Heterologous Expression Of Enzymes Involved In Bacillamide C Biosynthesis By Bacillus Atrophaeus C89 Associated With Marine Sponge

Marine sponge bacterial symbionts are important sources of bioactive natural products.Bacillamide C has been produced by sponge-symbiont Bacillus atrophaeus C89.Analogues of bacillamides exhibit cytotoxic activity against many cancer cells and bacillamides are potential natural algicide since they have algicidal activity.The biosynthesis of bacillamide C,which was speculated to be synthesized by nonribosomal peptide synthetases(NRPSs),requires four enzymes: NRPS,upstream oxidase,downstream amino acid decarboxylase(AADC)and Sfp-type phosphopantetheinyl Transferase(PPTase).Our previous experiments validated that the recomibant AADC had the ability to catalyze L-tryptophan into tryptamine in vitro.Here the heterologous expression of NRPS,AADC and Sfp-type PPTase in vivo laid a foundation for the study on the biosynthesis pathway of bacillamide C and the production of bacillamides heterologously.In order to identify the Sfp-type PPTases catalyzing PCPs of NRPS in the biosynthesis of bacillamide C,we found all the PPTase genes in the genome of B.atrophaeus C89 and the Sfp-type PPTase Bap was identified by BLAST and amino acid sequences alignment.The bap gene was heterologously expressed in sfp gene mutant strains Bacillus subtilis 168 and the recombinant strains B.subtilis 168-bap synthesized the nonribosomal polypeptide surfactin.This study illustrated that Bap actived all the PCPs in surfactin synthetases NRPS and validated the capacity of Bap in activating PCPs in NRPSs.To explore biosynthesis of bacillamide C,we amplified the whole nrps-aadc gene cluster and integrated it to the genome of high-efficiency surrogate host Streptomyces lividans SBT18.An extra secondary metabolite was detected in the fermentation products of recombinant strain compared to that of control strain by HPLC.The compound was identified to be N-acetyltryptamine by LC-MS and NMR.We supposed that the decarboxylation from L-tryptophan to tryptamine was performed firstly by AADC and then the tryptamine was transformed into N-acetyltryptamine by the catalyzation of N-acetyltransferase in host cells.This study proved that recombinant AADC could catalyze tryptophan into tryptamine.NRPS compounds in the recombinant strain S.lividans weren't detected,so in order to confirm the function of NRPS,we ligated nrps gene with p ET28 a to form a recombinant p ET28 N and co-transformed it with psv20 which contained the broad substrate selectivity PPTase gene sfp to the surrogate host Escherichia coli BL21(DE3).SDS-PAGE showed that the NRPS was expressed in soluble state in E.coli.This study laid a foundation for the following co-expression of nrps and aadc gene in E.coli and for the revelation of bacillamide C biosynthesis pathway.