Phenotypic and comparative transcriptomics analysis of RDS1 overexpression reveal tolerance of Saccharomyces cerevisiae to furfural.

The yeast Saccharomyces cerevisiae able to tolerate lignocellulose-derived inhibitors like furfural. Yeast strain performance tolerance has been measured by the length of the lag phase for cell growth in response to the furfural inhibitor challenge. The aims of this work were to obtain RDS1 yeast tolerant strain against furfural through overexpression using a method of in vivo homologous recombination. Here, we report that the overexpressing RDS1 recovered more rapidly and displayed a lag phase at about 12 h than its parental strain. Overexpressing RDS1 strain encodes a novel aldehyde reductase with catalytic function for reduction of furfural with NAD(P)H as the co-factor. It displayed the highest specific activity (24.8 U/mg) for furfural reduction using NADH as a cofactor. Fluorescence microscopy revealed improved accumulation of reactive oxygen species resistance to the damaging effects of inhibitor in contrast to the parental. Comparative transcriptomics revealed key genes potentially associated with stress responses to the furfural inhibitor, including specific and multiple functions involving defensive reduction-oxidation reaction process and cell wall response. A significant change in expression level of log2 (fold change >1) was displayed for RDS1 gene in the recombinant strain, which demonstrated that the introduction of RDS1 overexpression promoted the expression level. Such signature expressions differentiated tolerance phenotypes of RDS1 from the innate stress response of its parental strain. Overexpression of the RDS1 gene involving diversified functional categories is accountable for stress tolerance in yeast S. cerevisiae to survive and adapt the furfural during the lag phase.

Contribution of YPRO15C Overexpression to the Resistance of Saccharomyces cerevisiae BY4742 Strain to Furfural Inhibitor.

Lignocellulosic biomass is still considered a feasible source of bioethanol production. Saccharomyces cerevisiae can adapt to detoxify lignocellulose-derived inhibitors, including furfural. Tolerance of strain performance has been measured by the extent of the lag phase for cell proliferation following the furfural inhibitor challenge. The purpose of this work was to obtain a tolerant yeast strain against furfural through overexpression of YPR015C using the in vivo homologous recombination method. The physiological observation of the overexpressing yeast strain showed that it was more resistant to furfural than its parental strain. Fluorescence microscopy revealed improved enzyme reductase activity and accumulation of oxygen reactive species due to the harmful effects of furfural inhibitor in contrast to its parental strain. Comparative transcriptomic analysis revealed 79 genes potentially involved in amino acid biosynthesis, oxidative stress, cell wall response, heat shock protein, and mitochondrial-associated protein for the YPR015C overexpressing strain associated with stress responses to furfural at the late stage of lag phase growth. Both up- and down-regulated genes involved in diversified functional categories were accountable for tolerance in yeast to survive and adapt to the furfural stress in a time course study during the lag phase growth. This study enlarges our perceptions comprehensively about the physiological and molecular mechanisms implicated in the YPR015C overexpressing strain's tolerance under furfural stress. Construction illustration of the recombinant plasmid. a) pUG6-TEF1p-YPR015C, b) integration diagram of the recombinant plasmid pUG6-TEF1p-YPR into the chromosomal DNA of Saccharomyces cerevisiae.

Draft Whole-Genome Sequence of the Black Yeast Aureobasidium pullulans NRRL 62031.

The black-yeast-like Aureobasidium is discussed as a versatile cell factory for many biotechnological applications. This article describes the 25.05-Mb draft genome sequence of Aureobasidium pullulans NRRL 62031, which was isolated in Thailand. The genome sequence provides evidence for a plethora of synthesis pathways for valuable secondary metabolites.

Cloning and functional characterization of xylitol dehydrogenase genes from Issatchenkia orientalis and Torulaspora delbrueckii.

Saccharomyces cerevisiae can obtain xylose utilization capacity via integration of heterogeneous xylose reductase (XR) and xylitol dehydrogenase (XDH) genes into its metabolic pathway, and XYL2 which encodes the XDH plays an essential role in this process. Herein, we reported that two hypothetical XYL2 genes from the multistress-tolerant yeasts of Issatchenkia orientalis and Torulaspora delbrueckii were cloned, and they encoded two XDHs, IoXyl2p and TdXyl2p, respectively, with the activities for oxidation of xylitol to xylulose. Comparative studies demonstrated that IoXyl2p and TdXyl2p, like the SsXyl2p from Scheffersomyces stipitis, were probably localized to the cytoplasm and strictly dependent on NAD+ rather than NADP+ as the cofactor for catalyzing the oxidation reaction of xylitol. IoXyl2p had the highest specific activity, maximum velocity (Vmax), affinity to xylitol (Km), and catalytic efficiency (kcat/Km) among the three XDHs. The optimum temperature for oxidation of xylitol were at 45 °C by IoXyl2p and at 35 °C by TdXyl2p and SsXyl2p, and the optimum pH of IoXyl2p, TdXyl2p and SsXyl2p for oxidation of xylitol was 8.0, 8.5 and 7.5, respectively. Mg2+ promoted the activities of IoXyl2p and TdXyl2p, but slightly inhibited the activity of SsXyl2p. Most metal ions had much weaker inhibition effects on IoXyl2p and TdXyl2p than SsXyl2p. IoXyl2p displayed the strongest salt resistance among the three XDHs. To summarize, IoXyl2p from I. orientalis and TdXyl2p from T. delbrueckii characterized in this study are considered to be the attractive candidates for the construction of genetically engineered S. cerevisiae for efficiently fermentation of carbohydrate in lignocellulosic hydrolysate.

Functions of aldehyde reductases from Saccharomyces cerevisiae in detoxification of aldehyde inhibitors and their biotechnological applications.

Bioconversion of lignocellulosic biomass to high-value bioproducts by fermentative microorganisms has drawn extensive attentions worldwide. Lignocellulosic biomass cannot be efficiently utilized by microorganisms, such as Saccharomyces cerevisiae, but has to be pretreated prior to fermentation. Aldehyde compounds, as the by-products generated in the pretreatment process of lignocellulosic biomass, are considered as the most important toxic inhibitors to S. cerevisiae cells for their growth and fermentation. Aldehyde group in the aldehyde inhibitors, including furan aldehydes, aliphatic aldehydes, and phenolic aldehydes, is identified as the toxic factor. It has been demonstrated that S. cerevisiae has the ability to in situ detoxify aldehydes to their corresponding less or non-toxic alcohols. This reductive reaction is catalyzed by the NAD(P)H-dependent aldehyde reductases. In recent years, detoxification of aldehyde inhibitors by S. cerevisiae has been extensively studied and a huge progress has been made. This mini-review summarizes the classifications and structural features of the characterized aldehyde reductases from S. cerevisiae, their catalytic abilities to exogenous and endogenous aldehydes and effects of metal ions, chemical protective additives, and salts on enzyme activities, subcellular localization of the aldehyde reductases and their possible roles in protection of the subcellular organelles, and transcriptional regulation of the aldehyde reductase genes by the key stress-response transcription factors. Cofactor preference of the aldehyde reductases and their molecular mechanisms and efficient supply pathways of cofactors, as well as biotechnological applications of the aldehyde reductases in the detoxification of aldehyde inhibitors derived from pretreatment of lignocellulosic biomass, are also included or supplemented in this mini-review.

YKL071W from Saccharomyces cerevisiae encodes a novel aldehyde reductase for detoxification of glycolaldehyde and furfural derived from lignocellulose.

Aldehydes generated as by-products during the pretreatment of lignocellulose are the key inhibitors to Saccharomyces cerevisiae, which is considered as the most promising microorganism for industrial production of biofuel, xylitol as well as other special chemicals from lignocellulose. S. cerevisiae has the inherent ability to in situ detoxify aldehydes to corresponding alcohols by multiple aldehyde reductases. Herein, we report that an uncharacterized open reading frame YKL071W from S. cerevisiae encodes a novel "classical" short-chain dehydrogenase/reductase (SDR) protein with NADH-dependent enzymatic activities for reduction of furfural (FF), glycolaldehyde (GA), formaldehyde (FA), and benzaldehyde (BZA). This enzyme showed much better specific activities for reduction of GA and FF than FA and BZA, and displayed much higher Km and Kcat/Km but lower Vmax and Kcat for reduction of GA than FF. For this enzyme, the optimum pH was 5.5 and 6.0 for reduction of GA and FF, and the optimum temperature was 30 °C for reduction of GA and FF. Both pH and temperature affected stability of this enzyme in a similar trend for reduction of GA and FF. Cu2+, Zn2+, Ni2+, and Fe3+ had severe inhibition effects on enzyme activities of Ykl071wp for reduction of GA and FF. Transcription of YKL071W in S. cerevisiae was significantly upregulated under GA and FF stress conditions, and its transcription is most probably regulated by transcription factor genes of YAP1, CAD1, PDR3, and STB5. This research provides guidelines to identify more uncharacterized genes with reductase activities for detoxification of aldehydes derived from lignocellulose in S. cerevisiae.

YLL056C from Saccharomyces cerevisiae encodes a novel protein with aldehyde reductase activity.

The short-chain dehydrogenase/reductase (SDR) family, the largest family in dehydrogenase/reductase superfamily, is divided into "classical," "extended," "intermediate," "divergent," "complex," and "atypical" groups. Recently, several open reading frames (ORFs) were characterized as intermediate SDR aldehyde reductase genes in Saccharomyces cerevisiae. However, no functional protein in the atypical group has been characterized in S. cerevisiae till now. Herein, we report that an uncharacterized ORF YLL056C from S. cerevisiae was significantly upregulated under high furfural (2-furaldehyde) or 5-(hydroxymethyl)-2-furaldehyde concentrations, and transcription factors Yap1p, Hsf1p, Pdr1/3p, Yrr1p, and Stb5p likely controlled its upregulated transcription. This ORF indeed encoded a protein (Yll056cp), which was grouped into the atypical subgroup 7 in the SDR family and localized to the cytoplasm. Enzyme activity assays showed that Yll056cp is not a quinone or ketone reductase but an NADH-dependent aldehyde reductase, which can reduce at least seven aldehyde compounds. This enzyme showed the best Vmax, Kcat, and Kcat/Km to glycolaldehyde, but the highest affinity (Km) to formaldehyde. The optimum pH and temperature of this enzyme was pH 6.5 for reduction of glycolaldehyde, furfural, formaldehyde, butyraldehyde, and propylaldehyde, and 30 °C for reduction of formaldehyde or 35 °C for reduction of glycolaldehyde, furfural, butyraldehyde, and propylaldehyde. Temperature and pH affected stability of this enzyme and this influence varied with aldehyde substrate. Metal ions, salts, and chemical protective additives, especially at high concentrations, had different influence on enzyme activities for reduction of different aldehydes. This research provided guidelines for study of more uncharacterized atypical SDR enzymes from S. cerevisiae and other organisms.