Investigation On The Azalomycin F Polyketide Backbone Biosynthesis

Azalomycin F series compounds are a kind of 36-membered macrocyclic antibiotics,which are produced by Streptomyces sp.211726 that was isolated from mangrove rhizosphere soil in Hainan,China.These compounds showed a broad range of anti-bacterial activity,anti-fungal activity and cytotoxicity against human colon tumor cell line 116,which indicates that azalomycin F would be valuable in human tumor treatments.Besides,their anti-Fusarium oxyspOrum sp.Cubense antivity shows a good potential in chemical control of banana wilt disease.Although the biological activities of azalomycin F series compounds are obtained,the biosynthetic mechanism has not been yet revealed.This study aims to elucidate the azalomycin F polyketide backbone biosynthesis process,as well as provide knowledge and engineering blocks for combinatorial biosynthesis and synthetic biology of novel polyketide compounds.Firstly,E.coli-Streptomyces conjugation systems for Streptomyces sp.211726 were tested,and an optimized system of plasmid pYH7,pSET 152 and pIB 139 was obtained for genetic manipulation of Streptomyces sp.211726.This work provided foundation for following gene manipulation in this strain.Secondly,the whole draft genome was sequenced via combined whole genome shot gun sequencing and cosmid sequencing,and secondary metabolic related genes were annotated and analyzed.According to the chemical structure profile of azalomycin F,the biosynthesis process of its carbon backbone forming was predicted and the corresponding modules and domains were also assumed by classical polyketide synthase theory.A putative gene cluster for azalomycin F biosynthesis was located.To complete the cluster,a fosmid library based on vector pCClFOS was constructed and screened.A fosmid,which is covering the cluster,was sequenced and complete the whole azalomycin F biosynthetic gene cluster.To fully confirm the correction of this cluster,a large fragment deletion was established in wild-type Streptomyces sp.211726,resulting the abolishment of azalomycin F series compounds,which proved that this cluster is corresponding for the azalomycin F biosynthesis.Thirdly,every module and domain were predicted in azalomycin F biosynthetic gene cluster according to their amino acids characters.It was failed to correspond polyketide synthase module and domain organization to chemical structure in neither direction.To explain the paradox between genes and structure,four putative model for non-colinearity in polyketide synthases were raised and analyzed.Finally,inactivation of DH1 domain in Streptomyces sp.211726 resulted in total abolishment of azalomycin F production,and acculation of novel azalomycin F derivatives.LC-ESI-HRMS and NMR analysis of the new compounds proved that they were two round extension products with two adjacent hydroxyl groups.At the same time,in vitro reconstitution of both wild-type AzlA and AzlA(?DH1)drew the same conclusion with in vivo inactivation,which strongly proved that module 1 was used twice in the first and second elongation and ER1 domain in it was "toggled" off and on.Therefore,the clarification of azalomycin F biosynthetic mechanism will provide not only theoretical basis for directed modification and optimization by using synthetic biology or combinatorial biosynthesis approaches,but also building blocks of bioengineering of valuable products in future.