代谢工程改造大肠杆菌合成L-异亮氨酸的研究

Study on metabolic engineering of Escherichia coli for L-isoleucine production

以L-苏氨酸生产菌Escherichia coli THRD为出发菌株,利用基因重组技术替换ilv LXGMEDA启动子并过表达解除L-异亮氨酸反馈抑制的ilv A和ilv IH,以期获得L-异亮氨酸生产菌。将ilv LXGMEDA启动子替换为强启动子Ptrc并敲除ilv LXGM后获得ILE01菌株,于该菌株中分别过表达解除L-异亮氨酸反馈抑制的ilv IH及共表达解除L-异亮氨酸反馈抑制的ilv A和ilv IH,获得菌株ILE02和ILE03,其L-异亮氨酸产量分别达到1.75 g/L和2.19 g/L。针对ILE03α-酮丁酸积累量过高的问题,通过改变操纵子中ilv A和ilv IH的顺序调节其转录水平,获得菌株ILE04,其L-异亮氨酸产量达2.85 g/L。利用ILE04于5 L发酵罐中进行发酵实验,L-异亮氨酸产量、发酵强度及转化率分别为5.23 g/L、0.17 g/(L·h)及4.6%。

L-threonine-producing strain Escherichia coli THRD was used as starting strain to construct an L-isoleucine producing strain via metabolic engineering. Native promoter of ilvLXGMEDA was replaced with strong promoter Ptrc, resulting in ILE01. In order to relieve feedback inhibition, overexpression of ilvlH and ilvA together with ilvlH in ILE01 resulted in ILE02 and ILE03, of which L-isoleucine production was 1.75 g/L and 2.19 g/L, respectively. To reduce α-keto butyric acid accumulation in ILE03, ilvA and ilvIH transcription level was regulated isoleucine ILE04, in and 4.6% via the change of the genes position in the artificial operon to obtain ILE04 and the L- production of ILE04 was 2.85 g/L. Fermentation was performed in 5-L fermentator with which L-isoleueine production, productivity and conversion were 5.23 g/L, 0.17 g/（L·h） respectively.