Elucidation of the unusual methylenomycin biosynthetic pathway in Streptomyces coelicolor

Lead Research Organisation: University of Warwick
Department Name: Chemistry

Abstract

There is an urgent need for improved and new antibiotics due to the increasing emergence of multi-drug resistant pathogens such as vancomycin and methicillin resistant Staphylococcus aureus (VMRSA). One approach to meeting this need is the engineering of natural synthetic (biosynthetic) pathways to antibiotics in producing organisms to generate novel derivatives with potentially superior properties for medical applications. Fundamental knowledge of the genes, enzymes, intermediates and reactions involved in antibiotic biosynthetic pathways is an essential prerequisite for biosynthetic pathway engineering. Methylenomycins belong to an unusual group of antibiotics that have a broad spectrum of activity against Gram-positive and Gram-negative bacteria. Recently the genes required for assembly of the methylenomycins in the microorganism Streptomyces coelicolor have been identified and it has been shown that the methylenomycins are assembled from a unique combination of molecular building blocks. Analysis of the enzymes encoded by the biosynthetic genes indicates that the pathway for methylenomycin biosynthesis is highly novel and does not show similarities to the known pathways for biosynthesis of other antibiotics. This research project aims to investigate the unique methylenomycin biosynthetic pathway by identifying and determining the molecular structure of intermediates on the pathway and examining the role of each of the biosynthetic genes and enzymes. The potential for engineering the pathway to produce novel derivatives of the methylenomycins will also be explored.

Technical Summary

The methylenomycins are an unusual group of cyclopentanoid antibiotics produced by Streptomyces species. They possess a broad spectrum of activity against Gram-positive bacteria and some Gram negative strains, as well as antitumour activity against Lewis lung carcinoma. The sequence of the mmy gene cluster, which directs methylenomycin biosynthesis in S. coelicolor, recently became available as a result of a BBSRC-funded project to determine the complete sequence of the giant linear plasmid SCP1. Analysis of the enzymes encoded by this cluster indicates that the pathway for methylenomycin biosynthesis is highly unusual. In parallel work it has been shown that the carbon atoms of the methylenomycins are derived from a unique combination of primary metabolic precursors - acetyl CoA, malonyl CoA and a pentulose. This project aims to elucidate the intermediates on the pathway for methylenomycin biosynthesis, the reactions that give rise to these intermediates, and the enzymes that catalyse these reactions. A variety of approaches will be utilised in this multidisciplinary project including comparative metabolic profiling of biosynthetic mutants and wild type S. coelicolor to identify accumulated metabolites and feeding of isotopically labelled precursors to establish linkage between the metabolites accumulated and the methylenomycin biosynthetic pathway. The genetic engineering of the methylenomycin biosynthetic pathway to generate novel derivatives of the antibiotics will also be explored. The output of this project will be fundamental new insights into pathways for antibiotic biosynthesis in microorganisms which may ultimately lead to the discovery of new mechanistic enzymology for complex natural product biosynthesis, novel biocatalysts for biotransformation of highly functionalised organic molecules and the knowledge required to rationally engineer the methylenomycin biosynthetic pathway to produce new antibiotics.

Publications

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Description 1. MmyF (flavin reductase) and MmyO (flavin-dependent monooxygenase) were identified as the enzymes that catalyse selective epoxidation of one of the enones in methylenomycin C to form methylenomycin A, shedding light on the likely reaction mechanism.

2. The structures of five novel methylenomycin-related metabolites accumulated in the mmyE mutant were determined, shedding light on the biosynthetic steps between acetoacetyl-MmyA and methylenomycin C and the role of the MmyE enzyme in methylenomycin C biosynthesis.

3. Five novel 2-alkyl-4-hydroxymethylfuran-3-carboxylic acids (the methylenomycin furans) were identified as the metabolic products of the MmfL, MmfH and MmfP enzymes and shown to act as specific inducers of methylenomycin biosynthesis in S. coelicolor. The recognition that similar molecules are likely made by several other Streptomyces species has suggested a new paradigm for regulation of antibiotic production by diffusible signalling molecules in Streptomyces.
Exploitation Route Our findings have been taken forward by a PhD student who has made additional considerable progress. They have also been exploited for the discovery of novel natural products. See for example: J.D. Sidda et al. Discovery of a family of gamma-aminobutyrate ureas via rational derepression of a silent bacterial gene cluster. Chem. Sci. 2014, 5, 86-89.
Sectors Agriculture, Food and Drink,Chemicals,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology