Copy number variants impact phenotype-genotype relationships for adaptation of industrial yeast Saccharomyces cerevisiae.

The industrial yeast Saccharomyces cerevisiae possesses a plastic genome enabling its adaptation to varied environment conditions. A more robust ethanologenic industrial yeast strain NRRL Y-50049 was obtained through laboratory adaptation that is resistant to 2-furaldehyde (furfural) and 5-hydroxymethyl-2-furaldehyde (HMF), a major class of toxic chemicals associated with lignocellulose-to-biofuel conversion. A significant amount of knowledge has been achieved in characterizing its tolerant phenotypes and molecular mechanisms of the resistance. Recent findings on a limited number of nonsynonymous SNP (single nucleotide polymorphism) detected in NRRL Y-50049 compared with its progenitor NRRL Y-12632 raised doubt of SNP roles in the tolerance adaptation. The genotype-phenotype relationship for yeast adaptation to the toxic chemicals is yet unclear. Here, we examine copy number variant (CNV) of the adapted strain NRRL Y-50049 to address phenotype-genotype relationships. As a background information, CNV of model strain S288C of the reference genome was also examined versus the industrial-type strain NRRL Y-12632. More than 200 CNVs, mostly duplication events, were detected in NRRL Y-12632 compared with the laboratory model strain S288C. Such enriched genetic background supports its more diversified phenotype response for the industrial yeast than the laboratory strain S288C. Comparing the two industrial strains, we found extra nine CNVs in the mitochondrial genome and 28 CNVs in the nuclear genome of NRRL Y-50049 versus its progenitor NRRL Y-12632. Continued DNA recombination event and high rate of CNV observed in NRRL Y-50049 versus its progenitor suggests that CNV is more impactful than SNP in association with phenotype-genotype relationships of yeast adaptation to the toxic chemical stress. COX1 and COB loci were defined as DNA recombination hotspots in the mitochondrial genome for the industrial yeast based on the high frequency of CNVs observed in these loci. KEY POINTS: • COX1 and COB loci are identified as DNA recombination hotspots for the industrial yeast. • The industrial yeast type strain NRRL Y-12632 possesses more CNVs vs the reference genome S288C. • CNV is more important than SNP on phenotype-genotype relationships for yeast adaptation.

Reasons for 2-furaldehyde and 5-hydroxymethyl-2-furaldehyde resistance in Saccharomyces cerevisiae: current state of knowledge and perspectives for further improvements.

Common toxic compounds 2-furaldehyde (furfural) and 5-hydroxymethyl-2-furaldehyde (HMF) are formed from dehydration of pentose and hexose, respectively, during decomposition of lignocellulosic biomass polymers. Furfural and HMF represent a major class of aldehyde toxic chemicals that inhibit microbial growth and interfere with subsequent fermentation for production of renewable fuels and chemicals. Understanding mechanisms of yeast tolerance aids development of tolerant strains as the most economic means to overcome the toxicity. This review updates current knowledge on yeast resistance to these toxic chemicals obtained from rapid advances in the past few years. Findings are largely exemplified by an adapted strain NRRL Y-50049 compared with its progenitor, the industrial yeast Saccharomyces cerevisiae type strain NRRL Y-12632. Newly characterized molecular phenotypes distinguished acquired resistant components of Y-50049 from innate stress response of its progenitor Y-12632. These findings also raised important questions on how to address more deeply ingrained changes in addition to local renovations for yeast adaptation. An early review on understandings of yeast tolerance to these inhibitory compounds is available and its contents omitted here to avoid redundancy. Controversial and confusing issues on identification of yeast resistance to furfural and HMF are further clarified aiming improved future research. Propositions and perspectives on research understanding molecular mechanisms of yeast resistance and future improvements are also presented. KEY POINTS: • Distinguished adapted resistance from innate stress response in yeast. • Defined pathway-based molecular phenotypes of yeast resistance. • Proposed genomic insight and perspectives on yeast resistance and adaptation.

A glimpse of potential transposable element impact on adaptation of the industrial yeast Saccharomyces cerevisiae.

The adapted industrial yeast strain Saccharomyces cerevisiae NRRL Y-50049 is able to in situ detoxify major toxic aldehyde compounds derived from sugar conversion of lignocellulosic biomass while producing ethanol. Pathway-based studies on its mechanisms of tolerance have been reported previously, however, little is known about transposable element (TE) involvement in its adaptation to inhibitory compounds. This work presents a comparative dynamic transcription expression analysis in response to a toxic treatment between Y-50049 and its progenitor, an industrial type strain NRRL Y-12632, using a time-course study. At least 77 TEs from Y-50049 showed significantly increased expression compared with its progenitor, especially during the late lag phase. Sequence analysis revealed significant differences in TE sequences between the two strains. Y-50049 was also found to have a transposons of yeast 2 (Ty2) long terminal repeat-linked YAT1 gene showing significantly higher copy number changes than its progenitor. These results raise awareness of potential TE involvement in the adaptation of industrial yeast to the tolerance of toxic chemicals.

Pathway-based signature transcriptional profiles as tolerance phenotypes for the adapted industrial yeast Saccharomyces cerevisiae resistant to furfural and HMF.

The industrial yeast Saccharomyces cerevisiae has a plastic genome with a great flexibility in adaptation to varied conditions of nutrition, temperature, chemistry, osmolarity, and pH in diversified applications. A tolerant strain against 2-furaldehyde (furfural) and 5-hydroxymethyl-2-furaldehyde (HMF) was successfully obtained previously by adaptation through environmental engineering toward development of the next-generation biocatalyst. Using a time-course comparative transcriptome analysis in response to a synergistic challenge of furfural-HMF, here we report tolerance phenotypes of pathway-based transcriptional profiles as components of the adapted defensive system for the tolerant strain NRRL Y-50049. The newly identified tolerance phenotypes were involved in biosynthesis superpathway of sulfur amino acids, defensive reduction-oxidation reaction process, cell wall response, and endogenous and exogenous cellular detoxification. Key transcription factors closely related to these pathway-based components, such as Yap1, Met4, Met31/32, Msn2/4, and Pdr1/3, were also presented. Many important genes in Y-50049 acquired an enhanced transcription background and showed continued increased expressions during the entire lag phase against furfural-HMF. Such signature expressions distinguished tolerance phenotypes of Y-50049 from the innate stress response of its progenitor NRRL Y-12632, an industrial type strain. The acquired yeast tolerance is believed to be evolved in various mechanisms at the genomic level. Identification of legitimate tolerance phenotypes provides a basis for continued investigations on pathway interactions and dissection of mechanisms of yeast tolerance and adaptation at the genomic level.

Protein expression analysis revealed a fine-tuned mechanism of in situ detoxification pathway for the tolerant industrial yeast Saccharomyces cerevisiae.

Inhibitory compounds liberated from lignocellulose pretreatment are representative toxic chemicals that repress microbial growth and metabolism. A tolerant strain of the industrial yeast Saccharomyces cerevisiae is able to detoxify a major class of toxic compounds while producing ethanol. Knowledge on the yeast tolerance was mostly obtained by gene expression analysis and limited protein expression evidence is yet available underlying the yeast adaptation. Here we report a comparative protein expression profiling study on Y-50049, a tolerant strain compared with its parental industrial type strain Y-12632. We found a distinctive protein expression of glucose-6-phosphate dehydrogenase (Zwf1) in Y-50049 but not in Y-12632, in the relatively conserved glycolysis and pentose phosphate pathway (PPP) in response to a combinational challenge of 2-furaldehyde (furfural) and 5-hydroxymethyl-2-furaldehyde (HMF). A group of proteins with aldehyde reduction activity was uniquely induced expressed in Y-50049 but not in Y-12632. Such evidence allowed fine-tuning a mechanism of the renovated in situ detoxification by Y-50049. As the key protein, Zwf1 drove the glucose metabolism in favor of the oxidative branch of the PPP facilitating in situ detoxification of the toxic chemicals by Y-50049. The activated expression of Zwf1 generated the essential cofactor nicotinamide adenine dinucleotide phosphate (NADPH) enabling reduction of furfural and HMF through a group of aldehyde reduction enzymes. In return, the activate aldehyde reductions released desirable feedbacks of NADP+ stimulating continued oxidative activity of Zwf1. Thus, a well-maintained cofactor regeneration cycle was established to restore the cofactor imbalance caused by furfural-HMF. Challenges and perspectives on adaptation of significantly differential expressions of ribosomal proteins and other unique proteins are also discussed.

Improved cellulosic ethanol production from corn stover with a low cellulase input using a ß-glucosidase-producing yeast following a dry biorefining process.

A low-cost and sustainable cellulosic ethanol production is vital for fermentation-based industrial applications. Reducing the expenses of cellulose-deconstruction enzymes is one of the significant challenges to economic cellulose-to-ethanol conversion. Here, we report the improved ethanol production from corn stover after dry biorefining using a natural ß-glucosidase-producing strain Clavispora NRRL Y-50464 with a low cellulase dose of 5 mg protein/g glucan under separate enzymatic hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) conditions. Strain Clavispora NRRL Y-50464 exhibited a superior ethanol fermentation performance over Saccharomyces cerevisiae DQ1 under both conditions. It produced an ethanol titer of 38.1 g/L within 96 h at a conversion efficiency of 55.5% with 25% solids loading (w/w) via SSF without addition of extra ß-glucosidase supplement. Improved performance of Y-50464 on a bioreactor with a helical stirring apparatus confirmed its advantage over the conventional bioreactors originally designed for liquid fermentations in cellulosic ethanol conversion by SSF. The results of this study suggested that the strain Clavispora NRRL Y-50464 has a potential as a candidate for lower-cost cellulosic ethanol production from lignocellulosic materials.

ChiNet uncovers rewired transcription subnetworks in tolerant yeast for advanced biofuels conversion.

Analysis of rewired upstream subnetworks impacting downstream differential gene expression aids the delineation of evolving molecular mechanisms. Cumulative statistics based on conventional differential correlation are limited for subnetwork rewiring analysis since rewiring is not necessarily equivalent to change in correlation coefficients. Here we present a computational method ChiNet to quantify subnetwork rewiring by statistical heterogeneity that enables detection of potential genotype changes causing altered transcription regulation in evolving organisms. Given a differentially expressed downstream gene set, ChiNet backtracks a rewired upstream subnetwork from a super-network including gene interactions known to occur under various molecular contexts. We benchmarked ChiNet for its high accuracy in distinguishing rewired artificial subnetworks, in silico yeast transcription-metabolic subnetworks, and rewired transcription subnetworks for Candida albicans versus Saccharomyces cerevisiae, against two differential-correlation based subnetwork rewiring approaches. Then, using transcriptome data from tolerant S. cerevisiae strain NRRL Y-50049 and a wild-type intolerant strain, ChiNet identified 44 metabolic pathways affected by rewired transcription subnetworks anchored to major adaptively activated transcription factor genes YAP1, RPN4, SFP1 and ROX1, in response to toxic chemical challenges involved in lignocellulose-to-biofuels conversion. These findings support the use of ChiNet in rewiring analysis of subnetworks where differential interaction patterns resulting from divergent nonlinear dynamics abound.

GRE2 from Scheffersomyces stipitis as an aldehyde reductase contributes tolerance to aldehyde inhibitors derived from lignocellulosic biomass.

Scheffersomyces (Pichia) stipitis is one of the most promising yeasts for industrial bioethanol production from lignocellulosic biomass. S. stipitis is able to in situ detoxify aldehyde inhibitors (such as furfural and 5-hydroxymethylfurfural (HMF)) to less toxic corresponding alcohols. However, the reduction enzymes involved in this reaction remain largely unknown. In this study, we reported that an uncharacterized open reading frame PICST_72153 (putative GRE2) from S. stipitis was highly induced in response to furfural and HMF stresses. Overexpression of this gene in Saccharomyces cerevisiae improved yeast tolerance to furfural and HMF. GRE2 was identified as an aldehyde reductase which can reduce furfural to FM with either NADH or NADPH as the co-factor and reduce HMF to FDM with NADPH as the co-factor. This enzyme can also reduce multiple aldehydes to their corresponding alcohols. Amino acid sequence analysis indicated that it is a member of the subclass "intermediate" of the short-chain dehydrogenase/reductase (SDR) superfamily. Although GRE2 from S. stipitis is similar to GRE2 from S. cerevisiae in a three-dimensional structure, some differences were predicted. GRE2 from S. stipitis forms loops at D133-E137 and T143-N145 locations with two α-helices at E154-K157 and E252-A254 locations, different GRE2 from S. cerevisiae with an α-helix at D133-E137 and a ß-sheet at T143-N145 locations, and two loops at E154-K157 and E252-A254 locations. This research provided guidelines for the study of other SDR enzymes from S. stipitis and other yeasts on tolerant mechanisms to aldehyde inhibitors derived from lignocellulosic biomass.

Direct enzyme assay evidence confirms aldehyde reductase function of Ydr541cp and Ygl039wp from Saccharomyces cerevisiae.

The aldehyde reductase gene ARI1 is a recently characterized member of an intermediate subfamily within the short-chain dehydrogenase/reductase (SDR) superfamily that clarified mechanisms of in situ detoxification of 2-furaldehyde and 5-hydroxymethyl-2-furaldehyde by Saccharomyces cerevisiae. Uncharacterized open reading frames (ORFs) are common among tolerant candidate genes identified for lignocellulose-to-advanced biofuels conversion. This study presents partially purified proteins of two ORFs, YDR541C and YGL039W, and direct enzyme assay evidence against aldehyde-inhibitory compounds commonly encountered during lignocellulosic biomass fermentation processes. Each of the partially purified proteins encoded by these ORFs showed a molecular mass of approximately 38 kDa, similar to Ari1p, a protein encoded by aldehyde reductase gene. Both proteins demonstrated strong aldehyde reduction activities toward 14 aldehyde substrates, with high levels of reduction activity for Ydr541cp toward both aromatic and aliphatic aldehydes. While Ydr541cp was observed to have a significantly higher specific enzyme activity at 20 U/mg using co-factor NADPH, Ygl039wp displayed a NADH preference at 25 U/mg in reduction of butylaldehyde. Amino acid sequence analysis identified a characteristic catalytic triad, Ser, Tyr and Lys; a conserved catalytic motif of Tyr-X-X-X-Lys; and a cofactor-binding sequence motif, Gly-X-X-Gly-X-X-Ala, near the N-terminus that are shared by Ydr541cp, Ygl039wp, Yol151wp/GRE2 and Ari1p. Findings of aldehyde reductase genes contribute to the yeast gene annotation and aids development of the next-generation biocatalyst for advanced biofuels production.

Discovery of new strains for furfural degradation using adaptive laboratory evolution in Saccharomyces cerevisiae.

In industrial production, the excessive discharge of furfural can pose harm to soil microorganisms, aquatic animals and plants, as well as humans. Therefore, it is crucial to develop efficient and cost-effective methods for degrading furfural in the environment. Currently, the use of Saccharomyces cerevisiae for furfural degradation in water has shown effectiveness, but there is a need to explore improved efficiency and tolerance in S. cerevisiae for this purpose. In this study, we isolated and evolved highly efficient furfural degradation strains, namely YBA_08 and F60C. These strains exhibited remarkable capabilities, degrading 59% and 99% furfural in the YPD medium after 72 h of incubation, significantly higher than the 31% achieved by the model strain S288C. Through analysis of the efficient degradation mechanism in the evolutionary strain F60C, we discovered a 326% increase in the total amount of NADH and NADPH. This increase likely promotes faster furfural degradation through intracellular aldehyde reductases. Moreover, the decrease in NADPH content led to a 406% increase in glutathione content at the background level, which protects cells from damage caused by reactive oxygen species. Mutations and differential expression related to cell cycle and cell wall synthesis were observed, enabling cell survival in the presence of furfural and facilitating rapid furfural degradation and growth recovery. Based on these findings, it is speculated that strains YBA_08 and F60C have the potential to contribute to furfural degradation in water and the production of furfuryl alcohol, ethanol, and FDCA in biorefinery processes.

Kinetic mechanism of an aldehyde reductase of Saccharomyces cerevisiae that relieves toxicity of furfural and 5-hydroxymethylfurfural.

An effective means of relieving the toxicity of furan aldehydes, furfural (FFA) and 5-hydroxymethylfurfural (HMF), on fermenting organisms is essential for achieving efficient fermentation of lignocellulosic biomass to ethanol and other products. Ari1p, an aldehyde reductase from Saccharomyces cerevisiae, has been shown to mitigate the toxicity of FFA and HMF by catalyzing the NADPH-dependent conversion to corresponding alcohols, furfuryl alcohol (FFOH) and 5-hydroxymethylfurfuryl alcohol (HMFOH). At pH 7.0 and 25°C, purified Ari1p catalyzes the NADPH-dependent reduction of substrates with the following values (k(cat) (s(-1)), k(cat)/K(m) (s(-1)mM(-1)), K(m) (mM)): FFA (23.3, 1.82, 12.8), HMF (4.08, 0.173, 23.6), and dl-glyceraldehyde (2.40, 0.0650, 37.0). When acting on HMF and dl-glyceraldehyde, the enzyme operates through an equilibrium ordered kinetic mechanism. In the physiological direction of the reaction, NADPH binds first and NADP(+) dissociates from the enzyme last, demonstrated by k(cat) of HMF and dl-glyceraldehyde that are independent of [NADPH] and (K(ia)(NADPH)/k(cat)) that extrapolate to zero at saturating HMF or dl-glyceraldehyde concentration. Microscopic kinetic parameters were determined for the HMF reaction (HMF+NADPH↔HMFOH+NADP(+)), by applying steady-state, presteady-state, kinetic isotope effects, and dynamic modeling methods. Release of products, HMFOH and NADP(+), is 84% rate limiting to k(cat) in the forward direction. Equilibrium constants, [NADP(+)][FFOH]/[NADPH][FFA][H(+)]=5600×10(7)M(-1) and [NADP(+)][HMFOH]/[NADPH][HMF][H(+)]=4200×10(7)M(-1), favor the physiological direction mirrored by the slowness of hydride transfer in the non-physiological direction, NADP(+)-dependent oxidation of alcohols (k(cat) (s(-1)), k(cat)/K(m) (s(-1)mM(-1)), K(m) (mM)): FFOH (0.221, 0.00158, 140) and HMFOH (0.0105, 0.000104, 101).

Cellulosic Ethanol Production Using a Dual Functional Novel Yeast.

Reducing the cost of cellulosic ethanol production, especially for cellulose hydrolytic enzymes, is vital to growing a sustainable and efficient cellulosic ethanol industry and bio-based economy. Using an ethanologenic yeast able to produce hydrolytic enzymes, such as Clavispora NRRL Y-50464, is one solution. NRRL Y-50464 is fast-growing and robust, and tolerates inhibitory compounds 2-furaldehyde (furfural) and 5-hydroxymethyl-2-furaldehyde (HMF) associated with lignocellulose-to-fuel conversion. It produces three forms of ß-glucosidase isozymes, BGL1, BGL2, and BGL3, and ferment cellobiose as the sole carbon source. These ß-glucosidases exhibited desirable enzyme kinetic parameters and high levels of enzyme-specific activity toward cellobiose and many oligosaccharide substrates. They tolerate the product inhibition of glucose and ethanol, and are stable to temperature and pH conditions. These characteristics are desirable for more efficient cellulosic ethanol production by simultaneous saccharification and fermentation. NRRL Y-50464 provided the highest cellulosic ethanol titers and conversion rates at lower cellulase loadings, using either pure cellulose or agricultural residues, as so far reported in the literature. This review summarizes NRRL Y-50464 performance on cellulosic ethanol production from refined cellulose, rice straw, and corn stover processed in various ways, in the presence or absence of furfural and HMF. This dual functional yeast has potential to serve as a prototype for the development of next-generation biocatalysts. Perspectives on continued strain development and process engineering improvements for more efficient cellulosic ethanol production from lignocellulosic materials are also discussed.

Comparative transcription profiling analyses of maize reveals candidate defensive genes for seedling resistance against corn earworm.

As maize seedlings germinate into the soil, they encounter an environment teeming with insects seeking rich sources of nutrition. Maize presumably has developed a number of molecular mechanisms to ensure survival at the beginning of its life cycle. Comparative transcription analysis using microarrays was utilized to document the expression of a number of genes with potential defensive functions in seedling tissue. In addition to elevated levels of the genes involved in the biosynthesis of DIMBOA (2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one), an anti-insect resistance molecule, other highly expressed genes in the seedling encode the following putative defensive proteins: defensin, hydroxyproline and proline-rich protein, thaumatin-like protein, lipase, cystatin, protease inhibitor, and a variety of proteases. The potential resistance genes identified occurred mainly on chromosomes 1 and 5 in the B73 genome. Analysis of promoters of seven DIMBOA biosynthetic genes identified three transcription factor binding sites that are possibly involved in regulation of the DIMBOA biosynthetic pathway. The results indicate that maize employs a wide variety of potential resistance mechanisms in seedling tissue to resist a possible insect attack.

Repression of xylose-specific enzymes by ethanol in Scheffersomyces (Pichia) stipitis and utility of repitching xylose-grown populations to eliminate diauxic lag.

During the fermentation of lignocellulosic hydrolyzates to ethanol by native pentose-fermenting yeasts such as Scheffersomyces (Pichia) stipitis NRRL Y-7124 (CBS 5773) and Pachysolen tannophilus NRRL Y-2460, the switch from glucose to xylose uptake results in a diauxic lag unless process strategies to prevent this are applied. When yeast were grown on glucose and resuspended in mixed sugars, the length of this lag was observed to be a function of the glucose concentration consumed (and consequently, the ethanol concentration accumulated) prior to the switch from glucose to xylose fermentation. At glucose concentrations of 95 g/L, the switch to xylose utilization was severely stalled such that efficient xylose fermentation could not occur. Further investigation focused on the impact of ethanol on cellular xylose transport and the induction and maintenance of xylose reductase and xylitol dehydrogenase activities when large cell populations of S. stipitis NRRL Y-7124 were pre-grown on glucose or xylose and then presented mixtures of glucose and xylose for fermentation. Ethanol concentrations around 50 g/L fully repressed enzyme induction although xylose transport into the cells was observed to be occurring. Increasing degrees of repression were documented between 15 and 45 g/L ethanol. Repitched cell populations grown on xylose resulted in faster fermentation rates, particularly on xylose but also on glucose, and eliminated diauxic lag and stalling during mixed sugar conversion by P. tannophilus or S. stipitis, despite ethanol accumulations in the 60 or 70 g/L range, respectively. The process strategy of priming cells on xylose was key to the successful utilization of high mixed sugar concentrations because specific enzymes for xylose utilization could be induced before ethanol concentration accumulated to an inhibitory level.

Molecular mechanisms of yeast tolerance and in situ detoxification of lignocellulose hydrolysates.

Pretreatment of lignocellulose biomass for biofuel production generates inhibitory compounds that interfere with microbial growth and subsequent fermentation. Remediation of the inhibitors by current physical, chemical, and biological abatement means is economically impractical, and overcoming the inhibitory effects of lignocellulose hydrolysate poses a significant technical challenge for lower-cost cellulosic ethanol production. Development of tolerant ethanologenic yeast strains has demonstrated the potential of in situ detoxification for numerous aldehyde inhibitors derived from lignocellulose biomass pretreatment and conversion. In the last decade, significant progress has been made in understanding mechanisms of yeast tolerance for tolerant strain development. Enriched genetic backgrounds, enhanced expression, interplays, and global integration of many key genes enable yeast tolerance. Reprogrammed pathways support yeast functions to withstand the inhibitor stress, detoxify the toxic compounds, maintain energy and redox balance, and complete active metabolism for ethanol fermentation. Complex gene interactions and regulatory networks as well as co-regulation are well recognized as involved in yeast adaptation and tolerance. This review presents our current knowledge on mechanisms of the inhibitor detoxification based on molecular studies and genomic-based approaches. Our improved understanding of yeast tolerance and in situ detoxification provide insight into phenotype-genotype relationships, dissection of tolerance mechanisms, and strategies for more tolerant strain development for biofuels applications.

Comparative transcriptome profiling analyses during the lag phase uncover YAP1, PDR1, PDR3, RPN4, and HSF1 as key regulatory genes in genomic adaptation to the lignocellulose derived inhibitor HMF for Saccharomyces cerevisiae.

BACKGROUND: The yeast Saccharomyces cerevisiae is able to adapt and in situ detoxify lignocellulose derived inhibitors such as furfural and HMF. The length of lag phase for cell growth in response to the inhibitor challenge has been used to measure tolerance of strain performance. Mechanisms of yeast tolerance at the genome level remain unknown. Using systems biology approach, this study investigated comparative transcriptome profiling, metabolic profiling, cell growth response, and gene regulatory interactions of yeast strains and selective gene deletion mutations in response to HMF challenges during the lag phase of growth. RESULTS: We identified 365 candidate genes and found at least 3 significant components involving some of these genes that enable yeast adaptation and tolerance to HMF in yeast. First, functional enzyme coding genes such as ARI1, ADH6, ADH7, and OYE3, as well as gene interactions involved in the biotransformation and inhibitor detoxification were the direct driving force to reduce HMF damages in cells. Expressions of these genes were regulated by YAP1 and its closely related regulons. Second, a large number of PDR genes, mainly regulated by PDR1 and PDR3, were induced during the lag phase and the PDR gene family-centered functions, including specific and multiple functions involving cellular transport such as TPO1, TPO4, RSB1, PDR5, PDR15, YOR1, and SNQ2, promoted cellular adaptation and survival in order to cope with the inhibitor stress. Third, expressed genes involving degradation of damaged proteins and protein modifications such as SHP1 and SSA4, regulated by RPN4, HSF1, and other co-regulators, were necessary for yeast cells to survive and adapt the HMF stress. A deletion mutation strain Δrpn4 was unable to recover the growth in the presence of HMF. CONCLUSIONS: Complex gene interactions and regulatory networks as well as co-regulations exist in yeast adaptation and tolerance to the lignocellulose derived inhibitor HMF. Both induced and repressed genes involving diversified functional categories are accountable for adaptation and energy rebalancing in yeast to survive and adapt the HMF stress during the lag phase of growth. Transcription factor genes YAP1, PDR1, PDR3, RPN4, and HSF1 appeared to play key regulatory rules for global adaptation in the yeast S. cerevisiae.

Stereochemistry of furfural reduction by a Saccharomyces cerevisiae aldehyde reductase that contributes to in situ furfural detoxification.

Ari1p from Saccharomyces cerevisiae, recently identified as an intermediate-subclass short-chain dehydrogenase/reductase, contributes in situ to the detoxification of furfural. Furfural inhibits efficient ethanol production by yeast, particularly when the carbon source is acid-treated lignocellulose, which contains furfural at a relatively high concentration. NADPH is Ari1p's best known hydride donor. Here we report the stereochemistry of the hydride transfer step, determined by using (4R)-[4-(2)H]NADPD and (4S)-[4-(2)H]NADPD and unlabeled furfural in Ari1p-catalyzed reactions and following the deuterium atom into products 2-furanmethanol or NADP(+). Analysis of the products demonstrates unambiguously that Ari1p directs hydride transfer from the si face of NADPH to the re face of furfural. The singular orientation of substrates enables construction of a model of the Michaelis complex in the Ari1p active site. The model reveals hydrophobic residues near the furfural binding site that, upon mutation, may increase specificity for furfural and enhance enzyme performance. Using (4S)-[4-(2)H]NADPD and NADPH as substrates, primary deuterium kinetic isotope effects of 2.2 and 2.5 were determined for the steady-state parameters k(cat)(NADPH) and k(cat)/K(m)(NADPH), respectively, indicating that hydride transfer is partially rate limiting to catalysis.

Mechanisms of ethanol tolerance in Saccharomyces cerevisiae.

Saccharomyces cerevisiae is a superb ethanol producer, yet is also sensitive to higher ethanol concentrations especially under high gravity or very high gravity fermentation conditions. Ethanol tolerance is associated with interplay of complex networks at the genome level. Although significant efforts have been made to study ethanol stress response in past decades, mechanisms of ethanol tolerance are not well known. With developments of genome sequencing and genomic technologies, our understanding of yeast biology has been revolutionarily advanced. More evidence of mechanisms of ethanol tolerance have been discovered involving multiple loci, multi-stress, and complex interactions as well as signal transduction pathways and regulatory networks. Transcription dynamics and profiling studies of key gene sets including heat shock proteins provided insight into tolerance mechanisms. A transient gene expression response or a stress response to ethanol does not necessarily lead to ethanol tolerance in yeast. Reprogrammed pathways and interactions of cofactor regeneration and redox balance observed from studies of tolerant yeast demonstrated the significant importance of a time-course study for ethanol tolerance. In this review, we focus on current advances of our understanding for ethanol-tolerance mechanisms of S. cerevisiae including gene expression responses, pathway-based analysis, signal transduction and regulatory networks. A prototype of global system model for mechanisms of ethanol tolerance is presented.

Evolutionarily engineered ethanologenic yeast detoxifies lignocellulosic biomass conversion inhibitors by reprogrammed pathways.

Lignocellulosic biomass conversion inhibitors, furfural and HMF, inhibit microbial growth and interfere with subsequent fermentation of ethanol, posing significant challenges for a sustainable cellulosic ethanol conversion industry. Numerous yeast genes were found to be associated with the inhibitor tolerance. However, limited knowledge is available about mechanisms of the tolerance and the detoxification of the biomass conversion inhibitors. Using a robust standard for absolute mRNA quantification assay and a recently developed tolerant ethanologenic yeast Saccharomyces cerevisiae NRRL Y-50049, we investigate pathway-based transcription profiles relevant to the yeast tolerance and the inhibitor detoxification. Under the synergistic inhibitory challenges by furfural and HMF, Y-50049 was able to withstand the inhibitor stress, in situ detoxify furfural and HMF, and produce ethanol, while its parental control Y-12632 failed to function till 65 h after incubation. The tolerant strain Y-50049 displayed enriched genetic background with significantly higher abundant of transcripts for at least 16 genes than a non-tolerant parental strain Y-12632. The enhanced expression of ZWF1 appeared to drive glucose metabolism in favor of pentose phosphate pathway over glycolysis at earlier steps of glucose metabolisms. Cofactor NAD(P)H generation steps were likely accelerated by enzymes encoded by ZWF1, GND1, GND2, TDH1, and ALD4. NAD(P)H-dependent aldehyde reductions including conversion of furfural and HMF, in return, provided sufficient NAD(P)(+) for NAD(P)H regeneration in the yeast detoxification pathways. Enriched genetic background and a well maintained redox balance through reprogrammed expression responses of Y-50049 were accountable for the acquired tolerance and detoxification of furfural to furan methanol and HMF to furan dimethanol. We present significant gene interactions and regulatory networks involved in NAD(P)H regenerations and functional aldehyde reductions under the inhibitor stress.

Multiple gene-mediated NAD(P)H-dependent aldehyde reduction is a mechanism of in situ detoxification of furfural and 5-hydroxymethylfurfural by Saccharomyces cerevisiae.

Furfural and 5-hydroxymethylfurfural (HMF) are representative inhibitors generated from biomass pretreatment using dilute acid hydrolysis that interfere with yeast growth and subsequent fermentation. Few yeast strains tolerant to inhibitors are available. In this study, we report a tolerant strain, Saccharomyces cerevisiae NRRL Y-50049, which has enhanced biotransformation ability to convert furfural to furan methanol (FM), HMF to furan di-methanol (FDM), and produce a normal yield of ethanol. Our recent identification of HMF and development of protocol to synthesize the HMF metabolic conversion product FDM allowed studies on fermentation metabolic kinetics in the presence of HMF and furfural. Individual gene-encoding enzymes possessing aldehyde reduction activities demonstrated cofactor preference for NADH or NADPH. However, protein extract from whole yeast cells showed equally strong aldehyde reduction activities coupled with either cofactor. Deletion of a single candidate gene did not affect yeast growth in the presence of the inhibitors. Our results suggest that detoxification of furfural and HMF by the ethanologenic yeast S. cerevisiae strain Y-50049 likely involves multiple gene mediated NAD(P)H-dependent aldehyde reduction. Conversion pathways of furfural and HMF relevant to glycolysis and ethanol production were refined based on our findings in this study.