Connection between FabR and unsaturated membrane content in FFA producing <i>E. coli</i>.

a.) Transcript levels of fabA and fabB determined by qPCR on samples harvested 4.6 hours after inoculation were normalized to RL08ara harboring pTrc99A-BTE-H204A and pBAD33*. In strains carrying fabR (RL08ara, pBAD33*, labeled RL08ara, and RL08ara ΔfabR, pBAD33*fabR, labeled fabR+), expression of BTE (+) reduced both fabA and fabB levels. In ΔfabR strains (RL08ara ΔfabR, pBAD33*) levels of fabB were increased in cells expressing both BTE-H204A (−) and BTE (+). Error bars represent propagated standard errors about the mean of biological triplicate samples. P-values were calculated by a t-test of the Cq values of fabA or fabB in strain RL08ara BTE-H204A Samples with P-values less <0.05 were marked with an asterisk. b.) The percentage of unsaturated C16–C18 and cyclopropane (C17Δ) fatty acids were calculated from fatty acid samples extracted from cultures 8 hours post-inoculation. Expression of BTE dramatically increased unsaturated content in strains harboring chromosomal fabR (RL08ara), and further increased unsaturated content in the ΔfabR strain. Overexpression of fabR on a plasmid in the ΔfabR strain restored unsaturated content to a lower level than present in RL08ara. Error bars represent standard errors about the mean of biological triplicate samples. * = P-value<0.05, ** = P-value<0.01 compared against RL08ara BTE− for BTE− cultures or RL08ara BTE+ for BTE+ cultures at the same sampling time. c.) The percentage of intact cells were calculated from histograms of cells stained with SYTOX Green. In BTE-H204A-expressing cultures, deletion of fabR had little effect on percent intact cells. In BTE-expressing cultures, a dramatic decrease in the number of intact cells was observed as a result of deletion of fabR. Samples from fabR+ cells exhibited altered histograms and were not quantified (#). Error bars represent standard errors about the mean of biological triplicate samples. ** = P-value<0.01 compared against RL08ara BTE− for BTE− cultures or RL08ara BTE+ for BTE+ cultures. d.) Effect of fabR deletion on C8–C14 (predominantly free) fatty acid titer produced in BTE-expressing cultures. Reduced titers were observed at both 8 h and 24 h growth in the ΔfabR strain. Error bars represent standard deviations about the mean of biological triplicate samples.