The Analysis On Amide Synthase Structure And The Discovery Of AHBA Containing Natural Products From Two Streptomyces Strain

Ansamycins are a group of type I polyketides characterized by the unique starter unit AHBA and have diverse biological activities. To better understand and utilize ansamycins, our group has been working on the biosynthesis of ansamycin and discovery of novel ansamycins. By now, the biosynthesis pathway of ansamycins is almost clear, despite of several unsolved problems, like the release mechanism of ansamycin polyketide chain from PKS. My work focuses on the verification of ansamycin release mechanism and unearthing new ansamycins,Inactivation of amide synthase leads to the complete loss of ansamycins and the accumulation of intermediates, indicating that amide synthase catalyses the release of ansamycin polyketide chain. However, for lack of natural substrate, the function of amide synthase still havn’t been proved by in vitro reaction. In this study, we tried to get the structure of amide synthase by protein heterologous expression and X-ray diffraction. Among the15chosen amide synthases,10proteins show favorable productivity and solubility. Soluble amide synthases were purified by Ni-Agarose affinity chromatography, ion exchange chromatography and gel-filtration chromatography then underwent crystalization condition screening and modification. In this way, we acquired the structure of Micromonospora sp.160111amide synthase160111AS. Compare160111AS structure with the structure of homology protein of the NAT superfamily, the result of which revealed that the structure characteristics of160111AS well confirmed its releasing function. To further verify the result, experiments like co-crystalization of160111AS with substrate analogues and crystallization of other amide synthases are in need.To discover new ansamycins, we screened soil samples by PCR with degenerate primers at AHBA synthase for Actinomycetes. Positive strain Streptomyces sp. XZQH13contain a silent ansamycin synthesis cluster ansp. We constitutively expressed the LAL family regulator gene in the cluster, and successfully anctivated ansp. Through HR-ESI-MS and NMR, the main product of ansp was identified as two known ansatrienins:thiazinotrienomycin G and hygdroxymycotrienins A. To the best of our knowledge, this is the first report showing a silent ansamycin biosynthetic cluster activated by the overexpression of LAL family regulator. Another strain Streptomyces sp. XZYN4has two AHBA synthase gene containing cluster, rify and nay, both of which are cryptic. According to bioimformatic analysis, rify may produce rifamycin homologues with novel methylation modification and the product of nay could be certain unknown compound with AHBA. Different strategies were tried to activate rify and nay, including comparison of metabolic profiling, constitutively expression and inactivation of regulator genes, fermentation with rare earth element and the Tyrosinase-Guided mutant selection method, while none of the clusters have been activated yet. Subsequent plan is the high-throughput screening of reported streptomyces gene cluster activation methods on the basis of TGMS method.In conclusion, our work includes:1) The analysis of amide synthase3D structure, we uncovered the structure of amide synthase, which help to confirm its function to release ansamycin polyketide chain.2) Discovery of AHBA containing natural product from two streptomyces strains. We activated the ansatrienin synthesis gene cluster of Streptomyces sp. XZQH13while the activation of rify and nay of Streptomyces sp. XZYN4is still ongoing. The analysis of amide synthase structure aims at uncovering the release mechanism of ansamycins, and discovery of new ansamycins also help to find new ansamycin biosynthesis mechanisms. Our work will increase our knowledge on ansamycin biosynthesis, and build the base for designing and producing manmade ansamycins in the future.