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 free 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. The production of these novel fatty acids was further improved through the over-expression of the acetyl-CoA carboxylase complex which catalyzes the first dedicated step in fatty acid biosynthesis, and expression of E.coli native and non-native thioesterases which alleviate feedback inhibition and further increase total fatty acid production.
These rational engineering approaches exhaust current knowledge of improving fatty acid production in E.coli. For this reason a novel genome-wide tool developed in the Gill Group was employed to identify further targets that can improve fatty acid production. The TRackable Multiplex Recombineering (TReMR) method uses strong promoters and ribosomal binding sites to increase gene expression or deletes existing ribosomal binding sites to decrease expression. These “up” and “down” chromosomal insertions were introduced in E.coli ΔfabA and growth selections were performed. Mutants were identified using microarray analysis and evaluated based on their contribution to improved growth in E.coli ΔfabA.
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