Data availability
Data supporting the findings of this study are available within the paper and its Supplementary Information or have been deposited in the indicated public repositories. The whole-genome sequence of Streptomyces sp. WAC05950 is available in the NCBI GenBank under accession number RQJB00000000.1. Sequenced Streptomyces genomes used in the analyses shown in Extended Data Fig. 1 are available in GenBank under BioProject PRJNA1461987 with the accession numbers GCA_057371075.1, GCA_057371055.1, GCA_057371035.1, GCA_057371015.1, GCA_057370995.1 and GCA_057370965.1. Publicly available Streptomyces genome assemblies used for genome mining, comparative genomics and phylogenetic analyses were obtained from the NCBI GenBank/RefSeq databases. Phylogenetic alignments, DIAMOND databases and analysis outputs generated in this study are available on Zenodo70 (https://doi.org/10.5281/zenodo.19899453). Source data are provided for Figs. 1–4 and Extended Data Figs. 5–10. Source data are provided with this paper.
Code availability
Custom Python scripts used for the extraction of AciB protein sequences, DIAMOND output parsing and biosynthetic gene cluster analyses are available on Zenodo70 (https://doi.org/10.5281/zenodo.19899453).
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Acknowledgements
We thank W. Guan for the pSUC01 plasmid.
Funding
This research was supported by funds to E.D.B., including Tier 1 Canada Research Chair award, a Foundation Grant from the Canadian Institutes of Health Research (CIHR; FRN 143215) and a grant from the Ontario Research Fund (RE09-047). G.D.W. is supported by CIHR grants FDN 148463 and PJT190298. M.X. is supported by the Tianjin Synthetic Biotechnology Innovation Capacity Improvement Project (TSBICIP-CXRC-065 and TSBICIPCXRC-076) and the Hundred Talents Program of Chinese Academy of Sciences. M.M.T. was supported by a CIHR Canada Graduate Scholarship (CGS-D). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.
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Extended data figures and tables
Extended Data Fig. 1 CORASON-based phylogenetic analysis of megacluster-containing Streptomyces strains from the WAC collection.
The tree was generated using AciB, a core enzyme from the acidomycin biosynthetic gene cluster, to compare homologous clusters across strains. Streptomyces sp. WAC05950 was used as the reference genome (yellow star).
Extended Data Fig. 2 TAR cloning of the megacluster and svn BGC.
a, Schematic representation of cloning the megacluster from the genome of WAC05950 into pCGW, resulting in pADSK. b, Schematic representation of subcloning and refactoring the svn BGC from pADSK using TAR. ermEp* promoter was introduced to overexpress the svn BGC, and fd terminator was introduced after svnA for transcription termination.
Extended Data Fig. 3 Metabolic profiling of the truncated version of the anti-biotin megacluster.
a, Schematic of the engineered and refactored pADSK plasmids. Each subcluster was colored accordingly to clarify the deletion region. The dashed lines indicate the deleted regions from the pADSK plasmid. The × in pADSK∆svn2 indicates the presence of an impaired aci BGC. b, Base peak chromatogram (BPC) profile of S. coelicolor M1154 strain expressing pADSK derivatives. Each compound was labelled according to the BPC as: A: dapamycin A; α: α-Me-KAPA; β: 2,5-di-(2-methylhexanoic acyl)-3-methylimidazole; γ: N-Acetyl-α-Me-KAPA; δ: 2,5-dimethyl-3,6-di-(2-methylhexanoic acyl)pyrazine; and *: acidomycin.
Extended Data Fig. 4 Target deletion of aciB and bioAB from the chromosome of WAC05950.
Schematic representations of aciB (a) and bioAB (b) gene deletions using pSUC01 plasmid-mediated homologous recombination. c, Gel electrophoresis of PCR products amplified from the genomic DNA of WAC05950 and ΔaciB and ΔbioAB mutant strains. d, Extracted ion chromatogram of acidomycin ([M+Na]+=240.0680) from the fermentation broth of WAC05950 and ΔaciB and ΔbioAB mutant strains. aciB deletion abolished the production of acidomycin in WAC05950ΔaciB, whereas WAC05950ΔbioAB retained acidomycin production. For gel source data, see Supplementary Fig. 1.
Extended Data Fig. 5 Co-production of megacluster-derived natural products in the wild-type producer.
Stravidin S2, acidomycin, dapamycin A, dapamycin B and α-Me-KAPA are co-produced in the WT megacluster-containing strain Streptomyces sp. WAC05950 grown in SMM medium over 7 days. EICs for each compound were extracted from the conditioned media. Purified natural products from the heterologous expression strain acted as standards for quantification. Data are representative of three biological replicates and presented as mean ± SEM.
Extended Data Fig. 6 Antimicrobial activity of the isolated compounds from the megacluster.
a, MIC values of the isolated compounds against Gram-negative and Gram-positive bacteria, and mycobacteria. b, Growth kinetics of E. coli BW25113 and M. smegmatis mc2155 in the absence (grey) and presence (blue) of 2X MIC of dapamycin B (16 µg/mL and 0.5 µg/mL, respectively). Data are representative of three biological replicates and presented as mean ± SEM. c, The organization of the biotin operon and its transcriptional regulation by BirA-biotinyl-5′-AMP complex in E. coli.
Extended Data Fig. 7 Activity suppression profiles of stravidin S2, acidomycin, dapamycin B and α-Me-KAPA by biotin and intermediates of its biosynthesis.
a-e, Checkerboard broth microdilution assays of stravidin S2, acidomycin, dapamycin B and α-Me-KAPA against KAPA, DAPA, DTB and biotin in E. coli BW25113 (a-c, e) and M. smegmatis mc2155 (d). Data are representative of at least three biological replicates.
Extended Data Fig. 8 Genetic evidence implicates BioD as the target of dapamycins.
a, Theophylline-induced overexpression of M. smegmatis mc2155 bioFADB in the corresponding auxotrophic E. coli BW25113 ΔbioFADB strains. M9 minimal agar with and without biotin supplementation served as positive and negative controls for growth of the auxotrophs, respectively. b, Activity of stravidin S2, dapamycin B, α-Me-KAPA and acidomycin against M. smegmatis overexpressing bioFAD in the presence or absence of theophylline. “−” and “+” indicate cultures grown without or with 4 mM theophylline, respectively. Ethambutol, ciprofloxacin, and amikacin served as negative controls. Data are shown as mean of two biological replicates.
Extended Data Fig. 9 Pairwise combinations of stravidin S2, acidomycin, dapamycin B and α-Me-KAPA in multiple Gram-negative species and M. smegmatis.
a, Checkerboard broth microdilution assays of stravidin S2 against acidomycin in A. baumannii ATCC 17978, K. pneumoniae ATCC 43816 and E. coli C0244. b, Checkerboard broth microdilution assays of dapamycin B against acidomycin, α-Me-KAPA and stravidin S2 in E. coli BW25113. c, Checkerboard broth microdilution assays of pairwise combinations between stravidin S2, acidomycin and α-Me-KAPA in M. smegmatis mc2155. Checkerboard and FICI data are representative of at least three biological replicates.
Extended Data Fig. 10 Expanded evaluation of megacluster-encoded compounds in vivo and in mouse serum assays.
a, b, Bacterial load measured in blood and organs following compound treatment of an E. coli C0244 systemic infection in CD-1 mice pre-treated with streptavidin (2 mg/kg, 1 h prior to infection). Groups of mice were treated by a single intraperitoneal (IP) injection of vehicle (grey; n = 18), stravidin S2 (50 mg/kg, pink; n = 8), acidomycin (50 mg/kg, orange; n = 6), dapamycin B (50 mg/kg, lavender; n = 8), α-Me-KAPA (50 mg/kg, green; n = 7), cefotaxime (50 mg/kg, light blue; n = 6) or meropenem (50 mg/kg, light pink; n = 6) 1 h after infection. The infection progressed for 6 h. Each point represents an individual mouse. The centre line delineates the median, the box limits mark the upper and lower quartiles, and the whiskers depict the range. One-way ANOVA with Holm-Šídák multiple comparisons test. NS, not significant. LOD, limit of detection. c, Checkerboard microdilution assays of a combination between stravidin S2 and α-Me-KAPA in E. coli C0244 in CD-1 mouse serum. Red regions represent higher cell density. Checkerboard data are representative of three biological replicates. d, Bacterial load measured in organs following compound treatment of an E. coli C0244 systemic infection in CD-1 mice pre-treated with streptavidin (2 mg/kg, 1 h prior to infection). Groups of mice were treated by a single intraperitoneal (IP) injection of vehicle (grey, n = 18), stravidin S2 (1 mg/kg, pink; n = 7), α-Me-KAPA (25 mg/kg, green; n = 4) or a combination of both compounds (teal, n = 6) 1 h after infection. The infection progressed for 6 h. Each point represents an individual mouse. The centre line delineates the median, the box limits mark the upper and lower quartiles, and the whiskers depict the range. One-way ANOVA with Holm-Šídák multiple comparisons test. NS, not significant.
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Gordzevich, R., Xu, M., Wang, W. et al. A Streptomyces megacluster encodes synergistic biotin-targeting antibiotics. Nature 655, 478–486 (2026). https://doi.org/10.1038/s41586-026-10647-9
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DOI: https://doi.org/10.1038/s41586-026-10647-9