Construction Of A Signal Peptide-probe Vector And Screening Of Strong Signal Peptides In Corynebacterium Glutamicum

Corynebacterium glutamicum is a generally recognized as safe(GRAS)production strain.It has the characteristics of uncomplicated culture conditions and fast growth.The genetic analysis of the whole genome data of Corynebacterium glutamicum,the maturity of the operating system and the understanding of its protein secretion mechanism lay the foundation for its development as a general cell factory for the production of recombinant proteins.In order to make Corynebacterium glutamicum more efficiently produce recombinant proteins,it is necessary to develop a "gene expression tool box" with excellent performance,which includes:host,vector,promoter,secretion pathway,signal peptide and other related gene expression element.The signal peptides in the "gene expression tool kit"was mainly studied in this study.The signal peptide is a small segment of the N-terminal peptide of the transporter precursor,which determines the transmembrane transport path of the transporter,and is eventually cleaved by the signal peptidase SPasel on the cell membrane and does not appear in the mature protein.The strength of the signal peptide determines the efficiency of protein secretion.The main research results of this thesis are as follows:.(1)The vector pAU19 was constructed.A signal peptide-probe vector clone/expression cassette with the strong promoter of tacM,the consensus sequence of C.glutamicum RBS,and the polyclonal site was designed and synthesized.The vector pAU19 was constructed by linking the clone/expression cassette with the shuttle vector pAU2 previously constructed in our laboratory.(2)The signal peptide-probe vector pAU20 was constructed.The alkaline serine protease AprE of Bacillus subtilis was used as the reporter protein,and its encoding gene was connected to the multiple cloning sites of the vector pAU19,resulting in the Corynebacterium glutamicum signal peptide-probe vector pAU20.(3)Recombinant signal peptide-probe vectors were constructed.A total of 16 sec-type signal peptides encoding fragments including bglS,amyE,Vpr,yvgO,yckD,yobB and yndA from Bacillus subtilis,BprA,epr,ywaD,PbpB,oppA and ydhT from Bacillus licheniformis,and cgr2063,cgr2070 and cgr2495 from Corynebacterium glutamicum were PCR-amplified,linked with the signal peptide-probe vector pAU20,and transferred into Escherichia coli,respectively,resulting in the recombinant signal peptide-probe vectors.The recombinant signal peptide-probe vectors were further confirmed by enzyme digestion and sequencing(4)The recombinant signal peptide-probe vectors were transferred into Corynebacterium glutamicum,resulting in obtained a series of the engineered Corynebacterium glutamicum strains.(5)The assay results of alkaline serine protease activity in the supernatant of shake flask fermentation culture of the engineered strains showed that the signal peptides activities of Vpr and BprA were significantly stronger than that of the endogenous strong signal peptide cgr2070 of Corynebacterium glutamicum,and the activities of oppA,Epr,cgr2063 and ywaD were slightly weaker than that of cgr2070.These signal peptides are all strong signal peptides in Corynebacterium glutamicum.