STTR Phase II: An On-Demand, Computational and Microfluidic-Driven Cell-Free Protein Engineering Platform

The broader impact/commercial potential of this Small Business Technology Transfer (STTR) will be to develop a platform to accelerate the engineering of biological products, including enzymes and pathways, for production of chemicals, additives, and therapeutics. Enzymes are used in household materials (detergents and cleaners) and in chemical processes (cheese production, and bioremediation of waste). Pathways can produce bioplastics from sugar by engineered gut bacteria, or artemisinin (an antimalarial) from sugar by yeast. These biological products are engineered in cells. Cellular engineering, however, requires extensive scientific expertise, financial and material resources, and time. This project will design an engineering platform that eliminates production in cells. The goal is to simplify engineering and decrease costs 20-100 fold, and decrease time 2-5 fold. This results in a faster time-to-market for novel biological products and additional information to inform the engineering process. This STTR Phase II project proposes to utilize cell-free systems to speed up enzyme and pathway (metabolic) engineering. Cell-free systems can catalyze reactions without cellular complexity and without the need to maintain cellular growth. The goal of the project is to continue to develop a platform that can take as input user enzyme and pathway engineering questions and produce as output assay data. The primary focus is on developing computational methods for identification and optimization of enzymes and pathways for testing, molecular biological methods for assembling DNA, microfluidic methods for ultra-high-throughput analysis of cell-free expressions (10e7 samples), and analytical methods for detection. A secondary focus is the demonstration of this platform through cytochrome P450 engineering. Success with the primary focus demonstrates feasibility of replacing cellular engineering with faster and higher-throughput cell-free engineering processes. Success with the secondary focus produces directly-relevant enzymes for metabolic engineering pathways utilizing cytochrome P450s (e.g., natural products, bio-catalysis). This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.