Title

Engineering of industrial biocatalysts

Conference Dates

September 15-19, 2019

Abstract

In the last 40 years advances in the protein engineering have prompted the application of biocatalysts in the synthesis of building blocks, fine and bulk active pharmaceutical chemicals for the agrochemical, food, biofuel and pharmaceutical industries. Computational chemistry methodologies are fueling the development of a new generation of rationally designed biocatalysts with enhanced selectivity and specificity at a fraction of the time and cost compared to traditional protocols such as directed evolution.

We present two examples of rational enzyme design. Our first example is the study of the phenylacetone monooxygenase (PAMO), the most stable and thermo-tolerant member of the Baeyer–Villiger monooxygenases family. We solved the catalytic mechanism of this enzyme for the native substrate phenylacetone as well as for a linear non-native substrate 2-octanone, using molecular dynamics simulations, quantum mechanics and quantum mechanics/molecular mechanics calculations.1 By studying relevant PAMO variants we provide a theoretical basis for the preference of the enzyme for the native aromatic substrate over non-native linear substrates.2

The second example regards an (S)-selective-transaminase from Vibrio fluvialis (S-TAm), which offers an environmentally sustainable synthesis route for the production of pure chiral amines.3 By applying a rational enzyme engineering protocol we altered this enzyme towards better acceptance of bulky ketones, starting with no detectable activity of the WT. Our best S-TAm variant improved the reaction rate by > 1716-fold and retained activity even at 50 °C. To obtain such an outstanding result we only screened 113 variants, a substantially lower number than those typically associated with directed evolution (104 to 107 clones).

Both studies provide fundamental insights into the rational engineering of enzymes for industrial applications.

(1) Carvalho, A. T. P.; Dourado, D. F. A. R.; Skvortsov, T.; Abreu, M. de; Ferguson, L. J.; Quinn, D. J.; Moody, T. S.; Huang, M. Catalytic Mechanism of Phenylacetone Monooxygenases for Non-Native Linear Substrates. Phys. Chem. Chem. Phys. 2017, 19 (39), 26851–26861. https://doi.org/10.1039/C7CP03640J.

(2) Carvalho, A. T. P.; Dourado, D. F. A. R.; Skvortsov, T.; Abreu, M. de; Ferguson, L. J.; Quinn, D. J.; Moody, T. S.; Huang, M. Spatial Requirement for PAMO for Transformation of Non-Native Linear Substrates. Phys. Chem. Chem. Phys. 2018, 20 (4), 2558–2570. https://doi.org/10.1039/C7CP07172H.

(3) Dourado, D. F. A. R.; Pohle, S.; Carvalho, A. T. P.; Dheeman, D. S.; Caswell, J. M.; Skvortsov, T.; Miskelly, I.; Brown, R. T.; Quinn, D. J.; Allen, C. C. R.; Huang, M; Moody, T. Rational Design of a (S)-Selective-Transaminase for Asymmetric Synthesis of (1S)-1-(1,1′-Biphenyl-2-Yl)Ethanamine. ACS Catal. 2016, 6 (11), 7749–7759. https://doi.org/10.1021/acscatal.6b02380

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