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.     

Lysine 21突变对树干毕赤氏酵母木糖还原酶辅酶依赖性的影响

木糖还原酶是重组酿酒酵母工程菌利用木糖生成乙醇代谢途径中的关键酶, 该关键酶在利用木糖时依赖NADPH而不是NADH是导致酿酒酵母代谢木糖生成乙醇的最终产率低的主要原因之一。为了改变树干毕赤氏酵母木糖还原酶的辅酶依赖性, 对它的第21位赖基酸Lys进行了突变。利用质粒载体pET28b分别将突变后的基因K21A-XYL1、K21R-XYL1及野生基因WT-XYL1在大肠杆菌E. coli BL21(DE3)中进行表达, 表达后的蛋白经His-Tag纯化柱纯化后测定酶学性质。结果表明: K21R突变子的辅酶依赖性没有改变, 但K21A突变子的辅酶依赖性由NADPH完全逆转为NADH。     

Lysine 21突变对树干毕赤氏酵母木糖还原酶辅酶依赖性的影响

木糖还原酶是重组酿酒酵母工程菌利用木糖生成乙醇代谢途径中的关键酶, 该关键酶在利用木糖时依赖NADPH而不是NADH是导致酿酒酵母代谢木糖生成乙醇的最终产率低的主要原因之一。为了改变树干毕赤氏酵母木糖还原酶的辅酶依赖性, 对它的第21位赖基酸Lys进行了突变。利用质粒载体pET28b分别将突变后的基因K21A-XYL1、K21R-XYL1及野生基因WT-XYL1在大肠杆菌E. coli BL21(DE3)中进行表达, 表达后的蛋白经His-Tag纯化柱纯化后测定酶学性质。结果表明: K21R突变子的辅酶依赖性没有改变, 但K21A突变子的辅酶依赖性由NADPH完全逆转为NADH。     

Identification of lysine-78 as an essential residue in the Saccharomyces cerevisiae xylose reductase

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

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.     

树干毕赤酵母和酿酒酵母混合糖发酵产乙醇

以树干毕赤酵母和酿酒酵母为发酵菌株,酸性蒸汽爆破玉米秸秆预水解液和纯糖模拟液为C源,采用固定化酵母细胞的方法,研究了酸爆玉米秸秆预水解液初始pH、N源种类及其浓度、3种发酵模式对树干毕赤酵母戊糖发酵的影响。结果表明:玉米秸秆预水解液适合发酵的初始pH范围为6.0～7.0;1.0 g/L的(NH4)2SO4作为N源,在40 g/L葡萄糖和25 g/L木糖培养基中发酵24 h,糖利用率达到99.47%,乙醇质量浓度为24.72 g/L,优于尿素和蛋白胨作为N源;3种模式的发酵体系中,以游离树干毕赤酵母和固定化酿酒酵母发酵性能最好,糖利用率和乙醇得率分别为99.43%和96.39%。     

Effect of pretreatment chemicals on xylose fermentation by Pichia stipitis

Pretreatment of biomass with dilute H₂SO₄ results in residual acid which is neutralized with alkalis such as Ca(OH)₂, NaOH and NH₄OH. The salt produced after neutralization has an effect on the fermentation of Pichia stipitis. Synthetic media of xylose (60 g total sugar/l) was fermented to ethanol in the presence and absence of the salts using P. stipitis CBS 6054. CaSO₄ enhanced growth and xylitol production, but produced the lowest ethanol concentration and yield after 140 h. Na₂SO₄ inhibited xylitol production, slightly enhanced growth towards the end of fermentation but had no significant effect on xylose consumption and ethanol concentration. (NH₄)₂SO₄ inhibited growth, had no effect on xylitol production, and enhanced xylose consumption and ethanol production.     

Nonhomologous end joining and homologous recombination DNA repair pathways in integration mutagenesis in the xylose-fermenting yeast Pichia stipitis

Pichia stipitis integrates linear homologous DNA fragments mainly ectopically. High rates of randomly occurring integration allow tagging mutagenesis with high efficiency using simply PCR amplificates of suitable selection markers from the P. stipitis genome. Linearization of an autonomously replicating vector caused a distinct increase of the transformation efficiency compared with the circular molecule. Cotransformation of a restriction endonuclease further enhanced the transformation efficiency. This effect was also observed with integrative vector DNA. In most cases vector integration in chromosomal targets did not depend on microhomologies, indicating that restriction-enzyme-mediated integration (REMI) does not play an essential role in P. stipitis. Small deletions were observed at the ends of the integrated vectors and in the target sites. Disruption of the PsKU80 gene increased the frequency of homologous integration considerably but resulted in a remarkable decrease of the transformation efficiency. These results suggest that in P. stipitis the nonhomologous end joining (NHEJ) pathway obviously predominates the homologous recombination pathway of double-strand break repair.     

Harnessing Candida tenuis and Pichia stipitis in whole-cell bioreductions of o-chloroacetophenone: Stereoselectivity,cell activity,in situ substrate supply and product removal

Generally, recombinant and native microorganisms can be employed as whole-cell catalysts. The application of native hosts, however, shortens the process development time by avoiding multiple steps of strain construction. Herein, we studied the NAD(P)H-dependent reduction of o-chloroacetophenone by isolated xylose reductases and their native hosts Candida tenuis and Pichia stipitis. The natural hosts were benchmarked against Escherichia coli strains co-expressing xylose reductase and a dehydrogenase for co-enzyme recycling. Xylose-grown cells of C. tenuis and P. stipitis displayed specific o-chloroacetophenone reductase activities of 366 and 90 U g_CDW^–1, respectively, in the cell-free extracts. Fresh biomass was employed in batch reductions of 100 mM o-chloroacetophenone using glucose as co-substrate. Reaction stops at a product concentration of about 15 mM, which suggests sensitivity of the catalyst towards the formed product. In situ substrate supply and product removal by the addition of 40% hexane increased catalyst stability. Optimisation of the aqueous phase led to a (S)-1-(2-chlorophenyl)ethanol concentration of 71 mM (ee > 99.9%) obtained with 44 g_CDW L^–1 of C. tenuis. The final difference in productivities between native C. tenuis and recombinant E. coli was < 1.7-fold. The optically pure product is a required key intermediate in the synthesis of a new class of chemotherapeutic substances (polo-like kinase 1 inhibitors).     

The expression of a Pichia stipitis xylose reductase mutant with higher K(M) for NADPH increases ethanol production from xylose in recombinant Saccharomyces cerevisiae

Xylose fermentation by Saccharomyces cerevisiae requires the introduction of a xylose pathway, either similar to that found in the natural xylose-utilizing yeasts Pichia stipitis and Candida shehatae or similar to the bacterial pathway. The use of NAD(P)H-dependent XR and NAD(+)-dependent XDH from P. stipitis creates a cofactor imbalance resulting in xylitol formation. The effect of replacing the native P. stipitis XR with a mutated XR with increased K(M) for NADPH was investigated for xylose fermentation to ethanol by recombinant S. cerevisiae strains. Enhanced ethanol yields accompanied by decreased xylitol yields were obtained in strains carrying the mutated XR. Flux analysis showed that strains harboring the mutated XR utilized a larger fraction of NADH for xylose reduction. The overproduction of the mutated XR resulted in an ethanol yield of 0.40 g per gram of sugar and a xylose consumption rate of 0.16 g per gram of biomass per hour in chemostat culture (0.06/h) with 10 g/L glucose and 10 g/L xylose as carbon source.     

Identification and characterization of d-arabinose reductase and d-arabinose transporters from Pichia stipitis

d-xylose and l-arabinose are the major constituents of plant lignocelluloses, and the related fungal metabolic pathways have been extensively examined. Although Pichia stipitis CBS 6054 grows using d-arabinose as the sole carbon source, the hypothetical pathway has not yet been clarified at the molecular level. We herein purified NAD(P)H-dependent d-arabinose reductase from cells grown on d-arabinose, and found that the enzyme was identical to the known d-xylose reductase (XR). The enzyme activity of XR with d-arabinose was previously reported to be only 1% that with d-xylose. The k_cat/K_m value with d-arabinose (1.27 min^?1 mM^?1), which was determined using the recombinant enzyme, was 13.6- and 10.5-fold lower than those with l-arabinose and d-xylose, respectively. Among the 34 putative sugar transporters from P. stipitis, only seven genes exhibited uptake ability not only for d-arabinose, but also for d-glucose and other pentose sugars including d-xylose and l-arabinose in Saccharomyces cerevisiae.     

树干毕赤酵母木糖还原酶基因的克隆及在大肠杆菌中的表达

根据NCBI中的木糖还原酶基因序列设计引物,利用高保真聚合酶克隆树干毕赤酵母木糖还原酶基因,加A后克隆到质粒pGM-T中,测序验证.然后将目的基因克隆到舍有强启动子的穿梭表达载体p424GPD中,构建含有XYL1基因的重组质粒p424GPD-XYL1.将p424GPD-XYL1转化到大肠杆菌中,提取总蛋白,聚丙烯酰胺凝胶电泳分析.酶活测定确定木糖还原酶基因XYL1在大肠杆菌中得到活性表达,表明表达载体构建成功.表达载体的成功构建为后续构建重组酿酒酵母利用木糖发酵奠定基础.     

Cre/LoxP重组系统的改造及在树干毕赤酵母中的应用

为了实现在P.stipitis中进行无痕基因敲除,以Cre/LoxP系统为研究对象,首先通过同源重组构建尿嘧啶营养缺陷型树干毕赤酵母(ura3-);同时通过定点突变pSH47-Hpt质粒的hpt基因和cre基因,将CDS区CTG突变为TTG;最后以乙醛脱氢酶基因为靶基因,验证突变后的Cre/LoxP系统在P.stipitis进行无痕基因敲除的可行性。结果表明:本文在P.stipitis中成功使用潮霉素B抗性标记,经过修饰后的Cre/LoxP敲除系统能够在P.stipitis中无痕敲除目的基因,为后续研究P.stipitis功能基因和改造代谢途径提供了一种试验方法和筛选标记。     

Determination of kinetic parameters of fermentation processes by a continuous unsteady-state method: application to the alcoholic fermentation of D-xylose by Pichia stipitis

A quick technique for determination of kinetic parameters of fermentation processes is proposed and applied to the transformation of D-xylose into ethanol by Pichia stipitis. The commonly used method to evaluate these parameters is based on achieving several steady states. In the proposed procedure, mu(m) and K(s) can be determined from only one steady state, by provoking a disturbance over it, after allowing the system to return to the original conditions. The main difference between the steady and unsteady state methods is the required fermentation time; while the former method lasted 350 h, the latter required a period 25 times lower. Kinetic and stoichiometric parameters were determined with both methods under anoxic and limited oxygen concentration conditions. Results from the two methods were compared, giving only 2% and 4.5% differences in the values of K(s) and mu(m) and a little over 4% for mu(m) were the deviations under the latter ones. (c) 1993 Wiley & Sons, Inc.     

Effect of enhanced xylose reductase activity on xylose consumption and product distribution in xylose-fermenting recombinant Saccharomyces cerevisiae

Recombinant Saccharomyces cerevisiae TMB3001, harboring the Pichia stipitis genes XYL1 and XYL2 (xylose reductase and xylitol dehydrogenase, respectively) and the endogenous XKS1(xylulokinase), can convert xylose to ethanol. About 30% of the consumed xylose, however, is excreted as xylitol. Enhanced ethanol yield has previously been achieved by disrupting the ZWF1 gene, encoding glucose-6-phosphate dehydrogenase, but at the expense of the xylose consumption. This is probably the result of reduced NADPH-mediated xylose reduction. In the present study, we increased the xylose reductase (XR) activity 4-19 times in both TMB3001 and the ZWF1-disrupted strain TMB3255. The xylose consumption rate increased by 70% in TMB3001 under oxygen-limited conditions. In the ZWF1-disrupted background, the increase in XR activity fully restored the xylose consumption rate. Maximal specific growth rates on glucose were lower in the ZWF1-disrupted strains, and the increased XR activity also negatively affected the growth rate in these strains. Addition of methionine resulted in 70% and 50% enhanced maximal specific growth rates for TMB3255 (zwfl Delta) and TMB3261 (PGK1-XYL1, zwf1 Delta), respectively. Enhanced XR activity did not have any negative effect on the maximal specific growth rate in the control strain. Enhanced glycerol yields were observed in the high-XR-activity strains. These are suggested to result from the observed reductase activity of the purified XR for dihydroxyacetone phosphate.     

Generation of the improved recombinant xylose-utilizing Saccharomyces cerevisiae TMB 3400 by random mutagenesis and physiological comparison with Pichia stipitis CBS 6054

The recombinant xylose-utilizing Saccharomyces cerevisiae TMB 3399 was constructed by chromosomal integration of the genes encoding D-xylose reductase (XR), xylitol dehydrogenase (XDH), and xylulokinase (XK). S. cerevisiae TMB 3399 was subjected to chemical mutagenesis with ethyl methanesulfonate and, after enrichment, 33 mutants were selected for improved growth on D-xylose and carbon dioxide formation in Durham tubes. The best-performing mutant was called S. cerevisiae TMB 3400. The novel, recombinant S. cerevisiae strains were compared with Pichia stipitis CBS 6054 through cultivation under aerobic, oxygen-limited, and anaerobic conditions in a defined mineral medium using only D-xylose as carbon and energy source. The mutation led to a more than five-fold increase in maximum specific growth rate, from 0.0255 h(-1) for S. cerevisiae TMB 3399 to 0.14 h(-1) for S. cerevisiae TMB 3400, whereas P. stipitis grew at a maximum specific growth rate of 0.44 h(-1). All yeast strains formed ethanol only under oxygen-limited and anaerobic conditions. The ethanol yields and maximum specific ethanol productivities during oxygen limitation were 0.21, 0.25, and 0.30 g ethanol g xylose(-1) and 0.001, 0.10, and 0.16 g ethanol g biomass(-1) h(-1) for S. cerevisiae TMB 3399, TMB 3400, and P. stipitis CBS 6054, respectively. The xylitol yield under oxygen-limited and anaerobic conditions was two-fold higher for S. cerevisiae TMB 3399 than for TMB 3400, but the glycerol yield was higher for TMB 3400. The specific activity, in U mg protein(-1), was higher for XDH than for XR in both S. cerevisiae TMB 3399 and TMB 3400, while P. stipitis CBS 6054 showed the opposite relation. S. cerevisiae TMB 3400 displayed higher specific XR, XDH and XK activities than TMB 3399. Hence, we have demonstrated that a combination of metabolic engineering and random mutagenesis was successful to generate a superior, xylose-utilizing S. cerevisiae, and uncovered distinctive physiological properties of the mutant.     

Insertional tagging of the Scheffersomyces stipitis gene HEM25 involved in regulation of glucose and xylose alcoholic fermentation

Amid known microbial bioethanol producers, the yeast Scheffersomyces (Pichia) stipitis is particularly promising in terms of alcoholic fermentation of both glucose and xylose, the main constituents of lignocellulosic biomass hydrolysates. However, the ethanol yield and productivity, especially from xylose, are still insufficient to meet the requirements of a feasible industrial technology; therefore, the construction of more efficient S. stipitis ethanol producers is of great significance. The aim of this study was to isolate the insertional mutants of S. stipitis with altered ethanol production from glucose and xylose and to identify the disrupted gene(s). Mutants obtained by random insertional mutagenesis were screened for their growth abilities on solid media with different sugars and for resistance to 3-bromopyruvate. Of more than 1,300 screened mutants, 17 were identified to have significantly changed ethanol yields during the fermentation. In one of the best fermenting strains (strain 4.6), insertion was found to occur within the ORF of a homolog to the Saccharomyces cerevisiae gene HEM25 (YDL119C), encoding a mitochondrial glycine transporter required for heme synthesis. The role of HEM25 in heme accumulation, respiration, and alcoholic fermentation in the yeast S. stipitis was studied using strain 4.6, the complementation strain Comp—a derivative from the 4.6 strain with expression of the WT HEM25 allele and the deletion strain hem25Δ. As hem25Δ produced lower amounts of ethanol than strain 4.6, we assume that the phenotype of strain 4.6 may be caused not only by HEM25 disruption but additionally by some point mutation.     

Multiple forms of xylose reductase in Pachysolen tannophilus CBS4044

Abstract Cell-free extracts of xylose-grown Pachysolen tannophilus exhibited xylose reductase activity with both NADPH and NADH. The ratio of the NADPH- and NADH-dependent activities varied with growth conditions. Affinity chromatography of cell-free extracts resulted in a separation of two xylose reductases. One was active with both NADPH and NADH, the other was specific for NADPH. Apart from this coenzyme specificity, the two enzymes also differed in their affinities for xylose and NADPH. The role of the two enzymes in xylose metabolism is discussed in relation to attempts to use P. tannophilus for the alcoholic fermentation of wood sugars.     

Mutational analysis of NADH-binding residues in triphenylmethane reductase from Citrobacter sp. strain KCTC 18061P

Triphenylmethane reductase (TMR) catalyzes the NADH-dependent reduction of triphenylmethane dyes. Sequence alignment revealed a region with a conserved GXXGXXG motif near its N-terminus, which corresponds to a conserved structural motif of known dinucleotide-binding proteins. To verify whether some of these glycine residues are important for the enzyme catalysis, these three glycine residues (Gly-7, Gly-10 and Gly-13) were individually replaced by alanine using site-directed mutagenesis. The secondary structures of these mutants, as measured by circular dichroism spectroscopy, did not show remarkable differences as compared with the wild type. The V(max)/K(m) values of mutants G7A and G13A for both Basic fuchsin and NADH were increased about three and twofold over that of the wild type, respectively, whereas the V(max)/K(m) value of mutant G10A were decreased about sixfold. These results suggest that these three glycine residues are involved in the interaction with both substrate and cofactor for the catalytic activity of TMR.