在Klebsiella pneumoniae醛脱氢酶失活菌中构建NADH再生系统  

Construction of NADH Regeneration System in Klebisella pneumoniae with Aldehyde Dehydrogenase Inactivated

作　　者：黄志华[1] 张延平[1] 黄星[1] 王宝光[1] 曹竹安[1] 

机构地区：[1]清华大学化学工程系生物化工研究所

出　　处：《中国生物工程杂志》2006年第12期75-80,共6页China Biotechnology

基　　金：国家重点基础研究发展计划(973计划)资助项目(2003CB716007)

摘　　要：生物法生产1,3-丙二醇(1,3-Propanediol,1,3-PD)是当前工业生物技术研究的热点之一,生产过程中,需要消耗还原当量NADH,NADH的有效供给决定了1,3-PD的产量和得率。采用PCR的方法从Candidaboidinii基因组中克隆编码fdh的基因,将该基因片段插入载体pMALTM-p2X,构建表达载体pMALTM-p2X-fdh,并转入醛脱氢酶失活菌KlebsiellapneumoniaeDA-1HB,获得重组菌KlebsiellapneumoniaeDAF-1。在IPTG浓度0.5mmol/L时,诱导3h后甲酸脱氢酶表达明显;发酵过程中甲酸脱氢酶比酶活达到4.82U/mg;与出发菌株K.pneumoniaeDA-1HB相比,重组菌DAF-1合成1,3-丙二醇的浓度提高了19.2%。The microbial production of 1,3-propanediol attracted much interest in the field of industry biotechnology. During the process of 1,3-propanediol production by Klebsiella pneumoniae, reducing equivalent NADH was consumed. Therefore, the available NADH would be critical for the yield of 1,3-propanediol. Formate/ formate dehydrogenase system was used for the generation of NADH in vivo and therefore the improvement of 1,3-Propanediol production. Formate Dehydrogenase gene （fdh） was amplified from Candida boidinii genome DNA and the purified PCR product was inserted into the vector pMD18-T Simple to obtain plasmid pMD18-T Simple-fdh, which was transformed into Escherichia coli DH5α, and recombinants were selected by blue-white selection. For effective regeneration of NADH in K. pneumoniae, the fdh gene was separated from the transformant DH5α （pMD18-T Simple-fdh） and inserted into pMAL^TM-p2X to construct expression vector pMAL^TM-p2X-fdh. The vector was then transformed into DA-1HB, an engineered strain of Klebsiella pneumoniae with the aldehyde dehydrogenase inactivated, and a recombinant strain Klebsiella pneumoniae DAF-1 was obtained. The activity of formate dehydrogenase reached 4.82 U/mg crude protein after K. pneumoniae DAF-1 was induced for 3 h by 0.5 mmol/L IPTG, much higher than that of the parent strain DA-1HB. As results of NADH regeneration in vivo, the yield of 1,3-propanediol was increased by 19.2 % in the anaerobic bioreactor, the yield of ethanol was increased by 41. 18 %, and the yield of 2,3-butanediol was increased by 209.7 %. It indicated that branches with consumption of NADH were strengthened by regeneration of NADH in K. pneumoniae.

关 键 词：醛脱氢酶 甲酸脱氢酶 KLEBSIELLA PNEUMONIAE NADH 1 3-丙二醇 

分 类 号：Q78[生物学—分子生物学]