Rational Membrane Engineering Increases E. coli Robustness and Production of Biorenewables

Production of fuels and chemicals by using renewable material is limited by toxicity from inhibitors in feedstock and end-products, many of which generally cause membrane damage to host strains. Herein, three different strategies were performed to construct robust strains with increased production of biorenewables by engineering membrane of E. coli.

Firstly, novel trans unsaturated fatty acids (TUFA) were produced and incorporated into the membrane of E. coli MG1655 by expression of cis-trans isomerase (Cti) from Pseudomonas aeruginosa. While the engineered strain was found to have no increase in membrane integrity, a significant decrease in membrane fluidity was observed. As a result, tolerance to exogenously added octanoic acid and octanoic acid production were both increased relative to the wild-type strain. This membrane engineering strategy to improve octanoic acid tolerance was found to require fine-tuning of TUFA abundance. Besides improving tolerance and production of carboxylic acids, TUFA production also enabled increased tolerance in E. coli to other bio-products, e.g. alcohols, organic acids, aromatic compounds, a variety of adverse industrial conditions, e.g. low pH, high temperature, and also elevated styrene, another versatile bio-chemical product. TUFA permitted enhanced growth due to alleviating bio-product toxicity, demonstrating the general effectiveness of this membrane engineering strategy towards improving strain robustness.

Secondly, since a phospholipid molecule generally consists of a hydrophilic phosphate head and two hydrophobic fatty acid tails and most previous engineering studies attempted to engineer membrane fatty acid tails, little was known about effect of phospholipids head engineering. To this end, we proposed the idea that engineering phospholipid head to booster the robustness of microbes. Specifically, key enzymes candidates which are responsible for different phospholipids biosynthesis were chosen and engineered in E. coli MG1655. Results showed that increase of phospholipid A significantly increased membrane integrity and membrane electrochemical potential. In the case of fatty acids, this membrane engineering was also found to increase both tolerance to exogenously added fatty acids by 29.1% at most and fatty acids production by 42% relative to the wild-type strain. There was a positive relationship between phospholipid A content and tolerance increase to fatty acids. Besides increasing tolerance and titer of carboxylic acids, phospholipid A production also enabled increased tolerance of E. coli to other important bio-products, e.g. ethanol, hexanol, toluene, styrene. Moreover, tolerance to several important inhibitors in hydrolysate of lignocellulose, e.g. furfural, hydroxymethylfurfural (HMF), acetate and a variety of adverse industrial conditions, e.g. low pH, high temperature, were also elevated by phospholipid A production.

Thirdly, besides phospholipids, membrane bound proteins are also important components of cell membrane, while we know little of their engineering effects on membrane physical properties and strain robustness. Herein, two membrane protein candidates, X and Y were engineered in E. coli MG1655. Results showed that, individual disruption of X and overexpression of Y significantly increased membrane integrity during fatty acids production. Besides its individual effect, the two engineering manipulations 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.