Development And Application Research Of Heterologous Protein Secretory Expression System In Corynebacterium Glutamicum

Corynebacterium glutamicum,a Gram-positive and?generally recognized as safe?GRAS bacterium,is a potential host for the production of heterologous proteins despite its traditional use in amino acids and organic acids production.The C.glutamicum could effectively secrete target proteins into culture medium due to its powerful secretion system,which simplified the downstream purification process.A few expression elements had been developed and used for heterologous proteins production in C.glutamicum.However,the state-of-art of C.glutamicum protein production is in its infancy due to its limited expression elements and high efficiency expression hosts compared to the widely used E.coli expression system.So it is necessary to explore more available expression elements to fulfill the heterologous proteins production in C.glutamicum,and further improve the protein production level by host optimization.In the present study,we successfully developed the high efficiency secretory expression system based on C.glutamicum CGMCC1.15647 as host,and developed high efficiency expression elements such as cspB2 signal peptide with its promoter,and investigation of host optimization on protein production level were performed,including integration of target gene into chromosome,disruption of the protease and cell surface layer protein,and coexisting plasmid expression.The main results were summarized as follows:?1?Construction of monocistronic and bicistronic expression system based on the highly active P_aph promoter,analyzed different?ribosome binding site?RBS sequences on EGFP expression,and get that the bicistronic expression structure with RBS sequence RM3 showed the highest fluorescence intensity.The expression level of EGFP,?-amylase and?variable domain of the heavy-chain antibody?VHH showed10%,50%and 10%increased after-10 region mutation of P_aph promoter.Using EGFP as reporter protein and RBS sequence RM3,we constructed monocistronic and bicistronic expression system to evaluate 12 promoters from B.subtilis 168 and get the construct BSB4 showed highest fluorescence intensity.The?single chain antibody fragment?ScFv production level achieved more than 100 mg/L using the BSB4 construct.?2?We identified the signal peptide of CspB2 in C.glutamicum CGMCC1.15647by MALDI-TOF,and applied it along with the strong native P_AH6 promoter and RBS sequence RM3 for the secretory production of?endoxylanase?XynA and VHH in this strain.The secretory production level of XynA in a flask was 486.2 U/mL and become1648.7 U/mL when in a 5 L jar,and the secretory expression level of VHH was1.4-fold improved compared with RM3V'.The production level of XynA in a flask level become 596.7 U/mL when the cspB2 signal peptide and P_cspB2 promoter was applied,and become 1849.03 U/mL after culture medium optimization?single-factor analysis,Plackett-Burman design,steepest ascent experiment,central composite design and response surface method?.The enzyme properties of the XynA studies showed that the optimal temperature and pH for XynA were pH 4.5 and 45°C,respectively.And the XynA was relatively stable in pH range of 4-11 and at before50°C.Cu²⁺and SDS reduced the XynA activity but Co²⁺and Mn²⁺increased its activity.?3?The heterologous proteins production level could be further improved by host optimization.The native cspB2 signal peptide with its P_cspB2 promoter function well for XynA expression in its parent C.glutamicum CGMCC1.15647 strain,while the expression level of XynA in C.glutamicum ATCC13032 was only 28.61%of the activity in C.glutamicum CGMCC1.15647.Disruption of the S-layer protein CspB2and protease ClpS increased the secretion level of XynA by 107.56 and 195.96 U/mL,respectively.The highest activity of XynA reached 2492.88 U/mL in deep 24-well plates level by chromosomal integration with coexisting plasmids in cspB2 and clpS disrupted mutant?cspB2?clpSInX+P19-X+pEC-X,which was 10.43-and 0.35-fold greater than that for the chromosomal integration strain and the wild-type C.glutamicum CGMCC1.15647 harboring pXMJ19-xynA,respectively.The highest XynA secretion level reached 3537.24 U/mL?1768.62 mg/L?of?cspB2?clpSInX+P19-X+pEC-X in a 5 L fermentor.In addition,we expressed pullulanase in C.glutamicum CGMCC1.15647,codon usage optimization and host mutation facilitated the secretory production level of pullulanase.The highest concentration of pullulanase achieved was 108.13 U/mL in a 5 L jar after 32 h of cultivation.