Altering the coenzyme preference of xylose reductase to favor utilization of NADH enhances ethanol yield from xylose in a metabolically engineered strain of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Background  

Metabolic engineering of Saccharomyces cerevisiae for xylose fermentation into fuel ethanol has oftentimes relied on insertion of a heterologous pathway that consists of xylose reductase (XR) and xylitol dehydrogenase (XDH) and brings about isomerization of xylose into xylulose via xylitol. Incomplete recycling of redox cosubstrates in the catalytic steps of the NADPH-preferring XR and the NAD⁺-dependent XDH results in formation of xylitol by-product and hence in lowering of the overall yield of ethanol on xylose. Structure-guided site-directed mutagenesis was previously employed to change the coenzyme preference of Candida tenuis XR about 170-fold from NADPH in the wild-type to NADH in a Lys²⁷⁴→Arg Asn²⁷⁶→Asp double mutant which in spite of the structural modifications introduced had retained the original catalytic efficiency for reduction of xylose by NADH. This work was carried out to assess physiological consequences in xylose-fermenting S. cerevisiae resulting from a well defined alteration of XR cosubstrate specificity.     

Expression of protein engineered NADP<Superscript>+</Superscript>-dependent xylitol dehydrogenase increases ethanol production from xylose in recombinant <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

A recombinant Saccharomyces cerevisiae strain transformed with xylose reductase (XR) and xylitol dehydrogenase (XDH) genes from Pichia stipitis has the ability to convert xylose to ethanol together with the unfavorable excretion of xylitol, which may be due to cofactor imbalance between NADPH-preferring XR and NAD⁺-dependent XDH. To reduce xylitol formation, we have already generated several XDH mutants with a reversal of coenzyme specificity toward NADP⁺. In this study, we constructed a set of recombinant S. cerevisiae strains with xylose-fermenting ability, including protein-engineered NADP⁺-dependent XDH-expressing strains. The most positive effect on xylose-to-ethanol fermentation was found by using a strain named MA-N5, constructed by chromosomal integration of the gene for NADP⁺-dependent XDH along with XR and endogenous xylulokinase genes. The MA-N5 strain had an increase in ethanol production and decrease in xylitol excretion compared with the reference strain expressing wild-type XDH when fermenting not only xylose but also mixed sugars containing glucose and xylose. Furthermore, the MA-N5 strain produced ethanol with a high yield of 0.49 g of ethanol/g of total consumed sugars in the nonsulfuric acid hydrolysate of wood chips. The results demonstrate that glucose and xylose present in the lignocellulosic hydrolysate can be efficiently fermented by this redox-engineered strain.     

Reversal of coenzyme specificity and improvement of catalytic efficiency of <Emphasis Type="Italic">Pichia</Emphasis><Emphasis Type="Italic">stipitis</Emphasis> xylose reductase by rational site-directed mutagenesis

A major problem when xylose is used for ethanol production is the intercellular redox imbalance arising from different coenzyme specificities of xylose reductase (XR) and xylitol dehydrogenase. The residue Lys21 in XR from Pichia stipitis was subjected to site-directed mutagenesis to alter its coenzyme specificity. The N272D mutant exhibited improved catalytic efficiency when NADH was the coenzyme. Both K21A and K21A/N272D preferred NADH to NADPH, their catalytic efficiencies for NADPH were almost zero. The catalytic efficiency of K21A/N272D for NADH was almost 9-fold and 2-fold that of K21A and the wild-type enzyme, respectively. Complete reversal of coenzyme specificity toward NADH and improved catalytic efficiency were achieved. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Qi-Kai Zeng, Hong-Li Du, Jing-Fang Wang have contributed equally to this work.     

Heterologous expression,purification, and characterization of xylose reductase from <Emphasis Type="Italic">Candida shehatae</Emphasis>

The xylose reductase (XR) gene (xyl1) from Candida shehatae was cloned and expressed in Escherichia coli, and purified as a His₆-tagged fusion protein. The recombinant XR had K_m values for NADH than NADPH of 150 μM and 20 μM, respectively. The optimal reaction was at pH 6.5 and 35°C. The enzyme was specific for d-xylese.     

<Emphasis Type="SmallCaps">l</Emphasis>-Arabinose pathway engineering for arabitol-free xylitol production in <Emphasis Type="Italic">Candida tropicalis</Emphasis>

Xylose reductase (XR) is a key enzyme in biological xylitol production, and most XRs have broad substrate specificities. During xylitol production from biomass hydrolysate, non-specific XRs can reduce l-arabinose, which is the second-most abundant hemicellulosic sugar, to the undesirable byproduct arabitol, which interferes with xylitol crystallization in downstream processing. To minimize the flux from l-arabinose to arabitol, the l-arabinose-preferring, endogenous XR was replaced by a d-xylose-preferring heterologous XR in Candida tropicalis. Then, Bacillus licheniformis araA and Escherichia coli araB and araD were codon-optimized and expressed functionally in C. tropicalis for the efficient assimilation of l-arabinose. During xylitol fermentation, the control strains BSXDH-3 and KNV converted 9.9 g l-arabinose l⁻¹ into 9.5 and 8.3 g arabitol l⁻¹, respectively, whereas the recombinant strain JY consumed 10.5 g l-arabinose l⁻¹ for cell growth without forming arabitol. Moreover, JY produced xylitol with 42 and 16% higher productivity than BSXDH-3 and KNV, respectively.     

Exploiting the NADPH pool for xylitol production using recombinant Saccharomyces cerevisiae

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.     

Ethanol Production from Xylose by a Recombinant Candida utilis Strain Expressing Protein-Engineered Xylose Reductase and Xylitol Dehydrogenase

The industrial yeast Candida utilis can grow on media containing xylose as sole carbon source, but cannot ferment it to ethanol. The deficiency might be due to the low activity of NADPH-preferring xylose reductase (XR) and NAD⁺-dependent xylitol dehydogenase (XDH), which convert xylose to xylulose, because C. utilis can ferment xylulose. We introduced multiple site-directed mutations in the coenzyme binding sites of XR and XDH derived from the xylose-fermenting yeast Candida shehatae to alter their coenzyme specificities. Several combinations of recombinant and native XRs and XDHs were tested. Highest productivity was observed in a strain expressing CsheXR K275R/N277D (NADH-preferring) and native CsheXDH (NAD⁺-dependent), which produced 17.4 g/L of ethanol from 50 g/L of xylose in 20 h. Analysis of the genes responsible for ethanol production from the xylose capacity of C. utilis indicated that the introduction of CsheXDH was essential, while overexpression of CsheXR K275R/N277D improved efficiency of ethanol production.     

Optimization of fed-batch fermentation for xylitol production by Candida tropicalis

Xylitol, a functional sweetener, was produced from xylose by biological conversion using Candida tropicalis ATCC 13803. Based on a two-substrate fermentation using glucose for cell growth and xylose for xylitol production, fed-batch fermentations were undertaken to increase the final xylitol concentration. The effects of xylose and xylitol on xylitol production rate were studied to determine the optimum concentrations for fed-batch fermentation. Xylose concentration in the medium (100 g l⁻¹) and less than 200 g l⁻¹ total xylose plus xylitol concentration were determined as optimum for maximum xylitol production rate and xylitol yield. Increasing the concentrations of xylose and xylitol decreased the rate and yield of xylitol production and the specific cell growth rate, probably because of an increase in osmotic stress that would interfere with xylose transport, xylitol flux to secretion to cell metabolism. The feeding rate of xylose solution during the fed-batch mode of operation was determined by using the mass balance equations and kinetic parameters involved in the equations in order to increase final xylitol concentration without affecting xylitol and productivity. The optimized fed-batch fermentation resulted in 187 g l⁻¹ xylitol concentration, 0.75 g xylitol g xylose⁻¹ xylitol yield and 3.9 g xylitol l⁻¹ h⁻¹ volumetric productivity. Journal of Industrial Microbiology & Biotechnology (2002) 29, 16–19 doi:10.1038/sj.jim.7000257 Received 15 October 2001/ Accepted in revised form 30 March 2002     

Production of xylitol in cell recycle fermentations of Candida tropicalis

Xylitol was produced a in two-substrate, batch fermentation with cell recycling of Candida tropicalis ATCC 13803. A series of cell-recycle experiments showed that the feeding of xylose, glucose and yeast extract in the xylitol production phase was most effective in enhancing xylitol productivity. The optimized cell recycle fermentation resulted in 0.82 g xylitol/g xylose yield, 4.94 g xylitol l^–1 h^–1 productivity, and final xylitol concentration of 189 g l^–1. These results were 1.3 times higher in volumetric xylitol productivity and 2.2 times higher in final product concentration compared with the corresponding values of the optimized two-substrate batch culture.     

Increase of xylitol yield by feeding xylose and glucose in Candida tropicalis

Candida tropicalis, a strain isolated from the sludge of a factory manufacturing xylose, produced a high xylitol concentration of 131 g/l from 150 g/l xylose at 45 h in a flask. Above 150 g/l xylose, however, volumetric xylitol production rates decreased because of a lag period in cell growth. In fed-batch culture, the volumetric production rate and xylitol yield from xylose varied substantially with the controlled xylose concentration and were maximum at a controlled xylose concentration of 60 g/l. To increase the xylitol yield from xylose, feeding experiments using different ratios of xylose and glucose were carried out in a fermentor. The maximum xylitol yield from 300 g/l xylose was 91% at a glucose/xylose feeding ratio of 15%, while the maximum volumetric production rate of xylitol was 3.98 g l⁻¹ h⁻¹ at a glucose/xylose feeding ratio of 20%. Xylitol production was found to decrease markedly as its concentration rose above 250 g/l. In order to accumulate xylitol to 250 g/l, 270 g/l xylose was added in total, at a glucose/xylose feeding ratio of 15%. Under these conditions, a final xylitol production of 251 g/l, which corresponded to a yield of 93%, was obtained from 270 g/l xylose in 55 h. Received: 20 April 1998 / Received revision: 29 May 1998 / Accepted: 19 June 1998     

Xylose reductase from Pichia stipitis with altered coenzyme preference improves ethanolic xylose fermentation by recombinant Saccharomyces cerevisiae

Background  

Xylose reductase (XR) and xylitol dehydrogenase (XDH) from Pichia stipitis are the two enzymes most commonly used in recombinant Saccharomyces cerevisiae strains engineered for xylose utilization. The availability of NAD⁺ for XDH is limited during anaerobic xylose fermentation because of the preference of XR for NADPH. This in turn results in xylitol formation and reduced ethanol yield. The coenzyme preference of P. stipitis XR was changed by site-directed mutagenesis with the aim to engineer it towards NADH-preference.     

Fermentation behavior of osmophilic yeast Candida tropicalis isolated from the nectar of Hibiscus rosa sinensis flowers for xylitol production

Eighteen yeast species belonging to seven genera were isolated from ten samples of nectar from Hibiscus rosa sinensis and investigated for xylitol production using d-xylose as sole carbon source. Amongst these isolates, no. 10 was selected as the best xylitol producer and identified as Candida tropicalis on the basis of morphological, biochemical and 26S rDNA sequencing. C. tropicalis produced 12.11 gl⁻¹ of xylitol in presence of 50 gl⁻¹ of xylose in 72 h at pH 5, 30°C and 200 rpm. The strain of C. tropicalis obtained through xylose enrichment technique has resulted in a yield of 0.5 gg⁻¹ with a xylitol volumetric productivity of 1.07 gl⁻¹h⁻¹ in the presence of 300 gl⁻¹ of xylose through batch fermentation. This organism has been reported for the first time from Hibiscus rosa sinensis flowers. Realizing, the importance of this high valued compound, as a sugar substitute, xylose enrichment technique was developed in order to utilize even higher concentrations of xylose as substrate for maximum xylitol production.     

Expression of different levels of enzymes from the Pichia stipitis XYL1 and XYL2 genes in Saccharomyces cerevisiae and its effects on product formation during xylose utilisation

Saccharomyces cerevisiae was transformed with the Pichia stipitis CBS 6054 XYL1 and XYL2 genes encoding xylose reductase (XR) and xylitol dehydrogenase (XDH) respectively. The XYL1 and XYL2 genes were placed under the control of the alcohol dehydrogenase 1 (ADH1) and phosphoglycerate kinase (PGK1) promoters in the yeast vector YEp24. Different vector constructions were made resulting in different specific activities of XR and XDH. The XR:XDH ratio (ratio of specific enzyme activities) of the transformed S. cerevisiae strains varied from 17.5 to 0.06. In order to enhance xylose utilisation in the XYL1-, XYL2-containing S. cerevisiae strains, the native genes encoding transketolase and transaldolase were also overexpressed. A strain with an XR:XDH ratio of 17.5 formed 0.82 g xylitol/g consumed xylose, whereas a strain with an XR:XDH ratio of 5.0 formed 0.58 g xylitol/g xylose. The strain with an XR:XDH ratio of 0.06, on the other hand, formed no xylitol and less glycerol and acetic acid compared with strains with the higher XR:XDH ratios. In addition, the strain with an XR:XDH ratio of 0.06 produced more ethanol than the other strains. Received: 12 March 1997 / Received revision: 17 April 1997 / Accepted: 27 April 1997     

Engineering of a matched pair of xylose reductase and xylitol dehydrogenase for xylose fermentation by Saccharomyces cerevisiae

Metabolic engineering of Saccharomyces cerevisiae for xylose fermentation has often relied on insertion of a heterologous pathway consisting of nicotinamide adenine dinucleotide (phosphate) NAD(P)H-dependent xylose reductase (XR) and NAD⁺-dependent xylitol dehydrogenase (XDH). Low ethanol yield, formation of xylitol and other fermentation by-products are seen for many of the S. cerevisiae strains constructed in this way. This has been ascribed to incomplete coenzyme recycling in the steps catalyzed by XR and XDH. Despite various protein-engineering efforts to alter the coenzyme specificity of XR and XDH individually, a pair of enzymes displaying matched utilization of NAD(H) and NADP(H) was not previously reported. We have introduced multiple site-directed mutations in the coenzyme-binding pocket of Galactocandida mastotermitis XDH to enable activity with NADP⁺, which is lacking in the wild-type enzyme. We describe four enzyme variants showing activity for xylitol oxidation by NADP⁺ and NAD⁺. One of the XDH variants utilized NADP⁺ about 4 times more efficiently than NAD⁺. This is close to the preference for NADPH compared with NADH in mutants of Candida tenuis XR. Compared to an S. cerevisiae-reference strain expressing the genes for the wild-type enzymes, the strains comprising the gene encoding the mutated XDH in combination a matched XR mutant gene showed up to 50% decreased glycerol yield without increase in ethanol during xylose fermentation.     

Enhanced Xylitol Production by Precultivation of Candida guilliermondii Cells in Sugarcane Bagasse Hemicellulosic Hydrolysate

The present work evaluated the key enzymes involved in xylitol production (xylose reductase [XR] and xylitol dehydrogenase [XDH]) and their correlation with xylose, arabinose, and acetic acid assimilation during cultivation of Candida guilliermondii FTI 20037 cells in sugarcane bagasse hemicellulosic hydrolysate. For this purpose, inocula previously grown either in sugarcane bagasse hemicellulosic hydrolysate (SBHH) or in semidefined medium (xylose as a substrate) were used. The highest xylose/acetic acid consumption ratio (1.78) and the lowest arabinose consumption (13%) were attained in the fermentation using inoculum previously grown in semidefined medium (without acetic acid and arabinose). In this case, the highest values of XR (1.37 U mg prot⁻¹) and XDH (0.91 U mg prot⁻¹) activities were observed. The highest xylitol yield (∼0.55 g g⁻¹) and byproducts (ethanol and glycerol) formation were not influenced by inoculum procedure. However, the cell previously grown in the hydrolysate was effective in enhancing xylitol production by keeping the XR enzyme activity at high levels (around 0.99 U·mg_prot⁻¹), reducing the XDH activity (34.0%) and increasing xylitol volumetric productivity (26.5%) with respect to the inoculum cultivated in semidefined medium. Therefore, inoculum adaptation to SBHH was shown to be an important strategy to improve xylitol productivity.     

The effect of xylose reductase genes on xylitol production by industrial Saccharomyces cerevisiae in fermentation of glucose and xylose

The co-production of xylitol and ethanol from agricultural straw has more economic advantages than the production of ethanol only. Saccharomyces cerevisiae, the most widely used ethanol-producing yeast, can be genetically engineered to ferment xylose to xylitol. In the present study, the effects of xylose-specificity, cofactor preference, and the gene copy number of xylose reductase (XR; encoding by XYL1 gene) on xylitol production of S. cerevisiae were investigated. The results showed that overexpression of XYL1 gene with a lower xylose-specificity and a higher NADPH preference favored the xylitol production. The copy number of XYL1 had a positive correlation with the XR activity but did not show a good correlation with the xylitol productivity. The overexpression of XYL1 from Candida tropicalis (CtXYL1) achieved a xylitol productivity of 0.83 g/L/h and a yield of 0.99 g/g-consumed xylose during batch fermentation with 43.5 g/L xylose and 17.0 g/L glucose. During simultaneous saccharification and fermentation (SSF) of pretreated corn stover, the strain overexpressing CtXYL1 produced 45.41 g/L xylitol and 50.19 g/L ethanol, suggesting its application potential for xylitol and ethanol co-production from straw feedstocks.     

Identification of a xylose reductase gene in the xylose metabolic pathway of <Emphasis Type="Italic">Kluyveromyces marxianus</Emphasis> NBRC1777

Kluyveromyces marxianus is thermotolerant yeast that is able to utilize a wider range of substrates and has greater thermal tolerance than most other yeast species. K. marxianus can assimilate xylose, but its ability to produce ethanol from xylose in oxygen-limited environments is poor. In the present study, the K. marxianus xylose reductase (KmXR) gene (Kmxyl1) was cloned and the recombinant enzyme was characterized to clarify the factors that limit xylose fermentation in K. marxianus NBRC1777. KmXR is a key enzyme in the xylose metabolism of K. marxianus, which was verified by disruption of the Kmxyl1 gene. The Km of the recombinant KmXR for NADPH is 65.67 μM and KmXR activity is 1.295 U/mg, which is lower than those of most reported yeast XRs, and the enzyme has no activity with coenzyme NADH. This result demonstrates that the XR from K. marxianus is highly coenzyme specific; combined with the extremely low XDH activity of K. marxianus with NADP⁺, the limitation of xylose fermentation is due to a redox imbalance under anaerobic conditions and low KmXR activity.     

Alcoholic fermentation of xylose and mixed sugars using recombinant <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> engineered for xylose utilization

Previously, a Saccharomyces cerevisiae strain was engineered for xylose assimilation by the constitutive overexpression of the Orpinomyces xylose isomerase, the S. cerevisiae xylulokinase, and the Pichia stipitis SUT1 sugar transporter genes. The recombinant strain exhibited growth on xylose, under aerobic conditions, with a specific growth rate of 0.025 h⁻¹, while ethanol production from xylose was achieved anaerobically. In the present study, the developed recombinant yeast was adapted for enhanced growth on xylose by serial transfer in xylose-containing minimal medium under aerobic conditions. After repeated batch cultivations, a strain was isolated which grew with a specific growth rate of 0.133 h⁻¹. The adapted strain could ferment 20 g l⁻¹ of xylose to ethanol with a yield of 0.37 g g⁻¹ and production rate of 0.026 g l⁻¹ h⁻¹. Raising the fermentation temperature from 30°C to 35°C resulted in a substantial increase in the ethanol yield (0.43 g g⁻¹) and production rate (0.07 g l⁻¹ h⁻¹) as well as a significant reduction in the xylitol yield. By the addition of a sugar complexing agent, such as sodium tetraborate, significant improvement in ethanol production and reduction in xylitol accumulation was achieved. Furthermore, ethanol production from xylose and a mixture of glucose and xylose was also demonstrated in complex medium containing yeast extract, peptone, and borate with a considerably high yield of 0.48 g g⁻¹.     

Improving ethanol and xylitol fermentation at elevated temperature through substitution of xylose reductase in Kluyveromyces marxianus

Thermo-tolerant yeast Kluyveromyces marxianus is able to utilize a wide range of substrates, including xylose; however, the xylose fermentation ability is weak because of the redox imbalance under oxygen-limited conditions. Alleviating the intracellular redox imbalance through engineering the coenzyme specificity of NADPH-preferring xylose reductase (XR) and improving the expression of XR should promote xylose consumption and fermentation. In this study, the native xylose reductase gene (Kmxyl1) of the K. marxianus strain was substituted with XR or its mutant genes from Pichia stipitis (Scheffersomyces stipitis). The ability of the resultant recombinant strains to assimilate xylose to produce xylitol and ethanol at elevated temperature was greatly improved. The strain YZB014 expressing mutant PsXR N272D, which has a higher activity with both NADPH and NADH as the coenzyme, achieved the best results, and produced 3.55 g l^?1 ethanol and 11.32 g l^?1 xylitol—an increase of 12.24- and 2.70-fold in product at 42 °C, respectively. A 3.94-fold increase of xylose consumption was observed compared with the K. marxianus YHJ010 harboring KmXyl1. However, the strain YZB015 expressing a mutant PsXR K21A/N272D, with which co-enzyme preference was completely reversed from NADPH to NADH, failed to ferment due to the low expression. So in order to improve xylose consumption and fermentation in K. marxianus, both higher activity and co-enzyme specificity change are necessary.     

A novel strictly NADPH-dependent Pichia stipitis xylose reductase constructed by site-directed mutagenesis

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.