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

蛋白质生产是一个不断增长、价值数十亿美元的行业，提供量身定做的酶，如用于制药、洗涤剂、纺织品制造和环保化学品生产的酶。利用细菌作为“蛋白质工厂”的新方法有可能极大地降低制造成本和新蛋白质产品的开发时间。实现这些好处的一个关键障碍是确定一种经济有效的方法，将所需的功能蛋白质从宿主细胞的剩余内容中分离出来。直接从细胞中分泌蛋白质产品是实现这一目标的一种很有前途的方法。这项工作建议在实验室菌株肠沙门氏菌中设计一个蛋白质分泌系统，以高效和低成本地生产蛋白质。成功将为许多行业提供一条敏捷设计和生产蛋白质的道路。此外，这项工作将培训各级学生从事生物技术职业，因为调查人员将参与通过博物馆、社区讲座和科学节将与这项工作相关的尖端科学带给普通公众的项目。蛋白质从细胞内部到外部的受控运输是一个挑战，大自然已经通过几种方式克服了这一挑战，但科学家们还没有完全探索。这项建议的重点是改造肠沙门氏菌的III型分泌系统，以高数量和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所获得的蛋白质产量，2)优化对分泌过程的控制，使其能够扩大到工业规模，以及3)开发一套分子生物学工具，用于高通量生产广泛的蛋白质。改造III型分泌系统以有效地生产所需的蛋白质将提高对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，因为他们构建了细胞功能的详细图景，而且对于希望设计出比以前更复杂的生物和细胞外相互作用更高水平的合成生物学家来说也是如此。这项工作将产生新的结构、方案和菌株，以便在工业和研究规模上与其他科学家共享蛋白质生产。

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.