The Study On A Polyketide Synthase Gene Cluster(Cluster 9) And The Regulatory Factor Aurd From The Aureothin Biosynthetic Gene Cluster

Polyketones are a wide variety of compounds and have drawn extensive attaention for their excellent biological activities such as antibacterial,antifungal,anti-tumor,immunosuppressive,and so on.Polyketides are catalyzed by polyketide synthases.Elucidation of the biosynthetic pathways of different polyketides will provide directions for the discovery and creation of new polyketide compounds in the future.In this study,two polyketide synthase gene clusters were studied to recover their function and regulation by research methods of genetics,molecular biology and biochemistry.1 A polyketide synthase gene cluster(cluster 9)from Streptomyces sp.SH-62The polyketide synthase gene cluster(cluster 9)derived from Streptomyces sp.SH-62 is a type I PKS.According to the low homology with natural product biosynthetic gene clusters in the database,it is supposed to be the cluster for a new natural product.The BAC plasmid containing cluster 9 was heterologously expressed in Streptomyces albus and produced 3 suspected metabolites.In order to investigate whether these 3 compounds were produced by cluster 9,two kinds of genome editing methods including PCR-targeting system mediated by ?-Red recombinases and CRISPR/Cas9-CodA(sm)system,were employed for gene knockout of the key PKS gene(pks)in cluster 9.Production of 3 compounds was not affected by deletion of pks,indicating that they were not produced by cluster 9.2 The regulatory factor AurD from the aureothin biosynthetic gene clusterAureothin is a nitroaryl-substituted metabolite with good biological activity.Only one potential transcriptional regulation factor aurD was found in the biosynthetic gene cluster of aureothin.Deletion of aurD abolished the biosynthesis of aureothin,but it was a pity that all attempts for gene complementation of aurD failed.Considering that overexpression of aurD may suppress cell growth,the aurD gene on the control of the constitutive strong promotor PermE* was returned to the knockout site of the original one in this study and the gene complementation mutant strain was achieved.Metabolite analysis of the complementation strain discovered that AurD could restart the production of aureothin,and the yield of aureothin was significantly improved by overexpression of the aurD gene under a strong promoter.AurD with an OmpR-like DNA binding domain and a bacterial transcriptional activation domain,belongs to the SARP family of transcriptional regulation proteins.To find its regulation mechanism,AurD protein was expressed in E.coli and Streptomyces hosts.However,the expression level of AurD was very low in Streptomyces and inclusion bodies were formed in E.coli.The expression system and conditions need to be optimized in the future.