Certain classes of fatty acids are valuable raw materials that can used to produce bio-based lubricants
however, their broader use has been limited by the high cost and limited availability. The project described in this report was focused on producing a fatty acid methyl ester (FAME) of a specific chain length from cellulosic sugars. Development of a biological pathway and engineered organisms for production of low-cost FAME from cellulosic sugar is a novel and potentially disruptive technology that is supportive of BETO?s mission to displace all fractions of a barrel of crude oil. The project was organized around four tasks that focus on A) production pathway engineering, B) host strain engineering, C) assembly of production pathway in host strain, and D) fermentation process development. Pathway engineering focused on discovery and improvement of two key pathway enzymes, namely 3-keto acetyl CoA synthase (KAS) and wax ester synthase (WES). Both enzymes were improved using a variety of enzyme engineering and mutagenesis approaches, and assembled into a host strain with relevant additional pathway genes and genome modifications. Assembled strains were evaluated in 2-L benchtop fermenters using a commercially produced cellulosic hydrolysate to supply carbon source for FAME production. These evaluations demonstrated that performance targets and final milestones were met or exceeded for sugar uptake rate, product titer, product specificity, and production rate.