Tian Kangming, Shi Guiyang, Lu Fuping, Singh Suren, Wang Zhengxiang
School of Biotechnology, Jiangnan University, Wuxi 214122, Jiangsu, China.
National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, Wuxi 214122, Jiangsu, China.
Sheng Wu Gong Cheng Xue Bao. 2013 Sep;29(9):1268-77.
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 (P(ldhA)) 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, P(ldhA)-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::P(ldhA)-BcoaLDH, deltaack-pta deltapps deltapflB deltadld deltapoxB deltaadhE deltafrdA and deltaldhA, 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/Lh and the yield of 93.7% in 27 h from glycerol. The optical purity of L-lactate in broth is above 99.95%.
将甘油高效转化为L-乳酸对油脂水解工业和生物可降解材料制造业的发展都有益。为构建一株L-乳酸生产菌，我们首先从凝结芽孢杆菌CICIM B1821中克隆了编码L-乳酸脱氢酶的基因BcoaLDH的编码区，以及来自大肠杆菌CICIM B0013的D-乳酸脱氢酶（LdhA）的启动子序列（P(ldhA)）。然后我们在体外组装这两个DNA片段，得到一个表达盒P(ldhA)-BcoaLDH。接着，通过替换编码FMN依赖型L-乳酸脱氢酶的lldD，将该表达盒染色体整合到ldhA突变株大肠杆菌CICIM B0013-080C中。由此产生了一株L-乳酸高产菌株，命名为大肠杆菌B0013-090B，其基因型为lldD::P(ldhA)-BcoaLDH、deltaack-pta deltapps deltapflB deltadld deltapoxB deltaadhE deltafrdA和deltaldhA。在最佳条件下，B0013-090B在27小时内从甘油中积累了132.4 g/L的L-乳酸，乳酸生产率为4.90 g/Lh，产率为93.7%。发酵液中L-乳酸的光学纯度高于99.95%。
