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

J. Will Medlin

Third Advisor

Theodore Randolph

Abstract

Developing fungible biofuels derived from renewable resources remains a major focus of efforts aimed at reducing dependence on petroleum and shrinking greenhouse gas emissions. To address this goal, fatty acid-derived fuels are emerging as a viable option with various, yet limited, successful methods for increased production via the fatty acid biosynthesis pathway. In vivo approaches as well as inorganic catalysis have been used to modify the carboxylic acid functional group of fatty acid molecules to allow for improved fuel characteristics, but these approaches typically suffer from inefficient utilization of carbon. To ameliorate this disadvantage we engineered an Escherichia coli unsaturated fatty acid (UFA) auxotroph in combination with a d5 desaturase from Bacillus subtilis to produce monounsaturated fatty acids that can subsequently be split at the unsaturated carbon bond through inorganic catalysis to result in a long-chain alkane and a short-chain alcohol without loss of carbon. We further identified novel metabolic targets in E. coli for increased fatty acid production by the use of a genome-wide tool able to up- or down-regulate expression of nearly every gene on the genome, together with a selection for resistance to the fatty acid biosynthesis inhibiting antibiotic cerulenin. To further aid in the identification of beneficial mutations for improved fatty acid production, a novel selection tool is being developed allowing for the coupling of fatty acid biosynthesis to a selectable phenotype. Together this work demonstrates the potential of using E. coli as a biorefining platform for fungible biofuel production and advances knowledge on ways of improving fatty acid biosynthesis by identifying novel targets and by creating a tool linking fatty acid production to a selectable phenotype.

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