外源蛋白表达过程中谷氨酸棒状杆菌转录组及代谢物的多变元分析

摘要

The engineering strain expressing EGFP (C.glutamicum EGFP) and wild type strain (C.glutamicum BZH001) were cultivated in parallel in 5 L fermenters under 30％ dissolved oxygen concentration.Samplings at 20 h were analyzed through RNA-sequencing (RNA-seq).Extracellular and intracellular metabolites and key enzymes' activity of both strains were investigated.Multivariate data analysis (MVDA) was used to deal with the RPKM data of RNA-seq and metabolism data.The principal component analysis (PCA) was used to analyze the principal component.And the S-plot of OPLS-DA was used to find the biomarkers of C.glutamicum BZH001 and C.glutamicum EGFP.Eight critical genes were found,and ATP,glutamic acid,acetate,intracellular glutamic acid,methionine and lactate were identified as the critical metabolites.The metabolism-like anaerobic conditions were as followed:the glycolysis was enhanced,the TCA cycle was shunted,and the contents of acetate and lactate increased.This research laid a foundation for optimizing C.glutamicum host structure to get high protein expression level.%以谷氨酸棒状杆菌为研究对象,在发酵罐水平30％溶解氧条件下对野生菌及表达EGFP的工程菌进行平行培养,对培养20 h的菌体进行转录组分析,并对不同时间点所取样品进行代谢物检测.利用多变元分析方法分析转录组和代谢物数据,其中主要利用主成分分析法进行关键因素分析,利用正交偏最小二乘法判别分析进行转录组数据分析,继而通过S-plot得到不同溶解氧下的生物标记物(biomarkers),最终找到了8个关键基因.代谢数据分析结果表明ATP、谷氨酸、乙酸、胞内谷氨酸、甲硫氨酸及乳酸是外源蛋白表达的关键代谢物;通过不同时间点代谢物含量变化分析发现外源蛋白表达的代谢变化类似于低氧环境下的代谢变化现象,即糖酵解增强,TCA溢流,回补途径增加,乙酸、乳酸生成增加.通过对比外源蛋白表达工程菌与野生菌的转录组数据及代谢物含量数据,为今后在代谢工程中菌株改造、优化代谢获得高效表达外源蛋白的谷氨酸棒状杆菌表达宿主奠定基础.