甲酸脱氢酶在Klebsiella pneumoniae中的表达和功能分析  

Expression and characterization of formate dehydrogenase gene in Klebisella pneumoniae

作　　者：黄志华[1] 张延平[1] 刘铭[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：《微生物学报》2007年第1期64-68,共5页Acta Microbiologica Sinica

基　　金：国家重点基础研究发展规划(973项目)(2003CB716007)~~

摘　　要：在甘油厌氧发酵生产1,3-丙二醇的过程中,需要消耗还原当量NADH,NADH的有效供给决定了1,3-丙二醇的产量和得率。采用PCR方法从Candidaboidinii基因组中克隆编码甲酸脱氢酶基因fdh,将fdh基因片段插入载体pMALTM-p2X中,构建表达载体pMALTM-p2X-fdh,并转入1,3-丙二醇生产菌Klebsiella pneumoniae YMU2,获得重组菌Klebsiella pneumoniae F-1。研究了重组质粒的稳定性和IPTG诱导fdh基因过量表达的条件。结果表明,重组质粒具有良好的稳定性;fdh基因表达的蛋白分子量为40.2kDa;IPTG诱导表达研究表明,在IPTG浓度为0.5mmol/L时,诱导4h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5.47U/mg;与出发菌株K.pneumoniae YMU2相比,重组菌F-1合成1,3-丙二醇的浓度提高了12.5%。Glycerol can be converted to 1,3-propanediol by the anaerobic fermentation of KlebsieUa pneumoniae, during which reducing equivalent NADH was consumed. Therefore, the availability of NADH would be critical for the yield of 1,3- propanediol. In this paper, formate/formate dehydrogenase system was used for the regeneration of in vivo NADH and the improvement of 1,3-propanediol production. Formate Dehydrogenase gene （fdh） was amplified from Candida boidinii genome by PCR and the purified PCR product was inserted into the vector pMD18-T Simple to construct plasmid pMD18-T Simple-fdh, which was transformed into Escherichia coli DH5α and recombinants were selected by blue-white selection. From the transformant the fdh gene was separated and inserted into pMAL^TM-p2X to construct expression vector pMAL^TM-p2X-fdh, which was transformed into Klebsiella pneumoniae YMU2 and a recombinant strain KlebsieUa pneumoniae F-1 was obtained. The plasmid stability of strain F-1 and the conditions of fdh expression induced by IPTG were studied. It was demonstrated that the plasmid had good stability, and 0.5mmol/L IPTG would induce the expression of protein encoded by fdh gene with the molecular weight of 40.2kDa. The enzyme activity reached 5.47U/mg crude protein when K. pneumoniae F-1 was induced for4h by 0.5mmol/L IPTG. Compared with that of the parent strain K. pneumoniae YMU2, the yield of 1,3-propanediol of recombinant strain F-I increased by 12.5% in the anaeribic bioreactor.

关 键 词：CANDIDA boidinii 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]