Introduction

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Methylenomycin A (1) is an unusual antibiotic produced by the model Actinomycete Streptomyces coelicolor A3(2) (Figure 1), (1−3) with a wide spectrum of antibiotic activity, including against diverse Gram-positive bacteria and Gram-negative Proteus spp. (4) The structurally related metabolites methylenomycin C (2), methylenomycin B (3) and xanthocidin (4) (Figure 1) have been isolated from several Streptomyces spp. (5−7)

Figure 1

Figure 1. Structures of methylenomycin A (1), methylenomycin C (2), methylenomycin B (3), and xanthocidin (4).

Incorporation experiments with isotope-labeled precursors have identified the metabolic origin of the methylenomycins and shown that 2 is a precursor of 1. (8−10) Furthermore, sequencing of the S. coelicolor A3(2) giant linear plasmid SCP1 identified the methylenomycin biosynthetic gene cluster. (11) Based on sequence comparisons, 13 proteins encoded by this gene cluster are proposed to play a role in methylenomycin biosynthesis (Figure 2 and Table S1). Three genes (mmfL, mmfP, and mmfH) flanking the cluster of biosynthetic genes direct the production of the methylenomycin furans (MMFs), a group of hormones that induce methylenomycin production. (10−14) The nine carbon atoms of 1 and 2 derive from two molecules of acetate and a molecule of ribose (Figure 2), (8−10) and MmyD shows 47% similarity to AvrD, which catalyzes the condensation of beta-ketoacyl thioesters with xylulose in syringolide biosynthesis. (15) Together, these observations led us to propose that MmyD catalyzes the condensation of acetoacetyl-MmyA (assembled from acetyl- and malonyl-CoA by MmyC and a malonyltransferase borrowed from primary metabolism) with a pentulose, forming a butenolide intermediate that gets elaborated to 2, which undergoes epoxidation catalyzed by MmyF and MmyO to form 1 (Figure 2). (10)

Figure 2

Figure 2. (A) Organization of the S. coelicolor methylenomycin biosynthetic gene cluster. The 13 genes implicated in methylenomycin biosynthesis are colored red. mmyD, mmyE, mmyO, and mmyF were deleted in this study; they encode enzymes with similarity to putative butenolide synthases, flavin-dependent oxidoreductases, flavin-dependent monooxygenases, and flavin reductases, respectively. (B) Proposed pathway for the biosynthesis of methylenomycins A (1) and C (2) in S. coelicolor. MCAT = malonyl-CoA acyl transferase from primary metabolism. The previously reported site(s) of 13C-labeled ribose and acetate incorporation are highlighted in pink and blue, respectively, (8−10) and are consistent with the proposal that MmyD catalyzes condensation of an acetoacetyl thioester with a pentulose.

Although the pathway we propose for the biosynthesis of 1 is plausible, experimental evidence to support the hypothetical roles played by proteins encoded by the mmy gene cluster has hitherto been lacking. Here, we report in-frame deletion of four putative biosynthetic genes (mmyD, mmyE, mmyO, and mmyF) in a cosmid containing the entire methylenomycin biosynthetic gene cluster, in parallel with the deletion of mmyR to boost methylenomycin titers. The cosmids were integrated into the chromosome of S. coelicolor M145, which lacks SCP1, and the production of methylenomycin-related metabolites in each strain was analyzed. The results of these experiments were consistent with the proposed roles of MmyD, MmyF, and MmyO in the methylenomycin biosynthesis, and further experiments confirmed that MmyF and MmyO catalyze the conversion of 2 to 1 using molecular oxygen. Two novel compounds, premethylenomycin C lactone 5 and premethylenomycin C 6 (Figure 4), accumulated in the mmyE mutant, suggest MmyE participates in the formation of the exomethylene group in 2, via the elimination of water from 6. Surprisingly, 5 and 6 were significantly more active than 1 and 2 against various Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA). The low MIC values for 5 against MRSA and Enterococcus faecium (1–2 μg/mL) suggest that it may provide a promising starting point for the development of new antibiotics to tackle antimicrobial resistance.

Results and Discussion

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Construction and Expression of Mutated mmy Gene Clusters

Cosmid C73–787, containing the entire mmy gene cluster in addition to an integrative cassette that enables it to insert into the phage C31 attB site of Streptomyces chromosomes, (12,16) was used to construct the desired mutants. S. coelicolor M145 containing this plasmid produces the methylenomycins. (12) To investigate the biosynthetic roles of mmyD, mmyE, mmyO, and mmyF, each of these genes was separately replaced in C73–787 by an apramycin resistance cassette using PCR-targeting (Figure S1 and Table S2). (17) The resistance cassette was then excised, leaving an 81 bp in-frame “scar” sequence between the start and stop codon of each gene.

MmyR represses the expression of the mmy gene cluster. (13) Consequently, the deletion of mmyR boosts methylenomycin A titers in S. coelicolor. (13) We therefore replaced mmyR with the apramycin resistance cassette in both the starting cosmid and each of the mutant cosmids (Figure S1). This also enabled selection for each construct upon transformation of E. coli ET12567/pUZ8002.

S. coelicolor M145 was transformed with each of the cosmids, and DNA integration was confirmed by PCR (Table S3 and Figures S2,S3). The resultant strains were grown on supplemented agar minimal medium containing a higher phosphate concentration than typical Streptomyces fermentation media, such as R2YE. Increased phosphate has been shown to inhibit prodiginine and actinorhodin production in S. coelicolor without affecting the production of the methylenomycins. (2) This increases the precursor supply for the biosynthesis of the methylenomycins (and related metabolites), facilitating purification and spectroscopic analysis. The profile of metabolites produced by each strain was analyzed by LC-MS.

Deletion of mmyD Abolishes Methylenomycin Production

As anticipated, S. coelicolor W89, containing the ΔmmyR derivative of C73–787, produced 1 and 2 in good titers (Figure 3). In contrast, the production of 1 and 2 was abolished in S. coelicolor W95, which contains the ΔmmyD/ΔmmyR derivative of C73–787, and no methylenomycin-related metabolites could be detected in this mutant (Figure 3). Transformation of W95 with an integrative plasmid containing mmyD under the control of the constitutive ermE* promoter, resulting in S. coelicolor W118, restored methylenomycin production (Table S3 and Figure S4). This is consistent with our previous proposal that MmyD catalyzes the condensation of a pentulose with acetoacetyl-MmyA at an early stage in methylenomycin biosynthesis. (10)

Figure 3

Figure 3. Extracted ion chromatograms at m/z = 139.0700 ± 0.005 (corresponding to the [M-CO2+H]+ ion of 1) and m/z = 167.070 ± 0.005 (corresponding to the [M + H]+ ions of 2 and 5 and the [M-H2O+H]+ ion of 6) from positive ion mode LC-MS analyses of neutral extracts (pH 5.5) of S. coelicolor M145 (bottom), W95 (second from bottom), W86 (third from bottom), W89 (third from top), W100 (second from top), and W108 (top).

Novel Metabolites Accumulate in the mmyE Mutant

The production of 1 and 2 was greatly reduced in S. coelicolor W86 (containing the ΔmmyE/ΔmmyR derivative of C73–787) and two new metabolites, absent from cultures of both S. coelicolor W89 and M145, were detected in neutral extracts (Figure 3). One exhibited a λmax of 245 nm and had the molecular formula C9H10O3 (m/z = 167.0701 and 189.0518; calculated m/z for C9H11O3+ and C9H10O3Na+ = 167.0708 and 189.0521, respectively; Figure S5). The other had a λmax of 236 nm and the molecular formula C9H12O4 (m/z = 167.0705, 185.0814, and 207.0632; calculated m/z for C9H11O3+, C9H13O4+ and C9H12O4Na+ = 167.0708, 185.0807, and 207.0629, respectively). In negative ion mode, the latter gave rise to an ion with m/z = 183.0650 corresponding to [M-H]− for a species with the molecular formula C9H12O4.

In acidified (pH 3) extracts of S. coelicolor W86, the compound with a molecular formula of C9H12O4 could not be detected (Figure S6). The compound with the molecular formula C9H10O3 was extracted from acidified large cultures using ethyl acetate and purified by preparative HPLC. Analysis of 1H, 13C, COSY, HSQC, HMBC, and ROESY NMR spectra (Figures S7–S12) showed this compound has structure 5 (Figure 4). The CD spectra of 1, 2, and 5 are very similar (Figure S13), leading us to conclude that all three compounds have identical absolute configurations.

The compound with the molecular formula C9H12O4 could not be isolated in sufficient quantity from neutral extracts of S. coeliocolor W86 to permit structure elucidation using NMR spectroscopy. However, compound 5 could be converted to this compound with NaOH in THF. 1H, 13C, COSY, HSQC, HMBC, and ROESY NMR spectroscopic analyses (Figures S14–S19) showed this compound has structure 6 (Figure 4).

Figure 4

Figure 4. Summary of correlations observed (blue = strong, red = weak) in HMBC and ROESY spectra of premethylenomycin C lactone (5) and the structure of premethylenomycin C (6).

Like other γ-hydroxy acids, accumulated 6 probably undergoes a slow spontaneous lactonization to form 5. As expected, this process is accelerated at a lower pH. Dehydration of 6 would yield 2. Thus, we propose the name premethylenomycin C for 6. Stereoelectronic constraints prevent 5, which we named premethylenomycin C lactone, from being converted directly to 2. The protein encoded by mmyT shows sequence similarity to type II thioesterases, and we propose that it catalyzes the hydrolytic ring opening of 5 to form 6.

Our data indicate that MmyE catalyzes the conversion of 6 to 2. MmyE has 32% sequence identity with PlmM, a flavin-dependent enoylreductase from Streptomyces sp. HK803 involved in the assembly of the cyclohexanecarboxyl-CoA starter unit for phoslactomycin biosynthesis. (18) Interestingly, flavoenzymes usually catalyze redox reactions, but the conversion of 6 to 2 does not involve a net change in oxidation state. Other flavoenzymes have been reported to catalyze nonredox reactions. (19) The bound flavin is proposed to play a purely structural role in these enzymes. The MmyE-catalyzed conversion of 6 to 2 likely proceeds via an E1cB mechanism involving a basic residue in the active site that generates an enolate intermediate, which undergoes elimination of hydroxide promoted by an acidic active site residue (Scheme 1).

Scheme 1

Scheme 1. MmyE-Catalyzed Conversion of 6 to 2 Likely Proceeds via an E1cB Mechanism Involving a Basic (B) and an Acidic (A-H) Active Site Residue

Methylenomycin A Is Not Produced by the mmyO and mmyF Mutants

The production of 1 but not 2 was abolished in S. coelicolor W100 and 108 (containing the ΔmmyO/ΔmmyR and ΔmmyF/ΔmmyR derivatives of C73–787, respectively) (Figure 3). Two novel metabolites with the same molecular formula (C9H12O3; Figure S20) were detected in extracts of cultures grown for more than 3 days. Both compounds were purified from ethyl acetate extracts of acidified supernatants from 7-day cultures of S. coelicolor W108. Analysis of 1H, COSY, HSQC, and HMBC NMR spectra (Figures S21–S28) showed the two compounds are diastereomers with structures 7 and 8. These are likely shunt metabolites derived from unselective reduction of exomethylene in 2 (Figure 5). They were named methylenomycins D1 (7) and D2 (8), respectively. Consistent with the shunt metabolite hypothesis, decreased levels of 2 were observed in cultures accumulating 7 and 8, and in cultures grown for more than 72 h, 2 could no longer be detected. Comparison of the CD spectra for 7 and 8 with those of 6, 2, and 1 suggested these metabolites all have the same absolute configuration at C1 (Figure S13).

Figure 5

Figure 5. Structures of methylenomycins D1 (7) and D2 (8) isolated from S. coelicolor W108 proposed to derive from 2.

MmyO and MmyF Convert Methylenomycin C to A

MmyO has 43% sequence identity to LimB, a monooxygenase that catalyzes FADH2–dependent epoxidation of the 1,2 double bond of limonene in Rhodococcus erythropolis, using molecular oxygen as cosubstrate. (20) MmyF shows sequence similarity to NADPH-dependent flavin reductases. The inability of S. coelicolor W108 (containing mmyO but not mmyF) to produce 1 indicates there is a strict requirement for MmyF to supply FADH2 to MmyO (Figure 3). Attempts to overproduce MmyO and MmyF in E. coli were unsuccessful. Thus, we elected to coexpress mmyO and mmyF in S. coelicolor M145. The two genes were amplified from C73_787 and cloned into pOSV556t under the control of the strong constitutive ermE* promoter (Figure S29). The resulting vector was integrated into the chromosome of S. coelicolor M145 via intergenic conjugation from E. coli ET12567 containing pUZ8002, resulting in S. coelicolor W110.

Purified 2 was fed to cultures of S. coelicolor W110 and M145. However, both died presumably because these strains are sensitive to 1. To circumvent this problem, mmr, a putative efflux pump that has been shown to confer methylenomycin resistance, was amplified from C73_787 (Table S4) and cloned into the multicopy plasmid pIJ86 under the control of the ermE* promoter (Figure S30). The resulting construct was introduced into S. coelicolor M145 and W110 via intergenic conjugation from E. coli, resulting in S. coelicolor W301 and W302, respectively. To examine methylenomycin resistance, filter paper discs saturated with 1 were placed on plates inoculated separately with S. coelicolor M145, W110, W301, and W302. After 5 days of incubation, a sizable zone of growth inhibition was observed for S. coelicolor M145 and W110, whereas the growth of S. coelicolor W301 and W302 was unaffected (Figure S30). This confirmed that S. coelicolor W301 and W302 are resistant to 1, the putative product of MmyO/MmyF catalysis.

S. coelicolor W301 and W302 were grown on AlaMM (pH 5.0) agar medium for 2 days, and 2 was added. After 3 days of further incubation, the agar was extracted with methanol, and the concentrated extracts were analyzed using LC-ESI-Q-ToF-MS. This showed that 1 was produced by S. coelicolor W301 (containing mmyO, mmyF, and mmr) but not W302 (containing just mmr), confirming MmyO and MmyF together catalyze the conversion of 2 to 1 (Figure 6).

Figure 6

Figure 6. Extracted ion chromatograms (EICs) at m/z = 183.065 ± 0.005 corresponding to [M + H]+ for 1 from UHPLC-ESI-Q-ToF-MS analyses of methanol extracts from S. coelicolor W302 (bottom panel) and W301 (middle panel) fed with 2. The top panel shows the EIC at m/z = 183.065 ± 0.005 for an authentic standard of 1.

To further characterize the reaction catalyzed by MmyO/MmyF, we investigated the origin of the oxygen atom in the epoxide group of 1, which we hypothesized derives from molecular oxygen. S. coelicolor W89 was grown under an 18O2 atmosphere and UHPLC-ESI-Q-ToF-MS analysis of culture extracts revealed just over 50% incorporation of a single 18O atom into 1, whereas no 18O incorporation into 2 was observed (Figure S31). The lower than 100% incorporation of 18O into 1 is likely due to the incomplete exclusion of air from the agar cultures. Because 1 is derived from 2 by converting a carbon–carbon double bond into an epoxide and no 18O2 is incorporated into 2, the epoxide oxygen of 1 must be derived from molecular oxygen.

The conversion of 2 to 1 likely proceeds via the reaction of MmyO-bound FADH2, generated by the MmyF-mediated reduction of FAD, with O2 (Scheme 2). The resulting C4a-peroxy-flavin can add to C5 of 2, creating an enolate intermediate that collapses via the nucleophilic attack of C4 on the peroxide. An analogous mechanism has been proposed for the epoxidation of electron-deficient double bonds by other flavoenzymes. (20)

Scheme 2

Scheme 2. Proposed Mechanism for the Conversion of 2 to 1

Substrate Tolerance of MmyO

To investigate whether MmyO has a broad substrate scope, we fed 5, 6, 7, and 8, all of which have the same cyclopentenone core as 2, to S. coelicolor W301 and W302 (as a negative control). LC-MS analysis of culture extracts (Figures S32,S33) showed that 6 was the only compound turned over by S. coelicolor W301 to a monooxygenated product. Reexamination of S. coelicolor W89 culture extracts showed that this strain produces the same monooxygenated derivative of 6. Thus, this metabolite was purified by HPLC from large-scale cultures of W89. HRMS showed this compound has the molecular formula C9H12O5 (m/z calculated for [C9H12O5Na]+ = 223.0576, measured m/z = 223.0577; Figure S34) consistent with structure 9, the anticipated product of the MmyO-catalyzed epoxidation of 6. Surprisingly, 1H, COSY, HSQC, and HMBC NMR spectroscopic analyses (Figures S35–S38) led us to conclude that this compound has structure 10, which can be formed from 9 via an intramolecular rearrangement (Scheme 3). Simple commercially available analogues of 2, such as cylopentenone and cyclohexanone, were also fed to S. coelicolor W301, but no monooxygenated derivatives could be detected.

Scheme 3

Scheme 3. Proposed Mechanism for Formation of 10 from 9, the Presumed Product of MmyO-Catalysed Epoxidation of 6

Antimicrobial Activity of Intermediates, Products, and Shunt Metabolites

The antimicrobial activity of 5, 6, 7, and 8 against diverse Gram-positive bacteria, Gram-negative bacteria, and Candida albicans was compared to that of 1 and 2. Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) for active compounds were determined using standard procedures. (21−23)

None of the compounds were active against Gram-negative bacteria (Table 1), presumably because they either are unable to penetrate the outer membrane or are rapidly exported via efflux. 7 and 8 had no detectable activity against Gram-positive bacteria or C. albicans up to a maximum concentration of 512 μg/mL (Table 1). This is consistent with a previous report that the reduction of the exomethylene group in methylenomycin A results in no biological activity at concentrations up to 400 μg/mL. (4c) The exomethylene group in 1 and 2 thus appears to be a key pharmacophore.

Table 1. Antimicrobial Activitya of Intermediates, Products, and Shunt Metabolites in Methylenomycin Biosynthesis

MIC (MBC) in μg/mL Organism125678 Gram-positive bacteria Staphylococcus aureus DSM 21979 (MRSA)256(256)512(512)1(1)16(16)– Staphylococcus aureus R34256(256)256(256)2(2)16(16)– Bacillus subtilis MarburgT192(192)192(192)1(1)9.8(9.8)– Streptomyces coelicolor M14564(64)64(64)1(1)8(8)– Streptomyces albus J1074256(256)128(128)0.5 (0.5)0.5 (0.5)– Enterococcus faecium U0317256(256)512(512)2(2)32(64)– Enterococcus faecium 64/3–2(2)8(16)– Yeast Candida albicans SC 5314256(256)384(384)9.8(64)9.8(64)–

a

A dash indicates that no activity was observed at concentrations up to 512 μg/mL. No activity was observed at concentrations up to 512 μg/mL against several Gram-negative bacteria, including Escherichia coli SY327, DSM29239, DSM26371, RVH1, BCC1391, Burkholderia metallica DSM23519 and DSM16087 Serratia plymuthica RVH1, Ralstonia mannitolilyticaBCC1391, Burkholderia metallica DSM23519 and Burkholderia ambifaria DSM16087.

In our hands, 2, which was previously reported to be inactive against B. subtilis, (5) had similarly modest levels of activity as 1 against Gram-positive bacteria and C. albicans (Table 1). Surprisingly, 6 was an order of magnitude more active across the board than 1 and 2 (Table 1). Even more surprisingly, 5 was two orders of magnitude more active than 1 and 2 against all Gram-positive bacteria tested and had a similar level of activity to 6 against C. albicans (Table 1). Given that 7 has no antimicrobial activity and the only structural difference between this and 6 is that the former lacks the primary hydroxyl group, it seems unlikely that the enone group common to compounds 2, 5, 6, 7, and 8 plays a role in the antimicrobial mechanism of action.

The MIC values for 5 of 1 and 2 μg/mL against the clinical isolates Staphylococcus aureus DSM 21979 and Enterococcus faecium U0317, respectively, are particularly noteworthy. S. aureus DSM 21979 is resistant to methicillin and aminoglycosides, whereas E. faecium U0317 is resistant to multiple classes of antibiotics, including chloramphenicol, macrolides, aminoglycosides, β-lactams, and tetracyclines. E. faecium U0317 and 64/3 are both susceptible to vancomycin, an antibiotic widely used for the treatment of enterococcal infections, with MBCs of 64 and 128 μg/mL, respectively. (24−26) Strikingly, 5 has an MBC of 2 μg/mL against both these strains.

The acquisition of vancomycin resistance is a significant problem for the treatment of E. faecium infections. (24−26) To investigate whether E. faecium 64/3 can evolve resistance to 5, using vancomycin as a control, it was subjected to sequential passage through increasing concentrations of the antibiotics over a period of 28 consecutive days. This resulted in mutants with an 8-fold increase in MIC for vancomycin (from 4 to 32 μg/mL), whereas the MIC for 5 remained unchanged (2 μg/mL). Thus, E. faecium appears to be unable to easily develop resistance to 5.

The fact that 5 displays excellent antimicrobial activity, despite lacking the exomethylene group responsible for the weaker activity of 1 and 2, demonstrates that it employs a different pharmacophore. γ-Hydroxy acids are known to spontaneously lactonize, although this is often slow under neutral conditions. Partial lactonization of 6 to form 5 during antimicrobial activity assays could explain the observation of lower but still significant activity for the latter. These considerations suggest that the γ-butyrolactone may be the pharmacophore in 5.

Conclusions

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In this study, we employed a genetic approach to investigate the biosynthetic role of putative enzymes encoded by four genes in the S. coelicolor methylenomycin BGC. Abolition of the production of all methylenomycin-related metabolites in the mmyD mutant is consistent with our previous proposal that this gene encodes an AvrD-like enzyme responsible for condensing a β-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 the mmyE mutant suggest they are biosynthetic intermediates. We propose that the lactone in 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 in 5 by MmyT, which shows sequence similarity to type II thioesterases, would yield 6 (Scheme 4). Conversion of 6 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 of 1 and 2 are still produced by the mmyE mutant suggests that another enzyme encoded by a gene outside the methylenomycin BGC can also catalyze the conversion of 6 to 2, albeit inefficiently. A series of experiments demonstrate that MmyO and MmyF together catalyze the final step in methylenomycin A (1) biosynthesis–epoxidation of the tetrasubstituted double bond in 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 epoxidize 6 to make unstable product 9 that undergoes a spectacular series of rearrangements to form 10. Finally, two novel methylenomycin-related metabolites 7 and 8 were observed to accumulate in mmyF and mmyO mutants in addition to the parent strain when grown for extended periods. These appear to result from nonspecific reduction of the exomethylene group in 2 (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

Scheme 4. Updated Proposed Pathway for Methylenomycin Biosynthesis in S. coelicolor A3(2), Based on the Isolation and Structural Characterization of Metabolites 5, 6, 7, 8, and 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 antibiotic 5, with the subsequent acquisition of the mmyT, mmyE, mmyO, and mmyF genes diverting the pathway first to 2 and then 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 of 5 against drug-resistant clinical isolates of S. aureus and E. faecium coupled with its relatively simple structure and the apparent difficulty of evolving resistance to this compound in the latter, are all notable. It suggests that 5 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 of 5 has been developed in collaboration with the Lupton group. (27) This should enable the creation of diverse analogues that can be used to probe the structure–activity relationship and mechanism of action.

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jacs.5c12501.

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Author Information

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Gregory L. Challis - Department of Chemistry, University of Warwick, Coventry CV4 7AL, U.K.; Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Clayton, Victoria 3800, Australia; ARC Centre of Excellence for Innovations in Peptide and Protein Science, Monash University, Clayton, Victoria 3800, Australia; https://orcid.org/0000-0001-5976-3545; Email: [email protected]

Christophe Corre - Department of Chemistry, University of Warwick, Coventry CV4 7AL, U.K.; School of Life Sciences, University of Warwick, Coventry CV4 7AL, U.K.

Gideon A. Idowu - Department of Chemistry, University of Warwick, Coventry CV4 7AL, U.K.; Present Address: Department of Chemistry, Federal University of Technology, PMB 704 Akure, Nigeria

Lijiang Song - Department of Chemistry, University of Warwick, Coventry CV4 7AL, U.K.

Melanie E. Whitehead - Department of Chemistry, University of Warwick, Coventry CV4 7AL, U.K.; Present Address: Nottingham University Hospitals; Queens Medical Center, Derby Road, Nottingham, NG7 2UH, U.K

C.C. and G.A.I. contributed equally.

The authors declare the following competing financial interest(s): G.L.C. is a Non-Executive Director, shareholder, and consultant of Erebagen Ltd.

Acknowledgments

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We thank Dr. Nikola Chmel for assistance with measuring CD spectra. This work was supported by a grant from the U.K. BBSRC (grant ref: BB/E008003/1) and the EU integrated project Actinogen (Contract No. 005224). GAI was the recipient of a Chancellor’s International Scholarship from the University of Warwick. GLC and CC were the recipients of a Wolfson Research Merit Award and a University Research Fellowship from the Royal Society (grant nos. WM130033 and UF090255).

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1. Miryam Naddaf. Powerful new antibiotic that can kill superbugs discovered in soil bacteria. Nature 2025, *5 *https://doi.org/10.1038/d41586-025-03595-3