Degree Type

Dissertation

Date of Award

2018

Degree Name

Doctor of Philosophy

Department

Chemical and Biological Engineering

Major

Chemical Engineering

First Advisor

Laura R. Jarboe

Second Advisor

Thomas J. Mansell

Abstract

Product toxicity is a common problem in microbial production of biorenewable fuels and chemicals. Historically, the field of metabolic engineering has relied on enrichment of expression libraries and strain evolution for improving tolerance. Reverse engineering of these strains can aid in the identification and development of rational design strategies for strain improvement, according to Orgel’s Second Rule that “evolution is cleverer than you are”.

Here, we investigated the evolved strain with increased short-chain fatty acid tolerance and 5-fold higher fatty acid titer relative to its parent strain to discover and understand the mechanisms of the phenotypic changes. Four mutations were identified in the evolved strains, as well as the chronological order of mutations during adaptive evolution. Then we studied each mutation and their synergistic interaction by characterizing the reconstructed strains, which had single mutation, double mutation or triple mutations replaced into the genome of the parent strain. The waaG mutation contributed to increased C8 tolerance, fatty acid titer, and membrane integrity. The increased fatty acid titer was mainly affected by the mutant rpoC gene. The decreased membrane fluidity and increased cell surface hydrophobicity of evolved strain were caused by the synergistic interaction of waaG, rpoC, and basR mutations. We also noticed each mutation was able to alter the membrane lipid composition differently. The association between mutations and phenotypic changes we identified could be used as rational engineering strategies for improving tolerance and production of microbial biocatalyst.

However, the rpoC and basR encode transcriptional regulators, and we were not able to fully understand the mechanisms of these two mutations by only genome-level reverse engineering. Thus, we further investigated these two mutations by transcriptome analysis. Compared evolved strain LAR1 to parent strain ML115 during fatty acid producing, twenty-nine genes made statistically significant changes in transcript abundance, which could be influenced by rpoC mutation. Also, nine genes were identified had differential transcript abundance, which could be impacted by the basR mutation. Characterization of these interesting genes is in progress to discover the mechanisms of increased short-chain fatty acids tolerance and production of evolved strain LAR1.

In order to deepen our understanding the association between fatty acid production and cell membrane properties, we characterized a small group of environmental E. coli isolates with significantly higher short-chain fatty acids production in minimal medium relative to lab strain MG1655. Consistent with previous studies, decreased membrane fluidity was associated with increased fatty acid production. The C16:1/C16:0 ratio (mol/mol) was suggested to be one of the important metrics, when rationally engineering membrane lipid composition for improving short-chain fatty acids production in E. coli. We also discovered the direct association between cell surface hydrophobicity and short-chain-fatty acids titer. In short, the findings of our work could provide new insights into the mechanisms of short-chain fatty acid tolerance and production, and rational engineering strategies for improving performance of biocatalyst.

Copyright Owner

Yingxi Chen

Language

en

File Format

application/pdf

File Size

186 pages

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