Membrane Engineering Strategies to Improve Production of Biorenewable Fuels and Chemicals

The production of fuels and chemicals from biomass in a manner that is economically competitive with petroleum-based processes is often hindered by microbial inhibition by the product and/or impurities in the biomass-derived sugar stream. In many of these cases, this inhibition of the microbial biocatalyst involves membrane damage and the threshold for this damage can differ for production vs exogenous challenge. We have demonstrated the effectiveness of various strategies for strengthening the Escherichia colimembrane.

                The first engineering strategy is enabling homeoviscous adaptation in E. coli via expression of the Pseudomonas aerugionase cis-trans isomerase (Cti) enzyme, as described in Tan et alMetabolic Engineering 2016. The Cti enzyme converts existing cis-unsaturated fatty acids (CUFA) to the trans unsaturated fatty acid (TUFA) form, resulting in a significant increase in membrane fluidity and improved tolerance to a variety of inhibitors. TUFA production has a dual effect on the protection provided against exogenously-supplied octanoic acid: some amount of TUFA production is helpful, but too much TUFA production dampens the protective effect. Strains with the appropriately tuned TUFA/CUFA ratio not only showed improved tolerance to exogenously provided octanoic acid, but also demonstrated significantly increased octanoic acid titers. Specifically, the titer was increased 41% in minimal media and 29% in rich media. This association of improved tolerance and improved production was also observed for styrene. The tolerance of n-butanol, hexane, toluene, elevated temperature (42C) and low pH (5.5) were also significantly increased.

                The second membrane engineering strategy involves altering the distribution of the native phospholipid head groups. One such engineering strategy was associated with increased tolerance of carboxylic acids, various biomass-associated inhibitors (such as furfural), aromatic monomers, low pH (6.0) and low temperature (20C). This increased tolerance of carboxylic acids was accompanied by a substantial increase in membrane integrity during carboxylic acid challenge. When this engineered strain was provided with appropriate fatty acid transporters, fatty acid production titers were increased nearly 40% relative to the corresponding control with the unaltered phospholipid distribution.

                Finally, by implementing Orgel’s Second Rule that “evolution is cleverer than you are”, we can find inspiration for other membrane engineering strategies. Two such membrane engineering strategies inspired by reverse engineering of evolved E. coli strains will be discussed.