Modification Of Bacillus Amyloliquefaciens Host Strain And Evaluation Of Protein Expression Performance

Bacillus amyloliquefaciens is a versatile Gram-positive bacterium without endotoxin,and it also grows rapidly.B.amyloliquefaciens has efficient protein secretion ability,making it an ideal host for the secretion and expression of target proteins,and has been designated for the production of enzyme in the food industry.However,there are still a lot of problems need to be solved in B.amyloliquefaciens.For example,effects of its own proteases and redundant proteins on the secretion and expression of target proteins have not been elucidated,and the genetic manipulation elements are relatively lacking.These problems restrict the wide application of B.amyloliquefaciens as a host chassis cells in the field of food enzyme preparations.Therefore,the purpose of this study is to develop a highly efficient B.amyloliquefaciens host chassis cell.In order to reduce the degradation of the target protein by the host’s own proteases,and minimize the secretion and expression stress of redundant proteins,11 important proteases and redundant protein genes(epr,npr E,apr E,apr X,mpr,pbp F,vpr,htr B,yktc1,hag,htr A)were knocked out in B.amyloliquefaciens HZ-12,and 11 gene-deficient mutants were obtained,including BAX-1,BAX-2,BAX-3,BAX-4,BAX-5,BAX-6,BAX-7,BAX-8,BAX-9,BAX-10 and BAX-11.The extracellular protease activity of the abovedefective mutants were tested by the milk plate method.The results showed that no protease activity was detected in the fermentation supernatant of the mutants except for BAX-1 and BAX-2,indicating that the extracellular proteases secreted by B.amyloliquefaciens HZ-12 were mainly neutral serine protease Npr E and alkaline serine protease Apr E.To further investigate the effects of chassis strains on recombinant protein expression,the alkaline protease gene apr E from the Bacillus subtilis D7 was heterologously expressed in mutants BAX-3,BAX-4,BAX-5,BAX-6,BAX-7,BAX-8,BAX-9 and BAX-10,and the effects of different host bacteria on the expression activity of alkaline protease Apr E were studied.The results showed that the alkaline protease activity in BAX-10/apr E was the highest value(666.83 U/m L),which was 57% and 30% higher than that of the HZ/apr E and BAX-3/apr E.These results indicate that BAX-10 is the optimal host for expression of apr E.To further increase the secreted level of Apr E by BAX-10/apr E the alkaline protease gene apr E was expressed in BAX-10 through different promoters(Pykz A and P43UTR12).The results showed that BAX-10/apr E with P43 promoter had the highest alkaline protease enzyme activity of.Using different strength terminators(Ter1,Ter3,Ter6,Ter14 and Ter18)to express apr E in BAX-10,the highest alkaline protease activity was reached in BAX-10/BSTer3 apr E,which increased by 10% compared with that of BAX-10/apr E.In order to further explore the effects of host on different enzyme proteins,the β-glucanase gene from B.amyloliquefaciens HZ-12 were expressed in defective mutant strains.The β-glucanase activity of BAX-9/bgl S strain reached the highest value(86.40U/m L),which was 43% higher than that of HZ/bgl S,while the β-glucanase activity of BAX-10/bgl S strain was not significantly different from that of HZ/bgl S strain.This is inconsistent with the expression of alkaline protease Apr E,indicating that different host bacteria have different effects on different enzyme proteins.This result is consistent with previous findings,and its influence mechanism is worth further exploring.In conclusion,effects of proteases and redundant proteins on the production of target protein were explored by knocking out the genes of proteases and redundant proteins from B.amyloliquefaciens HZ-12.Combined with the optimization of promoter and terminator,an engineered strain with significantly increased alkaline protease expression was obtained.Effects of different hosts on the expression of β-glucanase were also explored,and different influence patterns were found,indicating that different hosts have different effects on enzyme expression.In this study,a series of B.amyloliquefaciens mutants host chassis cells were constructed,and provided alternative hosts for the efficient expression of different target proteins.This study has a great significance and potential application value for the efficient expression enzymes by B.amyloliquefaciens in the food industry.