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BackgroundCost-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.ResultsWe 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.ConclusionCollectively, 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.

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Abstract 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 Background Over the last decades, many strategies to procure and improve xylose consumption in Saccharomyces cerevisiae have been reported. This includes the introduction of efficient xylose-assimilating enzymes, the improvement of xylose transport, or the alteration of the sugar signaling response. However, different strain backgrounds are often used, making it difficult to determine if the findings are transferrable both to other xylose-consuming strains and to other xylose-assimilation pathways. For example, the influence of anomerization rates between α- and β-xylopyranose in pathway optimization and sugar sensing is relatively unexplored. Results In this study, we tested the effect of expressing a xylose epimerase in S. cerevisiae strains carrying different xylose-consuming routes. First, XIs originating from three different species in isogenic S. cerevisiae strains were tested and the XI from Lachnoclostridium phytofermentans was found to give the best performance. The benefit of increasing the anomerization rate of xylose by adding a xylose epimerase to the XI strains was confirmed, as higher biomass formation and faster xylose consumption were obtained. However, the impact of xylose epimerase was XI-dependent, indicating that anomer preference may differ from enzyme to enzyme. The addition of the xylose epimerase in xylose reductase/xylitol dehydrogenase (XR/XDH)-carrying strains gave no improvement in xylose assimilation, suggesting that the XR from Spathaspora passalidarum had no anomer preference, in contrast to other reported XRs. The reduction in accumulated xylitol that was observed when the xylose epimerase was added may be associated with the upregulation of genes encoding endogenous aldose reductases which could be affected by the anomerization rate. Finally, xylose epimerase addition did not affect the sugar signaling, whereas the type of xylose pathway (XI vs. XR/XDH) did. Conclusions Although xylose anomer specificity is often overlooked, the addition of xylose epimerase should be considered as a key engineering step, especially when using the best-performing XI enzyme from L. phytofermentans. Additional research into the binding mechanism of xylose is needed to elucidate the enzyme-specific effect and decrease in xylitol accumulation. Finally, the differences in sugar signaling responses between XI and XR/XDH strains indicate that either the redox balance or the growth rate impacts the SNF1/Mig1p sensing pathway.

Background: Over the last decades, many strategies to procure and improve xylose consumption in Saccharomyces cerevisiae have been reported. This includes the introduction of efficient xylose-assimilating enzymes, the improvement of xylose transport, or the alteration of the sugar signaling response. However, different strain backgrounds are often used, making it difficult to determine if the findings are transferrable both to other xylose-consuming strains and to other xylose-assimilation pathways. For example, the influence of anomerization rates between α- and β-xylopyranose in pathway optimization and sugar sensing is relatively unexplored. Results: In this study, we tested the effect of expressing a xylose epimerase in S. cerevisiae strains carrying different xylose-consuming routes. First, XIs originating from three different species in isogenic S. cerevisiae strains were tested and the XI from Lachnoclostridium phytofermentans was found to give the best performance. The benefit of increasing the anomerization rate of xylose by adding a xylose epimerase to the XI strains was confirmed, as higher biomass formation and faster xylose consumption were obtained. However, the impact of xylose epimerase was XI-dependent, indicating that anomer preference may differ from enzyme to enzyme. The addition of the xylose epimerase in xylose reductase/xylitol dehydrogenase (XR/XDH)-carrying strains gave no improvement in xylose assimilation, suggesting that the XR from Spathaspora passalidarum had no anomer preference, in contrast to other reported XRs. The reduction in accumulated xylitol that was observed when the xylose epimerase was added may be associated with the upregulation of genes encoding endogenous aldose reductases which could be affected by the anomerization rate. Finally, xylose epimerase addition did not affect the sugar signaling, whereas the type of xylose pathway (XI vs. XR/XDH) did. Conclusions: Although xylose anomer specificity is often overlooked, the addition of xylose epimerase should be considered as a key engineering step, especially when using the best-performing XI enzyme from L. phytofermentans. Additional research into the binding mechanism of xylose is needed to elucidate the enzyme-specific effect and decrease in xylitol accumulation. Finally, the differences in sugar signaling responses between XI and XR/XDH strains indicate that either the redox balance or the growth rate impacts the SNF1/Mig1p sensing pathway. [ABSTRACT FROM AUTHOR]

Purpose: Xylitol is used in the food and pharmaceutical industries as a sweetener, and its consumption rate has remarkably increased over the years. Currently, xylitol is produced through a chemical reduction method. However, considering the global climate change issue, the development of eco-friendly, renewable, and sustainable substrates for the production of high-value platform chemicals such as xylitol is the need of the hour. Hence, the present study aimed to use a microbial process for xylitol production. Methods: Escherichia coli M15 platform was used to overexpress the D-xylose reductase (XR) gene from a mesophilic yeast, Candida tropicalis GRA1. The 37-KDa CtXR sequence exhibited a highly conserved active site structure, where a tetrad of residues (Tyr51, Lys80, His113, and Asp46) was located at the base of the substrate-binding pocket. To mitigate the rate-limiting step of cofactor supply in the bioconversion of xylose to xylitol, overexpression of XR genes coupled with auxiliary substrates was used toward in vivo cofactor regeneration. Results: In the presence of xylose as the only carbon source, the M15 CtXRΔ produced 2.1 g L−1 of xylitol. The xylitol titer showed a steady-state increase with the addition of auxiliary substrates glucose and glycerol to 3.4 and 6.4 g L−1, respectively. Further, M15 CtXRΔ as a whole-cell biocatalyst in alkali pretreated detoxified corncob hydrolysate produced xylitol titer of 3.7 g L−1. Conclusion: Therefore, we successfully produced xylitol from corncob hydrolysates using the engineered E. coli M15 as whole-cell biocatalysts. Further, this process can be up scaled for the synthesis of eco-friendly high-value green chemicals. [ABSTRACT FROM AUTHOR]

Xylose reductase (XR), an industrially important enzyme, catalyzes the hydrogenation of xylose into xylitol. Xylitol, a polyol sugar, has tremendous applications in different industries due to its significant properties. XR is mainly produced from yeasts and molds, and limited studies have been reported on bacteria. The present study explores the potential of newly isolated bacteria, i.e., Pseudomonas putida BSX-46 for XR synthesis through process scale-up by tailoring its nutritional and cultural requirements. A simple media containing only four ingredients was designed for the production of XR in a short incubation time of 24 h. A process for pretreatment of rice straw was developed to achieve hydrolysate with a good amount of xylose (140 g/kg of rice straw). The enhanced XR production of 213.14±0.47 IU/mg of cells was achieved at bioreactor level using waste rice straw hydrolysate as compared to 94.26±0.62 IU/mg of cells at flask level. The developed bioprocess using efficient bacterial source and economical raw material would provide a low-cost substitute for XR production from xylose-based agro-waste materials at the industrial level. [ABSTRACT FROM AUTHOR]

This study provides a new perspective for xylose reductase enzyme separation from the reaction mixtures—obtained in the production of xylitol—by means of machine learning technique for large-scale production. Two types of machine learning models, including an adaptive neuro-fuzzy inference system based on grid partitioning of the input space and a boosted regression tree were developed, validated, and tested. The models' inputs were cross-flow velocity, transmembrane pressure, and filtration time, whereas the membrane permeability (called membrane flux) and xylitol concentration were considered as the outputs. According to the results, the boosted regression tree model demonstrated the highest predictive performance in forecasting the membrane flux and the amount of xylitol produced with a coefficient of determination of 0.994 and 0.967, respectively, against 0.985 and 0.946 for the grid partitioning-based adaptive neuro-fuzzy inference system, 0.865 and 0.820 for the best nonlinear regression picked from among 143 different equations, and 0.815 and 0.752 for the linear regression. The boosted regression tree modeling approach demonstrated a superior capability of predictive accuracy of the critical separation performances in the enzymatic-based cross-flow ultrafiltration membrane for xylitol synthesis. [ABSTRACT FROM AUTHOR]

Xylitol production from lignocellulose hydrolysate is a sustainable and environment-friendly process. In this study, a systematic process of converting corncob waste into xylitol is described. First, the corncobs are hydrolyzed with acid to a hydrolysate. Second, Kluyveromyces marxianus YZJQ016 derived from K. marxianus YZJ074, constructed by overexpressing ScGAL2-N376F from Saccharomyces cerevisiae, CtXYL1 from Candida tropicalis, and KmZWF1 from K. marxianus, produces xylitol from the hydrolysate. A total of ten xylose reductase genes were evaluated, and CtXYL1 proved best by showing the highest catalytic activity under the control of the KmGAPDH promoter. A 5 L fermenter at 42°C produced 105.22 g/L xylitol using K. marxianus YZJQ016—the highest production reported to date from corncob hydrolysate. Finally, for crystallization of the xylitol, the best conditions were 50% (v/v) methanol as an antisolvent, at 25°C, with purity and yield of 99%–100% and 74%, respectively—the highest yield reported to date.

Many operating parameters of ultrafiltration (UF) are playing a crucial role when using a polyethersulfone membrane to separate xylose reductase (XR) enzyme from reaction mixtures during xylitol synthesis. The present study focuses on the separation of XR enzyme using a cross-flow ultrafiltration (UF) membrane. The filtration process was analyzed using the three effective variables such as filtration time, cross-flow velocity (CFV), and the transmembrane pressure (TMP), which were ranging from 0 to 100 min, 0.52 to 1.2 cm/s and 1–1.6 bar, respectively. Then, using the resistance in series model, the hydraulic resistance for alkali chemical cleaning during XR separation was estimated. During separation, increased TMP showed a positive-flux effect as a driving force, however, fouling and polarized layer were more prominent under higher TMP. Increased CFV, on the other hand, was found more efficient in fouling control. In terms of the membrane cleaning techniques, an alkaline solution containing 0.1 M sodium hydroxide was shown to be the most effective substance in removing foulants from the membrane surface in this investigation. Cleaning with an alkaline solution resulted in a maximum flux recovery of 93% for xylose reductase separation. This work may serve as a useful guide to better understand the optimization parameters during XR separation and alleviating UF membrane fouling induced during XR separation. [ABSTRACT FROM AUTHOR]

The aim was to develop a two-stage seeding strategy and its optimization to enhance the conversion of xylose to xylitol by Debaryomyces nepalensis NCYC 3413. To develop efficient seed, multi-response optimization was employed to obtain optimal inoculum age and volume where xylitol concentration, yield and productivity were maximized. The optimal conditions of inoculation age and volume were 5.86 h and 11.66 % (v/v), respectively. Maximized results were observed at 48 h as compared to 72 h pre-optimization. Xylitol concentration slightly improved from 56 g/L to 59.71 g/L (p-value = 0.043). Yield improved from 0.56 g/g to 0.66 g/g (p-value = 0.044), whereas, productivity showed a significant increase from 0.76 g/L.h to 1.24 g/L.h (p-value = 0.008). Xylose Reductase activity improved by 1.67-folds and Xylitol Dehydrogenase activity decreased by 1.3 folds. This work suggests a simple inoculum strategy that could expedite the enzyme system required for xylitol production, enabling a 1.7-fold increase in productivity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Abstract Background Xylitol 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. Results Pichia 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. Conclusions We 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.

The potent promoter and its transcriptional control make a significant contribution to strain optimization. Using transcriptome-based approach, a novel pentose-regulated promoter of the xylose reductase gene (PxyrA) of Aspergillus oryzae was identified. The promoter analysis showed that the PxyrA was tightly regulated by pentose sugars, which xylose and xylan were favorable inducers. The PxyrA function was highly efficient as compared with the maltose-inducible promoters of A. oryzae. It also exhibited the efficient transcription induction even though certain amounts of glucose and sucrose existed in the cultures. The expression control of PxyrA was dependent on xylose consumption capacity for fungal growth. The control mode of PxyrA offers a simple operation in simultaneous gene expression and cultivation optimization in Aspergilli. This study provides a prospective development of fungal production platform using cellulosic sugars by the xylose-utilizing strains for sustainable growing in circular economy.

Xylitol is an industrially important chemical due to its commercial applications. The use of xylitol as a sweetener as well as its utilization in biomedical applications has made it a high value specialty chemical. Although several species of yeast synthesize xylitol, this review focusses on the species of the genus Candida. The importance of the enzyme xylitol reductase present in Candida species as it relates to their ability to synthesize xylitol was examined. Another focus of this work was to review prior studies examining the ability of the Candida species to synthesize xylitol effectively from hydrolysates of agricultural residues and grasses. An advantage of utilizing such a hydrolysate as a substrate for yeast xylitol production would be decreasing the overall cost of synthesizing xylitol. The intent of this review was to learn if such hydrolysates could substitute for xylose as a substrate for the yeast when producing xylitol. In addition, a comparison of xylitol production by Candida species should indicate which hydrolysate of agricultural residues and grasses would be the best substrate for xylitol production. From studies analyzing previous hydrolysates of agricultural residues and grasses, it was concluded that a hydrolysate of sugarcane bagasse supported the highest level of xylitol by Candida species, although corncob hydrolysates also supported significant yeast xylitol production. It was also concluded that fewer studies examined yeast xylitol production on hydrolysates of grasses and that further research on grasses may provide hydrolysates with a higher xylose content, which could support greater yeast xylitol production. [ABSTRACT FROM AUTHOR]

Enzymatic production of bioxylitol from lignocellulosic biomass (LCB) provides a promising alternative to both chemical and fermentative routes. This study aimed to assess the impacts of catalytic variables on bioxylitol production from wood sawdust using xylose reductase (XR) enzyme and to optimize the bioprocess. Enzyme-based xylitol production was carried out in batch cultivation under various experimental conditions to obtain maximum xylitol yield and productivity. The response surface methodology (RSM) was followed to fine-tune the most significant variables such as reaction time, temperature, and pH, which influence the synthesis of bioxylitol from sawdust hydrolysate and to optimize them. The optimum time, temperature, and pH became were 12.25 h, 35 °C, and 6.5, respectively, with initial xylose 18.8 g/L, NADPH 2.83 g/L, XR 0.027 U/mg, and agitation 100 rpm. The maximum xylitol production was attained at 16.28 g/L with a yield and productivity of 86.6% (w/w) and 1.33 g/L·h, respectively. Optimization of catalytic parameters using sequential strategies resulted in 1.55-fold improvement in overall xylitol production. This study explores a novel strategy for using sawdust hemicellulose in bioxylitol production by enzyme technology. [ABSTRACT FROM AUTHOR]

Abstract Meyerozyma guilliermondii, a non-conventional yeast that naturally assimilates xylose, is considered as a candidate for biotechnological production of the sugar alternative xylitol. Because the genes of the xylose metabolism were yet unknown, all efforts published so far to increase the xylitol yield of this yeast are limited to fermentation optimization. Hence, this study aimed to genetically engineer this organism for the first time with the objective to increase xylitol production. Therefore, the previously uncharacterized genes of M. guilliermondii ATCC 6260 encoding for xylose reductase (XR) and xylitol dehydrogenase (XDH) were identified by pathway investigations and sequence similarity analysis. Cloning and overexpression of the putative XR as well as knockout of the putative XDH genes generated strains with about threefold increased xylitol yield. Strains that combined both genetic modifications displayed fivefold increase in overall xylitol yield. Enzymatic activity assays with lysates of XR overexpressing and XDH knockout strains underlined the presumed functions of the respective genes. Furthermore, growth evaluation of the engineered strains on xylose as sole carbon source provides insights into xylose metabolism and its utilization for cell growth.

Galactitol, a rare sugar alcohol, has promising potential in the food industry and pharmaceutical field. The available industrial production methods rely on harsh hydrogenation processes, which incur high costs and environmental concerns. It is urgent to develop environmentally friendly and efficient biosynthesis technologies. In this study, a xylose reductase named AnXR derived from Aspergillus niger CBS 513.88 was identified and characterized for the enzymatic properties. AnXR exhibited the highest activity at 25 ℃ and pH 8.0, and it belonged to the NADPH-dependent aldose reductase family. To engineer a strain for galactitol production, we deleted the galactokinase (GAL1) gene in Saccharomyes cerevisiae by using the recombinant gene technology, which significantly reduced the metabolic utilization of D-galactose by host cells. Subsequently, we introduced the gene encoding AnXR into this modified strain, creating an engineered strain capable of catalyzing the conversion of D-galactose into galactitol. Furthermore, we optimized the whole-cell catalysis conditions for the engineered strain, which achieved a maximum galactitol yield of 12.10 g/L. Finally, we tested the reduction ability of the strain for other monosaccharides and discovered that it could produce functional sugar alcohols such as xylitol and arabinitol. The engineered strain demonstrates efficient biotransformation capabilities for galactitol and other functional sugar alcohols, representing a significant advancement in environmentally sustainable production practices.

Xylose reductase (XR) is an intermediate inducible enzyme of xylose metabolism responsible for the reduction of xylose into xylitol. It is an intracellular enzyme present in various facultative bacteria, yeasts, molds and algae in their cytoplasm. The active site of enzyme is polar in nature which is responsible for acid–base catalysis. The enzyme is folded into (β/α)8 barrel and its substrate specificity is through C-terminal region. XR is an emerging industrially important food enzyme due to its major application in production of xylitol. Xylitol has shown remarkable applications in other industrial sectors such as food, biofuels, pharmaceutical, medical, odontological, textiles and cosmetics. This enzyme has been purified and characterized to understand its physico-chemical properties and stability. Further, robust microbial strains with tolerance towards high substrate concentration and toxic compounds are required to be developed for successful exploitation of XR at industrial level using agrowaste materials. [ABSTRACT FROM AUTHOR]

Background: Xylitol 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. Results: Pichia 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. Conclusions: We 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. [ABSTRACT FROM AUTHOR]

Cofactor imbalance obstructs the productivities of metabolically engineered cells. Herein, we employed a minimally perturbing system, xylose reductase and lactose (XR/lactose), to increase the levels of a pool of sugar phosphates which are connected to the biosynthesis of NAD(P)H, FAD, FMN, and ATP in Escherichia coli. The XR/lactose system could increase the amounts of the precursors of these cofactors and was tested with three different metabolically engineered cell systems (fatty alcohol biosynthesis, bioluminescence light generation, and alkane biosynthesis) with different cofactor demands. Productivities of these cells were increased 2-4-fold by the XR/lactose system. Untargeted metabolomic analysis revealed different metabolite patterns among these cells, demonstrating that only metabolites involved in relevant cofactor biosynthesis were altered. The results were also confirmed by transcriptomic analysis. Another sugar reducing system (glucose dehydrogenase) could also be used to increase fatty alcohol production but resulted in less yield enhancement than XR. This work demonstrates that the approach of increasing cellular sugar phosphates can be a generic tool to increase in vivo cofactor generation upon cellular demand for synthetic biology., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Xylose reductase (XR) is an enzyme catalyzes the oxidation of xylose into xylitol using NADH or NADPH or both as cofactors with different preferences. The sequences of the XR enzyme from Spathaspora passalidarum (SsXR) was amplified, cloned and expressed in E. coli. Enzyme kinetics of a purified Histag6-XR fused protein were determined. This enzyme was successfully amplified with a molecular size of 957 bp using PCR with specific primers. Followed by cloning into pETite plasmid to obtain pETite_SsXR recombinant molecules, which was successfully expressed in E. coli. This XR is a dimer and it has a homology to Debaryomycesnepalensis xylose reductase (DnXR) with a global model quality estimation (GMQE) score of 0.94, and QMEAN of 0.52. This XR is strictly an NADPH dependent enzyme with a Km (28.24 ± 2.38 µM). This SsXR kinetics with respect to its substrate (xylose) has Km, kcat, kcat/Km with 68.01 ± 12.22 mM, 3207.41 ± 120.04 min-1 and 49.06 ± 10.58mM-1/min-1, respectively. This XR could be used as a catalyst to convert Xylose into the important industrial alcohol sugar (Xylitol). [ABSTRACT FROM AUTHOR]

Xylitol is a natural sweetener with diverse pharmaceutical and industrial applications. Low concentration of xylitol in fruits and vegetables is not enough to fulfil its increasing market demand. Catalytic reduction of xylose to xylitol is although simpler and rapid but this is also cost intensive due to catalyst regeneration, requirement of purified substrate, by-product generation, and need of high energy. Therefore bio-production of xylitol is probably the most preferred solution at the moment. Since xylose reductase (XR) is the crucial enzyme for xylitol production, efforts were made to explore the potential of hyper XR producer Emericella sp. XLT-11, selected out of 228 microbial isolates (68 bacterial and 160 fungal). Statistical approach was followed to enhance the xylitol yield. Of total, ten factors tried using Placket Burman Design, only 5 exhibiting positive impact on xylitol production, were used for further screening through central composite design (CCD). Maximum XR activity was recorded with 3.25% xylose, 0.125% MgSO4 using 24 h old inoculum after 108 h of incubation at 32.5 °C. The enzyme activity showed 3.81 folds increase (126.08±1.53 U/mg protein) over the control (33.07 U/mg) with up to 64% xylose conversion after optimization. The notable factor that XR from Emericella sp. XLT-11 could retain up to 75% of enzyme activity, even at pH 10.0, can be exploited at a large scale after further R&D. Emericella sp. XLT-11 has emerged as one of the potential isolates for bioconversion of xylose to xylitol. Its stability at a higher pH range can be best utilized for xylitol production at commercial scale. [ABSTRACT FROM AUTHOR]

Xylitol is an industrially important chemical due to its commercial applications. The use of xylitol as a sweetener as well as its utilization in biomedical applications has made it a high value specialty chemical. Although several species of yeast synthesize xylitol, this review focusses on the species of the genus Candida. The importance of the enzyme xylitol reductase present in Candida species as it relates to their ability to synthesize xylitol was examined. Another focus of this work was to review prior studies examining the ability of the Candida species to synthesize xylitol effectively from hydrolysates of agricultural residues and grasses. An advantage of utilizing such a hydrolysate as a substrate for yeast xylitol production would be decreasing the overall cost of synthesizing xylitol. The intent of this review was to learn if such hydrolysates could substitute for xylose as a substrate for the yeast when producing xylitol. In addition, a comparison of xylitol production by Candida species should indicate which hydrolysate of agricultural residues and grasses would be the best substrate for xylitol production. From studies analyzing previous hydrolysates of agricultural residues and grasses, it was concluded that a hydrolysate of sugarcane bagasse supported the highest level of xylitol by Candida species, although corncob hydrolysates also supported significant yeast xylitol production. It was also concluded that fewer studies examined yeast xylitol production on hydrolysates of grasses and that further research on grasses may provide hydrolysates with a higher xylose content, which could support greater yeast xylitol production.

In recent years, many novel xylose-fermenting yeasts belonging to the new genus Spathaspora have been isolated from the gut of wood-feeding insects and/or wood-decaying substrates. We have cloned and expressed, in Saccharomyces cerevisiae, a Spathaspora arborariae xylose reductase gene (SaXYL1) that accepts both NADH and NADPH as co-substrates, as well as a Spathaspora passalidarum NADPH-dependent xylose reductase (SpXYL1.1 gene) and the SpXYL2.2 gene encoding for a NAD+-dependent xylitol dehydrogenase. These enzymes were co-expressed in a S. cerevisiae strain over-expressing the native XKS1 gene encoding xylulokinase, as well as being deleted in the alkaline phosphatase encoded by the PHO13 gene. The S. cerevisiae strains expressing the Spathaspora enzymes consumed xylose, and xylitol was the major fermentation product. Higher specific growth rates, xylose consumption and xylitol volumetric productivities were obtained by the co-expression of the SaXYL1 and SpXYL2.2 genes, when compared with the co-expression of the NADPH-dependent SpXYL1.1 xylose reductase. During glucose-xylose co-fermentation by the strain with co-expression of the SaXYL1 and SpXYL2.2 genes, both ethanol and xylitol were produced efficiently. Our results open up the possibility of using the advantageous Saccharomyces yeasts for xylitol production, a commodity with wide commercial applications in pharmaceuticals, nutraceuticals, food and beverage industries. [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 stability of Debaryomyces nepalensis NCYC 3413 xylose reductase, a homodimeric enzyme recombinantly expressed and purified from E. coli Rosetta cells, was studied at different pH ranging from 5.0 to 10.0. Deactivation kinetics at different pH were studied by analyzing residual activity of the recombinant enzyme over time at 40 °C whereas conformational changes and stability dependence were investigated by using circular dichroism and differential scanning calorimetry. Four osmolytes viz. glycerol, sucrose, trehalose and sorbitol were explored for their effect on the deactivation and melting temperatures of the enzyme under neutral and extreme pH conditions. The enzyme was found to be catalytically and structurally stable at pH 7.0 with half-life of 250 min and a melting temperature of 50 °C. It was found that alteration in both secondary and tertiary structures caused enzyme deactivation in acidic pH while increased deactivation rates at alkaline pH was attributed to the variation of tertiary structure over time. Estimated thermodynamic parameters also showed that the enzyme stability was highest at neutral pH with ΔH of 348 kcal/mole and ΔG40 of 9.53 kcal/mole. All four osmolytes were effective in enhancing enzyme stability by several folds at extreme pH with sorbitol being the most efficient, which increased enzyme half-life by 11-fold at pH 10.0 and 8-fold at pH 5.0. [ABSTRACT FROM AUTHOR]

Xylitol is a five‐carbon sugar alcohol that has a variety of uses in the food and pharmaceutical industries. In xylose assimilating yeasts, NAD(P)H‐dependent xylose reductase (XR) catalyzes the reduction of xylose to xylitol. In the present study, XR with varying cofactor specificities was overexpressed in Saccharomyces cerevisiae to screen for efficient xylitol production. Xylose consumption and xylitol yields were higher when NADPH‐dependent enzymes (Candida tropicalis XR and S. cerevisiae Gre3p aldose reductase) were expressed, indicating that heterologous enzymes can utilize the intracellular NADPH pool more efficiently than the NADH pool, where they may face competition from native enzymes. This was confirmed by overexpression of a NADH‐preferring C. tropicalis XR mutant, which led to decreased xylose consumption and lower xylitol yield. To increase intracellular NADPH availability for xylitol production, the promoter of the ZWF1 gene, coding for the first enzyme of the NADPH‐generating pentose phosphate pathway, was replaced with the constitutive GPD promoter in a strain expressing C. tropicalis XR. This change led to a ~12% increase in xylitol yield. Deletion of XYL2 and SOR1, whose gene products can use xylitol as substrate, did not further increase xylitol yield. Using wheat stalk hydrolysate as source of xylose, the constructed strain efficiently produced xylitol, demonstrating practical relevance of this approach. [ABSTRACT FROM AUTHOR]

Meyerozyma guilliermondii, a non-conventional yeast that naturally assimilates xylose, is considered as a candidate for biotechnological production of the sugar alternative xylitol. Because the genes of the xylose metabolism were yet unknown, all efforts published so far to increase the xylitol yield of this yeast are limited to fermentation optimization. Hence, this study aimed to genetically engineer this organism for the first time with the objective to increase xylitol production. Therefore, the previously uncharacterized genes of M. guilliermondii ATCC 6260 encoding for xylose reductase (XR) and xylitol dehydrogenase (XDH) were identified by pathway investigations and sequence similarity analysis. Cloning and overexpression of the putative XR as well as knockout of the putative XDH genes generated strains with about threefold increased xylitol yield. Strains that combined both genetic modifications displayed fivefold increase in overall xylitol yield. Enzymatic activity assays with lysates of XR overexpressing and XDH knockout strains underlined the presumed functions of the respective genes. Furthermore, growth evaluation of the engineered strains on xylose as sole carbon source provides insights into xylose metabolism and its utilization for cell growth. [ABSTRACT FROM AUTHOR]

With growing interest in alternative fuels to minimize carbon and particle emissions, research continues on the production of lignocellulosic ethanol and on the development of suitable yeast strains. However, great diversities and continued technical advances in pretreatment methods for lignocellulosic biomass complicate the evaluation of developed yeast strains, and strain development often lags industrial applicability. In this review, recent studies demonstrating developed yeast strains with lignocellulosic biomass hydrolysates are compared. For the pretreatment methods, we highlight hydrothermal pretreatments (dilute acid treatment and autohydrolysis), which are the most commonly used and effective methods for lignocellulosic biomass pretreatment. Rather than pretreatment conditions, the type of biomass most strongly influences the composition of the hydrolysates. Metabolic engineering strategies for yeast strain development, the choice of xylose-metabolic pathway, adaptive evolution, and strain background are highlighted as important factors affecting ethanol yield and productivity from lignocellulosic biomass hydrolysates. A comparison of the parameters from recent studies demonstrating lignocellulosic ethanol production provides useful information for future strain development. [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]

Xylitol is a valuable substance utilized by food and biochemical industries. NAD(P)H-dependent xylose reductase (XR)—encoded by the yeast KmXYL1 gene—is the key enzyme which facilitates reduction of xylose to xylitol. Multi-copy integration of a mutant KmXYL1 (mKmXYL1) gene was carried out using thermotolerant yeast Kluyveromyces marxianus KCTC17555ΔURA3, in order to enhance xylitol production. After multi-copy integration, the highest xylitol producing strain was isolated and named K. marxianus 17555-JBP2. This strain exhibited 440% higher xylitol production than the parental strain at 30 °C. Due to a multi-copy integration of the mKmXYL1 gene, various additional differences between K. marxianus 17555-JBP2 and the parental strain were observed, including a 66% increase in NAD(P)H-dependent XR activity at high temperature (45 °C). Quantitative real-time PCR and transcriptome analysis demonstrated that, relative to the parent strain, K. marxianus 17555-JBP2 exhibited two more copies of mKmXY1 genes and a 9.63-fold elevation in transcription of NAD(P)H-dependent XR. After optimization of bioreactor fermentation conditions (agitation speed), high-temperature (40 °C) xylitol productivity of K. marxianus 17555-JBP2 exhibited an 81% improvement relative to the parental strain. In this study, we demonstrated that the overexpression of endogenous XR could enhance xylitol productivity at 40 °C by thermotolerant K. marxianus. [ABSTRACT FROM AUTHOR]

Abstract Background Understanding the effects of oxygen levels on yeast xylose metabolism would benefit ethanol production. In this work, xylose fermentative capacity of Scheffersomyces 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.

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]

Xylitol is a functional sugar alcohol widely used as a sugar substitute. Chemical route of xylitol production is highly expensive, whereas microbial sources offer lower yield and conversion efficiency. In the present investigation, xylose reductase (XR) gene from Candida tropicalis GRA1 was cloned in pQE30Xa vector and transformed into E. coli M15. The resultant recombinant strain produced xylitol from xylose that harboured Xr gene transcribed under the control of lac operon. Such transcribed Xr protein converts xylose into xylitol using NADPH as a co-factor. Xylitol production was achieved by the recombinant E. coli M15 pQE30XrCt under shake-flask fermentation (2.1 g.l-1 in 24 h) with xylose as the sole substrate in the medium. Addition of co-substrate, glucose in the growth medium enhanced the xylitol production to the tune of 3.4g.l-1. Furthermore, the addition of co-substrate glycerol along with xylose enhanced a xylitol yield of 6.4g.l-1. [ABSTRACT FROM AUTHOR]

Highlights • Expression of a thermostable xylose reductase (XRTL) in two yeast hosts. • First report of ferulic acid as the most potent lignocellulose-derived inhibitor. • Preference to NADPH as cofactor and broad substrate specificity. • The engineered S. cerevisiae harbouring XRTL could successfully produce xylitol. • Potential for xylose utilization in lignocellulosic bioconversion applications. Abstract In this study, the xylose reductase gene (XRTL) from Thermomyces lanuginosus SSBP was expressed in Pichia pastoris GS115 and Saccharomyces cerevisiae Y294. The purified 39.2 kDa monomeric enzyme was optimally active at pH 6.5 and 50 °C and showed activity over a wide range of temperatures (30–70 °C) and pH (4.0–9.0), with a half-life of 1386 min at 50 °C. The enzyme preferred NADPH as cofactor and showed broad substrate specificity. The enzyme was inhibited by Cu2+, Fe2+ and Zn2+, while ferulic acid was found to be the most potent lignocellulosic inhibitor. Recombinant S. cerevisiae with the XRTL gene showed 34% higher xylitol production than the control strain. XRTL can therefore be used in a cell-free xylitol production process or as part of a pathway for utilization of xylose from lignocellulosic waste. [ABSTRACT FROM AUTHOR]

Highlights • High-yielding mutant C. tropicalis T31 was obtained after mutagenized by ARTP system. • High yield mutant is due to the increased expression of XYL1 gene. • A two-stage dissolved oxygen supply strategy was used to improve the xylitol yield. Abstract Xylitol is a sugar alcohol that is used as a sweetener in food and confections. Industrially, xylitol is manufactured by chemical hydrogenation of d -xylose, which requires expensive separation and purification steps as well as high pressure and temperature. The microbial production of xylitol has been examined as an alternative to the chemical process. In this study, a xylitol over-producing strain is breeded by mutagenesis of a newly isolated yeast Candida tropicalis with a new mutation breeding system named atmospheric and room temperature plasma. The highest yield strain T31 was screened among more than 200 mutants with a xylitol yield of 0.61 g/g, which represents a yield increase of 22%. Furthermore, a two-stage dissolved oxygen supply strategy was used in a fermentation process resulting the maximum xylitol yield 0.79 g/g, which makes it a promising candidate for xylitol production. Further biochemical analysis indicating the relative gene expression and the enzyme activity of xylose reductase were higher in mutants than those in the original strain, which partly explained the high yield of xylitol. Thus, this study provides a new strategy to breed the over-producing strains for the xylitol industry. [ABSTRACT FROM AUTHOR]

Xylitol is an important functional sugar alcohol which is widely used in industries. To efficiently produce xylitol from xylose by xylose reductase in a single step, the coupled system and the coexpression system using xylose reductase (XR) from Rhizopus oryzae and glucose dehydrogenase (GDH) from Exiguobacterium sibiricum were constructed, the latter of which was used for cofactor regeneration. One hundred fifty grams/liter of xylose could be fully converted to xylitol by employing the two-enzyme coupled system with cell-free extract, and xylitol productivity of 21.2 g/L/h was reached by biotransformation in vitro. The whole-cell biocatalyst coexpressing xylose reductase and glucose dehydrogenase was successfully used to synthesize xylitol via enzymatic reduction of xylose. After optimization of the induction condition, the enzyme activity reached 1533 U/L. Two hundred grams/liter of xylose could be completely converted to xylitol, and the highest xylitol productivity of 6.37 g/L/h was obtained under the optimal transformation conditions. Thus, recombinant Escherichia coli coexpressing xylose reductase and glucose dehydrogenase is a promising candidate for xylitol production from xylose. [ABSTRACT FROM AUTHOR]

Highlights • Active xylose reductase (XR) was displayed at the surface of S. cerevisiae cells using two different anchoring signals. • Catalitic constants of both displayed XRs were somewhat worse than that of the control enzyme. • Surface display in both ways lead to the thermal, and pH stabilization of the enzyme activity. • Capacity of cells lacking all four Pir proteins for the Pir4-XR recombinant form was higher than that of the wild type. • Replacing the GAL1 with the PHO5 promoter enabled better modulation of expression of the recombinant XR. Abstract In order to display xylose reductase at the surface of S. cerevisiae cells two different gene constructs have been prepared. In the first, xylose reductase gene GRE3 was fused with two parts of the CCW12 gene, the N-terminal one coding for the secretion signal sequence, and the C–terminal coding for the glycosylphosphatidylinositol anchoring signal. Transformed cells synthesized xylose reductase and incorporated it in the cell wall through the remnant of the glycosylphosphatidylinositol anchor. The other construct was prepared by fusing the GRE3 with the PIR4 gene coding for one of the proteins of the Pir-family containing the characteristic N-terminal repetitive sequence that anchors Pir proteins to β-1,3-glucan. In this way xylose reductase was covalently attached to glucan through its N-terminus. For the expression of the constructs either the GAL1 , or the PHO5 promoters have been used. Both strains displayed active xylose reductases and their enzyme properties were compared with the control enzyme bearing the secretion signal sequence but no anchoring signals, thus secreted into the medium. The enzyme displayed through the N-terminal fusion with PIR4 had higher affinity for xylose than the other construct, but they both expressed somewhat lower affinity than the control enzyme. Similarly, the K m values for NADPH of both immobilized enzymes were somewhat higher than the K m of the control XR. Both displayed enzymes, especially the one fused with Pir4, had higher thermal and pH stability than the control, while other enzymatic properties were not significantly impaired by surface immobilization. [ABSTRACT FROM AUTHOR]

Directed evolutionary approach and random mutagenesis were performed on thermotolerant yeast Kluyveromyces marxianus KCTC17694 for isolating a yeast strain producing ethanol from xylose efficiently. The isolated mutant strain, K. marxianus 17694-DH1, showed 290% and 131% improvement in ethanol concentration and ethanol production yield from xylose, respectively, as compared with the parental strain. Sequencing of the KmXYL1 gene of K. marxianus 17694-DH1 revealed substitutions of arginine and tryptophan with lysine and leucine at positions 25 and 202, respectively, as compared to the parental strain. In addition, sequencing of the KmXYL2 gene uncovered a substitution of glutamate with leucine at position 232. When enzymatic assays of xylose reductase (XR) and xylitol dehydrogenase (XDH) from the parental strain and K. marxianus 17694-DH1 were performed, XR activities were not significantly different whereas XDH activities were significantly improved in the mutant strain up to 50 °C of reaction temperatures. RNA-Seq based transcriptome analysis showed that alcohol dehydrogenases and glucose transporters were up-regulated while TCA cycle involved enzymes were down-regulated in K. marxianus 17694-DH1. [ABSTRACT FROM AUTHOR]

Aldose reductases (ARs) belonging to the aldo‐keto reductase (AKR) superfamily catalyze the conversion of carbonyl substrates into their respective alcohols. Here we report the crystal structures of the yeast Debaryomyces nepalensis xylose reductase (DnXR, AKR2B10) in the apo form and as a ternary complex with a novel NADP‐DTT adduct. Xylose reductase, a key enzyme in the conversion of xylose to xylitol, has several industrial applications. The enzyme displayed the highest catalytic efficiency for l‐threose (138 ± 7 mm−1·s−1) followed by d‐erythrose (30 ± 3 mm−1·s−1). The crystal structure of the complex reveals a covalent linkage between the C4N atom of the nicotinamide ring of the cosubstrate and the S1 sulfur atom of DTT and provides the first structural evidence for a protein mediated NADP–low‐molecular‐mass thiol adduct. We hypothesize that the formation of the adduct is facilitated by an in‐crystallo Michael addition of the DTT thiolate to the specific conformation of bound NADPH in the active site of DnXR. The interactions between DTT, a four‐carbon sugar alcohol analog, and the enzyme are representative of a near‐cognate product ternary complex and provide significant insights into the structural basis of aldose binding and specificity and the catalytic mechanism of ARs. Database: Structural data are available in the PDB under the accession numbers 5ZCI and 5ZCM. Crystal structure of the yeast Debaryomyces nepalensis xylose reductase shows a novel covalent adduct between the nicotinamide ring of NADPH and DTT (NADP‐DTT), a feature hitherto unobserved in a protein‐mediated complex. The structure of this aldo‐keto reductase (AKR) as a ternary complex with a four‐carbon sugar alcohol analog provides new insights into substrate recognition and catalysis of AKRs. [ABSTRACT FROM AUTHOR]

BACKGROUND Acetoin and xylitol are widely used as high‐value platform chemicals with numerous potential applications. The chiral enantiomers L‐(+)‐ and D‐(−)‐acetoin are used as pharmaceutical intermediates. Cell‐free biosynthesis has many applications in chemicals production, but efficient methods for production of optically pure acetoin were rarely reported. RESULTS: A novel two‐enzyme system composed of meso‐2,3‐butanediol dehydrogenase (BDH) and xylose reductase was constructed to co‐produce acetoin and xylitol with NAD+ regeneration. Four BDHs from four candidates, as well as xylose reductase from Candida tenuis were first purified and characterized. The best BDH was then selected according to titers and chiral purities of acetoin. After optimization of reaction conditions, the ratios of meso‐2,3‐butanediol to xylose and BDH to xylose reductase, 28.5 g L−1 D‐(−)‐acetoin with an optical purity of 95.2% was produced in 6 h. The yield and productivity of acetoin was 0.97 g g−1 and 4.75 g L−1 h−1. The titer of co‐product xylitol was 40.29 g L−1, and the yield and productivity of xylitol reached 0.98 g g−1 and 6.72 g L−1 h−1. CONCLUSIONS: To our best knowledge, this is the first report on the production of optically pure D‐(−)‐acetoin using xylose reductase to regenerate NAD+, as well as the highest acetoin titer among enzymatic synthesis methods. This work therefore provides an economical and green alternative for the in vitro production of D‐(−)‐acetoin. © 2018 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

Economical biofuel production from plant biomass requires the conversion of both cellulose and hemicellulose in the plant cell wall. The best industrial fermentation organism, the yeast Saccharomyces cerevisiae, has been developed to utilize xylose by heterologously expressing either a xylose reductase/xylitol dehydrogenase (XR/XDH) pathway or a xylose isomerase (XI) pathway. Although it has been proposed that the optimal means for fermenting xylose into biofuels would use XI instead of the XR/XDH pathway, no clear comparison of the best publicly-available yeast strains engineered to use XR/XDH or XI has been published. We therefore compared two of the best-performing engineered yeast strains in the public domain—one using the XR/XDH pathway and another using XI—in anaerobic xylose fermentations. We find that, regardless of conditions, the strain using XR/XDH has substantially higher productivity compared to the XI strain. By contrast, the XI strain has better yields in nearly all conditions tested.