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.

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.

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.

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.

Signature pathway expression of xylose utilization in the genetically engineered industrial yeast Saccharomyces cerevisiae.

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 non-oxidative 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.

Tolerant industrial yeast Saccharomyces cerevisiae posses a more robust cell wall integrity signaling pathway against 2-furaldehyde and 5-(hydroxymethyl)-2-furaldehyde.

Cell wall integrity signaling pathway in Saccharomyces cerevisiae is a conserved function for detecting and responding to cell stress conditions but less understood for industrial yeast. We examined gene expression dynamics for a tolerant industrial yeast strain NRRL Y-50049 in response to challenges of furfural and HMF through comparative quantitative gene expression analysis using pathway-based qRT-PCR array assays. All tested genes from Y-50049, except for MLP2, demonstrated more resistant and significantly increased gene expression than that from a laboratory strain BY4741. While all five sensor encoding genes WSC1, WSC2, WSC3, MID2 and MTL1 from both strains were activated in response to the furfural-HMF treatment, WSC3 from Y-50049 demonstrated the most increased expression over time compared with any other sensor genes. These results suggested the industrial yeast poses more robust cell wall integrity pathway, and gene WSC3 could have the special capability for signal transmission against furfural and HMF. Among five single nucleotide variations discovered in WSC3 from Y-50049, three were found to be non-synonymous mutations resulting in amino acid alterations of Ser158 → Tyr158, Val186 → Ile186, and Glu430 → Asp430. Our results suggest the industrial yeast as a more desirable delivery vehicle for the next-generation biocatalyst development.

Two New Native ß-Glucosidases from Clavispora NRRL Y-50464 Confer Its Dual Function as Cellobiose Fermenting Ethanologenic Yeast.

Yeast strain Clavispora NRRL Y-50464 is able to produce cellulosic ethanol from lignocellulosic materials without addition of external ß-glucosidase by simultaneous saccharification and fermentation. A ß-glucosidase BGL1 protein from this strain was recently reported supporting its cellobiose utilization capability. Here, we report two additional new ß-glucosidase genes encoding enzymes designated as BGL2 and BGL3 from strain NRRL Y-50464. Quantitative gene expression was analyzed and the gene function of BGL2 and BGL3 was confirmed by heterologous expression using cellobiose as a sole carbon source. Each gene was cloned and partially purified protein obtained separately for direct enzyme assay using varied substrates. Both proteins showed the highest specific activity at pH 5 and relatively strong affinity with a Km of 0.08 and 0.18 mM for BGL2 and BGL3, respectively. The optimum temperature was found to be 50°C for BGL2 and 55°C for BGL3. Both proteins were able to hydrolyze 1,4 oligosaccharides evaluated in this study. They also showed a strong resistance to glucose product inhibition with a Ki of 61.97 and 38.33 mM for BGL2 and BGL3, respectively. While BGL3 was sensitive showing a significantly reduced activity to 4% ethanol, BGL2 demonstrated tolerance to ethanol. Its activity was enhanced in the presence of ethanol but reduced at concentrations greater than 16%. The presence of the fermentation inhibitors furfural and HMF did not affect the enzyme activity. Our results suggest that a ß-glucosidase gene family exists in Clavispora NRRL Y-50464 with at least three members in this group that validate its cellobiose hydrolysis functions for lower-cost cellulosic ethanol production. Results of this study confirmed the cellobiose hydrolysis function of strain NRRL Y-50464, and further supported this dual functional yeast as a candidate for lower-cost cellulosic ethanol production and next-generation biocatalyst development in potential industrial applications.

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.

Transcriptome analysis of Zymomonas mobilis ZM4 reveals mechanisms of tolerance and detoxification of phenolic aldehyde inhibitors from lignocellulose pretreatment.

BACKGROUND: Phenolic aldehydes generated from lignocellulose pretreatment exhibited severe toxic inhibitions on microbial growth and fermentation. Numerous tolerance studies against furfural, 5-hydroxymethyl-2-furaldehyde (HMF), acetate, and ethanol were reported, but studies on inhibition of phenolic aldehyde inhibitors are rare. For ethanologenic strains, Zymomonas mobilis ZM4 is high in ethanol productivity and genetic manipulation feasibility, but sensitive to phenolic aldehyde inhibitors. Molecular mechanisms of tolerance for Z. mobilis toward phenolic aldehydes are not known. RESULTS: We took the first insight into genomic response of Z. mobilis ZM4 to the phenolic aldehyde inhibitors derived from lignocellulose pretreatment. The results suggest that the toxicity to cells is caused by the functional group of phenolic aldehyde, similar to furfural and HMF, rather than aromatic groups or phenolic hydroxyl groups. Transcriptome response against 4-hydroxybenzaldehyde, syringaldehyde, and vanillin, representing phenolic groups H, S, and G, respectively, was investigated. The atlas of the important genes responsible for significantly enhanced and repressed genes at the genomic level was illustrated. 272 genes with twofold greater expressions than non-treated controls and 36 gene clusters in response to challenges of these phenolic aldehydes were identified. Several reductases encoded by ZMO1116, ZMO1696, and ZMO1885 were found to play the key roles in reducing phenolic aldehydes into the corresponding phenolic alcohols. Reduction of phenolic aldehydes by overexpression of ZMO1116, ZMO1696, and ZMO1885 in Z. mobilis ZM4 resulted in the increased inhibitor conversion and ethanol productivity, especially for 4-hydroxybenzaldehyde and vanillin. Several transporter genes such as ZMO0282, ZMO0283, ZMO0798, ZMO0799, and ZMO0800 was also displayed significantly increased expressions against the phenolic aldehydes. CONCLUSIONS: The genes encoding reductases are with potentials on phenolic aldehydes-tolerant genes contributing to the reduction of phenolic aldehydes into the corresponding phenolic alcohols forms for Z. mobilis ZM4. Overexpression of the key genes improved the conversion ratio and ethanol productivity of 4-hydroxybenzaldehyde and vanillin with high toxicity. New knowledge obtained from this research aids understanding the mechanisms of bacterial tolerance and the development of the next-generation biocatalysts for advanced biofuels production.

Virulence Structure of Blumeria graminis f. sp. tritici and Its Genetic Diversity by ISSR and SRAP Profiling Analyses.

Blumeria graminis f. sp. tritici, which causes wheat powdery mildew, is an obligate biotrophic pathogen that can easily genetically adapt to its host plant. Understanding the virulence structure of and genetic variations in this pathogen is essential for disease control and for breeding resistance to wheat powdery mildew. This study investigated 17 pathogenic populations in Sichuan, China and classified 109 isolates into two distinct groups based on pathogenicity analysis: high virulence (HV, 92 isolates) and low virulence (LV, 17 isolates). Populations from Yibin (Southern region), Xichang (Western region), and Meishan (Middle region) showed lower virulence frequencies than populations from other regions. Many of the previously known resistance genes did not confer resistance in this study. The resistance gene Pm21 displayed an immune response to pathogenic challenge with all populations in Sichuan, and Pm13, Pm5b, Pm2+6, and PmXBD maintained resistance. AMOVA revealed significantly higher levels of variation within populations and lower levels of variation among populations within regions. High levels of gene flow were detected among populations in the four regions. Closely related populations within each region were distinguished by cluster analyses using ISSR and SRAP alleles. Both ISSR and SRAP allele profiling analyses revealed high levels of genetic diversity among pathogenic populations in Sichuan. Although ISSR and SRAP profiling analysis showed similar resolutions, the SRAP alleles appeared to be more informative. We did not detect any significant association between these alleles and the virulence or pathogenicity of the pathogen. Our results suggest that ISSR and SRAP alleles are more efficient for the characterization of small or closely related populations versus distantly related populations.

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.

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.

Genomic and transcriptome analyses reveal that MAPK- and phosphatidylinositol-signaling pathways mediate tolerance to 5-hydroxymethyl-2-furaldehyde for industrial yeast Saccharomyces cerevisiae.

The industrial yeast Saccharomyces cerevisiae is a traditional ethanologenic agent and a promising biocatalyst for advanced biofuels production using lignocellulose materials. Here we present the genomic background of type strain NRRL Y-12632 and its transcriptomic response to 5-hydroxymethyl-2-furaldehyde (HMF), a commonly encountered toxic compound liberated from lignocellulosic-biomass pretreatment, in dissecting the genomic mechanisms of yeast tolerance. Compared with the genome of laboratory model strain S288C, we identified more than 32,000 SNPs in Y-12632 with 23,000 missense and nonsense SNPs. Enriched sequence mutations occurred for genes involved in MAPK- and phosphatidylinositol (PI)- signaling pathways in strain Y-12632, with 41 and 13 genes containing non-synonymous SNPs, respectively. Many of these mutated genes displayed consistent up-regulated signature expressions in response to challenges of 30 mM HMF. Analogous single-gene deletion mutations of these genes showed significantly sensitive growth response on a synthetic medium containing 20 mM HMF. Our results suggest at least three MAPK-signaling pathways, especially for the cell-wall integrity pathway, and PI-signaling pathways to be involved in mediation of yeast tolerance against HMF in industrial yeast Saccharomyces cerevisiae. Higher levels of sequence variations were also observed for genes involved in purine and pyrimidine metabolism pathways.

Integrated phospholipidomics and transcriptomics analysis of Saccharomyces cerevisiae with enhanced tolerance to a mixture of acetic acid, furfural, and phenol.

A mixture of acetic acid, furfural, and phenol (AFP), three representative lignocellulose-derived inhibitors, significantly inhibited the growth and bioethanol production of Saccharomyces cerevisiae. In order to uncover the mechanisms behind the enhanced tolerance of an inhibitor-tolerant S. cerevisiae strain (T), we measured the plasma membrane properties, which significantly influence cellular adaptation to inhibitors, of T strain and its parental strain (P) with and without AFP treatment. We integrated data obtained from multi-statistics-assisted phospholipidomics and parallel transcriptomics by using LC-tandem MS and microarray analysis. With the AFP treatment, the transcriptional changes of fatty acid metabolic genes showed a strong correlation with the increase of fatty-acyl-chain length of phosphatidylcholine (PC) and phosphatidylinositol (PI). This suggests a possible compensatory mechanism to cope with the increase of plasma membrane permeability and fluidity in both strains. Moreover, the absence of phosphatidylserine (PS) and phosphatidylethanolamine (PE) species from the most variable phospholipid species group was a discriminative feature of the T strain. This resulted from the decrease of CHO1 and increase of CHO2 levels of the T strain upon AFP treatment. These novel findings reveal that the coordinated transcription and phospholipid composition changes contribute to the increased robustness of the T strain and highlight potential metabolic engineering targets for mutants with higher tolerance.

Engineered NADH-dependent GRE2 from Saccharomyces cerevisiae by directed enzyme evolution enhances HMF reduction using additional cofactor NADPH.

Furfural and 5-hydroxymethylfurfural (HMF) are inhibitors generated by lignocellulosic biomass pretreatment such as dilute acid hydrolysis that inhibit microbial growth and interfere with subsequent fermentation. It is possible to in situ detoxify these inhibitory compounds by aldehyde reductions using tolerant Saccharomyces cerevisiae. YOL151W (GRE2) is a commonly recognized up-regulated gene expressed under stress conditions that encodes reductase activities toward furfural and HMF using cofactor NADH. Applying a directed enzyme evolution approach, we altered the genetic code of GRE2 yielding two mutants with amino acid substitutions of Gln261 to Arg261 and Phe283 to Leu283; and Ile107 to Val107, Gln261 to Arg261, and Val285 to Asp285 for strain Y62-C11 and Y62-G6, respectively. Clones of these mutants showed faster growth rates and were able to establish viable cultures under 30 mM HMF challenges when compared with a wild type GRE2 clone when inoculated into synthetic medium containing this inhibitor. Compared with the wild type control, crude cell extracts of the two mutants showed 3- to 4-fold and 3- to 9-fold increased specific enzyme activity using NADH toward HMF and furfural reduction, respectively. While retaining its aldehyde reductase activities using the cofactor NADH, mutant Y62-G6 displayed significantly greater reductase activities using NADPH as the cofactor with 13- and 15-fold increase toward furfural and HMF, respectively, as measured by its partially purified protein. Using reverse engineering and site directed mutagenesis methods, we were able to confirm that the amino acid substitution of the Asp285 is responsible for the increased aldehyde reductase activities by utilizing the additional cofactor NADPH.

A new ß-glucosidase producing yeast for lower-cost cellulosic ethanol production from xylose-extracted corncob residues by simultaneous saccharification and fermentation.

This study reports a new yeast strain of Clavispora NRRL Y-50464 that is able to utilize cellobiose as sole source of carbon and produce sufficient native ß-glucosidase enzyme activity for cellulosic ethanol production using SSF. In addition, this yeast is tolerant to the major inhibitors derived from lignocellulosic biomass pre-treatment such as 2-furaldehyde (furfural) and 5-(hydroxymethyl)-2-furaldehyde (HMF), and converted furfural into furan methanol in less than 12h and HMF into furan-2,5-dimethanol within 24h in the presence of 15 mM each of furfural and HMF. Using xylose-extracted corncob residue as cellulosic feedstock, an ethanol production of 23 g/l was obtained using 25% solids loading at 37 °C by SSF without addition of exogenous ß-glucosidase. Development of this yeast aids renewable biofuels development efforts for economic consolidated SSF bio-processing.