Direct biosynthesis of medium-chain fatty acid methyl esters in a plant host using a rationally designed SABATH...

Fatty acid methyl esters (FAMEs) are recognized as promising biofuels. While engineering efforts have enabled FAME production in diverse microbes, no such work has been reported in plants. Here, we use an engineered SABATH methyltransferase for plant FAME production. PtJMT1, a jasmonic acid methyltransferase from Populus trichocarpa, displayed low activity toward octanoic acid in Escherichia coli. Guided by structural modeling of the active site, we designed a mutant enzyme predicted to have improved catalytic efficiency with fatty acid substrates. In vitro assays confirmed this prediction. The mutant enzyme displayed activity with several medium-chain fatty acids with highest activity toward octanoic acid. This mutant enzyme was denoted PtFTAMTmut, standing for a mutant fatty acid methyltransferase from P. trichocarpa. To evaluate in planta activity, PtFTAMTmut was transiently expressed in Nicotiana benthamiana. Expression of PtFTAMTmut alone in the cytosol or chloroplast did not yield FAMEs. However, coexpression of PtFTAMTmut with a chloroplast-targeted thioesterase (ChFatB2) enabled FAME production. This study provides the first report of engineered FAME biosynthesis in plants and demonstrates the utility of an engineered SABATH methyltransferase for direct biofuel production.