(102a) An Ultrahigh Throughput Strategy for Extracellular Metabolite Screening By Combining In Vivo Biosensor with Microdroplet-Based FACS

Saccharomyces cerevisiae has been widely used in the biotechnology industry for production of biofuels and value-added chemicals. Recently, a number of genome-wide engineering tools that can introduce modifications to all genes have been developed in S. cerevisiae. However, like other directed evolution methods, such genome-wide engineering strategies are generally limited by lack of efficient high throughput screening and selection methods. This drawback becomes more obvious when our target of improvement is an extracellular metabolite. In this study, we selected lactic acid as a target to develop an ultrahigh throughput screening strategy for extracellular metabolites by combining in vivo biosensor with microdroplet-based FACS system. A _L-LA biosensor was constructed in S. cerevisiae using a transcription factor LIdR and its corresponding operator sequence to link _L-LA concentration to GFP expression level. Microfluidic droplet was used to compartmentalize single mutants for _L-LA fermentation. FACS was applied to sort positive candidates from a pool of either single cells after breaking the droplet emulsion or double emulsion droplets at the speed of 10⁸ mutants/day. By using this system, it is possible to identify mutants with improved _L-LA production from a genome-wide mutation library (10⁷) within one day.