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Genome mining reveals uncommon alkylpyrones as type III PKS products from myxobacteria

  • Natural Products - Original Paper
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Journal of Industrial Microbiology & Biotechnology

Abstract

Type III polyketide synthases (PKSs) are comparatively small homodimeric enzymes affording natural products with diverse structures and functions. While type III PKS biosynthetic pathways have been studied thoroughly in plants, their counterparts from bacteria and fungi are to date scarcely characterized. This gap is exemplified by myxobacteria from which no type III PKS-derived small molecule has previously been isolated. In this study, we conducted a genomic survey of myxobacterial type III PKSs and report the identification of uncommon alkylpyrones as the products of type III PKS biosynthesis from the myxobacterial model strain Myxococcus xanthus DK1622 through a self-resistance-guided screening approach focusing on genes encoding pentapetide repeat proteins, proficient to confer resistance to topoisomerase inhibitors. Using promoter-induced gene expression in the native host as well as heterologous expression of biosynthetic type III PKS genes, sufficient amounts of material could be obtained for structural elucidation and bioactivity testing, revealing potent topoisomerase activity in vitro.

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References

  1. Austin MB, Noel JP (2003) The chalcone synthase superfamily of type III polyketide synthases. Nat Prod Rep 20(1):79–110. https://doi.org/10.1039/B100917F

    Article  CAS  PubMed  Google Scholar 

  2. Austin MB, Izumikawa M, Bowman ME et al (2004) Crystal structure of a bacterial type III polyketide synthase and enzymatic control of reactive polyketide intermediates. J Biol Chem 279(43):45162–45174. https://doi.org/10.1074/jbc.M406567200

    Article  CAS  PubMed  Google Scholar 

  3. Baumann S, Herrmann J, Raju R et al (2014) Cystobactamids: myxobacterial topoisomerase inhibitors exhibiting potent antibacterial activity. Angew Chem Int Ed 53(52):14605–14609. https://doi.org/10.1002/anie.201409964

    Article  CAS  Google Scholar 

  4. Bode HB, Ring MW, Schwär G et al (2009) Identification of additional players in the alternative biosynthesis pathway to isovaleryl-CoA in the myxobacterium Myxococcus xanthus. ChemBioChem 10(1):128–140. https://doi.org/10.1002/cbic.200800219

    Article  CAS  PubMed  Google Scholar 

  5. Chemler JA, Buchholz TJ, Geders TW et al (2012) Biochemical and structural characterization of germicidin synthase. Analysis of a type III polyketide synthase that employs acyl-ACP as a starter unit donor. J Am Chem Soc 134(17):7359–7366. https://doi.org/10.1021/ja2112228

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Cociancich S, Pesic A, Petras D et al (2015) The gyrase inhibitor albicidin consists of p-aminobenzoic acids and cyanoalanine. Nat Chem Biol 11(3):195–197. https://doi.org/10.1038/nchembio.1734

    Article  CAS  PubMed  Google Scholar 

  7. Collin F, Karkare S, Maxwell A (2011) Exploiting bacterial DNA gyrase as a drug target. Current state and perspectives. Appl Microbiol Biotechnol 92(3):479–497. https://doi.org/10.1007/s00253-011-3557-z

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Edayathumangalam R, Wu R, Garcia R et al (2013) Crystal structure of Bacillus subtilis GabR, an autorepressor and transcriptional activator of gabT. Proc Natl Acad Sci USA 110(44):17820–17825. https://doi.org/10.1073/pnas.1315887110

    Article  PubMed  PubMed Central  Google Scholar 

  9. Edgar RC (2004) MUSCLE: a multiple sequence alignment method with reduced time and space complexity. BMC Bioinform 5:113. https://doi.org/10.1186/1471-2105-5-113

    Article  CAS  Google Scholar 

  10. Edgar RC (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32(5):1792–1797. https://doi.org/10.1093/nar/gkh340

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Finn RD, Coggill P, Eberhardt RY et al (2016) The Pfam protein families database: towards a more sustainable future. Nucleic Acids Res 44(D1):D279–285. https://doi.org/10.1093/nar/gkv1344

    Article  CAS  PubMed  Google Scholar 

  12. Funa N, Ohnishi Y, Ebizuka Y et al (2002) Properties and substrate specificity of RppA, a chalcone synthase-related polyketide synthase in Streptomyces griseus. J Biol Chem 277(7):4628–4635. https://doi.org/10.1074/jbc.M110357200

    Article  CAS  PubMed  Google Scholar 

  13. Funa N, Funabashi M, Yoshimura E et al (2005) A novel quinone-forming monooxygenase family involved in modification of aromatic polyketides. J Biol Chem 280(15):14514–14523

    Article  CAS  PubMed  Google Scholar 

  14. Funa N, Funabashi M, Ohnishi Y et al (2005) Biosynthesis of hexahydroxyperylenequinone melanin via oxidative aryl coupling by cytochrome P-450 in Streptomyces griseus. J Bacteriol 187(23):8149–8155. https://doi.org/10.1128/JB.187.23.8149-8155.2005

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Funabashi M, Funa N, Horinouchi S (2008) Phenolic lipids synthesized by type III polyketide synthase confer penicillin resistance on Streptomyces griseus. J Biol Chem 283(20):13983–13991. https://doi.org/10.1074/jbc.M710461200

    Article  CAS  PubMed  Google Scholar 

  16. Gokulan K, O’Leary SE, Russell WK et al (2013) Crystal structure of Mycobacterium tuberculosis polyketide synthase 11 (PKS11) reveals intermediates in the synthesis of methyl-branched alkylpyrones. J Biol Chem 228(23):16484–16494. https://doi.org/10.1074/jbc.M113.468892

    Article  CAS  Google Scholar 

  17. Gross F, Luniak N, Perlova O et al (2006) Bacterial type III polyketide synthases: phylogenetic analysis and potential for the production of novel secondary metabolites by heterologous expression in pseudomonads. Arch Microbiol 185(1):28–38. https://doi.org/10.1007/s00203-005-0059-3

    Article  CAS  PubMed  Google Scholar 

  18. Hashimi SM, Wall MK, Smith AB et al (2007) The phytotoxin albicidin is a novel inhibitor of DNA gyrase. Antimicrob Agents Chemother 51(1):181–187. https://doi.org/10.1128/AAC.00918-06

    Article  CAS  PubMed  Google Scholar 

  19. Hayashi T, Kitamura Y, Funa N et al (2011) Fatty acyl-AMP ligase involvement in the production of alkylresorcylic acid by a Myxococcus xanthus type III polyketide synthase. ChemBioChem 12(14):2166–2176. https://doi.org/10.1002/cbic.201100344

    Article  CAS  PubMed  Google Scholar 

  20. Hegde SS, Vetting MW, Roderick SL et al (2005) A fluoroquinolone resistance protein from Mycobacterium tuberculosis that mimics DNA. Science 308(5727):1480–1483. https://doi.org/10.1126/science.1109745

    Article  CAS  PubMed  Google Scholar 

  21. Herrmann J, Fayad AA, Müller R (2017) Natural products from myxobacteria: novel metabolites and bioactivities. Nat Prod Rep 34(2):135–160. https://doi.org/10.1039/C6NP00106H

    Article  CAS  PubMed  Google Scholar 

  22. Hoffmann T, Krug D, Hüttel S et al (2014) Improving natural products identification through targeted LC-MS/MS in an untargeted secondary metabolomics workflow. Anal Chem 86(21):10780–10788. https://doi.org/10.1021/ac502805w

    Article  CAS  PubMed  Google Scholar 

  23. Huang T, Lin S (2017) Microbial natural products. A promising source for drug discovery. Appl Microbiol Biotechnol 1(2):1–3. https://doi.org/10.21767/2576-1412.100005

    Article  Google Scholar 

  24. Huo L, Rachid S, Stadler M et al (2012) Synthetic biotechnology to study and engineer ribosomal bottromycin biosynthesis. Chem Biol 19(10):1278–1287. https://doi.org/10.1016/j.chembiol.2012.08.013

    Article  CAS  PubMed  Google Scholar 

  25. Iniesta AA, García-Heras F, Abellón-Ruiz J et al (2012) Two systems for conditional gene expression in Myxococcus xanthus inducible by isopropyl-β-d-thiogalactopyranoside or vanillate. J Bacteriol 194(21):5875–5885. https://doi.org/10.1128/JB.01110-12

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Kashefi K, Hartzell PL (1995) Genetic suppression and phenotypic masking of a Myxococcus xanthus frzF-defect. Mol Microbiol 15(3):483–494

    Article  CAS  PubMed  Google Scholar 

  27. Kelley LA, Mezulis S, Yates CM et al (2015) The Phyre2 web portal for protein modeling, prediction and analysis. Nat Protoc 10(6):845–858. https://doi.org/10.1038/nprot.2015.053

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Koskiniemi H, Metsa-Ketela M, Dobritzsch D et al (2007) Crystal structures of two aromatic hydroxylases involved in the early tailoring steps of angucycline biosynthesis. J Mol Biol 372(3):633–648. https://doi.org/10.1016/j.jmb.2007.06.087

    Article  CAS  PubMed  Google Scholar 

  29. Mallika V, Sivakumar KC, Soniya EV (2011) Evolutionary implications and physicochemical analyses of selected proteins of type III polyketide synthase family. Evol Bioinform 7:41–53. https://doi.org/10.4137/EBO.S6854

    Article  CAS  Google Scholar 

  30. Mascotti ML, Juri Ayub M, Furnham N et al (2016) Chopping and changing: the evolution of the flavin-dependent monooxygenases. J Mol Biol 428(15):3131–3146. https://doi.org/10.1016/j.jmb.2016.07.003

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Minowa Y, Araki M, Kanehisa M (2007) Comprehensive analysis of distinctive polyketide and nonribosomal peptide structural motifs encoded in microbial genomes. J Mol Biol 368(5):1500–1517. https://doi.org/10.1016/j.jmb.2007.02.099

    Article  CAS  PubMed  Google Scholar 

  32. Miyanaga A, Funa N, Awakawa T et al (2008) Direct transfer of starter substrates from type I fatty acid synthase to type III polyketide synthases in phenolic lipid synthesis. Proc Natl Acad Sci USA 105(3):871–876

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Morita H, Yamashita M, Shi SP et al (2011) Synthesis of unnatural alkaloid scaffolds by exploiting plant polyketide synthase. Proc Natl Acad Sci USA 108(33):13504–13509. https://doi.org/10.1073/pnas.1107782108

    Article  PubMed  PubMed Central  Google Scholar 

  34. Ohnishi Y, Ishikawa J, Hara H et al (2008) Genome sequence of the streptomycin-producing microorganism Streptomyces griseus IFO 13350. J Bacteriol 190(11):4050–4060. https://doi.org/10.1128/JB.00204-08

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Panter F, Krug D, Baumann S et al (2018) Self-resistance guided genome mining uncovers new topoisomerase inhibitors from myxobacteria. Chem Sci 9(21):4898–4908. https://doi.org/10.1039/C8SC01325J

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Parvez A, Giri S, Giri GR et al (2018) Novel type III polyketide synthases biosynthesize methylated polyketides in Mycobacterium marinum. Sci Rep 8(1):6529. https://doi.org/10.1038/s41598-018-24980-1

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor

    Google Scholar 

  38. Sankaranarayanan R, Saxena P, Marathe UB et al (2004) A novel tunnel in mycobacterial type III polyketide synthase reveals the structural basis for generating diverse metabolites. Nat Struct Mol Biol 11(9):894–900. https://doi.org/10.1038/nsmb809

    Article  CAS  PubMed  Google Scholar 

  39. Satou R, Miyanaga A, Ozawa H et al (2013) Structural basis for cyclization specificity of two Azotobacter type III polyketide synthases. A single amino acid substitution reverses their cyclization specificity. J Biol Chem 288(47):34146–34157. https://doi.org/10.1074/jbc.m113.487272

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Schuz R, Heller W, Hahlbrock K (1983) Substrate specificity of chalcone synthase from Petroselinum hortense. Formation of phloroglucinol derivatives from aliphatic substrates. J Biol Chem 258(11):6730–6734

    CAS  PubMed  Google Scholar 

  41. Shah S, Heddle JG (2014) Squaring up to DNA. Pentapeptide repeat proteins and DNA mimicry. Appl Biochem Biotechnol 98(23):9545–9560. https://doi.org/10.1007/s00253-014-6151-3

    Article  CAS  Google Scholar 

  42. Shimizu Y, Ogata H, Goto S (2017) Type III polyketide synthases. Functional classification and phylogenomics. ChemBioChem 18(1):50–65. https://doi.org/10.1002/cbic.201600522

    Article  CAS  PubMed  Google Scholar 

  43. Sone Y, Nakamura S, Sasaki M et al. (2018) Identification and characterization of bacterial enzymes catalyzing the synthesis of 1,8-dihydroxynaphthalene, a key precursor of dihydroxynaphthalene melanin, from Sorangium cellulosum. Appl Environ Microbiol. AEM-00258-18. https://doi.org/10.1128/aem.00258-18

  44. Sucipto H, Pogorevc D, Luxenburger E et al (2017) Heterologous production of myxobacterial α-pyrone antibiotics in Myxococcus xanthus. Metab Eng 44:160–170. https://doi.org/10.1016/j.ymben.2017.10.004

    Article  CAS  PubMed  Google Scholar 

  45. Taylor JA, Burton NP, Maxwell A (2012) High-throughput microtitre plate-based assay for DNA topoisomerases. Methods Mol Biol 815:229–239. https://doi.org/10.1007/978-1-61779-424-7_17

    Article  CAS  PubMed  Google Scholar 

  46. Vetting MW, Hegde SS, Fajardo JE et al (2006) Pentapeptide repeat proteins. Biochemistry 45(1):1–10. https://doi.org/10.1021/bi052130w

    Article  CAS  PubMed  Google Scholar 

  47. Vetting MW, Hegde SS, Zhang Y et al (2011) Pentapeptide-repeat proteins that act as topoisomerase poison resistance factors have a common dimer interface. Acta Crystallogr Sect F 67(Pt 3):296–302. https://doi.org/10.1107/S1744309110053315

    Article  CAS  Google Scholar 

  48. Wang H, Li Z, Jia R et al (2016) RecET direct cloning and Redαβ recombineering of biosynthetic gene clusters, large operons or single genes for heterologous expression. Nat Protoc 11(7):1175–1190. https://doi.org/10.1038/nprot.2016.054

    Article  CAS  PubMed  Google Scholar 

  49. Wenzel SC, Müller R (2007) Myxobacterial natural product assembly lines: fascinating examples of curious biochemistry. Nat Prod Rep 24(6):1211–1224. https://doi.org/10.1039/b706416k

    Article  CAS  PubMed  Google Scholar 

  50. Wenzel SC, Müller R (2009) The impact of genomics on the exploitation of the myxobacterial secondary metabolome. Nat Prod Rep 26(11):1385–1407. https://doi.org/10.1039/b817073h

    Article  CAS  PubMed  Google Scholar 

  51. Witte SNR, Hug JJ, Géraldy M et al (2017) Biosynthesis, and total synthesis of pyrronazol B a secondary metabolite from Nannocystis pusilla. Chem Eur J 23(63):15917–15921. https://doi.org/10.1002/chem.201703782

    Article  CAS  PubMed  Google Scholar 

  52. Yu D, Xu F, Zeng J et al (2012) Type III polyketide synthases in natural product biosynthesis. IUBMB Life 64(4):285–295. https://doi.org/10.1002/iub.1005

    Article  CAS  PubMed  Google Scholar 

  53. Zha W, Rubin-Pitel SB, Zhao H (2006) Characterization of the substrate specificity of PhlD, a type III polyketide synthase from Pseudomonas fluorescens. J Biol Chem 281(42):32036–32047. https://doi.org/10.1074/jbc.M606500200

    Article  CAS  PubMed  Google Scholar 

  54. Zhang Y, Buchholz F, Muyrers JP et al (1998) A new logic for DNA engineering using recombination in Escherichia coli. Nat Genet 20:123–128. https://doi.org/10.1038/2417

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

The authors thank Alexander Popoff for recording the NMR spectra of the alkylpyrones, Viktoria Schmitt and Jennifer Herrmann for performing bioactivity assays, Ronald Garcia for microbiological assistance and Nestor Zaburannyi for bioinformatic support. Joachim J. Hug was supported by a PhD fellowship of the Boehringer Ingelheim Fonds.

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Authors and Affiliations

  1. Department Microbial Natural Products, Helmholtz-Institute for Pharmaceutical Research Saarland (HIPS), Helmholtz Centre for Infection Research (HZI), Campus E8.1, 66123, Saarbrücken, Germany

    Joachim J. Hug, Fabian Panter, Daniel Krug & Rolf Müller

  2. Department of Pharmacy, Saarland University, Campus E8.1, 66123, Saarbrücken, Germany

    Joachim J. Hug, Fabian Panter, Daniel Krug & Rolf Müller

  3. German Center for Infection Research (DZIF), Partner Site Hannover-Braunschweig, Germany

    Joachim J. Hug, Fabian Panter, Daniel Krug & Rolf Müller

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  1. Joachim J. Hug
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  2. Fabian Panter
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  3. Daniel Krug
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  4. Rolf Müller
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Correspondence to Rolf Müller.

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Dedicated to Professor Heinz G. Floss for his numerous contributions to the field of natural products.

This article is part of the Special Issue “Natural Product Discovery and Development in the Genomic Era 2019”.

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Hug, J.J., Panter, F., Krug, D. et al. Genome mining reveals uncommon alkylpyrones as type III PKS products from myxobacteria. J Ind Microbiol Biotechnol 46, 319–334 (2019). https://doi.org/10.1007/s10295-018-2105-6

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