The genus Streptomyces is known to be responsible for the production of more than two-thirds of the world’s antibiotics, through complex specialised metabolic pathways. However, given the high frequency of rediscovery of known antibiotics and the challenge of producing novel analogues via chemical synthesis, biosynthetic engineering has emerged as an attractive approach to optimising antibiotic natural products for clinical use. This technique utilises enzymes from antibiotic biosynthetic pathways to create novel antibiotic derivatives. However, its application requires an understanding of how antibiotics are biosynthesised. This work is focused on the methylenomycin antibiotics produced by Streptomyces coelicolor A 3 (2), a model Actinobacterium. The cluster of genes directing methylenomycin production and its regulation are carried on the giant linear plasmid SCP1. The sequencing of the entire 356-kb SCP1 plasmid allowed bioinformatics analyses to be applied to the assignment of putative roles in methylenomycin biosynthesis for several of the enzymes encoded within the methylenomycin biosynthetic gene cluster. However, experimental evidence to support the proposed roles of several of these enzymes has yet to be obtained, while the roles of some of the proteins encoded by the cluster remain unclear. Here, work towards understanding the biosynthesis as well as the mode of action of the methylenomycin antibiotics is reported. In particular, the roles of MmyO and MmyF in the epoxidation of methylenomycin C to produce methylenomycin A are demonstrated via feeding of methylenomycin C to a methylenomycin-resistant derivative of S. coelicolor M145 expressing mmyO and mmyF. A putative butenolide intermediate in the pathway, believed to derive from a MmyD-catalysed condensation of acetoacetyl-MmyA with a pentulose, was identified in S. coelicolor strains expressing the methylenomycin biosynthetic gene cluster. The pattern of incorporation of [U-13C]-D-ribose into the putative butenolide intermediate was similar to that observed for methylenomycin C, indicating the former could indeed be a precursor to the latter. A putative intermediate of the pathway, pre-methylenomycin C, accumulating in a mmyE mutant strain, and its lactone form, pre-methylenomycin C lactone, were shown to be 16 and 256 times, respectively, more potent than methylenomycin A, against methicillin-resistant Staphylococcus aureus (MRSA). Expression of the methylenomycin resistance determinant (mmr) in Streptomyces species also confers no resistance against these two putative intermediates unlike methylenomycin A, the final antibiotic product of the pathway. Investigations into the mechanism of action of methylenomycin antibiotics with luciferase and β-galactosidase pathway-specific promoter-reporter fusion strains strongly suggest that the methylenomycins exert their antibiotic effects in bacteria primarily by targeting the biosynthesis of cell wall peptidoglycan, consistent with their activities mainly against Gram-positive strains. This is the first report of the mode of action of methylenomycin family of antibiotics.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:731398 |
| Date | January 2017 |
| Creators | Idowu, Gideon Aina |
| Publisher | University of Warwick |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://wrap.warwick.ac.uk/96060/ |
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