Matching Protein Interfaces for Improved Medium-Chain Fatty Acid Production

Area of Science:

• Biotechnology
• Synthetic Biology
• Metabolic Engineering

Background:

• Medium-chain fatty acids (MCFAs) are crucial precursors for synthesizing valuable chemicals like α-olefins and dicarboxylic acids.
• Microbial production of MCFAs is often constrained by the efficiency and specificity of fatty acyl-ACP thioesterases within bacteria.

Purpose of the Study:

• To enhance microbial production of MCFAs by engineering a heterologous bacterial fatty acyl-ACP thioesterase for improved activity in Escherichia coli.
• To investigate strategies for increasing the titers of microbially produced chemicals through enzyme interface engineering.

Main Methods:

• Engineered a heterologous Acinetobacter baylyi fatty acyl-ACP thioesterase (AbTE) by introducing positively charged amino acids at its surface to improve electrostatic matching with endogenous E. coli fatty acid ACP (AcpP).
• Utilized nuclear magnetic resonance (NMR) titration to analyze the binding interactions between engineered AbTE variants and E. coli octanoyl-AcpP.
• Employed a previously developed G-protein coupled receptor (GPCR)-based sensor to detect and screen MCFAs secreted by E. coli expressing different AbTE variants.

Main Results:

• Engineered AbTE variants increased secreted MCFA titers by more than 3-fold compared to the wild-type enzyme.
• The most effective AbTE mutant produced 131 mg/L of MCFAs, constituting 80% of all secreted fatty acids after 72 hours.
• Demonstrated that GPCR-based sensors can effectively detect MCFAs directly in the supernatant of producer microbes, irrespective of the microbial strain.

Conclusions:

• Engineering the interface between heterologous enzymes and endogenous host proteins is an effective strategy for increasing the production titers of microbially synthesized chemicals.
• GPCR-based sensors offer a versatile tool for high-throughput screening and sensor-guided engineering of microbial cell factories for chemical production.