Engineering Outer Membrane Proteins to Increase E. coli Membrane Integrity and Production of Fatty Acids

Constructing microbial biocatalysts producing biorenewables at economically viable yields and titers is often hampered by toxicity of products. For production of fatty acids, membrane damage is deemed as the principal mechanism of toxicity, particularly in regards to membrane integrity. Previous engineering studies have attempted to engineer membrane fatty acids of phospholipids (“membrane wall”), with the goal of increasing E. coli fatty acid tolerance and production, have had mixed results. Herein, we used a novel approach to reconstruct the E. coli membrane by engineering membrane proteins (“membrane gates”). Two outer membrane proteins, FX and OP were chosen for engineering. Results showed that, OP mutant not only increased short chain fatty acids (SCFA) production, but also increased long chain fatty acids (LCFA) production. However, although FX and FY were considered as partners in transport of fatty acids, their engineering effects on FA production are totally different. Knockout of FY increased FA production, while FX mutant decreased LCFA production significantly by 34% (P<0.01). Instead, elevated expression level of FX contributed to LCFA production. Further membrane characterization results showed that, either disruption of OP or FX overexpression increased membrane integrity remarkably while disruption of FX decreased integrity during FA production process. There was a positive relationship between FX abundance and LCFA titer within a certain range. Besides its individual effect, the two engineering manipulations, disruption of OP and FX overexpression have a synergistic role in further increasing fatty acids production. These engineering expands the understanding of membrane proteins in strengthening membrane properties and strain robustness.