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Abstract Coelimycin P1 is novel yellow-pigmented, polyketide-alkaloid with an unprecedented 1,5-oxathiocane structure produced by Streptomyces coelicolor.

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Presentation on theme: "Abstract Coelimycin P1 is novel yellow-pigmented, polyketide-alkaloid with an unprecedented 1,5-oxathiocane structure produced by Streptomyces coelicolor."— Presentation transcript:

1 Abstract Coelimycin P1 is novel yellow-pigmented, polyketide-alkaloid with an unprecedented 1,5-oxathiocane structure produced by Streptomyces coelicolor M1157. It is a product of the cpk cryptic polyketide biosynthetic gene cluster which encodes a type I modular polyketide synthase (PKS) and associated tailoring enzymes. Bioinformatic analysis of the gene cluster indicates that coelimycin P1 is assembled using some previously undescribed biosynthetic mechanisms. One of these steps was proposed to involve reductive chain release of an ACP-bound polyketide intermediate from a type I modular PKS by a thioester reductase (TR) domain. This project has focussed on characterising the TR domain by expressing and purifying the recombinant protein from E. coli for use in biochemical assays to elucidate the mechanism of chain release. Initial experiments revealed that the TR domain preferentially utilizes NADH over NADPH to catalyse the reduction of a substrate analogue of acyl-ACPs (dodecanoyl-CoA). Subsequent analysis also indicates the formation of the alcohol from the thioester substrate analogue upon incubation with the purified recombinant enzyme. Probing the Biosynthesis of Coelimycin P1: a Novel Polyketide-Alkaloid from Streptomyces coelicolor Ufedo Ruby Awodi and Greg L. Challis Department of Chemistry, Univerisity of Warwick, Gibbet Hill, Coventry, CV4 7AL Proposed mechanism of chain release References 1.Berdy J. Microbial Metabolites: A Personal View. Journal of Antibiotics 2005, 58, J-P Gomez-Escribano, L. Song, D. J. Fox, M. J. Bibb and G. L. Challis. Identification of the yellow-pigmented polyketide product of the cryptic cpk gene cluster of Streptomyces coelicolor M145. Chem. Sci. 2012, 3, K. Scherlach and C. Hertweck. Triggering cryptic natural product biosynthesis in microorganisms. Org. Biomol. Chem. 2009, 7, Acknowledgement This study is supported by the University of Warwick, Chancellor’s Scholarship and the Department of Chemistry Departmental Scholarship. I would like to thank Prof. G.L. Challis for his support, guidance and excellent supervision. I would also like to thank all the members of the Chemical-Biology research cluster for helpful discussions especially in the lab. Introduction Reductive chain release of the thioester bond to form the aldehyde using NAD(P)H as a co-factor Subsequently, the tailoring enzymes modify the polyketide to yield coelimycin P Proposed Pathway for Coelimycin P1 Biosynthesis Microbial secondary metabolites have been known for their wide-ranging biological activity and fascinating chemical structures, especially since the golden era of antibiotic discovery [1]. However, frequent re-isolation of known compounds led many pharmaceutical companies to shift their focus from microbial natural product research [2]. Recent advances in whole-genome sequencing technology coupled with increasing understanding of the enzymatic logic of secondary metabolite biosynthesis has enabled the identification of a large number of gene clusters encoding enzymes likely responsible for the assembly of novel biologically active compounds [3]. These “cryptic” natural product biosynthetic gene clusters represent a vast pool of information with immense potential for applications in the field of natural product discovery. Discovery and Isolation of Coelimycin P1 Fig.1. A. Culture supernatants of S. coelicolor M1152 and M1157 grown in R3 medium. B. Structure of Coelimycin P1 A B Coelimycin P1 Fig.2 Coelimycin biosynthetic pathway showing module and domain organization of the type I modular PKS Coelimycin P1 was isolated using a novel genetic engineering strategy Biosynthetic gene clusters encoding known antibiotics were deleted A specific mutation in the rpoB gene induced production Structural elucidation showed that it possesses the ene-triene-one moeity responsible for its characteristic yellow color. 2. Substrate Consumption and Product Detection 1.Purification of TR domain and Biochemical Activity Assay Fig. 4. a. Recombinant His 6 -TR protein of expected size (46kDa) was expressed in E. coli and purified to homogeneity. b. The enzyme was incubated in the presence of dodecanoyl-CoA and either NADH or NADPH and the change in fluorescent emission at 462 nm was monitored. a b Conclusion and Future Work Purified TR protein showed activity in vitro and was found to consume dodecanoyl-CoA in the presence of NADH to form dodecanol. The enzyme appeared to preferentially utilize NADH over NADPH. Further experiments will be required to establish product formation by GC-MS and obtain crystals of the TR protein to determine its mechanism of catalysis. Similar assays will also be developed to investigate the activity of the tailoring enzymes. Results Aldehyde Alcohol NADH Fig 3: TR catalysed polyketide chain release. The aldehyde formed may be reduced further to the alcohol in the presence of an additional equivalent of NAD(P)H Fig. 5. a) Extracted-ion chromatogram of m/z 949 (dodecanoyl-CoA) from LC-MS analysis. TR reaction contains active enzyme while the control reaction contains the denatured (boiled) enzyme. b) TLC analysis of organic extract (hexane) showing the presence of dodecanol by comparison with standards. TR reaction Control


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