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

Area of Science:

• Biocatalysis and Metabolic Engineering
• Synthetic Biology
• Enzyme Engineering

Background:

• Engineering terpene synthases for specific products is difficult due to complex mechanisms and limited understanding of enzyme structure-function relationships.
• Sesquiterpene synthases (SdS) exhibit diverse cyclization pathways, posing challenges for targeted product formation.

Purpose of the Study:

• To engineer a selina-4(15),7(11)-diene synthase (SdS) to produce a predefined hydroxylated sesquiterpene.
• To utilize atomistic simulations and site-directed mutagenesis to guide enzyme engineering efforts.
• To demonstrate the feasibility of producing engineered sesquiterpenes via microbial fermentation.

Main Methods:

• Employed atomistic simulations to understand enzyme-carbocation interactions and guide mutagenesis.
• Utilized site-directed mutagenesis to create the SdS G305E variant.
• Optimized reaction conditions, including pH, to enhance product yield.
• Integrated the engineered enzyme and mevalonate pathway genes into BL21(DE3) cells for fermentation.

Main Results:

• The SdS G305E variant initially produced 20% selin-7(11)-en-4-ol.
• Optimization of pH to 6.0 increased selin-7(11)-en-4-ol production to 48%.
• Achieved a production scale of 10 mg/L of selin-7(11)-en-4-ol in batch fermentation.

Conclusions:

• Simulation-guided engineering is effective for creating terpene synthases that produce specific hydroxylated sesquiterpenes.
• The engineered SdS G305E variant demonstrates potential for biocatalytic production of complex sesquiterpenes.
• This approach opens avenues for the sustainable production of valuable natural products.