Date of Award

Spring 1-1-2011

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemical & Biochemical Engineering

First Advisor

Ryan T. Gill

Second Advisor

James W. Medlin

Third Advisor

Min Zhang

Abstract

Engineering organisms for improved performance using lignocellulose feedstocks is an important step toward a sustainable fuel and chemical industry. Cellulosic feedstocks contain carbon and energy in the form of cellulosic and hemicellulosic sugars. Pretreatment processes that hydrolyze lignocellulose into its component sugars often also result in the accumulation of growth inhibitory compounds, such as acetate and furfural among others. Engineering tolerance to these inhibitors is a necessary step for the efficient production of biofuels and biochemicals. For this end we use multiple genome-wide and targeted tools to alter the genetic makeup of E. coli so we can obtain the desired trait of growth on lignocellulosic hydrolysate and tolerance to inhibitory concentrations of acetate. Each of these tools used introduces mutations within a population. These populations are placed in a selection environment where the fittest survive. The change in population genotypes is then analyzed. We applied a recently reported strategy for engineering tolerance towards the goal of increasing Escherichia coli growth in elevated acetate concentrations (Lynch, Warnecke et al. 2007). We performed selections upon an E. coli genome library using a moderate selection pressure. These studies identified a range of high-fitness genes that are normally involved in membrane and extracellular processes, are key regulated steps in pathways, and are involved in pathways that yield specific amino acids and nucleotides. Supplementation of the products and metabolically-related metabolites of these pathways increased growth rate in acetate.

Directed evolution has been used successfully to increase tolerance to a variety of inhibitors on a variety of microorganisms. However, the number of unique and non-neutral mutations searched has been limited. With recent advances in DNA synthesis and recombination technologies, new advanced tools can be used. We report a two step strategy that can search a very large number of mutations that are more likely to improve the tolerance of the organism. First, the trackable multiplex recombineering (TRMR) tool searches a genome-wide library for single mutations which have a mutation which either turns up or down gene expression. Based on microarray analysis, a small number of targets are selected for recursive multiplex recombineering. We constructed and searched a library of mutations in the ribosomal binding site of targeted genes, including clones which have multiple mutations. We conducted this strategy in two inhibitory environments (acetate and lignocellulosic hydrolysate). For both cases, we successfully found single mutants from the first step, but in the second step, we found no tolerant mutants for acetate and multiple tolerant single mutants for the hydrolysate. A model was applied to predict the outcome of these selections with varying epistatic effects. This strategy is capable of searching a very large mutational space, but without prior knowledge of epistatic interaction, successful multiple mutants are not guaranteed.

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