Production of carboxylic acids in strain ML103+pXZ18Z+pZS-GFP does not significantly change the intracellular pH.

<p>Shake flasks of E. coli producing predominately C14:0 and C16:0 carboxylic acids in M9 media with 1.5% dextrose at 30°C. IPTG induces the pXZ18Z plasmid carrying a thioesterase and a β-hydroxyacyl-ACP dehydratase. The intracellular pH values are the average of four biological replicates and four technical replicates. The error bars indicate the standard deviation.</p

lrp mutations do not improve E.

<p>coli growth rate upon addition of octanoic acid. The specific growth rate of strains with altered lrp expression during the log phase in MOPS with 2% dextrose at 37°C, 150 rpm.</p

Supplementation with arginine or glutamate does not mitigate intracellular acidification.

<p>Measurements of the intracellular pH of E. coli MG1655 pJTD1 during C8 challenge while grown in the presence of supplemental arginine and glutamate. The cells were incubated for 3°C in MOPS media to allow utilization of the amino acid-dependent acid resistance systems. All concentrations are 10 mM.</p

cfa mutants with altered the membrane lipid profile, S:U ratio and the average lipid length.

<p>a: Membrane lipid profile of MG1655 and strains with altered cfa expression. Strains were incubated with 0–30 mM C8, pH = 7.0. Inset: specific growth rate in the log phase of E. coli with varying cfa expression. C16:0- palmitic acid, C16:1- palmitoleic acid, C17cyc- cyclopropane C17:0, C18:1- vaccenic acid, C18:0- stearic acid, C19cyc- cyclopropane C19:0. The complete lipid profiles are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089580#pone.0089580.s002" target="_blank">Figure S2</a> of the Supporting Information. Membrane properties are calculated from a to obtain: b: saturated:unsaturated lipid ratio and c: average lipid length.</p

Strains and plasmids used in this study.

<p>Strains and plasmids used in this study.</p

Octanoic acid challenge decreases the intracellular pH.

<p>E. coli MG1655 pJTD1 was grown to midlog in minimal media at pH(HCl). Values are the average of 4 biological replicates, with error bars indicating the standard deviation.</p

Transcription factor activities significantly changed.

<p>TFA with a significant change (p<0.05) using NCA algorithm with the RegulonDB regulatory links <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089580#pone.0089580-Salgado1" target="_blank">[29]</a>.</p

Transcriptomic Analysis of Carboxylic Acid Challenge in Escherichia coli: Beyond Membrane Damage

Carboxylic acids are an attractive biorenewable chemical. Enormous progress has been made in engineering microbes for production of these compounds though titers remain lower than desired. Here we used transcriptome analysis of Escherichia coli during exogenous challenge with octanoic acid (C8) at pH 7.0 to probe mechanisms of toxicity. This analysis highlights the intracellular acidification and membrane damage caused by C8 challenge. Network component analysis identified transcription factors with altered activity including GadE, the activator of the glutamate-dependent acid resistance system (AR2) and Lrp, the amino acid biosynthesis regulator. The intracellular acidification was quantified during exogenous challenge, but was not observed in a carboxylic acid producing strain, though this may be due to lower titers than those used in our exogenous challenge studies. We developed a framework for predicting the proton motive force during adaptation to strong inorganic acids and carboxylic acids. This model predicts that inorganic acid challenge is mitigated by cation accumulation, but that carboxylic acid challenge inverts the proton motive force and requires anion accumulation. Utilization of native acid resistance systems was not useful in terms of supporting growth or alleviating intracellular acidification. AR2 was found to be non-functional, possibly due to membrane damage. We proposed that interaction of Lrp and C8 resulted in repression of amino acid biosynthesis. However, this hypothesis was not supported by perturbation of lrp expression or amino acid supplementation. E. coli strains were also engineered for altered cyclopropane fatty acid content in the membrane, which had a dramatic effect on membrane properties, though C8 tolerance was not increased. We conclude that achieving higher production titers requires circumventing the membrane damage. As higher titers are achieved, acidification may become problematic