乳酸片球菌L-乳酸脱氢酶基因的克隆及在重组大肠杆菌JH12中的过量表达

生物技术通报 ›› 2014, Vol. 0 ›› Issue (4): 121-126.

乳酸片球菌L-乳酸脱氢酶基因的克隆及在重组大肠杆菌JH12中的过量表达

罗璇¹, 许丽媛², 王永泽², 赵锦芳², 赵筱², 王金华²

(1. 华中农业大学楚天学院食品与生物科技学院，武汉 430205；
2. 湖北工业大学发酵工程教育部重点实验室工业发酵湖北省协同创新中心，武汉 430068)

收稿日期:2013-11-12 出版日期:2014-04-29 发布日期:2014-04-29
作者简介:罗璇，女，讲师，研究方向：发酵工程与生物技术；E-mail：luoxuan20051982@126.com
基金资助:
国家自然科学基金项目(31070094)，湖北省教育厅科研项目(Q20121405)，湖北省科技厅科研项目(2011CDA008，2011CD-B076)

Cloning L-lactate Dehydrogenase Gene from Pediococcus acidilactici and Overexpression of It in Recombination Strain E.coli JH12

Luo Xuan¹, Xu Liyuan², Wang Yongze², Zhao Jinfang², Zhao xiao², Wang Jinhua²,

(1. College of Food Science and Biotechnology，Huazhong Agricultural University Chutian College，Wuhan 430205； 2. Key Laboratory of Fermentation Engineering(Ministry of Education)，Hubei Provincial Cooperative Innovation Center of Industrial Fermentation，College of Bioengineering，Hubei University of Ttechnology，Wuhan 430068)

Received:2013-11-12 Published:2014-04-29 Online:2014-04-29

摘要/Abstract

摘要： 以工程菌Escherichia coli SZ85基因组为模板，克隆得到乳酸片球菌(Pediococcus acidilactici)的L-乳酸脱氢酶基因(ldhL)，连接到pUcm-T载体后，双酶切然后将其连接到表达载体pET-28a上，重组质粒经筛选后转入染色体上含有ldhL基因(来源P. acidilactici)的工程菌Escherichia coli JH12。 P. acidilactici 的ldhL基因过量表达体系E. coli JH12(pET-28a-ldhL)能利用浓度7% 的木糖为碳源进行厌氧发酵，过量表达ldhL使L-乳酸产量提高了10 g/L，达64.86 g/L，糖酸转化率高达96%。

关键词: 重组大肠杆菌, 过量表达, L-乳酸

Abstract: Gene ldhL encoding L-lactate dehydrogenase was amplified by PCR technique using genome DNA of Escherichia coli SZ85 as temple. The PCR product was cloned into pUcm-T vector and double digested with restriction endonucleases, and then the DNA fragment of ldhL was inserted into pET-28a(+). The recombinants expression plasmid pET-ldhL was transformed into high purity L-lactate production of E. coli. JH12, which inserted ldhL of P. acidilactici into chromosome. The overexpression system of ldhL of Pediococcus acidilactici gene E. coli JH12(pET-28a-ldhL)could product L-lactic acid using 7% xylose. Overexpression of ldhL led to L-lactic acid yield of 64.86 g/L, which is 10 g/L higher than control without Overexpression of ldhL. Meanwhile, high sugar-acid ratio of 96% was achieved by overexpression of ldhL.

Key words: Recombination, E.coli, Overexpression, L-lactic acid

罗璇, 许丽媛, 王永泽, 赵锦芳, 赵筱, 王金华. 乳酸片球菌L-乳酸脱氢酶基因的克隆及在重组大肠杆菌JH12中的过量表达[J]. 生物技术通报, 2014, 0(4): 121-126.

Luo Xuan, Xu Liyuan, Wang Yongze, Zhao Jinfang, Zhao xiao, Wang Jinhua,. Cloning L-lactate Dehydrogenase Gene from Pediococcus acidilactici and Overexpression of It in Recombination Strain E.coli JH12[J]. Biotechnology Bulletin, 2014, 0(4): 121-126.

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