Signature pathway expression of xylose utilization in the genetically engineered industrial yeast Saccharomyces cerevisiae
Signature pathway expression of xylose utilization in the genetically engineered industrial yeast Saccharomyces cerevisiae
Quanzhou Feng 0 1
Z. Lewis Liu 0 1
Scott A. Weber 0 1
Shizhong Li 1
0 Bioenergy Research Unit, US Department of Agriculture, Agricultural Research Service, National Center for Agricultural Utilization Research , Peoria, IL , United States of America, 2 Institute of New Energy Technology, Tsinghua University , Haidian Qu, Beijing , China , 3 USDA-MOST Joint Research Center for Biofuels , Peoria, IL , United States of America
1 Editor: Shihui Yang, Hubei University , CHINA
Haploid laboratory strains of Saccharomyces cerevisiae are commonly used for genetic engineering to enable their xylose utilization but little is known about the industrial yeast which is often recognized as diploid and as well as haploid and tetraploid. Here we report three unique signature pathway expression patterns and gene interactions in the centre metabolic pathways that signify xylose utilization of genetically engineered industrial yeast S. cerevisiae NRRL Y-50463, a diploid yeast. Quantitative expression analysis revealed outstanding high levels of constitutive expression of YXI, a synthesized yeast codon-optimized xylose isomerase gene integrated into chromosome XV of strain Y-50463. Comparative expression analysis indicated that the YXI was necessary to initiate the xylose metabolic pathway along with a set of heterologous xylose transporter and utilization facilitating genes including XUT4, XUT6, XKS1 and XYL2. The highly activated transketolase and transaldolase genes TKL1, TKL2, TAL1 and NQM1 as well as their complex interactions in the nonoxidative pentose phosphate pathway branch were critical for the serial of sugar transformation to drive the metabolic flow into glycolysis for increased ethanol production. The significantly increased expression of the entire PRS gene family facilitates functions of the life cycle and biosynthesis superpathway for the yeast. The outstanding higher levels of constitutive expression of YXI and the first insight into the signature pathway expression and the gene interactions in the closely related centre metabolic pathways from the industrial yeast aid continued efforts for development of the next-generation biocatalyst. Our results further suggest the industrial yeast is a desirable delivery vehicle for new strain development for efficient lignocellulose-to-advanced biofuels production.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
Funding: The authors received no specific funding
for this work.
Competing interests: The authors claim no
competing interests. Strain Saccharomyces
cerevisiae NRRL Y-50463 is covered under United
States Patent entitled: ªYeast strains and method
for lignocellulose to ethanol productionº, Patent
No.: US 9102931. This does not alter our
The industrial yeast Saccharomyces cerevisiae is widely applied in starch-based fermentation
industries for ethanol production. The native S. cerevisiae is superb in glucose consumption
but limited in uptake and utilization of pentose such as xylose. This has been a major obstacle
adherence to PLOS ONE policies on sharing data
Abbreviations: 2C-A, acetaldehyde; 3C-G,
glyceraldehydes 3-phosphate; 3C-P, pyruvate; 4C,
erythrose 4-phosphate; 5C-R, ribose 5-phosphate;
5C-X, xylulose 5-phosphate; 6C-F, fructose
6phosphate; 6C-G, glucose 6-phosphate; 7C,
sedoheptulose 7-phosphate; ATP, adenosine
triphosphate; cDNA, complementary
deoxyribonucleic acid; Ct, cycle threshold; HMF,
highperformance liquid chromatography; mRNA,
messenger ribonucleic acid; NADH, nicotinamide
adenine dinucleotide; NADPH, nicotinamide
adenine dinucleotide phosphate; OD, optical
density; PCR, polymerase chain reaction; PRPP,
phosphoribosyl pyrophosphate; qRT-PCR,
quantitative real-time polymerase chain reaction;
TCA cycle, tricarboxylic acid cycle; XI, xylose
isomerase; YXI, yeast xylose isomerase.
for efficient cellulosic ethanol production from lignocellulosic materials. Although it is not a
natural xylose utilization yeast S. cerevisiae does pose a pathway for oxidizing xylose [
However, in this pathway, xylose was not recognized as a metabolic fermentation carbon
source but led to yeast starvation and respiratory response as observed in recombinant S.
cerevisiae strains [
]. Over the past decades a significant advance has been made to improve
xylose utilization for S. cerevisiae with improved ethanol yield ranging from 0.09 to 0.46 g g-1
as reviewed elsewhere [
]. However, the challenge remains since the limited rate of xylose
conversion and ethanol productivity for genetically engineered S. cerevisiae are not readily for
economic industrial applications [
Introduction of oxidoreductase reaction pathways from Scheffersomyces stipitis is
commonly applied to enable S. cerevisiae utilizing xylose for ethanol production [
]. In this
pathway, xylose is first oxidized into xylitol by xylose reductase (XR, XYL1, EC 184.108.40.206), and
xylitol is further reduced into xylulose by xylitol dehydrogenase (XDH, XYL2, EC 220.127.116.11).
Then xylulose is phosphorylated into xylulose-5-phosphate by xylulokinase (XKS1, EC
18.104.22.168) prior entering into the pentose phosphate pathway (Fig 1). However, this method
causes cofactor imbalance and increased xylitol production as a byproduct [
]. In this
pathway, the xylose-to-xylitol conversion releases NADP+ while the xylitol-to-xylulose
reduction reaction yields NADH. When electron acceptor is short under anaerobic conditions, yeast
cells are unable to maintain a sound redox balance. Even when a xylose-to-xylitol conversion
was coupled with NADH, the higher ratio of NADPH/NADH still led to more xylitol
accumulation since such a pathway was relatively weak [
Another popular approach is to use a bacterial pathway applying xylose isomerase gene XI/xylA
from bacterial Thermus thermophilus and Clostridium phytofermentans [
] or fungal species
from Piromyces and Orpinomyces [
]. This pathway initiates xylose metabolism through its
isomerization into xylulose by xylose isomerase (XI, EC 22.214.171.124) (Fig 1), which avoids cofactor
imbalance associated with the xylitol formation and reduction. However, the limited XI expression
in the yeast often results in lower xylose conversion and ethanol productivity. When grown on
mixed sugars of glucose and xylose, the recombinant strains preferred glucose, and the xylose
consumption was substantially slow with a low affinity of xylose transportation kinetics [
Sugar transporter is a necessary first step for carbohydrate utilization. S. cerevisiae has
plenty glucose transporters but lacks an efficient xylose transporter system. In order to
improve xylose uptake and utilization for S. cerevisiae, many heterologous xylose transporter
genes were evaluated, including those from Arabidopsis thanliana, Candida intermedia,
Debaryomyces hansenii, Neurospora crassa and S. stipitis [
]. However, most heterologous xylose
Fig 1. Xylose metabolic pathways. A schematic illustration of typical xylose metabolic pathways applied for genetic
engineering of Saccharomyces cerevisiae. Enzyme-encoding genes and EC numbers are presented as follows: xylose reductase
(XYL1, EC126.96.36.199), non-specific aldose reductase (ns-XR), xylitol dehydrogenase (XYL2, EC188.8.131.52), xylulokinase (XKS1,
EC184.108.40.206), and xylose isomerase (XI/YXI, EC220.127.116.11).
2 / 23
transporter genes showed poor expression in S. cerevisiae, and no satisfactory level of
improvement was observed on laboratory strains. On the other hand, the industrial yeast strains
appeared to have a different response. Using an industrial yeast strain of S. cerevisiae as a host,
overexpression of individual xylose transporter genes from S. stipitis improved the rate of
volumetric xylose consumption . A serial of new genotypes of an industrial yeast strain with an
individual xylose transporter gene from S. stipitis increased ethanol production from xylose
]. In a comparative study, all five industrial strains outperformed another five laboratory
strains engineered with the same pathway in both xylose consumption rate and ethanol
]. We previously developed a tolerant industrial yeast strain NRRL Y-50049 that is
able to in situ detoxify major class of toxic chemical compounds liberated from lignocellulose
biomass pretreatment such as 2-furaldehyde (furfural) and 5-(hydroxymethyl)-2-furaldehyde
]. We further enabled Y-50049 to utilize xylose by genetic engineering and
generated strain NRRL Y-50463. Strain Y-50463 contains a synthesized yeast xylose isomerase
gene YXI in its chromosome XV and a set of plasmid-carried heterologous genes including
XYL2, XKS1, XUT4 and XUT6 [
]. Strain Y-50463 is able to grow on xylose as its sole
carbon source and ferment ethanol on mixed sugars of glucose and xylose in the presence of
fermentation inhibitors furfural and HMF [
Due to the well known genetic background and readily available genetic tools of yeast
model strains, laboratory strains were widely used for studies on xylose utilization in S.
cerevisiae. For example, characterization on xylose induced effects against glucose on metabolism
and gene expression was reported using traditional XR-XDH pathway for laboratory strains
]. Transcriptome and proteome of a laboratory strain using the same XR-XDH pathway
was characterized for cells grown under aerobic batch culture conditions [
fermentation process occurs under anaerobic or oxygen-limited conditions. Characterization of
aerobic grown cells aids understanding cell growth response but has limited impact on ethanol
fermentation under anaerobic conditions. Recently, a transcriptome analysis of a
xylose-utilizing flocculating industrial yeast was reported [
]; however, it was again applied the
conventional XR-XDH pathway. XI pathway has a significant advantage over the traditional XR-XDH
pathway but relatively fewer information is available. Furthermore, single genes were often
identified but reports on gene interactions are rare. There is an especial lack of pathway-based
knowledge on xylose utilization in XI-pathway for genetically engineered industrial yeast S.
cerevisiae. The advanced development of qRT-PCR technology allowed more accurate
quantitative analysis of gene expression that surpasses high throughput method such as microarray.
Recently developed pathway-based qRT-PCR array provided an efficient platform for
comparative analysis of a subset of genes that suitable for more defined and closely related pathway
]. In this study, we explore the first insight into the important gene interactions
in the centre metabolic pathways of the genetically engineered industrial yeast Y-50463 using
comparative gene expression analysis. The quantitative expression analysis of Y-50463 on a
time-course study revealed a unique signature expression profile of the industrial yeast. Such a
signature pathway expression of Y-50463 underlines the pathway-based genetic interactions of
the improved xylose utilization for the genetically engineered industrial yeast. Knowledge
obtained by this investigation aids continued efforts for the next-generation biocatalyst
development for low-cost cellulosic ethanol production.
Materials and methods
An industrial yeast type strain S. cerevisiae NRRL Y-12632 obtained from ARS Cultural
Collection (Peoria, IL USA) was used in this study as a parental strain control. Strain Y-50463 is also
3 / 23
known as ATCC 18824, WRI74, CCRC 21447, DBVPG 6173, DSM 70449, IFO 10217, IGC
4455, JCM 7255 and NCYC 505 by varied collection centers [
]. A genetically engineered
industrial yeast strain NRRL Y-50463 from ARS Patent Culture Collection was the subject of
the investigation. Strain Y-50463 was a genetically modified strain from a fermentation
inhibitor-tolerant variant of strain Y-12632. It contains a synthesized yeast codon optimized xylose
isomerase gene YXI [
] in its chromosome XV and a set of heterologous xylose utilization
genes carried by a plasmid, including xylitol dehydrogenase (XYL2), xylulokinase (XKS1), and
two xylose transporter genes XUT4 and XUT6 from S. stipitis [
]. The lyophilized cultures
were recovered on YP medium and maintained on the YP medium supplemented with or
without 25 g/L D-xylose.
Culture conditions and sampling
Utilization and ethanol production of glucose and xylose was evaluated under aerobic and
oxygen-limited fermentation conditions separately on YP medium containing 25 g/L D- glucose
and 25 g/L D-xylose. For aerobic growth experiment, inoculum cells were prepared from YP
medium containing xylose only. Cultures with an initial OD600 reading at 0.1 were incubated
using a fleaker system [
] at 30ÊC with agitation at 250 rpm and cell growth was monitored by
absorbance at OD600. For oxygen-limited fermentation experiments, a cell mass at 5 g/L was
prepared on YP medium containing glucose only to save time building the cell mass required
and to facilitate an immediate fermentation process. Tubes on fleaker covers were sealed to
maintain oxygen-limited conditions for the fermentation. Two replicated experiments were
carried out for each of the aerobic and oxygen-limited test set separately. Cell samples were taken
periodically, frozen on dry ice, and then stored at -20ÊC until use for total RNA extraction. Cell
free supernatants from each time point were collected for metabolic profile analysis using a
Shimadzu high-performance liquid chromatography (HPLC) as previously described [
The qRT-PCR array
A set of 96-well qRT-PCR array was made containing 86 genes involved in glycolysis, pentose
phosphate pathway, and tricarboxylic acid (TCA) cycle. To ensure reproducibility and
comparability of qRT-PCR data, a standard mRNA quality control reference was applied for the
multiple-plate qRT-PCR array assay. Five external mRNA species, beta-2-microglobulin (B2M),
major latex protein (MSG), chlorophyll A-B binding protein of LHCI type III precursor
(CAB), ribulose bisphosphate carboxylase small chain 1 precursor (RBS1), and beta-actin
(ACTB), were synthesized in vitro following procedures described previously [
external mRNA reference was prepared in a mix consisting of accurately calibrated mRNA
transcripts of MSG, CAB, RBS1, and ACTB at 0.1, 1, 10, and 1000 pg per μl, respectively. A
standard curve was constructed for each qRT-PCR run using the mRNA reference as a
calibration standard. Reactions for the reference genes were placed on the top of each 96-well plate
with two replications. Reactions of the 86 target genes were arranged in the remaining wells on
the plate. Two plates of replicated reactions were made for each sample serving as technical
replications for each condition from strain Y-12632 and Y-50463 separately. Two biological
replications were carried out for all samples at each time-point.
Genes involved in pathways of glycolysis, pentose phosphate pathway and TCA cycle were
selected based on the KEGG database [
]. Primers for these genes and five heterologous
xylose-utilizing genes YXI, XYL2, XKS1, XUT4 and XUT6 were designed based on the YXI
] and Scheffersomyces stipitis genome sequence [
] with an aid of primer
screening procedure using Primer 3 software. Primers used in this study are presented (S1
Table) with designed amplicon length ranging from 100 to 150 bp for target genes.
4 / 23
Conditions and profiles of qRT-PCR
Total RNA was isolated and RNA integrity was verified by gel electrophoresis and NanoDrop
Spectrophotometer ND-100 (NanoDrop Technologies, Inc., Wilmington, DE) as previously
31, 37, 44
]. Reverse transcription reaction was prepared by adding 1 μl of external
mRNA reference consisting of a set of the above mentioned accurately calibrated mRNA
transcripts into 2 μg of a host total RNA, 0.5 μg of oligo (dT)18, and 1 μ of 10 mM of dNTP mix.
The volume was adjusted by water to 13 μl, then mixed well and incubated at 65ÊC for 5 min.
The reaction tubes were chilled on ice for at least 1 min. Then added 4 μl 5X first strand buffer,
1 μl of 0.1M DTT, 1 μl SuperScript III (200 U/μl) (Invitrogen, CA), and 1 μl RNaseOUT (40 U/
μl) (Invitrogen, CA) with a final volume of 20 μl. The reaction was incubated at 50ÊC for 1 h,
70ÊC for 15 min, and 4ÊC to end the reaction using a PCR cycler. SYBR Green iTaq PCR
master mix (BioRad Laboratories) was applied for each qRT-PCR prep. For each reaction, a total
of 25 μl was used consisting of 12.5 μl 2X SYBR Green MasterMix, 0.5 μl each of forward and
reverse primer (10 μM each), 0.25 μl cDNA template and 11.25 μl H2O. PCR was run on an
ABI Sequence Detection 7500 System using the following thermal profile: stage1: 95ÊC for 3
min; stage 2: 40 cycles of 95ÊC for 15 sec and 60ÊC for 45 sec; stage 3: 95ÊC for 15 sec, 60ÊC for
1 min and 95ÊC for 15 sec; stage 4: run dissociation curve with 95ÊC for 15 sec, 60ÊC for 1 min
and 95ÊC for 15 sec. Stat Collection was set at stage 2 step 2 (60ÊC for 45 sec). A PCR reaction
with a pair of primers for B2M and without a B2M template was used to serve as a negative
control. A laboratory protocol entitled ªQuantitative real-time RT-PCR assay applying
Calibrated mRNA reference (Ctrl Mix)º is available in protocols.io with the following DOI: http://
To guard a reproducible and comparable data analysis, the build-in Auto data acquisition
option with the instrument was quitted. Instead, a Manual option was applied in qRT-PCR
data acquisition for each PCR run. Mean value of the CAB (with 1 pg spiked-in mRNA in the
quality control mix) amplifications on each plate was designated as a sole reference to set up a
Manual cycle threshold (Ct) at 26 for each plate as previously described [
]. Data were
analyzed after normalization by this mRNA reference. A standard curve was generated for
each qRT-PCR plate. Upon completion of all reactions, a master equation was established and
used for quantitative data analysis following previously described procedures . The mRNA
mass for each gene was obtained by an anti-log conversion. For absolute quantification of gene
expression analysis, transcription number for each target genes was calculated using an
equation as previously described [
]. For each strain of Y-12632 and Y-50463, a transcription
copy number at various time-points was normalized with its own at the 0 h for aerobic growth
and oxygen-limited fermentation conditions separately. The differential expression at each
time-point was presented by fold changes of Y-50463 over Y-12632 using the above
normalized values in the comparative analysis.
Cell growth and sugar consumption under aerobic conditions
Under aerobic conditions on YP medium containing mixed sugars of glucose and xylose each
at 25g/L, both wild-type strain Y-12632 and the engineered strain Y-50463 showed a rapid
growth and reached to 1.4 OD reading at 24 h after incubation (Fig 2A). Then cell density of
strain Y-50463 was increased continuously reaching the highest OD of 1.85 at 96 h. In contrast,
there was no increased cell growth was observed for the wild-type Y-12632 until 120 h (Fig
5 / 23
Fig 2. Comparison of strain response. Comparison of cell growth (A) and sugar consumption of genetically engineered Saccharomyces
cerevisiae NRRL Y-50463 (B) and its parental wild type industrial yeast strain NRRL Y-12632 (C) on a medium containing mixed sugars
of glucose and xylose each at 25g/L under aerobic conditions; and ethanol production for Y-50463 (D) and Y-12632 (E) under
oxygenlimited fermentation conditions.
2A). Both strains showed a similar pattern of glucose consumption which was exhausted at 24
h as indicated by HPLC analysis (Fig 2B and 2C). Strain Y-50463 consumed a large portion of
xylose, but strain Y-12632 showed no significant xylose consumption and most xylose was
recovered remaining in the medium until 120 h. Under the aerobic growth conditions, there
was no significant ethanol production was observed.
Under oxygen-limited fermentation conditions, strain Y-50463 quickly exhausted glucose and
consumed xylose in a nearly linear pattern toward the end of the fermentation (Fig 2D). Since
6 / 23
a higher amount of cell mass at 5g/L was introduced to accelerate an immediate fermentation
process, no further cell mess was measured under the oxygen-limited fermentation conditions.
The highest concentration of ethanol production of 17 g/L was observed at 48 h. It produced
approximately 6 g/L xylitol. Its parental strain Y-12632 showed similar trend of glucose
consumption but no utilization of xylose throughout the fermentation by HPLC assay (Fig 2E).
The highest concentration of its ethanol production only reached to 9.4 g/L which was mainly
from glucose. The xylitol conversion was recovered at approximately 4 g/L at the end of the
Quantitative analysis of gene expression
Application of the universal external RNA reference safe guarded the reproducibility and
comparability of data obtained from the qRT-PCR array assays. Based on all qRT-PCR reactions, a
master equation was established (Fig 3) as follows:
where variable Y stands for the cycle value of the qRT-PCR; variable X represents the
quantified mass of mRNA (log pg); and constant 25.5397 or -3.3559 represents a constant Ct or
slope, respectively, for the qRT-PCR in this study. The slope is an important quality control
measurement indicating the amplification efficiency of the qRT-PCR. The slope of -3.3559 for
the highly fitted linear relationship obtained in this investigation represented a high quality of
qRT-PCR performance with an average amplification efficiency of 98.6% [
Fig 3. Master equation. A master equation generated based on all qRT-PCR reactions in this study using the universal
RNA reference performed on the ABI Sequence Detection 7500 System. The slope of -3.3559 indicated an
amplification efficiency of 0.986 for the qRT-PCR reactions in this study.
7 / 23
an anti-log conversion, a transcription number for each target gene was obtained for
comparative gene expression analysis between the two strains.
Heterologous gene expression
The yeast codon optimized YXI genetically integrated into the chromosome XV of strain
Y50463 displayed an extremely higher level of expression with 1.03 x 1010 transcriptions at 4 h
after incubation under aerobic growth conditions, which was 7000-fold increase than the wild
type control (Fig 4A). Xylitol dehydrogenase gene (XYL2) and xylose transporter gene XUT6
from S. stipitis carried in a plasmid showed a transcription level of 7.9 x 105 and 4.5 x 105
representing 45- and 5-fold increase than the control, respectively. However, no significant
expression was observed for xylulokinase gene (XKS1) and xylose transporter gene XUT4 compared
with the control strain Y-12632 at 4 h after incubation (Fig 4A). Expression levels of all these
genes were increased significantly 24 h after incubation. YXI maintained the highest level of
Fig 4. Hetrologous gene expression. Quantitative expression of five heterologous genes in the genetically engineered
Saccharomyces cerevisiae NRRL Y-50463 in comparison with its parental wild type industrial yeast strain NRRL
Y12632 by qRT-PCR analysis at 4 h (A) and 24 h (B) after incubation under aerobic growth conditions.
8 / 23
enhanced expression reaching to 1.4 x 1010 transcriptions, more than 13,000-fold increase
compared with the control (Fig 4B). XYL2 and XKS1 increased 250- and 100-fold, respectively.
Xylose transporter genes XUT4 displayed more than 600-fold increase and XUT6 only
increased 70-fold. A similar trend of expression was observed under the oxygen-limited
fermentation conditions (data not shown).
Early gene expression response to the mixed sugars of glucose-xylose
Comparative gene expression analysis was conducted under both aerobic and oxygen-limited
conditions. Cells grown on the medium containing both glucose (20 g/L) and xylose (25 g/L)
at 4 h after incubation were used for aerobic growth treatment. In a total of 86 genes tested, 27
genes from strain Y-50463 showed significantly higher levels of expression compared with the
parental strain Y-12632. Among which, 16 genes were identified to be involved in glycolysis, 5
in pentose phosphate pathway, and 6 in TCA cycle (Table 1). Several genes displayed extremely
higher levels of up-regulated expressions ranged from 5- to 20-fold increases, such as PRS4,
HXK2, PCK1, FBP1, and ADH7, compared with the parental control strain.
Since the glucose was depleted completely in the medium 4 h after fermentation under
oxygen-limited conditions, cell samples were taken at 2 h and used for gene expression analysis in
response to the mixed sugars. During the early hours of ethanol production in the presence of
glucose and xylose, most genes maintained normal or nearly normal levels of expression.
Under the oxygen-limited conditions, only six genes out of 42 from glycolysis and two genes
out of 19 from pentose phosphate pathway showed increased expressions (Table 2). Under
oxygen-limited conditions, a small number of genes showed similar repressed expressions
such as ADH4, ENO2, GND1 and GND2. These genes were also repressed under aerobic
growth conditions. Several other genes shared increased expressions under both conditions
such as ALD5, GPM2, GPM3, HXK2, PRS4 and PRS5 which are involved in glycolysis and
pentose phosphate pathways.
Gene expression response to xylose after depletion of glucose
Glucose content in the medium was completely depleted for both strains Y-50563 and
Y12632 24 h after incubation under both aerobic and oxygen-limited conditions. At this stage,
xylose was the only source of carbon for cell utilization. Under aerobic growth conditions,
most genes from the genetically engineered strain Y-50463 showed increased expressions in
response to xylose utilization compared with its parental strain Y-12632. There were 24 genes
out of 42 genes tested in glycolysis, 10 out of 19 tested in pentose phosphate pathway, and 17
out of 25 tested in TCA cycle, showed significantly higher levels of increased expression
(Table 1 and Fig 5). Except for a few, many remaining genes showed normal or nearly normal
levels of expression. A similar trend of expression patterns was observed at 24 h under
oxygenlimited fermentation conditions for strain Y-50463. While most genes displayed normal or
nearly normal expressions for xylose utilization, only 12 genes out of 42 genes tested, and 10
out of 19 showed significantly higher levels of increased expressions for glycolysis and pentose
phosphate pathways, respectively (Table 2 and Fig 5). However, many genes maintained a
sound expression level at 24 h and others showed improved expressions with the continued
xylose utilization at 48 h. No expression analysis was conducted for TCA cycle genes under the
oxygen-limited fermentation conditions.
Unlike observed for the early response to the mixed sugars, many genes showing increased
expressions were overlapped for both aerobic and oxygen-limited conditions when glucose
was depleted and xylose left as the only source of carbon. All genes showing significantly
increased expression under the oxygen-limited condition were also consistently displayed
9 / 23
Glycolysis / Gluconrogenesis
Pentose phosphate pathway
Numbers in bold indicate a significant differential expression ratio above 1.5.
PLOS ONE | https://doi.org/10.1371/journal.pone.0195633
11 / 23
12 / 23
Numbers in bold indicate a significant differential expression ratio above 1.5.
higher levels of expression under the aerobic conditions, except for SOL4 with a robust normal
expression, for strain Y-50463.
Signature expression of NRRL Y-50463
Since strain Y-50463 received five heterologous genes of YXI, XUT4, XUT6, XYL2 and XKS1 in
its chromosome or cytoplasm, the enriched genetic background and the expression of these
heterologous genes were naturally unique for the genetically engineered strain in contrast to
its wild-type parental strain Y-12632 (Figs 4 and 5). The high level of constitutive expression
by YXI was outstanding. Even for the other four genes showing relatively lower levels of
expression, they were distinctly presented in the yeast and changed the yeast performance.
These YXI-led five xylose-utilization facilitating genes signified a system response to adjust the
Y-50463 performance, which induced significantly altered interactive expression relationships
of the endogenous genes of the yeast.
Another outstanding pattern of the signature expression was observed for 10 genes, TAL1,
NQM1, TKL1, TKL2, PGI, FBP1, PFK1, PFK2, FBA1 and TPI1, involved in the non-oxidative
pentose phosphate pathway at 24 h under both aerobic and oxygen-limited conditions (Tables
1 and 2 and Fig 5). Among these, four genes TAL1, NQM1, TKL1 and TKL2 were especially
active in the pentose phosphate shunt pathway. These genes play significantly important roles
in cell metabolism with multiple functions involving in GO categories of cellular component,
molecular function and biological process (Table 3). Such a signature expression was distinct
in contrast to the early expression response under both aerobic and oxygen-limited conditions.
The other six genes important in this pathway were highly active and closely interactive with
the upper portion of the glycolysis.
13 / 23
Fig 5. Signature expression pathway. A schematic illustration of significant gene expression changes for the genetically engineered Saccharomyces cerevisiae
NRRL Y-50463 compared with its parental wild type industrial yeast strain NRRL Y-12632 for endogenous genes involved in glycolysis, pentose phosphate
pathway and TCA cycle at 24 h using xylose as the sole source of carbon when glucose was depleted. Arrows on the left and the top from the parallel lines
represent aerobic growth condition and those on the right side or at the bottom represent oxygen-limited fermentation condition. Blue or green colored
arrows indicate significantly greater gene expression for aerobic and oxygen-limited condition, respectively. Arrows in red indicate repressed expression and
arrows in black indicate gene expression at normal or nearly normal levels. Elements of the signature expression for strain NRRL Y-50463 were boxed in
varied colors and marked as I, II and III, respectively.
The third significant pattern of the signature expression for the genetically engineered yeast
strain was observed for the PRS gene family including PRS5, PRS4, PRS2, PRS1 and PRS3
which is directly connected to the pathway of phosphoribosyl pyrophosphate (PRPP) (Tables
1, 2 and 3). This signature expression existed for the yeast cells grown under both aerobic and
oxygen-limited conditions regardless of the stage of the early response or at 24 h. However, the
expression levels of those genes were even higher on xylose when glucose was completely
depleted (Fig 5).
Using pathway-based qRT-PCR array analyses, we demonstrated significantly higher levels of
constitutive expression of YXI and revealed the insight into the signature pathway expression
of the xylose utilization for the genetically engineered industrial yeast S. cerevisiae NRRL
Y50463 in this study. We identified three distinct signature expression patterns underlying the
Y-50463 performance for its enabled xylose utilization capability, involving the following three
groups of genes: I. A set of five heterologous genes engineered into Y-50463 including YXI,
XUT4, XUT6, XYL2 and XKS1 involved in xylose-to-xylulose-5-phosphate conversion (Fig 5).
II. Ten genes in the non-oxidative pentose phosphate pathway branch, especially for TKL1 and
TKL2 or TAL1 and NQM1 which encode for transketolase or transaldolase enzymes,
respectively for the serial of sugar transformation to drive the metabolic flow into glycolysis. III. The
entire PRS gene family consisting of PRS1, PRS2, PRS3, PRS4 and PRS5 which encode
5-phospho-ribosyl-1(alpha)-pyrophosphate synthetases for synthesis of PRPP, a central compound
for biosynthesis superpathway of nucleotide and amino acids. Knowledge obtained from the
industrial yeast aids continued efforts in development of the next-generation biocatalyst for
efficient lignocellulose-to-advanced biofuels conversion.
In the first element of the signature expression, the yeast codon optimized YXI genetically
integrated into the chromosome XV of Y-50463 displayed a significantly higher level of
constitutive expression in this study. Historically, XI-expressing S. cerevisiae strains suffered a lower
rate of xylose fermentation despite an improved yield of ethanol [
]. Since XI is often
expressed under promoters of multi-copy plasmids, its expression tends to be unstable
especially under continuous cultivations [
]. In this study, the chromosomally integrated YXI
with a robust promoter of ADH1safe guarded the YXI expression in Y-50463 [
chromosomal location of the target gene was suggested to impact its expression and regulation
]. In the design of Y-50463 used in this study, YXI was resided at the ADH1 locus in
chromosome XV (33). The superb high levels of YXI expression observed in this study can be
benefited from its specific robust location in the chromosome. Such a constitutive expression
of the YXI served a necessary driving force for xylose metabolism in Y-50463. Since xylose
uptake and flux are limited by the sugar transport, efficient xylose transporters are necessary to
improve the rate of xylose metabolism [
24, 27, 28
]. Introduction of a single xylose transporter
gene in combination with YXI into an industrial yeast strain has been demonstrated to
improve xylose uptake, volumetric consumption and increased ethanol production
]. In this study, two xylose transporter genes XUT4 and XUT6 appeared to facilitate
15 / 23
Genes involved in the signature expression are bolded.
PRS4 , PGI1, SFA1, TPI1, PRS2, HXK1, HXK2, PFK1, SOL4, GND2, FBA1, PRS1, TAL1,
ADH3, PGM2, ALD2, PFK2, SOL1, GPM3, PRS5, TKL1
PGI1, PGM2, PRS5, PRS2, PFK1, FBA1, NQM1, HXK2, ALD2, TAL1, GPM3, PRS4, ADH3,
SOL1, TPI1, SFA1, TKL1, PRS1, SOL4, PFK2, HXK1, GND2
SFA1, ADH3, GND2
PRS4, PRS2, PRS1, PRS5
HXK1, HXK2, PFK1, PFK2
PRS4, PRS2, HXK1, HXK2, NQM1, PFK1, PRS1, TAL1, PFK2, PRS5, TKL1
NQM1, TAL1, TKL1
the xylose transport function although their expression level was not compatible with that of
YXI. Since these xylose transporter genes were carried by a single plasmid, the lower levels of
the expression in contrast to the constitutive expression of YXI is expected due to a potential
low copy number. Although XUT4 and XUT6 facilitated xylose uptake and consumption in
this study, they are not the most efficient xylose transporter genes. Other xylose transporter
genes such as RGT2, SUT4 and XUT7 were found to be more efficient than XUT4 and XUT6
under the same conditions [
]. We suggest these more efficient xylose transporter genes to be
included for improvement in future genetic engineering efforts. In order to reduce xylitol
16 / 23
accumulations caused by endogenous xylose reductase activity, XYL2 was introduced into
Y50463 for the strain design [
]. However, such an approach did not completely eliminate
xylitol and a residue amount of xylitol was still observed in this study. The expression of XKS1 in
Y-50463 in this study was also relatively lower than expected. It is likely caused by the large
insert and the low copy number of the plasmid. Kinase reaction from xylulose into
xylulose5-phosphate is a key step to supply the basic intermediate into the non-oxidative pentose
phosphate pathway branch. A constitutive expression of XKS1 is needed for further improvement
through combined efforts of sequence optimization and chromosomal integration. It is
obvious that the introduction of a set the YXI-lead heterologous genes in Y-50463 changed gene
expression profiles of the yeast. Consequently, the altered gene interactions activated xylose
metabolism through the non-oxidative pentose phosphate pathway branch in Y-50463, which
enabled xylose to be transformed into downstream of glycolysis for increased ethanol
production (Fig 5).
The second element of the signature expression was concentrated in the non-oxidative
pentose phosphate pathway branch involving at least 10 genes. Most genes closely associated with
the upper portion of glycolysis were able to maintain a normal level of expression to function
in the presence of mixed sugars of glucose and xylose under both aerobic and oxygen-limited
conditions (Fig 5). Four genes of TAL1, NQM1, TKL1 and TKL2 were outstanding in the
presence of xylose when glucose was completely depleted. Therefore, they are accountable as the
most critical genes playing substantial roles for the acquired xylose metabolism in Y-50463
(Fig 6). TKL1 and TKL2 encode for transketolases that catalyze conversion of
xylulose-5-phosphate and ribose-5-phosphate to sedoheptulose-7-phosphate and glyceraldehyde-3-phosphate
]. TAL1 and NQM1 encode transaldolase enzymes that convert
sedoheptulose-7-phosphate and glyceraldehydes-3-phosphate to erythrose-4-phosphate and fructose-6-phosphate
]. Overexpression of genes involved in non-oxidative pentose phosphate pathway
including TAL1 and TKL1 was observed to improve cell growth and the rate of xylose
consumption in xylose-utilizing yeast stains [
]. TAL1 and TKL1 were suggested as essential
genes for xylose assimilation and utilization . A mutation with better xylose fermentation
was also found to have elevated protein expression of TKL1 [
]. Our results from this study
were consistent with previous observations, and further illustrated these gene interactions and
relationships in the non-oxidative pentose phosphate pathway branch. These transketolase
and transaldolase genes are actively involved in a serial of sugar transformation reactions
through complex interactions to facilitate the efficient metabolism of xylose for the engineered
Y-50463 (Fig 6). The enhanced non-oxidative pentose phosphate pathway metabolism drives
metabolic flow into the glycolysis. Evidently, functions and interactions between TAL1-NQM1
and TKL1-TKL2 are extremely critical for the xylose metabolism in Y-50463 using xylose as a
sole source of carbon for ethanol production.
It needs to point out that activation of these genes and interactions are initiated with
intermediate xylulose-5-phosphate but not xylose (Fig 6). Expression of TAL1 did not lead
consumption of xylose [
]. Therefore, the reduction branch form xylose to xylulose-5-phosphate
relies heavily on YXI, XUT4 and XKS1 (Figs 5 and 6). On the other hand, the active
non-oxidative pentose phosphate pathway metabolism also needs intermediate supply of
ribose-5-phosphate. In our study, this branch appeared to have an active transcription response.
The third signature expression element included five members of PRS gene family with
significantly enhanced gene expression response under both aerobic and oxygen-limited
conditions. These genes encode 5-phospho-ribosyl-1(alpha)-pyrophosphate synthetases which
synthesize PRPP for biosynthesis of nucleotide and many amino acids such as histidine,
tryptophan, tyrosine and alanine [
42, 58, 59
]. PRS genes are often repressed under fermentation
inhibitor challenges associated with declined cell growth [
]. The highly activated expression
17 / 23
Fig 6. Xylose transformation pathway. A schematic illustration of xylose transformation and metabolism through the
non-oxidative pentose phosphate pathway for the genetically engineered industrial yeast Saccharomyces cerevisiae
NRRL Y-50463. 2C-A stands for acetaldehyde; and 3C-G, glyceraldehydes 3-phosphate; 3C-P, pyruvate; 4C, erythrose
4-phosphate; 5C-R, ribose 5-phosphate; 5C-X, xylulose 5-phosphate; 6C-F, fructose 6-phosphate; 6C-G, glucose
6-phosphate; and 7C, sedoheptulose 7-phosphate. Expression fold changes against the wild type control at 24 h are
presented in green.
of this group of genes doubtlessly contributed to the sound life cycle and enhanced
biosynthesis functions for strain Y-50463. In addition, we also observed that under aerobic conditions,
most genes involved in the TCA cycle showed significantly increased expression at almost
every step of the reactions at 24 h when xylose was the sole source of carbon supply. This is
consistent with observations from another reported flocculating industrial yeast strain [
For a laboratory yeast strain, a lower level of oxygen enhanced gene expression related to
respiratory metabolism under controlled conditions [
]. The oxygen-limited condition carried in
this study allowed a lower level of oxygen which could lead similar reactions under such
conditions for Y-50463. In this study, the current xylose-to-ethanol production by Y-50463 has not
reached its maximum theoretical potential yet. Interfere by the uncontrolled endogenous
aldose reduction activities appeared exist. Thus, continued efforts of system management are
needed for global optimization to improve its efficiency of ethanol production from xylose.
Development of the next-generation biocatalyst remains a continued challenge for efficient
utilization of biomass sugars toward a sustainable biofuels production. The industrial yeast
strains are more robust and a recent genomic study showed more tolerant signaling pathways
of an industrial yeast strain than the model strain S288C . Adaptation is a commonly used
method for new strain development. The plastic genome of the industrial yeast allows efficient
18 / 23
yeast adaptation to varied environmental conditions associated with industrial applications
31, 62, 63
]. The rate of genome evolution for naturally collected yeast strains was also found
to be faster than the laboratory strains . Genetically engineered industrial strains have
been demonstrated to outperform the laboratory strains for ethanol productivity [
transcription levels of genes involved in xylose metabolism were also found to be higher than
the similarly engineered laboratory strains  The current study and previous reports
demonstrated the industrial yeast functions well as a host to engage new gene functions including
YXI and heterologous xylose transport genes [
]. We are confident the industrial yeast, in
general, can better serve as a desirable delivery vehicle for development of the next-generation
biocatalyst in production of fuels and chemicals from lignocellulose materials.
Compliance with ethical standards
Authors claim no conflict of interest. This research did not apply any human participants and/
or animals. Informed consent was obtained from all individual participants included in the
S1 Table. Primers applied for the comparative quantitative gene expression analysis using pathway-based qRT-PCR array assays in this study.
Mention of trade names or commercial products in this publication is solely for the purpose of
providing specific information and does not imply recommendation or endorsement by the
U.S. Department of Agriculture. USDA is an equal opportunity provider and employer.
Conceptualization: Z. Lewis Liu.
Data curation: Quanzhou Feng.
Formal analysis: Z. Lewis Liu.
Investigation: Quanzhou Feng, Scott A. Weber.
Methodology: Z. Lewis Liu.
Resources: Z. Lewis Liu, Shizhong Li.
Supervision: Shizhong Li.
Validation: Z. Lewis Liu.
Writing ± original draft: Z. Lewis Liu.
19 / 23
20 / 23
21 / 23
22 / 23
1. Gong C.S. , Chen L.F. , Flickinger M.C. , Chiang L.C. and Tsao G.T. ( 1981 ) Production of ethanol from Dxylose by using D-xylose isomerase and yeasts . Appl. Environ. Microbiol . 41 : 430 ± 436 . PMID: 16345717
2. van Zyl C. , Prior B.A. , Kilian S.G. and Kock J.L. ( 1989 ) D-xylose utilization by Saccharolilyces cereuisiae . J. Gen. Microbial . 135 : 2791 ± 2798 .
3. Toivari M.H. , Salusjarvi L. , Ruohonen L . and PenttilaÈ M. ( 2004 ) Endogenous xylose pathway in Saccharomyces cerevisiae . Appl. Environ. Microbiol . 70 : 3681 ± 3686 . https://doi.org/10.1128/AEM.70.6. 3681 - 3686 . 2004 PMID: 15184173
4. Jin Y.S. , Laplaza J.M. and Jeffries T.W. ( 2004 ) Saccharomyces cerevisiae engineered for xylose metabolism exhibits a respiratory response . Appl. Environ. Microbiol . 70 : 6816 ± 6825 . https://doi.org/10. 1128/AEM.70.11. 6816 - 6825 . 2004 PMID: 15528549
5. Salusjarvi L. , Pitkanen J.P. , Aristidou A. , Ruohonen L. and Penttila M. ( 2006 ) Transcription analysis of recombinant Saccharomyces cerevisiae reveals novel responses to xylose . Appl. Biochem. Biotechnol . 128 : 237 ± 261 . PMID: 16632884
6. Liu Z.L. , Saha B.C. and Slininger P.J. ( 2008 ) Lignocellulosic biomass conversion to ethanol by Saccharomyces . In Bioenergy pp 17 ±36 ed by Wall JD , Harwood CS and Demain A . ASM Press. Washington DC.
7. Matsushika A. , Liu Z.L. , Sawayama S. and Moon J. ( 2012 ) Improving biomass sugar utilization by engineered Saccharomyces cerevisiae . In Microbial stress tolerance for biofuels: Systems biology pp 137 ± 160 ed by Liu ZL. Springer. Verlag Berlin Heidelberg.
8. Moyses D.N. , Reis V.C.B. , de Almeida J.R.M ., de Moraes L.M.P. and Torresm F.A.G. ( 2016 ) Xylose fermentation by Saccharomyces cerevisiae: challenges and prospects . Int. J. Mol. Sci . 17 :207. https:// doi.org/10.3390/ijms17030207 PMID: 26927067
9. Ho N.W.Y. , Chen Z. and Brainard A.P. ( 1998 ) Genetically engineered Saccharomyces yeast capable of effective cofermentation of glucose and xylose . Appl. Environ. Microbiol . 64: 1852 ± 1859 . PMID: 9572962
10. Jin Y.S. , Lee T.H. , Choi Y.D. , Ryu Y.W. and Seo J.H. ( 2000 ) Conversion of xylose to ethanol by recombinant Saccharomyces cerevisiae containing genes for xylose reductase and xylitol dehydrogenase from Pichia stipitis . J. Micorbiol. Biotechnol . 10 : 564 ± 567 .
11. Eliasson A. , Hofmeyr J-H.S. , Pedler S. and Hahn-HaÈgerdal B. ( 2001 ) The xylose reductase/xylitol dehydrogenase/xylulokinase ratio affects product formation in recombinant xylose-utilising Saccharomyces cerevisiae . Enzyme Microb. Technol . 29 : 288 ± 297 .
12. Toivari M.H. , Aristidou A. , Ruohonen L . and PenttilaÈ M. ( 2001 ) Conversion of xylose to ethanol by recombinant Saccharomyces cerevisiae: importance of xylulokinase (XKS1) and oxygen availability . Metab. Eng . 3 : 236 ± 249 . https://doi.org/10.1006/mben. 2000 .0191 PMID: 11461146
13. Matsushika A. , Inoue H. , Kodaki T. and Sawayama S. ( 2009 ) Ethanol production from xylose in engineered Saccharomyces cerevisiae strains: current state and perspectives . Appl Microbiol . Biotechnol. 84 : 37 ± 53 . https://doi.org/10.1007/s00253-009-2101 -x PMID : 19572128
14. Bruinenberg P.M. , de Bot P.H.M. , van Dijken J.P. and Scheffers WA. ( 1983 ) The role of redox balances in the anaerobic fermentation of xylose by yeasts . Appl. Microbiol. Biotechnol . 18 : 287 ± 292 .
15. Kuyper M. , Winkler A .A., van Dijken J.P. and Pronk J.T. ( 2004 ) Minimal metabolic engineering of Saccharomyces cerevisiae for efficient anaerobic xylose fermentation: a proof of principle . FEMS Yeast Res . 4 : 655 ± 664 . https://doi.org/10.1016/j.femsyr. 2004 . 01 .003 PMID: 15040955
16. van Maris A.J. , Abbott D.A. , Bellissimi E ., van den Brink J., Kuyper M. , Luttik M.A. et al. ( 2006 ) Alcoholic fermentation of carbon sources in biomass hydrolysates by Saccharomyces cerevisiae: current status . Antonie Leeuwenhoek . 90 : 391 ± 418 . https://doi.org/10.1007/s10482-006-9085-7 PMID: 17033882 17 . Walfridsson M. , Bao X. , Anderlund M. , Lilius G. , Bulow L. and Hahn-Hagerdal B. ( 1996 ) Ethanolic fermentation of xylose with Saccharomyces cerevisiae harboring the Thermus thermophilus xylA gene, which expresses an active xylose (glucose) isomerase . Appl. Environ. Microbiol . 62 : 4648 ± 4651 . PMID: 8953736
18. Brat D. , Boles E. and Wiedemann B. ( 2009 ) Functional expression of a bacterial xylose isomerase in Saccharomyces cerevisiae . Appl. Environ. Microbiol . 75 : 2304 ± 2311 . https://doi.org/10.1128/AEM. 02522-08 PMID: 19218403
19. Kuyper M. , Harhangi H.R. , Stave A.K. , Winkler A.A. , Jetten M.S. , de Laat W.T. et al. ( 2003 ) High-level functional expression of a fungal xylose isomerase: the key to efficient ethanolic fermentation of xylose by Saccharomyces cerevisiae . FEMS Yeast Res . 4 : 69 ± 78 . PMID: 14554198
20. Madhavan A. , Tamalampudi S. , Ushida K. , Kanai D. , Katahira S. , Srivastava A. et al. ( 2009 ) Xylose isomerase from polycentric fungus Orpinomyces: gene sequencing, cloning, and expression in Saccharomyces cerevisiae for bioconversion of xylose to ethanol . Appl. Microbiol. Biotechnol. 82 : 1067 ± 1078 . https://doi.org/10.1007/s00253-008 -1794-6 PMID: 19050860
21. Kuyper M. , Hartog M.M. , Toirkens M.J. , Almering M.J. , Winkler A .A., van Dijken J.P. et al. ( 2005 ) Metabolic engineering of a xylose-isomerase-expressing Saccharomyces cerevisiae strain for rapid anaerobic xylose fermentation . FEMS Yeast Res . 5 : 399 ± 409 . https://doi.org/10.1016/j.femsyr. 2004 . 09 .010 PMID: 15691745
22. Hamacher T. , Becker J. , GaÂrdonyi M. , Hahn-HaÈgerdal B. and Boles E. ( 2002 ) Characterization of the xylose-transporting properties of yeast hexose transporters and their influence on xylose utilization . Microbiology . 148 : 2783 ± 2788 . https://doi.org/10.1099/ 00221287 -148-9-2783 PMID: 12213924
23. Sedlak M. and Ho N.M.Y. ( 2004 ) Characterization of the effectiveness of hexose transporters for transporting xylose during glucose and xylose co-fermentation by a recombinant Saccharomyces cerevisiae . Yeast . 21 : 671 ± 684 . https://doi.org/10.1002/yea.1060 PMID: 15197732
24. Runquist D. , Fonseca C. , Radstrom P. , Spencer M. and Hahn B. ( 2009 ) Expression of the Gxf1 transporter from Candida intermedia improves fermentation performance in recombinant xylose-utilizing Saccharomyces cerevisiae . Appl. Microbiol. Biotechnol . 82 : 123 ± 130 . https://doi.org/10.1007/s00253- 008-1773-y PMID: 19002682
25. Runquist D. , Hahn-Hgerdal B. and Rdstrm P. ( 2010 ) Comparison of heterologous xylose transporters in recombinant Saccharomyces cerevisiae . Biotechnol. Biofuels . 3 :5. https://doi.org/10.1186/ 1754 -6834- 3-5 PMID: 20236521
26. Fonseca C. , Olofsson K. , Ferreira C. , Runquist D. , Fonseca L. , Hahn B. et al. ( 2011 ) The glucose/ xylose facilitator gxf1 from Candida intermedia expressed in a xylose-fermenting industrial strain of Saccharomyces cerevisiae increases xylose uptake in SSCF of wheat straw . Enzyme Microb. Technol . 48 : 518 ± 525 . https://doi.org/10.1016/j.enzmictec. 2011 . 02 .010 PMID: 22113025
27. Young E. , Poucher A. , Comer A. , Bailey A. and Alper H. ( 2011 ) Functional survey for heterologous sugar transport proteins, using Saccharomyces cerevisiae as a host . Appl. Environ. Microbiol . 77 : 3311 ± 3319 . https://doi.org/10.1128/AEM.02651-10 PMID: 21421781
28. Moon J. , Liu Z.L. , Ma M. and Slininger P.J. ( 2013 ) New genotypes of industrial yeast Saccharomyces cerevisiae engineered with YXI and heterologous xylose transporters improve xylose utilization and ethanol production . Biocat. Agr. Biotechnol . 2 : 247 ± 254 .
29. Matsushika A. , Inoue H. , Murakami K. , Takimura O. and Sawayama S. ( 2009 ) Bioethanol production performance of five recombinant strains of laboratory and industrial xylose-fermenting Saccharomyces cerevisiae . Bioresour Technol . 100 : 2392 ± 2398 . https://doi.org/10.1016/j.biortech. 2008 . 11 .047 PMID: 19128960
30. Liu Z.L. , Slininger P.J. and Gorsich S.W. ( 2005 ) Enhanced biotransformation of furfural and hydroxymethylfurfural by newly developed ethanologenic yeast strains . Appl. Biochem. Biotechnol . 121 ± 124 : 451 ± 460 . PMID: 15917621
31. Liu Z.L. , Ma M. and Song M. ( 2009 ) Evolutionarily engineered ethanologenic yeast detoxifies lignocellulosic biomass conversion inhibitors by reprogrammed pathways . Mol. Genet . Genomics. 282 : 233 ± 244 . https://doi.org/10.1007/s00438-009 -0461-7 PMID: 19517136
32. Liu Z.L. and Moon J. ( 2011 ) Complete codons of Saccharomyces cerevisiae transgenic strain NRRL Y50463 xylose isomerase (YXI) mRNA . https://www.ncbi.nlm.nih.gov/nuccore/jf261697.
33. Ma M. , Liu Z.L. and Moon J. ( 2012 ) Genetic Engineering of Inhibitor-Tolerant Saccharomyces cerevisiae for Improved Xylose Utilization in Ethanol Production . Bioenergy Res . 5 : 459 ± 469 .
34. Alff-Tuomala S. , Salusjarvi L. , Barth D. , Oja M. , Penttila M. , Pitkanen J.-P. et al. ( 2016 ) Xylose-induced dynamic effects on metabolism anf gen eexpression in engineered Saccharomyces cerevisiae in anaerobic glucose-xylose cultures . Appl. Micorbiol. Biotechnol 100 : 969 ± 985 .
35. Salusjavi L , Kanhainen M , Soliyman R , Pitkanen J-P , Penttila M , Ruohonen L ( 2008 ) Regulation of xylose metabolism in recombinant Saccharomyces cerevisiae . Microb. Cell Fact . 7 :18. https://doi.org/ 10.1186/ 1475 -2859-7-18 PMID: 18533012
36. Zeng W.-Y., Tang Y.-Q. , Guo M. , Xia Z._Y. and Kida K. ( 2016 ) Transcriptomes of a xylose-utilizing industrial flocculating Saccharomyces cerevisiae strain cultured in mediua containing different sugar sources . AMB Express . 6 :51. https://doi.org/10.1186/s13568-016-0223-y PMID: 27485516
37. Liu Z.L. , Palmquist D.E. , Ma M. , Liu J. and Alexander N. ( 2009 ) Application of a master equation for quantitative mRNA analysis using qRT-PCR . J. Biotechnol . 143 : 10 ± 16 . https://doi.org/10.1016/j. jbiotec. 2009 . 06 .006 PMID: 19539678
38. Mannazzu I. , Clementi F. and Ciani M. ( 2002 ) Strategies and criteria for the isolation and selection of autochthonous starters , in Biodiversity and Biotechnology of Wine Yeasts Ciani M., ed), Research Signpost , pp. 19 ± 35 .
39. Bradbury J.E. , Richards K.D. , Niederer H.A. , Lee S.A. , Dunbar P.R. and Gardner R.C. ( 2006 ) A homozygous diploid subset of commercial wine yeast strains . Antonie van Leeuwenhoek . 89 : 27 ± 37 . https:// doi.org/10.1007/s10482-005 -9006-1 PMID: 16328862
40. Liu Z.L. and Cotta M.A. ( 2015 ) Technical assessment of cellulosic ethanol production using β-glucosidase producing yeast Clavispora NRRL Y-50464. Bioenerg. Res . 8 : 1203 ± 1211 .
41. Liu Z.L. and Slininger P.J. ( 2007 ) Universal external RNA controls for microbial gene expression analysis using microarray and qRT-PCR . J. Microbiol . Methods. 68 : 486 ± 496 . https://doi.org/10.1016/j. mimet. 2006 . 10 .014 PMID: 17173990
42. Kanehisa M. , Sato Y. , Kawashima M. , Furumichi M. and Tanabe M. ( 2016 ) KEGG as a reference resource for gene and protein annotation . Nucleic Acids Res . 44 : D457±D462 . https://doi.org/10.1093/ nar/gkv1070 PMID: 26476454
43. Jeffries T.W. , Grigoriev I.V. , Grimwood J. , Laplaza J.M. , Aerts A. , Salamov A. et al. ( 2007 ) Genome sequence of the lignocellulose-bioconverting and xylose-fermenting yeast Pichia stipitis . Nat. Biotechnol . 25 : 319 ± 326 . https://doi.org/10.1038/nbt1290 PMID: 17334359
44. ERCC. ( 2005 ) The External RNA Control Consortium: a progress report . Nature Methods . 2 : 731 ± 734 . https://doi.org/10.1038/nmeth1005-731 PMID: 16179916
45. Staroscik , A. ( 2004 ) http://www.uri.edu/research/gsc/resources/cndna.html.
46. Applied Biosystem. ( 2006 ) Amplification efficiency of Tagman gene expression assays . Application Note 5pp. Publication 127AP05-03.
47. Karhumaa K. , Fromanger R. , Hahn-Hagerdal B. and Gorwa-Grauslund M.F. ( 2007 ) High activity of xylose reductase and xylitol dehydrogenase improves xylose fermentation by recombinant Saccharomyces cerevisiae . Appl. Microbiol. Biotechnol . 73 : 1039 ± 1046 . https://doi.org/10.1007/s00253-006 - 0575-3 PMID: 16977466
48. Regenberg B. , Grotkjaer T. , Winther O. , Fausboll A. , Akesson M. , Bro C. et al. ( 2006 ) Growth-rate regulated genes have profound impact on interpretation of transcriptome profiling in Saccharomyces cerevisiae . Genome Biol . 7 : R107 . https://doi.org/10.1186/gb-2006 -7-11-r107 PMID: 17105650
49. Fletcher S.F. , Kwee I.L. , Nakada T. , Largman C. and Martin B.M. ( 1992 ) DNA sequence of the yeast transketolase gene . Biochem . 31 : 1892 ± 1896 .
50. Byrne K.P. and Wolfe K.H. ( 2005 ) The yeast gene browser: Combining curated homology and syntenic context reveals gene fate in polyploidy species . Genome Res . 15 : 1456 ± 1461 . https://doi.org/10.1101/ gr.3672305 PMID: 16169922
51. Schaaff I. , Hohmann S. and Zimmermann F.K. ( 1990 ) Molecular analysis of the structural gene for yeast transaldolase . Eur. J. Biochem . 188 : 597 ± 603 . PMID: 2185015
52. Huang H. , Rong H. , Li X. , Tong S. , Zhu Z. , Niu L . et al. ( 2008 ) The crital structure and identification of NQM1/YGR043C, a transaldolase from Saccharomyces cerevisiae . Proteins . 73 : 1076 ± 1081 . https:// doi.org/10.1002/prot.22237 PMID: 18831051
53. Walfridsson M. , Hallborn J. , Penttila M. , Keranen S. and Hahn-Hagerdal B. ( 1995 ) Xylose-metabolizing Saccharomyces cerevisiae strains overexpressing the TKL1 and TAL1 genes encoding the pentose phosphate pathway enzymes transketolase and transaldolase . Appl. Environ. Microbiol . 61 : 4184 ± 4190 . PMID: 8534086
54. Johansson B. and Hahn-Hagerdal B. ( 2002 ) The non-oxidative pentose phosphate pathway controls the fermentation rate of xylulose but not of xylose in Saccharomyces cerevisiae TMB3001 . FEMS Yeast Res . 2 : 277 ± 282 . PMID: 12702276
55. Karhumaa K. , Hahn-Hagerdal B. and Gorwa-Grauslund M.F. ( 2005 ) Investigation of limiting metabolic steps in the utilization of xylose by recombinant Saccharomyces cerevisiae using metabolic engineering . Yeast . 22 : 359 ± 368 . https://doi.org/10.1002/yea.1216 PMID: 15806613
56. Matsushika A. , Goshimaa T. , Fujii T. , Inouea H. , Sawayama S. and Yanoa S. ( 2012 ) Characterization of non-oxidative transaldolase and transketolase enzymes in the pentose phosphate pathway with regard to xylose utilization by recombinant Saccharomyces cerevisiae . Enzyme Microbiol. Technol . 51 : 16 ± 25 .
57. Salusjarvi L. , Poutanen M. , Pitkanen J.P. , Koivistoinen H. , Aristidou A. , Kalkkinen N . et al. ( 2003 ) Proteome analysis of recombinant xylose-fermenting Saccharomyces cerevisiae . Yeast . 20 : 295 ± 314 . https://doi.org/10.1002/yea.960 PMID: 12627397
58. Carter A.T. , Narbad A. , Pearson B.M. , Beck K-F. , Baum B. , Logghe M. et al. ( 1994 ) Phosphoribosylpyrophosphate synthetase (PRS): a new gene family in Saccharomyces cerevisiae . Yeast . 10 : 1031 ± 1044 . https://doi.org/10.1002/yea.320100805 PMID: 7992503
59. Hernando Y. , Carter A. , Parr A. , Hove-Jensen B. and Schweizer M. ( 1999 ) Genetic analysis and enzyme activity suggest the existence of more than one minimal functional unit capable of synthesizing phosphoribosyl pyrophosphate in Saccharomyces cerevisiae . J. Biol. Chem . 274 : 12480 ± 12487 . PMID: 10212224
60. Rintala E. , Toivari M. , Pitkanen J.-P. , Wiebe M.G. , Ruohonen L . et al. ( 2009 ) Low oxygen levels as a trigger for enhancement of respiratory metabolism in Saccharomyces cerevisiae . BMC Genomics . 10 :461. https://doi.org/10.1186/ 1471 -2164-10-461 PMID: 19804647 Zhou Q ., Liu Z.L. , Ning K. , Wang A. , Zeng X. and Xu J. ( 2014 ) Genomic and transcriptome analyses reveal that MAPK- and phosphatidylinositol-signaling pathways mediate tolerance to 5-hydroxymethyl2-furaldehyde for industrial yeast Saccharomyces cerevisiae . Sci. Reports . 4 : 6556 .
Liu Z.L. ( 2006 ) Genomic adaptation of ethanologenic yeast to biomass conversion inhibitors . Appl. Microbiol. Biotechnol . 73 : 27 ± 36 . https://doi.org/10.1007/s00253-006-0567-3 PMID: 17028874 Argueso J.L. , Carazzolle M.F. , Mieczkowski P.A. , Duarte F.M. , Netto O.V.C. , Missawa S.K . et al. ( 2009 ) Genome structure of a Saccharomyces cerevisiae strain widely used in bioethanol production . Genome Res . 19 : 2258 ± 2270 . https://doi.org/10.1101/gr.091777.109 PMID: 19812109 Ronald J., Tang H. and Brem R.B. ( 2006 ) Genomewide evolutionary rates in laboratory and wild yeast . Genetics . 174 : 541 ± 544 . https://doi.org/10.1534/genetics.106.060863 PMID: 16816417 Matsushika A ., Goshima T. and Hshino T. ( 2014 ) Transcription analysis of recombinant industrial and laboratory Saccharomyces cerevisiae strains reveals the olecular basis for fermentation of glucose and xylose . Microbial Cell Factories . 13 :16. https://doi.org/10.1186/ 1475 -2859-13-16 PMID: 24467867