Directed evolution of a Baeyer-Villiger monooxygenase to enhance enantioselectivity

Applied Microbiology and Biotechnology
Anett Kirschner, Uwe T Bornscheuer

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 approximately 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 approximately 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 approximately 75-90). In a second step, beneficial mutations were recombined by consecutive cycles of QuikChange site-dir...Continue Reading

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Citations

Aug 29, 2009·Applied Microbiology and Biotechnology·Yong Cheol ParkGeorge N Bennett
Aug 7, 2010·Applied Microbiology and Biotechnology·Kristian GeitnerUwe T Bornscheuer
May 6, 2011·Chemical Reviews·Hannes LeischPeter C K Lau
May 11, 2011·Chemical Reviews·Gernot A StrohmeierMandana Gruber-Khadjawi
Jan 14, 2012·Journal of the American Chemical Society·Iakov PolyakWalter Thiel
Aug 13, 2013·Journal of the American Chemical Society·Manfred T Reetz
Oct 8, 2009·Journal of the American Chemical Society·Manfred T Reetz, Sheng Wu
Jul 5, 2011·Applied and Environmental Microbiology·Hanna M DudekMarco W Fraaije
Feb 23, 2010·Current Opinion in Microbiology·Dominique Böttcher, Uwe T Bornscheuer
Nov 17, 2009·Trends in Biotechnology·Linda G OttenIsabel W C E Arends
Sep 19, 2012·Critical Reviews in Biotechnology·Ashwani Kumar, Suren Singh
Aug 10, 2016·Applied Microbiology and Biotechnology·Anett Schallmey, Marcus Schallmey
May 4, 2017·Chemical Reviews·Jonathan LathamJason Micklefield
May 16, 2017·Chembiochem : a European Journal of Chemical Biology·Kathleen BalkeUwe T Bornscheuer
Mar 16, 2017·Chemical Communications : Chem Comm·Jian-Bo WangManfred T Reetz
Jun 7, 2014·Chemical Communications : Chem Comm·Dirk HoltmannFrank Hollmann
Jul 19, 2012·Chemistry : a European Journal·Zhi-Gang ZhangManfred T Reetz
Mar 31, 2019·Applied and Environmental Microbiology·Yuan-Yang LiuGao-Wei Zheng
Jun 27, 2012·Organic & Biomolecular Chemistry·Kathleen BalkeUwe T Bornscheuer
Mar 29, 2013·Journal of Biomolecular Screening·Hanna M DudekMarco W Fraaije
Oct 14, 2016·Chembiochem : a European Journal of Chemical Biology·Andy BeierUwe T Bornscheuer
Nov 24, 2011·Chembiochem : a European Journal of Chemical Biology·Jan G E van LeeuwenDick B Janssen

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