Establishment Of The Highly Effective Expression System In Bacillus Amyloliquefaciens K11 And Optimization Of Its Efficient Expression Elements

As the producer of many important industrial enzymes,Bacillus amyloliquefaciens has the strong ability of protein secretion.It also exhibits excellent prospects as heterologous host to express feeding enzymes with high efficiency.However,the application of B.amyloliquefaciens as heterologous expression host is severely limited by several factors,e.g.,presence of restriction and modification system,poor plasmid stability,lack of expression elements and high activity of endogenous proteases.The previous study of the applicant had been given a good solution to the problem of poor genetic transformation and plasmid stability in Bacillus amyloliquefaciens K11 strain.Using B.amyloliquefaciens K11 and alkaline protease gene BcaprE as the expression host and model gene,the gene expression levels mediated by combinations of promoters PamyQ,PaprE and Pnpr and signal peptides SPamyQ,SPaprE and SPnpr were assessed on shake flask level.The PamyQ-SPaprE was found to be the best secretory expression cassette,giving the highest yields of extracellular BcaprE(13800U/mL).Using the same expression system,the maltogenic a-amylase Gs-MAase and neutral protease BaNPR were successfully produced with the yields of 19.0 U/mL and 17495 U/mL,respectively.After knocking-out the endogenous neutral protease-encoding gene Banpr,the yields of BcaprE and Gs-MAase were further improved by 25.4%and 19.4%,respectively.Moreover,the yields of BcaprE were further improved to 30200 U/mL in a 15-L fermenter following optimization of the fermentation conditions.In order to obtain the library of secretory expression cassette with different intensities for B.amyloliquefaciens,and to explore the efficient secretory mechanism mediated by efficient secretory expression elements,the efficient secretory expression cassette PamyQ-SPaprE was mutated randomly.The random mutation library of secretory expression cassette was constructed by error-prone PCR,and 1352 clones were obtained.The 1352 transformants were subjected to plate assay and shaker flask fermentation,and a total of 20 mutant strains with better expression and secretion intensity than the wild type were screened.The constructed secretory expression cassette library not only provides more platforms for the expression of exogenous proteins in B.amyloliquefaciens expression system,but also lays a foundation for the in-depth study on the efficient secretion and expression mechanism of B.amyloliquefaciens.In the present study the genetically engineered B.amyloliquefaciens strain 7-6 containing PamyQ-SPaprE library as the secretory expression cassette was developed.This highly efficient expression system shows universal applicability,high plasmid stability and high-yield secretion capacity,and represents an excellent industrial strain for the production of heterologous proteins.