Properties of the NAD(P)H-dependent xylose reductase from the xylose-fermenting yeast Pichia stipitis.
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ABSTRACT:
SUBMITTER: Verduyn C
PROVIDER: S-EPMC1144764 | biostudies-other | 1985 Mar
REPOSITORIES: biostudies-other
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Project description:During growth on d-xylose the yeast Candida tenuis produces one aldose reductase that is active with both NADPH and NADH as coenzyme. This enzyme has been isolated by dye ligand and anion-exchange chromatography in yields of 76%. Aldose reductase consists ofa single 43 kDa polypeptide with an isoelectric point of 4.70. Initial velocity, product inhibition and binding studies are consistent with a compulsory-ordered, ternary-complex mechanism with coenzyme binding first and leaving last. The catalytic efficiency (kcat/Km) in d-xylose reduction at pH 7 is more than 60-fold higher than that in xylitol oxidation and reflects significant differences in the corresponding catalytic centre activities as well as apparent substrate-binding constants. The enzyme prefers NADP(H) approx. 2-fold to NAD(H), which is largely due to better apparent binding of the phosphorylated form of the coenzyme. NADP+ is a potent competitive inhibitor of the NADH-linked aldehyde reduction (Ki 1.5 microM), whereas NAD+ is not. Unlike mammalian aldose reductase, the enzyme from C. tenuis is not subject to oxidation-induced activation. Evidence of an essential lysine residue located in or near the coenzyme binding site has been obtained from chemical modification of aldose reductase with pyridoxal 5'-phosphate. The results are discussed in the context of a comparison of the enzymic properties of yeast and mammalian aldose reductase.
| S-EPMC1218722 | biostudies-other
Project description:The alcohol dehydrogenase (ADH) system plays a critical role in sugar metabolism involving in not only ethanol formation and consumption but also the general "cofactor balance" mechanism. Candida maltosa is able to ferment glucose as well as xylose to produce a significant amount of ethanol. Here we report the ADH system in C. maltosa composed of three microbial group I ADH genes (CmADH1, CmADH2A and CmADH2B), mainly focusing on its metabolic regulation and physiological function.Genetic analysis indicated that CmADH2A and CmADH2B tandemly located on the chromosome could be derived from tandem gene duplication. In vitro characterization of enzymatic properties revealed that all the three CmADHs had broad substrate specificities. Homo- and heterotetramers of CmADH1 and CmADH2A were demonstrated by zymogram analysis, and their expression profiles and physiological functions were different with respect to carbon sources and growth phases. Fermentation studies of ADH2A-deficient mutant showed that CmADH2A was directly related to NAD regeneration during xylose metabolism since CmADH2A deficiency resulted in a significant accumulation of glycerol.Our results revealed that CmADH1 was responsible for ethanol formation during glucose metabolism, whereas CmADH2A was glucose-repressed and functioned to convert the accumulated ethanol to acetaldehyde. To our knowledge, this is the first demonstration of function separation and glucose repression of ADH genes in xylose-fermenting yeasts. On the other hand, CmADH1 and CmADH2A were both involved in ethanol formation with NAD regeneration to maintain NADH/NAD ratio in favor of producing xylitol from xylose. In contrast, CmADH2B was expressed at a much lower level than the other two CmADH genes, and its function is to be further confirmed.
| S-EPMC2909261 | biostudies-literature
Project description:BACKGROUND: The NAD(P)H-dependent Pichia stipitis xylose reductase (PsXR) is one of the key enzymes for xylose fermentation, and has been cloned into the commonly used ethanol-producing yeast Saccharomyces cerevisiae. In order to eliminate the redox imbalance resulting from the preference of this enzyme toward NADPH, efforts have been made to alter the coenzyme specificity of PsXR by site-directed mutagenesis, with limited success. Given the industrial importance of PsXR, it is of interest to investigate further ways to create mutants of PsXR that prefers NADH rather than NADPH, by the alternative directed evolution approach. RESULTS: Based on a homology model of PsXR, six residues were predicted to interact with the adenine ribose of NAD(P)H in PsXR and altered using a semi-rational mutagenesis approach (CASTing). Three rounds of saturation mutagenesis were carried to randomize these residues, and a microplate-based assay was applied in the screening. A best mutant 2-2C12, which carried four mutations K270S, N272P, S271G and R276F, was obtained. The mutant showed a preference toward NADH over NADPH by a factor of about 13-fold, or an improvement of about 42-fold, as measured by the ratio of the specificity constant kcat/Kmcoenzyme. Compared with the wild-type, the kcatNADH for the best mutant was only slightly lower, while the kcatNADPH decreased by a factor of about 10. Furthermore, the specific activity of 2-2C12 in the presence of NADH was 20.6 U.mg-1, which is highest among PsXR mutants reported. CONCLUSION: A seemingly simplistic and yet very effective mutagenesis approach, CASTing, was applied successfully to alter the NAD(P)H preference for Pichia stipitis xylose reductase, an important enzyme for xylose-fermenting yeast. The observed change in the NAD(P)H preference for this enzyme seems to have resulted from the altered active site that is more unfavorable for NADPH than NADH in terms of both Km and kcat. There are potentials for application of our PsXR in constructing a more balanced XR-XDH pathway in recombinant xylose-fermenting S. cerevisiae strains.
| S-EPMC2213684 | biostudies-literature
Project description:Two strains of a D-xylose-fermenting yeast species were isolated from rotten wood samples collected from the Baotianman Nature Reserve in Henan Province, central China. These strains formed hat-shaped ascospores in conjugated and deliquescent asci. Multilocus phylogenetic analysis that included the nearly complete small subunit (SSU), the internal transcribed spacer (ITS) region and the D1/D2 domain of the large subunit (LSU) rRNA genes, as well as RNA polymerase II largest subunit (RPB1) gene demonstrated that the two strains represent a novel yeast species closely related to Scheffersomyces segobiensis. A sequence comparison of xylose reductase (XYL1) gene, which was recently recommended for rapid identification of cryptic species in the Scheffersomyces clade, revealed a significant sequence divergence of 25 nucleotides between the novel strains and their closest relative S. segobiensis, supporting their classification as a distinct species. Furthermore, these new strains can be clearly distinguished from S. segobiensis by a number of morphological and physiological characteristics. Therefore, a novel yeast species, Scheffersomyces henanensis sp. nov., is proposed to accommodate these strains. The type strain is BY-41T ( = CICC 1974T = CBS 12475T).
| S-EPMC3960237 | biostudies-literature
Project description:Three strains of a d-xylose-fermenting yeast species were isolated from the host beetle Dorcus titanus collected from two different localities in Henan Province, Central China. These strains formed two hat-shaped ascospores in conjugated and deliquescent asci. Multilocus phylogenetic analysis that included the nearly complete small subunit (SSU), the internal transcribed spacer (ITS) region and the D1/D2 domains of the large subunit (LSU) rDNAs, as well as RNA polymerase II largest subunit (RPB1) gene demonstrated that these strains represent a novel yeast species belonging to the genus Scheffersomyces. The phylogenetic analysis based on the nucleotide sequences of the xylose reductase (XYL1) gene supported the view that the new strains could be grouped as a unique species. Although this new species is highly similar to Scheffersomyces stipitis-like yeasts in terms of nrDNA sequences and morphological and physiological characteristics, the species can be clearly differentiated from its close relatives on the basis of the sequences of XYL1 and RPB1. Therefore, a novel yeast species, Scheffersomyces titanus sp. nov., is proposed to accommodate these strains. The type strain is NYNU 14712(T) (CICC 33061(T) = CBS 13926(T)).
| S-EPMC4997322 | biostudies-literature
Project description:Novel bioreactor beads for simultaneous saccharification and fermentation (SSF) of lime-pretreated rice straw (RS) into ethanol were prepared. Genetically modified Saccharomyces cerevisiae cells expressing genes encoding xylose reductase, xylitol dehydrogenase, and xylulokinase were immobilized in calcium alginate beads containing inorganic lightweight filler particles to reduce specific gravity. For SSF experiments, the beads were floated in slurry composed of lime-pretreated RS and enzymes and incubated under CO2 atmosphere to reduce the pH for saccharification and fermentation. Following this reaction, beads were readily picked up from the upper part of the slurry and were directly transferred to the next vessel with slurry. After 240 h of incubation, ethanol production by the beads was equivalent to that by free cells, a trend that was repeated in nine additional runs, with slightly improved ethanol yields. Slurry with pre-saccharified lime-pretreated RS was subjected to SSF with floating beads for 168 h. Although higher cell concentrations in beads resulted in more rapid initial ethanol production rates, with negligible diauxic behavior for glucose and xylose utilization, no improvement in the ethanol yield was observed. A fermentor-scale SSF experiment with floating beads was successfully performed twice, with repeated use of the beads, resulting in the production of 40.0 and 39.7 g/L ethanol. There was no decomposition of the beads during agitation at 60 rpm. Thus, this bioreactor enables reuse of yeast cells for efficient ethanol production by SSF of lignocellulosic feedstock, without the need for instruments for centrifugation or filtration of whole slurry.
| S-EPMC8056895 | biostudies-literature
Project description:BackgroundXylitol is a five-carbon sugar alcohol that has numerous beneficial health properties. It has almost the same sweetness as sucrose but has lower energy value compared to the sucrose. Metabolism of xylitol is insulin independent and thus it is an ideal sweetener for diabetics. It is widely used in food products, oral and personal care, and animal nutrition as well. Here we present a two-stage strategy to produce bio-xylitol from D-xylose using a recombinant Pichia pastoris expressing a heterologous xylose reductase gene. The recombinant P. pastoris cells were first generated by a low-cost, standard procedure. The cells were then used as a catalyst to make the bio-xylitol from D-xylose.ResultsPichia pastoris expressing XYL1 from P. stipitis and gdh from B. subtilis demonstrated that the biotransformation was very efficient with as high as 80% (w/w) conversion within two hours. The whole cells could be re-used for multiple rounds of catalysis without loss of activity. Also, the cells could directly transform D-xylose in a non-detoxified hemicelluloses hydrolysate to xylitol at 70% (w/w) yield.ConclusionsWe demonstrated here that the recombinant P. pastoris expressing xylose reductase could transform D-xylose, either in pure form or in crude hemicelluloses hydrolysate, to bio-xylitol very efficiently. This biocatalytic reaction happened without the external addition of any NAD(P)H, NAD(P)+, and auxiliary substrate as an electron donor. Our experimental design & findings reported here are not limited to the conversion of D-xylose to xylitol only but can be used with other many oxidoreductase reactions also, such as ketone reductases/alcohol dehydrogenases and amino acid dehydrogenases, which are widely used for the synthesis of high-value chemicals and pharmaceutical intermediates.
| S-EPMC7898734 | biostudies-literature
Project description:Cellulosic biomass is an abundant and underused substrate for biofuel production. The inability of many microbes to metabolize the pentose sugars abundant within hemicellulose creates specific challenges for microbial biofuel production from cellulosic material. Although engineered strains of Saccharomyces cerevisiae can use the pentose xylose, the fermentative capacity pales in comparison with glucose, limiting the economic feasibility of industrial fermentations. To better understand xylose utilization for subsequent microbial engineering, we sequenced the genomes of two xylose-fermenting, beetle-associated fungi, Spathaspora passalidarum and Candida tenuis. To identify genes involved in xylose metabolism, we applied a comparative genomic approach across 14 Ascomycete genomes, mapping phenotypes and genotypes onto the fungal phylogeny, and measured genomic expression across five Hemiascomycete species with different xylose-consumption phenotypes. This approach implicated many genes and processes involved in xylose assimilation. Several of these genes significantly improved xylose utilization when engineered into S. cerevisiae, demonstrating the power of comparative methods in rapidly identifying genes for biomass conversion while reflecting on fungal ecology.
| S-EPMC3156214 | biostudies-literature
Project description:In this study, we evaluated the potential of yeasts isolated from Amazon to produce second-generation ethanol from sugarcane bagasse delignified with alkaline hydrogen peroxide and hydrolysed with commercial enzyme preparation. The best efficiency savings in glucose and release of xylose were determined by considering the solids and enzyme loads. Furthermore, we selected Spathaspora passalidarum UFMG-CM-Y473 strain with the best fermentative parameters. Fermentations used bagasse hydrolysate without any nutritional supplementation, a significant difference from previous studies, which is closer to industrial conditions. Ethanol yield of 0.32 g/g and ethanol productivity of 0.34 g/L h were achieved after the consumption of 78% of the sugar. This hydrolysis/fermentation technology package could represent the input of an additional 3180 L of ethanol per hectare in areas of average sugarcane productivity such as 60 ton/ha. Thus, we concluded that Sp. passalidarum UFMG-CM-Y473 has a clear potential for the production of second-generation ethanol from delignified and enzyme-hydrolysed bagasse.
| S-EPMC6041217 | biostudies-literature
Project description:BackgroundIt remains a challenge for recombinant S. cerevisiae to convert xylose in lignocellulosic biomass hydrolysates to ethanol. Although industrial diploid strains are more robust compared to laboratory haploid strains, however, industrial diploid S. cerevisiae strains have been less pursued in previous studies. This work aims to construct fast xylose-fermenting yeast using an industrial ethanol-producing diploid S. cerevisiae strain as a host.ResultsFast xylose-fermenting yeast was constructed by genome integration of xylose-utilizing genes and adaptive evolution, including 1) Piromyces XYLA was introduced to enable the host strain to convert xylose to xylulose; 2) endogenous genes (XKS1, RKI1, RPE1, TKL1, and TAL1) were overexpressed to accelerate conversion of xylulose to ethanol; 3) Candida intermedia GXF1, which encodes a xylose transporter, was introduced at the GRE3 locus to improve xylose uptake; 4) aerobic evolution in rich xylose media was carried out to increase growth and xylose consumption rates. The best evolved strain CIBTS0735 consumed 80 g/l glucose and 40 g/l xylose in rich media within 24 hours at an initial OD600 of 1.0 (0.63 g DCW/l) and produced 53 g/l ethanol.ConclusionsBased on the above fermentation performance, we conclude that CIBTS0735 shows great potential for ethanol production from lignocellulosic biomass.
| S-EPMC3878346 | biostudies-literature