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.

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).

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.

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.

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.

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 novel NADPH-dependent aldehyde reductase gene from Saccharomyces cerevisiae NRRL Y-12632 involved in the detoxification of aldehyde inhibitors derived from lignocellulosic biomass conversion.

Aldehyde inhibitors such as furfural, 5-hydroxymethylfurfural, anisaldehyde, benzaldehyde, cinnamaldehyde, and phenylaldehyde are commonly generated during lignocellulosic biomass conversion process for low-cost cellulosic ethanol production that interferes with subsequent microbial growth and fermentation. In situ detoxification of the aldehyde inhibitors is possible by the tolerant ethanologenic yeast that involves multiple genes including numerous functional reductases. In this study, we report a novel aldehyde reductase gene clone Y63 from ethanologenic yeast Saccharomyces cerevisiae NRRL Y12632, representing the uncharacterized ORF YGL157W, which demonstrated NADPH-dependent reduction activities toward at least 14 aldehyde substrates. The identity of gene clone Y63 is the same with YGL157W of SGD since a variation of only 35 nucleotides in genomic sequence and three amino acid residues were observed between the two that share the same length of 347 residues in size. As one among the highly induced genes, YGL157W of Y-12632 showed significantly high levels of transcript abundance in response to furfural and HMF challenges. Based on the deduced amino acid sequence and the most conserved functional motif analyses including closely related reductases from five other yeast species to this date, YGL157W was identified as a member of the subclass 'intermediate' of the SDR (short-chain dehydrogenase/reductase) superfamily with the following typical characteristics: the most conserved catalytic site to lie at Tyr(169)-X-X-X-Lys(173); an indispensable reduction catalytic triad at Ser(131), Tyr(169), and Lys(173), and an approved cofactor-binding motif at Gly(11)-X-X-Gly(14)-X-X-Ala(17) near the N-terminus. YGL039W, YDR541C, and YOL151W (GRE2) appeared to be the similar type of enzymes falling into the same category of the intermediate subfamily.

Arabidopsis vegetative storage protein is an anti-insect acid phosphatase.

Indirect evidence previously suggested that Arabidopsis (Arabidopsis thaliana) vegetative storage protein (VSP) could play a role in defense against herbivorous insects. To test this hypothesis, other AtVSP-like sequences in Arabidopsis were identified through a Basic Local Alignment Search Tool search, and their transcriptional profiles were investigated. In response to methyl jasmonate application or phosphate starvation, AtVSP and AtVSP-like genes exhibited differential expression patterns, suggesting distinct roles played by each member. Arabidopsis VSP2 (AtVSP2), a gene induced by wounding, methyl jasmonate, insect feeding, and phosphate deprivation, was selected for bacterial expression and functional characterization. The recombinant protein exhibited a divalent cation-dependent phosphatase activity in the acid pH range. When incorporated into the diets of three coleopteran and dipteran insects that have acidic gut lumen, recombinant AtVSP2 significantly delayed development of the insects and increased their mortality. To further determine the biochemical basis of the anti-insect activity of the protein, the nucleophilic aspartic acid-119 residue at the conserved DXDXT signature motif was substituted by glutamic acid via site-directed mutagenesis. This single-amino acid alteration did not compromise the protein's secondary or tertiary structure, but resulted in complete loss of its acid phosphatase activity as well as its anti-insect activity. Collectively, we conclude that AtVSP2 is an anti-insect protein and that its defense function is correlated with its acid phosphatase activity.