瑞氏木霉木糖代谢关键酶基因在不同碳源条件下的表达

用20种不同的碳源(包括单一碳源和混合碳源)分别培养瑞氏木霉(Trichoderma reesei)QM9414。通过一系列Northern杂交分析检测瑞氏木霉木糖还原酶(XR),木糖醇脱氢酶(XDH)以及转醛醇酶(TAL)mRNA的表达情况。实验结果证实,槐糖和木二糖是xr和xdh表达的强诱导物,阿拉伯糖和乳糖也有较强的诱导作用。葡萄糖在培养基中的存在阻遏该二基因的表达。当葡萄糖耗尽以后,培养基中不存在任何诱导物的情况下,xr和xdh以一定的基础水平进行转录。相比较,tal基因在每种碳源上都是强表达。     

发状念珠藻醛酮还原酶基因NfAKR的克隆与表达

以发状念珠藻细胞为试材,采用PCR技术克隆了醛酮还原酶基因的开放阅读框(ORF)序列,命名为NfAKR。对基因序列特征进行了生物信息学分析,根据其编码氨基酸序列预测了NfAKR蛋白的三维结构,同时探讨了PEG-6000胁迫下NfAKR的表达特性。结果表明:NfAKR基因的编码序列长912bp,编码304个氨基酸,预测其编码蛋白的相对分子量为33.51kD,理论等电点为4.94,具有醛酮还原酶超家族保守结构域。NfAKR蛋白主要由10个α-螺旋和11β-折叠组成,中间形成一个疏水穴,作为酶的催化活性中心。NfAKR与点形念珠藻处在同一进化枝上,具有较近的亲缘关系。qRT-PCR分析显示,PEG-6000胁迫下NfAKR基因上调表达,当PEG-6000浓度为8%时,其相对表达量为5.66并达到峰值。依据NfAKR基因响应干旱胁迫上调表达的特性,推测醛酮还原酶可能参与发状念珠藻抵御干旱胁迫过程。     

米曲霉木糖醇脱氢酶的分子模建与对接

[目的]研究米曲霉木糖醇脱氢酶基因的结构与功能.[方法]克隆测序来源于米曲霉的木糖醇脱氢酶(XDH)基因,利用Swiss-MODEL和Modeller对XDH进行三级结构模建,通过PROCHECK和Prosa2003对得到的4个目标模型进行评价,从中得到一个最佳模型.在同源建模的基础上,通过分子对接软件MolsoftICM-Pro,对辅因子进行对接,预测了XDH与NAD+、Zn2+作用的相关残基.寻找底物木糖醇与XDH结合的可能活性口袋,用Molsoft模拟XDH与木糖醇的对接,预测了酶与底物作用的关键氨基酸残基.[结果]结构分析显示,米曲霉XDH含有醇脱氢酶家族锌指纹结构和典型醇脱氢酶Rossmann折叠的辅酶结合域,属于Medium-chain脱氢酶(MDR)家族.通过对接研究,预测了XDH与NAD+之间形成氢键的氨基酸有Asp206、Arg211、Ser255、Ser301和Arg303,这些氨基酸位于结合域,与Zn2+形成氢键的氨基酸有His72和Glu73,位于催化域,与天然底物木糖醇形成氢键的氨基酸有Ile46、Ile349、Lys350和Thr351,位于催化域.[结论]所得信息对XDH分子定向改造、拓展米曲霉工业应用范围有重要意义.     

树干毕赤酵母(Pichia Stipitis)木糖还原酶(xyl1)基因克隆与序列分析

根据木糖还原酶基因序列相似的特点,设计一对引物获得Pichia stipitis CICC1960的一段基因片段,此片段长度为957bp,共编码318个氨基酸.利用生物信息学软件对该序列进行了同源性分析、氨基酸组成分析、疏水性分析、磷酸化位点预测、CDS 分析及二、三级结构预测.结果表明该片段为Pichia stipitis CICC1960木糖还原酶基因序列.     

利用不同启动子控制木糖代谢关键酶基因构建可共代谢葡萄糖木糖重组酿酒酵母

利用不同强度的启动子调控木糖代谢关键酶活性,构建稳定代谢葡萄糖和木糖产乙醇的重组酿酒酵母。以本实验室专利菌株Saccharomyces cerevisiae Y5为宿主菌,将树干毕赤酵母Pichia stipitis CBS6054的木糖还原酶基因XYL1和木糖醇脱氢酶基因XYL2置于磷酸甘油酸激酶基因启动子(PGKp)控制下,酿酒酵母Y5内源的木酮糖激酶基因XKS1分别由己糖激酶基因启动子(HXK2p)及其内源启动子(XKS1p)控制。这3个基因连同各自表达元件导入宿主细胞中,打通其木糖上游代谢途径。酶活测定结果显示,HXK2p对木酮糖激酶表现出更强的启动效率。重组菌Y5-X3-1中木糖还原酶/木糖醇脱氢酶/木酮糖激酶(XR/XDH/XK)的酶活比值为1∶5∶4,其木糖消耗量是宿主菌的5倍,最高乙醇产量为24.35 g/L,达到理论值的73%。结果表明,通过调节XYL1、XYL2及XKS1启动子的强度,调控其表达水平,进而改变3种酶的活性水平,对于提高重组酿酒酵母利用木糖发酵产乙醇有明显效果。     

CAZy-AA3家族酶及其在生物传感器中的应用研究进展

碳水化合物活性酶数据库（CAZy）中位于“辅助活性”（auxiliary activities,AA）3家族的酶属于葡萄糖-甲醇-胆碱氧化还原酶大家族。它们以黄素腺嘌呤二核苷酸（FAD）作为辅酶,通过反应产物（H₂O₂或对苯二酚）协助其他AA家族酶发挥作用,或辅助糖苷水解酶降解木质纤维素。根据结构序列相似性,AA3家族酶进一步细分为4个亚家族,包括 AA3_1（纤维二糖脱氢酶）、AA3_2（芳醇氧化酶、葡萄糖氧化还原酶）、AA3_3（醇氧化酶）、AA3_4（吡喃糖氧化还原酶）。AA3家族酶因其独特的结构、广泛的用途,近几十年来受到人们的广泛关注。本文系统综述了CAZy-AA3家族酶来源、分子结构及改造,对部分AA3家族酶在生物传感器中的最新研究进展进行了重点综述,并对未来研究方向进行了展望。     

不同宿主来源的重组酿酒酵母混合糖代谢比较

【目的】研究不同工业酿酒酵母宿主背景对重组酵母木糖利用效率的影响。【方法】将木糖利用途径的木糖还原酶(XR)、木糖醇脱氢酶(XDH)和木酮糖激酶(XK)编码基因串联后分别转入3株不同的工业酿酒酵母中,得到重组酵母ZQ1、ZQ5和ZQ7。分别对3个木糖途径代谢基因的表达水平、酶活和重组菌株的木糖发酵效率进行比较。【结果】重组菌株在木糖代谢基因转录、酶活性和木糖利用性能方面有很大差异,其中ZQ5木糖代谢能力最强,ZQ7其次,ZQ1木糖利用能力最弱。ZQ7在初始木糖浓度为20 g/L时木糖利用速率快于ZQ5,表明木糖浓度对重组菌发酵性能评价具有影响。【结论】不同菌株的遗传背景和木糖浓度对重组菌木糖利用的影响很大,评价重组酵母的木糖利用需考虑宿主的遗传背景和底物浓度的影响。     

一个新的茶树黄酮醇合酶基因的克隆和表达分析

Cs—COR113（GenBank登录号：FE942098）为一受冷诱导的茶树黄酮醇合酶基因的cDNA片段,采用RACE技术克隆了这一基因的全长cDNA,命名为CsFLS（GenBank登录号：FJ577509）。CsFLScDNA序列全长为1303bp,5＇-UTR和3’-UTR分别长91bp和175bp,包含一个编码336个氨基酸的完整开放阅读框。序列分析显示,CsFLS与烟草、矮牵牛、欧芹、拟南芥的黄酮醇合酶的同源性分别为74％、75％、75％和63％,含有2-酮戊二酸依赖性双加氧酶家族2个保守的基序,以及与黄酮醇合酶正确折叠有关的2个保守的甘氨酸残基。CsFLS的表达受低温诱导,但不受ABA诱导。     

野生罂粟COR基因克隆及转化

以野生罂粟幼叶总RNA为模板,采用RT-PCR技术克隆到可待因酮还原酶基因COR的cDNA序列,所获得的cDNA序列全长966 bp,具有完整的ORF,编码321个氨基酸.Blast分析表明,该片段与GenBank中的可待因酮还原酶基因(COR)家族相似性很高,其中与基因COR1.1的一致性最高可达98.96%,该片段命名为COR(GenBank,登录号为FJ624147).以中间载体pHANNIBAL和植物表达载体pART27为基础,构建了CaMV-35S启动子驱动的含可待因酮还原酶基因片段反向重复序列的RNAi双元表达载体pARC,转化烟草获得转基因植株.     

巴西橡胶树的脂酰辅酶A还原酶cDNA克隆及其序列分析

从巴西橡胶树差减cDNA文库中筛选到一个与脂酰辅酶A还原酶同源性较高的基因片段,根据该基因片段序列信息,设计特异引物,采用RACE进行差异片段的5’和3’端的扩增,获得长度为1365bp的cDNA克隆R28（GenBank登陆号：AY461413）。序列分析表明,该基因包含1149bp的开放阅读框,5＇-UTR为96bp,3＇-UTR为128bp,编码382个氨基酸,推测其蛋白质的分子量为43．5kDa,等电点为8．97,有一个跨膜螺旋N（187至215位氨基酸）和1个由17个氨基酸组成的信号肽（1至17位氨基酸）。R28含有脂酰辅酶A还原酶的保守（NADP结合蛋白保守区）,推测该基因是一个脂酰辅酶A还原酶基因。     

The structure of apo and holo forms of xylose reductase,a dimeric aldo-keto reductase from Candida tenuis

Xylose reductase is a homodimeric oxidoreductase dependent on NADPH or NADH and belongs to the largely monomeric aldo-keto reductase superfamily of proteins. It catalyzes the first step in the assimilation of xylose, an aldose found to be a major constituent monosaccharide of renewable plant hemicellulosic material, into yeast metabolic pathways. It does this by reducing open chain xylose to xylitol, which is reoxidized to xylulose by xylitol dehydrogenase and metabolically integrated via the pentose phosphate pathway. No structure has yet been determined for a xylose reductase, a dimeric aldo-keto reductase or a family 2 aldo-keto reductase. The structures of the Candida tenuis xylose reductase apo- and holoenzyme, which crystallize in spacegroup C2 with different unit cells, have been determined to 2.2 A resolution and an R-factor of 17.9 and 20.8%, respectively. Residues responsible for mediating the novel dimeric interface include Asp-178, Arg-181, Lys-202, Phe-206, Trp-313, and Pro-319. Alignments with other superfamily members indicate that these interactions are conserved in other dimeric xylose reductases but not throughout the remainder of the oligomeric aldo-keto reductases, predicting alternate modes of oligomerization for other families. An arrangement of side chains in a catalytic triad shows that Tyr-52 has a conserved function as a general acid. The loop that folds over the NAD(P)H cosubstrate is disordered in the apo form but becomes ordered upon cosubstrate binding. A slow conformational isomerization of this loop probably accounts for the observed rate-limiting step involving release of cosubstrate. Xylose binding (K(m) = 87 mM) is mediated by interactions with a binding pocket that is more polar than a typical aldo-keto reductase. Modeling of xylose into the active site of the holoenzyme using ordered waters as a guide for sugar hydroxyls suggests a convincing mode of substrate binding.     

The positive effect of the decreased NADPH-preferring activity of xylose reductase from Pichia stipitis on ethanol production using xylose-fermenting recombinant Saccharomyces cerevisiae

We focused on the effects of a mutation of xylose reductase from Pichia stipitis (PsXR) on xylose-to-ethanol fermentation using recombinant Saccharomyces cerevisiae transformed with PsXR and PsXDH (xylitol dehydrogenase from P. stipitis) genes. Based on inherent NADH-preferring XR and several site-directed mutagenetic studies using other aldo-keto reductase enzymes, we designed several single PsXR mutants. K270R showing decreased NADPH-preferring activity without a change in NADH-preferring activity was found to be a potent mutant. Strain Y-K270R transformed with K270R PsXR and wild-type PsXDH showed a 31% decrease in unfavorable xylitol excretion with 5.1% increased ethanol production as compared to the control in the fermentation of 15 g l(-1) xylose and 5 g l(-1) glucose.     

瑞氏木霉木糖醇脱氢酶基因的分离与鉴定

将在木聚糖上生长的瑞氏木霉(Trichoderma reesei)RutC-30的cDNA文库全部质粒转化已携带有毕赤氏酵(Pithia stipitis)木糖还原酶基因的重组酿酒酵母(Saccharomycescerevisiae)菌株H475,在H475中构建了瑞氏木霉的cDNA表达亚文库。在以木糖为唯一碳源的选择性酵母合成培养基上,从该亚文库中筛选到瑞氏木霉木糖醇脱氢酶cDNA基因．该基因片段长为1．3kb。Southern、Norhern印迹杂交分析和蛋白质凝胶电泳结果表明该基因确实来源于瑞氏木霉,所编码蛋白质分子量约为40kDa。携带有毕赤氏酵母木糖还原酶和瑞氏木霉木糖醇脱氢酶基因的重组酵母能够在以木糖为唯一碳源的培养基上生长,并能将90％以上的木糖转化为木糖醇、乙醇和其它副产品。     

Genetic analysis of D-xylose metabolism by endophytic yeast strains of Rhodotorula graminis and Rhodotorula mucilaginosa

Two novel endophytic yeast strains, WP1 and PTD3, isolated from within the stems of poplar (Populus) trees, were genetically characterized with respect to their xylose metabolism genes. These two strains, belonging to the species Rhodotorula graminis and R. mucilaginosa, respectively, utilize both hexose and pentose sugars, including the common plant pentose sugar, D-xylose. The xylose reductase (XYL1) and xylitol dehydrogenase (XYL2) genes were cloned and characterized. The derived amino acid sequences of xylose reductase (XR) and xylose dehydrogenase (XDH) were 32%～41% homologous to those of Pichia stipitis and Candida. spp., two species known to utilize xylose. The derived XR and XDH sequences of WP1 and PTD3 had higher homology (73% and 69% identity) with each other. WP1 and PTD3 were grown in single sugar and mixed sugar media to analyze the XYL1 and XYL2 gene regulation mechanisms. Our results revealed that for both strains, the gene expression is induced by D-xylose, and that in PTD3 the expression was not repressed by glucose in the presence of xylose.     

Heterologous expression of Aspergillus oryzae xylose reductase and xylitol dehydrogenase genes facilitated xylose utilization in the yeast Saccharomyces cerevisiae

The cDNA encoding a putative xylose reductase (xyrA) from Aspergillus oryzae was cloned and coexpressed in the yeast Saccharomyces cerevisiae with A. oryzae xylitol dehydrogenase cDNA (xdhA). XyrA exhibited NADPH-dependent xylose reductase activity. The S. cerevisiae strain, overexpressing the xyrA, xdhA, endogenous XKS1, and TAL1 genes, grew on xylose as sole carbon source, and produced ethanol.     

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.     

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.     

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 bifunctional enzymes with xylose reductase and xylitol dehydrogenase activity in Saccharomyces cerevisiae alters product formation during xylose fermentation.

To enhance metabolite transfer in the two initial sequential steps of xylose metabolism in yeast, two structural genes of Pichia stipitis, XYL1 and XYL2 encoding xylose reductase (XR) and xylitol dehydrogenase (XDH), respectively, were fused in frame. Four chimeric genes were constructed, encoding fusion proteins with different orders of the enzymes and different linker lengths. These genes were expressed in Saccharomyces cerevisiae. The fusion proteins exhibited both XR and XDH activity when XYL1 was fused downstream of XYL2. The specific activity of the XDH part of the complexes increased when longer peptide linkers were used. Bifunctional enzyme complexes, analyzed by gel filtration, were found to be tetramers, hexamers, and octamers. No degradation products were detected by Western blot analysis. S. cerevisiae strains harboring the bifunctional enzymes grew on minimal-medium xylose plates, and oxygen-limited xylose fermentation resulted in xylose consumption and ethanol formation. When a fusion protein, containing a linker of three amino acids, was coexpressed with native XR and XDH monomers in S. cerevisiae, enzyme complexes consisting of chimerical and native subunits were formed. The total activity of these complexes showed XR and XDH activities similar to the activities obtained when the monomers were expressed individually. Strains which coexpressed chimerical subunits together with native XR and XDH monomers consumed less xylose and produced less xylitol. However, the xylitol yield was lower in these strains than in strains expressing only native XR and XDH monomers, 0.55 and 0.62, respectively, and the ethanol yield was higher. The reduced xylitol yield was accompanied by reduced glycerol and acetate formation suggesting enhanced utilization of NADH in the XR reaction.