Cloning And Preliminary Analysis Of The Ficellomycin Biosynthetic Gene Cluster

Ficellomycin is a dipeptide-like antibiotic produced by the soil bacterium S.ficellus(ATCC 19752).This agent can selectively impair semiconservative DNA replication and cause the accumulation of deficient okazaki fragments,which lack the capability to be further integrated into larger DNA fragments while has no effect on normal cell metabolism.As for the special action mode of ficellomycin,it has large potential for developing novel antibiotics especially for some antitumor drugs with low toxicity and fewer side-effects.In this study,the biosynthetic gene cluster of ficellomycin will be isolated and analysed preliminarily.In order to get the complete biosynthetic gene cluster,an S.ficellus cosmid gene library was constructed.The library contains 2688 clones with an average insert size of 35kb and low repetition rate.After calculation,the library can cover more than 99%of genome information.According to the molecular structure of ficellomycin,it is speculated that it may be synthesized by non-ribosomal peptide synthetases(NRPSs).Therefore,this study mainly focused on the screening of clones that carried NRPSs encoding genes.To date five positive clones,pOJ446-N1 to N5,were screened and sequenced by construction of subclones.Through sequence alignment and splicing,the whole nucleotide sequence of the three NRPS gene clusters were obtained.Sequence analysis shows that there is a frameshift mutation in the A domain of NRPS3 gene cluster,which leads to the gene cluster can not be translated into a functional NRPSs.So this gene cluster has nothing to do with the synthesis of ficellomycin.NRPS1 and NRPS2 constitute a linkage gene cluster,among which the NRPS2 gene cluster can encode one-module NRPSs,while the NRPS2 gene cluster can encode twomodule NRPSs,consistent with the dipeptide structure of the ficellomycin.Besides,in the upstream of the two gene clusters,there are other genes which will encode transcriptional regulator,resistance protein,acyl-CoA synthetase and other fectors,respectively.And these factors may also be involved in the biosynthesis of ficellomycin.It is known that functionally related genes in prokaryotic genome generally constitute a linkage gene cluster.So it is hypothesized that the linkage gene cluster might be related to the biosynthesis of ficellomycin.In order to further verify the relationship between the linkage gene cluster and the synthesis of ficellomycin,the recombinant plasmid pOJ446-nl containing the gene cluster was transferred into a type strain,S.Coelicolor,by conjugation.Then the recombinant srains were fermented and the activity of the fermentation broth was detected.The result showed that the fermentation broth of the recombinant Streptomyces had an inhibitory effect on ficellomycin-sensitive strain,Penicillium oxalicum,which preliminary indicates that the linkage gene cluster is responsible for the biosynthesis of ficellomycin.This study will lay the foundation for the study of the biosynthesis and metabolic regulation mode of ficellomycin.