Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Discovery and biosynthetic pathway analysis of cyclopentane–β-lactone globilactone A

Nature Synthesis volume 3, pages 99–110 (2024)Cite this article

Subjects

Abstract

Naturally occurring β-lactone compounds are a class of strained four-membered heterocycles and can act as highly reactive electrophiles. Despite intensive studies for several decades, fewer than 30 β-lactones, many of which have high clinical potential, have been characterized from microorganisms. Here we report the discovery of a β-lactone compound, globilactone A through heterologous expression of a polyketide synthase/non-ribosomal peptide synthetase-like biosynthetic gene cluster (glo) in Streptomyces albus J1074. Biosynthetic pathway studies revealed that the polyketide synthase part can synthesize a polyunsaturated polyketide chain. While the downstream non-ribosomal peptide synthetase-like module, comprising condensation, FkbH, peptidyl carrier protein and thioester reductase domains (C–FkbH–PCP–R), incorporates a three-carbon pyruvate unit, and mediates formation of two carbon–carbon bonds between the polyketide and pyruvate to give a cyclopentane intermediate tethered on acyl carrier protein. A downstream esterase, GloD, plays a direct role in the β-lactone ring formation and releases the cyclopentane–β-lactone from the assembly line.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Structures and biosynthesis of β-lactones.
Fig. 2: LC–MS chromatograms of metabolite extracts from recombinant strains.
Fig. 3: The proposed biosynthetic pathway of cyclopentane–β-lactone core formation.
Fig. 4: In vitro reconstitution of 3.
Fig. 5: FkbH (GloB) catalysed β-elimination to form pyruvoyl-S-PCP.
Fig. 6: Genome mining of homologous glo gene clusters.

Similar content being viewed by others

Data availability

Data supporting the findings of this work are available within the paper and its Supplementary Information files. The DNA sequence of gene cluster glo has been deposited in GenBank with the accession number OP620071 (https://www.ncbi.nlm.nih.gov/nuccore/OP620071.1/).The source data underlying Figs. 4a–d and 5c,d; Extended Data Figs. 2, 6a,b, 8a–c, 9a–c and 10a,b are provided as a Source Data file. Source data are provided with this paper.

References

  1. Lowe, C. & Vederas, J. C. Naturally occuring β-lactones: occurence, synthesis and properties. A review. Org. Prep. Proced. Int. 27, 305–346 (1995).

    Article  CAS  Google Scholar 

  2. Robinson, S. L., Christenson, J. K. & Wackett, L. P. Biosynthesis and chemical diversity of β-lactone natural products. Nat. Prod. Rep. 36, 458–475 (2019).

    Article  CAS  PubMed  Google Scholar 

  3. Nishimura, T. et al. JBIR-155, a specific class D β-lactamase inhibitor of microbial origin. Org. Lett. 23, 4415–4419 (2021).

    Article  CAS  PubMed  Google Scholar 

  4. Shi, Y. M. et al. Global analysis of biosynthetic gene clusters reveals conserved and unique natural products in entomopathogenic nematode-symbiotic bacteria. Nat. Chem. 14, 701–712 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. De Pascale, G., Nazi, I., Harrison, P. H. & Wright, G. D. β-Lactone natural products and derivatives inactivate homoserine transacetylase, a target for antimicrobial agents. J. Antibiot. 64, 483–487 (2011).

    Article  Google Scholar 

  6. Gulder, T. A. & Moore, B. S. Salinosporamide natural products: potent 20S proteasome inhibitors as promising cancer chemotherapeutics. Angew. Chem. Int. Ed. 49, 9346–9367 (2010).

    Article  CAS  Google Scholar 

  7. Lall, M. S., Ramtohul, Y. K., James, M. N. G. & Vederas, J. C. Serine and threonine β-lactones: a new class of hepatitis A virus 3C cysteine proteinase inhibitors. J. Org. Chem. 67, 1536–1547 (2002).

    Article  CAS  PubMed  Google Scholar 

  8. Pemble, C. W. IV, Johnson, L. C., Kridel, S. J. & Lowther, W. T. Crystal structure of the thioesterase domain of human fatty acid synthase inhibited by Orlistat. Nat. Struct. Mol. Biol. 14, 704–709 (2007).

    Article  CAS  PubMed  Google Scholar 

  9. Derosa, G. & Maffioli, P. Anti-obesity drugs: a review about their effects and their safety. Expert Opin. Drug Saf. 11, 459–471 (2012).

    Article  CAS  PubMed  Google Scholar 

  10. Torgerson, J. S., Hauptman, J., Boldrin, M. N. & Sjöström, L. XENical in the prevention of diabetes in obese subjects (XENDOS) study: a randomized study of orlistat as an adjunct to lifestyle changes for the prevention of type 2 diabetes in obese patients. Diabetes Care 27, 155–161 (2004).

    Article  CAS  PubMed  Google Scholar 

  11. Roth, P. et al. A phase III trial of marizomib in combination with temozolomide-based radiochemotherapy versus temozolomide-based radiochemotherapy alone in patients with newly diagnosed glioblastoma. J. Clin. Oncol. 39, 2004 (2021).

    Article  Google Scholar 

  12. Christenson, J. K. et al. β-Lactone synthetase found in the olefin biosynthesis pathway. Biochemistry 56, 348–351 (2017).

    Article  CAS  PubMed  Google Scholar 

  13. Schaffer, J. E., Reck, M. R., Prasad, N. K. & Wencewicz, T. A. β-Lactone formation during product release from a nonribosomal peptide synthetase. Nat. Chem. Biol. 13, 737–744 (2017).

    Article  CAS  PubMed  Google Scholar 

  14. Kreitler, D. F., Gemmell, E. M., Schaffer, J. E., Wencewicz, T. A. & Gulick, A. M. The structural basis of N-acyl-α-amino-β-lactone formation catalyzed by a nonribosomal peptide synthetase. Nat. Commun. 10, 3432–3445 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  15. Feng, K.-N. et al. A hydrolase-catalyzed cyclization forms the fused bicyclic β-lactone in vibralactone. Angew. Chem. Int. Ed. 59, 7209–7213 (2020).

    Article  CAS  Google Scholar 

  16. Bauman, K. D. et al. Enzymatic assembly of the salinosporamide γ-lactam–β-lactone anticancer warhead. Nat. Chem. Biol. 18, 538–546 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Shi, J. et al. Genome mining and biosynthesis of kitacinnamycins as a STING activator. Chem. Sci. 10, 4839–4846 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Shi, J. et al. Genome mining and enzymatic total biosynthesis of purincyclamide. Org. Lett. 21, 6825–6829 (2019).

    Article  CAS  PubMed  Google Scholar 

  19. Shi, J. et al. Discovery and biosynthesis of guanipiperazine from a NRPS-like pathway. Chem. Sci. 12, 2925–2930 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Blin, K. et al. antiSMASH 5.0: updates to the secondary metabolite genome mining pipeline. Nucleic Acids Res. 47, W81–W87 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Chan, Y. A., Podevels, A. M., Kevany, B. M. & Thomas, M. G. Biosynthesis of polyketide synthase extender units. Nat. Prod. Rep. 26, 90–114 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Sun, Y., Hong, H., Gillies, F., Spencer, J. B. & Leadlay, P. F. Glyceryl-S-acyl carrier protein as an intermediate in the biosynthesis of tetronate antibiotics. ChemBioChem 9, 150–156 (2008).

    Article  CAS  PubMed  Google Scholar 

  23. Xu, Z. F. et al. Discovery and biosynthesis of bosamycins from Streptomyces sp. 120454. Chem. Sci. 11, 9237–9245 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Li, L. et al. The mildiomycin biosynthesis: initial steps for sequential generation of 5-hydroxymethylcytidine 5′-monophosphate and 5-hydroxymethylcytosine in Streptoverticillium rimofaciens ZJU5119. ChemBioChem 9, 1286–1294 (2008).

    Article  CAS  PubMed  Google Scholar 

  25. Liu, D. Z. et al. Vibralactone: a lipase inhibitor with an unusual fused β-lactone produced by cultures of the basidiomycete Boreostereum vibrans. Org. Lett. 8, 5749–5752 (2006).

    Article  CAS  PubMed  Google Scholar 

  26. Morinaka, A. et al. OP0595, a new diazabicyclooctane: mode of action as a serine β-lactamase inhibitor, antibiotic and β-lactam ‘enhancer’. J. Antimicrob. Chemother. 70, 2779–2786 (2015).

    Article  CAS  PubMed  Google Scholar 

  27. Kim, B. S., Cropp, T. A., Beck, B. J., Sherman, D. H. & Reynolds, K. A. Biochemical evidence for an editing role of thioesterase II in the biosynthesis of the polyketide pikromycin. J. Biol. Chem. 277, 48028–48034 (2002).

    Article  CAS  PubMed  Google Scholar 

  28. Geyer, K., Hartmann, S., Singh, R. R. & Erb, T. J. Multiple functions of the type II thioesterase associated with the phoslactomycin polyketide synthase. Biochemistry 61, 2662–2671 (2022).

    Article  CAS  PubMed  Google Scholar 

  29. Kotowska, M. & Pawlik, K. Roles of type II thioesterases and their application for secondary metabolite yield improvement. Appl. Microbiol. Biotechnol. 98, 7735–7746 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Romo, A. J. et al. The amipurimycin and miharamycin biosynthetic gene clusters: unraveling the origins of 2-aminopurinyl peptidyl nucleoside antibiotics. J. Am. Chem. Soc. 141, 14152–14159 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Etzbach, L., Plaza, A., Garcia, R., Baumann, S. & Müller, R. Cystomanamides: structure and biosynthetic pathway of a family of glycosylated lipopeptides from myxobacteria. Org. Lett. 16, 2414–2417 (2014).

    Article  CAS  PubMed  Google Scholar 

  32. Auerbach, D., Yan, F., Zhang, Y. & Müller, R. Characterization of an unusual glycerate esterification process in vioprolide biosynthesis. ACS Chem. Biol. 13, 3123–3130 (2018).

    Article  CAS  PubMed  Google Scholar 

  33. Agarwal, V. et al. Chemoenzymatic synthesis of acyl coenzyme a substrates enables in situ labeling of small molecules and proteins. Org. Lett. 17, 4452–4455 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Yi, D., Acharya, A., Gumbart, J. C., Gutekunst, W. R. & Agarwal, V. Gatekeeping ketosynthases dictate initiation of assembly line biosynthesis of pyrrolic polyketides. J. Am. Chem. Soc. 143, 7617–7622 (2021).

    Article  CAS  PubMed  Google Scholar 

  35. Calderone, C. T., Bumpus, S. B., Kelleher, N. L., Walsh, C. T. & Magarvey, N. A. A ketoreductase domain in the PksJ protein of the bacillaene assembly line carries out both α- and β-ketone reduction during chain growth. Proc. Natl Acad. Sci. USA 105, 12809–12814 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Scharf, D. H. et al. Epidithiol formation by an unprecedented twin carbon-sulfur lyase in the gliotoxin pathway. Angew. Chem. Int. Ed. 51, 10064–10068 (2012).

    Article  CAS  Google Scholar 

  37. Agarwal, A., Kahyaoglu, C. & Hansen, D. B. Characterization of HmqF, a protein involved in the biosynthesis of unsaturated quinolones produced by Burkholderia thailandensis. Biochemistry 51, 1648–1657 (2012).

    Article  CAS  PubMed  Google Scholar 

  38. He, H. Y. et al. Quartromicin biosynthesis: two alternative polyketide chains produced by one polyketide synthase assembly line. Chem. Biol. 19, 1313–1323 (2012).

    Article  CAS  PubMed  Google Scholar 

  39. Süssmuth, R. D. & Mainz, A. Nonribosomal peptide synthesis—principles and prospects. Angew. Chem. Int. Ed. 56, 3770–3821 (2017).

    Article  Google Scholar 

  40. Roche, E. D. & Walsh, C. T. Dissection of the EntF condensation domain boundary and active site residues in nonribosomal peptide synthesis. Biochemistry 42, 1334–1344 (2003).

    Article  CAS  PubMed  Google Scholar 

  41. Samel, S. A., Schoenafinger, G., Knappe, T. A., Marahiel, M. A. & Essen, L. O. Structural and functional insights into a peptide bond-forming bidomain from a nonribosomal peptide synthetase. Structure 15, 781–792 (2007).

    Article  CAS  PubMed  Google Scholar 

  42. Barajas, J. F. et al. Comprehensive structural and biochemical analysis of the terminal myxalamid reductase domain for the engineered production of primary alcohols. Chem. Biol. 22, 1018–1029 (2015).

    Article  CAS  PubMed  Google Scholar 

  43. Kopp, F., Mahlert, C., Grünewald, J. & Marahiel, M. A. Peptide macrocyclization: the reductase of the nostocyclopeptide synthetase triggers the self-assembly of a macrocyclic imine. J. Am. Chem. Soc. 128, 16478–16479 (2006).

    Article  CAS  PubMed  Google Scholar 

  44. He, H. Y., Yuan, H., Tang, M. C. & Tang, G. L. An unusual dehydratase acting on glycerate and a ketoreducatse stereoselectively reducing α-ketone in polyketide starter unit biosynthesis. Angew. Chem. Int. Ed. 53, 11315–11319 (2014).

    Article  CAS  Google Scholar 

  45. Bretschneider, T. et al. Vinylogous chain branching catalysed by a dedicated polyketide synthase module. Nature 502, 124–128 (2013).

    Article  CAS  PubMed  Google Scholar 

  46. Geoghegan, K. F. et al. Spontaneous α-N-6-phosphogluconoylation of a ‘His tag’ in Escherichia coli: the cause of extra mass of 258 or 178 Da in fusion proteins. Anal. Biochem. 267, 169–184 (1999).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This research was financially supported by MOST (2022YFC2303100, 2022YFC2804100 and 2018YFA0902000), NSFC (81925033, 22193071, 22107048, 22077062, 81991522 and 81991524). We thank H. Zheng (China Pharmaceutical University) for his generous gift of expression plasmid NDM-1.

Author information

Authors and Affiliations

  1. State Key Laboratory of Pharmaceutical Biotechnology, Department of Neurology, Nanjing Drum Tower Hospital, School of Life Sciences, Chemistry and Biomedicine Innovation Center (ChemBIC), Nanjing University, Nanjing, China

    Zi Fei Xu, Ye Lei Zhou, Sheng Tao Bo, Jing Shi, Lang Xiang, Meng Yu Xi, Bo Zhang, Ren Xiang Tan & Hui Ming Ge

  2. State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing, China

    Shou Qi Zhang & Zheng Ren Xu

  3. Department of Chemistry, The Herbert Wertheim UF Scripps Institute for Biomedical Innovation and Technology, University of Florida, Jupiter, FL, USA

    Dong Yang & Ben Shen

  4. Natural Products Discovery Center, The Herbert Wertheim UF Scripps Institute for Biomedical Innovation and Technology, University of Florida, Jupiter, FL, USA

    Dong Yang & Ben Shen

  5. Department of Molecular Medicine, The Herbert Wertheim UF Scripps Institute for Biomedical Innovation and Technology, University of Florida, Jupiter, FL, USA

    Ben Shen

Authors
  1. Zi Fei Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ye Lei Zhou
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Sheng Tao Bo
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Shou Qi Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jing Shi
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Lang Xiang
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Meng Yu Xi
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Bo Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Zheng Ren Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Dong Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Ben Shen
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Ren Xiang Tan
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Hui Ming Ge
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

Z.F.X., Y.L.Z. and S.T.B. carried out experiments. L.X., M.Y.X., J.S., B.Z. and R.X.T. assisted in NMR and MS data measurement and analysis. S.Q.Z., Z.R.X., D.Y. and B.S. contributed materials. Z.F.X. and H.M.G. wrote the paper. H.M.G. supervised the work. All authors discussed the results and analysed the data.

Corresponding authors

Correspondence to Ren Xiang Tan or Hui Ming Ge.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature Synthesis thanks the anonymous reviewers for their contribution to the peer review of this work. Primary Handling Editor: Thomas West, in collaboration with the Nature Synthesis team.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Enzymatic mechanisms of known β-lactone ring biosynthesis.

cis-3-octyl-4-nonyloxetan-2-one (a), obafluorin (b), vibralactone (c), salinosporamide A (d).

Extended Data Fig. 2 Inhibitory activitiy against the class A β-lactamase of 1.

An IC50 value against class A β-lactamase of 1 was 15.05 μM.

Source data

Extended Data Fig. 3 Biochemical characterization of GloE.

a Reaction scheme for the GloE activity assay. b HPLC analysis of the enzymatic assays of GloE with 4. c HPLC analysis of the enzymatic assays of GloE with 1.

Extended Data Fig. 4 Labeling patterns of globilactone C (3) and metabolic intermediates after feeding 13C-labeled glucoses.

Heavy bars denote contiguous 13C labels of connected atoms, dots denote single 13C labels.

Extended Data Fig. 5 Chemoenzymatic synthesis of 8-S-ACP (GloB) and 12-S-GloC GloD.

Reaction scheme depicting chemoenzymatic synthesis and subsequent acylation assay to generate 8-S-ACP (GloB), 8-S-ACP-C-FkbH (GloB) (a) and 13-S-GloC-GloD (b).

Extended Data Fig. 6 In vitro reconstitution of 10.

a LC-MS analysis of the one-pot enzymatic total biosynthesis of 10. i) boiled holo-GloC-GloD, 8-S-ACP-C-FkbH (GloB), 1,3-BPG, NADPH and N-acetyl-L-cysteine; ii) holo-GloC-GloD, 8-S-ACP-C-FkbH (GloB), 1,3-BPG and N-acetyl-L-cysteine; iii) holo-GloC-GloD, 8-S-ACP-C-FkbH (GloB), 1,3-BPG, NADPH and N-acetyl-L-cysteine. b high-resolution MS (HRMS) data of 10.

Source data

Extended Data Fig. 7 Chemoenzymatic synthesis of 11-S-ACP (GloB) and 11-S-ACP-C-FkbH (GloB).

Reaction scheme depicting chemoenzymatic synthesis and subsequent acylation assay to generate 11-S-ACP-C-FkbH (GloB).

Extended Data Fig. 8 Mass spectrometric analysis of production of acylated ACP.

a holo-ACP (GloB). b ACP-tethered linear substrate (8-S-ACP) from pantetheine-activated (8c) via in vitro CoA biosynthesis and Sfp-mediated loading. c holo-GloC-GloD, holo-ACP-C-FkbH (GloB), 1,3-BPG, NADPH and 8-S-ACP. Additional peaks marked by asterisks represent adventitious gluconylation (+178 Da) of N-terminal His6-tag during expression of the recombinant protein in E. coli.

Source data

Extended Data Fig. 9 MS2 data localized the active site to the ACP, where the serine (S) is the site of 4′-Ppant and mass shifts were confirmed through measurement of the 4′-Ppant elimination product.

a holo-ACP (GloB). b ACP-tethered linear substrate (8-S-ACP) from pantetheine-activated (8c) via in vitro CoA biosynthesis and Sfp-mediated loading. c holo-GloC-GloD, holo-ACP-C-FkbH (GloB), 1,3-BPG, NADPH and 8-S-ACP.

Source data

Extended Data Fig. 10 LC-MS analysis of ACP-C-FkbH (GloB) and GloD active site mutagenesis.

a LC-MS analysis of the one-pot enzymatic total biosynthesis of 3. b Mass spectrometric analysis of production of acylated ACP (12). Additional peaks marked by asterisks represent adventitious gluconylation (+178 Da) of N-terminal His6-tag during expression of the recombinant protein in E. coli.

Source data

Supplementary information

Source data

Source Data Fig. 4

Statistical source data.

Source Data Fig. 5

Statistical source data.

Source Data Extended Data Fig./Table 2

Statistical source data.

Source Data Extended Data Fig./Table 6

Statistical source data.

Source Data Extended Data Fig./Table 8

Statistical source data.

Source Data Extended Data Fig./Table 9

Statistical source data.

Source Data Extended Data Fig./Table 10

Statistical source data.

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Xu, Z.F., Zhou, Y.L., Bo, S.T. et al. Discovery and biosynthetic pathway analysis of cyclopentane–β-lactone globilactone A. Nat. Synth 3, 99–110 (2024). https://doi.org/10.1038/s44160-023-00414-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s44160-023-00414-3

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing