Online

Tomko, Timothy

Mechanical Engineering

2015

MS

Microorganisms are capable of producing advanced biofuels that can be used as 'drop-in' alternatives to conventional liquid fuels. However, vital physiological processes and membrane properties are often disrupted by the presence of biofuel and limit the production yields. In order to make microbial biofuels a competitive fuel source, finding mechanisms of improving resistance to the toxic effects of biofuel production is vital. This investigation aims to identify resistance mechanisms from microorganisms that have evolved to withstand hydrocarbon-rich environments, such as those that thrive near natural oil seeps and in oil-polluted waters. In this study, screened the genomes of two types of bacteria, Pseudomonas aeruginosa and Marinobacter aquaeolei, looking for genes that impart biofuel tolerance when expressed in Escherichia coli. Both of these microbes have adapted in their respective natural environments to contain mechanisms for dealing with environmental stress. For initial work, P. aeruginosa was used to test our experimental design and procedure, and we validated our methods by identifying a gene, ohr from P. aeruginosa, that increased tolerance to the bio-jet fuel precursor limonene in Escherichia coli. Using genomic DNA from M. aquaeolei, we constructed a transgenic library that we expressed in E. coli. We exposed cells to inhibitory levels of pinene, a monoterpene that can serve as a jet fuel precursor with chemical properties similar to existing tactical fuels. Using a sequential strategy of a fosmid library followed by a plasmid library, we were able to isolate a region of DNA from the M. aquaeolei genome that conferred pinene tolerance when expressed in E. coli. We determined that a single gene, yceI, was responsible for the tolerance improvements. Overexpression of this gene placed no additional burden on the host. We also tested tolerance to other monoterpenes and showed that yceI selectively improves tolerance.