Antonie van Leeuwenhoek

, Volume 94, Issue 1, pp 75–84 | Cite as

Evolutionary ecology and multidisciplinary approaches to prospecting for monooxygenases as biocatalysts

  • Andrew J. HolmesEmail author
  • Nicholas V. Coleman
Original Paper


New techniques to explore microbial diversity have led to resurgent interest in prospecting for natural products (bioprospecting or biodiscovery). Although many bioprospecting projects may share little in common at first glance, the vast majority share one particular challenge. Their targets are rare to very rare members of complex natural assemblages. Despite the advances made in bringing new organisms into cultivation and application of culture-independent techniques to isolation of novel genes there remain systematic biases against relatively rare organisms with specific growth requirements. These can frequently be overcome by application of multidisciplinary approaches that take into account principles of evolutionary ecology. Our experiences with prospecting for soluble di-iron monooxygenases (SDIMO) indicate that conventional approaches to organism isolation and metagenomic cloning systematically under-sample diversity in this enzyme family. This reflects that SDIMO-containing organisms are typically relatively low-abundance members of natural assemblages (thus biased against by direct cloning) and SDIMOs have discrete physiological roles in each organism (thus are not amenable to generic enrichment culture strategies). We have sought to overcome this by a PCR-based survey of gene diversity to guide evaluation of subsequent culture or cloning studies. A surprising outcome of this survey was that conventional PCR approaches using degenerate primers also systematically under-sampled diversity, but nested PCR strategies revealed unprecedented diversity. We conclude that many PCR-based gene-prospecting studies are likely to have under-estimated the impact of target:competitor ratios on their success.


Soluble di-iron monooxygenase Biocatalysis Bioprospecting Alkene monooxygenase Mycobacterium 


  1. Amaral JA, Knowles R (1995) Growth of methanotrophs in methane and oxygen counter gradients. FEMS Microbiol Lett 126:215–220CrossRefGoogle Scholar
  2. Bull AT, Stach JEM (2007) Marine actinobacteria: new opportunities for natural product search and discovery. Trends Microbiol 15:491–499PubMedCrossRefGoogle Scholar
  3. Champreda V, Zhou NY, Leak DJ (2004) Heterologous expression of alkene monooxygenase components from Xanthobacter autotrophicus Py2 and reconstitution of the active complex. FEMS Microbiol Lett 239:309–318PubMedCrossRefGoogle Scholar
  4. Champreda V, Choi YJ, Zhou NY, Leak DJ (2006) Alteration of the stereo- and regioselectivity of alkene monooxygenase based on coupling protein interactions. Appl Microbiol Biotechnol 71:840–847PubMedCrossRefGoogle Scholar
  5. Chion C, Askew SE, Leak DJ (2005) Cloning, expression, and site-directed mutagenesis of the propene monooxygenase genes from Mycobacterium sp. strain M156. Appl Environ Microbiol 71:1909–1914CrossRefGoogle Scholar
  6. Coleman NV, Bui NB, Holmes AJ (2006) Soluble di-iron monooxygenase gene diversity in soils, sediments and ethene enrichments. Environ Microbiol 8:1228–1239PubMedCrossRefGoogle Scholar
  7. Duetz WA, Dejong C, Williams PA, Vanandel JG (1994) Competition in chemostat culture between pseudomonas strains that use different pathways for the degradation of toluene. Appl Environ Microbiol 60:2858–2863PubMedGoogle Scholar
  8. Fiet SV, van Beilen JB, Witholt B (2006) Selection of biocatalysts for chemical synthesis. Proc Natl Acad Sci USA 103:1693–1698CrossRefGoogle Scholar
  9. Futamata H, Harayama S, Watanabe K (2001) Group-specific monitoring of phenol hydroxylase genes for a functional assessment of phenol-stimulated trichloroethylene bioremediation. Appl Environ Microbiol 67:4671–4677PubMedCrossRefGoogle Scholar
  10. Green JL, Holmes AJ, Westoby M, Oliver I, Briscoe D, Dangerfield M, Gillings M, Beattie AJ (2004) Spatial scaling of microbial eukaryote diversity. Nature 432:747–750PubMedCrossRefGoogle Scholar
  11. Groves JT (2006) High-valent iron in chemical and biological oxidations. J Inorg Biochem 100:434–447PubMedCrossRefGoogle Scholar
  12. Halsey KH, Sayavedra-Soto LA, Bottomley PJ, Arp DJ (2006) Site-directed amino acid substitutions in the hydroxylase at subunit of butane monooxygenase from Pseudomonas butanovora: implications for substrates knocking at the gate. J Bacteriol 188:4962–4969PubMedCrossRefGoogle Scholar
  13. Handelsman J, Rondon MR, Brady SF, Clardy J, Goodman RM (1998) Molecular biological access to the chemistry of unknown soil microbes: a new frontier for natural products. Chem Biol 5:R245–R249PubMedCrossRefGoogle Scholar
  14. Holmes AJ, Roslev P, McDonald IR, Iversen N, Henriksen K, Murrell JC (1999) Characterization of methanotrophic bacterial populations in soils showing atmospheric methane uptake. Appl Environ Microbiol 65:3312–3318PubMedGoogle Scholar
  15. Horner-Devine MC, Lage M, Hughes JB, Bohannan BJM (2004) A taxa-area relationship for bacteria. Nature 432:750–753PubMedCrossRefGoogle Scholar
  16. Janssen PH (2006) Identifying the dominant soil bacterial taxa in libraries of 16S rRNA and 16S rRNA genes. Appl Environ Microbiol 72:1719–1728PubMedCrossRefGoogle Scholar
  17. Leahy JG, Batchelor PJ, Morcomb SM (2003) Evolution of the soluble diiron monooxygenases. FEMS Microbiol Rev 27:449–479PubMedCrossRefGoogle Scholar
  18. McClay K, Boss C, Keresztes I, Steffan RJ (2005) Mutations of toluene-4-monooxygenase that alter regiospecificity of indole oxidation and lead to production of novel indigoid pigments. Appl Environ Microbiol 71:5476–5483PubMedCrossRefGoogle Scholar
  19. McDonald IR, Kenna EM, Murrell JC (1995) Detection of methanotrophic bacteria in environmental-samples with the pcr. Appl Environ Microbiol 61:116–121PubMedGoogle Scholar
  20. Minz D, Flax JL, Green SJ, Muyzer G, Cohen Y, Wagner M, Rittmann BE, Stahl DA (1999) Diversity of sulfate-reducing bacteria in oxic and anoxic regions of a microbial mat characterized by comparative analysis of dissimilatory sulfite reductase genes. Appl Environ Microbiol 65:4666–4671PubMedGoogle Scholar
  21. Notomista E, Lahm A, Di Donato A, Tramontano A (2003) Evolution of bacterial and archaeal multicomponent monooxygenases. J Mol Evol 56:435–445PubMedCrossRefGoogle Scholar
  22. Park J, Kim D, Kim S, Kim J, Bae K, Lee C (2007) The analysis and application of a recombinant monooxygenase library as a biocatalyst for the Baeyer–Villiger reaction. J Microbiol Biotechnol 17:1083–1089PubMedGoogle Scholar
  23. Pollard DJ, Woodley JM (2007) Biocatalysis for pharmaceutical intermediates: the future is now. Trends Biotechnol 25:66–73PubMedCrossRefGoogle Scholar
  24. Sieber V, Martinez CA, Arnold FH (2001) Libraries of hybrid proteins from distantly related sequences. Nat Biotechnol 19:456–460PubMedCrossRefGoogle Scholar
  25. Stirling DI, Dalton H (1979) Properties of the methane mono-oxygenase from extracts of methylosinus-trichosporium Ob3b and evidence for its similarity to the enzyme from methylococcus-capsulatus (Bath). Eur J Biochem 96:205–212PubMedCrossRefGoogle Scholar
  26. Tee KL, Schwaneberg U (2007) Directed evolution of oxygenases: screening systems, success stories and challenges. Comb Chem High Throughput Screen 10:197–217PubMedCrossRefGoogle Scholar
  27. Urlacher VB, Eiben S (2006) Cytochrome P450 monooxygenases: perspectives for synthetic application. Trends Biotechnol 24:324–330PubMedCrossRefGoogle Scholar
  28. Urlacher VB, Schmid RD (2006) Recent advances in oxygenase-catalyzed biotransformations. Curr Opin Chem Biol 10:156–161PubMedCrossRefGoogle Scholar
  29. van Beilen JB, Funhoff EG (2005) Expanding the alkane oxygenase toolbox: new enzymes and applications. Curr Opin Biotechnol 16:308–314PubMedCrossRefGoogle Scholar
  30. van Beilen JB, Duetz WA, Schmid A, Witholt B (2003) Practical issues in the application of oxygenases. Trends Biotechnol 21:170–177PubMedCrossRefGoogle Scholar
  31. van Berkel WJH, Kamerbeek NM, Fraaije MW (2006) Flavoprotein monooxygenases, a diverse class of oxidative biocatalysts. J Biotechnol 124:670–689PubMedCrossRefGoogle Scholar
  32. Wackett LP (2002) Mechanism and applications of Rieske non-heme iron dioxygenases. Enzyme and Microb Technol 31:577–587CrossRefGoogle Scholar
  33. Wagner M, Loy A, Klein M, Lee N, Ramsing NB, Stahl DA, Friedrich MW (2005) Functional marker genes for identification of sulfate-reducing prokaryotes. Methods Enzymol 397:469–489PubMedCrossRefGoogle Scholar
  34. Watanabe K, Teramoto M, Futamata H, Harayama S (1998) Molecular detection, isolation, and physiological characterization of functionally dominant phenol-degrading bacteria in activated sludge. Appl Environ Microbiol 64:4396–4402PubMedGoogle Scholar
  35. Watanabe K, Futamata H, Harayama S (2002) Understanding the diversity in catabolic potential of microorganisms for the development of bioremediation strategies. Antonie Van Leeuwenhoek Int J Gen Mol Microbiol 81:655–663CrossRefGoogle Scholar
  36. Yeates C, Holmes AJ, Gillings MR (2000) Novel forms of ring-hydroxylating dioxygenases are widespread in pristine and contaminated sails. Environ Microbiol 2:644–653PubMedCrossRefGoogle Scholar
  37. Zehr JP, Jenkins BD, Short SM, Steward GF (2003) Nitrogenase gene diversity and microbial community structure: a cross-system comparison. Environ Microbiol 5:539–554PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2008

Authors and Affiliations

  1. 1.School of Molecular and Microbial BiosciencesThe University of SydneySydneyAustralia

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