| Heterologous expression is an effective strategy to improve the production of target proteins.Recently,many industrial enzymes(proteases,a-amylase,etc.)have been successfully produced in bacteria and fungi expression systems.Bacillus.licheniformis is regard as a non-toxicity and promising host strain with high yields of secreted proteins.In this research,we focused on the signal peptidases and signal peptide peptidases,which are the important secretion components during the protein transportion.We have successfully constructed a highly efficient platform ?0F-3GS for protein secretion,both nattokinase and a-amylase yield were improved in the recombinant strain.Futhermore we have constructed a number of expression vectors for screening the efficient promoters for Bacillus licheniformis expression system.Signal peptidase is crucial element in the heterologous protein production by cleaving the signal peptides from the preproteins to form the mature proteins.To verify the function of signal peptidase sipV in protein secretion,gene sipV was overexpressed in B.licheniformis AOF-3 by homologous combination.The nattokinase expression plasmid pHY300-P43-NK was further transformed into B.licheniforrmis ?0F-3GV,resulting in the mutant B.licheniforrmis?0F-3GV/pHY300-P43-NK.The nattokinase activity of this reconbinant strain reached to 35.60 FU/mL,increase by 16%compared with that of B.licheniformis ?0F-3/pHY300-P43-NK.Signal peptide peptidases could cleave the remnant signal peptides in the cellular membrane,after the shearing of signal peptide by signal peptidase.In this research,the signal peptide peptidases SppA and TepA mediated by P43 promoter were further overexpressed in B.licheniformis ?0F-3 by inserting the relevant expression cassettes into the chromosome by homologous recombination,and the recombinant strains named ?0F-3GS and ?0F-3GA,respectively.Then,the nattokinase and a-amylase expression vectors were electro-transferred into the ? 0F-3GS and ? 0F-3GA,and nattokinase and a-amylase activities were analyzed to evaluated the effects of overexpression of signal peptide peptidase on the protein secretion.Based on our results,the specific activities of nattokinase and a-amylase were increased by 18%and 43%respectively in the sppA overexpression strain,compared with those produced by B.licheniformis? 0F-3.Meanwhile,in the tepA overexpression strain,only a 12%improvement of specific a-amylase activity was obtained and overexpression of tepA had no effect on the concentrations of nattokinase.These results suggest that SppA plays the main functional SPP for protein secretion in B.licheniformis.Basing on the laboratory transcriptome data of B.licheniformis DW2,we selected 15 promoters to replace P43 promoter of the nattokinase expression vector.The fermentation of B.licheniformis ?0F-3 containing expression vector having different promoters showed that the higher nattokinase activity promoters are Prpsn,PcstA and Pveg.The promoter Pveg mediated the highest nattokinase activity,reached 37.84 FU/mL,however,it had no significant difference compare to ?0F-3/pHY300-P43-NK(34.39 FU/mL).Based on the existing laboratory nattokinase and a-amylase expression vectors pHY300-P43-NK and pHY-P43-amyL,dual-promoter vectors Pykza-P43-NK,PasiB-P43-NK,PylB-P43-NK,Pykza-P43-amyL,PgsiB-P43-amyL,PylB-P43-amyL were constructed.Those dual-promoter vectors contain the ?B promoter Pykza,PgsiB,Pylb respectively.Similarly,those dual-promoters were electro-transferred into the ?0F-3GS for fermentation.Our results showed that the specific activities of a-amylase with dual-promoters were all higher than the control,the recombinant strain ?0F-3GS/PylB-P43-amyL reached the highest activities 296.35 U/mL,which was increased by 101%,compare with that of ?0F-3GS/pHY-P43-amyL,and 272%compare with that of the original strain ? 0F-3/pHY-P43-amyL.However,the dual-promoter had no effect on the nattokinase production. |
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