产L-异亮氨酸谷氨酸棒杆菌的代谢工程构建  

Construction of Metabolic Engineering of Corynebacterium glutamicum for Producing L-Isoleucine

作　　者：张佳蓉 李彦臻 芦楠 张成林[1] ZHANG Jiarong;LI Yanzhen;LU Nan;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：《天津科技大学学报》2025年第2期13-21,共9页Journal of Tianjin University of Science & Technology

基　　金：国家自然科学基金项目(31770053);天津市研究生科研创新项目(2022BKYZ006,2022BKYZ018)。

摘　　要：L-异亮氨酸作为必需氨基酸具有重要的生理功能,被广泛应用于食品、医药、农业等领域。针对目前发酵工业中L-异亮氨酸生产菌株谷氨酸棒杆菌(Corynebacterium glutamicum)存在营养缺陷、发酵周期长等不足,利用代谢工程策略从头构建L-异亮氨酸合成菌株。首先,过表达解除反馈抑制的天冬氨酸激酶、苏氨酸脱水酶以及乙酰乳酸合酶编码基因lysC^(A279T)、ilvA^(G418A/T1147G)和ilvBN^(A139T/T140A)以强化L-异亮氨酸合成通路,所获菌株ILE-3的L-异亮氨酸产量达到3.5 g/L。然后,过表达丙酮酸羧化酶编码基因pyc以增强前体物L-天冬氨酸的供应。在此基础上过表达L-异亮氨酸输出蛋白编码基因brnFE以促进其外排,获得菌株ILE-6。该菌株在5 L发酵罐经发酵48 h后,ILE-6的L-异亮氨酸产量和转化率分别达到21.3 g/L和14.1%。该菌株遗传背景清晰、无营养缺陷、发酵周期短,其所用策略可为分支链氨基酸生产菌株的代谢工程构建提供参考。L-isoleucine,as an essential amino acid,possesses distinctive physiological functions and has been widely used in food,pharmaceutical and agricultural industry.However,the current Corynebacterium glutamicum strains used for scaledup production of L-isoleucine exhibit auxotrophy and the fermentation period is relatively long.In this study,an L-isoleucine producing strain was developed with the use of metabolic engineering strategies.Initially,the feedback resistant aspartate kinase,threonine dehydratase and acetolactate synthase encoding genes lysC^(A279T),ilvA^(G418A/T1147G) and ilvBN^(A139T/T140A) were overexpressed to enhance the L-isoleucine synthetic pathway.As a result,3.5 g/L of L-isoleucine was produced by the generated strain ILE-3.Then,the pyruvate carboxylase encoding gene pyc was overexpressed to improve L-aspartate supply.Fi-nally,brnFE was overexpressed to promote the efflux of L-isoleucine.After fermentation for 48 h in a 5L fermentor,the Lisoleucine production titer and yield achieved 21.3 g/L and 14.1%by the final strain ILE-6.The strain ILE-6 possesses clear genetic background,non-auxotroph and short fermentation period.The strategies applied in the study thus could provide reference for metabolic engineering strains for producing branched chain amino acids.

关 键 词：L-异亮氨酸 L-苏氨酸 代谢工程 谷氨酸棒杆菌 代谢流 

分 类 号：Q815[生物学—生物工程]