S7: High Throughput Screen for Fatty Acid O-Methyl Transferase with Activity on Long-Chain Fatty Acids

The direct production of fatty acyl methyl esters (FAMEs) or biodiesel from H₂ and CO₂ is both novel and economically advantageous compared to alternative approaches via triglyceride feedstocks. We are using a genetically modified strain of Cupriavidus necator to carry out this production, incorporating an evolved fatty acid O-methyltransferase (FA-OMT) protein that utilizes S-adenosylmethionine as the methyl donor. We developed a high throughput, fluorescent activated cell sorter (FACS)-based screen to identify FA-OMT mutants with increased activity on long chain fatty acids such as palmitate. Mutants are identified by their ability to convert the fatty acid to FAME and generate S-adenosyl homocysteine (SAHC), which in turn is hydrolyzed by SAHC hydrolase releasing homocysteine to react with a thiol reactive dye creating a detectable fluorescent product. A culture of cells expressing the FA-OMT mutants and SAHC hydrolase were suspended in an aqueous environment with the dye and palmitate substrate, and were encapsulated in oil droplets to segregate individual cells. These were sorted to isolate clones with increased fluorescence due to increased FA-OMT-dependent production of homocysteine. This method yielded several unique mutants with improved FA-OMT activity on palmitate, and their increased ability to produce FAME is being evaluated.