Evolution-Guided Biosynthesis of Terpenoid Inhibitors

Area of Science:

• Biochemistry
• Synthetic Biology
• Enzyme Engineering

Background:

• Terpenoids are a diverse class of natural products with significant biological activity, including medicinal applications.
• The evolutionary pathways for generating novel functional terpenoids remain largely unknown and challenging to replicate in engineered systems.

Purpose of the Study:

• To evolve a terpene synthase for the production of enzyme inhibitors targeting protein tyrosine phosphatase 1B (PTP1B).
• To utilize a synthetic biochemical system linking PTP1B inhibition to antibiotic resistance in Escherichia coli (E. coli).

Main Methods:

• Employing site saturation mutagenesis on gamma-humulene synthase (GHS), a promiscuous enzyme.
• Linking enzyme inhibition to antibiotic resistance in E. coli to select for improved mutants.
• Analyzing mutant libraries to identify beneficial mutations and understand structure-function relationships.

Main Results:

• Mutants of GHS exhibited improved fitness in E. coli by reducing enzyme toxicity and/or increasing inhibitor production.
• A specific combination of two mutations led to a >50-fold enhancement in the production of a PTP1B-inhibiting terpene alcohol.
• Identification of a key mutation site that facilitates efficient hydroxylation, a crucial step in inhibitor synthesis.

Conclusions:

• The inherent plasticity of terpene synthases allows for efficient exploration of diverse chemical structures.
• This study provides a framework for activity-guided evolution of terpene synthases to generate novel functional molecules.
• The developed system demonstrates a powerful approach for discovering new enzyme inhibitors and understanding enzyme evolution.