Construction And Expression Of A Typical Non-ribosomal Peptide Module Of Brevibacillus Laterosporus S62-9

Brevibacillin(BBL),an antimicrobial peptide produced by Brevibacillus laterosporus S62-9,is characterized by the broad antibacterial spectrum,good heat resistance,unsuitable residue,and no drug resistance,thus having good application prospects in the field of food preservative and antibacteria.This peptides are the kind of nonribosomal peptides(NRPs)composed of modules with nonribosomal peptide synthetases at their core,and their synthesis is determined by related gene clusters.In this study,we will construct and heterologously express a core module of NRPs of the synthetic antibacterial peptides in vitro.By detecting the amino acid activity of the adenosine domain of non-ribosomal peptide synthetase,and exploring the role of NRPs in the synthesis of antibacterial peptides,it will lay the foundation for the future construction of the whole module of antibacterial peptides and large-scale synthesis of BBL in vitro.The main results of this research are as follows:(1)The synthetic gene cluster of BBL contains two core elements,non-ribosomal peptide synthetase adenylation domain(NRPS-A)gene and acyl carrier protein domain(acyl aarrier protein domain,ACP),and other modified elements including epimerases,methyltransferases,and Germany.Using genome mining technology,we found that the core element of the antibacterial peptide synthesis gene cluster module was C-A-PCP.Because the synthetic gene cluster of BBL is lacking of a condensation domain C,we cloned a C domain gene from Brevibacillus sp.The theoretical molecular weight of the C domain,A domain and ACP structure are 35.8 kDa,59.9 kDa and 9.6 kDa respectively.(2)Using the A domain as the test gene and pET-21b and pET-22b as the vectors,we constructed four recombinant expression vectors pET21b-N1,pET21b-B2,pET22b-N1,and pET22b-N3,and then transformed them into E.coli BL21,E.coli Rosetta(DE3),E.coli Origami B(DE3)receptor cells respectively.After IPTTG induction,only the trasformant cell withpET22b-N3 transforming into E.coli Origami B(DE3)could expressed the active fusion protein in a soluble form,while the others were inclusion bodies.Therefore,the pET-22b was selected as the expression vector,and E.coli Origami B(DE3)served as the heterologous expression receptor.(3)We used the domain C,domain ACP and C-A-ACP synthetic module and the pET-22b vector constructed three recombinant expression vectors pET-22b-S4,pET-22b-B5,and pET-22b-S4-N3-B5,and then transformed them into E.coli Origami B(DE3)receptor cells,and finally the domain C,ACP structure and C-A-ACP synthetic module genes produced three inclusion bodies without biological activity and their molecular weights were approximately 35.8 kDa,9.6 kDa,and 105.3 kDa respectively.(4)The substrate specificity and basic enzymatic kinetic parameters of the expressed domain A fusion protein were detected using pyrophosphate kit.The result showed that the fusion protein of the adenylation domain had specific lysine activity and had very low activity against other tested amino acids,indicating that the adenylation domain could specifically upload the lysine and perform its phosphorylation on the adenosine,and thus activated lysine would be transferred and condensed into peptide bonds.The optimal temperature for adenosine domain is 35?,the optimal pH is 7,Km value is 8.793 ?mol/L,and Vmax value is 6.811 ?mol/L·min.