Simulation-guided engineering enables a functional switch in selinadiene synthase toward hydroxylation

Srivastava, Prabhakar L. ORCID: https://orcid.org/0000-0002-8219-6419, Johns, Sam T., Voice, Angus, Morley, Katharine, Escorcia, Andrés M., Miller, David J., Allemann, Rudolf K. ORCID: https://orcid.org/0000-0002-1323-8830 and van der Kamp, Marc W. 2024. Simulation-guided engineering enables a functional switch in selinadiene synthase toward hydroxylation. ACS Catalysis 14 (14) , 11034–11043. 10.1021/acscatal.4c02032

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Abstract

Engineering sesquiterpene synthases to form predefined alternative products is a major challenge due to their diversity in cyclization mechanisms and our limited understanding of how amino acid changes affect the steering of these mechanisms. Here, we use a combination of atomistic simulation and site-directed mutagenesis to engineer a selina-4(15),7(11)-diene synthase (SdS) such that its final reactive carbocation is quenched by trapped active site water, resulting in the formation of a complex hydroxylated sesquiterpene (selin-7(11)-en-4-ol). Initially, the SdS G305E variant produced 20% selin-7(11)-en-4-ol. As suggested by modeling of the enzyme-carbocation complex, selin-7(11)-en-4-ol production could be further improved by varying the pH, resulting in selin-7(11)-en-4-ol becoming the major product (48%) at pH 6.0. We incorporated the SdS G305E variant along with genes from the mevalonate pathway into bacterial BL21(DE3) cells and demonstrated the production of selin-7(11)-en-4-ol at a scale of 10 mg/L in batch fermentation. These results highlight opportunities for the simulation-guided engineering of terpene synthases to produce predefined complex hydroxylated sesquiterpenes.

Item Type:	Article
Date Type:	Publication
Status:	Published
Schools:	Chemistry
Publisher:	American Chemical Society
ISSN:	2155-5435
Funders:	BBSRC
Date of First Compliant Deposit:	17 July 2024
Date of Acceptance:	1 July 2024
Last Modified:	22 Jul 2024 11:00
URI:	https://orca.cardiff.ac.uk/id/eprint/170640

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