共表达xyl1、xyl2和tal1重组酿酒酵母的构建及木糖发酵研究

将来自树干毕赤酵母的木糖还原酶基因(xyl1)、木糖醇脱氢酶基因(xyl2)和酿酒酵母自身的木糖转醛酶基因(tal1),通过串联共表达的方法构建到表达载体pAUR123上,构建了1株重组酿酒酵母。该菌在打通木糖向木酮糖转化通路基础上,超表达木糖转醛酶。该菌以木糖为唯一碳源进行限氧发酵,能初步利用木糖。结果表明,木糖的利用率为77.4%,但乙醇产率仅为0.04 mg/mL。同时探讨了3种酶共表达对酿酒酵母发酵木糖生成乙醇的影响,发现3种酶对木糖的利用起关键性的作用,其过量表达导致木糖醇大量积累,乙醇得率降低。     

木糖发酵酵母Pichia stipitis木糖还原酶基因XYL1在酿酒酵母中的表达

通过PCR方法克隆得到树干毕赤氏酵母木糖还原酶 (XR)基因XYL1。将该基因连入酵母表达载体pYX2 12的强启动子磷酸丙糖异构酶 (TPI)启动子下 ,得到融合表达载体pYX XYL1。通过电转化方法将 pYX XYL1转入酿酒酵母SaccharonmycescerevisiaeW 30 3-1A中 ,酶活测定表明 ,在酿酒酵母中树干毕赤氏酵母木糖还原酶 (XR)基因XYL1得到活性表达 ,2酿酒酵母转化子粗酶液中木糖还原酶活分别为 0 .89U/mg(蛋白 )和 0 .83U/mg(蛋白 ) ,为供体菌的 1 5倍多。与基因供体菌不同 ,木糖还原酶基因在酿酒酵母中表达不需木糖诱导 ,为组成型表达。树干毕赤氏酵母木糖还原酶 (XR)基因XYL1的成功表达为后续的利用木糖的酿酒酵母菌株的构建奠定了基础     

工业酿酒酵母木糖代谢途径的建立及酒精发酵初步研究

以酵母AS2.1190为出发菌株,把含有木糖还原酶基因(XYL1)、木糖醇脱氢酶基因(XYL2)以及木酮糖激酶基因(XKS1)的质粒载体pYMIKP-xy127线性化后多拷贝整合进入其基因组,筛选得到基因工程菌株GZ4-127,并对此工程菌株进行葡萄糖、木糖共发酵试验.结果显示GZ4-127比出发菌株的菌体密度提高5%,木糖消耗提高2倍,酒精产率提高12%,说明工程菌已能够有效地利用木糖生产乙醇.     

稳定高效表达木糖还原酶基因工业酿酒酵母的构建及木糖醇发酵的初步研究

将树干毕赤氏酵母(Pichia stipitis)木糖还原酶基因XYL1连接到适用于酿酒酵母工业菌株的多拷贝整合载体pYMIKP中,构建得到表达质粒pYMIKP-XYL1,转化酿酒酵母工业菌株Saccharomyces cerevisiae6508。在G418平板上筛选转化子,得到含高拷贝木糖还原酶基因的酿酒酵母重组菌株XGH2,,该菌株的木糖还原酶比活力为0.8 U/mg(蛋白),比出发菌株提高了80倍以上,表明外源基因在工业菌株中实现了高效表达。摇瓶发酵结果显示,重组菌株XGH2木糖消耗为27.9 g/L,木糖消耗率为51%;木糖醇产量为30.2 g/L,木糖醇的转化率大于1.0 g/g木糖。     

Pichia stipitis木糖醇脱氢酶基因XYL2在酿酒酵母中的表达

通过PCR方法克隆得到树干毕赤氏酵母木糖醇脱氢酶(XDH)基因XYL2.将该基因连入酵母表达载体pYX212的强启动子磷酸丙糖异构酶(TPI)启动子下,得到融合表达载体pYX-XYL2.通过电转化方法将pYX-XYL2转入酿酒酵母Saccharomyces cerevisiae W303-1A中,酶活测定表明在酿酒酵母中树干毕赤氏酵母木糖醇脱氢酶基因XYL2得到活性表达,酿酒酵母转化子粗酶液中木糖醇脱氢酶比活为每毫克蛋白0.6 U左右,约为供体菌的2.4倍.与基因供体菌不同,木糖醇脱氢酶基因在酿酒酵母中表达不需木糖诱导,为组成型表达.     

代谢改造热带假丝酵母利用木糖生产D-阿拉伯糖醇

为探究酵母菌以木糖为底物生产D-阿拉伯糖醇的可行性,该研究以热带假丝酵母(Candida tropicalis)CU-208为出发菌株,通过代谢工程构建利用木糖生产D-阿拉伯糖醇的菌株。在验证了热带假丝酵母内源D-阿拉伯糖醇脱氢酶(D-arabitol dehydrogenase, ARD)功能的基础上,进行如下代谢改造：先敲除木酮糖激酶(xyluki-nase, XKS)基因,以阻断木糖进入磷酸戊糖途径,同时过表达上游木糖醇脱氢酶(xylitol dehydrogenase, XDH)基因,构建菌株FYM01;再敲除ARD基因ard,减少菌株对D-阿拉伯糖醇的消耗,构建菌株FYM02;然后,过表达来源于圆红冬孢酵母菌的外源D-阿拉伯糖醇脱氢酶(D-arabitol dehydrogenase, ADH)基因,从木酮糖合成D-阿拉伯糖醇,构建菌株FYM04;最后,过表达内源葡萄糖-6-磷酸脱氢酶(glucose-6-phosphate dehydrogenase, ZWF)基因,以增强辅酶NADPH供应,构建菌株FYM05。结果发现,基因ard参与代谢D-阿拉伯糖醇;代谢改造获得的菌株...     

代谢木糖生产乙醇的基因工程菌研究进展

木糖广泛存在于林业及农业废弃物中 ,木糖发酵生产乙醇是再生资源的有效利用。文中报道了近几年来在利用基因工程菌发酵木糖生产乙醇方面的研究进展。重点介绍了大肠杆菌、酿酒酵母、树干毕赤酵母及运动发酵单胞菌的基因改造情况。     

直接发酵木糖为酒精的新酵母基因工程菌的选育

裂殖酵母,Schizosacchatomyces porebe不能发酵木糖,也不能用它来增殖细胞。但是,这种酵母能发酵木酮糖为酒精,产率和转化率都很高。这表明,它缺少异构酶基因,如果将其它微生物的这种基因克隆到酵母中去,它就能直接发酵木糖。本文介绍了Schizosaceh.porebe的五碳糖磷酸代谢途径,这种代谢途径使得它成为用于这一目的的理想菌株。含有异构酶基因的DNA片段来自Clark和Carbon的大肠杆菌基因库,它的大小为2.4kb,PstⅠ-抗性。将这个基因克隆到上述酵母中去,得到的酵母基因工程菌株不仅能利用木糖作为唯一碳源和能源,还能发酵木糖为酒精,它具有在工业上应用的潜力     

热带假丝酵母木糖还原酶在酿酒酵母细胞表面展示

利用酿酒酵母(Saccharomyces cerevisiae)表面展示系统,将来源于热带假丝酵母(Candida tropicalis)的木糖还原酶基因xyl1嵌入带有His-Tag的酿酒酵母α-凝集素展示载体pICAS-His,构建重组质粒pICAS- His-Ctxyl1,并转化到酿酒酵母宿主菌酿酒酵母MT8—1,通过流式细胞仪快速检测和筛选,得到重组菌株MT8- 1/pICAS-His—Ctxyl1。将重组酵母用于葡萄糖(15g/L)和木糖(5g/L)的混合糖发酵研究,结果表明,重组酿酒酵母MT8/1/pICAS-His—Ctxyl1细胞具有良好的生长和产酶特性,同时能转化木糖生产木糖醇,在培养基中2.5g/ L木糖转化生成2.5g/L木糖醇,转化率达98.7%。     

木糖还原酶基因整合表达载体构建

木糖还原酶是酵母代谢木糖发酵的关键酶之一。根据已报道的木糖还原酶基因的全序列设计引物,从休哈塔假丝酵母中克隆得到1 110 bp大小的片段。将木糖还原酶基因亚克隆到酿酒酵母的整合表达载体p406ADH1中,醋酸锂转化法将线性化重组质粒p YX-AK-xyl1转化到酿酒酵母W5,对阳性转化子进行酶活测定,结果表明,以辅酶DANH和NADPH为底物诱导,转化子均表现出活性,酶活分别为6.513 U/mg和7.080 U/mg,是受体菌W5酶活的1.4倍(NADH)和1.3倍(NADPH),其酶活比为0.919。     

Endogenous NADPH-dependent aldose reductase activity influences product formation during xylose consumption in recombinant Saccharomyces cerevisiae

Introduction of the xylose pathway from Pichia stipitis into Saccharomyces cerevisiae enables xylose utilization in recombinant S. cerevisiae. However, xylitol is a major by-product. An endogenous aldo-keto reductase, encoded by the GRE3 gene, was expressed at different levels in recombinant S. cerevisiae strains to investigate its effect on xylose utilization. In a recombinant S. cerevisiae strain producing only xylitol dehydrogenase (XDH) from P. stipitis and an extra copy of the endogenous xylulokinase (XK), ethanol formation from xylose was mediated by Gre3p, capable of reducing xylose to xylitol. When the GRE3 gene was overexpressed in this strain, the xylose consumption and ethanol formation increased by 29% and 116%, respectively. When the GRE3 gene was deleted in the recombinant xylose-fermenting S. cerevisiae strain TMB3001 (which possesses xylose reductase and XDH from P. stipitis, and an extra copy of endogenous XK), the xylitol yield decreased by 49% and the ethanol yield increased by 19% in anaerobic continuous culture with a glucose/xylose mixture. Biomass was reduced by 31% in strains where GRE3 was deleted, suggesting that fine-tuning of GRE3 expression is the preferred choice rather than deletion.     

Investigation of limiting metabolic steps in the utilization of xylose by recombinant Saccharomyces cerevisiae using metabolic engineering

A Saccharomyces cerevisiae screening strain was designed by combining multiple genetic modifications known to improve xylose utilization with the primary objective of enhancing xylose growth and fermentation in xylose isomerase (XI)-expressing strains. Strain TMB 3045 was obtained by expressing the XI gene from Thermus thermophilus in a strain in which the GRE3 gene coding for aldose reductase was deleted, and the genes encoding xylulokinase (XK) and the enzymes of the non-oxidative pentose phosphate pathway (PPP) [transaldolase (TAL), transketolase (TKL), ribose 5-phosphate ketol-isomerase (RKI) and ribulose 5-phosphate epimerase (RPE)] were overexpressed. A xylose-growing and fermenting strain (TMB 3050) was derived from TMB 3045 by repeated cultivation on xylose medium. Despite its low XI activity, TMB 3050 was capable of aerobic xylose growth and anaerobic ethanol production at 30 degrees C. The aerobic xylose growth rate reached 0.17 l/h when XI was replaced with xylose reductase (XR) and xylitol dehydrogenase (XDH) genes expressed from a multicopy plasmid, demonstrating that the screening system was functional. Xylose growth had not previously been detected in strains in which the PPP genes were not overexpressed or when overexpressing the PPP genes but having XR and XDH genes chromosomally integrated. This demonstrates the necessity to simultaneously increase the conversion of xylose to xylulose and the metabolic steps downstream of xylulose for efficient xylose utilization in S. cerevisiae.     

转木糖还原酶基因XYL1酿酒酵母的构建及产木糖醇能力研究

将人工合成的树干毕赤酵母（Pichia stipitis）的木糖还原酶基因XYL1插入酿酒酵母（Saccharomyces cerevisiae）表达载体pYES2中,然后将重组质粒pYES2-XYL1导入酿酒酵母INVSc1中,构建转木糖还原酶基因XYL1酿酒酵母菌株INVSc1/pYES2-XYL1,最后采用营养缺陷培养基筛选转木糖还原酶基因酿酒酵母并对其产木糖醇的能力进行检测。结果表明,成功获得2株转木糖还原酶基因XYL1酿酒酵母菌株INVSc1/pYES2-XYL1-01、INVSc1/pYES2-XYL1-02,当两菌株以50 g/L木糖及10 g/L半乳糖为碳源发酵5 d后,木糖醇产量分别高达（13.68±2.37）g/L、（12.09±1.45）g/L,显著高于非转基因酿酒酵母INVSc1的木糖醇产量（1.08±0.37）g/L（P＜0.05）,说明XYL1基因的导入显著提高了酿酒酵母INVSc1生产木糖醇的能力（P＜0.05）。为采用基因工程酿酒酵母制备食用木糖醇提供了理论及技术基础。     

瑞氏木霉纤维素酶基因在酿酒酵母中的表达研究

从瑞氏木霉（Trichoderma reesei）cDNA文库中克隆到外切葡聚糖纤维二糖水解酶Ⅰ（CBHI）eDNA基因,将该基因分别连接到酿酒酵母（Saccharomyces cerevisiae）表达载体pAJ401和pVT100_U上,构建了重组质粒pAJ401-cbhl和pVTIOO_U-cbhl。分别电转化2个重组质粒转移至酿酒酵母H158中,得到重组酵母HPC和HAC,实现了CBHI的分泌型表达。再将质粒pAJ401-cbhl转入已含有瑞氏木霉内切葡聚糖酶Ⅰ基因egl的重组酵母Hlm中,构建了同时表达cbhl和egl的重组酵母菌株HMEPC。比较HMEPC和Hlm对纤维素底物的降解,前者滤纸酶活比后者提高14．3％,表明CBHI与EGI对滤纸降解有协同作用。     

重组枯草芽孢杆菌PaE菌株的构建及碳代谢阻遏效应研究

通过基因重组的方法,将枯草芽孢杆菌168的α-淀粉酶基因amyE（除掉启动子）整合在Paxo1质粒上,然后将重组表达质粒Paxo1-amyE导入枯草芽孢杆菌168菌株基因组中的半乳糖苷酶基因lacZ位点。通过转化筛选和重组基因分析,获得含有重组α-淀粉酶基因的工程菌株PaE。经过添加4 种不同的碳源发酵实验检测,在外加2 g/100 mL木糖诱导的情况下,重组菌株的α-淀粉酶产量均有增加。而且重组菌株在一定程度上能够克服葡萄糖等还原性糖引起的碳代谢阻遏现象,提高了α-淀粉酶产量。