(608g) High-Throughput Screens and Selections for Enzyme Function in Non-Model Bacteria

Enzyme activities vary depending on the environments in which they are used. Heterologous enzymes must function in a new cytoplasmic environment, which can be altered through mutations to the host. Therefore, increasing enzyme activity in vivo benefits from co-optimization of both the enzyme and its new host. Expanding potential engineering targets to include the host genetic background offers new routes to increase productivity, but also dramatically increases the combinatorial complexity to be searched. This task is further complicated when enzymes are used in non-model hosts with limited genetic tools. In this talk, I will describe three approaches for enzyme discovery and in vivo optimization in non-model bacteria. First, we have used high-throughput selections to rapidly identify novel enzymes and pathways for assimilation of lignin-derived aromatic compounds. Next, we have developed selection strategies to optimize heterologous enzymes in non-model hosts, by coupling enzyme activity to bacterial competition in microfluidic droplets. Finally, we are using quantitative trait-locus mapping in bacteria as a genome-wide high-throughput screen to identify genetic variants in the host that affect enzyme activity.