Reiterative Recombination for Combinatorial Optimization of Yeast Metabolic Flux for Terpenoid ProductionView all posters
Columbia University, United States
The plasticity of the metabolic network makes microorganism-based biosynthesis of natural products a promising platform for rapid, efficient and highly versatile drug and commodity production. Reprogramming cells for these increasingly sophisticated applications requires the construction of customized multi-gene pathways and their introduction into host organisms. Moreover, the ability to create libraries of pathway variants in order to optimize function using directed evolution approaches is essential. Our DNA assembly system, “Reiterative Recombination”, employs endonuclease-induced homologous recombination in a cyclical format that allows for stepwise elongation of the construct of interest. Here, we exploit Reiterative Recombination as a straightforward and general technology for combinatorial mutagenesis of metabolic flux. We demonstrate preliminary results for the construction of terpenoid pathway in S. cerevisiae using Reiterative Recombination. Furthermore, we present progress towards the application of Reiterative Recombination to optimize metabolic flux for terpenoid production. Together these results establish Reiterative Recombination as a simple and powerful library mutagenesis technique and advance our efforts to engineer the cell for fully in vivo directed evolution. More broadly, our network-oriented experimental approach expands the toolkit available for engineering living cells toward the routine production of valuable natural products in yeast.