动态调控谷氨酸棒状杆菌合成4-羟基异亮氨酸  

Dynamic regulation of Corynebacterium glutamicum for biosynthesis of 4-hydroxyisoleucine

作　　者：来文梅 谭书煜 史锋[1,2] LAI Wenmei;TAN Shuyu;SHI Feng(State Key Laboratory of Food Science and Technology,Jiangnan University,Wuxi 214122,China;Key Laboratory of Industrial Biotechnology,Ministry of Education,Jiangnan University,Wuxi 214122,China)

机构地区：[1]食品科学与技术国家重点实验室(江南大学),江苏无锡214122 [2]工业生物技术教育部重点实验室(江南大学),江苏无锡214122

出　　处：《食品与发酵工业》2022年第17期42-48,共7页Food and Fermentation Industries

基　　金：江南大学食品与科学国家重点实验室自主研究课题项目(SKLF-ZZA-201904)。

摘　　要：4-羟基异亮氨酸(4-hydroxyisoleucine,4-HIL)在治疗Ⅱ型糖尿病方面极具潜力。异亮氨酸双加氧酶(isoleucine dioxygenase,IDO)能够将L-异亮氨酸(L-isoleucine,Ile)转变成4-HIL。为提高重组谷氨酸棒杆菌的4-HIL产量同时降低副产物L-赖氨酸(L-lysine,Lys)的合成,首先将Lys-OFF核糖体开关整合到Lys合成途径的关键基因dapA序列上游,得到D-RS菌株。该菌株能够根据胞内外Lys的浓度,动态弱化Lys合成,使Lys的含量降低了46.7%。其次,在该菌株中再利用Ile激活型传感器Lrp-P_(brnFE)N去控制密码子优化后的ido基因的表达,使4-HIL的产量提高至116.3 mmol/L。最后,为了进一步提高4-HIL产量,利用强启动子P_(brnFE 7)动态控制odhI和vgb的表达,增强α-酮戊二酸和O_(2)的供应。最终得到了1株摇瓶发酵4-HIL产量高达166.0 mmol/L,Lys含量降至6.5 mmol/L的重组菌株D-RS-_(0)I_(7)O_(7)V。研究所采用的动态调控策略为4-HIL的高效合成提供了一种新思路。4-Hydroxyisoleucine(4-HIL)has great potential in the treatment of type II diabetes.Isoleucine dioxygenase(IDO)can convert L-isoleucine(Ile)into 4-HIL.To improve the product of 4-HIL and reduce the by-product L-lysine(Lys)synthesis in recombinant Corynebacterium glutamicum,the Lys-OFF riboswitch was integrated into the upstream of dapA gene,the key gene of Lys synthesis pathway,and the D-RS strain was obtained.The strain could dynamically minimize the synthesis to Lys by the concentration of intracellular and extracellular Lys,and decreased the content of Lys by 46.7%.Secondly,the Ile-activated biosensor Lrp-P_(brnFE) N was used to control the expression of codon-optimized ido in D-RS strain,and the titer of 4-HIL was increased to 116.3 mmol/L.In order to further improve the titer of 4-HIL,the strong promoter P_(brnFE)7 was used to dynamically control the expression of odhI and vgb to enhance the supply ofα-ketoglutarate and oxygen.Finally,a recombinant strain D-RS-_(0)I_(7)O_(7)V with the 4-HIL titer up to 166.0 mmol/L and Lys content down to 6.5 mmol/L after fermentation in shake flasks was obtained.The dynamic regulation strategy provides a new idea for the efficient biosynthesis of 4-HIL.

关 键 词：4-羟基异亮氨酸 谷氨酸棒杆菌 赖氨酸 Lrp-P_(brnFEN) Lys-OFF核糖体开关 

分 类 号：TQ922[轻工技术与工程—发酵工程]