In a report titled โDiscovery of Late Intermediates in Methylenomycin Biosynthesis Active against DrugโResistant GramโPositive Bacterial Pathogens,โ published October 27th, 2025, in the Journal of the American Chemical Society (ACS), researchers uncovered previously overlooked intermediate compounds in the biosynthetic pathway of the antibiotic Methylenomycin Aโproduced by the soil bacterium Streptomyces coelicolor. They found that one intermediate, premethylenomycinโฏC lactone, is one to two orders of magnitude more potent against resistant Gramโpositive pathogens such as Staphylococcus aureus and Enterococcus faecium than the final antibiotic product. Genetic deletion of biosynthetic enzymes (mmyD, mmyE, mmyF, mmyO) helped map the pathway and confirmed how these intermediates arise. The findings highlight a new strategy in antibiotic discovery: mining intermediate metabolites from known biosynthetic pathways to combat antimicrobial resistance. The ACS writes:
The methylenomycins are highly functionalized cyclopentanone antibiotics produced byย Streptomyces coelicolorย A3(2). A biosynthetic pathway to the methylenomycins has been proposed based on sequence analysis of the proteins encoded by the methylenomycin biosynthetic gene cluster and the incorporation of labeled precursors. However, the roles played by putative biosynthetic enzymes remain experimentally uninvestigated. Here, the biosynthetic functions of enzymes encoded byย mmyD,ย mmyO,ย mmyF,ย andย mmyEย were investigated by creating in-frame deletions in each gene and investigating the effect on methylenomycin production. No methylenomycin-related metabolites were produced by theย mmyDย mutant, consistent with the proposed role of MmyD in an early biosynthetic step. The production of methylenomycin A, but not methylenomycin C, was abolished in theย mmyFย andย mmyOย mutants, consistent with the corresponding enzymes catalyzing the epoxidation of methylenomycin C, as previously proposed. Expression ofย mmyFย andย mmyOย in aย S. coelicolorย M145 derivative engineered to expressย mmr, which confers methylenomycin resistance, enabled the resulting strain to convert methylenomycin C to methylenomycin A, confirming this hypothesis. A novel metabolite (premethylenomycin C), which readily cyclizes to form the corresponding butanolide (premethylenomycin C lactone), accumulated in theย mmyEย mutant, indicating the corresponding enzyme is involved in introducing the exomethylene group into methylenomycin C. Remarkably, both premethylenomycin C and its lactone precursor were one to two orders of magnitude more active against various Gram-positive bacteria, including antibiotic-resistantย Staphylococcus aureusย andย Enterococcus faeciumย isolates, than methylenomycins A and C, providing a promising starting point for the development of novel antibiotics to combat antimicrobial resistance. […]
In this study, we employed a genetic approach to investigate the biosynthetic role of putative enzymes encoded by four genes in theย S. coelicolorย methylenomycin BGC. Abolition of the production of all methylenomycin-related metabolites in theย mmyDย mutant is consistent with our previous proposal that this gene encodes an AvrD-like enzyme responsible for condensing a ฮฒ-keto-ACP thioester with a pentulose at an early stage in methylenomycin biosynthesis (Scheme 4). The structures of two novel methylenomycin-related metabolites, premethylenomycin C lactone (5) and premethylenomycin C (6), accumulated in theย mmyEย mutant suggest they are biosynthetic intermediates. We propose that the lactone inย 5, which is formed by the MmyD-catalyzed reaction, is carried through subsequent steps catalyzed by MmyG, MmyK, MmyQ, MmyY, and MmyX, resulting in the assembly of the cyclopentanone (Scheme 4). Hydrolysis of the lactone inย 5ย by MmyT, which shows sequence similarity to type II thioesterases, would yieldย 6ย (Scheme 4). Conversion ofย 6ย to methylenomycin C (2) is proposed to be catalyzed by MmyE, which appears to be a redox-inactive flavoenzyme (Scheme 4). The observation that small amounts ofย 1ย andย 2ย are still produced by theย mmyEย mutant suggests that another enzyme encoded by a gene outside the methylenomycin BGC can also catalyze the conversion ofย 6ย toย 2, albeit inefficiently. A series of experiments demonstrate that MmyO and MmyF together catalyze the final step in methylenomycin A (1) biosynthesisโepoxidation of the tetrasubstituted double bond inย 2. MmyO, which we propose is a flavin-dependent monooxygenase supplied with reduced flavin by the reductase MmyF, appears to have a narrow substrate tolerance, although it can epoxidizeย 6ย to make unstable productย 9ย that undergoes a spectacular series of rearrangements to formย 10. Finally, two novel methylenomycin-related metabolitesย 7ย andย 8ย were observed to accumulate inย mmyFย andย mmyOย mutants in addition to the parent strain when grown for extended periods. These appear to result from nonspecific reduction of the exomethylene group inย 2ย (Scheme 4). Overall, these studies afford considerable additional insight into methylenomycin biosynthesis, providing several testable new hypotheses and indicating that future efforts should focus on the mid-pathway roles played by MmyG, MmyK, MmyQ, MmyX, and MmyY.
Scheme 4. Updated Proposed Pathway for Methylenomycin Biosynthesis in S. coelicolorย A3(2), Based on the Isolation and Structural Characterization of Metabolitesย 5,ย 6,ย 7,ย 8, andย 10. Dashed Arrows Indicate Transformations Still Requiring Direct Evidence to Confirm They Are Catalyzed by the Enzymes Indicated
Antimicrobial activity assays of the novel methylenomycin-related metabolites discovered in this work showed that the key biosynthetic intermediate premethylenomycin C lactone (5) is two orders of magnitude more active against diverse Gram-positive bacteria than methylenomycin A (1), the ultimate metabolic product. This suggests that the methylenomycin BGC may initially have evolved to make the potent antibioticย 5, with the subsequent acquisition of theย mmyT, mmyE, mmyO, andย mmyFย genes diverting the pathway first toย 2ย and thenย 1, which may have an alternative biological function. Identification and testing of intermediates in the biosynthesis of other metabolites with weak or no antimicrobial activity may therefore provide a fruitful new approach to antibiotic discovery. The activity ofย 5ย against drug-resistant clinical isolates ofย S. aureusย andย E. faeciumย coupled with its relatively simple structure and the apparent difficulty of evolving resistance to this compound in the latter, are all notable. It suggests thatย 5ย may provide a useful starting point for the development of novel antibiotics to tackle infections caused by multidrug-resistant Gram-positive bacteria. To this end, an expedient and versatile synthesis ofย 5ย has been developed in collaboration with the Lupton group.ย (27)ย This should enable the creation of diverse analogues that can be used to probe the structureโactivity relationship and mechanism of action.