大肠杆菌合成4-羟基异亮氨酸的代谢工程研究  

Metabolic engineering of Escherichia coli for synthesis of 4-hydroxyisoleucine

作　　者：王浚哲 闫倩玉 徐皓然 孟燕 张成林[1] WANG Junzhe;YAN Qianyu;XU Haoran;MENG Yan;ZHANG Chenglin(College of Biotechnology,Tianjin University of Science and Technology,Tianjin 300457,China)

机构地区：[1]天津科技大学生物工程学院,天津300457

出　　处：《中国酿造》2023年第4期46-52,共7页China Brewing

基　　金：国家自然科学基金项目(32170049);国家重点研发计划(2021YFC210800);大学生创新创业计划项目(202110057132,202110057314)。

摘　　要：为进一步提高大肠杆菌(Escherichia coli)合成4-羟基异亮氨酸的性能,以前期构建的重组菌株Escherichia coli IEOH-9为出发菌株进行代谢工程改造。首先,过表达解除反馈抑制作用的天冬氨酸激酶编码基因lysCC1055T以强化L-异亮氨酸代谢通量;然后,过表达吡啶核苷酸转氢酶编码基因pntAB以提高胞内烟酰胺腺嘌呤二核苷酸磷酸(NADPH)水平;在此基础上,分别敲除丙酮酸激酶编码基因pykF和pykA以增强磷酸烯醇式丙酮酸合成草酰乙酸的代谢流;最后过表达柠檬酸合酶编码基因glt A以提高前体物α-酮戊二酸的供应。结果表明,基因lysCC1055T、pnt AB过表达菌株E. coli IEOH-10、E. coli IEOH-11的4-羟基异亮氨酸产量分别达到17.3 g/L、19.4 g/L;敲除基因pykF的效果优于pykA,基因pykF敲除菌株Escherichia coli IEOH-13的4-羟基异亮氨酸产量为21.5 g/L;过表达基因glt A菌株IEOH-14的4-羟基异亮氨酸产量增加至24.7 g/L,较出发菌株IEOH-9提高57.3%,生物量无明显差异(P>0.05)。To further improve the performance of Escherichia coli for 4-hydroxyisoleucine production,the recombinant strain E.coli IEOH-9 constructed earlier was modified by metabolic engineering.Firstly,the aspartate kinase encoding gene lysCC1055T was overexpressed to remove feedback inhibition and enhance the metabolic flow for L-isoleucine synthesis.Then,the pyridine nucleotide transhydrogenase encoding gene pntAB was overexpressed to improve the intracellular nicotinamide adenine dinucleotide phosphate hydride(NADPH)level.On this basis,the pyruvate kinase encoding gene pykF and pykA was knocked out respectively,to enhance the metabolic flow of oxaloacetate synthesis from phosphoenolpyruvic acid.Finally,the citrate synthase encoding gene gltA was overexpressed to elevate the supply ofα-ketobutyrate.The results showed that,the 4-hydroxyisoleucine yield of the E.coli IEOH-10 overexpressed gene lysCC1055T and E.coli IEOH-11 overexpressed gene pntAB reached 17.3 g/L and 19.4 g/L,respectively.The effect of knocking out gene pykF was better than that of knocking out gene pykA,and the 4-hydroxyisoleucine yield of the strain E.coli IEOH-13 knocked out gene pykF was 21.5 g/L.The 4-hydroxyisoleucine yield of the strain E.coli IEOH-14 overexpressed gene gltA was increased to 24.7 g/L,which was 57.3%higher than that of E.coli IEOH-9,and their biomass were not significantly different(P>0.05).

关 键 词：大肠杆菌 4-羟基异亮氨酸 基因敲除 过表达 L-异亮氨酸羟化酶 L-异亮氨酸 Α-酮戊二酸 

分 类 号：TQ922[轻工技术与工程—发酵工程]