(129a) Rapid Directed Evolution of Enzymes and Regulatory Factors through an in Vivo Continuous Evolution (ICE) Approach in Yeast

Cellular systems have been engineered for a vast array of applications, ranging from production of biofuels and specialty chemicals to growth on non-native, renewable substrates.  Each of these applications typically involves a directed evolution approach in order to optimize individual components such as enzymes and regulatory factors.  However, traditional directed evolution techniques are limited in library size and workflow capacity due to the in vitro step in this process.  Here, we describe a new technique for continuous evolution of genes, synthetic parts, and pathways in yeast whereby mutations are created in vivo.  In this approach, the library size scales with culture volume, allowing for an unprecedentedly large number of variants to be created and screened simultaneously.  We demonstrate the effectiveness of this approach for the rapid evolution of both enzymes and regulatory factors, achieving desired phenotypes both faster and with less labor intensive intervention compared with traditional approaches.  These phenotypes span both tolerance and enzyme activity related traits.  We further demonstrate how the process of ICE can be interfaced with high throughput metabolite detection to enable a more rapid selection of metabolically engineered strains.  These results demonstrate the capacity of ICE to significantly accelerate strain engineering efforts and enable advanced studies of gene evolution in eukaryotes.