代谢工程构建谷氨酸棒杆菌合成5-氨基乙酰丙酸  

Metabolic engineering of Corynebacterium glutamicum chassis for biosynthesis of 5-aminolevulinic acid

作　　者：魏敏华 李宇虹 张佳蓉 孟静 孟燕 王浚哲 张成林[1] WEI Minhua;LI Yuhong;ZHANG Jiarong;MENG Jing;MENG Yan;WANG Junzhe;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：《食品与发酵工业》2022年第14期9-15,共7页Food and Fermentation Industries

基　　金：国家自然科学基金面上项目(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：非蛋白氨基酸5-氨基乙酰丙酸是合成四氢吡咯化合物的前体物,被广泛应用于医药、农业和畜牧业。为提升5-氨基乙酰丙酸合成效率,该研究以谷氨酸棒杆菌Corynebacterium glutamicum ATCC13032为出发菌株,首先敲除L-谷氨酸输出蛋白编码基因NCgl1221阻断其分泌,菌株5-氨基乙酰丙酸产量达到0.62 g/L。然后利用脱氧核糖核酸(deoxyribonucleic acid,DNA)脚手架体系组装关键酶谷氨酰-tRNA还原酶和谷氨酸-1-半醛氨基转移酶,当二者比例为2∶1时最有利于5-氨基乙酰丙酸的合成,产量为0.84 g/L;为增强三羧酸(tricarboxylic acid,TCA)循环,分别通过过表达pyc、ppc和pckA强化回补途径,结合过表达gltA强化柠檬酸合成,结果表明以共表达pckA和gltA的效果最佳;在此基础上通过敲除aceA并过表达pntAB以增强α-酮戊二酸和NADPH供应,所获菌株ALA-10的5-氨基乙酰丙酸产量为1.47 g/L。该研究可为提升5-氨基乙酰丙酸的发酵合成效率提供参考。As a non-proteinogenic amino acid and precursor for synthesis of various tetrapyrrole compounds,5-aminolevulinic acid has been widely used in medicine,agriculture and husbandry.In this study,Corynebacterium glutamicum ATCC13032 was used as a chassis for efficient 5-aminolevulinic acid biosynthesis.The MscCG protein is the exporter of L-glutamate.To prevent L-glutamate secretion,NCgl1221 was deleted,resulting in a production of 0.62 g/L 5-aminolevulinic acid.The reactions in microorganisms are mediated by simple diffusion and random collisions of metabolites and enzymes,which lowers the local metabolite concentration around the enzymes and results in the inefficiency of metabolic reactions.Furthermore,the cell growth will be inhibited by the accumulation of toxic intermediates.The DNA scaffold system can organize enzymes spatially and temporally and increases the local concentration of intermediates.So,this system was used to assemble glutamyl-tRNA reductase and glutamate-1-semialdehyde aminotransferase.The ratio of the two enzymes was set as 1∶1,1∶2 and 2∶1,respectively.The highest production of 5-aminolevulinic acid(0.84 g/L)was achieved when the ratio of the two enzymes is 2∶1.To enhance TCA cycle forα-ketoglutarate supply,the anaplerotic pathwaywas strengthened by overexpressing pyc,ppc and pckA through individually integrating the genes together with the strong promoter P_(tuf) to genome DNA.The production of 5-aminolevulinic acid by the resulted strains was increased by 14.2%,27.4%and 33.3%,indicating the feasibility of enhancing anaplerotic pathway.Meanwhile,gltA was co-overexpressed with pckA,resulting in 12.5%increase in 5-aminolevulinic acid production(1.26 g/L).As a bypass of TCA cycle,the glyoxylate cycle competes isocitrate withα-ketoglutarate synthesis.Meanwhile,the biosynthesis of 5-aminolevulinic acid requires NADPH.Therefore,a copy transhydrogenase encoding gene pntAB driven P_(tuf) was integrated at aceA locus,aiming at increasing blocking the glyoxylate cycle to improveα-ketoglutarate

关 键 词：谷氨酸棒杆菌 5-氨基乙酰丙酸 DNA脚手架 回补途径 NADPH 

分 类 号：TQ922[轻工技术与工程—发酵工程]