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

这种小型企业技术转让(STTR)的更广泛的影响/商业潜力将是开发一个平台，以加速生物产品的工程，包括用于生产化学品、添加剂和治疗药物的酶和途径。酶用于家用材料(洗涤剂和清洁剂)和化学过程(奶酪生产和废物的生物修复)。Path可以通过改造肠道细菌从糖中生产生物塑料，或者通过酵母从糖中生产青蒿素(一种抗疟疾药物)。这些生物产品是在细胞中进行工程改造的。然而，细胞工程需要广泛的科学知识、财力和物力以及时间。该项目将设计一个工程平台，消除单元格中的生产。其目标是简化工程，将成本降低20-100倍，将时间缩短2-5倍。这导致了新生物制品更快地上市，并为工程过程提供了更多信息。这个STTR第二阶段项目建议利用无细胞系统来加速酶和途径(代谢)工程。无细胞系统可以催化反应，不需要细胞的复杂性，也不需要保持细胞的生长。该项目的目标是继续开发一个平台，可以将用户酶和途径工程问题作为输入，并产生作为输出的化验数据。主要的重点是开发用于鉴定和优化用于测试的酶和途径的计算方法，用于组装DNA的分子生物学方法，用于无细胞表达的超高通量分析的微流控方法(10e7样本)，以及用于检测的分析方法。第二个焦点是通过细胞色素P450工程对该平台的演示。主要焦点的成功证明了用更快和更高吞吐量的无细胞工程过程取代细胞工程的可行性。次级重点的成功为利用细胞色素P450的代谢工程途径产生了直接相关的酶(例如，天然产品、生物催化)。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

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.