Feasibility Studies of New Strategy for Ultra-High-Throughput Screening (uHTS) of Novel Enzyme By in Vitro Compartmentalization (IVC) Using Microbeads from Metagenomic Resources

In general, it is very difficult to screen for novel enzymes using traditional methods owing to limitations in resources and screening methods. This study is the first to demonstrate direct screening for novel enzymes activity using an ultra-HTS (uHTS) system by in vitro compartmentalization (IVC) using microbeads from metagenomic resources. Recently, a screening protocol for robotic HTS system have been enabled us to perform activity verifications on more than 10⁴ clones per day and identify four distinct clones with enzyme activity. However, we demonstrate to process screening on more than 10⁷ clones per hour by single cell level. In the present study, we can generate very easily microbeads without microfluidic system and show a rapid, simple, and efficient method for screening enzyme including hydrolase using an uHTS system by using fluorescence-activated cell sorting (FACS) analysis. This IVC method using agarose-based microbeads can detect and screen various enzyme/pathway in the single cell level without diffusion of fluorescent substrate in IVC using water-oil-water (w/o/w) emulsion method. This new uHTS system is a model system for ultra-speedy, sensitive, multiplex screening of enzyme/pathway from various genetic libraries and holds promise for providing new enzymes/pathways for bioindustrial applications.