S156: A Functional Genomics Analysis of Free Fatty Acid Production in Escherichia coli

This talk will describe a functional genomics study of free fatty acid (FFA) production in E. coli that was motivated by severe physiological stresses observed in engineered strains. For example, cultures expressing a thioesterase displayed a 1-2 order of magnitude reduction in viability, increased membrane permeability, and heterogeneity in cell size when compared with cells expressing a non-functional thioesterase variant (His to Ala mutation). These effects were more severe in cells which endogenously produced FFA compared to cells that were exposed to exogenous FFA. To identify the underlying cause of these observations, samples for fatty acids, extracellular metabolites, transcriptomics, and proteomics were collected from two orthogonal sets of experimental conditions: a.) shake-flask, glucose, IPTG induction and b.) fermentor, glycerol, arabinose induction. Under condition a.), over 25% of the transcriptome was differentially expressed in FFA overproducing cells in comparison to cells expressing a non-functional thioesterase. The intersection of the two conditions significantly reduced the number of potential leads. After analysis, several hypotheses were generated. To test each, additional genetic changes were engineered into the original FFA overproducing strain. The viability and FFA titer of each were determined and compared to appropriate control strains. In general, mutations which removed important genes exacerbated the observed stresses, but overexpression resulted in minimal improvements in fitness and FFA titer. From these experiments, we have concluded that the E. coli genome does not encode a response capable of mitigating the physiological effects of FFA overproduction. Additional metabolic engineering efforts will be required to achieve maximal titers.