产L-乳酸的运动发酵单胞菌代谢工程菌株的构建  

Construction of Metabolically-engineered Zymomonas mobilis Strain for L-lactic Acid Production

作　　者：姜坤妤[1] 苏喆 王勇 潘超强[1] 朱燕[4] 郑秀[4] 高强[1] 

机构地区：[1]天津科技大学生物工程学院工业微生物教育部暨天津市重点实验室,天津300457 [2]天津市药品检验所,天津300070 [3]中法合营王朝葡萄酿酒有限公司,天津300402 [4]天津科技大学后勤服务集团,天津300457

出　　处：《生物技术通报》2011年第6期170-174,共5页Biotechnology Bulletin

基　　金：国家“973”项目(2007CB714305,2011CB707401);国家“863”项目(2008AA10Z336);科技部国际合作计划(2007DFB31620);天津市自然科学基金重点项目(08JCZDJC15100)

摘　　要：以运动发酵单胞菌(Zymomonas mobilis)CP4基因组DNA为模板,采用PCR技术克隆得到其丙酮酸脱氢酶基因(pdc)同源下游p3片段,并连接到广谱宿主载体pBBR1MCS3-Ppdc-ldhL中构建了重组质粒pBBR1MCS3-Ppdc-ldhL-p3,将此重组质粒转化到受体菌Z mobilis CP4中,分别以Ppdc和p3片段作为同源上游和下游片段,利用同源双交换重组技术将重组质粒中的ldhL基因置换了Z.mobilis染色体中的pdc基因,得到重组菌株Z.mobilis CP4(△pdc::ldhL)。测得重组菌株乳酸产量为10.8g/L,明显高于出发菌株,说明初步成功构建了产L-乳酸的运动发酵单胞菌代谢工程菌株。The down-stream p3 fragment of Zymomonas mobilis CP4 pdc gene was achieved by PCR using its host genome as the template. The resulting p3 fragment was inserted into vector pBBR1MCS3-Ppdc-ldhL to construct the plasmid pBBR1MCS3-Ppdc-ldhL- p3 which was transformed into 7,. mobilis CP4 host by eleetroporation. Using the Ppdc and p3fragments as the upstream and downstream parts respectively in the homologous recombination, the pdc gene in Z. mobilis CP4 genome was replaced by ldhL gene from the recombi- nant plasmid to construct the recombinant strain Z. mobilis CP4（ Apdc ：： ldhL）. Finally,the engineered strain produced 10.8 g/L L-lactic acid,which was obviously higher than its parent strain Z. mobilis CP4. This result demonstrated that the metabolically-engineered Z. mobilis strain for L-lactic acid production was successfully constructed.

关 键 词：运动发酵单胞菌 同源重组L-乳酸乳酸脱氢酶 丙酮酸脱氢酶 

分 类 号：TQ921.3[轻工技术与工程—发酵工程]