(686a) Rapid Evolution of Enzymes and Regulatory Factors Through An in Vivo Continuous Evolution (ICE) Approach in Yeast

Engineered cellular 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 rely upon the optimization of enzymes and regulatory factors.  Typically, a directed evolution approach is undertaken to improve performance at both the individual component and the final strain level.  However, traditional directed evolution techniques introduce mutations through in vitro procedures, which fundamentally limit the overall library size and thus can constrain success.  Here, we describe a new technique for continuous evolution of desired genes and synthetic parts that reduces the practice of library generation to a simple cloning step followed by cell outgrowth.  In this technique, library sizes are not limited by transformation efficiency and instead scale with culture volume, which is the fundamental upper limit to the size of any evolving population.  The utility of this approach is demonstrated with both model proteins as well as key metabolic and regulatory factors to demonstrate the benefits of continuous evolution over traditional approaches.   These results show the capability of ICE to significantly accelerate strain engineering efforts and enable advanced studies of gene evolution in eukaryotes.