Genome Mining For The Pentangular Polyphenols

Pentangular polyphenols, characterized with their pentangular ring core structure, are a subfamily of aromatic polyketides(PKs) derived from C24 to C30 polyketide chains. They are aromatic natural products with complex and diverse structures and amazing bioactivities. The aim of this research is to discover novel pentangular polyphenols from actinomycetes through genome mining strategy. The stduy can be divided into two parts, one is genome mining pentangular polyphenos from Streptosporangium sp. CGMCC 4.7309; the other is to set up homology-dependent PCR approach to obtain potential pentangular polyphenol producers and activate slient pantangular polyphenol clusters for novel compounds.Genome sequencing datas in NCBI showed that Streptosporangium has a strong potential to produce diverse secondary metabolites. Therefore, Streptosporangium sp. CGMCC 4.7309 was selected for whole genome sequencing. The anti SMASH analysis showed that S. sp. CGMCC 4.7309 contains more than 20 secondary metabolite gene clusters, with one putative type II PKS gene cluster for pentangular polyphenols. The gene cluster of pentangular polyphenol contained 33 open reading frames, whose functions were predicted by BLAST. Phylogenetic analysis of KSβ revealed the length of pentangular polyphenol producded by S. sp. CGMCC 4.7309 would be C26. Multiple sequence alignment of the active sites of the Baeyer-Villiger oxidase Hex18 and asparagine synthetase-like Hex6 allowed us to predict the core structure of the pentangular polyphenol, hexaricins. The thesis set up the genetic system of Streptosporangium unprecedentedly. Then, the ketoreductase Hex30, which plays an essential role in the early biosynthetic steps, was knocked out. The pentangular polyphenols produced by S. sp. CGMCC 4.7309 were then discovered by metabolic profile comparison. After isolation and purification, three compounds were obtained: Hexaricin A, Hexaricin B and new compound Hexaricin C. Their structures were determined by LC-MS and NMR analyses, and stereochemistries of the three compounds were determined by specific rotation and circular dichroism analyses.Homology-dependent PCR screening strategy has achieved a great success in genome mining of natural products. Pentangular polyphenols are biosynthesized by type II polyketide synthases. Previous PCR screenings were carried out with primers for KSβ genes, which were not specific for pentangular polyphenols. Two specific monooxygenases exist in the known pentangular polyphenol gene clusters, which could be used to design PCR probes for screening. Six degenerated primer pairs were designed based on the homology of those monooxygenases. Three pairs of primers were used in this study to check more than 318 bacterias, and 14 positive strains were obtained. One pair of primers were finally selected for screening all positive strains. Among them, three Streptomyces champavatii CGMCC 4.1615, Streptomyces sp. 1.093 and Streptomyces sp. B81 were chosen for genome sequencing, and all three strains contain pentangular polyphenol gene clusters. Pentangular polyphenol gene cluster in strain 4.1615 is almost the same as the fredericamycin gene cluster; pentangular polyphenol gene clusters in strain 1.093 and B81 show high similarity to xantholipin biosynthetic gene cluster, indicating that they may produce compounds similar to xantholipin. By overexpression of an activator gene cds12 in B81, the cryptic pentangular polyphenol gene cluster was activated and produced a proposed pentangular poypgenol. The purification method of this compound was set up. Structure elucidation of this compound is still in progress.