Genome-Engineering Tools for Fungible Diesel and Co-Product Production

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. In particular, TRackable Multiplex Recombineering (TReMR), allows for the rapid mapping of phenotypic landscapes that facilitates the more effective searching of combinatorial genomic diversity. TReMR replaces every promoter in the E. coli genome with a molecular barcoded heterologous promoter that either turns on or turns off translation of the downstream gene. Each unique molecular barcode can be tracked in the transformant population, thus allowing rapid and quantitative mapping of modifications in gene expression to selectable and screenable traits. Successes in the application of this approach to the microbial production of commercially-relevant chemicals require the development of genetic screens and selections that can couple the production of such molecules to increased fitness or a detectable phenotype. We are currently incorporating TReMR with heterologous desaturases, auxotrophic selections, and engineered genetic switches that monitor intracellular fatty acid levels to rapidly identify engineered strains proficient in the production of specific monounsaturated fatty acids useful in the development of sustainable and fungible biofuels. Subsequent catalytic processing of these fatty acid molecules across an engineered carbon-carbon double bond to produce linear alkanes and associated short-chain organic acids has the potential to improve cost and carbon balances while producing fungible diesel molecules. 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.