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

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％。

Abstract:

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 (PldhA) 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, PldhA-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::PldhA-BcoaLDH, Dack-pta Dpps DpflB Ddld DpoxB DadhE DfrdA and DldhA, 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%.