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.
Highlights
There has been a substantial interest in using microbial fatty acid biosynthesis as a platform for a variety of biorenewable chemicals [1,2,3,4]
We performed transcriptome analysis of E. coli MG1655 during midlog growth in MOPS minimal media with and without 10 mM octanoic acid, where octanoic acid serves as a representative carboxylic acid
Considering the fact that E. coli inhibition by carboxylic acids is associated with both membrane damage and induction of the acid response, we proposed that increasing the abundance of these cyclopropane fatty acids in the cell membrane could serve as a method of increasing carboxylic acid tolerance
Summary
There has been a substantial interest in using microbial fatty acid biosynthesis as a platform for a variety of biorenewable chemicals [1,2,3,4]. There are challenges associated with harnessing the fatty acid biosynthesis pathway for producing chemicals at industrially relevant titers, productivities, and yields It has been noted by multiple researchers that product toxicity is a major problem for the microbial production of carboxylic acids [5,6,7,8,9]. Transcriptome analysis is one method for identifying these mechanisms [7,10,13,14] It is relatively well-established that one of the major effects of short chain carboxylic acid toxicity is membrane damage, largely due to the hydrophobic nature of the carbon chain [15]. We used transcriptomic analysis of exogenous octanoic acid challenge to identify and quantify other mechanisms of inhibition, as well as exploring strategies for improving tolerance
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