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.

  • Review Article
  • Published:

Directed evolution drives the next generation of biocatalysts

Nature Chemical Biology volume 5, pages 567–573 (2009)Cite this article

Abstract

Enzymes are increasingly being used as biocatalysts in the generation of products that have until now been derived using traditional chemical processes. Such products range from pharmaceutical and agrochemical building blocks to fine and bulk chemicals and, more recently, components of biofuels. For a biocatalyst to be effective in an industrial process, it must be subjected to improvement and optimization, and in this respect the directed evolution of enzymes has emerged as a powerful enabling technology. Directed evolution involves repeated rounds of (i) random gene library generation, (ii) expression of genes in a suitable host and (iii) screening of libraries of variant enzymes for the property of interest. Both in vitro screening–based methods and in vivo selection–based methods have been applied to the evolution of enzyme function and properties. Significant developments have occurred recently, particularly with respect to library design, screening methodology, applications in synthetic transformations and strategies for the generation of new enzyme function.

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

Figure 1: Biocatalysis in practice.
Figure 2: Location of 'hot spots' in an enzyme by random mutagenesis coupled with high-throughput screening.
Figure 3: Two complementary biocatalytic approaches to the synthesis of the side chain of atorvastatin.
Figure 4
Figure 5
Figure 6

Similar content being viewed by others

References

  1. Schoemaker, H. et al. Dispelling the myths – biocatalysis in industrial synthesis. Science 299, 1694–1697 (2003).

    CAS  PubMed  Google Scholar 

  2. Patel, R.N. Chemo-enzymatic synthesis of pharmaceutical intermediates. Expert Opin. Drug Discov. 3, 187–245 (2008).

    CAS  PubMed  Google Scholar 

  3. Pollard, D.J. & Woodley, J.M. Biocatalysis for pharmaceutical intermediates: the future is now. Trends Biotechnol. 25, 66–73 (2007).

    CAS  PubMed  Google Scholar 

  4. Panke, S., Held, M. & Wubbolts, M. Trends and innovations in industrial biocatalysis for the production of fine chemicals. Curr. Opin. Biotechnol. 15, 272–279 (2004).

    CAS  PubMed  Google Scholar 

  5. Fox, R.J. & Clay, M.D. Catalytic effectiveness, a measure of enzyme proficiency for industrial applications. Trends Biotechnol. 27, 137–140 (2009).

    CAS  PubMed  Google Scholar 

  6. Stemmer, W.P. Rapid evolution of a protein in vitro by DNA shuffling. Nature 370, 389–391 (1994).

    CAS  PubMed  Google Scholar 

  7. Crameri, A. et al. DNA shuffling of a family of genes from diverse species accelerates directed evolution. Nature 391, 288–291 (1998).

    CAS  PubMed  Google Scholar 

  8. Moore, J.C. & Arnold, F.H. Directed evolution of a para-nitrobenzyl esterase for aqueous-organic solvents. Nat. Biotechnol. 14, 458–467 (1996).

    CAS  PubMed  Google Scholar 

  9. Reetz, M.T., Zonta, A., Schimossek, K., Liebeton, K. & Jaeger, K.-E. Creation of enantioselective biocatalysts for organic chemistry by in vitro evolution. Angew. Chem. Int. Ed. 36, 2830–2832 (1997).

    CAS  Google Scholar 

  10. Arnold, F.H. Directed evolution: creating biocatalysts for the future. Chem. Eng. Sci. 51, 5091–5102 (1996).

    CAS  Google Scholar 

  11. Tracewell, C.A. & Arnold, F.H. Directed enzyme evolution: climbing fitness peaks one amino acid at a time. Curr. Opin. Chem. Biol. 13, 3–9 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Alexeeva, M., Carr, R. & Turner, N.J. Directed evolution of enzymes: new biocatalysts for asymmetric synthesis. Org. Biomol. Chem. 1, 4133–4137 (2003).

    CAS  PubMed  Google Scholar 

  13. Bershtein, S. & Tawfik, D.S. Advances in laboratory evolution of enzymes. Curr. Opin. Chem. Biol. 12, 151–158 (2008).

    CAS  PubMed  Google Scholar 

  14. Turner, N.J. Directed evolution of enzymes new biocatalysts for organic synthesis. Chim. Oggi 26, 9–10 (2008).

    CAS  Google Scholar 

  15. Reetz, M.T. Directed evolution of enzymes for asymmetric syntheses. in Asymmetric Synthesis (eds. Christmann, M. & Bräse, S.) 207–211 (Wiley-VCH, Weinheim, Germany, 2007).

    Google Scholar 

  16. Johannes, T.W. & Zhao, H. Directed evolution of enzymes and biosynthetic pathways. Curr. Opin. Microbiol. 9, 261–267 (2006).

    CAS  PubMed  Google Scholar 

  17. Sylvestre, J., Chautard, H., Cedrone, F. & Delcourt, M. Directed evolution of biocatalysts. Org. Process Res. Dev. 10, 562–571 (2006).

    CAS  Google Scholar 

  18. Hibbert, E.G. et al. Directed evolution of biocatalytic processes. Biomol. Eng. 22, 11–19 (2005).

    CAS  PubMed  Google Scholar 

  19. Valetti, F. & Gilardi, G. Directed evolution of enzymes for product chemistry. Nat. Prod. Rep. 21, 490–511 (2004).

    CAS  PubMed  Google Scholar 

  20. Lutz, S. & Patrick, W.M. Novel methods for directed evolution of enzymes: quality, not quantity. Curr. Opin. Biotechnol. 15, 291–297 (2004).

    CAS  PubMed  Google Scholar 

  21. Montiel, C. & Bustos-Jaimes, I. Trends and challenges in directed evolution. Curr. Chem. Biol. 2, 50–59 (2008).

    CAS  Google Scholar 

  22. Fox, R.J. et al. Improving catalytic function by ProSAR-driven enzyme evolution. Nat. Biotechnol. 25, 338–344 (2007).

    CAS  PubMed  Google Scholar 

  23. Fox, R.J. & Huisman, G.W. Enzyme optimization: moving from blind evolution to statistical exploration of sequence-function space. Trends Biotechnol. 26, 132–138 (2008).

    CAS  PubMed  Google Scholar 

  24. Grate, J. Directed evolution of three biocatalysts to produce the key chiral building block for atorvastatin, the active ingredient in Lipitor. United States Environmental Protection Agency <http://www.epa.gov/greenchemistry/pubs/pgcc/winners/grca06.html> (2006).

  25. Park, S. et al. Focusing mutations into the P. fluorescens esterase binding site increases enantioselectivity more effectively than distant mutations. Chem. Biol. 12, 45–54 (2005).

    CAS  PubMed  Google Scholar 

  26. Morley, K.L. & Kazlauskas, R.J. Improving enzyme properties: when are closer mutations better? Trends Biotechnol. 23, 231–237 (2005).

    CAS  PubMed  Google Scholar 

  27. Reetz, M.T., Kahakeaw, D. & Lohmer, R. Addressing the numbers problem in directed evolution. ChemBioChem 9, 1797–1804 (2008).

    CAS  PubMed  Google Scholar 

  28. Reetz, M.T., Wang, L.-W. & Bocola, M. Directed evolution of enantioselective enzymes: iterative cycles of CASTing for probing protein-sequence space. Angew. Chem. Int. Ed. 45, 1236–1241 (2006).

    CAS  Google Scholar 

  29. Reetz, M.T. et al. Expanding the substrate scope of enzymes: combining mutations obtained by cASTing. Chem. Eur. J. 12, 6031–6038 (2006).

    CAS  PubMed  Google Scholar 

  30. Muñoz, E. & Deem, M.W. Amino acid alphabet size in protein evolution experiments: better to search a small library thoroughly or a large library sparsely? Protein Eng. Des. Sel. 21, 311–317 (2008).

    PubMed  PubMed Central  Google Scholar 

  31. Reetz, M.T. & Wu, S. Greatly reduced amino acid alphabets in directed evolution: making the right choice for saturation mutagenesis at homologous enzyme positions. Chem. Commun. (Camb.) 5499–5501 (2008).

  32. Hult, K. & Berglund, P. Enzyme promiscuity: mechanism and applications. Trends Biotechnol. 25, 231–238 (2007).

    CAS  PubMed  Google Scholar 

  33. Taglieber, A. et al. Alternate-site enzyme promiscuity. Angew. Chem. Int. Ed. 46, 8597–8600 (2007).

    CAS  Google Scholar 

  34. Bornscheuer, U.T. & Kazlauskas, R.J. Reaction specificity of enzymes: catalytic promiscuity in biocatalysis: using old enzymes to form new bonds and follow new pathways. Angew. Chem. Int. Ed. 43, 6032–6040 (2004).

    CAS  Google Scholar 

  35. Peisajovich, S.G. & Tawfik, D.S. Protein engineers turned evolutionists. Nat. Methods 4, 991–994 (2007).

    CAS  PubMed  Google Scholar 

  36. Bershtein, S., Goldin, K. & Tawfik, D.S. Intense neutral drifts yield robust and evolvable consensus proteins. J. Mol. Biol. 379, 1029–1044 (2008).

    CAS  PubMed  Google Scholar 

  37. Aharoni, A. et al. The 'evolvability' of promiscuous protein functions. Nat. Genet. 37, 73–76 (2004).

    PubMed  Google Scholar 

  38. Gupta, R.D. & Tawfik, D.S. Directed enzyme evolution via small and effective neutral drift libraries. Nat. Methods 5, 939–942 (2008).

    CAS  PubMed  Google Scholar 

  39. Bloom, J.D., Romero, P.A., Lu, Z. & Arnold, F.H. Neutral genetic drift can alter promiscuous protein functions, potentially aiding functional evolution. Biol. Direct 2, 1–19 (2007).

    Google Scholar 

  40. Sakai, A. et al. Evolution of enzymatic activities in the enolase superfamily: stereochemically distinct mechanisms in two families of cis,cis-muconate lactonizing enzymes. Biochemistry 48, 1445–1453 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Grogan, G. Emergent mechanistic diversity of enzyme-catalysed β-diketone cleavage. Biochem. J. 388, 721–730 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Hamed, R.B., Batchelar, E.T., Clifton, I.J. & Schofield, C.J. Mechanisms and structures of crotonase superfamily enzymes-how nature controls enolate and oxyanion reactivity. Cell. Mol. Life Sci. 65, 2507–2527 (2008).

    CAS  PubMed  Google Scholar 

  43. Hasnaoui-Dijoux, G., Majerić Elenkov, M., Lutje Spelberg, J.H., Hauer, B. & Janssen, D.B. Catalytic promiscuity of halohydrin dehalogenase and its application in enantioselective epoxide ring opening. ChemBioChem 9, 1048–1051 (2008).

    CAS  PubMed  Google Scholar 

  44. Terao, Y., Miyamoto, K. & Ohta, H. Introduction of single mutation changes arylmalonate decarboxylase to racemase. Chem. Commun. (Camb.) 3600–3602 (2006).

  45. Seebeck, F.P., Guainazzi, A., Amoreira, C., Baldridge, K.K. & Hilvert, D. Stereoselectivity and expanded substrate scope of an engineered PLP-dependent aldolase. Angew. Chem. Int. Ed. 45, 6824–6826 (2006).

    CAS  Google Scholar 

  46. Jochens, H. et al. Converting an esterase into an epoxide hydrolase. Angew. Chem. Int. Ed. 48, 3532–3535 (2009).

    CAS  Google Scholar 

  47. Keefe, A.D. & Szostak, J. Functional proteins from a random sequence library. Nature 410, 715–718 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Park, H.S. et al. Design and evolution of new catalytic activity with an existing protein scaffold. Science 311, 535–538 (2006).

    CAS  PubMed  Google Scholar 

  49. Röthlisberger, D. et al. Kemp elimination catalysts by computational enzyme design. Nature 453, 190–195 (2008).

    PubMed  Google Scholar 

  50. Jiang, L. et al. De novo computational design of retro-aldol enzymes. Science 319, 1387–1391 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Sterner, R., Merkl, R. & Raushel, F.M. Computational design of enzymes. Chem. Biol. 15, 421–423 (2008).

    CAS  PubMed  Google Scholar 

  52. Damborsky, J. & Brezovsky, J. Computational tools for designing and engineering biocatalysts. Curr. Opin. Chem. Biol. 13, 26–34 (2009).

    CAS  PubMed  Google Scholar 

  53. Smith, A.J.T. et al. Structural reorganization and preorganization in enzyme active sites: comparisons of experimental and theoretically ideal active site geometries in the multistep serine esterase reaction cycle. J. Am. Chem. Soc. 130, 15361–15373 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  54. Seelig, B. & Szostak, J.W. Selection and evolution of enzymes from a partially randomized non-catalytic scaffold. Nature 448, 828–831 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  55. Leemhuis, H., Kelly, R.M. & Dijkhuizen, L. Directed evolution of enzymes: library screening strategies. IUBMB Life 61, 222–228 (2009).

    CAS  PubMed  Google Scholar 

  56. Belder, D., Ludwig, M., Wang, L.-W. & Reetz, M.T. Enantioselective catalysis and analysis on a chip. Angew. Chem. Int. Ed. 45, 2463–2466 (2006).

    CAS  Google Scholar 

  57. Mastrobattista, E. et al. High-throughput screening of enzyme libraries: in vitro evolution of a β-galactosidase by fluorescence-activated sorting of double emulsions. Chem. Biol. 12, 1291–1300 (2005).

    CAS  PubMed  Google Scholar 

  58. Griffiths, A.D. & Tawfik, D.S. Directed evolution of an extremely fast phosphotriesterase by in vitro compartmentalization. EMBO J. 22, 24–35 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  59. Fernandez-Gacio, A., Uguen, M. & Fastrez, J. Phage display as a tool for the directed evolution of enzymes. Trends Biotechnol. 21, 408–414 (2003).

    CAS  PubMed  Google Scholar 

  60. Lipovsek, D. et al. Selection of horseradish peroxidase variants with enhanced enantioselectivity by yeast surface display. Chem. Biol. 14, 1176–1185 (2007).

    CAS  PubMed  Google Scholar 

  61. Qian, Z., Fields, C.J. & Lutz, S. Investigating the structural and functional consequences of circular permutation on lipase B from Candida antarctica. ChemBioChem 8, 1989–1996 (2007).

    CAS  PubMed  Google Scholar 

  62. Enright, A. et al. Stereoinversion of β- and γ-substituted-α-amino acids using a chemoenzymatic oxidation-reduction procedure. Chem. Commun. (Camb.) 2636–2637 (2003).

  63. Roff, G.J., Lloyd, R.C. & Turner, N.J. A versatile chemo-enzymatic route to enantiomerically pure β-branched-α-amino acids. J. Am. Chem. Soc. 126, 4098–4099 (2004).

    CAS  PubMed  Google Scholar 

  64. Alexeeva, M., Enright, A., Dawson, M.J., Mahmoudian, M. & Turner, N.J. Deracemisation of α-methylbenzylamine using an enzyme obtained by in vitro evolution. Angew. Chem. Int. Ed. 41, 3177–3180 (2002).

    CAS  Google Scholar 

  65. Carr, R. et al. Directed evolution of an amine oxidase possessing both broad substrate specificity and high enantioselectivity. Angew. Chem. Int. Ed. 42, 4807–4810 (2003).

    CAS  Google Scholar 

  66. Carr, R. et al. Directed evolution of an amine oxidase for the preparative deracemisation of cyclic secondary amines. ChemBioChem 6, 637–639 (2005).

    CAS  PubMed  Google Scholar 

  67. Dunsmore, C.J., Carr, R., Fleming, T. & Turner, N.J. A chemo-enzymatic route to enantiomerically pure cyclic tertiary amines. J. Am. Chem. Soc. 128, 2224–2225 (2006).

    CAS  PubMed  Google Scholar 

  68. Eve, T.S.C., Wells, A.S. & Turner, N.J. Enantioselective oxidation of O-methyl-N-hydroxylamines using MAO-N as catalyst. Chem. Commun. (Camb.) 1530–1531 (2007).

  69. Bailey, K.R., Ellis, A.J., Reiss, R., Snape, T.J. & Turner, N.J. A template-based mnemonic for monoamine oxidase (MAO-N) catalyzed reactions and its application to the chemo-enzymatic deracemisation of the alkaloid (±)-crispine A. Chem. Commun. (Camb.) 3640–3642 (2007).

  70. Atkin, K.E. et al. The structure of monoamine oxidase from Aspergillus niger provides a molecular context for improvements in activity obtained by directed evolution. J. Mol. Biol. 384, 1218–1231 (2008).

    CAS  PubMed  Google Scholar 

  71. Jennewein, S. et al. Directed evolution of an industrial biocatalyst: 2-deoxy-D-ribose 5-phosphate aldolase. Biotechnol. J. 1, 537–548 (2006).

    CAS  PubMed  Google Scholar 

  72. Ran, N. & Frost, J.W. Directed evolution of 2-keto-3-deoxy-6-phosphogalactonate aldolase to replace 3-deoxy-D-arabino-heptulosonic acid 7-phosphate synthase. J. Am. Chem. Soc. 129, 6130–6139 (2007).

    CAS  PubMed  Google Scholar 

  73. Hsu, C.-C., Hong, Z., Wada, M., Franke, D. & Wong, C.-H. Directed evolution of D-sialic acid aldolase to L-3-deoxy-manno-2-octulosonic acid (L-KDO) aldolase. Proc. Natl. Acad. Sci. USA 102, 9122–9126 (2005).

    CAS  PubMed  Google Scholar 

  74. Williams, G.J., Woodhall, T., Farnsworth, L.M., Nelson, A. & Berry, A. Creation of a pair of stereochemically complementary biocatalysts. J. Am. Chem. Soc. 128, 16238–16247 (2006).

    CAS  PubMed  Google Scholar 

  75. Smith, M.E.B., Hibbert, E.G., Jones, A.B., Dalby, P.A. & Hailes, H.C. Enhancing and reversing the stereoselectivity of Escherichia coli transketolase via single-point mutations. Adv. Synth. Catal. 350, 2631–2638 (2008).

    CAS  Google Scholar 

  76. Tee, K.L. & Schwaneberg, U. A screening system for the directed evolution of epoxygenases: importance of position 184 in P450 BM3 for stereoselective styrene epoxidation. Angew. Chem. Int. Ed. 45, 5380–5383 (2006).

    CAS  Google Scholar 

  77. Reetz, M.T. et al. Directed evolution of an enantioselective epoxide hydrolase: uncovering the source of enantioselectivity at each evolutionary stage. J. Am. Chem. Soc. 131, 7334–7343 (2009).

    CAS  PubMed  Google Scholar 

  78. Liu, Z. et al. Laboratory evolved biocatalysts for stereoselective syntheses of substituted benzaldehyde cyanohydrins. ChemBioChem 9, 58–61 (2008).

    CAS  PubMed  Google Scholar 

  79. Koch, D.J., Chen, M.M., van Beilen, J.B. & Arnold, F.H. In vivo evolution of butane oxidation by terminal alkane hydroxylases AlkB and CYP153A6. Appl. Environ. Microbiol. 75, 337–344 (2009).

    CAS  PubMed  Google Scholar 

  80. Landwehr, M. et al. Enantioselective alpha-hydroxylation of 2-arylacetic acid derivatives and buspirone catalyzed by engineered cytochrome P450 BM-3. J. Am. Chem. Soc. 128, 6058–6059 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  81. Escalettes, F. & Turner, N.J. Directed evolution of galactose oxidase: generation of enantioselective secondary alcohol oxidases. ChemBioChem 9, 857–860 (2008).

    CAS  PubMed  Google Scholar 

  82. Truppo, M.D., Escalettes, F. & Turner, N.J. Rapid determination of both the activity and enantioselectivity of ketoreductases. Angew. Chem. Int. Ed. 49, 2639–2641 (2008).

    Google Scholar 

  83. Klein, G. et al. Tailoring the active site of chemzymes by using a chemogenetic-optimization procedure: towards substrate-specific artificial hydrogenases based on the biotin-avidin technology. Angew. Chem. Int. Ed. 44, 7764–7767 (2005).

    CAS  Google Scholar 

  84. Ward, T.R. Artificial enzymes made to order: combination of computational design and directed evolution. Angew. Chem. Int. Ed. 47, 7802–7803 (2008).

    CAS  Google Scholar 

  85. Reetz, M.T., Peyralans, J.J.-P., Maichele, A., Fu, Y. & Maywald, M. Directed evolution of hybrid enzymes: evolving enantioselectivity of an achiral Rh-complex anchored to a protein. Chem. Commun. (Camb.) 4318–4320 (2006).

  86. Jing, Q., Okrasa, K. & Kazlauskas, R.J. Stereoselective hydrogenation of olefins using rhodium-substituted carbonic anhydrase - a new reductase. Chem. Eur. J. 15, 1370–1376 (2009).

    CAS  PubMed  Google Scholar 

  87. Pordea, A. et al. Artificial metalloenzyme for enantioselective sulfoxidation based on vanadyl-loaded streptavidin. J. Am. Chem. Soc. 130, 8085–8088 (2008).

    CAS  PubMed  Google Scholar 

  88. Rousselot-Pailley, P. et al. The protein environment drives selectivity for sulfide oxidation by an artificial metalloenzyme. ChemBioChem 10, 545 (2009).

    CAS  PubMed  Google Scholar 

  89. Pierron, J. et al. Artificial metalloenzymes for asymmetric allylic alkylation on the basis of the biotin-avidin technology. Angew. Chem. Int. Ed. 47, 701–705 (2008).

    CAS  Google Scholar 

  90. Umeno, D., Tobias, A.V. & Arnold, F.H. Diversifying carotenoid biosynthetic pathways by directed evolution. Microbiol. Mol. Biol. Rev. 69, 51–78 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  91. Chatterjee, R. & Yuan, L. Directed evolution of metabolic pathways. Trends Biotechnol. 24, 28–38 (2006).

    CAS  PubMed  Google Scholar 

  92. Fischbach, M.A., Lai, J.R., Roche, E.D., Walsh, C.T. & Liu, D.R. Directed evolution can rapidly improve the activity of chimeric assembly-line enzymes. Proc. Natl. Acad. Sci. USA 104, 11951–11956 (2007).

    CAS  PubMed  Google Scholar 

  93. Chica, R.A., Doucet, N. & Pelletier, J.N. Semi-rational approaches to engineering enzyme activity: combining the benefits of directed evolution and rational design. Curr. Opin. Biotechnol. 16, 378–384 (2005).

    CAS  PubMed  Google Scholar 

  94. Kazlauskas, R.J. & Lutz, S. Engineering enzymes by intelligent design. Curr. Opin. Chem. Biol. 13, 1–2 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. School of Chemistry, University of Manchester, Manchester Interdisciplinary Biocentre, Manchester, UK

    Nicholas J Turner

Authors
  1. Nicholas J Turner
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Nicholas J Turner.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Turner, N. Directed evolution drives the next generation of biocatalysts. Nat Chem Biol 5, 567–573 (2009). https://doi.org/10.1038/nchembio.203

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchembio.203

This article is cited by

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