Indigo Formation and Rapid NADPH Consumption Provide Robust Prediction of Raspberry Ketone Synthesis by Engineered Cytochrome P450 BM3

Pelletier, Joelle N.; Rousseau, Olivier; Ebert, Maximilian C. C. J. C.; Quaglia, Daniela; Fendri, Ali; Parisien, Adem H.; Besna, Jonathan N.; Iyathurai, Saathanan

Abstract

Natural raspberry ketone has a high value in the flavor, fragrance and pharmaceutical industries. Its extraction is costly, justifying the search for biosynthetic routes. We hypothesized that cytochrome P450 BM3 (P450 BM3) could be engineered to catalyze the hydroxylation of 4‐phenyl‐2‐butanone, a naturally sourceable precursor, to raspberry ketone. The synthesis of indigo by variants of P450 BM3 has previously served as a predictor of promiscuous oxidation reactions. To this end, we screened 53 active‐site variants of P450 BM3 using orthogonal high‐throughput workflows to identify the most streamlined route to all indigo‐forming variants. Among the three known and 13 new indigo‐forming variants, eight hydroxylated 4‐phenyl‐2‐butanone to raspberry ketone. Previously unreported variant A82Q displayed the highest initial rates and coupling efficiencies in synthesis of indigo and of raspberry ketone. It produced the highest total concentration of raspberry ketone despite producing less total indigo than previously reported variants. Its productivity, although modest, clearly demonstrates the potential for development of a biocatalytic route to raspberry ketone. In addition to validating indigo as a robust predictor of this promiscuous activity, we demonstrate that monitoring rapid NADPH consumption serves as an alternative predictor of a promiscuous reactivity in P450 BM3.

Citation

Pelletier, J. N., Rousseau, O., Ebert, M. C. C. J. C., Quaglia, D., Fendri, A., Parisien, A. H., …Iyathurai, S. (2020). Indigo Formation and Rapid NADPH Consumption Provide Robust Prediction of Raspberry Ketone Synthesis by Engineered Cytochrome P450 BM3. ChemCatChem, 12(3), 837-845. https://doi.org/10.1002/cctc.201901974

• Journal Article Type: Article
• Acceptance Date: Nov 5, 2019
• Online Publication Date: Dec 2, 2019
• Publication Date: Feb 6, 2020
• Deposit Date: Jun 18, 2020
• Publicly Available Date: Dec 3, 2020
• Journal: ChemCatChem
• Print ISSN: 1867-3880
• Electronic ISSN: 1867-3899
• Publisher: Wiley
• Peer Reviewed: Peer Reviewed
• Volume: 12
• Issue: 3
• Pages: 837-845
• DOI: https://doi.org/10.1002/cctc.201901974
• Keywords: Physical and Theoretical Chemistry; Inorganic Chemistry; Organic Chemistry; Catalysis
• Public URL: https://nottingham-repository.worktribe.com/output/4672563
• Publisher URL: https://chemistry-europe.onlinelibrary.wiley.com/doi/abs/10.1002/cctc.201901974
• Additional Information: This is the peer reviewed version of the following article: O. Rousseau, M. C. C. J. C. Ebert, D. Quaglia, A. Fendri, A. H. Parisien, J. N. Besna, S. Iyathurai, J. N. Pelletier, ChemCatChem 2020 , 12 , 837, which has been published in final form at https://doi.org/10.1002/cctc.201901974. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions.