S155: Metabolic engineering of mono-unsaturated fatty acid production as a biorefining platform with the use of genome-wide tools and novel selection systems

Renewable transportation fuels produced by microorganisms must meet various criteria to be considered viable alternatives to petroleum-based fuels. Chemical properties, infrastructure compatibility and sensible production routes are essential considerations. Microbial fatty acid derivatives are promising biofuel candidates as they have numerous desirable properties and are readily produced by Escherichia coli. The goal of this work is to produce specific monounsaturated fatty acids for subsequent catalytic processing into alkane and alcohol fuels. Unsaturated fatty acid biosynthesis depends on the function of the fabA gene, responsible for introducing a double bond to the growing fatty acyl chain. Deletion of this essential gene and simultaneous expression of a plant desaturase, capable of introducing specific double bonds to saturated acyl-chains, created a selection system for novel unsaturated fatty acids.  Further, with the limited availability of approaches to increase production of total fatty acids in E.coli, two new tools were applied to this problem in order to identify beneficial genetic changes for the desired phenotype.  First, a genome-wide tool developed in the Gill laboratory (TRackable Multiplex Recombineering – TRMR) was employed to select for resistance to the fatty acid inhibiting antibiotic cerulenin as a proxy for increased flux through the fatty acid biosynthesis pathway.  Second, a novel plasmid-based tool was developed allowing for the screening and selection of unsaturated fatty acid production in E.coli, a previously unselectable phenotype.