1. Transformation of PCR product into SR8-trp was
158
performed using Yeast EZ-transformation Kit (BIO 101). Positive transformants were selected
159
on YP medium containing 20 g/L glucose and 300 µg/mL Geneticin G418. Diagnostic PCR was
160
performed to confirm successful deletion, yielding the strain S-msn2Δ. The overexpression
161
plasmids were transformed respectively to the strain SR8-trp, SR8-4 or S-msn2Δ using Yeast
162
EZ-transformation Kit (BIO 101).
163
Protein expression experiments and data analysis. A yeast GFP (Green Fluorescent
164
Protein) clone collection of S. cerevisiae BY4742 strains (Life Technology, no. 95702),
165
developed by oligonucleotide directed homologous recombination to tag each open reading
166
frame (ORF) with Aequorea victoria GFP (S65T) in its chromosomal location at the 3’ end (38),
167
were used in protein expression analysis in response to acetic acid stress. Pre-cultured yeast cells
168
with GFP fusion protein Whi2 were grown in SC+glucose (SCD) media to early exponential
8
169
phase, and then were inoculated to 200 µL SCD medium (OD600 = 0.2) in clear bottom black 96-
170
well plates (Costar, Bethesda, MD) containing different concentrations of acetic acid (0 g/L, 2
171
g/L and 3 g/L, pH=4.0). The plates were then incubated in a Microplate Reader (SynergyTM HT
172
Multi-Mode, Biotech, Winooski, VT) at 30°C with fast shaking, and OD600 and GFP signal
173
(fluorescence readings, excitation at 485nm and emission at 528 nm) were simultaneously
174
measured every one hour for six hours. All experiments were performed in triplicate.
175
The protein expression levels based on GFP signals were calculated using the method
176
previously described (39, 40). The raw data from the OD600 and GFP measurements were
177
corrected by considering the background signals in control with medium only, and the protein
178
expression per cell unit for each measurement was calculated by normalizing GFP data to cell
179
number (OD600) as P=GFP/OD600. The expression of PGK1 gene product was used as the
180
reference, and the P values for each protein of interest were normalized based on mean P level of
181
PGK1 corresponding to the same condition on the same plate. The relative expression (RE) for
182
the protein of interest at each time point under each acetic acid exposure condition versus the
183
control condition without acetic acid was calculated as RE = Paa/Pc, where Paa referred to
184
normalized P in the experiment condition with acetic acid and Pc represented corrected and
185
normalized P in the control condition without acetic acid.
186
Reverse transcription quantitative PCR (RT-qPCR). The wild type strain in early
187
exponential phase was grown in SC medium containing glucose (20 g/L) and acetic acid (2 g/L)
188
or in medium without acetic acid (control condition). After 6 hour incubation, cells from
189
triplicate experiments were harvested by centrifugation for 30 s at 1000 rpm under 4ºC and RNA
190
was immediately extracted by using PureLink® RNA Mini Kit (Life Technology, NY) according
191
to the manufacturer’s instructions. RNA was reverse transcribed into complementary DNA
9
192
(cDNA) using the iScriptTM cDNA synthesis kit (Bio-Rad Laboratories, CA). The cDNA was
193
then used for qPCR analysis using the primers in Table S1 on a CFX Connect Real-time System
194
(Bio-Rad Laboratories, CA) and by using iQ™ SYBR® Green Supermix (Bio-Rad Laboratories,
195
CA). To confirm amplification specificity, PCR products were subjected to melting curve
196
analysis and gel electrophoresis. All the measurements were performed in triplicate for each
197
biological triplicate (n=3x3). Gene expression was calculated by quantification method (41) as (1
198
+ E)Cq / (1 + Eref)Cq,ref, E stands for application efficiency and Cq stands for quantification cycle
199
value.
200
Fermentation experiments. Batch fermentation experiments under oxygen-limited
201
conditions were performed in 125mL Erlenmeyer flasks containing 20 mL medium, and
202
anaerobic batch fermentation experiments were performed in 160 mL serum bottles containing
203
20 mL medium sealed with butyl rubber stoppers, both at 30°C and 100 rpm.The anaerobic
204
fermentation media were prepared by flushing with nitrogen which had passed through a heated,
205
reduced copper column to remove trace oxygen. Pre-cultured yeast cells in SCD medium were
206
centrifuged and washed with sterilized water, and then inoculated into fermentation media
207
containing glucose/xylose and acetic acid. The initial cell densities were adjusted to OD600=1.
208
The initial pH of the media was adjusted to 4.0. For anaerobic fermentation experiments,
209
ergosterol and Tween 80 were added to final concentrations of 0.01 g/L and 0.42 g/L,
210
respectively (42). Culture samples were taken from fermentation experiments to measure the
211
OD600 and concentrations of metabolites. For anaerobic fermentation experiments, samples were
212
taken by sterile syringe and 26G BD needles. Yeast cell dry weight was determined using a
213
microwave method as described previously (43). All of the fermentation experiments were set up
214
in duplicate.
10
215
As for fermentation with cellulosic hydrolysates, the corn stover hydrolysate was
216
prepared by NREL (http://www.nrel.gov/biomass/pdfs/47764.pdf) through dilute acid
217
pretreatment. The hydrolysate liquid fraction contained 10.9 g/L acetic acid, 115 g/L xylose and
218
17 g/L glucose. The hydrolysate was mixed with YP medium at 50% v/v ratio to result in a
219
hydrolysate mixture containing around 3.5 g/L acetic acid, 40 g/L xylose, and an adjusted
220
glucose concentration of 20 g/L. The pH was adjusted to 4.0. The fermentation experiments were
221
set up in flasks under oxygen limited conditions as described above.
222
Analytical methods. Cell growth was monitored by measuring OD600 using a UV-visible
223
spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA). Glucose, xylose, xylitol,
224
glycerol, acetic acid, and ethanol were quantified by high performance liquid chromatography
225
(Agilent Technologies 1200 series) equipped with a refractive index detector and a Rezex ROA-
226
Organic Acid H+ (8%) column (Phenomenex Inc., CA). The column was eluted with 0.005 N
227
H2SO4 as the mobile phase under the flow rate of 0.6 mL/min at 50 °C.
228
Results
229
Screening of transformants exhibiting improved acetic acid resistance. With the goal
230
to obtain transformants that grew faster under acetic acid stress in both glucose and xylose
231
consuming conditions, a method was developed that combined selection on agar plates and
232
screening in liquid medium containing toxic levels of acetic acid. The plates inoculated with the
233
genomic-library transformants had colonies grown on glucose or xylose containing agar plates
234
with up to 3 g/L acetic acid (pH 4.0) within 3-5 days. In contrast, plates inoculated with the
235
control strain S-C1 (Table 1) did not have any colony grown at acetic acid concentrations above
236
2 g/L through two week incubation. Then 130 fast-growing transformants were isolated from
11
237
plates containing 2.5 g/L or 3 g/L acetic acid (65 from glucose-containing plates and 65 from
238
xylose-containing plates).
239
We further screened the isolated transformants in liquid SC medium to identify strains
240
with superior resistance to acetic acid under both glucose-consuming and xylose-consuming
241
conditions. Among the 65 transformants isolated from xylose-containing plates, ten
242
transformants (#7, #32, #34, #35, #36, #38, #41, #49, #52 and #59) grew much faster than the
243
control strain S-C1 in liquid medium containing glucose+acetic acid (see Fig. S1A in the
244
supplemental material), and five out of these ten (#34, #35, #38, #52 and #59) also exhibited
245
significant improvement in liquid medium containing xylose+acetic acid (see Fig. S1B in the
246
supplemental material). As for the 65 transformants isolated from glucose-containing plates,
247
while some transformants showed improved cell growth in medium containing glucose+acetic
248
acid, no significant improvement was observed when these transformants were grown in medium
249
containing xylose+acetic acid (data not shown). A possible reason might be that the yeast was
250
more sensitive to acetic acid when grown on xylose than on glucose; similar observation was
251
also reported in a previous study (44).
252
Plasmids from each of the five selected transformants were isolated and re-transformed
253
into the parental strain respectively to confirm the effects. Four plasmids (#34, #38, #52 and #59)
254
showed positive effects in the re-transformed strains comparable to that in the original
255
transformants in terms of cell growth and sugar consumption under acetic acid stress. The #35
256
plasmid did not result in improved acetic acid resistance in the re-transformed strain, indicating
257
that the increased resistance in the original transformant might be due to genome mutations.
258
Sequence analysis of the four confirmed plasmids revealed that three (#34, #38 and #59) had
259
similar genome coordinates of the identified targets (chrXV: 409,259-412,369 for #34 and #38,
12
260
chrXV: 410,675-412,722 for #59), while the plasmid #52 did not harbor any intact open reading
261
frame (chrIX: 4597-5728).
262
All the three inserts harbored a complete sequence of the gene WHI2 (chrXV: 410,870-
263
412,330). The WHI2 gene product in S. cerevisiae is a 55 kDa cytoplasmatic globular scaffold
264
protein (38). Whi2 is involved in coordinating cell growth and proliferation and plays an
265
important role in nutrient-dependent cell cycle arrest (45-48). The protein has also been reported
266
to be required for activation of general stress response in the yeast (49-51).
267
Enhanced acetic acid resistance by overexpression of WHI2 gene. The effect of
268
overexpressing WHI2 on acetic acid resistance in S. cerevisiae has not been reported before, and
269
the role of Whi2 in acetic acid stress response is unclear. To determine the effect of WHI2 as a
270
gene perturbation target for improving acetic acid resistance, we introduced a multicopy plasmid
271
overexpressing WHI2 under the control of the constitutive GPD (TDH3) promoter and CYC1
272
terminator (Table 1) into S. cerevisiae strain SR8-trp or BY4742 (i.e. WHI2 overexpression in
273
different strain backgrounds), yielding strains S-WHI2 and BY2-WHI2 (Table 1), and
274
corresponding control strains S-C1 and BY2-C1 harbored the plasmid without WHI2 insert. The
275
strains S-WHI2 had noticeably higher cell growth under acetic acid stress than the control strain
276
S-C1, according to the results of yeast spotting assays on SC agar plates containing glucose (20
277
g/L) and different concentrations of acetic acid (Fig. 1A). When grown on plates with xylose as
278
the substrate, the strain S-WHI2 also had substantially higher acetic acid resistance than the
279
control strain (Fig. 1B). Overexpression of WHI2 in another strain background (BY-WHI2) also
280
showed improvement (see Fig. S2 in the supplemental material).
281 282
Batch experiments were conducted to characterize the glucose/xylose fermentation performances of the strain S-WHI2 versus the control strain S-C1under acetic acid stress. Fig. 2
13
283
shows ethanol production and sugar consumption profiles of the two strains. In glucose
284
fermentation under oxygen limited conditions, glucose (20 g/L) was completely consumed
285
within 39 hours by S-WHI2 with the presence of 2.5 g/L acetic acid, while the control strain S-
286
C1 consumed only 3.2 g/L glucose (Fig. 2A). The specific sugar consumption rate and specific
287
ethanol productivity of the strain S-WHI2 were both over five times higher than those of the
288
control (see Table S2 in the supplemental material). The pH of the fermentation medium was
289
4.0 initially and decreased to 3.3 at the end of S-WHI2 fermentation; a toxic level of protonated
290
form of acetic acid prevailed during the fermentation process. It was noted that the fermentation
291
profiles of S-WHI2 and S-C1 had no significant difference under the condition without acetic
292
acid (with pH below 4.0 during fermentation) (Fig. 2B), suggesting that the improvement
293
brought by WHI2 overexpression is associated with cellular response to acetic acid stress. It was
294
noted that the ethanol yields in glucose fermentation with acetic acid were higher than the yields
295
without acetic acid for both S-WHI2 and S-C1 (see Table S2 in supplemental material),
296
probably due to stimulating effect on glucose fermentation by low concentrations of weak acids
297
as reported in previous studies (52, 53). Similar level of improvement in the fermentation by the
298
strain S-WHI2 versus S-C1 was also observed under strict anoxic conditions (see Table S2 in
299
the supplemental material).
300
Additionally, xylose fermentation under acetic acid stress also had significant
301
improvement by overexpression of WHI2. The strain S-WHI2 consumed xylose with a specific
302
rate of 0.245±0.004 g/g dry cell wt/hr under oxygen limited condition, while the control strain
303
did not consume xylose in experimental time frame (Fig. 2C). Xylose fermentation by the two
304
strains in the absence of acetic acid had similar profiles (Fig. 2D), suggesting that WHI2
305
overexpression did not have significant impact on xylose fermentation pathway itself. Xylose
14
306
fermentation under anaerobic conditions proceeded slowly due to intrinsic limitation of the fungi
307
xylose assimilating pathway. No cell growth was observed with 2.5 g/L acetic acid, and thus
308
lower level of acetic acid (1.5 g/L) was used in the experiment. There were significant
309
improvements (t-test, P
