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Directed evolution of a Baeyer–Villiger monooxygenase to enhance enantioselectivity

Abstract

The Baeyer–Villiger monooxygenase (BVMO) BmoF1 from Pseudomonas fluorescens DSM 50106 was shown before to enantioselectively oxidize different 4-hydroxy-2-ketones to the corresponding hydroxyalkyl acetates, being the first example of a BVMO-catalyzed kinetic resolution of aliphatic acyclic ketones. However, the wild-type enzyme exhibited only moderate E values (E ∼ 55). Thus, the enantioselectivity was enhanced by means of directed evolution and optimization of reaction conditions since it was found that higher E values (E ∼ 70 for wild-type BmoF1) could already be obtained when performing biotransformations in shake flasks rather than small tubes. In a first step, random mutations were introduced by error-prone polymerase chain reaction, and BmoF1 mutants (>3,500 clones) were screened for improved activity and enantioselectivity using a microtiter-plate-based screening method. Mutations S136L and L252Q were found to increase conversion compared to wild type, while several mutations (H51L, F225Y, S305C, and E308V) were identified enhancing the enantioselectivity to a varying extent (E ∼ 75–90). In a second step, beneficial mutations were recombined by consecutive cycles of QuikChange® site-directed mutagenesis resulting in a double mutant (H51L/S136L) showing both improved conversion and enantioselectivity (E ∼ 86).

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References

  1. Alphand V, Furstoss R (2000) Microbial transformations 44. Optimisation of a new Baeyer–Villiger activity: application to stereospecific oxidation of 3-phenylcyclobutanone. J Mol Catal B: Enzym 9:209–217

  2. Baeyer A, Villiger V (1899) Einwirkung des Caro’schen Reagens auf Ketone. Ber Dtsch Chem Ges 32:3625–3633

  3. Bocola M, Schulz F, Leca F, Vogel A, Fraaije MW, Reetz MT (2005) Converting phenylacetone monooxygenase into phenylcyclohexanone monooxygenase by rational design: towards practical Baeyer–Villiger monooxygenases. Adv Synth Catal 347:979–986

  4. Brzostowicz PC, Blasko MS, Rouviere PE (2002) Identification of two gene clusters involved in cyclohexanone oxidation in Brevibacterium epidermidis strain HCU. Appl Microbiol Biotechnol 58:781–789

  5. Brzostowicz PC, Walters DM, Thomas SM, Nagarajan V, Rouviere PE (2003) mRNA differential display in a microbial enrichment culture: simultaneous identification of three cyclohexanone monooxygenases from three species. Appl Environ Microbiol 69:334–342

  6. Carrea G, Redigolo B, Riva S, Colonna S, Gaggero N, Battistel E, Bianchi D (1992) Effects of substrate structure on the enantioselectivity and stereochemical course of sulfoxidation catalyzed by cyclohexanone monooxygenase. Tetrahedron: Asymmetry 3:1063–1068

  7. Cedrone F, Bhatnagar T, Baratti JC (2005) Colorimetric assays for quantitative analysis and screening of epoxide hydrolase activity. Biotechnol Lett 27:1921–1927

  8. Chen C-S, Fujimoto Y, Girdaukas G, Sih CJ (1982) Quantitative analyses of biochemical kinetic resolutions of enantiomers. J Am Chem Soc 104:7294–7299

  9. Cirino PC, Mayer KM, Umeno D (2003) Generating mutant libraries using error-prone PCR. In: Arnold FH, Georgiou G (eds) Directed evolution library creation. Humana, Totowa, pp 3–9

  10. Clouthier CM, Kayser MM, Reetz MT (2006) Designing new Baeyer–Villiger monooxygenases using restricted CASTing. J Org Chem 71:8431–8437

  11. Criegee R (1948) Die Umlagerung der Dekalin-peroxydester als Folge von kationischem Sauerstoff. Justus Liebigs Ann Chem 560:127–135

  12. de Gonzalo G, Torres Pazmino DE, Ottolina G, Fraaije MW, Carrea G (2006) 4-Hydroxyacetophenone monooxygenase from Pseudomonas fluorescens ACB as an oxidative biocatalyst in the synthesis of optically active sulfoxides. Tetrahedron: Asymmetry 17:130–135

  13. Eggert T, Funke SA, Rao NM, Acharya P, Krumm H, Reetz MT, Jaeger K-E (2005) Multiplex-PCR-based recombination as a novel high-fidelity method for directed evolution. ChemBioChem 6:1062–1067

  14. Gutiérrez M-C, Sleegers A, Simpson HD, Alphand V, Furstoss R (2003) The first fluorogenic assay for detecting a Baeyer–Villigerase activity in microbial cells. Org Biomol Chem 1:3500–3506

  15. Kamerbeek NM, Moonen MJH, van der Ven JGM, van Berkel WJH, Fraaije MW, Janssen DB (2001) 4-Hydroxyacetophenone monooxygenase from Pseudomonas fluorescens ACB. Eur J Biochem 268:2547–2557

  16. Kamerbeek NM, Janssen DB, Van Berkel WJH, Fraaije MW (2003a) Baeyer–Villiger monooxygenases, an emerging family of flavin-dependent biocatalysts. Adv Synth Catal 345:667–678

  17. Kamerbeek NM, Olsthoorn JJ, Fraaije MW, Janssen DB (2003b) Substrate specificity and enantioselectivity of 4-hydroxyacetophenone monooxygenase. Appl Environ Microbiol 69:419–426

  18. Kirschner A, Bornscheuer UT (2006) Kinetic resolution of 4-hydroxy-2-ketones catalyzed by a Baeyer–Villiger monooxygenase. Angew Chem Int Ed 45:7004–7006

  19. Kirschner A, Altenbuchner J, Bornscheuer UT (2007) Cloning, expression and characterization of a Baeyer–Villiger monooxygenase from Pseudomonas fluorescens DSM 50106 in E. coli. Appl Microbiol Biotechnol 73:1065–1072

  20. Kostichka K, Thomas SM, Gibson KJ, Nagarajan V, Cheng Q (2001) Cloning and characterization of a gene cluster for cyclododecanone oxidation in Rhodococcus ruber SC1. J Bacteriol 183:6478–6486

  21. Krebsfanger N, Zocher F, Altenbuchner J, Bornscheuer UT (1998) Characterization and enantioselectivity of a recombinant esterase from Pseudomonas fluorescens. Enzyme Microb Technol 22:641–646

  22. Malito E, Alfieri A, Fraaije MW, Mattevi A (2004) Crystal structure of a Baeyer–Villiger monooxygenase. Proc Natl Acad Sci 101:13157–13162

  23. May O, Nguyen PT, Arnold FH (2000) Inverting enantioselectivity by directed evolution of hydantoinase for improved production of l-methionine. Nat Biotechnol 18:317–320

  24. Mihovilovic MD (2006) Enzyme mediated Baeyer–Villiger oxidations. Curr Org Chem 10:1265–1287

  25. Mihovilovic MD, Müller B, Stanetty P (2002) Monooxygenase-mediated Baeyer–Villiger oxidations. Eur J Org Chem 2002:3711–3730

  26. Mihovilovic MD, Rudroff F, Grötzl B (2004) Enantioselective Baeyer–Villiger oxidations. Curr Org Chem 8:1057–1069

  27. Miyazaki K, Takenouchi M (2002) Creating random mutagenesis libraries using megaprimer PCR of whole plasmids. BioTechniques 33:1033–1038

  28. Moonen MJH, Westphal AH, Rietjens IMCM, van Berkel WJH (2005) Enzymatic Baeyer–Villiger oxidation of benzaldehydes. Adv Synth Catal 347:1027–1034

  29. Reetz MT, Zonta A, Schimossek K, Liebeton K (1997) Creation of enantioselective biocatalysts for organic chemistry by in vitro evolution. Angew Chem Int Ed 36:2830–2832

  30. Reetz MT, Brunner B, Schneider T, Schulz F, Clouthier CM, Kayser MM (2004a) Directed evolution as a method to create enantioselective cyclohexanone monooxygenases for catalysis in Baeyer–Villiger reactions. Angew Chem Int Ed 43:4075–4078

  31. Reetz MT, Daligault F, Brunner B, Hinrichs H, Deege A (2004b) Directed evolution of cyclohexanone monooxygenases: enantioselective biocatalysts for the oxidation of prochiral thioethers. Angew Chem Int Ed 43:4078–4081

  32. Schmidt M, Hasenpusch D, Kaehler M, Kirchner U, Wiggenhorn K, Langel W, Bornscheuer UT (2006) Directed evolution of an esterase from Pseudomonas fluorescens yields a mutant with excellent enantioselectivity and activity for the kinetic resolution of a chiral building block. ChemBioChem 7:805–809

  33. Schmidt M, Henke E, Heinze B, Kourist R, Hidalgo A, Bornscheuer UT (2007) A versatile esterase from Bacillus subtilis: cloning, expression, characterization, and its application in biocatalysis. Biotechnol J 2:249–253

  34. Sicard R, Chen LS, Marsaioli AJ, Reymond J-L (2005) A fluorescense-based assay for Baeyer–Villiger monooxygenases, hydrolases and lactonases. Adv Synth Catal 347:1041–1050

  35. Strukul G (1998) Transition metal catalysis in the Baeyer–Villiger oxidation of ketones. Angew Chem Int Ed 37:1199–1209

  36. Torres Pazmino DE, Snajdrova R, Rial DV, Mihovilovic MD, Fraaije MW (2007) Altering the substrate specificity and enantioselectivity of phenylacetone monooxygenase by structure-inspired enzyme redesign. Adv Synth Catal 349:1361–1368

  37. van Beilen JB, Mourlane F, Seeger MA, Kovac J, Li Z, Smits THM, Fritsche U, Witholt B (2003) Cloning of Baeyer–Villiger monooxygenases from Comamonas, Xanthobacter and Rhodococcus using polymerase chain reaction with highly degenerate primers. Environ Microbiol 5:174–182

  38. Wahler D, Reymond J-L (2002) The adrenalin test for enzymes. Angew Chem Int Ed 41:1229–1232

  39. Walsh CT, Chen YCJ (1988) Enzymatic Baeyer–Villiger oxidations by flavin-dependent monooxygenases. Angew Chem Int Ed 27:333–343

  40. Watts AB, Beecher J, Whitcher CS, Littlechild JA (2002) A method for screening Baeyer–Villiger monooxygenase activity against monocyclic ketones. Biocatal Biotransform 20:209–214

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Acknowledgements

We thank the Fonds der Chemischen Industrie (Frankfurt, Germany) and the Studienstiftung des Deutschen Volkes (Bonn, Germany) for stipends to Anett Kirschner.

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Correspondence to Uwe T. Bornscheuer.

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Kirschner, A., Bornscheuer, U.T. Directed evolution of a Baeyer–Villiger monooxygenase to enhance enantioselectivity. Appl Microbiol Biotechnol 81, 465–472 (2008). https://doi.org/10.1007/s00253-008-1646-4

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Keywords

  • Baeyer–Villiger monooxygenase
  • Directed evolution
  • Enzyme catalysis
  • Enantioselectivity
  • β-hydroxyketones