代谢工程大肠杆菌利用甘油高效合成L-乳酸  

High-efficiency L-lactate production from glycerol by metabolically engineered Escherichia coli

作　　者：田康明[1] 石贵阳[2] 路福平[3] Suren Singh 王正祥[3] 

机构地区：[1]江南大学生物工程学院,江苏无锡214122 [2]江南大学粮食发酵工艺与技术国家工程实验室,江苏无锡214122 [3]天津科技大学生物工程学院,天津3004574 [4]德班理工大学生物工程与食品技术学院应用科学系

出　　处：《生物工程学报》2013年第9期1268-1277,共10页Chinese Journal of Biotechnology

基　　金：国际科技合作项目(No.CS06-L11)资助~~

摘　　要：以甘油为碳源高效合成L-乳酸有助于推进油脂水解产业和生物可降解材料制造业的共同发展。为此,首先分别从凝结芽胞杆菌Bacillus coagulans CICIM B1821和大肠杆菌Escherichia coli CICIM B0013中克隆了L-乳酸脱氢酶基因BcoaLDH和D-乳酸脱氢酶(LdhA)的启动子片段PldhA。将两条DNA片段连接组成了表达盒PldhA-BcoaLDH。然后将上述表达盒通过同源重组删除FMN为辅酶的L-乳酸脱氢酶编码基因lldD的同时克隆入ldhA基因缺失菌株E.coli CICIM B0013-080C(ack-pta pps pflB dld poxB adhE frdA ldhA)的染色体上,获得了L-乳酸高产菌株E.coli CICIM B0013-090B(B0013-080C,lldD::PldhA-BcoaLDH)。考察了菌株CICIM B0013-090B不同培养温度下代谢利用甘油和合成L-乳酸的特征后,建立并优化了一种新型L-乳酸变温发酵工艺。在7 L发酵罐上,发酵27 h,积累L-乳酸132.4 g/L,产酸强度4.90 g/(L·h),甘油到L-乳酸的得率为93.7%,L-乳酸的光学纯度达到99.95%。High-efficient conversion of glycerol to L-lactate is beneficial for the development of both oil hydrolysis industry and biodegradable materials manufacturing industry. In order to construct an L-lactate producer, we first cloned a coding region of gene BcoaLDH encoding an L-lactate dehydrogenase from Bacillus coagulans CICIM B1821 and the promoter sequence （PtdhA） of the D-lactate dehydrogenase （LdhA） from Escherichia coli CICIM B0013. Then we assembled these two DNA fragments in vitro and yielded an expression cassette, Pttha-BcoaLDH. Then, the cassette was chromosomally integrated into an ldhA mutant strain, Escherichia coli CICIM B0013-080C, by replacing lldD encoding an FMN-dependent L-lactate dehydrogenase. An L-lactate higher-producer strain, designated as E. coli B0013-090B, possessing genotype of lldD：：Ptdh.4-BcoaLDH, Aack-pta Apps ApflB Adld ApoxB AadhE AfrdA and AldhA, was generated. Under the optimal condition, 132.4 g/L L-lactate was accumulated by B0013-090B with the lactate productivity of 4.90 g/L＇h and the yield of 93.7% in 27 h from glycerol. The optical purity of L-lactate in broth is above 99.95%.

关 键 词：L-乳酸 甘油 L-乳酸脱氢酶 途径工程 大肠杆菌 

分 类 号：TQ921.3[轻工技术与工程—发酵工程]