TY - JOUR

T1 - Simulation-Guided Engineering Enables a Functional Switch in Selinadiene Synthase toward Hydroxylation

AU - Srivastava, Prabhakar L

AU - Johns, Sam T

AU - Voice, Angus

AU - Morley, Katharine

AU - Escorcia, Andrés M

AU - Miller, David J

AU - Allemann, Rudolf K

AU - van der Kamp, Marc W

N1 - © 2024 The Authors. Published by American Chemical Society.

PY - 2024/7/19

Y1 - 2024/7/19

N2 - 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.

AB - 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.

U2 - 10.1021/acscatal.4c02032

DO - 10.1021/acscatal.4c02032

M3 - Article

C2 - 39050902

VL - 14

SP - 11034

EP - 11043

JO - ACS Catalysis

JF - ACS Catalysis

SN - 2155-5435

IS - 14

ER -