Effect Of Spore Pigment Biosynthesis And PhoR-PhoP Two-component System On The Production Of Avermectins In Streptomyces Avermitilis

Avermectins,a series of 16-membered macrocyclic lactones produced by Streptomyces avermitilis NRRL8165.Avermectins are the most effective agriculture pesticides and antiparasitic agents and used widely in medical,veterinary and agricultural fields.Besides avermectins,S.avermitilis can also produce many other kinds of secondary metabolites,such as spore pigment,melanin and oligomycin.Spore pigments are polycyclic aromatic polyketides.Analysis of polyketides pathway showed that spore pigment and avermectin share the same precursor,acetyl-CoA.In this paper,the flanking fragments of the spore pigment biosynthetic genes(whiE_a) was PCR amplified,cloned and used to construct the gene replacement plasmid pHL643,phL643 was conjugated into S. avermitilis NRRL8165 followed by screening for double crossover event,yielding three apramycin resistance and thiostrepton sensitive isolates named ZJ1-1,ZJ1-2 and ZJ1-3, which were deficient in biosynthesis of the grey spore pigment.Fermentation of the mutant strains in shaking flasks and HPLC analyses showed that the production of avermectins increased by 47%compared with that of the wild type,indicating that the spore pigment biosynthesis competes with the avermectins biosynthetic pathway.The biosynthesis of many different types of antibiotics and other secondary metabolites is regulated by phosphate.Recently,it was shown that phosphate control morphological differentiation and secondary metabolism in Streptomyces lividans.is mediated by the two-component phoR-phoP system.Deletion mutants lacking phoR-phoP, overproduced large amounts of actinorhodin and undecylprodigiosin.In the S.avermitilis genome,phoR-phoP homologue has been identified.To investigate the function of it,a gene replacement plasmid pHL645 was constructed and conjugated into S.avermitilis NRRL8165.Tree phoR-phoP gene replacement mutant strains(named ZJ2) was obtained. ZJ2 growed poor on YMS solid medium,whereas growed well on YMS solid medium supplemented with phosphate.HPLC analysis of the ZJ2 fermentation culture showed that ZJ2 could synthesize avermectins only in fermentation medium supplemented with phosphate and production of avermectins is very low.