1887

Microbiology Society logo

Volume 150, Issue 10

Crotonyl-coenzyme A reductase provides methylmalonyl-CoA precursors for monensin biosynthesis by in an oil-based extended fermentation Free

Abstract

It is demonstrated that crotonyl-CoA reductase (CCR) plays a significant role in providing methylmalonyl-CoA for monensin biosynthesis in oil-based 10-day fermentations of . Under these conditions L1, a derivative of a high-titre producing industrial strain C730.1 in which has been insertionally inactivated, produces only 15 % of the monensin yield. Labelling of the coenzyme A pools using [H]--alanine and analysis of intracellular acyl-CoAs in the L1 and C730.1 strains demonstrated that loss of led to lower levels of the monensin precursor methymalonyl-CoA, relative to coenzyme A. Expression of a heterologous gene from fully restored monensin production to the L1 mutant. Using C730.1 and an oil-based extended fermentation an exceptionally efficient and comparably intact incorporation of ethyl [3,4-C]acetoacetate into both the ethylmalonyl-CoA- and methylmalonyl-CoA-derived positions of monensin was observed. No labelling of the malonyl-CoA-derived positions was observed. The opposite result was observed when the incorporation study was carried out with the L1 strain, demonstrating that insertional inactivation has led to a reversal of carbon flux from an acetoacetyl-CoA intermediate. These results dramatically contrast similar analyses of the L1 mutant in glucose-soybean medium which indicate a role in providing ethylmalonyl-CoA but not methylmalonyl-CoA, thus causing a change in the ratio of monensin A and monensin B analogues, but not the overall monensin titre. These results demonstrate that the relative contributions of different pathways and enzymes to providing polyketide precursors are thus dependent upon the fermentation conditions. Furthermore, the generally accepted pathways for providing methylmalonyl-CoA for polyketide production may not be significant for the high-titre monensin producer in oil-based extended fermentations. An alternative pathway, leading from the fatty acid catabolite acetyl-CoA, via the CCR-catalysed reaction is proposed.

  • Received:
  • Accepted:
  • Revised:
  • Published Online:
Keyword(s): CCR, crotonyl-CoA reductase , ICM, isobutyryl-CoA mutase , MCM, methylmalonyl-CoA mutase and PKS, polyketide synthase
© SGM
Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.27251-0
2004-10-01
2024-05-09
Loading full text...

Full text loading...

/deliver/fulltext/micro/150/10/mic1503463.html?itemId=/content/journal/micro/10.1099/mic.0.27251-0&mimeType=html&fmt=ahah

References

  1. Baltz, R. H.(1998). Genetic manipulation of antibiotic-producing Streptomyces. Trends Microbiol 6, 76–83.[CrossRef] [Google Scholar]
  2. Bramwell, H., Hunter, I. S., Coggins, J. R. & Nimmo, H. G.(1996). Propionyl-CoA carboxylase from Streptomyces coelicolor A3(2): cloning of the gene encoding the biotin-containing subunit. Microbiology 142, 649–655.[CrossRef] [Google Scholar]
  3. Chan, M. & Sim, T. S.(1998). Malate synthase from Streptomyces clavuligerus NRRL3585: cloning, molecular characterization and its control by acetate. Microbiology 144, 3229–3237.[CrossRef] [Google Scholar]
  4. Cortes, J., Haydock, S. F., Roberts, G. A., Bevitt, D. J. & Leadlay, P. F.(1990). An unusually large multifunctional polypeptide in the erythromycin-producing polyketide synthase of Saccharopolyspora erythraea. Nature 348, 176–178.[CrossRef] [Google Scholar]
  5. Cronan, J. E., Jr(1980). Beta-alanine synthesis in Escherichia coli. J Bacteriol 141, 1291–1297. [Google Scholar]
  6. Cropp, T. A., Smogowicz, A. A., Hafner, E. W., Denoya, C. D., McArthur, H. A. & Reynolds, K. A.(2000). Fatty acid biosynthesis in a branched chain α-keto acid dehydrogenase mutant of Streptomyces avermitilis. Can J Microbiol 46, 506–514.[CrossRef] [Google Scholar]
  7. Dayem, L. C., Carney, J. R., Santi, D. V., Pfeifer, B. A., Khosla, C. & Kealey, J. T.(2002). Metabolic engineering of a methylmalonyl-CoA mutase-epimerase pathway for complex polyketide biosynthesis in Escherichia coli. Biochemistry 41, 5193–5201.[CrossRef] [Google Scholar]
  8. Donadio, S., Staver, M. J., McAlpine, J. B., Swanson, S. J. & Katz, L.(1991). Modular organization of the genes required for complex polyketide biosynthesis. Science 252, 675–679.[CrossRef] [Google Scholar]
  9. Gandecha, A. R., Large, S. L. & Cundliffe, E.(1997). Analysis of four tylosin biosynthetic genes from the tylLM region of the Streptomyces fradiae genome. Gene 184, 197–203.[CrossRef] [Google Scholar]
  10. Goh, L. L., Koh, R., Loke, P. & Sim, T. S.(2003). Thermostable malate synthase of Streptomyces thermovulgaris. J Ind Microbiol Biotechnol 30, 577–581.[CrossRef] [Google Scholar]
  11. Han, L. & Reynolds, K. A.(1997). A novel alternate anaplerotic pathway to the glyoxylate cycle in streptomycetes. J Bacteriol 179, 5157–5164. [Google Scholar]
  12. Hopwood, D. A.(1993). Genetic engineering of Streptomyces to create hybrid antibiotics. Curr Opin Biotechnol 4, 531–537.[CrossRef] [Google Scholar]
  13. Hopwood, D. A.(1997). Genetic contributions to understanding polyketide synthases. Chem Rev 97, 2465–2498.[CrossRef] [Google Scholar]
  14. Hopwood, D. A., Bibb, M. J., Chater, K. F. & 8 other authors(1985).Genetic Manipulation of Streptomycetes, a Laboratory Manual. Norwich, UK: John Innes Institute.
  15. Hunaiti, A. R. & Kolattukudy, P. E.(1982). Isolation and characterization of an acyl-coenzyme A carboxylase from an erythromycin-producing Streptomyces erythreus. Arch Biochem Biophys 216, 362–371.[CrossRef] [Google Scholar]
  16. Hunaiti, A. A. & Kolattukudy, P. E.(1984). Source of methylmalonyl-coenzyme A for erythromycin synthesis: methylmalonyl-coenzyme A mutase from Streptomyces erythreus. Antimicrob Agents Chemother 25, 173–178.[CrossRef] [Google Scholar]
  17. Huttner, S., Mecke, D. & Frohlich, K. U.(1997). Gene cloning and sequencing, and enzyme purification of the malate synthase of Streptomyces arenae. Gene 188, 239–246.[CrossRef] [Google Scholar]
  18. Jackowski, S. & Rock, C. O.(1984). Metabolism of 4′-phosphopantetheine in Escherichia coli. J Bacteriol 158, 115–120. [Google Scholar]
  19. Kakavas, S. J., Katz, L. & Stassi, D.(1997). Identification and characterization of the niddamycin polyketide synthase genes from Streptomyces caelestis. J Bacteriol 179, 7515–7522. [Google Scholar]
  20. Katz, L.(1997). Manipulation of modular polyketide synthases. Chem Rev 97, 2557–2575.[CrossRef] [Google Scholar]
  21. Katz, L. & Donadio, S.(1993). Polyketide synthesis: prospects for hybrid antibiotics. Annu Rev Microbiol 47, 875–912.[CrossRef] [Google Scholar]
  22. Liu, H. & Reynolds, K. A.(1999). Role of crotonyl coenzyme A reductase in determining the ratio of polyketides monensin A and monensin B produced by Streptomyces cinnamonensis. J Bacteriol 181, 6806–6813. [Google Scholar]
  23. Liu, H. & Reynolds, K. A.(2001). Precursor supply for polyketide biosynthesis: the role of crotonyl-CoA reductase. Metab Eng 3, 40–48.[CrossRef] [Google Scholar]
  24. Loke, P. & Sim, T. S.(2000). Molecular cloning, heterologous expression, and functional characterisation of a malate synthase gene from Streptomyces coelicolor A3(2). Can J Microbiol 46, 764–769.[CrossRef] [Google Scholar]
  25. Loke, P., Goh, L. L., Seng Soh, B., Yeow, P. & Sim, T. S.(2002). Purification and characterization of recombinant malate synthase enzymes from Streptomyces coelicolor A3(2) and S. clavuligerus NRRL3585. J Ind Microbiol Biotechnol 28, 239–243.[CrossRef] [Google Scholar]
  26. Marsh, E. N.(1999). Coenzyme B12 (cobalamin)-dependent enzymes. Essays Biochem 34, 139–154. [Google Scholar]
  27. O'Hagan, D., Rogers, S. V., Duffin, G. R. & Reynolds, K. A.(1995). The biosynthesis of monesin-A: thymine, 3-aminoisobutyrate and methacrylate metabolism in Streptomyces cinnamonensis. J Antibiot (Tokyo) 48, 1280–1287.[CrossRef] [Google Scholar]
  28. Oliynyk, M., Stark, C. B., Bhatt, A. & 7 other authors(2003). Analysis of the biosynthetic gene cluster for the polyether antibiotic monensin in Streptomyces cinnamonensis and evidence for the role of monB and monC genes in oxidative cyclization. Mol Microbiol 49, 1179–1190.[CrossRef] [Google Scholar]
  29. Palaniappan, N., Kim, B. S., Sekiyama, Y., Osada, H. & Reynolds, K. A.(2003). Enhancement and selective production of phoslactomycin B, a protein phosphatase IIa inhibitor, through identification and engineering of the corresponding biosynthetic gene cluster. J Biol Chem 278, 35552–35557.[CrossRef] [Google Scholar]
  30. Rangaswamy, V., Mitchell, R., Ullrich, M. & Bender, C.(1998). Analysis of genes involved in biosynthesis on coronafacic acid, the polyketide component of the phytotoxin coronatine. J Bacteriol 180, 3330–3338. [Google Scholar]
  31. Reynolds, K. & Robinson, J. A.(1985). Biosynthesis of Monensin. The intramolecular rearrangement of Isobutyryl-CoA to n-Butyryl-CoA. J Chem Soc Chem Commun, 1831. [Google Scholar]
  32. Reynolds, K., Gani, D. & Robinson, J. A.(1986). Biosynthesis of Monensin. Evidence for a vicinal interchange rearrangement linking n-Butyryl-CoA and Isobutyryl-CoA. J Chem Soc Chem Commun, 1334. [Google Scholar]
  33. Reynolds, K., O'Hagan, D., Gani, D. & Robinson, J. A.(1988). Butyrate metabolism in Streptomycetes. Characterization of a vicinal interchange rearrangement linking isobutyrate and butyrate in Streptomyces cinnamonensis. J Chem Soc Perkin Trans I, 3195–3207. [Google Scholar]
  34. Rodríguez, E. & Gramajo, H.(1999). Genetic and biochemical characterization of the α and β components of a propionyl-CoA carboxylase complex of Streptomyces coelicolor A3(2). Microbiology 145, 3109–3119. [Google Scholar]
  35. Smith, L. M., Meijer, W. G., Dijkhuizen, L. & Goodwin, P.(1996). A protein having similarity with methylmalonyl-CoA mutase is required for the assimilation of methanol and ethanol by Methylobacterium extorquens AM1. Microbiology 142, 657–684.[CrossRef] [Google Scholar]
  36. Soh, B. S., Loke, P. & Sim, T. S.(2001). Cloning, heterologous expression and purification of an isocitrate lyase from Streptomyces clavuligerus NRRL 3585. Biochim Biophys Acta 1522, 112–117.[CrossRef] [Google Scholar]
  37. Stark, W. M., Knox, N. G. & Westhead, J. E.(1967). Monensin, a new biologically active compound. II. Fermentation studies. Antimicrob Agents Chemother 7, 353–358. [Google Scholar]
  38. Tang, L., Zhang, Y. X. & Hutchinson, C. R.(1994). Amino acid catabolism and antibiotic synthesis: valine is a source of precursors for macrolide biosynthesis in Streptomyces ambofaciens and Streptomyces fradiae. J Bacteriol 176, 6107–6119. [Google Scholar]
  39. Thomä, N. H. & Leadlay, P. F.(1998). Mechanistic and structural studies on methylmalonyl-CoA mutase. Biochem Soc Trans 26, 293–298. [Google Scholar]
  40. Vrijbloed, J. W., Zerbe-Burkhardt, K., Ratnatilleke, A., Grubelnik-Leiser, A. & Robinson, J. A.(1999). Insertional inactivation of methylmalonyl coenzyme A (CoA) mutase and isobutyryl-CoA mutase genes in Streptomyces cinnamonensis: influence on polyketide antibiotic biosynthesis. J Bacteriol 181, 5600–5605. [Google Scholar]
  41. Wallace, K. K., Zhao, B., McArthur, H. A. I. & Reynolds, K. A.(1995). In vivo analysis of straight-chain and branched-chain fatty acid biosynthesis in three actinomycetes. FEMS Microbiol Lett 131, 227–234.[CrossRef] [Google Scholar]
  42. Wu, K., Chung, L., Revill, W. P., Katz, L. & Reeves, C. D.(2000). The FK520 gene cluster of Streptomyces hygroscopicus var. ascomyceticus (ATCC 14891) contains genes for biosynthesis of unusual polyketide extender units. Gene 251, 81–90.[CrossRef] [Google Scholar]
  43. Zhang, W. & Reynolds, K. A.(2001). MeaA, a putative coenzyme B12-dependent mutase, provides methylmalonyl coenzyme A for monensin biosynthesis in Streptomyces cinnamonensis. J Bacteriol 183, 2071–2080.[CrossRef] [Google Scholar]
  44. Zhang, W., Yang, L., Jiang, W., Zhao, G., Yang, Y. & Chiao, J.(1999a). Molecular analysis and heterologous expression of the gene encoding methylmalonyl-coenzyme A mutase from rifamycin SV-producing strain Amycolatopsis mediterranei U32. Appl Biochem Biotechnol 82, 209–225.[CrossRef] [Google Scholar]
  45. Zhang, Y. X., Denoya, C. D., Skinner, D. D. & 7 other authors(1999b). Genes encoding acyl-CoA dehydrogenase (AcdH) homologues from Streptomyces coelicolor and Streptomyces avermitilis provide insights into the metabolism of small branched-chain fatty acids and macrolide antibiotic production. Microbiology 145, 2323–2334. [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.27251-0
Loading
Crotonyl-coenzyme A reductase provides methylmalonyl-CoA precursors for monensin biosynthesis by Streptomyces cinnamonensis in an oil-based extended fermentation
Microbiology 150, 3463 (2004); https://doi.org/10.1099/mic.0.27251-0
/content/journal/micro/10.1099/mic.0.27251-0
/content/journal/micro/10.1099/mic.0.27251-0
Loading

Data & Media loading...

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