Activation Of The Silent Neoansamycin Biosynthetic Gene Cluster And Hygrocin Biosynthesis In Streptomyces Sp. LZ35

There is a constant need for new drugs to combat the increase of resistance of pathogenic microorganisms and infectious diseases. Natural products continue to provide a diverse and unique source of bioactive lead compounds for drug discovery. Actinomycetes and especially the genus Streptomyces are the main producers of secondary metabolites. Genomics data has revealed that most of microorganisms have the potential to produce a far larger number of natural products than have been isolated previously. In search for new drugs, the genome mining approach proved to be a powerful tool in the identification of cryptic secondary metabolite pathways. Ansamycins is a family of macrocyclic lactam compounds, mainly produced by Actinomycetes. These compounds demonstrate extensive biological and pharmaceutical activities, and are used as antibiotics, anticancer gents, and enzyme inhibitors. In the post-genome era, it’s significant that genome mining for novel ansamycins to offer new lead compound for drugs.Genome sequence of Streptomyces sp.LZ35has revealed three ansamycins biosynthetic gene clusters, two of which for producing geldanamycin and hygrocin, the other for producing cryptic ansamycin compounds. According to bioinformatics analysis, the cryptic ansamycin gene cluster is related to a new octaketide naphthalenic ansamycin compounds. Initially, we failed to isolate the compound by the optimization of culture conditions, maybe in the laboratory standard conditions, the cluster is either silent or expressed at a very low level. To trigger expression of the cluster, three different strategies were adopted in this work, respectively. The first one is introduction of a constitutive promoter ermEp*into the upstream of the polyketide synthase genes, RT-PCR analysis showed that the cluster is partially activated, and leading to the production of tetraketide intermediates. The second one is knocking out the TetR family negative regulators, the resulted mutant had no discernible effect on the metabolite profile. The third one is constitutive overexpression of the luxR family regulatory gene naml, transcriptional analysis showed total activation of the silent ansamycin biosynthetic genes, and led to the identification of three novel ansamycins, named neoansamycins. Interestingly, the neoansamycin A showed IC5010μM against the growth of HeLa and HepG2cell line, respectively, exhibiting moderate anti-tumor activity. The structure of neoansamycins contains an atypical extender unit, ethylnalonyl-CoA and pentalmalonyl-CoA, inactivation of nam8terminated the generation of neoansamycin, indcated that nam8maybe involved in the biosynthesis of pentalmalonyl-CoA. Nam7is homologous to Rif-orfl9, disruption of nam7gene resulted in neoansamycin elimination and concurrent production of a putative analog, maybe a derivative of neoansamycin with a benzenic ring system.Hygrocin is another naphthalenic ansamycin compounds produced by strain LZ35and its biosynthesis have not been reported in the literature. We analyzed the sequence of hgc gene cluster and locate it in the genome, the cluster was confirmed by disruption of hgcA leading to no hygrocin production. We carried out the following work:(1) The biosynthesis of AHBA precursor and ethyhnalonyl-CoA extendr unit. The hygrocin biosynthetic pathway shared the precursor with other secondary metabolites pathway, such as the AHBA precursor in geldanamycin gene cluster, and ethylmabnyl-CoA extendr unit in other biosynthetic pathways in strain LZ35.(2) Amide synthase functional verification. Amide synthase is a key enzyme, which is respond for ansamycin cyclization and release from the PKS. We have investigated the substrate selectivity of amide synthase. In hgcF disruption mutant, cross-complementation of amide synthase gene from other ansamycin gene cluster, only the amide synthase gene from ansalactam and divergolides can replace the hgcF to release and cyclization hygrocin derivatives.(3) The genes involved in tailoring process. To investigate the roles of oxidase gene played in the hygrocin gene cluster, seven genes were inactivated, the hgc2, hgc3, hgc4disruption mutants abolished the production of hygrocin, confirming their involvement in the hygrocin biosynthesis. Cross-complementation of rif-orfl9indicated that the Hgc2may involvement in naphthalene ring formation in biosynthesis of hygrocin. The Hgc3functions in the oxidative cleavage C5/C6of hygrocin via a Baeyer-Viliger type oxidation reaction. The Hgc4encodes a cytochrome P450hydroxylases, involved in the hydroxylation of the hygrocin polyketide moiety at C7. Gene inactivation, complementation and constitutive overexpression indicated that Hgcl is a positive regulator for hygrocin biosynthesis.In summary, we have identified a unique novel structural class of ansamycin named neoansamycins used genome mining strategy, which demonstrated that the feasibility and effectiveness of gene cluster and pathway analysis provide important information to guide isolation of compounds specified by particular gene clusters. And it also proved that many novel structural ansamycins produced by microbiology are waiting for us to exploit. The information of biosynthesis hygrocin presented here expands our knowledge of the biosynthesis of ansamycins, and pave the way for further rational engineering new ansamycin with combinatorial biosynthesis and synthetic biology.