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Background: Cost-effective production of biofuels from lignocellulose requires the fermentation of d-xylose. Many yeast species within and closely related to the genera Spathaspora and Scheffersomyces (both of the order Serinales) natively assimilate and ferment xylose. Other species consume xylose inefficiently, leading to extracellular accumulation of xylitol. Xylitol excretion is thought to be due to the different cofactor requirements of the first two steps of xylose metabolism. Xylose reductase (XR) generally uses NADPH to reduce xylose to xylitol, while xylitol dehydrogenase (XDH) generally uses NAD+ to oxidize xylitol to xylulose, creating an imbalanced redox pathway. This imbalance is thought to be particularly consequential in hypoxic or anoxic environments. Results: We screened the growth of xylose-fermenting yeast species in high and moderate aeration and identified both ethanol producers and xylitol producers. Selected species were further characterized for their XR and XDH cofactor preferences by enzyme assays and gene expression patterns by RNA-Seq. Our data revealed that xylose metabolism is more redox balanced in some species, but it is strongly affected by oxygen levels. Under high aeration, most species switched from ethanol production to xylitol accumulation, despite the availability of ample oxygen to accept electrons from NADH. This switch was followed by decreases in enzyme activity and the expression of genes related to xylose metabolism, suggesting that bottlenecks in xylose fermentation are not always due to cofactor preferences. Finally, we expressed XYL genes from multiple Scheffersomyces species in a strain of Saccharomyces cerevisiae. Recombinant S. cerevisiae expressing XYL1 from Scheffersomyces xylosifermentans, which encodes an XR without a cofactor preference, showed improved anaerobic growth on xylose as the primary carbon source compared to S. cerevisiae strain expressing XYL genes from Scheffersomyces stipitis. Conclusion: Collectively, our data do not support the hypothesis that xylitol accumulation occurs primarily due to differences in cofactor preferences between xylose reductase and xylitol dehydrogenase; instead, gene expression plays a major role in response to oxygen levels. We have also identified the yeast Sc. xylosifermentans as a potential source for genes that can be engineered into S. cerevisiae to improve xylose fermentation and biofuel production. [ABSTRACT FROM AUTHOR]

High temperature fermentation can substantially reduce the contaminations and condensation costs. Candida glycerinogenes was more resistant to high temperature than Saccharomyces cerevisiae. Quantitative real-time PCR results showed that 7 of 33 candidates served as potential high temperature inducible promoters in C. glycerinogenes. Fluorescence analysis indicated that P Cgcwp1 showed the highest activity at 42°C. P Cgaac and P Cgpot1 showed medium–high expression, while P Cghsp12 , P Cgatp1 , P Cgino1 and P Cgscl were modest or weaker expression. Above seven promoters were further used to control the expression of xylose reductase gene from Neurospora crassa in C. glycerinogenes. Compared with 30°C, the xylitol yields of the seven recombinant strains were all improved at 42°C, and P Cgcwp1 showed the highest xylitol production which was increased by 204% and reached 15.4 g/L. These results showed a potential to high temperature fermentation on the stress tolerant C. glycerinogenes and some of novel high temperature inducible promoters. [ABSTRACT FROM AUTHOR]

The key catabolic enzymes of D-xylose, an important structural component of different agricultural wastes, were studied in cells of mutant strains of the xylose-assimilating yeast Pachysolen tannophilus. The evaluation of catalytic activity and cofactor specificity of xylose reductase (EC 1.1.1.307) and xylitol dehydrogenase (EC 1.1.1.9) confirmed the dependence of intracellular catabolic pathway for D-xy lose on the NAD×H/NADP×H ratio, formed under microaerobic conditions. The study of total activity of some NAD+/NAP×H-dependent dehydrogenases revealed the metabolic characteristics of the yeast cells, which could ensure selective ethanol or xylitol production. Thus, the efficient involvement of D-xylose into the Embden-Meyerhof-Parnas pathway provided not only the high activities of xylose reductase and xylitol dehydrogenase, but also of 1-glycerophosphate dehydrogenase (EC 1.1.1.8) and lactate dehydrogenase (EC 1.1.1.27), respectively. The inhibition of activity of these enzymes led to selective production of xylitol from D-xylose. On the base of the experimental results, the principles of metabolic engineering of xylose-assimilating yeasts were formulated. The possibility of bioethanol and xylitol production from different agricultural wastes using xyloseassimilating yeasts are discussed. [ABSTRACT FROM AUTHOR]

Xylose assimilation and fermentation are important traits for second generation ethanol production. However, some genomic features associated with this pentose sugar's metabolism remain unknown in yeasts. Comparative genomics studies have led to important insights in this field, but we are still far from completely understanding endogenous yeasts' xylose metabolism. In this work, we carried out a deep evolutionary analysis suited for comparative genomics of xylose-consuming yeasts, searching for of positive selection on genes associated with glucose and xylose metabolism in the xylose-fermenters' clade. Our investigation detected positive selection fingerprints at this clade not only among sequences of important genes for xylose metabolism, such as xylose reductase and xylitol dehydrogenase, but also in genes expected to undergo neutral evolution, such as the glycolytic gene phosphoglycerate mutase. In addition, we present expansion, positive selection marks, and convergence as evidence supporting the hypothesis that natural selection is shaping the evolution of the little studied methylglyoxal reductases. We propose a metabolic model suggesting that selected codons among these proteins caused a putative change in cofactor preference from NADPH to NADH that alleviates cellular redox imbalance. These findings provide a wider look into pentose metabolism of yeasts and add this previously overlooked piece into the intricate puzzle of oxidative imbalance. Although being extensively discussed in evolutionary works the awareness of selection patterns is recent in biotechnology researches, rendering insights to surpass the reached status quo in many of its subareas. [ABSTRACT FROM AUTHOR]

Background: Understanding the effects of oxygen levels on yeast xylose metabolism would benefit ethanol production. In this work, xylose fermentative capacity of Schefersomyces stipitis, Spathaspora passalidarum, Spathaspora arborariae and Candida tenuis was systematically compared under aerobic, oxygen-limited and anaerobic conditions. Results: Fermentative performances of the four yeasts were greatly influenced by oxygen availability. S. stipitis and S. passalidarum showed the highest ethanol yields (above 0.44 g g-1) under oxygen limitation. However, S. passalidarum produced 1.5 times more ethanol than S. stipitis under anaerobiosis. While C. tenuis showed the lowest xylose consumption rate and incapacity to produce ethanol, S. arborariae showed an intermediate fermentative performance among the yeasts. NAD(P)H xylose reductase (XR) activity in crude cell extracts correlated with xylose consumption rates and ethanol production. Conclusions: Overall, the present work demonstrates that the availability of oxygen influences the production of ethanol by yeasts and indicates that the NADH-dependent XR activity is a limiting step on the xylose metabolism. S. stipitis and S. passalidarum have the greatest potential for ethanol production from xylose. Both yeasts showed similar ethanol yields near theoretical under oxygen-limited condition. Besides that, S. passalidarum showed the best xylose consumption and ethanol production under anaerobiosis. [ABSTRACT FROM AUTHOR]

Background: An advantageous but challenging approach to overcome the limited supply of petroleum and relieve the greenhouse effect is to produce bulk chemicals from renewable materials. Fatty alcohols, with a billion-dollar global market, are important raw chemicals for detergents, emulsifiers, lubricants, and cosmetics production. Microbial production of fatty alcohols has been successfully achieved in several industrial microorganisms. However, most of the achievements were using glucose, an edible sugar, as the carbon source. To produce fatty alcohols in a renewable manner, non-edible sugars such as xylose will be a more appropriate feedstock. Results: In this study, we aim to engineer a Saccharomyces cerevisiae strain that can efficiently convert xylose to fatty alcohols. To this end, we first introduced the fungal xylose utilization pathway consisting of xylose reductase (XR), xylitol dehydrogenase (XDH), and xylulose kinase (XKS) into a fatty alcohol-producing S. cerevisiae strain (XF3) that was developed in our previous studies to achieve 1-hexadecanol production from xylose at 0.4 g/L. We next applied promoter engineering on the xylose utilization pathway to optimize the expression levels of XR, XDH, and XKS, and increased the 1-hexadecanol titer by 171%. To further improve the xylose-based fatty alcohol production, two optimized S. cerevisiae strains from promoter engineering were evolved with the xylose as the sole carbon source. We found that the cell growth rate was improved at the expense of decreased fatty alcohol production, which indicated 1-hexadecanol was mainly produced as a non-growth associated product. Finally, through fed-batch fermentation, we successfully achieved 1-hexadecanol production at over 1.2 g/L using xylose as the sole carbon source, which represents the highest titer of xylose-based 1-hexadecanol reported in microbes to date. Conclusions: A fatty alcohol-producing S. cerevisiae strain was engineered in this study to produce 1-hexadecanol from xylose. Although the xylose pathway we developed in this study could be further improved, this proof-of-concept study, for the first time to our best knowledge, demonstrated that the xylose-based fatty alcohol could be produced in S. cerevisiae with potential applications in developing consolidated bioprocessing for producing other fatty acid-derived chemicals. [ABSTRACT FROM AUTHOR]

Xylose, recovered from Meranti wood sawdust (MWS), can be used as a promising and alternative carbon source for yeast growth as well as for the production of xylose reductase (XR). This enzyme has potential applications in the bioproduction of various high value products, especially xylitol. The aim of this study was to isolate XR from adapted yeast Candida tropicalis and to characterize it. The XR enzyme was prepared from C. tropicalis strain, grown in MWS hydrolysate-based medium, by ultrasonic homogenization. The isolated XR was characterized based on enzyme activity, stability, and kinetic constants. The activity of NADPH-dependent crude XR measured was 11.16 U/mL. It was stable at pH 5.0–7.0 and temperature of 25–40 °C for 24 h, and retained above 95% of its original activity after 4 months of storage at –80 °C. The apparent K m values of XR for xylose and NADPH were 81.78 mM and 7.29 μM while the V max for xylose and NADPH were 178.57 and 12.5 μM/min, respectively. The low K m,app and high V max,app values of XR for xylose as a substrate indicates a strong binding affinity for xylose and good productivity of the reaction. [ABSTRACT FROM AUTHOR]

Background Bioplastics, like polylactic acid (PLA), are renewable alternatives for petroleum-based plastics. Lactic acid, the monomer of PLA, has traditionally been produced biotechnologically with bacteria. With genetic engineering, yeast have the potential to replace bacteria in biotechnological lactic acid production, with the benefits of being acid tolerant and having simple nutritional requirements. Lactate dehydrogenase genes have been introduced to various yeast to demonstrate this potential. Importantly, an industrial lactic acid producing process utilising yeast has already been implemented. Utilisation of D-xylose in addition to D-glucose in production of biochemicals such as lactic acid by microbial fermentation would be beneficial, as it would allow lignocellulosic raw materials to be utilised in the production processes. Results The yeast Candida sonorensis, which naturally metabolises D-xylose, was genetically modified to produce L-lactic acid from D-xylose by integrating the gene encoding L-lactic acid dehydrogenase (ldhL) from Lactobacillus helveticus into its genome. In microaerobic, CaCO3-buffered conditions a C. sonorensis ldhL transformant having two copies of the ldhL gene produced 31 g 1-1 lactic acid from 50 g 1-1 D-xylose free of ethanol. Anaerobic production of lactic acid from D-xylose was assessed after introducing an alternative pathway of D-xylose metabolism, i.e. by adding a xylose isomerase encoded by XYLA from Piromyces sp. alone or together with the xylulokinase encoding gene XKS1 from Saccharomyces cerevisiae. Strains were further modified by deletion of the endogenous xylose reductase encoding gene, alone or together with the xylitol dehydrogenase encoding gene. Strains of C. sonorensis expressing xylose isomerase produced L-lactic acid from Dxylose in anaerobic conditions. The highest anaerobic L-lactic acid production (8.5 g1-1) was observed in strains in which both the xylose reductase and xylitol dehydrogenase encoding genes had been deleted and the xylulokinase encoding gene from S. cerevisiae was overexpressed. Conclusions Integration of two copies of the ldhL gene in C. sonorensis was sufficient to obtain good Llactic acid production from D-xylose. Under anaerobic conditions, the ldhL strain with exogenous xylose isomerase and xylulokinase genes expressed and the endogenous xylose reductase and xylitol dehydrogenase genes deleted had the highest L- lactic acid production. [ABSTRACT FROM AUTHOR]

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). [ABSTRACT FROM AUTHOR]

Fermentation of xylose in lignocellulosic hydrolysates by Saccharomyces cerevisiae has been achieved through heterologous expression of the xylose reductase (XR)-xylitol dehydrogenase (XDH) pathway. However, the fermentation efficiency is far from the requirement for industrial application due to high yield of the byproduct xylitol, low ethanol yield, and low xylose consumption rate. Through evolutionary engineering, an improved xylose-utilizing strain SyBE005 was obtained with 78.3 % lower xylitol production and a 2.6-fold higher specific ethanol production rate than those of the parent strain SyBE004, which expressed an engineered NADP-preferring XDH. The transcriptional differences between SyBE005 and SyBE004 were investigated by quantitative RT-PCR. Genes including XYL1, XYL2, and XKS1 in the initial xylose metabolic pathway showed the highest up-regulation in SyBE005. The increased expression of XYL1 and XYL2 correlated with enhanced enzymatic activities of XR and XDH. In addition, the expression level of ZWF1 in the oxidative pentose phosphate pathway increased significantly in SyBE005, indicating an elevated demand for NADPH from XR. Genes involved in the TCA cycle ( LAT1, CIT1, CIT2, KGD1, KGD, SDH2) and gluconeogenesis ( ICL1, PYC1) were also up-regulated in SyBE005. Genomic analysis revealed that point mutations in transcriptional regulators CYC8 and PHD1 might be responsible for the altered expression. In addition, a mutation (Y89S) in ZWF1 was identified which might improve NADPH production in SyBE005. Our results suggest that increasing the expression of XYL1, XYL2, XKS1, and enhancing NADPH supply are promising strategies to improve xylose fermentation in recombinant S. cerevisiae. [ABSTRACT FROM AUTHOR]

We constructed a xylose-fermenting recombinant strain of thermotolerant yeast Kluyveromyces marxianus, DMB3-7. Both xylose consumption and ethanol production were remarkably increased in DMB3-7 compared to the control strain at 30°C. Furthermore, DMB3-7 produced ethanol from xylose at both 42°C and 45°C, above which xylose metabolic activity decreased. [ABSTRACT FROM AUTHOR]

Xylitol is a high value sugar alcohol with anticariogenic properties that is used as an ideal sweetener for diabetic patients. Industrially, xylitol is manufactured by catalytic reduction of pure xylose, which has some disadvantages. The fermentation process has been studied as an alternative, but its viability is dependent on the optimization of several variables. This fermentation process on an industrial-scale is not feasible due to decreased productivity. Compared to the fermentation process, enzymatic method is expected to make a substantial increase in productivity. Enzymatic xylitol production from xylose exist in lignocellulosics is an attractive and promising alternative method to the chemical process. The enzymatic method might be able to overcome the disadvantages of the chemical process. This article reviews the literature on the processes for xylitol production and identifies further ways for improved xylitol production to compete with the current chemical process. [ABSTRACT FROM AUTHOR]

The article presents a study that examines the ethanol production from xylose by a recombinant industrial yeast Candida utilis (C. utilis). It notes that xylose is one of the most abundant sugars derived from lignocellulose where fungi and bacteria dwells on it aerobically. Furthermore , analysis of the genes responsible for ethanol production has confirmed that the introduction of CsheXDH was important as overexpression of such improved the efficiency of ethanol production.

At present, yeast is the main microorganism involved in ethanol production using xylose as carbon source. The filamentous fungi have been less studied compared to yeast. Duchenne tube and ethanol yield assay methods were applied to screen different microbes for xylose fermentation. The microorganism producing the highest amount of ethanol was identified by microscopy and 18s rRNA amplification and analysis. Fermentation assays of this microorganism were studied by the Potassium dichromate method. The screening results showed that the organism code named cs -- 28 was Fusarium oxysporum. It was found that this fungus could ferment xylose, glucose, sucrose and corn stalk to produce ethanol. The optimal nitrogen source was yeast extract. The ethanol production rate was highest at initial pH 6.0 and 30 °C, when initial xylose concentration was 20 g · L-1. Thus, cs -- 28 can be used as a new strain for transformation of cellulose materials to ethanol. Moreover, characterization of cs -- 28 on xylose fermentation can be applied in technological advancement. [ABSTRACT FROM AUTHOR]

Abstract: Xylose reductase (XR) and xylitol dehydrogenase (XDH) are the key enzymes for xylose fermentation and have been widely used for construction of a recombinant xylose fermenting yeast. The effective recycling of cofactors between XR and XDH has been thought to be important to achieve effective xylose fermentation. Efforts to alter the coenzyme specificity of XR and HDX by site-directed mutagenesis have been widely made for improvement of efficiency of xylose fermentation. We previously succeeded by protein engineering to improve ethanol production by reversing XDH dependency from NAD+ to NADP+. In this study, we applied protein engineering to construct a novel strictly NADPH-dependent XR from Pichia stipitis by site-directed mutagenesis, in order to recycle NADPH between XR and XDH effectively. One double mutant, E223A/S271A showing strict NADPH dependency with 106% activity of wild-type was generated. A second double mutant, E223D/S271A, showed a 1.27-fold increased activity compared to the wild-type XR with NADPH and almost negligible activity with NADH. [ABSTRACT FROM AUTHOR]

Xylose reductase (XR) is the enzyme that catalyzes the first step of xylose metabolism. Although XRs from various yeasts have been characterized, little is known about this enzyme in Debaryomyces hansenii. In the present study, response surface analysis was used to determine the optimal conditions for D. hansenii UFV-170 XR activity. The influence of pH and temperature, ranging from 4.0 to 8.0 and from 25 to 55°C, respectively, was evaluated by a 22 central composite design face-centered. The F-test (ANOVA) and the Student’s t test were performed to evaluate the statistical significance of the model and the regression coefficients, respectively. The NADPH-dependent XR activity varied from 0.502 to 2.53 U mL−1, corresponding to 0.07–0.352 U mg−1, whereas the NADH-dependent one was almost negligible. The model predicted with satisfactory correlation ( R2 = 0.940) maximum volumetric activity of 2.27 U mL−1 and specific activity of 0.300 U mg−1 at pH 5.3 and 39°C, which were fairly confirmed by additional tests performed under these conditions. The enzyme proved very stable at low temperature (4°C), keeping its activity almost entirely after 360 min, which corresponded to the half-time at 39°C. On the other hand, at temperatures ≥50°C it was lost almost completely after only 20 min. [ABSTRACT FROM AUTHOR]

We varied the promoter strength of xylose reductase (XR) gene and the copy number of xylulokinase (XK) gene to determine how XR and XK activities affect the xylose-fermenting abilities of recombinant Saccharomyces cerevisiae expressing xylitol dehydrogenase (XDH). The most enhanced ethanol yield and lowered xylitol yield occurred in strain I-PGK/AUR, which has high activity of both XR and XDH and moderate XK activity. [Copyright &y& Elsevier]

The purpose of this study was to help lay the foundation for further development of xylose-fermenting Saccharomyces cerevisiae yeast strains through an approach that combined metabolic engineering and random mutagenesis in a recombinant haploid strain that overexpressed only two genes of the xylose pathway. Previously, S. cerevisiae strains, overexpressing heterologous genes encoding xylose reductase, xylitol dehydrogenase and the endogenous XKS1 xylulokinase gene, were randomly mutagenised to develop improved xylose-fermenting strains. In this study, two gene cassettes ( ADH1 p - PsXYL1-ADH1 T and PGK1 p - PsXYL2-PGK1 T ) containing the xylose reductase ( PsXYL1) and xylitol dehydrogenase ( PsXYL2) genes from the xylose-fermenting yeast, Pichia stipitis, were integrated into the genome of a haploid S. cerevisiae strain (CEN.PK 2-1D). The resulting recombinant strain (YUSM 1001) over-expressing the P. stipitis XYL1 and XYL2 genes (but not the endogenous XKS1 gene) was subjected to ethyl methane sulfonate (EMS) mutagenesis. The resulting mutants were screened for faster growth rates on an agar medium containing xylose as the sole carbon source. A mutant strain (designated Y-X) that showed 20-fold faster growth in xylose medium in shake-flask cultures was isolated and characterised. In anaerobic batch fermentation, the Y-X mutant strain consumed 2.5-times more xylose than the YUSM 1001 parental strain and also produced more ethanol and glycerol. The xylitol yield from the mutant strain was lower than that from the parental strain, which did not produce glycerol and ethanol from xylose. The mutant also showed a 50% reduction in glucose consumption rate. Transcript levels of XYL1, XYL2 and XKS1 and the GPD2 glycerol 3-phosphate dehydrogenase gene from the two strains were compared with real-time reverse transcription polymerase chain reaction (RT-PCR) analysis. The mutant showed 10–40 times higher relative expression of these four genes, which corresponded with either the higher activities of their encoded enzymes or by-product formation during fermentation. Furthermore, no mutations were observed in the mutant’s promoter sequences or the open reading frames of some of its key genes involved in carbon catabolite repression, glycerol production and redox balancing. The data suggest that the enhancement of the xylose fermentation properties of the Y-X mutant was made possible by increased expression of the xylose pathway genes, especially the XKS1 xylulokinase gene. [ABSTRACT FROM AUTHOR]

Xylitol is a well-known sugar substitute with low-calorie and anti-cariogenic characteristics. An effort of biological production of xylitol from xylose was made in repeated fed-batch and cell-recycle fermentations of recombinant Saccharomyces cerevisiae BJ3505/δXR harboring the xylose reductase gene from Pichia stipitis. Batch fermentation with 20g/l xylose and 18g/l glucose resulted in 9.52g/l dry cell mass, 20.1g/l xylitol concentration and approximately 100% conversion yield. Repeated fed-batch operation to remove 10% of culture broth and to supplement an equal volume of 200g/l xylose was designed to improve xylitol production. In spite of a sudden drop of cell concentration, an increase in dry cell mass led to high accumulation of xylitol at 48.7g/l. To overcome loss of xylitol-producing biocatalysts in repeated fed-batch fermentation, cell-recycle equipment of hollow fiber membrane was implemented into a xylitol production system. Cell-recycle operation maintained concentration of the recombinant cells high inside a bioreactor. Final dry cell mass of 22.0g/l, 116g/l xylitol concentration, 2.34g/lh overall xylitol productivity were obtained in cell-recycle fermentation supplemented with xylose and yeast extract solution, which were equivalent to 2.3-, 5.8- and 3.8-fold increases compared with the corresponding values of batch-type xylitol production parameters. [Copyright &y& Elsevier]

The activities of xylitol dehydrogenase and xylose reductase in the yeasts Candida shehatae, C. didensiae, C. intermediae, C. tropicalis, Kluyveromyces marxianus, Pichia stipitis, P. guillermondii, Pachysolen tannophilus, and Torulopsis molishiama were studied at different oxygen transfer rates (OTRs) to the fermentation medium (0, 5, and 140 mmol O2/(l h)). The activities of these enzymes were maximum in the yeasts P. stipitis and C. shehatae. The xylitol dehydrogenase of all the yeasts was NAD+-dependent, irrespective of the intensity of aeration. The xylose reductase of the yeasts C. didensiae, C. intermediae, C. tropicalis, Kl. marxianus, P. guillermondii, and T. molishiama was NADPH-dependent, whereas the xylose reductase of P. stipitis, C. shehatae, and Pa. tannophilus was specific for both NADPH and NADH. The effect of OTR on the activities of the different forms of xylitol dehydrogenase and xylose reductase in xylose-assimilating yeasts is discussed. [ABSTRACT FROM AUTHOR]

The activity and the cofactor specificity of xylose reductase and xylitol dehydrogenase were studied in extracts of yeasts from the genera Candida, Kluyveromyces, Pachysolen, Pichia, and Torulopsis grown under microaerobic conditions. It was found that xylitol dehydrogenase in all of the yeast species studied is specific for NAD+; xylose reductase in the xylitol-producing species C. didensiae, C. intermediae, C. parapsilosis, C. silvanorum, C. tropicalis, Kl. fragilis, Kl. marxianus, P. guillermondii, and T. molishiama is specific for NADPH; and xylose reductase in the ethanol-producing species P. stipitis, C. shehatae, and Pa. tannophilus is specific for both NADPH and NADH. [ABSTRACT FROM AUTHOR]

Yeast xylose reductases are hypothesized as hybrid enzymes as their primary sequences contain elements of both the aldo–keto reductases (AKR) and short chain dehydrogenase/reductase (SDR) enzyme families. During catalysis by members of both enzyme families, an essential Lys residue H-bonds to a Tyr residue that donates proton to the aldehyde substrate. In the Saccharomyces cerevisiae xylose reductase, Tyr49 has been identified as the proton donor. However, the primary sequence of the enzyme contains two Lys residues, Lys53 and Lys78, corresponding to the conserved motifs for SDR and AKR enzyme families, respectively, that may H-bond to Tyr49. We used site-directed mutagenesis to substitute each of these Lys residues with Met. The activity of the K53M variant was slightly decreased as compared to the wild-type, while that of the K78M variant was negligible. The results suggest that Lys78 is the essential residue that H-bonds to Tyr49 during catalysis and indicate that the active site residues of yeast xylose reductases match those of the AKR, rather than SDR, enzymes. Intrinsic enzyme fluorescence spectroscopic analysis suggests that Lys78 may also contribute to the efficient binding of NADPH to the enzyme. [Copyright &y& Elsevier]

Several compounds were examined for their inhibitory effects on xylose reductase from the yeast Pichia stipitis NRC 2548. Mercuric chloride, cupric chloride, menadione sodium bisulfite, and sodium bisulfite inhibited enzyme activity in a sigmoidal dose-dependent manner, whereas quercetin and rutin were observed to have nonsigmoidal dose-response curves. Diphenylhydantoin, hydantoin, and valproic acid had no effect on xylose reductase activity. Mercuric chloride was the most potent inhibitor tested, with an IC (the concentration that inhibited enzyme activity by 50%) of 4.7xl0M. Three distinct inhibition patterns were observed amongst selected inhibitors. Mercuric chloride and quercetin were noncompetitive inhibitors of xylose reductase with respect to substrate and cofactor. Sodium bisulfite was an uncompetitive inhibitor with respect to substrate and cofactor, whereas menadione sodium bisulfite was a competitive inhibitor with respect to substrate, but noncompetitive to the cofactor. [ABSTRACT FROM AUTHOR]

The relationship between the degree of aerobiosis, xylitol production and the initial two key enzymes of d-xylose metabolism were investigated in the yeasts Pichia stipitis, Candida shehatae and C. tenuis. Anoxic conditions severely curtailed growth and retarded ethanol productivity. This, together with the inverse relationship between xylitol accumulation and aeration level, suggested a degree of redox imbalance. The ratios of NADH- to NADPH-linked xylose reductase were similar in all three yeasts and essentially independent of the degree of aerobiosis, and thus did not correlate with their differing capacities for ethanol production, xylitol accumulation or growth under the different conditions of aerobiosis. Under anoxic conditions the enzyme activity of Pichia stipitis decreased significantly, which possibly contributed to its weaker anoxic fermentation of xylose compared to C. shehatae. [ABSTRACT FROM AUTHOR]

Meyerozyma caribbica is prominently used for the production of xylitol from agro waste hydrolysate of the corncob. M. caribbica was isolated from fermented apple juice, the nicotinamide adenine dinucleotide phosphate (NADP) dependent xylose reductase (XR) enzyme act as a catalyst for the production of xylitol from xylose during xylose fermentation by M. caribbica. The enzyme XR was extracted, partially purified, sequenced and molecular weight was determined by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF). This study explores the in silico and in vitro comparative analysis of the cofactor NADP binding mechanism in rossmann fold of isolated M. caribbica and other xylose fermenting Debaryomycetaceae family yeast. The rossmaan fold analysis of M. caribbica and Debaryomycetaceae family of yeast revealed, xylose fermenting organisms such as Scheffersomyces stipitis, Debaryomyces hansenii, Debaryomyces nepalensis, Candida parapsilosis, Candida maltos a have divergence structure of rosmann fold. Whereas M. caribbica and Candida tenuis has unique structure of rossmann fold. Also, the identified rossmann fold of Debaryomycetaceae family yeast and isolated M. caribbica are conserved to interact with cofactor NADP by H 2 bonding, which displays the proximity evolutionary relationship of the enzyme in Debaryomycetaceae family yeast. Annotation analysis showed the isolated M. caribbica has the ability of aldopentose fermentation for xylitol production. Image 1 • Isolation of xylose fermenting yeast M. caribbica for xylitol production. • The isolated yeast were identified using 16s rRNA gene sequences. • M. caribbica and C. tenuis have close evolution relationship in rossmann fold. • Acidophilic yeast M. caribbica acts as the potential strain for xylose fermentation. [ABSTRACT FROM AUTHOR]