Development Of A CRISPR/Cas9 Genome Editing System In Corynebacterium Glutamicum And Genetic Modification Of The Genome For Recombinant Protein Expression

Corynebacterium glutamicum(C.glutamicum)is a gram-positive,non-sporulating,and non-pathogenic bacterium.Due to its excellent cultural characteristics,it is one of the most used bacterium in industrial fermentation,has been used for production of amion acid,nucleic acids,and organic acids for more than 50 years.Furthermore,C.glutamicum is demonstrated as an important host for the production of various heterologous proteins including industrial enzymes and pharmaceutical proteins.However,C.glutamicum has some intrinsic disadvantages,such as a lower levels of protein expression and few gene editing tools being available.Therefore,in this study,a CRISPR/Cas9 system was develeped for rapid and efficient genome editing in C.glutamicum.In addition,the clpC,porB,and mepA genes were deleted using this CRISPR/Cas9 system,and ?clpC?porB?mepA was constructed as a potentially suitable host for the production of recombinant proteins.N-terminal pro-brain natriuretic peptide(NT-proBNP),which is an important protein used for the diagnosis of preclinical and mild heart failure was attempted to express in C.glutamicum.Finally,the mechanism of why deletions of the mepA and porB genes are able to enhance protein expression was studied.The major conclusions are as follows:(1)Construction of the CRISPR/Cas9 system for gene editing in C.glutamicum.The recent development of the CRISPR/Cas9 system provides a simple,sequencespecific platform for genome engineering.The system has been widely applied in both prokaryotes and eukaryotes.In this research,a two-plasmid CRISPR/Cas9 system and an all-in-one CRISPR/Cas9 system were constructed in C.glutamicum for rapid and efficient genome editing,including gene deletion and insertion.The efficiency of NHEJ in C.glutamicum is much lower than that in eukaryotic organisms,a repair arm was added to to assess editing efficiency by fixing the DSB through homolog-directed repair.The strong Ptac promoter was choosen to drive the expression of Cas9,which is IPTGnducible.The SD sequence in front of the Cas9 gene ATG was indispensable for Cas9 protein expression.Compared with published genome modification methods,methods based on the CRISPR/Cas9 system can rapidly and efficiently achieve genome editing.Our research provides a powerful tool for facilitating the study of gene function,metabolic pathways,and enhanced productivity in C.glutamicum.(2)Genetic engineering of C.glutamicum for production of heterologous protein.Omics analysis revealed that the porB and mepA genes undergo substantial changes under different dissolved oxygen conditions(0%,30% and 50%).Further investigation demonstrated that the deletion of each of these genes was helpful for enhancing the GFP expression level in C.glutamicum.ClpC is potential targets for the modification of protease expression in C.glutamicum to enhance its production of recombinant protein.Therefore,it was speculated that deleting these three genes might be a viable strategy for host modification to enhance the recombinant protein expression ability of C.glutamicum.In this study,the CRISPR/Cas9 system was used to edit the C.glutamicum genome and to generate clpC,porB,and mepA deletion mutants.Compared with the wild-type C.glutamicum,the ?clpC?porB?mepA mutant exhibited a 2-fold increase in the GFP expression and a 2.5-fold increase in the scFv expression.The method used here for the modification of the C.glutamicum host could also be generally applicable to other microorganisms.(3)Expression of B-type natriuretic peptide expression in C.glutamicum.For researching the expression model of NT-proBNP in C.glutamicum,a fast protein expression system was constructed using GFP as a model protein.The results showed constitutive intracellular patterns were the best way for NT-proBNP expression compared with inducible intracellular,constitutive secretory and inducible secretory patterns.Furthermore,after evaluation of various plasmids and hosts,NT-proBNP was successfully expressed in ?clpC?porB?mepA,with 118.8 mg/g DCW of soluble protein achieved,to the best of our knowledge,this is the first report on the production of NT-proBNP in C.glutamicum.This will greatly facilitate the application of this important platform organism.(4)Mechanism of deletions of mepA and porB genes enhanced the protein expression in C.glutamicum.Lysozyme and NaCl sensitivity experiments were performed in ?porB,?mepA,?porB?mepA,the porB overexpression strain OporB,and the mepA overexpression strain OmepA.The results show that overexpression of porB and mepA increased the sensitivity of the strains to NaCl and lysozymes.A representative photo of the porB and mepA overexpression strains also demonstrates that the mepA overexpression strain had an elongated colony morphology.The results indicated the porB and mepA deletion mutants reduced the death rate of C.glutamicum in response to stress.Moreover,mepA deletion mutants exhibited a different ATP generation rate compared with the wild-type C.glutamicum.Finally,the results of omics analysis and real-time quantitative PCR revealed that the mepA and porB genes involed in a complex transcriptional regulatory network with sigma factors and two component systems in C.glutamicum.Those results could at least partially explain why deletions of the mepA and porB genes are able to enhance protein expression and the reason requires further study.