Z. Lewis Liu

Furfural induces reactive oxygen species accumulation and cellular damage in Saccharomyces cerevisiae

BackgroundBiofuels offer a viable alternative to petroleum-based fuel. However, current methods are not sufficient and the technology required in order to use lignocellulosic biomass as a fermentation substrate faces several challenges. One challenge is the need for a robust fermentative microorganism that can tolerate the inhibitors present during lignocellulosic fermentation. These inhibitors include the furan aldehyde, furfural, which is released as a byproduct of pentose dehydration during the weak acid pretreatment of lignocellulose. In order to survive in the presence of furfural, yeast cells need not only to reduce furfural to the less toxic furan methanol, but also to protect themselves and repair any damage caused by the furfural. Since furfural tolerance in yeast requires a functional pentose phosphate pathway (PPP), and the PPP is associated with reactive oxygen species (ROS) tolerance, we decided to investigate whether or not furfural induces ROS and its related cellular damage in yeast.ResultsWe demonstrated that furfural induces the accumulation of ROS in Saccharomyces cerevisiae. In addition, furfural was shown to cause cellular damage that is consistent with ROS accumulation in cells which includes damage to mitochondria and vacuole membranes, the actin cytoskeleton and nuclear chromatin. The furfural-induced damage is less severe when yeast are grown in a furfural concentration (25 mM) that allows for eventual growth after an extended lag compared to a concentration of furfural (50 mM) that prevents growth.ConclusionThese data suggest that when yeast cells encounter the inhibitor furfural, they not only need to reduce furfural into furan methanol but also to protect themselves from the cellular effects of furfural and repair any damage caused. The reduced cellular damage seen at 25 mM furfural compared to 50 mM furfural may be linked to the observation that at 25 mM furfural yeast were able to exit the furfural-induced lag phase and resume growth. Understanding the cellular effects of furfural will help direct future strain development to engineer strains capable of tolerating or remediating ROS and the effects of ROS.

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.

Enhanced Biotransformation of Furfural and Hydroxymethylfurfural by Newly Developed Ethanologenic Yeast Strains

Furfural and hydroxymethylfurfural (HMF) are representative inhibitors among many inhibitive compounds derived from biomass degradation and saccharification for bioethanol fermentation. Most yeasts, including industrial strains, are susceptible to these inhibitory compounds, especially when multiple inhibitors are present. Additional detoxification steps add cost and complexity to the process and generate additional waste products. To promote efficient bioethanol production, we studied the mechanisms of stress tolerance, particularly to fermentation inhibitors such as furfural and HMF. We recently reported a metabolite of 2,5-bis-hydroxymethylfuran as a conversion product of HMF and characterized a dose-dependent response of ethanologenic yeasts to inhibitors. In this study, we present newly adapted strains that demonstrated higher levels of tolerance to furfural and HMF. Saccharomyces cerevisiae 307-12H60 and 307-12H120 and Pichia stipitis 307 10H60 showed enhanced biotransformation ability to reduce HMF to 2,5-bis-hydroxymethylfuran at 30 and 60 mM, and S. cerevisiae 307-12-F40 converted furfural into furfuryl alcohol at significantly higher rates compared to the parental strains. Strains of S. cerevisiae converted 100% of HMF at 60 mM and S. cerevisiae 307-12-F40 converted 100% of furfural into furfuryl alcohol at 30 mM. The results of this study suggest a possible in situ detoxification of the inhibitors by using more inhibitor-tolerant yeast strains for bioethanol fermentation. The development of such tolerant strains provided a basis and useful materials for further studies on the mechanisms of stress tolerance.

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.

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.

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. Biotechnol. Bioeng. 2011; 108:1801–1815.

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

AbstractBackgroundPhenolic 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.ResultsWe 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.ConclusionsThe 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.

Stereochemistry of Furfural Reduction by a Saccharomyces cerevisiae Aldehyde Reductase That Contributes to In Situ Furfural Detoxification

ABSTRACT 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 Ari1ps best known hydride donor. Here we report the stereochemistry of the hydride transfer step, determined by using (4R)-[4-2H]NADPD and (4S)-[4-2H]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)-[4-2H]NADPD and NADPH as substrates, primary deuterium kinetic isotope effects of 2.2 and 2.5 were determined for the steady-state parameters kcatNADPH and kcat/KmNADPH, respectively, indicating that hydride transfer is partially rate limiting to catalysis.

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).

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. Copyright