GOALI: Rewiring Escherichia coli for the efficient synthesis of chondroitin polysaccharides

目标：重新连接大肠杆菌以有效合成软骨素多糖

• 批准号: 1604547
• 负责人: Mattheos Koffas
• 金额: $ 42万
• 依托单位: Rensselaer Polytechnic Institute
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-01 至 2022-10-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1604547&HistoricalAwards=false
• 关键词: GOALI; Rewiring; Escherichia; coli; efficient

1604547 Koffas, Mattheos There is a growing interest in developing methodologies for the robust, high-yield production of bioengineered natural polysaccharides that can be used as nutraceuticals or pharmaceuticals, such as chondroitin sulfate. Such methods, using recombinant microorganisms will allow, for the first time, the production of animal-free polysaccharides and will eliminate the risks associated with animal-derived products that include viral infections, product inconsistencies and possible adulteration as well as reliance on environmentally non-friendly processes. This project is a translational and multi-disciplinary research effort with the ultimate goal to facilitate the efficient and safe production of kilogram quantities of non-animal sourced bioengineered chondroitin sulfate. It seeks to unravel the role of precursor availability on the production of large molecules and to translate this knowledge to a highly efficient production platform of polysaccharides. The project objective is to enhance the production levels of non-sulfated chondroitin through standard metabolic engineering methods, stoichiometry-based modeling and metabolic pathway balancing (specific aim 1). In specific aim 2, the investigators will demonstrate CRISPR interference (CRISPRi) as a metabolic engineering tool, enabling inducible knockdown and tunable repression of multiple genes simultaneously for controlling metabolic fluxes in E. coli. In specific aim 3, a scalable methodology will be developed for in vitro enzymatic production of USP chondroitin sulfate A (CSA, chondroitin-4-sulfate) using chondroitin substrate harvested from E. coli fermentation. The societal impact of the proposed project is expected to be broad, as the importance of having a non-animal-derived chondroitin cannot be overstated. Recombinant protein biotherapeutics have had a revolutionary impact on healthcare. A number of both basic and applied research areas are expected to be impacted as a result of this project, including: (i) identification of metabolic steps that play a key role in biosynthesis of chondroitin in E. coli; (ii) application of CRISPRi for metabolic engineering, further facilitating its application as a synthetic biology tool, where it could also be utilized for construction of complex gene circuits, logic gates, and biosensors (iii) the synthesis of bioengineered chondroitin sulfate will become reality and will pave the way for the animal-free synthesis of several other polysaccharides of pharmaceutical importance. The proposed project will be performed in a scientifically diverse environment with graduate students working together with industrial investigators to perform the experimental tasks with the aid of undergraduate students. In addition, a focused outreach effort by the PI and co-PI will include their continued participation in the Rensselaer Summer High School Research Program and the Scientific Research Program in Shaker High School as well as enrollment in new outreach opportunities such as the New Visions Math, Engineering, Technology and Science program.This award by the Biotechnology and Biochemical Engineering Program of the CBET Division is co-funded by the GOALI Program of the Division of Industrial Innovation and Partnerships.

人们对开发用于稳健、高产率生产可用作营养品或药物的生物工程天然多糖（如硫酸软骨素）的方法越来越感兴趣。使用重组微生物的这种方法将首次允许生产不含动物的多糖，并将消除与动物源性产品相关的风险，包括病毒感染、产品不一致和可能的掺假以及对环境不友好工艺的依赖。该项目是一项转化和多学科的研究工作，其最终目标是促进公斤数量的非动物来源的生物工程硫酸软骨素的高效和安全生产。它旨在揭示前体可用性对大分子生产的作用，并将这些知识转化为多糖的高效生产平台。该项目的目标是通过标准的代谢工程方法，基于化学计量的建模和代谢途径平衡来提高非硫酸软骨素的生产水平（具体目标1）。在具体目标2中，研究人员将证明CRISPR干扰（CRISPRi）作为一种代谢工程工具，能够同时诱导多个基因的敲低和可调抑制，以控制大肠杆菌的代谢通量。杆菌在具体目标3中，将开发一种可扩展的方法，用于使用从E.大肠杆菌发酵预计该项目的社会影响将是广泛的，因为拥有非动物来源的软骨素的重要性不能被夸大。重组蛋白生物治疗对医疗保健产生了革命性的影响。该项目将影响许多基础和应用研究领域，包括：（i）确定在E.大肠杆菌;（ii）CRISPRi在代谢工程中的应用，进一步促进其作为合成生物学工具的应用，其中它也可以用于构建复杂的基因电路、逻辑门和生物传感器（iii）生物工程硫酸软骨素的合成将成为现实，并将为无动物合成其他几种具有药物重要性的多糖铺平道路。拟议的项目将在科学多样化的环境中进行，研究生将与工业调查人员一起工作，在本科生的帮助下执行实验任务。此外，PI和co-PI的重点外展工作将包括继续参与Rensselaer夏季高中研究计划和Shaker高中的科学研究计划，以及参加新的外展机会，如新视野数学，工程，技术和科学计划。由CBET部门的生物技术和生物化学工程计划颁发的这一奖项是共同的，由工业创新和伙伴关系司的GOALI计划资助。