Date of Award

Spring 1-1-2014

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

First Advisor

Ryan T. Gill

Second Advisor

James W. Medlin

Third Advisor

Theodore W. Randolph

Fourth Advisor

Min Zhang

Fifth Advisor

Corrella S. Detweiler

Abstract

As engineers, we are interested in designing controlled, predictable, and maintainable strategies for performing or improving tasks. Genome engineering aims to use these same principles to design or re-design biological systems for targeted purposes. Strategies for genome engineering are no longer primarily limited by the cost of DNA synthesis or sequencing as they have been in the past. Instead, strategies are limited by not having methods to inform efficient and directed design. In these studies, we present an example of overcoming this current limitation by using various tools to identify genetic manipulations of interest, and then subsequently use these findings to motivate the directed design of cells for novel phenotypes. Initial studies are focused on elucidating genetic manipulations that confer furfural tolerance. Furfural is a key microbial inhibitor found in lignocellulosic hydrolysate, which is the proposed renewable sugar source for fermentation of sustainable biofuels that do not rely on food-based feedstocks. We transition towards engineering biofuel tolerance based upon identifications made in the furfural studies.

Using libraries of 103 to 105 unique members with defined and trackable mutations, we tested, in parallel, their effect on growth in the presence of furfural. We used two different search strategies (multiSCale Analysis of Library Enrichments and TRackable Multiplex Recombineering) to map genotype-to-phenotype relationships for furfural tolerance. Improved growth was confirmed for six novel furfural tolerance alleles: lpcA (lipopolysaccharide biosynthesis), groESL (chaperonin), dicA (regulator of cell division proteins), rna (ribonuclease), ahpC (alkylhydroperoxide reductase subunit), and yhjH (involved in flagellar motility regulation). The diversity of beneficial mutations found here highlights the breadth of changes that can be made to confer the same phenotype.

Building upon one of the most tolerant genes elucidated for furfural tolerance (lpcA), we informed the directed design of mutants with altered lipopolysaccharide biosynthesis to confer tolerance to hydrophobic biofuels, like n-butanol. Using a recursive recombineering approach to create libraries of increasingly mutated strains, we isolated clones capable of up to 50% growth improvements in n-butanol. We also initiated use of a new method for tracking multiple mutations across the genome, which has the potential to further reduce DNA sequencing costs by an order of magnitude.

Together, these studies identify novel mutations which confer industrially relevant phenotypes that can be used in future cellulosic biofuel production efforts. We show mutations identified for furfural tolerance can be redirected to improve biofuel tolerant phenotypes, suggesting a unified approach towards engineering both feedstock and product tolerance. Our findings also discuss broader applications to genome engineering, both in the importance of library and selection design, and the propagation of random mutations during commonly used engineering strategies that convolute the mapping of genotype-to-phenotype relationships.

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