基于基因转录和代谢物分析的异亮氨酸生产菌谷氨酸棒状杆菌YILW的理性改造（英文）

Rational Design of Corynebacterium glutamicum YILW for Isoleucine Production Based on Gene Transcription and Metabolite Analysis

为获得异亮氨酸生产菌谷氨酸棒状杆菌(Corynebacterium glutamicum)YILW的理性改造策略,考察该菌株与出发菌株C.glutamicum ATCC 13032异亮氨酸合成途径中关键酶及代谢产物的差异。结果表明,C.glutamicum YILW丙酮酸羧化酶编码基因pyc的下调表达使得其胞内草酰乙酸含量降低,过表达该基因显著增加胞内草酰乙酸含量及异亮氨酸产量(分别从1.32μmol/g(细胞干质量,下同)和5.18 g/L提高至3.32μmol/g和5.81 g/L),但副产物赖氨酸及胞内2-酮丁酸积累量提高。针对该问题采用强启动子替换手段过表达ilv BNC操纵子,使得其异亮氨酸产量提高至6.63 g/L。为进一步增加异亮氨酸合成,过表达输出载体编码基因brn E和brn F,其产量提高至7.31 g/L,较出发菌株C.glutamicum YILW提高41.1%,转化率提高40.0%。由此可见,在基因转录及代谢物分析结果指导下理性过表达pyc、ilv BNC操纵子及brn E和brn F能够显著提高异亮氨酸产量并降低副产物浓度。

This study aimed to rationally identify new targets for improving isoleucine production.The transcription levelsof the key genes and intermediate metabolite levels involved in the isoleucine synthesis pathway of Corynebacteriumglutamicum ATCC13032and C.glutamicum YILW,a isoleucine-producing strain derived from the parental strain ATCC13032,were compared.The gene pyc was down-regulated,which might consequently lead to reduced supply of oxaloacetate.Then pyc was overexpressed in C.glutamicum YILW(denoted as YILW-1),resulting in increased oxaloacetate concentrationand isoleucine production(from1.32to3.32μmol/g(md)and from5.18to5.81g/L)but higher accumulation of lysine andintracellular2-ketobutyrate as byproduct.The ilvBNC operon was further overexpressed in YILW-1(denoted as YILW-2),resulting in production of up to6.63g/L isoleucine.To enhance exportation and consequently further increase the productionof isoleucine,the isoleucine exporter genes brnE and brnF was overexpressed in YILW-2(denoted as YILW-3),leadingto increased production of isoleucine(7.31g/L)by10.3%as compared to that of YILW-2.The strategy resulted in41.1%higher isoleucine production(from5.18to7.31g/L)and40.0%higher yield(from0.10to0.14g/g glucose)together withlower by-product lelvels by YILW-3as compared to C.glutamicum YILW.It could be concluded that overexpression of thepyc,ilvBNC operon as well as brnE and brnF based on transcription and metabolite pool analysis could significantly elevateisoleucine production and decrease by-product concentration levels.