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.     

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.     

The yeast <Emphasis Type="Italic">Scheffersomyces amazonensis</Emphasis> is an efficient xylitol producer

This study assessed the efficiency of Scheffersomyces amazonensis UFMG-CM-Y493^T, cultured in xylose-supplemented medium (YPX) and rice hull hydrolysate (RHH), to convert xylose to xylitol under moderate and severe oxygen limitation. The highest xylitol yields of 0.75 and 1.04 g g^?1 in YPX and RHH, respectively, were obtained under severe oxygen limitation. However, volumetric productivity in RHH was ninefold decrease than that in YPX medium. The xylose reductase (XR) and xylitol dehydrogenase (XDH) activities in the YPX cultures were strictly dependent on NADPH and NAD⁺ respectively, and were approximately 10% higher under severe oxygen limitation than under moderate oxygen limitation. This higher xylitol production observed under severe oxygen limitation can be attributed to the higher XR activity and shortage of the NAD⁺ needed by XDH. These results suggest that Sc. amazonensis UFMG-CM-Y493^T is one of the greatest xylitol producers described to date and reveal its potential use in the biotechnological production of xylitol.     

Effect of acetic acid present in bagasse hydrolysate on the activities of xylose reductase and xylitol dehydrogenase in <Emphasis Type="Italic">Candida guilliermondii</Emphasis>

The first two steps in xylose metabolism are catalyzed by NAD(P)H-dependent xylose reductase (XR) (EC 1.1.1.21) and NAD(P)-dependent xylitol dehydrogenase (XDH) (EC 1.1.1.9), which lead to xylosexylitolxylulose conversion. Xylitol has high commercial value, due to its sweetening and anticariogenic properties, as well as several clinical applications. The acid hydrolysis of sugarcane bagasse allows the separation of a xylose-rich hemicellulosic fraction that can be used as a substrate for Candida guilliermondii to produce xylitol. However, the hydrolysate contains acetic acid, an inhibitor of microbial metabolism. In this study, the effect of acetic acid on the activities of XR and XDH and on xylitol formation by C. guilliermondii were studied. For this purpose, fermentations were carried out in bagasse hydrolysate and in synthetic medium. The activities of XR and XDH were higher in the medium containing acetic acid than in control medium. Moreover, none of the fermentative parameters were significantly altered during cell culture. It was concluded that acetic acid does not interfere with xylitol formation since the increase in XR activity is proportional to XDH activity, leading to a greater production of xylitol and its subsequent conversion to xylulose.     

Evaluation of cotton stalk hydrolysate for xylitol production

Cotton stalk is a widely distributed and abundant lignocellulosic waste found in Turkey. Because of its rich xylose content, it can be a promising source for the production of xylitol. Xylitol can be produced by chemical or biotechnological methods. Because the biotechnological method is a simple process with great substrate specificity and low energy requirements, it is more of an economic alternative for the xylitol production. This study aimed to use cotton stalk for the production of xylitol with Candida tropicalis Kuen 1022. For this purpose, the combined effects of different oxygen concentration, inoculum level and substrate concentration were investigated to obtain high xylitol yield and volumetric xylitol production rate. Candida tropicalis Kuen 1022 afforded different concentrations of xylitol depending on xylose concentration, inoculum level, and oxygen concentration. The optimum xylose, yeast concentration, and airflow rate for cotton stalk hydrolysate were found as 10.41 g L^?1, 0.99 g L^?1, and 1.02 vvm, respectively, and under these conditions, xylitol yield and volumetric xylitol production rate were obtained as 36% and 0.06 g L^?1 hr^?1, respectively. The results of this study show that cotton stalk can serve as a potential renewable source for the production of xylitol.     

Carbon fluxes of xylose-consuming <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> strains are affected differently by NADH and NADPH usage in HMF reduction

Industrial Saccharomyces cerevisiae strains able to utilize xylose have been constructed by overexpression of XYL1 and XYL2 genes encoding the NADPH-preferring xylose reductase (XR) and the NAD⁺-dependent xylitol dehydrogenase (XDH), respectively, from Pichia stipitis. However, the use of different co-factors by XR and XDH leads to NAD⁺ deficiency followed by xylitol excretion and reduced product yield. The furaldehydes 5-hydroxymethyl-furfural (HMF) and furfural inhibit yeast metabolism, prolong the lag phase, and reduce the ethanol productivity. Recently, genes encoding furaldehyde reductases were identified and their overexpression was shown to improve S. cerevisiae growth and fermentation rate in HMF containing media and in lignocellulosic hydrolysate. In the current study, we constructed a xylose-consuming S. cerevisiae strain using the XR/XDH pathway from P. stipitis. Then, the genes encoding the NADH- and the NADPH-dependent HMF reductases, ADH1-S110P-Y295C and ADH6, respectively, were individually overexpressed in this background. The performance of these strains, which differed in their co-factor usage for HMF reduction, was evaluated under anaerobic conditions in batch fermentation in absence or in presence of HMF. In anaerobic continuous culture, carbon fluxes were obtained for simultaneous xylose consumption and HMF reduction. Our results show that the co-factor used for HMF reduction primarily influenced formation of products other than ethanol, and that NADH-dependent HMF reduction influenced product formation more than NADPH-dependent HMF reduction. In particular, NADH-dependent HMF reduction contributed to carbon conservation so that biomass was produced at the expense of xylitol and glycerol formation.     

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.     

Evaluation of fermentative potential of <Emphasis Type="Italic">Kluyveromyces marxianus</Emphasis> ATCC 36907 in cellulosic and hemicellulosic sugarcane bagasse hydrolysates on xylitol and ethanol production

This paper evaluates the fermentative potential of Kluyveromyces marxianus grown in sugarcane bagasse cellulosic and hemicellulosic hydrolysates obtained by acid hydrolysis. Ethanol was obtained from a single glucose fermentation product, whereas xylose assimilation resulted in xylitol as the main product and ethanol as a by-product derived from the metabolism of this pentose. Fermentation performed in a simulated hydrolysate medium with a glucose concentration similar to that of the hydrolysate resulted in ethanol productivity (Qp?=?0.86 g L^?1 h^?1) that was tenfold higher than the one observed in the cellulosic hydrolysate. However, the use of hemicellulosic hydrolysate favored xylose assimilation in comparison with simulated medium with xylose and glucose concentrations similar to those found in this hydrolysate, without toxic compounds such as acetic acid and phenols. Under this condition, xylitol yield was 53.8 % higher in relation to simulated medium. Thus, the total removal of toxic compounds from the hydrolysate is not necessary to obtain bioproducts from lignocellulosic hydrolysates.     

Metabolism of glucose and xylose as single and mixed feed in <Emphasis Type="Italic">Debaryomyces nepalensis</Emphasis> NCYC 3413: production of industrially important metabolites

Efficient conversion of hexose and pentose (glucose and xylose) by a single strain is a very important factor for the production of industrially important metabolites using lignocellulose as the substrate. The kinetics of growth and polyol production by Debaryomyces nepalensis NCYC 3413 was studied under single and mixed substrate conditions. In the presence of glucose, the strain produced ethanol (35.8 ± 2.3 g/l), glycerol (9.0 ± 0.2 g/l), and arabitol (6.3 ± 0.2 g/l). In the presence of xylose, the strain produced xylitol (38 ± 1.8 g/l) and glycerol (18 ± 1.0 g/l) as major metabolites. Diauxic growth was observed when the strain was grown with different combinations of glucose/xylose, and glucose was the preferred substrate. The presence of glucose enhanced the conversion of xylose to xylitol. By feeding a mixture of glucose at 100 g/l and xylose at 100 g/l, it was found that the strain produced a maximum of 72 ± 3 g/l of xylitol. A study of important enzymes involved in the synthesis of xylitol (xylose reductase (XR) and xylitol dehydrogenase (XDH)), glycerol (glycerol-3-phosphate dehydrogenase (G3PDH)) and ethanol (alcohol dehydrogenase (ADH)) in cells grown in the presence of glucose and xylose revealed high specific activity of G3PDH and ADH in cells grown in the presence of glucose, whereas high specific activity of XR, XDH, and G3PDH was observed in cells grown in the presence of xylose. To our knowledge, this is the first study to elaborate the glucose and xylose metabolic pathway in this yeast strain.     

High activity of xylose reductase and xylitol dehydrogenase improves xylose fermentation by recombinant <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Xylose fermentation performance was studied of a previously developed Saccharomyces cerevisiae strain TMB 3057, carrying high xylose reductase (XR) and xylitol dehydrogenase (XDH) activity, overexpressed non-oxidative pentose phosphate pathway (PPP) and deletion of the aldose reductase gene GRE3. The fermentation performance of TMB 3057 was significantly improved by increased ethanol production and reduced xylitol formation compared with the reference strain TMB 3001. The effects of the individual genetic modifications on xylose fermentation were investigated by comparing five isogenic strains with single or combined modifications. All strains with high activity of both XR and XDH had increased ethanol yields and significantly decreased xylitol yields. The presence of glucose further reduced xylitol formation in all studied strains. High activity of the non-oxidative PPP improved the xylose consumption rate. The results indicate that ethanolic xylose fermentation by recombinant S. cerevisiae expressing XR and XDH is governed by the efficiency by which xylose is introduced in the central metabolism.     

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     

Point mutation of the xylose reductase (XR) gene reduces xylitol accumulation and increases citric acid production in Aspergillus carbonarius

Aspergillus carbonarius accumulates xylitol when it grows on d-xylose. In fungi, d-xylose is reduced to xylitol by the NAD(P)H-dependent xylose reductase (XR). Xylitol is then further oxidized by the NAD⁺-dependent xylitol dehydrogenase (XDH). The cofactor impairment between the XR and XDH can lead to the accumulation of xylitol under oxygen-limiting conditions. Most of the XRs are NADPH dependent and contain a conserved Ile-Pro-Lys-Ser motif. The only known naturally occurring NADH-dependent XR (from Candida parapsilosis) carries an arginine residue instead of the lysine in this motif. In order to overcome xylitol accumulation in A. carbonarius a Lys-274 to Arg point mutation was introduced into the XR with the aim of changing the specificity toward NADH. The effect of the genetic engineering was examined in fermentation for citric acid production and xylitol accumulation by using d-xylose as the sole carbon source. Fermentation with the mutant strain showed a 2.8-fold reduction in xylitol accumulation and 4.5-fold increase in citric acid production compared to the wild-type strain. The fact that the mutant strain shows decreased xylitol levels is assumed to be associated with the capability of the mutated XR to use the NADH generated by the XDH, thus preventing the inhibition of XDH by the high levels of NADH and ensuring the flux of xylose through the pathway. This work shows that enhanced production of citric acid can be achieved using xylose as the sole carbon source by reducing accumulation of other by-products, such as xylitol.     

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.     

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.     

Xylitol production by genetically modified industrial strain of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> using glycerol as co-substrate

Xylitol is commercially used in chewing gum and dental care products as a low calorie sweetener having medicinal properties. Industrial yeast strain of S. cerevisiae was genetically modified to overexpress an endogenous aldose reductase gene GRE3 and a xylose transporter gene SUT1 for the production of xylitol. The recombinant strain (XP-RTK) carried the expression cassettes of both the genes and the G418 resistance marker cassette KanMX integrated into the genome of S. cerevisiae. Short segments from the 5′ and 3′ delta regions of the Ty1 retrotransposons were used as homology regions for integration of the cassettes. Xylitol production by the industrial recombinant strain was evaluated using hemicellulosic hydrolysate of the corn cob with glucose as the cosubstrate. The recombinant strain XP-RTK showed significantly higher xylitol productivity (212 mg L^?1 h^?1) over the control strain XP (81 mg L^?1 h^?1). Glucose was successfully replaced by glycerol as a co-substrate for xylitol production by S. cerevisiae. Strain XP-RTK showed the highest xylitol productivity of 318.6 mg L^?1 h^?1 and titre of 47 g L^?1 of xylitol at 12 g L^?1 initial DCW using glycerol as cosubstrate. The amount of glycerol consumed per amount of xylitol produced (0.47 mol mol^?1) was significantly lower than glucose (23.7 mol mol^?1). Fermentation strategies such as cell recycle and use of the industrial nitrogen sources were demonstrated using hemicellulosic hydrolysate for xylitol production.     

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.     

Xylan-hydrolyzing thermotolerant <Emphasis Type="Italic">Candida tropicalis</Emphasis> HNMA-1 for bioethanol production from sugarcane bagasse hydrolysate

Sugarcane bagasse is one of the low-cost substrates used for bioethanol production. In order to solubilize sugars in hemicelluloses like xylan, a new thermotolerant isolate of Candida tropicalis HNMA-1 with xylan-hydrolyzing ability was identified and characterized. The strain showed relative tolerance to high temperature. Our results demonstrated 0.211 IU ml^?1 xylanase activity at 40 °C compared to 0.236 IU ml^?1 at 30 °C. The effect of high temperature on the growth and fermentation of xylose and sugarcane bagasse hydrolysate were also investigated. In both xylose or hydrolysate medium, increased growth was recorded at 40 °C. Meanwhile, the efficiency of ethanol fermentation was adversely affected by temperature since yields of 0.088 g g^?1 and 0.076 g g^?1 in the xylose medium, in addition to 0.090 g g^?1 and 0.078 g g^?1 in the hydrolysate medium were noticed at 30 °C and 40 °C, respectively. Inhibitory compounds in the hydrolysate medium demonstrated negative effects on fermentation and productivity, with maximum ethanol concentration attained after 48 h in the hydrolysate, as opposed to 24 h in the xylose medium. Our data show that the newly thermotolerant isolate, C. tropicalis HNMA-1, is able to efficiently ferment xylose and hydrolysate, and also has the capacity for application in ethanol production from hemicellulosic sources.     

Fermentation behavior of an osmotolerant yeast D. hansenii for Xylitol production

Realizing the importance of xylitol as a high‐valued compound that serves as a sugar substitute, a new, one step thin layer chromatographic procedure for quick, reliable, and efficient determination of xylose and xylitol from their mixture was developed. Two hundred and twenty microorganisms from the laboratory stock cultures were screened for their ability to produce xylitol from D ‐xylose. Amongst these, an indigenous yeast isolate no.139 (SM‐139) was selected and identified as Debaryomyces hansenii on the basis of morphological and biochemical characteristics and (26S) D1/D2 r DNA region sequencing. Debaryomyces hansenii produced 9.33 gL^?1 of xylitol in presence of 50.0 gL^?1 of xylose in 84 h at pH 5.5, 30°C, 200 rpm. In order to utilize even higher concentrations of xylose for maximum xylitol production, a xylose enrichment technique was developed. The strain of Debaryomyces hansenii was obtained through xylose enrichment technique in a statistically optimized medium containing 0.3% yeast extract, 0.2% peptone, 0.03% MgSO₄.7H₂O along with 1% methanol. The culture was inoculated with 6% inoculum and incubated at 30°C and 250 rpm. A yield of 0.6 gg^?1 was obtained with a xylitol volumetric productivity of 0.65 g/L h^?1 in the presence of 200 gL^?1 of xylose although up to 300 gL^?1 of xylose could be tolerated through batch fermentation. Through this technique, even higher concentrations of xylose as substrate could be potentially utilized for maximum xylitol production. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

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.