(620s) In Vivo Continuous Evolution of Genes and Pathways in Yeast

Engineered microbial systems have the potential to solve pressing issues facing society, including the production of chemicals from renewable sources, degradation of environmental toxins, and the treatment of disease.  However, the engineering efforts leading to these outcomes almost always require alterations to native host metabolism, often by modifying both enzyme activities and expression levels on a pathway scale.  In the absence of detailed information about a biological system, directed evolution remains the most general method to generate improvements to performance, but is limited by low-throughput workflows and small library sizes, especially in workhorse eukaryotic systems such as yeast.  Here, we establish an in vivo continuous evolution system in yeast by synthetically activating the endogenous retrotransposon Ty1 and optimizing the system to enable libraries of 10⁸ per liter per round of evolution by simply growing cells.  This approach enables a rapid and generic increase in directed evolution throughput for synthetic parts, regulatory factors, single enzymes, and complete metabolic pathways.  We demonstrate the utility of this approach by applying it to the engineering of the global transcriptional regulator Spt15p, the catalytic enzyme Ura3p, and a multi-gene xylose catabolic pathway, obtaining rapid and significant improvements to performance in each case.  These improvements take place over days instead of weeks.  Taken together, this work establishes a platform for rapid directed evolution in eukaryotes that is capable of identifying novel beneficial mutations to coding regions, regulatory elements, and entire pathways.