摘要
谷氨酸棒杆菌(Corynebacterium glutamicum)由于其无内毒素以及有完善的分泌系统可用于分泌生产重组医药蛋白,被认为是很有潜力的外源蛋白表达宿主。外源蛋白分泌表达涉及蛋白合成、运输以及分泌等多个耗能过程,能量代谢的扰动引起宿主细胞整体代谢水平的变化。该研究以C.glutamicum CGMCC1.15647菌株为研究对象,利用非靶向代谢组学比较分泌表达外源蛋白对菌株自身代谢的影响,发现在分泌表达外源蛋白组中共有176个差异代谢物,其中79个代谢物显著上调表达,97个代谢物显著下调表达,关键代谢物为D-甘露糖-6-磷酸、叶酸、肌苷、L-色氨酸、L-苯丙氨酸等。差异代谢物通路分析表示,分泌表达外源蛋白会导致中心代谢加剧和多种氨基酸特别是芳香族氨基酸代谢的变化,以满足蛋白合成和分泌对能量的需求,同时保护胞内蛋白以维持菌体正常生长。该文从代谢水平上探究C.glutamicum CGMCC1.15647分泌外源蛋白过程中的关键代谢物及代谢调控网络,对于优化C.glutamicum细胞中代谢流使之趋向于有利于蛋白分泌表达有重要的研究意义。
Corynebacterium glutamicum is considered as a promising host for protein expression due to its absence of endotoxin and its well-established secretion system for the production of recombinant pharmaceutical proteins.The protein secretory expression involves multiple energy-consuming processes such as translation,transport and secretion and so on,causing genome-wide metabolic burden to host.In this study,the variable domain of heavy chain of heavy chain antibody(VHH)as a model protein was expressed in C.glutamicum CGMCC1.15647 strain for investigating the influence mechanism of secreted expression of exogenous proteins using non-targeted metabolomics.The metabolic analysis revealed that compared to the control(G1),97 metabolites were significantly(VIP>1,P<0.05)up-regulated and 79 metabolites were significantly down-regulated in the group(G2)with secretory expression of VHH.Analysis of the KEGG metabolic pathway detected that the differentially expressed metabolites were mainly related to amino acid metabolism,nucleotide metabolism and carbon metabolism.The key metabolites were D-mannose-6-phosphate,folic acid,inosine,L-tryptophan,and L-phenylalanine so on,and D-mannose-6-phosphate was the most significantly changed metabolite with a decrease of more than 76%.The secretion and expression of exogenous protein VHH required large amounts of energy while attracting amino acids to the synthesis pathway,as evidenced by the intensification of central metabolism to meet the bi-directional requirements of production and growth,and the significant down-regulation of amino acid-related metabolites in pyrimidine metabolism due to the massive flow of amino acids toward protein synthesis.By regulating the TCA cycle,nucleotide metabolism and amino acid metabolism in vivo to increase energy supply in response to the cellular stress caused by exogenous protein synthesis and secretion.Interestingly,it was found that under VHH expression conditions,the aromatic amino acid content was greatly altered,thus affecting the synthesis of cell membranes and cell walls to facilitate substance absorption and protein secretion.Overall,we investigated the key metabolites and metabolic regulatory network in the process of protein secretory expression in C.glutamicum CGMCC1.15647 at the metabolic level,which is important for optimizing the metabolic flow in C.glutamicum cells in favor of protein secretion and expression.
作者
黄丹妮
邹宇
刘秀霞
杨艳坤
白仲虎
HUANG Danni;ZOU Yu;LIU Xiuxia;YANG Yankun;BAI Zhonghu(National Engineering Research Center of Cereal Fermentation and Food Biomanufacturing,Jiangnan University,Wuxi 214122,China;Key Laboratory of Industrial Biotechnology,Ministry of Education,Jiangnan University,Wuxi 214122,China;School of Biotechnology,Jiangnan University,Wuxi 214122,China)
出处
《食品与发酵工业》
CAS
CSCD
北大核心
2023年第20期1-8,I0001,I0002,共10页
Food and Fermentation Industries
基金
国家自然科学基金(21878124,22078128)。
关键词
谷氨酸棒杆菌
分泌表达
代谢组学
表达宿主
外源蛋白
Corynebacterium glutamicum
secretory expression
metabolomics
expression host
exogenous protein
