Biomanufacturing in Cell-Free Systems

Pseudomonas putida engineered for high-level muconate production exhibited severe growth inhibition and slow muconate production. We developed a muconate biosensor to screen through a library of variant strains adapted to grow on glucose. We coupled the screening to Fluorescence activated cell sorting to select for strains that had improved growth and muconate titers. Whole genome sequencing analysis of the evolved clones showed two different sets of mutations both indicating the role of sugar deregulation. With the evolved strains, we observed 60% improvement in muconate titers in fed batch reactor with 0.21 g/L/h productivity with 35% yield from glucose. We are currently focusing on prototyping such a model in a cell free bioreactor so as to improve muconate yields.