SBIR-STTR Award

The broader impact/commercial potential of this Small Business Technology Transfer (STTR) project will be the development of a platform technology for high-throughput protein expression. The current standard for expressing panels of proteins involves extensive bioinformatics, cloning, in vivo expression, and assays. This method takes significant expertise in disparate fields, and weeks to months of time to perform successfully. Furthermore, it can be difficult to express complex proteins due to toxicity or purification difficulty, requiring labor-intensive diagnosis of expression and purification conditions. The proposed platform allows characterization of hundreds of protein sequences at significant cost and time savings by providing a combined ex vivo computational, expression, and assay system. This allows rapid access to biological data, and on-demand protein sequence prototyping. The methods developed as part of this platform also will allow greater access to biological engineering for K-12 and undergraduate students, requiring little capital or prior biological experience. By reducing costs and time for protein engineering, and by working in a simple system that requires no knowledge of bioinformatics, cloning, cell culturing, and biochemical characterization, biologists and non-biologists alike will be able to conduct relevant biological engineering research and rapidly test protein design hypotheses. This STTR Phase I project proposes to develop a high-throughput and computationally assisted platform to rapidly collect biochemical data on a diverse set of proteins. Using this platform, researchers will be able to conduct expression of hundreds of relevant protein variants from a single reference protein. The yields are micromolar-values, providing up to 50ug/50uL per run. Therefore, enough protein can be generated for detailed biochemical characterization and activity assays. The proposed platform is an all-encompassing ex-vivo computational, expression, and assay system. In this project, engineering and prototyping of cytochrome P450 enzymes, important industrial and pharmaceutical catalysts, will be demonstrated with an end-Phase II goal to prototype 1,000 diverse cytochrome P450 enzymes from design to characterization in less than a week.

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.