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

1604547 Koffas，Mattheos人们越来越有兴趣开发方法，以稳健、高产地生产可用作营养食品或药物的生物工程天然多糖，如硫酸软骨素。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不利于环境的工艺的依赖。该项目是一项翻译和多学科的研究工作，最终目标是促进高效和安全地生产公斤数量的非动物来源的生物工程硫酸软骨素。它试图揭示前体可获得性在大分子生产中的作用，并将这一知识转化为高效的多糖生产平台。该项目的目标是通过标准代谢工程方法、基于化学计量学的建模和代谢途径平衡(具体目标1)，提高非硫酸软骨素的生产水平。在具体目标2中，研究人员将证明CRISPR干扰(CRISPRi)是一种代谢工程工具，能够同时诱导敲除和可调抑制多个基因，以控制大肠杆菌中的代谢通量。在具体目标3中，将开发一种可扩展的方法，用于使用从大肠杆菌发酵中获得的软骨素底物在体外酶法生产USP软骨素硫酸盐A(CSA，软骨素-4-硫酸盐)。拟议项目的社会影响预计将是广泛的，因为拥有一种非动物来源的软骨素的重要性怎么强调都不为过。重组蛋白质生物疗法对医疗保健产生了革命性的影响。预计该项目将影响许多基础和应用研究领域，包括：(I)鉴定在大肠杆菌中软骨素生物合成中起关键作用的代谢步骤；(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，它还可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，并将为其他几种具有药用价值的多糖的无动物合成铺平道路。拟议的项目将在一个科学多样化的环境中进行，研究生与工业调查人员合作，在本科生的帮助下执行实验任务。此外，主办方和合作者将继续参与Rensselaer暑期高中研究计划和Shaker高中的科研计划，以及参加新的推广机会，如新视野数学、工程、技术和科学计划。该奖项由CBET部门生物技术和生化工程计划共同资助，由工业创新与伙伴关系部门的GOALI计划资助。

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.