Lignocellulosic biomass is an appealing feedstock for the production of biorenewable fuels and chemicals, and thermochemical processing is a promising method for depolymerizing it into sugars. However, trace compounds in this pyrolytic sugar syrup are inhibitory to microbial biocatalysts. This study demonstrates that hydrophobic inhibitors damage the cell membrane of ethanologenic Escherichia coli KO11+lgk. Adaptive evolution was employed to identify design strategies for improving pyrolytic sugar tolerance and utilization. Characterization of the resulting evolved strain indicates that increased resistance to the membrane-damaging effects of the pyrolytic sugars can be attributed to a glutamine to leucine mutation at position 29 of carbon storage regulator CsrA. This single amino acid change is sufficient for decreasing EPS protein production and increasing membrane integrity when exposed to pyrolytic sugars.