Highly Active C₈-Acyl-ACP Thioesterase Variant Isolated by a Synthetic Selection Strategy

Area of Science:

• Biotechnology
• Metabolic Engineering
• Enzyme Engineering

Background:

• Microbial production of medium-chain fatty acids (MCFAs) like octanoic acid is valuable for the oleochemical industry.
• Current limitations include the lack of highly active and specific enzymes for controlling fatty acid chain length in microbial hosts.
• Thioesterases, crucial for fatty acid biosynthesis, exhibit varying substrate specificities across organisms, complicating precise chain length control.

Purpose of the Study:

• To identify and engineer thioesterases with high activity and specificity for producing octanoic acid via microbial fermentation.
• To overcome challenges in microbial production of MCFAs with narrow chain length distributions.

Main Methods:

• Developed a novel genetic selection system in *Escherichia coli* based on lipoic acid auxotrophy to screen for desired thioesterase activity.
• Randomly mutagenized the *Cuphea palustris* FatB1 thioesterase (C8-specific) and screened variants using the developed selection.
• Optimized the expression of the selected mutant thioesterase in *E. coli*.

Main Results:

• Identified a mutant FatB1 thioesterase with significantly enhanced activity and specificity for octanoyl-ACP hydrolysis.
• Achieved a production titer of 1.7 g/L of octanoic acid with >90% specificity using a single chromosomal copy of the engineered thioesterase in *E. coli*.
• In vitro assays confirmed a 15-fold increase in catalytic efficiency (kcat) for the mutant enzyme compared to the wild-type.

Conclusions:

• The engineered thioesterase enables efficient and specific microbial production of octanoic acid.
• The high specific activity allows for lower enzyme expression, freeing cellular resources for pathway optimization and increasing overall productivity.
• This work provides a valuable tool for metabolic engineers aiming to produce specific MCFAs through microbial fermentation.