Novel approaches for the evolutionary engineering of pathways in saccharomyces cerevisiae




Abatemarco, Joseph T.

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Modern biotechnological tools are making microbial production of chemicals, fuels, and pharmaceuticals increasingly practical and economically feasible. The field of metabolic engineering aims to enable this production by hijacking cellular systems to modify metabolism, converting each cell into an efficient chemical reactor. Traditionally, this has been accomplished through combining various knockouts and/or overexpressions of metabolic genes, but directed evolution strategies are often critical for improving metabolic pathways beyond native activity. Due to the complexity of cellular metabolism, simply evolving single genetic parts in a stepwise fashion can be limiting. In this work, we develop novel and powerful methods for applying directed evolution to the engineering of metabolic pathways in the yeast Saccharomyces cerevisiae. First, we develop a method for in vivo Continuous Evolution (ICE), which uses a synthetic retrotransposon element to allow generation of the largest mutant libraries of any in vivo mutational generation approach in yeast. This method is then validated by using it to rapidly evolve a variety of diverse genetic systems, including single enzymes, global transcriptional regulators, and multi-gene pathways. Next, we apply a modeling approach to create novel biosensors that can rapidly screen for production and secretion phenotypes in these large mutant libraries. These biosensors are then imported into microfluidic droplet systems to apply this screen in a high-throughput manner, creating a novel platform for screening libraries finally commensurate with the high level of diversity generation previously enabled. This method is validated by evolving an overproduction and secretion phenotype, resulting in strains that produce significantly elevated levels of aromatic amino acids. Finally, we develop and characterize new tools to enable the expression and evolution of multiple genes in a single genetic cassette. Taken together, these novel technologies significantly advance the state of the art for evolutionary engineering of metabolic pathways and will enable the evolution of pathways of enzymes for rapidly improving production of a number of desirable high-value biochemicals.


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