High Quality, Error-Free DNA for Genome Engineering and Constructing Genetic Circuits for Cell-Free Systems

Microbes can be engineered to produce biologically-derived replacements for gasoline, diesel, and jet fuel. However, a major technical challenge is that cells can only tolerate limited concentrations of typical fuels. Hydrocarbons are toxic because they damage the cell membrane and increase membrane fluidity. Thus, without further engineering there is a fundamental upper limit to how much biofuel microbes can produce, and cells must trade off production against survival. However, there are many natural examples of microbes that thrive in oil rich environments, such as around natural oil seeps or near oil spills. These microbes harbor mechanisms for tolerating the toxic effects of hydrocarbons. By using library-based approaches, we have identified several novel strategies for improving microbial biofuel tolerance by selecting for genes that enable survival. For example, expression of efflux pumps can dramatically improve tolerance in production strains, and we have further improved survival by designing synthetic feedback systems to control pump expression. This talk will discuss our efforts to enhance microbial synthesis of next-generation biofuels by developing tools for improving microbial tolerance of biofuel production conditions.