Swamp eel aldehyde reductase is involved in response to nitrosative stress via regulating NO/GSH levels.

Aldehyde reductase (ALR) plays key roles in the detoxification of toxic aldehyde. In this study, the authors cloned the swamp eel ALR gene using rapid amplification of cDNA ends-PCR (RACE-PCR). The recombinant protein (rALR) was expressed in Escherichia coli and purified using a Ni2+ -NTA chelating column. The rALR protein exhibited efficient reductive activity towards several aldehydes, ketones and S-nitrosoglutathione (GSNO). A spot assay suggested that the recombinant E. coli strain expressing rALR showed better resistance to formaldehyde, sodium nitrite and GSNO stress, suggesting that swamp eel ALR is crucial for redox homeostasis in vivo. Consequently, the authors investigated the effect of rALR on the oxidative parameters of the liver in swamp eels challenged with Aeromonas hydrophila. The hepatic glutathione (GSH) content significantly increased, and the hepatic NO content and levels of reactive oxygen species and reactive nitrogen species significantly decreased when rALR was administered. In addition, the mRNA expression of hepatic Alr, HO1 and Nrf2 was significantly upregulated, whereas the expression levels of NF-κB, IL-1ß and NOS1 were significantly downregulated in the rALR-administered group. Collectively, these results suggest that ALR is involved in the response to nitrosative stress by regulating GSH/NO levels in the swamp eel.

Effects of aldo-keto reductase family 1 member A on osteoblast differentiation associated with lactate production in MC3T3-E1 preosteoblastic cells.

Aldo-keto reductase family 1 member A (AKR1A) is an NADPH-dependent aldehyde reductase widely expressed in mammalian tissues. In this study, induced differentiation of MC3T3-E1 preosteoblasts was found to increase AKR1A gene expression concomitantly increased NOx- (nitrite + nitrate), increased glucose uptake, increased [NAD(P)+]/[NAD(P)H] and lactate production but decreased reactive oxygen species (ROS) without changes in endothelial nitric oxide synthase (eNOS) expression in differentiated osteoblasts (OBs). A study using gain- and loss-of-function MC3T3-E1 cells indicated that AKR1A is essential for modulating OB differentiation and gene expression of collagen 1 A1, receptor activator of nuclear factor kappa-B ligand, and osteoprotegerin in OBs. Immunofluorescence microscopy also revealed that changes in AKR1A expression altered extracellular collagen formation in differentiated OBs. Consistently, analyses of alkaline phosphatase activity and calcium deposits of matrix mineralization by Alizarin Red S staining verified that AKR1A is involved in the regulation of OB differentiation and bone matrix formation. In addition, AKR1A gene alterations affected the levels of NOx-, eNOS expression, glucose uptake, [NAD(P)+]/[NAD(P)H] dinucleotide redox couples, lactate production, and ROS in differentiated OBs. Herein, we report that AKR1A-mediated denitrosylation may play a role in the regulation of lactate metabolism as well as redox homeostasis in cells, providing an efficient way to quickly gain energy and to significantly reduce oxidative stress for OB differentiation.

Structural and Biochemical Analysis of the Furan Aldehyde Reductase YugJ from Bacillus subtilis.

NAD(H)/NADP(H)-dependent aldehyde/alcohol oxidoreductase (AAOR) participates in a wide range of physiologically important cellular processes by reducing aldehydes or oxidizing alcohols. Among AAOR substrates, furan aldehyde is highly toxic to microorganisms. To counteract the toxic effect of furan aldehyde, some bacteria have evolved AAOR that converts furan aldehyde into a less toxic alcohol. Based on biochemical and structural analyses, we identified Bacillus subtilis YugJ as an atypical AAOR that reduces furan aldehyde. YugJ displayed high substrate specificity toward 5-hydroxymethylfurfural (HMF), a furan aldehyde, in an NADPH- and Ni2+-dependent manner. YugJ folds into a two-domain structure consisting of a Rossmann-like domain and an α-helical domain. YugJ interacts with NADP and Ni2+ using the interdomain cleft of YugJ. A comparative analysis of three YugJ structures indicated that NADP(H) binding plays a key role in modulating the interdomain dynamics of YugJ. Noticeably, a nitrate ion was found in proximity to the nicotinamide ring of NADP in the YugJ structure, and the HMF-reducing activity of YugJ was inhibited by nitrate, providing insights into the substrate-binding mode of YugJ. These findings contribute to the characterization of the YugJ-mediated furan aldehyde reduction mechanism and to the rational design of improved furan aldehyde reductases for the biofuel industry.

The open reading frame 02797 from Candida tropicalis encodes a novel NADH-dependent aldehyde reductase.

Owing to its high-temperature tolerance, robustness, and wide use of carbon sources, Candida tropicalis is considered a good candidate microorganism for bioconversion of lignocellulose to ethanol. It also has the intrinsic ability to in situ detoxify aldehydes derived from lignocellulosic hydrolysis. However, the aldehyde reductases that catalyze this bioconversion in C. tropicalis remain unknown. Herein, we found that the uncharacterized open reading frame (ORF), CTRG_02797, from C. tropicalis encodes a novel and broad substrate-specificity aldehyde reductase that reduces at least seven aldehydes. This enzyme strictly depended on NADH rather than NADPH as the co-factor for catalyzing the reduction reaction. Its highest affinity (Km), maximum velocity (Vmax), catalytic rate constant (Kcat), and catalytic efficiency (Kcat/Km) were observed when reducing acetaldehyde (AA) and its enzyme activity was influenced by different concentrations of salts, metal ions, and chemical protective additives. Protein localization assay demonstrated that Ctrg_02797p was localized in the cytoplasm in C. tropicalis cells, which ensures an effective enzymatic reaction. Finally, Ctrg_02797p was grouped into the cinnamyl alcohol dehydrogenase (CADH) subfamily of the medium-chain dehydrogenase/reductase family. This research provides guidelines for exploring more uncharacterized genes with reduction activity for detoxifying aldehydes.

Crystal Structure and Biophysical Analysis of Furfural-Detoxifying Aldehyde Reductase from Clostridium beijerinckii.

Many aldehydes, such as furfural, are present in high quantities in lignocellulose lysates and are fermentation inhibitors, which makes biofuel production from this abundant carbon source extremely challenging. Cbei_3974 has recently been identified as an aldo-keto reductase responsible for partial furfural resistance in Clostridium beijerinckii Rational engineering of this enzyme could enhance the furfural tolerance of this organism, thereby improving biofuel yields. We report an extensive characterization of Cbei_3974 and a single-crystal X-ray structure of Cbei_3974 in complex with NADPH at a resolution of 1.75 Å. Docking studies identified residues involved in substrate binding, and an activity screen revealed the substrate tolerance of the enzyme. Hydride transfer, which is partially rate limiting under physiological conditions, occurs from the pro-R hydrogen of NADPH. Enzyme isotope labeling revealed a temperature-independent enzyme isotope effect of unity, indicating that the enzyme does not use dynamic coupling for catalysis and suggesting that the active site of the enzyme is optimally configured for catalysis with the substrate tested.IMPORTANCE Here we report the crystal structure and biophysical properties of an aldehyde reductase that can detoxify furfural, a common inhibitor of biofuel fermentation found in lignocellulose lysates. The data contained here will serve as a guide for protein engineers to develop improved enzyme variants that would impart furfural resistance to the microorganisms used in biofuel production and thus lead to enhanced biofuel yields from this sustainable resource.

YKL107W from Saccharomyces cerevisiae encodes a novel aldehyde reductase for detoxification of acetaldehyde, glycolaldehyde, and furfural.

The aldehyde reductases from the short-chain dehydrogenase/reductase (SDR) family were identified as a series of critical enzymes for the improved tolerance of Saccharomyces cerevisiae to the aldehydes by catalyzing the detoxification reactions of aldehydes. Herein, we report that a novel aldehyde reductase Ykl107wp deduced from YKL107W from S. cerevisiae belongs to the classical SDR group and can catalyze the reduction reactions of acetaldehyde (AA), glycolaldehyde (GA), furfural (FF), formaldehyde (FA), and propionaldehyde (PA) but cannot reduce the six representative ketones. Ykl107wp displayed the best maximum velocity (Vmax), catalytic rate constant (Kcat), catalytic efficiency (Kcat/Km), and highest affinity (Km) to acetaldehyde. The optimum pH of Ykl107wp was 6.0 for the reduction of AA and 7.0 for the reduction of GA and FF, and the optimum temperatures were 40, 35, and 30 °C for the reduction of AA, GA, and FF, respectively. Ykl107wp for the reduction of AA was greatly affected by metal ions, chemical additives, and salts and showed poor thermal and pH stability, but its stability was slightly affected by a substrate. Ykl107wp was localized in endoplasmic reticulum and prevented the yeast cells from damage caused by furfural via the detoxification of furfural to furfural alcohol. This research provides guidelines for the study of uncharacterized classical SDR aldehyde reductases and exploration of their protective mechanisms on the corresponding organelles.

Cloning and characterization of enoate reductase with high ß-ionone to dihydro-ß-ionone bioconversion productivity.

BACKGROUND: Dihydro-ß-ionone is a principal aroma compound and has received considerable attention by flavor and fragrance industry. The traditional method of preparing dihydro-ß-ionone has many drawbacks, which has restricted its industrial application. Therefore, it is necessary to find a biotechnological method to produce dihydro-ß-ionone. RESULTS: In this study, the enoate reductase with high conversion efficiency of ß-ionone to dihydro-ß-ionone, DBR1, was obtained by screening four genetically engineered bacteria. The product, dihydro-ß-ionone, was analyzed by GC and GC-MS. The highest dihydro-ß-ionone production with 308.3 mg/L was detected in the recombinant strain expressing DBR1 which was later on expressed and purified. Its optimal temperature and pH were 45 °C and 6.5, respectively. The greatest activity of the purified enzyme was 356.39 U/mg using ß-ionone as substrate. In the enzymatic conversion system, 1 mM of ß-ionone was transformed into 91.08 mg/L of dihydro-ß-ionone with 93.80% of molar conversion. CONCLUSION: DBR1 had high selectivity to hydrogenated the 10,11-unsaturated double bond of ß-ionone as well as high catalytic efficiency for the conversion of ß-ionone to dihydro-ß-ionone. It is the first report on the bioconversion of ß-ionone to dihydro-ß-ionone by using enoate reductase.

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.

Construction of an alternative glycerol-utilization pathway for improved ß-carotene production in Escherichia coli.

Glycerol, which is an inevitable by-product of biodiesel production, is an ideal carbon source for the production of carotenoids due to its low price, good availability and chemically reduced status, which results in a low requirement for additional reducing equivalents. In this study, an alternative carbon-utilization pathway was constructed in Escherichia coli to enable more efficient ß-carotene production from glycerol. An aldehyde reductase gene (alrd) and an aldehyde dehydrogenase gene (aldH) from Ralstonia eutropha H16 were integrated into the E. coli chromosome to form a novel glycerol-utilization pathway. The ß-carotene specific production value was increased by 50% after the introduction of alrd and aldH. It was found that the glycerol kinase gene (garK), alrd and aldH were the bottleneck of the alternative glycerol metabolic pathway, and modulation of garK gene with an mRS library further increased the ß-carotene specific production value by 13%. Finally, co-modulation of genes in the introduced aldH-alrd operon led to 86% more of ß-carotene specific production value than that of the strain without the alternative glycerol-utilization pathway and the glycerol-utilization rate was also increased. In this work, ß-carotene production of E. coli was significantly improved by constructing and optimizing an alternative glycerol-utilization pathway. This strategy can potentially be used to improve the production of other isoprenoids using glycerol as a cheap and abundant substrate, and therefore has industrial relevance.

De Novo Transcriptome Sequencing of the Deep-Sea-Derived Fungus Dichotomomyces cejpii and Analysis of Gliotoxin Biosynthesis Genes.

Gliotoxin, produced by fungi, is an epipolythiodioxopiperazine (ETP) toxin with bioactivities such as anti-liver fibrosis, antitumor, antifungus, antivirus, antioxidation, and immunoregulation. Recently, cytotoxic gliotoxins were isolated from a deep-sea-derived fungus, Dichotomomyces cejpii. However, the biosynthetic pathway for gliotoxins in D. cejpii remains unclear. In this study, the transcriptome of D. cejpii was sequenced using an Illumina Hiseq 2000. A total of 19,125 unigenes for D. cejpii were obtained from 9.73 GB of clean reads. Ten genes related to gliotoxin biosynthesis were annotated. The expression levels of gliotoxin-related genes were detected through quantitative real-time polymerase chain reaction (qRT-PCR). The GliG gene, encoding a glutathione S-transferase (DC-GST); GliI, encoding an aminotransferase (DC-AI); and GliO, encoding an aldehyde reductase (DC-AR), were cloned and expressed, purified, and characterized. The results suggested the important roles of DC-GST, DC-AT, and DC-AR in the biosynthesis of gliotoxins. Our study on the genes related to gliotoxin biosynthesis establishes a molecular foundation for the wider application of gliotoxins from D. cejpii in the biomedical industry in the future.

Pcal_1311, an alcohol dehydrogenase homologue from Pyrobaculum calidifontis, displays NADH-dependent high aldehyde reductase activity.

Genome sequence of Pyrobaculum calidifontis, a hyperthermophilic archaeon, harbors three open-reading frames annotated as alcohol dehydrogenases. One of them, Pcal_1311, does not display a significantly high homology with any of the characterized alcohol dehydrogenases. Highest homology of 38% was found with the characterized counterpart from Geobacillus stearothermophilus. To examine the biochemical properties of Pcal_1311, we have cloned and functionally expressed the gene in Escherichia coli. Purified recombinant Pcal_1311 catalyzed the NAD(H)-dependent oxidation of various alcohols and reduction of aldehydes, with a marked preference for substrates with functional group at the terminal carbon. Highest activity for the oxidation reaction (3 µmol min-1 mg-1) was found with 1,4-butanediol and for the reduction reaction (150 µmol min-1 mg-1) with glutaraldehyde. Both the oxidation and reduction activities increased with the increase in temperature up to 80 °C. Recombinant Pcal_1311 was highly stable and retained more than 90% activity even after incubation of 180 min at 90 °C. In addition to the thermostabilty, Pcal_1311 was highly stable in the presence of known denaturants including urea and guanidine hydrochloride. The high stability, particularly thermostability, and the NADH-dependent aldehyde reduction activity make Pcal_1311 a unique member in the alcohol dehydrogenase family.

Identification of new potent inhibitor of aldose reductase from Ocimum basilicum.

Recent efforts to develop cure for chronic diabetic complications have led to the discovery of potent inhibitors against aldose reductase (AKR1B1, EC 1.1.1.21) whose role in diabetes is well-evident. In the present work, two new natural products were isolated from the ariel part of Ocimum basilicum; 7-(3-hydroxypropyl)-3-methyl-8-ß-O-d-glucoside-2H-chromen-2-one (1) and E-4-(6'-hydroxyhex-3'-en-1-yl)phenyl propionate (2) and confirmed their structures with different spectroscopic techniques including NMR spectroscopy etc. The isolated compounds (1, 2) were evaluated for in vitro inhibitory activity against aldose reductase (AKR1B1) and aldehyde reductase (AKR1A1). The natural product (1) showed better inhibitory activity for AKR1B1 with IC50 value of 2.095±0.77µM compare to standard sorbinil (IC50=3.14±0.02µM). Moreover, the compound (1) also showed multifolds higher activity (IC50=0.783±0.07µM) against AKR1A1 as compared to standard valproic acid (IC50=57.4±0.89µM). However, the natural product (2) showed slightly lower activity for AKR1B1 (IC50=4.324±1.25µM). Moreover, the molecular docking studies of the potent inhibitors were also performed to identify the putative binding modes within the active site of aldose/aldehyde reductases.

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.

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.

Identification of long chain specific aldehyde reductase and its use in enhanced fatty alcohol production in E. coli.

Long chain fatty alcohols have wide application in chemical industries and transportation sector. There is no direct natural reservoir for long chain fatty alcohol production, thus many groups explored metabolic engineering approaches for its microbial production. Escherichia coli has been the major microbial platform for this effort, however, terminal endogenous enzyme responsible for converting fatty aldehydes of chain length C14-C18 to corresponding fatty alcohols is still been elusive. Through our in silico analysis we selected 35 endogenous enzymes of E. coli having potential of converting long chain fatty aldehydes to fatty alcohols and studied their role under in vivo condition. We found that deletion of ybbO gene, which encodes NADP(+) dependent aldehyde reductase, led to >90% reduction in long chain fatty alcohol production. This feature was found to be strain transcending and reinstalling ybbO gene via plasmid retained the ability of mutant to produce long chain fatty alcohols. Enzyme kinetic study revealed that YbbO has wide substrate specificity ranging from C6 to C18 aldehyde, with maximum affinity and efficiency for C18 and C16 chain length aldehyde, respectively. Along with endogenous production of fatty aldehyde via optimized heterologous expression of cyanobaterial acyl-ACP reductase (AAR), YbbO overexpression resulted in 169mg/L of long chain fatty alcohols. Further engineering involving modulation of fatty acid as well as of phospholipid biosynthesis pathway improved fatty alcohol production by 60%. Finally, the engineered strain produced 1989mg/L of long chain fatty alcohol in bioreactor under fed-batch cultivation condition. Our study shows for the first time a predominant role of a single enzyme in production of long chain fatty alcohols from fatty aldehydes as well as of modulation of phospholipid pathway in increasing the fatty alcohol production.

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.

Improving alkane synthesis in Escherichia coli via metabolic engineering.

Concerns about energy security and global petroleum supply have made the production of renewable biofuels an industrial imperative. The ideal biofuels are n-alkanes in that they are chemically and structurally identical to the fossil fuels and can "drop in" to the transportation infrastructure. In this work, an Escherichia coli strain that produces n-alkanes was constructed by heterologous expression of acyl-acyl carrier protein (ACP) reductase (AAR) and aldehyde deformylating oxygenase (ADO) from Synechococcus elongatus PCC7942. The accumulation of alkanes ranged from 3.1 to 24.0 mg/L using different expressing strategies. Deletion of yqhD, an inherent aldehyde reductase in E. coli, or overexpression of fadR, an activator for fatty acid biosynthesis, exhibited a nearly twofold increase in alkane titers, respectively. Combining yqhD deletion and fadR overexpression resulted in a production titer of 255.6 mg/L in E. coli, and heptadecene was the most abundant product.

YNL134C from Saccharomyces cerevisiae encodes a novel protein with aldehyde reductase activity for detoxification of furfural derived from lignocellulosic biomass.

Furfural and 5-hydroxymethylfurfural (HMF) are the two main aldehyde compounds derived from pentoses and hexoses, respectively, during lignocellulosic biomass pretreatment. These two compounds inhibit microbial growth and interfere with subsequent alcohol fermentation. Saccharomyces cerevisiae has the in situ ability to detoxify furfural and HMF to the less toxic 2-furanmethanol (FM) and furan-2,5-dimethanol (FDM), respectively. Herein, we report that an uncharacterized gene, YNL134C, was highly up-regulated under furfural or HMF stress and Yap1p and Msn2/4p transcription factors likely controlled its up-regulated expression. Enzyme activity assays showed that YNL134C is an NADH-dependent aldehyde reductase, which plays a role in detoxification of furfural to FM. However, no NADH- or NADPH-dependent enzyme activity was observed for detoxification of HMF to FDM. This enzyme did not catalyse the reverse reaction of FM to furfural or FDM to HMF. Further studies showed that YNL134C is a broad-substrate aldehyde reductase, which can reduce multiple aldehydes to their corresponding alcohols. Although YNL134C is grouped into the quinone oxidoreductase family, no quinone reductase activity was observed using 1,2-naphthoquinone or 9,10-phenanthrenequinone as a substrate, and phylogenetic analysis indicates that it is genetically distant to quinone reductases. Proteins similar to YNL134C in sequence from S. cerevisiae and other microorganisms were phylogenetically analysed.

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.

Reductive detoxification of acrolein as a potential role for aldehyde reductase (AKR1A) in mammals.

Aldehyde reductase (AKR1A), a member of the aldo-keto reductase superfamily, suppresses diabetic complications via a reduction in metabolic intermediates; it also plays a role in ascorbic acid biosynthesis in mice. Because primates cannot synthesize ascorbic acid, a principle role of AKR1A appears to be the reductive detoxification of aldehydes. In this study, we isolated and immortalized mouse embryonic fibroblasts (MEFs) from wild-type (WT) and human Akr1a-transgenic (Tg) mice and used them to investigate the potential roles of AKR1A under culture conditions. Tg MEFs showed higher methylglyoxal- and acrolein-reducing activities than WT MEFs and also were more resistant to cytotoxicity. Enzymatic analyses of purified rat AKR1A showed that the efficiency of the acrolein reduction was about 20% that of glyceraldehyde. Ascorbic acid levels were quite low in the MEFs, and while the administration of ascorbic acid to the cells increased the intracellular levels of ascorbic acid, it had no affect on the resistance to acrolein. Endoplasmic reticulum stress and protein carbonylation induced by acrolein treatment were less evident in Tg MEFs than in WT MEFs. These data collectively indicate that one of the principle roles of AKR1A in primates is the reductive detoxification of aldehydes, notably acrolein, and protection from its detrimental effects.