title: Transcriptomic analysis of carboxylic acid challenge in Escherichia coli: beyond membrane damage.
abstract: 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.
authors.affiliation: Department of Chemical and Biological Engineering, Iowa State University, Ames, Iowa, United States of America.
authors.forename: Liam A
authors.initials: LA
authors.lastname: Royce
authors.affiliation: Department of Electrical and Computer Engineering, Iowa State University, Ames, Iowa, United States of America.
authors.forename: Erin
authors.initials: E
authors.lastname: Boggess
authors.affiliation: Department of Electrical and Computer Engineering, Iowa State University, Ames, Iowa, United States of America.
authors.forename: Yao
authors.initials: Y
authors.lastname: Fu
authors.affiliation: Interdepartmental Microbiology Program, Iowa State University, Ames, Iowa, United States of America.
authors.forename: Ping
authors.initials: P
authors.lastname: Liu
authors.affiliation: Department of Chemical and Biological Engineering, Iowa State University, Ames, Iowa, United States of America.
authors.forename: Jacqueline V
authors.initials: JV
authors.lastname: Shanks
authors.affiliation: Department of Electrical and Computer Engineering, Iowa State University, Ames, Iowa, United States of America.
authors.forename: Julie
authors.initials: J
authors.lastname: Dickerson
authors.affiliation: Department of Chemical and Biological Engineering, Iowa State University, Ames, Iowa, United States of America ; Interdepartmental Microbiology Program, Iowa State University, Ames, Iowa, United States of America.
authors.forename: Laura R
authors.initials: LR
authors.lastname: Jarboe
mesh_terms: D002264:Carboxylic Acids / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial
chemical_list: D002264:Carboxylic Acids; D029968:Escherichia coli Proteins