| Corynebacterium glutamicum is widely used in the production of amino acids and organic acids due to its many advantages in industrial production,and has also been developed for the expression of high value-added foreign proteins.Although its entire genome has been sequenced,some key genes that affect protein expression have not yet been studied.In the preliminary research of this project,by analyzing the differences in transcriptome data between wild bacteria and engineered bacteria expressing exogenous proteins,several key genes that may affect the expression of exogenous proteins were unearthed,including NCgl2632,NCgl0909,NCgl1522,NCgl0303,NCgl0457,NCgl0833,NCgl0838,NCgl2261,NCgl2993.NCgl2632 is predicted to encode acetyl-Co A acyltransferase,which affects the intracellular acetyl-Co A metabolic flux;NCgl0909 is predicted to encode the ABC transporter in Corynebacterium glutamicum,transporting specific substrates;NCgl1522 encodes the transmembrane protein subunit of the protein secretion system,affecting the secretion of protein out of the cell.NCgl0303,NCgl0457,NCgl0303,NCgl0457,NCgl0833,NCgl0838,NCgl2261,NCgl2993 are predicted to encode ribosomal proteins in Corynebacterium glutamicum,which affect protein synthesis.Previous studies have only investigated the function of NCgl0909 and its effect on exogenous protein expression,while other genes have not been studied in depth.For the above genes,this thesis investigated the effects of these genes on exogenous protein expression by constructing single knockout and overexpression strains as well as multigene modified strains.Specifically,the following research results are available.（1）The function of the NCgl2632 gene was initially investigated by bioinformatics analysis and activity assay.The effect of overexpression and knockout of the key genes on exogenous protein expression was examined using the enhanced green fluorescence protein（EGFP）and the variable domain of heavy chin of heavy-chin antibody（VHH）as model proteins.It was found that the knockdown genotype of NCgl2632,and the overexpression genotype of NCgl1522 were relatively more favorable for exogenous protein expression.（2）Several different overexpression and knockout of key genes were combined to generate several polygenic modified strains considering three aspects in terms of energy metabolism,material transport,and transmembrane transport.The effect of modification of these strains on growth was investigated.The ability of different polygenic modified strains to express exogenous proteins was further examined using the model proteins EGFP and VHH,respectively.Meanwhile,a promising protein human Mut T homolog 1（hMTH1）was selected to construct an expression plasmid and thus fermentation validation and activity assay were performed,which can be used as a new model protein to verify the ability of strains to produce exogenous proteins in the future.Finally,it was found that among several multigene modified strains,C.g 15647ΔNCgl2632ΔNCgl0909 have the most significant enhancement of exogenous protein expression ability,with 112%higher VHH protein expression 134%higher hMTH1 protein expression and 34.4%higher hMTH1 activity compared to the wild strain.（3）Finally,the optimization of the distribution of the carbon and nitrogen levels of the medium for the optimized high-producing protein strain was carried out,and the most suitable medium ratio for the fermentation of the strain was obtained:glucose concentration of 120g·L-1,corn flour concentration of 5g·L-1,ammonium sulfate concentration of 30g·L-1,and urea concentration of 10g·L-1.In this paper,the Corynebacterium glutamicum host was comprehensively modified by multi-gene co-knockout or overexpression,to obtain a host with elevated exogenous protein expression was obtained.A preliminary attempt was made to validate the expression of hMTH1 in high-yielding strains,and finally the ratio optimization of fermentation medium was carried out,laying the foundation for further in-depth research in the next step. |
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