Integrative expression vectors for overexpression of xylitol dehydrogenase (XYL2) in Osmotolerant yeast, Candida glycerinogenes WL2002-5
Integrative expression vectors for overexpression of xylitol dehydrogenase (XYL2) in Osmotolerant yeast, Candida glycerinogenes WL2002-5
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DOI：
10.1007/s10295-014-1530-4
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发表时间：
2014
影响因子：
3.4
通讯作者：
Cheng Zhang;H. Zong;B. Zhuge;Xinyao Lu;H. Fang;J. Zhuge
中科院分区：
文献类型：
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作者：
Cheng Zhang;H. Zong;B. Zhuge;Xinyao Lu;H. Fang;J. Zhuge
酵母菌是生产重组蛋白的优良宿主，产甘油假丝酵母WL2002-5是一种产甘油极高的耐渗酵母，为其提供了一个诱人的真核表达平台。构建了分别带有PCgGAP和PCgGPD启动子的含有绿霉素抗性编码序列和GFP编码序列的整合载体PURGAP-gfp和PURGPD-gfp。分别构建了毕赤酵母GAPDH基因启动子PPpGAP-gfp和酿酒酵母启动子PScGAP-gfp重组载体PURPpGAP-gfp。转化后，用荧光定量实时聚合酶链式反应(FQ-RTPCR)检测OFC基因组中gfp基因的拷贝数。1.利用不同的启动子，在含有盐和葡萄糖的渗透胁迫下，表达不同水平的gfpA。在本研究中，表达树干毕赤酵母木糖醇脱氢酶(XYL2)基因的产甘油酵母WL2002-5具有将木糖转化为磷酸戊糖途径生产甘油的能力。构建了两株高表达XYL2的PURGAPX重组菌株PURGPDX，用于分批发酵同时发酵葡萄糖和木糖的混合物。比较了TOC。WL2002-5菌株对木糖的甘油产量提高了95.9%，PURGPDX菌株提高了121.1%。
Yeasts are excellent hosts for the production of recombinant proteins.Candida glycerinogenesWL2002-5, an osmotolerant yeast with extremely high glycerol productivity, provides an attractive eukaryotic expression platform. The integrative vectors PURGAP-gfpand PURGPD-gfpharbouring phleomycin-resistance coding sequence and GFP coding sequence with PCgGAP, PCgGPDpromoter, respectively, were constructed. The recombinant plasmid PURPpGAP-gfpwith the promoter PPpGAPbased on the sequence ofPichia pastorisGAPDH gene and the plasmid PURScGAP-gfpwith the promoter PScGAPfromSaccharomyces cerevisiaewere constructed. After transformation, the copy number ofgfpgene, which determined using fluorescent quantitative real-time polymerase chain reaction (FQ-RTPCR) in genome ofC. glycerinogenesis 1. Expressions ofgfpat different levels were conducted using different promoters by osmotic stress containing NaCl or glucose for the recombinant strains. In this study,C. glycerinogenesWL2002-5, expressing xylitol dehydrogenase (XYL2) gene fromPichia stipitis, has the ability to produce glycerol from xylose entered into pentose phosphate pathway. Two recombinant strains of PURGAPX, PURGPDX withXYL2overexpression were constructed to ferment a mixture of glucose and xylose simultaneously in batch fermentation. Compared toC. glycerinogenesWL2002-5 strain, glycerol production from xylose in strains PURGAPX, PURGPDX were increased by 95.9 and 121.1 %, respectively.