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.
[1] CHAI M, DENG C, CHEN Q, et al.Synthetic biology toolkits and metabolic engineering applied in Corynebacterium glutamicum for biomanufacturing[J].ACS Synthetic Biology, 2021, 10(12):3237-3250.
[2] SUN M M, GAO A X, LI A, et al.Bicistronic design as recombinant expression enhancer:Characteristics, applications, and structural optimization[J].Applied Microbiology and Biotechnology, 2021, 105(20):7709-7720.
[3] WANG Y L, GAO X, LIU X X, et al.Construction of a 3A system from BioBrick parts for expression of recombinant hirudin variants Ⅲ in Corynebacterium glutamicum[J].Applied Microbiology and Biotechnology, 2020, 104(19):8257-8266.
[4] MENG L H, GAO X, LIU X X, et al.Enhancement of heterologous protein production in Corynebacterium glutamicum via atmospheric and room temperature plasma mutagenesis and high-throughput screening[J].Journal of Biotechnology, 2021, 339:22-31.
[5] YU X Y, LIU X X, GAO X, et al.Development of a novel platform for recombinant protein production in Corynebacterium glutamicum on ethanol[J].Synthetic and Systems Biotechnology, 2022, 7(2):765-774.
[6] LARSSON C, PÅHLMAN I L, GUSTAFSSON L.The importance of ATP as a regulator of glycolytic flux in Saccharomyces cerevisiae[J].Yeast, 2000, 16(9):797-809.
[7] UNREAN P.Pathway analysis of Pichia pastoris to elucidate methanol metabolism and its regulation for production of recombinant proteins[J].Biotechnology Progress, 2014, 30(1):28-37.
[8] KOEBMANN B J, WESTERHOFF H V, SNOEP J L, et al.The glycolytic flux in Escherichia coli is controlled by the demand for ATP[J].Journal of Bacteriology, 2002, 184(14):3909-3916.
[9] ZOU S P, ZHANG Z, ZHAO K, et al.Metabolic engineering of Escherichia coli for improved D-pantothenic acid biosynthesis by enhancing NADPH availability[J].Biochemical Engineering Journal, 2022, 187:108603.
[10] SUN X, LI Q G, WANG Y, et al.Isoleucyl-tRNA synthetase mutant based whole-cell biosensor for high-throughput selection of isoleucine overproducers[J].Biosensors and Bioelectronics, 2021, 172:112783.
[11] MUYLDERMANS S.Nanobodies:Natural single-domain antibodies[J].Annual Review of Biochemistry, 2013, 82:775-797.
[12] YU G, XU C F, ZHANG D N, et al.MetOrigin:Discriminating the origins of microbial metabolites for integrative analysis of the gut microbiome and metabolome[J].iMeta, 2022, 1(1):e10.
[13] XIE S S, TAN Y H, SONG W X, et al.N-glycosylation of a cargo protein C-terminal domain recognized by the type IX secretion system in Cytophaga hutchinsonii affects protein secretion and localization[J].Applied and Environmental Microbiology, 2022, 88(1):e01606-21.
[14] STACK T M M, GERLT J A.Discovery of novel pathways for carbohydrate metabolism[J].Current Opinion in Chemical Biology, 2021, 61:63-70.
[15] ZAHOOR A, LINDNER S N, WENDISCH V F.Metabolic engineering of Corynebacterium glutamicum aimed at alternative carbon sources and new products[J].Computational and Structural Biotechnology Journal, 2012, 3:e201210004.
[16] WU X Y, GUO X Y, ZHANG B, et al.Recent advances of L-ornithine biosynthesis in metabolically engineered Corynebacterium glutamicum[J].Frontiers in Bioengineering and Biotechnology, 2020, 7:440.
[17] STÄBLER N, OIKAWA T, BOTT M, et al.Corynebacterium glutamicum as a host for synthesis and export of D-amino acids[J].Journal of Bacteriology, 2011, 193(7):1702-1709.
[18] LIU Y, LI X Y, LU S H, et al.Comparative metabolic responses induced by pyridine and imidazole in Blakeslea trispora[J].Frontiers in Bioengineering and Biotechnology, 2019, 7:347.
[19] DING M Z, CHENG J S, XIAO W H, et al.Comparative metabolomic analysis on industrial continuous and batch ethanol fermentation processes by GC-TOF-MS[J].Metabolomics, 2009, 5(2):229-238.
[20] LV L X, YAN R, SHI H Y, et al.Integrated transcriptomic and proteomic analysis of the bile stress response in probiotic Lactobacillus salivarius LI01[J].Journal of Proteomics, 2017, 150:216-229.
[21] CHEN Y N, CHI W C, TRINH N N, et al.Transcriptome profiling and physiological studies reveal a major role for aromatic amino acids in mercury stress tolerance in rice seedlings[J].PLoS One, 2014, 9(5):e95163.
[22] WOO H M, NOACK S, SEIBOLD G M, et al.Link between phosphate starvation and glycogen metabolism in Corynebacterium glutamicum, revealed by metabolomics[J].Applied and Environmental Microbiology, 2010, 76(20):6910-6919.
[23] SUMAN E, D′SOUZA S, JACOB P, et al.Anti-biofilm and anti-adherence activity of Glm-U inhibitors[J].Indian Journal of Medical Sciences, 2011, 65(9):387-392.
