Discovery And Exploration Of Synthetic Products Of NRPS Gene Cluster REZU6 In Rhodococcus Erythropolis D-1

Nonribosomal peptides are very important natural products.Many microbial nonribosomal peptides have been widely used clinically,such as penicillin,cephalosporin,vancomycin,caspofungin,tacrolimus,etc.However,in recent years,it has become more and more difficult to discover new structures of nonribosomal peptides.The reasons include: the research object is relatively single,and most of the nonribosomal peptides are derived from Streptomyces;on the other hand,under laboratory culture conditions,many natural products synthetic genes are silent,and a large number of potential non-ribosomal peptides cannot be isolated.It has been reported that the Rhodococcus genome contains a large number of nonribosomal peptide synthetase(NRPS)gene clusters,so Rhodococcus is an important source of potential nonribosomal peptides.In this paper,the new NRPS gene cluster in the genome of Rhodococcus erythrinus D-1 was discovered by bioinformatics.And the gene cluster REZU6 has not been reported yet.We designed two ways to obtain the nonribosomal peptide synthesized by the gene cluster.The gene cluster REZU6 contains two regulatory genes,araC and marR,which were highly expressed and knocked out respectively to activate the gene cluster.We fermented the wild-type strain and mutant strains in TSB and M9 liquid medium,and compared the difference of fermentation products by HPLC,it was found that the mar R-overexpression strain in the M9 liquid medium had one more peak than the wild-type;the different peaks were collected and analyzed by LC-MS in order to obtain the molecular structure information of the nonribosomal peptide,however,the LC-MS results showed that the product corresponding to this peak was not our target product.On the other hand,we analyzed the NRPS sequence encoded by the gene cluster through bioinformatics software,and predicted that the backbone structure of non-ribosomal peptides might be Orn-Thr-ValLeu-Phe or Orn-Thr-Val-Phe-Val;We designed and chemically synthesized five peptides and characterized their siderophore activity and antibacterial activity.The results showed that none of the five peptides had siderophore activity,cyclic peptides have a certain inhibitory effect on Saccharomyces cerevisiae and Streptomyces coelicolor at high concentrations.This result suggests that the molecular structure of the cyclic peptide may be the closest to the nonribosomal peptide synthesized by the NRPS.