Skip to content
1887

Microbiology Society logo Microbiology

Volume 171, Issue 6

Role of a FAD-dependent monooxygenase in diazo group functionalization of kinamycin in Open Access

Abstract

Kinamycin biosynthesis is a complex process that has been extensively studied over the years, yet specific enzymatic steps continue to be unveiled. A diazo group present in the molecule is responsible for the promising antitumour activity of kinamycins, but its installation in the specific strain has yet to be characterized. In this study, we explore the diazo functionalization of kinamycin in this strain. A FAD-dependent monooxygenase is identified, which is essential for kinamycin biosynthesis. In its absence, stealthin C accumulates instead, likely as a pathway shunt product. Furthermore, as a result of the position of the gene encoding this monooxygenase, named , we also propose new boundaries of the kinamycin biosynthetic gene cluster, resulting in a large cluster spanning over 72 kb. This work paves the way for the continued understanding of the biosynthetic steps that are characteristic of diazo-containing natural products and provides new biocatalysts for molecular engineering and accelerates bioactive compounds production.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): diazo group , FAD-dependent monooxygenase , kinamycin , natural product and Streptomyces
Funding
This study was supported by the:
  • AgreenSkillsPlus (Award FP7609398.0000)
    • Principle Award Recipient: M. VicenteCláudia
  • Agence Nationale de la Recherche (Award ANR-13-BSV6-0009)
    • Principle Award Recipient: BertrandAigle
  • Agence Nationale de la Recherche (Award ANR-11-LABX-0002-01)
    • Principle Award Recipient: BertrandAigle
© 2025 The Authors
  • This is an open-access article distributed under the terms of the Creative Commons Attribution License. This article was made open access via a Publish and Read agreement between the Microbiology Society and the corresponding author’s institution.
Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.001576
2025-06-20
2025-07-10
Loading full text...

Full text loading...

/deliver/fulltext/micro/171/6/mic001576.html?itemId=/content/journal/micro/10.1099/mic.0.001576&mimeType=html&fmt=ahah

References

  1. Watve MG, Tickoo R, Jog MM, Bhole BD. How many antibiotics are produced by the genus Streptomyces?. Arch Microbiol 2001; 176:386–390 [View Article] [PubMed]
     [Google Scholar]
  2. Caffrey P. Dissecting complex polyketide biosynthesis. Comput Struct Biotechnol J 2012; 3:e201210010 [View Article] [PubMed]
     [Google Scholar]
  3. Grasso LL, Martino DC, Alduina R, Grasso LL, Martino DC et al. Production of antibacterial compounds from actinomycetes. IntechOpen 2016 Epub ahead of print 11 February 2016 [View Article]
     [Google Scholar]
  4. Genilloud O. The re-emerging role of microbial natural products in antibiotic discovery. Antonie Van Leeuwenhoek 2014; 106:173–188 [View Article] [PubMed]
     [Google Scholar]
  5. Nivina A, Yuet KP, Hsu J, Khosla C. Evolution and diversity of assembly-line polyketide synthases. Chem Rev 2019; 119:12524–12547 [View Article] [PubMed]
     [Google Scholar]
  6. Helfrich EJN, Ueoka R, Chevrette MG, Hemmerling F, Lu X et al. Evolution of combinatorial diversity in trans-acyltransferase polyketide synthase assembly lines across bacteria. Nat Commun 2021; 12:1422 [View Article] [PubMed]
     [Google Scholar]
  7. Nawrat CC, Moody CJ. Natural products containing a diazo group. Nat Prod Rep 2011; 28:1426–1444 [View Article] [PubMed]
     [Google Scholar]
  8. He H-Y, Niikura H, Du Y-L, Ryan KS. Synthetic and biosynthetic routes to nitrogen-nitrogen bonds. Chem Soc Rev 2022; 51:2991–3046 [View Article] [PubMed]
     [Google Scholar]
  9. Herzon SB, Woo CM. The diazofluorene antitumor antibiotics: structural elucidation, biosynthetic, synthetic, and chemical biological studies. Nat Prod Rep 2012; 29:87–118 [View Article] [PubMed]
     [Google Scholar]
  10. O’Hara KA, Dmitrienko GI, Hasinoff BB. Kinamycin F downregulates cyclin D3 in human leukemia K562 cells. Chem Biol Interact 2010; 184:396–402 [View Article] [PubMed]
     [Google Scholar]
  11. Hasinoff BB, Wu X, Yalowich JC, Goodfellow V, Laufer RS et al. Kinamycins A and C, bacterial metabolites that contain an unusual diazo group, as potential new anticancer agents: antiproliferative and cell cycle effects. Anticancer Drugs 2006; 17:825–837 [View Article] [PubMed]
     [Google Scholar]
  12. Woo CM, Ranjan N, Arya DP, Herzon SB. Analysis of diazofluorene DNA binding and damaging activity: DNA cleavage by a synthetic monomeric diazofluorene. Angew Chem Int Ed Engl 2014; 53:9325–9328 [View Article] [PubMed]
     [Google Scholar]
  13. Pang X, Aigle B, Girardet J-M, Mangenot S, Pernodet J-L et al. Functional angucycline-like antibiotic gene cluster in the terminal inverted repeats of the Streptomyces ambofaciens linear chromosome. Antimicrob Agents Chemother 2004; 48:575–588 [View Article] [PubMed]
     [Google Scholar]
  14. Bunet R, Song L, Mendes MV, Corre C, Hotel L et al. Characterization and manipulation of the pathway-specific late regulator AlpW reveals Streptomyces ambofaciens as a new producer of kinamycins. J Bacteriol 2011; 193:1142–1153 [View Article] [PubMed]
     [Google Scholar]
  15. Gould SJ, Hong ST, Carney JR. Cloning and heterologous expression of genes from the kinamycin biosynthetic pathway of Streptomyces murayamaensis. J Antibiot 1998; 51:50–57 [View Article] [PubMed]
     [Google Scholar]
  16. Lin H-C, Chang S-C, Wang N-L, Ceng L-R. FL-120A-D’, new products related to kinamycin from Streptomyces chattanoogensis subsp. taitungensis subsp. nov. I. Taxonomy, fermentation and biological properties. J Antibiot 1994; 47:675–680 [View Article]
     [Google Scholar]
  17. Liu X, Liu D, Xu M, Tao M, Bai L et al. Reconstitution of kinamycin biosynthesis within the heterologous host Streptomyces albus J1074. J Nat Prod 2018; 81:72–77 [View Article] [PubMed]
     [Google Scholar]
  18. Ito S, Matsuya T, Omura S, Otani M, Nakagawa A et al. A new antibiotic, kinamycin. J Antibiot 1970; 23:315–317 [View Article] [PubMed]
     [Google Scholar]
  19. Aigle B, Pang X, Decaris B, Leblond P. Involvement of AlpV, a new member of the Streptomyces antibiotic regulatory protein family, in regulation of the duplicated type II polyketide synthase alp gene cluster in Streptomyces ambofaciens. J Bacteriol 2005; 187:2491–2500 [View Article] [PubMed]
     [Google Scholar]
  20. Bunet R, Mendes MV, Rouhier N, Pang X, Hotel L et al. Regulation of the synthesis of the angucyclinone antibiotic alpomycin in Streptomyces ambofaciens by the autoregulator receptor AlpZ and its specific ligand. J Bacteriol 2008; 190:3293–3305 [View Article] [PubMed]
     [Google Scholar]
  21. Wang B, Ren J, Li L, Guo F, Pan G et al. Kinamycin biosynthesis employs a conserved pair of oxidases for B-ring contraction. Chem Commun 2015; 51:8845–8848 [View Article]
     [Google Scholar]
  22. Laufer RS, Dmitrienko GI. Diazo group electrophilicity in kinamycins and lomaiviticin A: potential insights into the molecular mechanism of antibacterial and antitumor activity. J Am Chem Soc 2002; 124:1854–1855 [View Article] [PubMed]
     [Google Scholar]
  23. Waldman AJ, Pechersky Y, Wang P, Wang JX, Balskus EP. The cremeomycin biosynthetic gene cluster encodes a pathway for diazo formation. Chembiochem Eur J Chem Biol 2015; 16:2172–2175 [View Article] [PubMed]
     [Google Scholar]
  24. Sugai Y, Katsuyama Y, Ohnishi Y. A nitrous acid biosynthetic pathway for diazo group formation in bacteria. Nat Chem Biol 2016; 12:73–75 [View Article] [PubMed]
     [Google Scholar]
  25. Wang K-KA, Ng TL, Wang P, Huang Z, Balskus EP et al. Glutamic acid is a carrier for hydrazine during the biosyntheses of fosfazinomycin and kinamycin. Nat Commun 2018; 9:3687 [View Article]
     [Google Scholar]
  26. Zhao Y, Liu X, Xiao Z, Zhou J, Song X et al. O-methyltransferase-like enzyme catalyzed diazo installation in polyketide biosynthesis. Nat Commun 2023; 14:5372 [View Article]
     [Google Scholar]
  27. Kieser T, Bibb M, Buttner M, Chater K, Hopwood DA. Practical Streptomyces Genetics United Kigdom: The John Innes Foundation; 2000
     [Google Scholar]
  28. Sambrook J, Russell D. Molecular Cloning: A Laboratory Manual, 3rd ed. New York: Cold Spring Harbor Laboratory Press; 2001
     [Google Scholar]
  29. Camacho C, Coulouris G, Avagyan V, Ma N, Papadopoulos J et al. BLAST+: architecture and applications. BMC Bioinformatics 2009; 10:421 [View Article] [PubMed]
     [Google Scholar]
  30. Gilchrist CLM, Booth TJ, van Wersch B, van Grieken L, Medema MH et al. cblaster: a remote search tool for rapid identification and visualization of homologous gene clusters. Bioinform Adv 2021; 1:vbab016 [View Article] [PubMed]
     [Google Scholar]
  31. Gilchrist CLM, Chooi Y-H. clinker & clustermap.js: automatic generation of gene cluster comparison figures. Bioinformatics 2021; 37:2473–2475 [View Article] [PubMed]
     [Google Scholar]
  32. Gust B, Challis GL, Fowler K, Kieser T, Chater KF. PCR-targeted Streptomyces gene replacement identifies a protein domain needed for biosynthesis of the sesquiterpene soil odor geosmin. Proc Natl Acad Sci USA 2003; 100:1541–1546 [View Article] [PubMed]
     [Google Scholar]
  33. Gregory MA, Till R, Smith MCM. Integration site for Streptomyces phage phiBT1 and development of site-specific integrating vectors. J Bacteriol 2003; 185:5320–5323 [View Article] [PubMed]
     [Google Scholar]
  34. Thibessard A, Leblond P. Complete genome sequence of Streptomyces ambofaciens DSM 40697, a paradigm for genome plasticity studies. Genome Announc 2016; 4: [View Article]
     [Google Scholar]
  35. Wang P, Hong GJ, Wilson MR, Balskus EP. Production of stealthin C involves an S-N-type smiles rearrangement. J Am Chem Soc 2017; 139:2864–2867 [View Article] [PubMed]
     [Google Scholar]
  36. Kersten RD, Lane AL, Nett M, Richter TKS, Duggan BM et al. Bioactivity‐guided genome mining reveals the lomaiviticin biosynthetic gene cluster in Salinispora tropica. ChemBioChem 2013; 14:955–962 [View Article]
     [Google Scholar]
  37. Kawai S, Sugaya Y, Hagihara R, Tomita H, Katsuyama Y et al. Complete biosynthetic pathway of alazopeptin, a tripeptide consisting of two molecules of 6‐Diazo‐5‐oxo‐ l ‐norleucine and one molecule of alanine. Angew Chem Int Ed 2021; 60:10319–10325 [View Article]
     [Google Scholar]
  38. Ma G-L, Candra H, Pang LM, Xiong J, Ding Y et al. Biosynthesis of tasikamides via pathway coupling and diazonium-mediated hydrazone formation. J Am Chem Soc 2022; 144:1622–1633 [View Article] [PubMed]
     [Google Scholar]
  39. Kawai S, Hagihara R, Shin-Ya K, Katsuyama Y, Ohnishi Y. Bacterial avenalumic acid biosynthesis includes substitution of an aromatic amino group for hydride by nitrous acid dependent diazotization. Angew Chem Int Ed Engl 2022; 61:e202211728 [View Article] [PubMed]
     [Google Scholar]
  40. Shikai Y, Kawai S, Katsuyama Y, Ohnishi Y. In vitro characterization of nonribosomal peptide synthetase-dependent O-(2-hydrazineylideneacetyl)serine synthesis indicates a stepwise oxidation strategy to generate the α-diazo ester moiety of azaserine. Chem Sci 2023; 14:8766–8776 [View Article] [PubMed]
     [Google Scholar]
  41. Pan G, Gao X, Fan K, Liu J, Meng B et al. Structure and function of a C-C bond cleaving oxygenase in atypical angucycline biosynthesis. ACS Chem Biol 2017; 12:142–152 [View Article] [PubMed]
     [Google Scholar]
  42. Gould SJ, Melville CR, Cone MC, Chen J, Carney JR. Synthesis, isolation, and incorporation of stealthin c, an aminobenzo[b]fluorene. J Org Chem 1997; 62:320–324 [View Article]
     [Google Scholar]
  43. Winter JM, Jansma AL, Handel TM, Moore BS. Formation of the pyridazine natural product azamerone by biosynthetic rearrangement of an aryl diazoketone. Angew Chem Int Ed 2009; 48:767–770 [View Article]
     [Google Scholar]
  44. Wang B, Guo F, Ren J, Ai G, Aigle B et al. Identification of Alp1U and Lom6 as epoxy hydrolases and implications for kinamycin and lomaiviticin biosynthesis. Nat Commun 2015; 6: [View Article]
     [Google Scholar]
  45. Hagihara R, Katsuyama Y, Sugai Y, Onaka H, Ohnishi Y. Novel desferrioxamine derivatives synthesized using the secondary metabolism-specific nitrous acid biosynthetic pathway in Streptomyces davawensis. J Antibiot (Tokyo) 2018; 71:911–919 [View Article] [PubMed]
     [Google Scholar]
  46. Aigle B, Lautru S, Spiteller D, Dickschat JS, Challis GL et al. Genome mining of Streptomyces ambofaciens. J Ind Microbiol Biotechnol 2014; 41:251–263 [View Article] [PubMed]
     [Google Scholar]
/content/journal/micro/10.1099/mic.0.001576
Loading
Role of a FAD-dependent monooxygenase in diazo group functionalization of kinamycin in Streptomyces ambofaciens
Microbiology 171, 001576 (2025); https://doi.org/10.1099/mic.0.001576
/content/journal/micro/10.1099/mic.0.001576
/content/journal/micro/10.1099/mic.0.001576
Loading

Data & Media loading...

Supplements

Supplementary material 1

PDF

Most cited Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error