S102: Genome engineering fungible diesel and co-products

A fundamental goal in metabolic engineering is to develop approaches for the more effective searching of genome-space for the purpose of re-engineering relevant traits through combinatorial gene modifications. Recent advances in synthetic biology and multiplex recombineering have dramatically expanded capabilities towards this goal.  Here, we will describe our efforts in this area and the application to the development of sustainable and fungible biofuels processes. In particular, we have envisioned a new approach for biodiesel production that entails the engineering of desaturated fatty-acid metabolism in E. coli. Working with our collaborator (Prof. W. Medlin), we are investigating the hydrogenolysis of this desaturated fatty-acid molecule across an engineered carbon-carbon double bond in order to produce a C12-14 linear alkane and an associated short-chain organic acid. This process has the potential to improve cost and carbon balances while producing a fungible diesel molecule. Our strain engineering strategy involves the modification of genes previously shown to increase flux down the fatty-acid pathway in E. coli as well as the use of desaturated fatty-acid auxotrophic strains in order to select for increased levels of our desired product. We are combining a variety of directed evolution tools towards this end, including evolution of our heterologous delta-4 desaturase, new genome-engineering tools developed in our lab, as well as regulatory switches that report on intracellular levels of fatty-acids. We will describe the current status of this research with a specific emphasis on our efforts to apply new genome-search tools to the goal of improving strain development efforts.