The overall objective of this proposal is to develop an integrated approach to genome engineering based on the application of synthetic biology and genomics methods pioneered in the laboratories of the investigatory team. The proposed approach brings together advances in DNA synthesis, genomics, and recombineering to enable the quantitative evaluation of billions of rationally designed genetic alterations simultaneously and combinatorially. Specifically, >10,000 unique DNA oligomers are synthesized in parallel, recombined at high-frequency into the Escherichia coli genome, and quantitatively tracked using deep sequencing or molecular barcoding strategies. This process can be performed recursively and automatically in order to rapidly accumulate beneficial modifications into an optimized strain. Here, we propose to apply these advances within the context of engineering a broadly significant model system: the production of biodiesel in E. coli. As part of a larger effort, the platform molecule produced by E. coli will be secreted, extracted, and provided for inorganic catalysis to produce a C12/C14 diesel molecule mixture and a C4 next generation biofuel (i.e.butanol). This approach has the potential to improve economics and carbon-balances compared to alternative biofuels approaches, which should broaden the overall impact of these efforts.
The overall objective of this research was to develop an integrated approach to genome engineering based on the application of synthetic biology and genomics methods pioneered in the laboratories of the investigatory team. The research brought together advances in DNA synthesis, the ability to use synthetic DNA to rapidly modify bacterial genomes, and DNA sequencing to demonstrate a new strategy for genome engineering. This strategy was demonstrated in a variety of studies, all of which were directed at enabling the sustainable produciton of next-generation biofuels. Specific outcomes of this research included i) the full or partial support for the training of 4 PhD student and 3 Postdoctoral researchers in areas of national need (sustianble fuels), two of which were from groups traditionally underreprsented in STEM subjects, ii) the publication of a range of peer reviewed manuscripts, one of which appeared in the Proceedings of the National Academy of Sciences, and iii) the dissemination of our research results at 13 different meetings/conferences (which include both academic and industrial representatives). This research also generated new understanding of how to engineer microbes for improved performance in industrially relevant contexts, such as fatty-acid based biodiesel and cellulosic biofuels production. In particular, our data described the first attempt to combine strategies for linear mapping of genes to traits at the genome scale with strategies for evaluating such mappings to guide the evaluation of combinatorial designs. Such understanding fills an important gap in current commercial practices in strain and metabolic engineering, and has the potential to improve the sustainable production of various fuels and chemical molecules.
