Advanced Tools for the Discovery and Engineering of Enzymes for Biocatalytic Applications
Uwe T. Bornscheuer
Institute of Biochemistry, Dept. of Biotechnology & Enzyme Catalysis,
Greifswald University, Germany
This lecture will highlight principle strategies and current challenges in enzyme discovery and protein engineering aiming to enhance their usefulness in biocatalytic applications [1]. This also includes the combination of enzymes with chemocatalysts [2] and their incorporation into retrosynthetic concepts [3]. Examples will cover amine transaminases (ATA), Baeyer-Villiger- (BVMO) as well as P450-monooxygenases. For the synthesis of chiral amines, Prof. Bornscheuer and his group engineered (S)-selective ATA for the acceptance of bulky ketones in the asymmetric synthesis of chiral amines [4]. For BVMOs, they could recently engineer these enzymes to efficiently accept the cofactor NADH instead of NADPH [5]. Furthermore, they demonstrated how they could invert their regioselectivity exemplified for different enzymes and substrate types [6]. In BVMO- as well as P450-catalyzed reactions, uncoupling – the undesired formation of H2O2 – can represent a major issue. Prof. Bornscheuer and his group have recently developed a sensitive assay to quantify H2O2-formation and hence to calculate desired product formation by following NAD(P)H consumption [7]. Finally, a new class of P450 monooxygenases from marine bacteria will be presented, which play a central role in the degradation of algal carbohydrates [8].
[1] Bornscheuer, U.T., et al., Nature, 485, 185-194 (2012); Kazlauskas, R.J., Bornscheuer, U.T., Nat. Chem. Biol., 5, 526-529 (2009); Lutz, S., Bornscheuer, U.T. (Eds.) Protein Engineering Handbook, Wiley-VCH, Weinheim (2009, 2012); Höhne, M., Bornscheuer, U.T. (Eds.) Protein Engineering, Meth. Mol. Biol., 1685, Humana Press, New York; Bornscheuer, U.T., Phil. Trans. R. Soc. A., 376, 20170063 (2018);Badenhorst C.P.S., Bornscheuer, U.T., Trends Biochem. Sci., 43, 180-198 (2018).
[2] Rudroff, F., et al., Nat. Catal. 1, 12-22 (2018).
[3] de Souza, R.O.M.A., Miranda, L.S.M., Bornscheuer, U.T., Chem. Eur. J., 23, 12040-12063 (2017).
[4] Pavlidis, I., et al., Nature Chem., 8, 1076-1082 (2016); Weiß, M.S. et al., Org. Biol. Chem., 14, 10249-10254 (2016); Weiß, M.S., et al., ChemBioChem, 18, 1022-1026 (2017).
[5] Beier, A., et al., ChemBioChem, 17, 2312-2315 (2016).
[6] Balke, K., Beier, A., Bornscheuer, U.T., Biotechnol. Adv., 36, 247-263 (2018); Balke, K., et al., ACS Chem. Biol., 11, 38-43 (2016); Balke, K., Bäumgen, M., Bornscheuer, U.T., ChemBioChem, 8, 1627-1638 (2017).
[7] Morlock, L.K., Böttcher, D., Bornscheuer, U.T., Appl. Microbiol. Biotechnol., 102, 985-994 (2018).
[8] Reisky, L., et al., Nat. Chem. Biol., online (2018).