Figure 1.
Unsaturated fatty acid biosynthesis and regulation.
Unsaturated fatty acid biosynthesis begins with the isomerization of trans-2-decenoyl-ACP to cis-3-decenoyl-ACP by FabA. Instead of being reduced, this intermediate is condensed with malonyl-ACP by FabB. The resulting unsaturated β-ketoacyl-ACP is processed analogous to its saturated counterpart until unsaturated C16 and C18 acyl-ACPs are made and incorporated into phospholipids. Unsaturated fatty acid biosynthesis is feedback-inhibited at the transcriptional level by FabR, which exhibits increased repression of transcription of fabA and fabB when bound to enoyl-ACP species [16], [17]. Expression of a thioesterase cleaves acyl-ACPs to generate FFA. BTE expression preferentially cleaves saturated C12-acyl-ACPs (solid blue arrow) and minorly cleaves unsaturated C12- (dashed blue arrow) and saturated and unsaturated C14-acyl-ACPs, thereby depleting saturated, long chain acyl-ACPs, the key regulatory signal for controlling fatty acid biosynthesis. As a result, flux through the saturated (prior to C10) and unsaturated pathway increases. Inset: the four arrows represent the elongation (FabB/FabF) (dashed arrow) in which the acyl chain represented by R grows by 2 carbons, ketoreduction (FabG), dehydration (FabZ), and enoyl reduction (FabI) reactions that comprise one round of fatty acid elongation and reduction.
Table 1.
Strains and plasmids used in this study.
Figure 2.
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 <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.
Figure 3.
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<0.05, ** = P-value<0.01 compared to BTE+ 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-value<0.05 for Cq 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<0.01 compared to BTE+ 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<0.05, ** = P-value<0.01 compared to BTE+ GeoTE− cultures at same sampling time.
Table 2.
Viability analysis of strains expressing combinations of BTE and GeoTE.