Understanding and Addressing Carboxylic Acid Toxicity
Carboxylic acids are an attractive biorenewable chemical in terms of their flexibility and usage as precursors for drop-in replacements of industrial chemicals. It has been demonstrated that such carboxylic acids can be fermentatively produced using engineered microbes, including Escherichia coli and Saccharomyces cerevisiae. However, like many other desirable biorenewable fuels and chemicals, carboxylic acids become inhibitory to these microbes at concentrations below the desired product titer.
Using carboxylic acids as a model inhibitory product, we have performed a variety of studies to understand and address carboxylic acid toxicity and tolerance in E. coli and S. cerevisiae. Transcriptomic and phenotypic characterization during exogenous challenge and production highlight the fact that the cell membrane is targeted by these compounds, though the specifics of the damage and the resulting responses differ. Strategies to increase carboxylic acid tolerance by altering the membrane composition have had mixed success.
The membrane permeability of these carboxylic acids results in acidification of the E. coli cell interior, which we have quantified during both exogenous challenge and production. Modeling of this effect suggests that it may result in a transient inversion of the cell’s proton motive force. Traditional strategies for dealing with acid stress, such as utilization of the native acid resistance systems, are not effective, possibly due to their dependence on membrane-associated proteins.
An E. coli strain evolved for increased octanoic acid tolerance is a significantly better carboxylic acid producer than its parent strain. Characterization and reverse engineering of this strain provide insight into mechanisms of carboxylic acid tolerance.
This work serves as a general framework for addressing product toxicity and is not specific to carboxylic acids. It is expected that membrane damage, and our strategies for quantifying this damage, are relevant to a range of biorenewable fuels and chemicals.