Modulating E. coli membrane content via co-expression of saturated and unsaturated acyl-ACP targeting thioesterases.

a.) The percentage of unsaturated C16–C18 and cyclopropane (C17Δ) fatty acids were calculated from FAMEs made by base-catalyzed methylation of fatty acids extracted from cultures expressing combinations of BTE and GeoTE at 8 h and 24 h post-inoculation. GeoTE-expressing cells (GeoTE+ BTE−) and GeoTE/BTE co-expressing cells (GeoTE+ BTE+) have a reduced unsaturated content relative to BTE-expressing cells (BTE+ GeoTE−). Error bars represent standard deviations about the mean of biological triplicate samples. * = P-value+ GeoTE− cultures at the same sampling time. b.) Transcript levels of fabA and fabB determined by qPCR on samples harvested 1 hour hour post-induction were normalized to BTE− GeoTE− (RL08ara pBAD33-BTE-H204A pBAD18-GeoTE-H173A) samples. Levels of fabA and fabB were decreased in cells expressing only BTE. Conversely, levels of fabA or fabB were statistically the same or higher in cells expressing GeoTE or GeoTE and BTE. Error bars represent propagated standard errors about the mean of biological triplicate samples. * = P-valueq values compared against fabA or fabB in cultures expressing only non-functional thioesterases (BTE− GeoTE−). c.) The percentage of intact cells were calculated from histograms of cells stained with SYTOX Green 8 h post-inoculation. Cultures expressing only BTE were the least intact. Cultures expressing only GeoTE, and both GeoTE and BTE were over 50% intact. Error bars represent standard errors about the mean of biological triplicate samples. ** = P-value+ GeoTE−. d.) FFA titers from strains expressing combinations of BTE and GeoTE. FFA titers were determined at 8 and 24 h post-inoculation. Cultures expressing only GeoTE exhibited the highest titer at 8 h, with a minor increase observed after 24 h. Nearly equivalent titers were reached after 24 h in cultures expressing only BTE, or co-expressing BTE and GeoTE. Error bars represent standard deviations about the mean of biological triplicate samples. * = P-value+ GeoTE− cultures at same sampling time.