Hydrolase BioH knockout in E. coli enables efficient fatty acid methyl ester bioprocessing

Area of Science:

• Biotechnology
• Microbial Engineering
• Renewable Feedstocks

Background:

• Fatty acid methyl esters (FAMEs) from plant oils are valuable renewable feedstocks.
• Microbial production and functionalization of FAMEs offer routes to biofuels and polymer building blocks.
• FAME hydrolysis by microbes yields free fatty acids, which are often degraded, limiting bioprocessing efficiency.

Purpose of the Study:

• To identify the key enzyme responsible for FAME hydrolysis in E. coli.
• To evaluate the impact of eliminating this enzyme on FAME biotransformation processes.
• To establish a broadly applicable strategy for improving microbial FAME bioprocessing.

Main Methods:

• Screening of E. coli K-12 strains to identify the FAME hydrolyzing enzyme.
• Construction and characterization of E. coli ΔbioH knockout strains.
• Assessing FAME hydrolysis rates and biocatalytic activity in wild-type and knockout strains.
• Evaluating biotransformations in two-liquid phase systems.

Main Results:

• BioH was identified as the primary enzyme for medium-chain FAME hydrolysis in E. coli.
• E. coli ΔbioH strains exhibited up to 22-fold reduced FAME hydrolysis rates.
• Knockout strains maintained growth and biocatalytic activity, enabling high specific rates for FAME oxyfunctionalization.
• Biotransformations in two-liquid phase systems showed reduced fatty acid accumulation and substrate loss.

Conclusions:

• Deletion of BioH is a broadly applicable strategy to enhance microbial bioprocessing of FAMEs.
• This approach minimizes FAME hydrolysis and subsequent free fatty acid degradation.
• Improved FAME bioprocessing can lead to more efficient production of biofuels and bio-based materials.