Connection between FabR and unsaturated membrane content in FFA producing E. coli.

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 16–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− 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− 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.