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Optimisation of Cytochrome P450 BM3 Assisted by Consensus-Guided Evolution

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Abstract

Cytochrome P450 enzymes have attracted much interest over the years given their ability to insert oxygen into saturated carbon-hydrogen bonds, a difficult feat to accomplish by traditional chemistry. Much of the activity in this field has centered on the bacterial enzyme CYP102A1, or BM3, from Bacillus megaterium, as it has shown itself capable of hydroxylating/acting upon a wide range of substrates, thereby producing industrially relevant pharmaceuticals, fine chemicals, and hormones. In addition, unlike most cytochromes, BM3 is both soluble and fused to its natural redox partner, thus facilitating its use. The industrial use of BM3 is however stifled by its instability and its requirement for the expensive NADPH cofactor. In this work, we added several mutations to the BM3 mutant R966D/W1046S that enhanced the turnover number achievable with the inexpensive cofactors NADH and NBAH. These new mutations, A769S, S847G, S850R, E852P, and V978L, are localized on the reductase domain of BM3 thus leaving the oxidase domain intact. For NBAH-driven reactions by new mutant NTD5, this led to a 5.24-fold increase in total product output when compared to the BM3 mutant R966D/W1046S. For reactions driven by NADH by new mutant NTD6, this enhanced total product output by as much as 2.3-fold when compared to the BM3 mutant R966D/W1046S. We also demonstrated that reactions driven by NADH with the NTD6 mutant not only surpassed total product output achievable by wild-type BM3 with NADPH but also retained the ability to use this latter cofactor with greater total product output as well.

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This research was supported by Natural Sciences and Engineering Research Council Innov-UC program (#CUI21-430895-12) and discovery program (#CUI21-430895-12).

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  1. Department of Chemical Engineering, Université Laval, Québec, Québec, G1V 0A6, Canada

    Thierry Vincent, Bruno Gaillet & Alain Garnier

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Thierry Vincent: term, conceptualization, methodology, validation, formal analysis, investigation, resources, Writing – Original Draft, writing—review and editing, visualization, project administration.

Bruno Gaillet: writing— review and editing, supervision, funding acquisition

Alain Garnier: conceptualization, formal analysis, resources, writing—review and editing, supervision, project administration, funding acquisition

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Vincent, T., Gaillet, B. & Garnier, A. Optimisation of Cytochrome P450 BM3 Assisted by Consensus-Guided Evolution. Appl Biochem Biotechnol 193, 2893–2914 (2021). https://doi.org/10.1007/s12010-021-03573-1

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