Preliminary Functional Study On Two Genes Orf1 And Orf3 Of The Azinomycin B Biosynthetic Gene Cluster

Azinomycin B is a complex natural product with potential antitumor activity,which has unique chemical structure,remarkable antitumor activity and novel mechanism of action.Recently,the azinomycin B biosynthetic gene cluster was cloned and sequenced,and formation of its 3-methoxy-5-methyl naphthoic acid moiety was elucidated.However,the biosynthesis of epoxide domain and aziridino[1,2a]pyrrolidine ring of azinomycin B is still unclear.In this study,we found deletion of orf2,orf4,orf5 and orf6 had no effect on the biosynthesis of azinomycin B,while the azinomycins yield significantly decreased when orf3 was deleted.In order to rule out possible polar effects caused by gene deletion,complementation of orf3 was performed,which showed that the production of azinomycin B was restored in somehow.Heterologous expression of orf3 in the azinomycins sensitive strain S.albus caused it became more sensitive to azinomycin B.In addition,the prokaryotic expression system of Orf3 was established and highly efficient expression of the soluble protein Orf3 was achieved after optimization of inductions.Many attempts to delete orf1 failed,which indicated that orf1 was essential for the survival of the producer strain Streptomyces sahachiroi.By heterologous expression of orfl in S.albus,we found that orfl made the host strain became resistance to azinomycin B,which indicated that orfl is a putative azinomycins resistant gene.Orf1 was purified from a prokaryotic expression system and electrophoretic mobility shift assays showed that Orfl could bind DNA without sequence specificity.We also made semi-quantitative RT-PCR experiment to monitor transcription changes of orfl in different incubation times and mutant strains.It showed that in the wild type strain orfl expressed at high levels before the production of azinomycin B,while its expression level decreased when azinomycin B reached peak production.In the mutant strain without production of azinomycin B,the transcription levels of orf1 stayed stable in all the time.