(191n) Reverse Engineering of Short-Chain Fatty Acid-Tolerance and Production in Escherichia coli

Product toxicity is a common problem in the microbial production of biorenewable fuels and chemicals. According to Orgel’s Second Rule that “evolution is cleverer than you are”, directed evolution is a promising approach to address this issue. Additionally, reverse engineering of the evolved strains can aid in the identification and development of rational design strategies for future strain improvement.

Directed evolution was previously used to improve the tolerance of E. coli to exogenously supplied octanoic acid at neutral pH in minimal media. The evolved strain had increased tolerance not only to short-chain fatty acids (C6, C8, C10), but also to n-butanol and isobutanol. This evolved strain was able to attain fatty acid titers five-fold higher than the parent strain when expressing the Anaerococcus tetradius thioesterase. In this work, we use reverse engineering to identify and understand the mechanisms of beneficial phenotypes of the evolved strain. Firstly, four interesting mutations were identified and investigated through gene replacement. With cell characterization study, we determined if each mutation effected octanoic acid tolerance and altered the cell membrane (membrane leakage, membrane fluidity, cell surface hydrophobicity). Two of the mutant genes are regulators, so we also subjected these strains to transcriptome analysis. According to the transcriptome analysis results, many of the genes with differential expression are related to cell membrane. We plan to use the TRMR method to target the genes which contribute to the beneficial phenotypes of the evolved strain. In short, our work provides design strategies which are able to directly or indirectly alter the cell membrane and to increase short-chain fatty acids tolerance and production in E. coli.