NADPH缺陷型Corynebacterium glutamicum重组菌株的构建及其性能分析  

Construction and performance analysis of NADPH-auxotrophic Corynebacterium glutamicum recombinant

作　　者：阮浩哲 刘立明[1,2] 张伟国[1] 徐建中[1] Haozhe Ruan;Liming Liu;Weiguo Zhang;Jianzhong Xu(The Key Laboratory of Industrial Biotechnology of Ministry of Education,School of Biotechnology,Jiangnan University,Wuxi 214122,Jiangsu Province,China;State Key Laboratory of Food Science and Technology,Jiangnan University,Wuxi 214122,Jiangsu Province,China)

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

出　　处：《微生物学报》2021年第8期2442-2456,共15页Acta Microbiologica Sinica

基　　金：国家自然科学基金(31601459);国家双一流轻工业技术与工程一级学科计划(LITE2018-08)。

摘　　要：【目的】改造谷氨酸棒杆菌(Corynebacterium glutamicum)中NADPH合成途径,阻断胞内NADPH的合成,获得1株NADPH营养缺陷型菌株。【方法】通过失活L-赖氨酸高产菌C.glutamicum Lys-χ中葡萄糖-6-磷酸脱氢酶(Zwf)和苹果酸酶(MalE)并将NADP^(+)依赖型异柠檬酸脱氢酶(NADP^(+)-Icdcg)替换成变形链球菌(Streptococcus mutans)中的NAD^(+)-Icdsm,阻断胞内NADPH的合成。随后结合辅因子工程,引入大肠杆菌(Escherichia coli)中膜结合吡啶核苷酸转氢酶(PntAB)并通过不同强度启动子控制PntAB的表达水平。最后,分析不同重组菌中胞内氧化还原水平和L-赖氨酸生产强度的变化。【结果】重组菌C.glutamicum Lys-χΔZMICg::ISm(即Lys-x1)胞内检测不到NADPH,为1株NADPH营养缺陷型菌株。该重组菌只在以葡萄糖酸为碳源的基础培养基中生长和积累L-赖氨酸,而以葡萄糖、丙酮酸、α-酮戊二酸和草酰乙酸为碳源时无法生长。此外,表达E.coli中的PntAB可回补重组菌Lys-χ1胞内NADPH的水平,但由于不同强度启动子控制PntAB表达水平不同,重组菌胞内NADPH水平也不同,并影响L-赖氨酸的生产强度。【结论】重组菌Lys-χ1可作为有效的底盘细胞,用于考察不同的NADPH再生策略,获得不同胞内NADPH水平的重组菌株,为进一步阐明NADPH调控微生物细胞生理代谢功能的机制提供研究基础。[Objective]The biosynthetic pathway of NADPH in Corynebacterium glutamicum was modified to block NADPH production,thus constructing an NADPH-auxotrophic C.glutamicum recombinant.[Methods]To block NADPH production in cell,we firstly inactivated the glucose-6-phosphate dehydrogenase(Zwf)and malic enzyme(MalE)in an L-lysine high-producing strain C.glutamicum Lys-x,and replaced the native NADP^(+)-dependent isocitrate dehydrogenase(NADP^(+)-IcdCg)with NAD^(+)-IcdSm from Streptococcus mutans.Then,we introduced the proton-pumping nicotinamide nucleotide transhydrogenase(PntAB)from Escherichia coli with different expression level controlled by different strength promoters into NADPH-auxotrophic C.glutamicum recombinant.Lastly,we analyzed the changes of intracellular redox level and production intensity of L-lysine in the recombinant strains with different PntAB expression levels.[Results]There was no detectable NADPH in the recombinant strain C.glutamicum Lys-χΔZMICg::ISm(i.e.,Lys-χ1),indicating that strain Lys-χ1 was an NADPH-auxotrophic C.glutamicum.Strain Lys-χ1 grew well and accumulated L-lysine in the basic medium with gluconolactone as carbone source,whereas it could not grow with glucose,pyruvate,α-ketoglutaric acid and oxalacetic acid as carbon source.In addition,overexpression of PntAB in strain Lys-χ1 replenished the intracellular NADPH level,but the NADPH level in different recombinant strains was different because there was the different expression level of PntAB under the different intensity of promoters.And these affected the cell growth and L-lysine production.[Conclusion]The strain Lys-χ1 could be used as a chassis cell for the capacity of strategies to regenerate NADPH in vivo,thus obtaining recombinant strains with different intracellular NADPH levels.Therefoere,this study provided a basis for investigating the regulatory mechanism of NADPH on physiology and metabolism of microbial cells.

关 键 词：谷氨酸棒杆菌 NADPH缺陷体 NADPH再生 L-赖氨酸合成 氧化还原水平 启动子工程 

分 类 号：Q78[生物学—分子生物学]