葡萄糖酸氧化杆菌木糖醇脱氢酶基因的克隆与表达

【目的】获得葡萄糖酸氧化杆菌(Gluconobacter oxydans CGMCC 1.637)的木糖醇脱氢酶基因,研究其酶学性质及碳源特别是D-阿拉伯醇和木糖醇对该酶活性的影响。【方法】通过已报道序列的木糖醇脱氢酶的保守区设计引物,用聚合酶链式反应(polymerase chain reaction,PCR)扩增获得目的基因片段。根据获得的片段序列设计引物克隆目的基因的5’和3’片段,将所获得的片段拼接,获得完整的木糖醇脱氢酶基因。通过构建工程菌获得重组蛋白,并利用氧化还原反应测定重组酶的活性。用含不同碳源的培养基培养G.oxydans CGMCC 1.637,并测定其破胞上清液木糖醇脱氢酶氧化木糖醇的活性;用不同碳源培养的G.oxydans CGMCC 1.637转化木酮糖,用高效液相色谱法测定木糖醇的产量。【结果】获得一个新的798bp的木糖醇脱氢酶基因,所编码的木糖醇脱氢酶含265个氨基酸,属于短链脱氢酶家族。酶学性质研究发现,该木糖醇脱氢酶催化木糖醇氧化的最适合条件为35℃、pH 10.0,最高活性为23.27 U/mg,催化木酮糖还原为木糖醇的最适条件为30℃、pH 6.0。最高活性为255.55 U/mg;该木糖醇脱氢酶的对木糖醇的Km和Vmax分别为78.97 mmol/L和40.17 U/mg。碳源诱导实验表明,d-山梨醇对G.oxydans CGMCC 1.637木糖醇脱氢酶的活性有明显的促进作用,而葡萄糖、果糖、木糖、木糖醇、D-阿拉伯醇对木糖醇脱氢酶活性有明显的抑制作用。而在转化实验中,用d-甘露糖培养的G.oxydans CGMCC 1.637的转化能力明显高于其他碳源培养的G.oxydans CGMCC 1.637的转化能力,其中,用阿拉伯醇培养的G.oxydans CGMCC 1.637的转化能力最低,仅为对照的35%。【结论】克隆自G.oxydans CGMCC 1.637的木糖醇脱氢酶基因是一个新的基因,用阿拉伯醇培养的G.oxydans CGMCC 1.637破胞液木糖醇脱氢酶活性低;且阿拉伯醇对G.oxydans CGMCC 1.637木酮糖的还原能力具有抑制作用。     

弗氏柠檬酸菌dhaT基因的克隆表达、序列分析、酶的纯化及特性研究

1,3-丙二醇（1,3-propanediol,1,3-PD）是一种重要的化工原料,越来越受到广泛的关注。以弗氏柠檬酸菌(Citrobacter freundii)基因组DNA为模板,通过PCR得到1,3-丙二醇氧化还原酶(1,3-propanediol dehydrogenase,PDOR) 的基因dhaT,序列显示与来源于C.freundii DSM 30040 (Genbank U09771)相应基因的相似性为78%。将此基因构建于表达载体pSE380,得到重组质粒pSE-dhaT。重组质粒转化到宿主菌E.coli JM109中进行了表达,重组酶通过镍柱及Sephacral S-300进行纯化,重组酶SDS-PAGE结果显示有非常明显的单一的42kDa特异性蛋白条带出现。以丙醛为底物测定重组酶还原反应的最适温度为37℃、最适pH为8.0,对丙醛的Km值为10.05mmol/L,最大反应速度Vmax为37.27umol/ min /mg;以1,3-PD为底物测定重组酶氧化反应的最适温度为25℃、最适pH为10.5,对1,3-PD的Km值为1.28mmol/L,最大反应速度Vmax为25.55umol/min/mg。重组酶的还原反应比活为49.50U/mg,氧化反应比活为79.72U/mg。该酶同样具有假定的结合Fe2+的G-X-X-H-X-X-A-H-X-X-G-X-X-X-X-X-P-H-G模体保守结构。此研究为工程菌高效生产1,3-PD奠定了基础。     

Gluconobacter oxydans NH-10中短链D-阿拉伯糖醇脱氢酶的研究

以氧化葡萄糖酸杆菌(Gluconobacter oxydans)NH-10基因组DNA为模板,扩增得到D-阿拉伯糖醇脱氢酶基因arDH,将其克隆到大肠杆菌表达载体JM109(DE3)中进行诱导表达。SDS-PAGE电泳分析ArDH的分子量约为30 kDa,是一个短链脱氢酶,既能催化D-阿拉伯糖醇氧化为D-木酮糖,又能催化D-木酮糖还原为D-阿拉伯糖醇。催化氧化反应时,对D-阿拉伯糖醇的K_m为60.67 mmol/L,V_max为0.803 U/mg;它能同时依赖于NAD⁺和NADP⁺,但是更加偏好辅酶NAD⁺;最适pH为12.0。还原反应对D-木酮糖的 K_m为36.39 mmol/L,V_max为1.71 U/mg;最优pH为7.0,最适温度均为30℃。     

在含D-木糖的培养基上利用Petromyces albertensis的生长生产木糖醇

在含D-木糖培养基上培养Petromyces albertensis生产木糖醇和D—木酮糖,用高液相色谱鉴定其发酵产物.从含醋酸铵和酵母膏,初pH7.0的D-木糠培养基内获得了大量的木糖醇.木糖醇的大量产生及其酶相关的产物是在培养10天后进行观察的.D—木糖(100g/L)培养基中增补1%(V/V)甲醇,获得最高木糖醇产量为39.8g/L和D-木酮糖2.8g/L.     

产1,3-丙二醇新型重组大肠杆菌的构建

利用PCR技术从大肠杆菌(Escherichia coli )中扩增出1.16 kb的编码1,3-丙二醇氧化还原酶同工酶的基因yqhD,将其连接到表达载体pEtac,得到重组载体pEtac-yqhD,重组载体在大肠杆菌JM109中得到高效表达。SDS_PAGE分析显示融合表达产物的分子量均为43 kD,同核酸序列测定所推导的值相符。对含有yqh-D的基因工程菌进行表达研究表明：37 ℃,以1.0 mmol /L IPTG诱导4 h,1,3-丙二醇氧化还原酶同工酶的酶活力达到120 u/mg蛋白,而对照菌株的酶活力为0.5 u/mg蛋白。再将含甘油脱水酶基因dhaB和含1,3-丙二醇氧化还原酶同工酶基因yqhD的重组质粒共转化大肠杆菌JM109得到重组大肠杆菌JM109（pUCtac-dhaB, pEtac-yqhD）,该菌株在好氧条件下,以1.0mmol/L IPTG诱导可将50 g/L甘油转化为38.0 g/L 1,3-丙二醇。首次发现1,3-丙二醇氧化还原酶同工酶在好氧条件下表现出较高的活性。     

S-腺苷甲硫氨酸合成酶的组成型表达、产物纯化及鉴定

将大肠杆菌(E.coli K12) S 腺苷甲硫氨酸合成酶(SAMS)基因克隆至质粒pBR322中,获得的重组质粒pBR322-SAMS转入大肠杆菌JM109菌株,构建了能高效组成型表达SAMS的重组菌E.coli JM109 (pBR322-SAMS)。将重组大肠杆菌破碎后上清液经20%~40%硫酸铵分级盐析、Phenyl-Sepharose Fast Flow疏水层析和Q Sepharose Fast Flow离子交换层析,即可得到纯度提高5倍,比活为48.7 μ/mg的SAMS,三步纯化的总回收率为62%,纯度达到92%。SAMS表达量为1 176μ/L,占到菌体可溶性总蛋白的20%。重组酶的最适反应pH为8.5,4℃下在pH 7.5的缓冲液中保温10h酶活性几乎不改变。重组酶反应的最适温度为55℃ ,酶活性稳定的温度范围为20~35℃。重组酶的KmL Met为0.22mmol/L,Vmax L-Met为1.07mmol/(L·h),Km ATP为0.52 mmol/L,Vmax ATP为1.05 mmol/( L·h)。     

大肠杆菌甘油激酶基因(glpK)和甘油脱氢酶基因(gldA)的敲除及其对甘油产量的影响

利用Red重组系统构建了大肠杆菌JM109甘油激酶基因(glpK)和甘油脱氢酶基因(gldA)缺失的双突变菌株JM109B,然后将表达酿酒酵母3-磷酸甘油脱氢酶基因(GPD1)和3-磷酸甘油酯酶基因(HOR2)的质粒pSE-gpd1-hor2转化到JM109B突变菌株中,在含1%葡萄糖的摇瓶发酵培养基中37℃发酵24 h,甘油的最高产量为5.61 g/L,是原始菌株JM109/pSE-gpd1-hor2甘油产量的1.59倍;在30 L发酵罐中发酵28 h,甘油的最高产量为103.12 g/L,是原始菌株JM109/pSE-gpd1-hor2甘油产量的1.59倍,是原始菌株BL21/pSE-gpd1-hor2甘油产量的1.41倍,葡萄糖转化率为50.39%。     

一种基因工程改造的NADH高亲和力木糖还原酶突变基因可促进酿酒酵母发酵木糖生成乙醇

在导入表达毕赤酵母(Pichia stipitis)木糖还原酶(xylose reductase,XR)和木糖醇脱氢酶(xylitol dehydrogenase,XDH)基因的重组酿酒酵母中,木糖还原酶活性主要依赖辅酶NADPH,木糖醇脱氢酶活性依赖辅酶 NAD+,两者的辅助因子不同导致细胞内电子氧化还原的不平衡,是造成木糖醇积累,影响木糖代谢和乙醇产量的主要原因之一.将经过基因工程改造获得的NADH高亲和力的木糖还原酶突变基因m1,与毕赤酵母木糖醇脱氢酶(PsXDH)基因xyl2共转染酿酒酵母AH109,以转染毕赤酵母木糖还原酶(PsXR)基因xyl1和xyl2重组质粒的酵母细胞为对照菌株,在SC/-Leu/-Trp营养缺陷型培养基中进行筛选,获得的阳性转化子分别命名为AH-M-XDH和AH-XR-XDH.重组酵母在限制氧通气条件下对木糖和葡萄糖进行共发酵摇瓶培养,HPLC检测发酵底物的消耗和代谢产物的产出情况.结果显示,与对照菌株AH-XR-XDH相比,AH-M-XDH的木糖利用率明显提高,乙醇得率增加了16%,木糖醇产生下降了41.4%.结果证实,通过基因工程改造的木糖代谢关键酶,可用于酿酒酵母发酵木糖生产乙醇,其能通过改善酿酒酵母细胞内氧化还原失衡的问题,提高木糖利用率和乙醇产率.     

Molecular cloning, gene expression, purification and characterization of fermentative D-lactate dehydrogenase from S.marcescens H3010

通过PCR技术从粘质沙雷氏菌H3010基因组DNA中扩增出该D-乳酸脱氢酶基因,连接至pET-28a(+)表达载体,转入大肠杆菌BL21 (DE3)中进行了重组表达,优化了酶纯化的条件,并对其酶学性质进行初步研究.结果表明,获得的该酶编码基因全长993 bp,编码330个氨基酸,大小为37 kDa.经优化表达及纯化条件后重组酶纯度可达90％.酶学性质研究发现,该重组酶最适反应温度为60℃,最适酶促反应pH为7.5(0.2 mol/L磷酸盐缓冲液),37℃下测得对底物丙酮酸的动力学参数Km =3.39 mmol/L,Vmax =6.87 mmol/( mg · min),对辅酶NADH的动力学参数Km=1.43 mmol/L,Vmax=1.61 mmol/( mg· min).为酶法生产D-乳酸及利用代谢工程构建产D-乳酸的基因工程菌打下基础.     

木糖醇脱氢酶基因高拷贝表达载体构建及在酿酒酵母中的表达

木糖醇脱氢酶（xylitol dehydrogenase, XDH）可以氧化木糖醇生成木酮糖,处于木糖代谢的节点位置。利用PCR方法克隆得到了休哈塔假丝酵母（Candida shehatae） 20335的木糖醇脱氢酶基因、质粒pKT0150的ADH1终止子序列和G418抗性基因（KanR）,以及酿酒酵母(Saccharomyces cerevisiae) W5特定的2.2 kb的rDNA片段。以酿酒酵母整合载体p406ADH1为骨架,利用基因工程手段构建一个多拷贝整合表达载体pLX-AGRX。将重组载体pLX-AGRX线性化转入到酿酒酵母W5后,通过高浓度G418筛选和PCR双重鉴定,证实重组载体pLX-AGRX已整合到酿酒酵母W5基因组上,测定木糖醇脱氢酶酶活可达65.957 4 U/mg。     

Cloning of the xylitol dehydrogenase gene from Gluconobacter oxydans and improved production of xylitol from D-arabitol

Xylitol dehydrogenase (XDH) was purified from the cytoplasmic fraction of Gluconobacter oxydans ATCC 621. The purified enzyme reduced D-xylulose to xylitol in the presence of NADH with an optimum pH of around 5.0. Based on the determined NH2-terminal amino acid sequence, the gene encoding xdh was cloned, and its identity was confirmed by expression in Escherichia coli. The xdh gene encodes a polypeptide composed of 262 amino acid residues, with an estimated molecular mass of 27.8 kDa. The deduced amino acid sequence suggested that the enzyme belongs to the short-chain dehydrogenase/reductase family. Expression plasmids for the xdh gene were constructed and used to produce recombinant strains of G. oxydans that had up to 11-fold greater XDH activity than the wild-type strain. When used in the production of xylitol from D-arabitol under controlled aeration and pH conditions, the strain harboring the xdh expression plasmids produced 57 g/l xylitol from 225 g/l D-arabitol, whereas the control strain produced 27 g/l xylitol. These results demonstrated that increasing XDH activity in G. oxydans improved 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.     

Decreased xylitol formation during xylose fermentation in Saccharomyces cerevisiae due to overexpression of water-forming NADH oxidase

The recombinant xylose-fermenting Saccharomyces cerevisiae strain harboring xylose reductase (XR) and xylitol dehydrogenase (XDH) from Scheffersomyces stipitis requires NADPH and NAD(+), creates cofactor imbalance, and causes xylitol accumulation during growth on d-xylose. To solve this problem, noxE, encoding a water-forming NADH oxidase from Lactococcus lactis driven by the PGK1 promoter, was introduced into the xylose-utilizing yeast strain KAM-3X. A cofactor microcycle was set up between the utilization of NAD(+) by XDH and the formation of NAD(+) by water-forming NADH oxidase. Overexpression of noxE significantly decreased xylitol formation and increased final ethanol production during xylose fermentation. Under xylose fermentation conditions with an initial d-xylose concentration of 50 g/liter, the xylitol yields for of KAM-3X(pPGK1-noxE) and control strain KAM-3X were 0.058 g/g xylose and 0.191 g/g, respectively, which showed a 69.63% decrease owing to noxE overexpression; the ethanol yields were 0.294 g/g for KAM-3X(pPGK1-noxE) and 0.211 g/g for the control strain KAM-3X, which indicated a 39.33% increase due to noxE overexpression. At the same time, the glycerol yield also was reduced by 53.85% on account of the decrease in the NADH pool caused by overexpression of noxE.     

混合糖发酵重组酿酒酵母的菌株构建和菊芋秸秆同步糖化发酵研究

【目的】构建可用于纤维素乙醇高效生产的混合糖发酵重组酿酒酵母菌株,并利用菊芋秸秆为原料进行乙醇发酵。【方法】筛选在木糖中生长较好的酿酒酵母YB-2625作为宿主菌,构建木糖共代谢菌株YB-2625 CCX。进一步通过r DNA位点多拷贝整合的方式,以YB-2625 CCX为出发菌株构建木糖脱氢酶过表达菌株,并筛选得到优势菌株YB-73。采用同步糖化发酵策略研究YB-73的菊芋秸秆发酵性能。【结果】YB-73菌株以90 g/L葡萄糖和30 g/L木糖为碳源进行混合糖发酵,乙醇产量比出发菌株YB-2625 CCX提高了13.9%,副产物木糖醇产率由0.89 g/g降低至0.31 g/g,下降了64.6%。利用重组菌YB-73对菊芋秸秆进行同步糖化发酵,48 h最高乙醇浓度达到6.10%(体积比)。【结论】通过转入木糖代谢途径以及r DNA位点多拷贝整合过表达木糖脱氢酶基因可有效提高菌株木糖发酵性能,并用于菊芋秸秆的纤维素乙醇生产。这是首次报道利用重组酿酒酵母进行菊芋秸秆原料的纤维素乙醇发酵。     

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.     

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。     

Effect of the reversal of coenzyme specificity by expression of mutated Pichia stipitis xylitol dehydrogenase in recombinant Saccharomyces cerevisiae

AIMS: To determine the effects on xylitol accumulation and ethanol yield of expression of mutated Pichia stipitis xylitol dehydrogenase (XDH) with reversal of coenzyme specificity in recombinant Saccharomyces cerevisiae. METHODS AND RESULTS: The genes XYL2 (D207A/I208R/F209S) and XYL2 (S96C/S99C/Y102C/D207A/I208R/F209S) were introduced into S. cerevisiae, which already contained the P. stipitis XYL1 gene (encoding xylose reductase, XR) and the endogenously overexpressed XKS1 gene (encoding xylulokinase, XK). The specific activities of mutated XDH in both strains showed a distinct increase in NADP(+)-dependent activity in both strains with mutated XDH, reaching 0.782 and 0.698 U mg(-1). In xylose fermentation, the strain with XDH (D207A/I208R/F209S) had a large decrease in xylitol and glycerol yield, while the xylose consumption and ethanol yield were decreased. In the strain with XDH (S96C/S99C/Y102C/D207A/I208R/F209S), the xylose consumption and ethanol yield were also decreased, and the xylitol yield was increased, because of low XDH activity. CONCLUSIONS: Changing XDH coenzyme specificity was a sufficient method for reducing the production of xylitol, but high activity of XDH was also required for improved ethanol formation. SIGNIFICANCE AND IMPACT OF THE STUDY: The difference in coenzyme specificity was a vital parameter controlling ethanolic xylose fermentation but the XDH/XR ratio was also important.     

带有木糖还原酶基因和木糖醇脱氢酶基因的重组酿酒酵母的构建

采用双载体系统,将携带有瑞氏木霉木糖醇脱氢酶基因的表达质粒pAJ401－Xdh1转化已带有树干毕赤氏酵母木糖还原酶基因的重组酿酒酵母H475,构建了同时带有毕赤氏酵母木糖还原酶基因和瑞氏木霉木糖醇脱氢酶基因的重组酿酒酵母HX1。研究了重组酿酒酵母HX1对木糖的转化利用情况。     

Ethanol production from xylose by recombinant Saccharomyces cerevisiae expressing protein engineered NADP+-dependent xylitol dehydrogenase

Effects of reversal coenzyme specificity toward NADP+ and thermostabilization of xylitol dehydrogenase (XDH) from Pichia stipitis on fermentation of xylose to ethanol were estimated using a recombinant Saccharomyces cerevisiae expressing together with a native xylose reductase from P. stipitis. The mutated XDHs performed the similar enzyme properties in S. cerevisiae cells, compared with those in vitro. The significant enhancement(s) was found in Y-ARSdR strain, in which NADP+-dependent XDH was expressed; 86% decrease of unfavorable xylitol excretion with 41% increased ethanol production, when compared with the reference strain expressing the wild-type XDH.     

Elevation of glucose 6-phosphate dehydrogenase activity increases xylitol production in recombinant Saccharomyces cerevisiae

To increase the NAD(P)H-dependent xylitol production in recombinant Saccharomyces cerevisiae harboring the xylose reductase gene from Pichia stipitis, the activity of glucose 6-phosphate dehydrogenase (G6PDH) encoded by the ZWF1 gene was amplified to increase the metabolic flux toward the pentose phosphate pathway and NADPH regeneration. Compared with the control strain, the specific G6PDH activity was enhanced approximately 6.0-fold by overexpression of the ZWF1 gene. Amplification in the G6PDH activity clearly improved the NAD(P)H-dependent xylitol production in the recombinant S. cerevisiae strain. With the aid of an elevated G6PDH level, maximum xylitol concentration of 86 g/l was achieved with productivity of 2.0 g/l h in the glucose-limited fed-batch cultivation, corresponding to 25% improvement in volumetric xylitol productivity compared with the recombinant S. cerevisiae strain containing the xylose reductase gene only.