Analysis On The Stress Factors Of Heterologous Protein Expression In Corynebacterium Glutamicum Based On Transcriptomics And Proteomics

Corynebacterium glutamicum（C.glutamicum）was an important industrial microorganism,which was widely used in the production of various biochemical products.Because of its simple culture conditions and had no endotoxin,C.glutamicum had great advantages in the production of pharmaceutical proteins.But there are still problems such as low expression efficiency of heterologous proteins and less expression vectors,leading to the limited application of C.glutamicum protein expression system.In order to explore the beneficial targets for improving the expression of heterologous proteins in C.glutamicum.In this study,C.glutamicum with high Enhanced Green Fluorescence Protein（EGFP）expression（H2 group）was used as the object to analyze the stress mechanism of high heterologous proteins expression in C.glutamicum by transcriptomics and proteomics techniques.Through GO and KEGG cluster analysis,the effects of protein expression on the transcriptomics and proteomics of C.glutamicum were further analyzed.And they provided a theoretical target for the high expression of heterologous proteins in C.glutamicum.In this paper,the important roles and mechanisms of glc B,put P,Cgl2108 and inf C genes in heterologous protein expression were elucidated by functional analysis and gene overexpression.Finally,intracellular soluble glycoprotein gD of porcine pseudorabies virus（gD）and secreted protein variable domain of heavy chain of heavy chain antibody（VHH）were expressed in the optimized strains to verify the universality of heterologous protein expression.The following are specific research results:（1）Transcriptomics analysis of C.glutamicum under heterologous protein expression stress.In order to explore the stress factors of heterologous protein expression in C.glutamicum and screen out the beneficial targets for the expression of recombinant proteins,the engineering strain（H2 group）with high expression of EGFP and the control strain（H1 group）were selected for parallel culture at the shake flask level,and the bacterial cells cultured for 20 h were subjected to the omics analysis.By comparing the transcriptomics changes of H2 vs H1,the differentially expressed genes（DEGs）were screened under the condition of|log₂（Fold Change）|≥1 and p-value<0.05.A total of 160 DEGs were screened out in the H2 vs H1 comparison group,of which 132 were up-regulated and 28 were down-regulated.The expression levels of10 DEGs were detected by q PCR random screening,which were basically consistent with the results of RNA-Seq.The transcriptomics level showed that the DEG were mainly enriched in TCA cycle,amino acid transport,transcription translation,DNA repair and other pathways.（2）Proteomics analysis of C.glutamicum under heterologous protein expression stress.Fold Change≥1.2 or≤0.83,and p-value<0.05 were used as the screening criteria to obtain differentially expressed proteins（DEPs）.A total of 226 DEPs were screened out in the H2group vs H1 group,of which 142 were up-regulated and 84 were down-regulated.In order to ensure that cells have sufficient energy for life activities when expressing heterologous proteins,proteomics data show that cell metabolism has migrated:the expression level of malate synthase（encoded by glc B）was up-regulated,and the glyoxylate pathway was enhanced;in terms of transport,the expression level of proline transporter（encoded by put P）was up-regulated.Proteomics showed that DEPs were mainly enriched in energy metabolism,amino acid transport and metabolism,and DNA repair-related functions.Through the combined analysis of transcriptomics,proteomics and their interaction network,12 up-regulated DEPs were screened,and these differentially overexpressed strains were constructed.Through the detection of EGFP fluorescence intensity,it was found that the protein expression levels of over-expressed strains Cg-p EC-XK99E-glc B,Cg-p EC-XK99E-put P,Cg-p EC-XK99E-Cgl2108 and Cg-p EC-XK99E-inf C had increased by 44%,19%,28%and 42%,respectively.（3）Construction of gD expression system and preliminary functional research on the stress-related genes of heterologous protein expression.The soluble expression of gD was performed in C.glutamicum,and the expression level of gD in C.glutamicum was increased by optimizing the promoter,chassis strain and fermentation conditions.The expression products were analyzed and identified by SDS-PAGE and Western Blot,and the protein products were purified by Ni⁺affinity chromatography column.The heterologous protein expression levels of the DEG overexpression strains were verified by intracellular protein gD and secreted protein VHH,and the expression level of gD-p EC-XK99E-glc B had increased by23%,while that of other overexpression strains was not significantly increased.The expression levels of VHH-p EC-XK99E-glc B,VHH-p EC-XK99E-put P,VHH-p EC-XK99E-Cgl2108 and VHH-p EC-XK99E-inf C had increased by 56%,23%,34%and 17%,respectively.