Engineering the third wave of biocatalysis


Over the past ten years, scientific and technological advances have established biocatalysis as a practical and environmentally friendly alternative to traditional metallo- and organocatalysis in chemical synthesis, both in the laboratory and on an industrial scale. Key advances in DNA sequencing and gene synthesis are at the base of tremendous progress in tailoring biocatalysts by protein engineering and design, and the ability to reorganize enzymes into new biosynthetic pathways. To highlight these achievements, here we discuss applications of protein-engineered biocatalysts ranging from commodity chemicals to advanced pharmaceutical intermediates that use enzyme catalysis as a key step.

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Figure 1: The evolution of enzyme discovery and protein engineering strategies used to identify desired catalysts.
Figure 2: Free energy (Δ G ) connects design goals to the required structural changes via protein engineering strategies.
Figure 3: Different enzymatic routes to the synthesis of the key side chain of atorvastatin (Lipitor).
Figure 4: Biocatalysis advances synthetic chemistry.


  1. 1

    Buchholz, K., Kasche, V. & Bornscheuer, U. T. Biocatalysts and Enzyme Technology 2nd edn (Wiley-VCH, 2012)

    Google Scholar 

  2. 2

    Drauz, K., Gröger, H., May, O. (eds) Enzyme Catalysis in Organic Synthesis Vols 1–3, 3rd edn (Wiley-VCH, 2012)

    Google Scholar 

  3. 3

    Bornscheuer, U. T. & Kazlauskas, R. J. Hydrolases in Organic Synthesis - Regio- and Stereoselective Biotransformations 2nd edn (Wiley-VCH, 2006)

    Google Scholar 

  4. 4

    Liese, A., Seelbach, K., Wandrey, C. (eds) Industrial Biotransformations 2nd edn (Wiley-VCH, 2006)

    Google Scholar 

  5. 5

    Wenda, S., Illner, S., Mell, A. & Kragl, U. Industrial biotechnology—the future of green chemistry? Green Chem. 13, 3007–3047 (2011)

    CAS  Google Scholar 

  6. 6

    Rosenthaler, L. Durch Enzyme bewirkte asymmetrische Synthese. Biochem. Z. 14, 238–253 (1908)

    CAS  Google Scholar 

  7. 7

    Sedlaczek, L. Biotransformations of steroids. Crit. Rev. Biotechnol. 7, 187–236 (1988)

    CAS  PubMed  PubMed Central  Google Scholar 

  8. 8

    Estell, D. A., Graycar, T. P. & Wells, J. A. Engineering an enzyme by site-directed mutagenesis to be resistant to chemical oxidation. J. Biol. Chem. 260, 6518–6521 (1985)This paper puts forward the basis for the first application of protein engineering in industrial biotechnology.

    CAS  Google Scholar 

  9. 9

    Jensen, V. J. & Rugh, S. Industrial scale production and application of immobilized glucose isomerase. Methods Enzymol. 136, 356–370 (1987)

    CAS  Google Scholar 

  10. 10

    Bruggink, A., Roos, E. C. & de Vroom, E. Penicillin acylase in the industrial production of β-lactam antibiotics. Org. Process Res. Dev. 2, 128–133 (1998)

    CAS  Google Scholar 

  11. 11

    Griengl, H., Schwab, H. & Fechter, M. The synthesis of chiral cyanohydrins by oxynitrilases. Trends Biotechnol. 18, 252–256 (2000)

    CAS  PubMed  PubMed Central  Google Scholar 

  12. 12

    Hills, G. Industrial use of lipases to produce fatty acid esters. Eur. J. Lipid Sci. Technol. 105, 601–607 (2003)

    CAS  Google Scholar 

  13. 13

    Nagasawa, T., Nakamura, T. & Yamada, H. Production of acrylic acid and methacrylic acid using Rhodococcus rhodochrous J1 nitrilase. Appl. Microbiol. Biotechnol. 34, 322–324 (1990)

    CAS  Google Scholar 

  14. 14

    Francis, J. C. & Hansche, P. E. Directed evolution of metabolic pathways in microbial populations. I. Modification of the acid phosphatase pH optimum in S. cerevisiae.. Genetics 70, 59–73 (1972)

    CAS  PubMed  PubMed Central  Google Scholar 

  15. 15

    Nakamura, C. E. & Whited, G. M. Metabolic engineering for the microbial production of 1,3-propanediol. Curr. Opin. Biotechnol. 14, 454–459 (2003)

    CAS  PubMed  PubMed Central  Google Scholar 

  16. 16

    Liang, J. et al. Development of a biocatalytic process as an alternative to the (-)-DIP-Cl-mediated asymmetric reduction of a key intermediate of montelukast. Org. Process Res. Dev. 14, 193–198 (2010)

    CAS  Google Scholar 

  17. 17

    Savile, C. K. et al. Biocatalytic asymmetric synthesis of chiral amines from ketones applied to sitagliptin manufacture. Science 329, 305–309 (2010)

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  18. 18

    Desai, A. A. Sitagliptin manufacture: a compelling tale of green chemistry, process intensification, and industrial asymmetric catalysis. Angew. Chem. Int. Ed. 50, 1974–1976 (2011)This highlight article compares in detail two processes for the production of sitagliptin, one catalysed by rhodium and one catalysed by a transaminase.

    CAS  Google Scholar 

  19. 19

    O’Maille, P. E. et al. Quantitative exploration of the catalytic landscape separating divergent plant sesquiterpene synthases. Nature Chem. Biol. 4, 617–623 (2008)

    Google Scholar 

  20. 20

    Atsumi, S., Hanai, T. & Liao, J. C. Non-fermentative pathways for synthesis of branched-chain higher alcohols as biofuels. Nature 451, 86–89 (2008)This paper describes the efficient diversion of amino-acid metabolism to the production of alcohols.

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  21. 21

    Lee, S. K., Chou, H., Ham, T. S., Lee, T. S. & Keasling, J. D. Metabolic engineering of microorganisms for biofuels production: from bugs to synthetic biology to fuels. Curr. Opin. Biotechnol. 19, 556–563 (2008)

    CAS  PubMed  PubMed Central  Google Scholar 

  22. 22

    Steen, E. J. et al. Microbial production of fatty-acid-derived fuels and chemicals from plant biomass. Nature 463, 559–562 (2010)

    ADS  CAS  Google Scholar 

  23. 23

    Bohmert-Tatarev, K., McAvoy, S., Daughtry, S., Peoples, O. P. & Snell, K. D. High levels of bioplastic are produced in fertile transplastomic tobacco plants engineered with a synthetic operon for the production of polyhydroxybutyrate. Plant Physiol. 155, 1690–1708 (2011)

    CAS  PubMed  PubMed Central  Google Scholar 

  24. 24

    McKenna, R. & Nielsen, D. R. Styrene biosynthesis from glucose by engineered E. coli. Metab. Eng. 13, 544–554 (2011)

    CAS  PubMed  PubMed Central  Google Scholar 

  25. 25

    Kazlauskas, R. J. & Bornscheuer, U. T. Finding better protein engineering strategies. Nature Chem. Biol. 5, 526–529 (2009)

    CAS  Google Scholar 

  26. 26

    Lutz, S., Bornscheuer, U. T. (eds) Protein Engineering Handbook (Wiley-VCH, 2009)

    Google Scholar 

  27. 27

    Turner, N. J. Directed evolution drives the next generation of biocatalysts. Nature Chem. Biol. 5, 567–573 (2009)

    CAS  Google Scholar 

  28. 28

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

    ADS  Google Scholar 

  29. 29

    Schmid, A. et al. Industrial biocatalysis today and tomorrow. Nature 409, 258–268 (2001)

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  30. 30

    Arnold, F. H. Combinatorial and computational challenges for biocatalyst design. Nature 409, 253–257 (2001)

    ADS  CAS  Google Scholar 

  31. 31

    Schoemaker, H. E., Mink, D. & Wubbolts, M. G. Dispelling the myths—biocatalysis in industrial synthesis. Science 299, 1694–1697 (2003)

    ADS  CAS  Google Scholar 

  32. 32

    Keasling, J. D. Manufacturing molecules through metabolic engineering. Science 330, 1355–1358 (2010)

    ADS  CAS  Google Scholar 

  33. 33

    Madison, L. L. & Huisman, G. W. Metabolic engineering of poly(3-hydroxy-alkanoates): from DNA to plastic. Microbiol. Mol. Biol. Rev. 63, 21–53 (1999)

    CAS  PubMed  PubMed Central  Google Scholar 

  34. 34

    Wetterstrand, K. A. DNA sequencing costs: data from the NHGRI large-scale genome sequencing program. National Human Genome Research Project〉 (2011)

  35. 35

    Lorenz, P. & Eck, J. Metagenomics and industrial applications. Nature Rev. Microbiol. 3, 510–516 (2005)

    CAS  Google Scholar 

  36. 36

    Fowler, D. M. et al. High-resolution mapping of protein sequence- function relationships. Nature Methods 7, 741–746 (2010)

    CAS  PubMed  PubMed Central  Google Scholar 

  37. 37

    Richmond, K. E. et al. Amplification and assembly of chip-eluted DNA (AACED): a method for high-throughput gene synthesis. Nucleic Acids Res. 32, 5011–5018 (2004)

    CAS  PubMed  PubMed Central  Google Scholar 

  38. 38

    Gibson, D. G. et al. Creation of a bacterial cell controlled by a chemically synthesized genome. Science 329, 52–56 (2010)

    ADS  CAS  Google Scholar 

  39. 39

    Lutz, S. Beyond directed evolution—semi-rational protein engineering and design. Curr. Opin. Biotechnol. 21, 734–743 (2010)

    CAS  PubMed  PubMed Central  Google Scholar 

  40. 40

    Höhne, M., Schätzle, S., Jochens, H., Robins, K. & Bornscheuer, U. T. Rational assignment of key motifs for function guides in silico enzyme identification. Nature Chem. Biol. 6, 807–813 (2010)In this paper, careful analysis of key motifs of 5,000 pyridoxal-5-phosphate-dependent transaminase sequences in public databases identified 20 novel enzymes for which substrate preference (ketone, not α-keto acid) and enantiopreference (( R ), not ( S )) could be predicted and experimentally confirmed.

    Google Scholar 

  41. 41

    Jochens, H. & Bornscheuer, U. T. Natural diversity to guide focused directed evolution. ChemBioChem 11, 1861–1866 (2010)

    CAS  PubMed  PubMed Central  Google Scholar 

  42. 42

    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  PubMed Central  Google Scholar 

  43. 43

    Horsman, G. P., Liu, A. M. F., Henke, E., Bornscheuer, U. T. & Kazlauskas, R. J. Mutations in distant residues moderately increase the enantioselectivity of Pseudomonas fluorescens esterase towards methyl 3-bromo-2-methylpropanoate and ethyl 3-phenylbutyrate. Chemistry 9, 1933–1939 (2003)

    CAS  PubMed  PubMed Central  Google Scholar 

  44. 44

    Esvelt, K. M., Carlson, J. C. & Liu, D. R. A system for the continuous directed evolution of biomolecules. Nature 472, 499–503 (2011)

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  45. 45

    Moore, J. C., Pollard, D. J., Kosjek, B. & Devine, P. N. Advances in the enzymatic reduction of ketones. Acc. Chem. Res. 40, 1412–1419 (2007)

    CAS  PubMed  PubMed Central  Google Scholar 

  46. 46

    Strohmeier, G. A., Pichler, H., May, O. & Gruber-Khadjawi, M. Application of designed enzymes in organic synthesis. Chem. Rev. 111, 4141–4164 (2011)This is a review about protein engineering to create biocatalysts for industrial applications.

    CAS  PubMed  PubMed Central  Google Scholar 

  47. 47

    Wiese, A., Wilms, B., Syldatk, C., Mattes, R. & Altenbuchner, J. Cloning, nucleotide sequence and expression of a hydantoinase and carbamoylase gene from Arthrobacter aurescens DSM 3745 in Escherichia coli and comparison with the corresponding genes from Arthrobacter aurescens DSM 3747. Appl. Microbiol. Biotechnol. 55, 750–757 (2001)

    CAS  PubMed  PubMed Central  Google Scholar 

  48. 48

    Martinez-Gomez, A. I. et al. Recombinant polycistronic structure of hydantoinase process genes in Escherichia coli for the production of optically pure D-amino acids. Appl. Environ. Microbiol. 73, 1525–1531 (2007)

    CAS  PubMed  PubMed Central  Google Scholar 

  49. 49

    Panke, S. & Wubbolts, M. Advances in biocatalytic synthesis of pharmaceutical intermediates. Curr. Opin. Chem. Biol. 9, 188–194 (2005)

    CAS  PubMed  PubMed Central  Google Scholar 

  50. 50

    Breuer, M. et al. Industrial methods for the production of optically active intermediates. Angew. Chem. Int. Ed. 43, 788–824 (2004)

    CAS  Google Scholar 

  51. 51

    DeSantis, G. et al. Creation of a productive, highly enantioselective nitrilase through gene site saturation mutagenesis (GSSM). J. Am. Chem. Soc. 125, 11476–11477 (2003)This paper describes how a single amino-acid substitution can create a nitrilase with high enantioselectivity at 3 M substrate concentration, for synthesis of an intermediate for atorvastatin.

    CAS  PubMed  PubMed Central  Google Scholar 

  52. 52

    Ma, S. K. et al. A green-by-design biocatalytic process for atorvastatin intermediate. Green Chem. 12, 81–86 (2010)

    CAS  Google Scholar 

  53. 53

    Anastas, P., Warner, J. (eds) Green Chemistry: Theory and Practice (Oxford Univ. Press, 1998)

    Google Scholar 

  54. 54

    Yang, T. H. et al. Biosynthesis of polylactic acid and its copolymers using evolved propionate CoA transferase and PHA synthase. Biotechnol. Bioeng. 105, 150–160 (2010)

    CAS  PubMed  PubMed Central  Google Scholar 

  55. 55

    Bernath, K. et al. In vitro compartmentalization by double emulsions: sorting and gene enrichment by fluorescence activated cell sorting. Anal. Biochem. 325, 151–157 (2004)

    CAS  PubMed  PubMed Central  Google Scholar 

  56. 56

    Becker, S. et al. Single-cell high-throughput screening to identify enantioselective hydrolytic enzymes. Angew. Chem. Int. Ed. 47, 5085–5088 (2008)

    CAS  Google Scholar 

  57. 57

    Fernández-Alvaro, E. et al. A combination of in vivo selection and cell sorting for the identification of enantioselective biocatalysts. Angew. Chem. Int. Ed. 50, 8584–8587 (2011)

    Google Scholar 

  58. 58

    Whittle, E. & Shanklin, J. Engineering delta 9–16:0-acyl carrier protein (ACP) desaturase specificity based on combinatorial saturation mutagenesis and logical redesign of the castor delta 9–18:0-ACP desaturase. J. Biol. Chem. 276, 21500–21505 (2001)

    CAS  PubMed  PubMed Central  Google Scholar 

  59. 59

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

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  60. 60

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

    CAS  Google Scholar 

  61. 61

    Yoshikuni, Y., Ferrin, T. E. & Keasling, J. D. Designed divergent evolution of enzyme function. Nature 440, 1078–1082 (2006)

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  62. 62

    Bava, K. A., Gromiha, M. M., Uedaira, H. & Kitajima, K. &. Sarai, A. ProTherm, version 4.0: thermodynamic database for proteins and mutants. Nucleic Acids Res. 32, D120–D121 (2004)

    CAS  PubMed  PubMed Central  Google Scholar 

  63. 63

    Guo, H. H., Choe, J. & Loeb, L. A. Protein tolerance to random amino acid change. Proc. Natl Acad. Sci. USA 101, 9205–9210 (2004)This paper shows that about 34% of random amino-acid replacements inactivate a protein’s functions, indicating the importance of starting with a stabilized protein for protein engineering experiments.

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  64. 64

    Drummond, D. A., Bloom, J. D., Adami, C., Wilke, C. O. & Arnold, F. H. Why highly expressed proteins evolve slowly. Proc. Natl Acad. Sci. USA 102, 14338–14343 (2005)

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  65. 65

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

    CAS  PubMed  PubMed Central  Google Scholar 

  66. 66

    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, 17 (2007)

    PubMed  PubMed Central  Google Scholar 

  67. 67

    Wells, J. A. Additivity of mutational effects in proteins. Biochemistry 29, 8509–8517 (1990)

    CAS  PubMed  PubMed Central  Google Scholar 

  68. 68

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

    CAS  Google Scholar 

  69. 69

    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. Edn Engl. 36, 2830–2832 (1997)

    CAS  Google Scholar 

  70. 70

    Weinreich, D. M., Delaney, N. F., Depristo, M. A. & Hartl, D. L. Darwinian evolution can follow only very few mutational paths to fitter proteins. Science 312, 111–114 (2006)

    ADS  CAS  PubMed  Google Scholar 

  71. 71

    Reetz, M. T. & Sanchis, J. Constructing and analyzing the fitness landscape of an experimental evolutionary process. ChemBioChem 9, 2260–2267 (2008)

    CAS  Google Scholar 

  72. 72

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

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  73. 73

    Meyer, D. et al. Conversion of pyruvate decarboxylase into an enantioselective carboligase with biosynthetic potential. J. Am. Chem. Soc. 133, 3609–3616 (2011)

    CAS  Google Scholar 

  74. 74

    Siegel, J. B. et al. Computational design of an enzyme catalyst for a stereoselective bimolecular Diels-Alder reaction. Science 329, 309–313 (2010)

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  75. 75

    Randolph, J., Yagodkin, A., Lamaitre, M., Azhayev, A. & Mackie, H. Codon based mutagenesis using trimer phosphoramidites. Nucleic Acids Symp. Ser. 52, 479 (2008)

    CAS  Google Scholar 

  76. 76

    Rana, S., Yeh, Y. C. & Rotello, V. M. Engineering the nanoparticle-protein interface: applications and possibilities. Curr. Opin. Chem. Biol. 14, 828–834 (2010)

    CAS  PubMed  PubMed Central  Google Scholar 

  77. 77

    Dueber, J. E. et al. Synthetic protein scaffolds provide modular control over metabolic flux. Nature Biotechnol. 27, 753–759 (2009)

    CAS  Google Scholar 

  78. 78

    McDonald, T. J. et al. Wiring-up hydrogenase with single-walled carbon nanotubes. Nano Lett. 7, 3528–3534 (2007)

    ADS  CAS  Google Scholar 

  79. 79

    Mouse Genome Sequencing Consortium Initial sequencing and comparative analysis of the mouse genome. Nature 420, 520–562 (2002)

    Google Scholar 

  80. 80

    Savile, C. K. & Lalonde, J. J. Biotechnology for the acceleration of carbon dioxide capture and sequestration. Curr. Opin. Biotechnol. 22, 818–823 (2011)

    CAS  PubMed  PubMed Central  Google Scholar 

  81. 81

    Morikawa, S. et al. Highly active mutants of carbonyl reductase S1 with inverted coenzyme specificity and production of optically active alcohols. Biosci. Biotechnol. Biochem. 69, 544–552 (2005)

    CAS  PubMed  PubMed Central  Google Scholar 

  82. 82

    Patel, R. N., Chu, L. & Mueller, R. Diastereoselective microbial reduction of (S)-[3-chloro-2-oxo-1-(phenylmethyl)propyl]carbamic acid, 1,1-dimethylethyl ester. Tetrahedr. Asymm. 14, 3105–3109 (2003)

    CAS  Google Scholar 

  83. 83

    Liang, J. et al. Highly enantioselective reduction of a small heterocyclic ketone: biocatalytic reduction of tetrahydrothiophene-3-one to the corresponding (R)-alcohol. Org. Process Res. Dev. 14, 188–192 (2010)

    CAS  Google Scholar 

  84. 84

    Urano, N. et al. Directed evolution of an aminoalcohol dehydrogenase for efficient production of double chiral aminoalcohols. J. Biosci. Bioeng. 111, 266–271 (2011)

    CAS  PubMed  PubMed Central  Google Scholar 

  85. 85

    Gooding, O. W. et al. Development of a practical biocatalytic process for (R)-2-methylpentanol. Org. Process Res. Dev. 14, 119–126 (2010)

    CAS  Google Scholar 

  86. 86

    Cobley, C. J., Hanson, C. H., Lloyd, M. C. & Simmonds, S. The combination of hydroformylation and biocatalysis for the large-scale synthesis of (S)-allysine ethylene acetal. Org. Process Res. Dev. 15, 284–290 (2011)

    CAS  Google Scholar 

  87. 87

    Xie, X., Watanabe, K., Wojcicki, W. A., Wang, C. C. & Tang, Y. Biosynthesis of lovastatin analogs with a broadly specific acyltransferase. Chem. Biol. 13, 1161–1169 (2006)

    CAS  PubMed  PubMed Central  Google Scholar 

  88. 88

    Truppo, M. D. & Hughes, G. Development of an improved immobilized CAL-B for the enzymatic resolution of a key intermediate to odanacatib. Org. Process Res. Dev. 15, 1033–1035 (2011)

    CAS  Google Scholar 

  89. 89

    Brocklehurst, C. E., Laumen, K., Vecchia, L. L., Shaw, D. & Vögtle, M. Diastereoisomeric salt formation and enzyme-catalyzed kinetic resolution as complementary methods for the chiral separation of cis-/trans-enantiomers of 3-aminocyclohexanol. Org. Process Res. Dev. 15, 294–300 (2011)

    CAS  Google Scholar 

  90. 90

    Iding, H. et al. in Asymmetric Catalysis on Industrial Scale (eds Blaser, H. U. & Federsel, H. J. ) 377–396 (Wiley-VCH, 2010)

    Google Scholar 

  91. 91

    Chen, Y. J. et al. Enzymatic preparation of an (S)-amino acid from a racemic amino acid. Org. Process Res. Dev. 15, 241–248 (2011)

    Google Scholar 

  92. 92

    Rousseau, A. L. et al. Scale-up of a chemo-biocatalytic route to (2R,4R)- and (2S,4S)-monatin. Org. Process Res. Dev. 15, 249–257 (2011)

    CAS  Google Scholar 

  93. 93

    Greenberg, W. A. et al. Development of an efficient, scalable, aldolase-catalyzed process for enantioselective synthesis of statin intermediates. Proc. Natl Acad. Sci. USA 101, 5788–5793 (2004)

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  94. 94

    Horinouchi, N. et al. Biochemical retrosynthesis of 2′-deoxyribonucleosides from glucose, acetaldehyde, and a nucleobase. Appl. Microbiol. Biotechnol. 71, 615–621 (2006)

    CAS  PubMed  PubMed Central  Google Scholar 

  95. 95

    Hibi, M. et al. Characterization of Bacillus thuringiensis L-isoleucine dioxygenase for production of useful amino acids. Appl. Environ. Microbiol. 77, 6926–6930 (2011)

    CAS  PubMed  PubMed Central  Google Scholar 

  96. 96

    de Lange, B. et al. Asymmetric synthesis of (S)-2-indoline carboxylic acid by combining biocatalysis and homogeneous catalysis. ChemCatChem 3, 289–292 (2011)

    CAS  Google Scholar 

  97. 97

    Asano, Y., Mihara, Y. & Yamada, H. A new enzymatic method of selective phosphorylation of nucleosides. J. Mol. Catal. B 6, 271–277 (1999)

    CAS  Google Scholar 

  98. 98

    Hermann, M. et al. Alternative pig liver esterase (APLE) - cloning, identification and functional expression in Pichia pastoris of a versatile new biocatalyst. J. Biotechnol. 133, 301–310 (2008)

    CAS  PubMed  PubMed Central  Google Scholar 

  99. 99

    Ikenaka, Y. et al. Thermostability reinforcement through a combination of thermostability-related mutations of N-carbamyl-D-amino acid amidohydrolase. Biosci. Biotechnol. Biochem. 63, 91–95 (1999)

    CAS  PubMed  PubMed Central  Google Scholar 

  100. 100

    Ro, D. K. et al. Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature 440, 940–943 (2006)

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

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We thank H.-P. Meyer and R. Fox for discussions. R.J.K. thanks the US National Science Foundation (CBET-0932762) and the Korea Science and Engineering Foundation funded by the Ministry of Education, Science and Technology (WCU programme R32-2008-000-10213-0). U.T.B. and S.L. thank the German Research Foundation (SPP 1170, Bo1864/4-1) and, respectively, the US National Science Foundation (CBET-0730312) for financial support.

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U.T.B., R.J.K. and S.L. drafted the text; K.R., G.W.H. and J.C.M. collected the examples for industrial applications; and the authors together wrote and edited the review. All authors contributed equally.

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Correspondence to U. T. Bornscheuer.

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Competing interests

K.R. is an employee of Lonza AG, G.W.H. is an employee of Codexis Inc. and J.C.M. is an employee of Merck & Co. Inc. All three companies are working on biocatalysis.

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Bornscheuer, U., Huisman, G., Kazlauskas, R. et al. Engineering the third wave of biocatalysis. Nature 485, 185–194 (2012).

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