FMRG: Bio: Enabling Cell-Free Engineering and Biomanufacturing of Bacteriophages as a Universal Platform for Tailorable Bioactive Materials

FMRG：生物：使噬菌体的无细胞工程和生物制造成为可定制生物活性材料的通用平台

• 批准号: 2228971
• 负责人: Vincent Noireaux
• 金额: $ 243.06万
• 依托单位: University of Minnesota-Twin Cities
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-10-01 至 2026-09-30
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2228971&HistoricalAwards=false
• 关键词: FMRG; Bio; Enabling; Cell; Free

Purified protein synthesis machinery can create proteins outside the cell. Cell-free synthesis (CFS) can produce proteins that would otherwise disrupt or kill the cell that produced it. Vaccines have been produced using CFS. Synthesizing more complex structures, like bacteriophages, remains to be investigated and accomplished. Phages are a resource of a variety of bioactive materials. Their natural function is to infect specific bacteria. They could be reprogrammed to kill bacteria resistant to antibiotics. They could kill food-borne pathogens and treat bacterial infections, to name two important applications. The objective of this project is to develop the CFS technology required to produce phages flexibly and with high fidelity. Educational efforts involving the Cold Spring Harbor Laboratory DNA Learning Center will focus on developing and delivering curricular material. Topics critical to integrating CFS of phages into the biomanufacturing ecosystem will be emphasized. Developed for high school and college students, these materials will bolster the development of an agile bioeconomy workforce.The CFS of phages has not been exploited commercially. Basic research advances are needed that will enable the cell-free biomanufacturing of phages. The project will deliver knowledge on phage synthesis across species and on phage genomes. This fundamental knowledge will guide the engineering of phages with broader host ranges, by developing a one-pot next generation sequencing (NGS)-based approach for determining gene essentiality in phage and identification of permissive loci. The objective of the project is to lay the foundation for the cell-free engineering and biomanufacturing of phages, the largest family of antimicrobials. Efforts will address four limitations to the current production of phages: (i) the need for bioreactors of dangerous pathogens such as Salmonella or Listeria to produce; (ii) the likelihood of generating phage-resistant pathogenic strains; (iii) the need for many purification steps, which drives up the cost of phage production; and (iv) bottlenecks surrounding large DNA transformation into cells to produce the phage. The tools and knowledge developed can be readily used to tackle societal problems such as the emergence of antibiotic-resistant microbes. The phages included in the project are related to the pathogens Salmonella and Listeria monocytogenes (Lmo), two of the most significant pathogens in the U.S. To explore alternative solutions, several E. coli CFS systems will be optimized as effective, safe platforms for phage synthesis, complementary to a Gram-positive CFS system prepared from pathogen substitutes for Lmo.This Future Manufacturing award was supported by the Chemical, Bioengineering, Environmental and Transport Systems Division (ENG/CBET), the Division of Engineering Education and Centers (ENG/EEC), the Office of the Assistant Director (ENG/OAD), and the Division of Undergraduate Education (EHR/DUE).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.

纯化的蛋白质合成机器可以在细胞外创造蛋白质。无细胞合成(CFS)可以产生蛋白质，否则会扰乱或杀死产生它的细胞。已经使用CFS生产了疫苗。合成更复杂的结构，如噬菌体，仍有待研究和完成。噬菌体是多种生物活性物质的来源。它们的天然功能是感染特定的细菌。它们可能会被重新编程，以杀死对抗生素具有抗药性的细菌。它们可以杀死食源性病原体和治疗细菌感染，这是两个重要的应用。该项目的目标是开发所需的CFS技术，以灵活和高保真地生产噬菌体。冷泉港实验室DNA学习中心的教育工作将集中在开发和提供课程材料上。将强调将噬菌体的CFS整合到生物制造生态系统中的关键主题。这些材料是为高中生和大学生开发的，将支持灵活的生物经济工作力量的发展。噬菌体的CFS尚未进行商业开发。需要基础研究进展，使噬菌体的无细胞生物制造成为可能。该项目将提供有关跨物种的噬菌体合成和噬菌体基因组的知识。这一基础知识将通过开发一种基于一锅下一代测序(NGS)的方法来确定噬菌体中的基因重要性和鉴定允许的基因座，从而指导具有更广泛宿主范围的噬菌体工程。该项目的目标是为噬菌体的无细胞工程和生物制造奠定基础，噬菌体是最大的抗菌剂家族。将努力解决目前噬菌体生产的四个限制：(1)生产沙门氏菌或李斯特菌等危险病原体的生物反应器的需要；(2)产生抗噬菌体致病菌株的可能性；(3)需要许多纯化步骤，这推高了噬菌体生产成本；(4)大DNA转化为细胞生产噬菌体的瓶颈。开发的工具和知识可以很容易地用于解决社会问题，如出现具有抗生素耐药性的微生物。项目中包含的噬菌体与美国最重要的两种病原体--沙门氏菌和单核细胞增生性李斯特氏菌(LMO)有关。为了探索替代解决方案，几种大肠杆菌CFS系统将被优化为有效、安全的噬菌体合成平台，以补充由病原体替代品制备的革兰氏阳性CFS系统。这个未来的制造奖项得到了化学、生物工程、环境和运输系统司(ENG/CBET)、工程教育和中心司(ENG/EEC)、助理主任办公室(ENG/OAD)、这一奖项反映了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。