Engineering a secretion-based, high-titer protein production process in bacteria

在细菌中设计基于分泌的高滴度蛋白质生产过程

• 批准号: 1706125
• 负责人: Danielle Tullman Ercek
• 金额: $ 30.54万
• 依托单位: Northwestern University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-07-01 至 2021-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1706125&HistoricalAwards=false
• 关键词: Engineering; secretion; based; titer; protein

Protein production is a growing, multi-billion dollar industry that supplies tailored enzymes, such as those used in pharmaceuticals, detergents, textile manufacturing, and environmentally-friendly chemical production. New methods, utilizing bacteria as "protein factories", have the potential to dramatically reduce manufacturing costs and the development time for new protein products. A critical barrier to realizing these benefits is identification of a cost-effective way to separate desired functional proteins from the remaining contents of their host cells. Secreting protein products directly out of the cell is a promising method toward this goal. This work proposes to engineer a protein secretion system in a laboratory strain of bacteria, Salmonella enterica, for efficient and low-cost production of proteins. Success would produce a path for agile design and production of proteins for a number of industries. Moreover, this work will train students at all levels for careers in biotechnology, as the investigators will participate in programs bringing cutting-edge science related to this work to the general public via museums, community talks, and science festivals. Controlled transport of proteins from the inside of the cell to the outside is a challenge that nature has overcome in several ways, but that scientists have yet to fully explore. This proposal focuses on engineering the type III secretion system of Salmonella enterica to secrete non-native proteins at high quantity and low cost. The specific objectives are to 1) increase the protein yields achieved with bacterial production and secretion, 2) optimize control over the secretion process to enable scale up to industrial scales, and 3) develop a suite of molecular biology tools for the high-throughput production of a broad set of proteins. Transforming the type III secretion system to enable efficient production of desired proteins will improve understanding of the mechanisms governing this process. Such information is critical not only to molecular biologists as they construct a detailed picture of cellular function, but also to synthetic biologists who wish to engineer organisms with greater levels of complexity and extracellular interaction than previously possible. This work will result in new constructs, protocols, and strains to be shared with other scientists for protein production at both the industrial and research scales.

蛋白质生产是一个不断增长的、价值数十亿美元的行业，它提供量身定制的酶，例如用于制药、洗涤剂、纺织制造和环保化学品生产的酶。利用细菌作为“蛋白质工厂”的新方法有可能大大降低制造成本和新蛋白质产品的开发时间。实现这些益处的关键障碍是确定一种经济有效的方法，将所需的功能蛋白从宿主细胞的剩余内容物中分离出来。直接从细胞中分泌蛋白质产物是实现这一目标的一种有希望的方法。这项工作提出在实验室菌株肠沙门氏菌中设计一种蛋白质分泌系统，以高效和低成本生产蛋白质。成功将为许多行业的敏捷设计和蛋白质生产开辟一条道路。此外，这项工作将培养各级学生从事生物技术的职业，因为研究人员将参与通过博物馆、社区讲座和科学节向公众介绍与这项工作相关的尖端科学的项目。控制蛋白质从细胞内部到细胞外部的运输是一个挑战，自然界已经以多种方式克服了这个挑战，但科学家们还没有充分探索。本研究的重点是对肠沙门氏菌III型分泌系统进行工程化改造，使其能够大量、低成本地分泌非天然蛋白。具体目标是：1)提高细菌生产和分泌的蛋白质产量；2)优化对分泌过程的控制，使其能够扩大到工业规模；3)开发一套分子生物学工具，用于高通量生产多种蛋白质。转化III型分泌系统，使所需蛋白质的有效生产将提高对控制这一过程的机制的理解。这些信息不仅对分子生物学家至关重要，因为他们可以构建细胞功能的详细图像，而且对合成生物学家也至关重要，因为他们希望设计出比以前更复杂的生物体和细胞外相互作用。这项工作将产生新的结构、方案和菌株，与其他科学家分享，用于工业和研究规模的蛋白质生产。