Degree Type

Dissertation

Date of Award

2013

Degree Name

Doctor of Philosophy

Department

Chemical and Biological Engineering

First Advisor

Laura R. Jarboe

Abstract

Many chemical products available today provide essential tools that make the world economy viable. However, the current reliance on fossil fuels is unsustainable and possibly negatively affects the environment. Therefore, a new method of synthesis for biorenewable and sustainable chemicals is needed. In this treatise, significant advancement in the production of short chain carboxylic acids during bacterial fermentation is described. This work offers new insights into carboxylic acid toxicity and methods for improvement of microbial tolerance that is important for improving productivity, titer, and yields for economical production of biorenewable chemicals.

The methods described include metabolic engineering techniques for E. coli strain construction: gene knockouts, gene overexpression, directed evolution for tolerance and E. coli biocatalyst characterization techniques: specific growth rate, membrane fluidity, membrane leakage, membrane lipid analysis, hydrophobicity, carboxylic acid production, transcriptome analysis, intracellular pH, γ-amino butyric acid determination, proton motive force simulation, genome reverse engineering, and biocatalyst zeta potential. These methods allow greater insight into the mechanisms of toxicity of carboxylic acids and how bacteria cells improve tolerance.

Hexanoic, octanoic (C8), and decanoic acids are completely inhibitory to Escherichia coli MG1655 in minimal medium. This growth inhibition is pH-dependent and is accompanied by a significant change in the fluorescence polarization (fluidity) and integrity. This inhibition and sensitivity to membrane fluidization, but not to damage of membrane integrity, can be at least partially mitigated during short-term adaptation to octanoic acid. This short-term adaptation was accompanied by a change in membrane lipid composition and a decrease in cell surface hydrophobicity. Specifically, the saturated/unsaturated lipid ratio decreased and the average lipid length increased. A fatty acid-producing strain exhibited an increase in membrane leakage as the product titer increased, but no change in membrane fluidity. These results highlight the importance of the cell membrane as a target for future metabolic engineering efforts for enabling resistance and tolerance of desirable biorenewable compounds, such as carboxylic acids.

Transcriptome analysis of Escherichia coli during exogenous challenge with C8 at pH 7.0 suggested that C8 challenge causes intracellular acidification and membrane damage. Network component analysis identified transcription factors with altered activity. We conclude that the membrane permeability of carboxylic acids enables acidification of the cell interior despite maintenance of the media pH at neutral. This acidification 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 the inverted proton motive force imposed by carboxylic acids requires anion accumulation. Utilization of native acid resistance systems was not useful in terms of supporting growth or alleviating intracellular acidification. The glutamate-dependent acid resistance system was found to be non-functional, possibly due to membrane damage. 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.

The microbial directed evolution method by sequential transfers was utilized to engineer an E. coli cell for an enhanced carboxylic acid tolerance phenotype. The C8-tolerant LAR1 strain overexpressing a short chain thioesterase specific for C8 more than doubled the production titer from 300 mg/L to 650 mg/L. The LAR1 strain displayed an altered physiology by a change in the membrane compared to the parent strain ML115. LAR1 had a lower saturated:unsaturated lipid ratio (S/U), higher average lipid length, a lower membrane fluidity, and a more negative surface charge. LAR1 did not show an improvement in the intracellular pH. Both the evolved strains and the parent strain were resequenced using Illumina short reads in order to identify the mutations responsible for the tolerance phenotype. Two mutations were found: two copies of the essential gene rpoC (rpoC-A1256C and rpoC∆(1-305) and basR-G82T. The rpoC mutations may give a hypermutability phenotype that allows LAR1 to adapt the stressful environments. The basR-G82T mutation increased the surface charge, as measured by zeta potential. These findings are quite significant as it may explain fundamental differences of bacterial physiology that can relate to changes in behavior.

DOI

https://doi.org/10.31274/etd-180810-2130

Copyright Owner

Liam Royce

Language

en

File Format

application/pdf

File Size

215 pages

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