Scalable Production of Precisely Engineered Proteins Using an Expanded Genetic Code

蛋白质是一种生物聚合物，在自然界中具有广泛的功能，包括加速生命所需的生化反应、跨膜运输分子、提供结构支持和控制信号传递过程。除了它们的天然功能外，蛋白质还被广泛应用于化学、生物技术和医学领域--例如，用作胰岛素等治疗药物，或用作洗涤剂中的生物催化剂或分解塑料。尽管它们的结构和功能具有显著的多样性，但绝大多数蛋白质只由20个标准构建块组成，这是典型的氨基酸。这些氨基酸只包含一组狭窄的功能基序，这最终限制了我们开发具有新功能和改进功能的蛋白质的能力。为了解决这一根本限制，一种名为遗传密码扩展(GCE)的强大蛋白质工程技术已经开发出来，允许从&gt；20个氨基酸构建块生产蛋白质。利用这项技术，现在可以选择性地将数百种具有新功能侧链的非正则氨基酸(NCAA)引入蛋白质中，从而导致新一代生物催化剂、先进材料和新生物疗法的发展。然而，尽管GCE取得了巨大的进展和巨大的商业潜力，但由于现有技术的局限性，GCE从学术实验室转化为商业蛋白质产品的工作一直受到阻碍。目前的翻译障碍包括含有NCAA的蛋白质产量低，以及需要大量过量的NCAA，这最终导致令人望而却步的高生产成本。在这项提案中，我们将开发一个完全集成的工程生物学平台，将GCE转化为商业应用。我们将汇聚学术界和工业界的多学科研究人员，开发能够生物合成新的功能性NCAA的工程菌株，并将它们有效地引入蛋白质中，以比现有平台低得多的成本提供精确功能化的蛋白质。为了证明我们的技术，我们将与我们的工业合作伙伴阿斯利康、葛兰素史克和Prozomix合作，将我们的工程菌株应用于下一代生物催化剂和蛋白质疗法的大规模生产。展望未来，在该提案中开发的多功能GCE平台和工程生物学工具将使各种功能化蛋白质的可扩展生产成为可能，以响应新出现的社会需求。

Proteins are biopolymers that perform a vast array of functions in nature, including speeding up the biochemical reactions needed for life, transporting molecules across membranes, providing structural support and controlling signalling processes. Beyond their natural functions, proteins are also used widely across chemistry, biotechnology and medicine - for example as therapeutics such as insulin or as biocatalysts used in laundry detergents or to break down plastics. Despite their remarkable structural and functional diversity, the vast majority of proteins are made from only 20 standard building blocks, the canonical amino acids. These amino acids only contain a narrow set of functional motifs, which ultimately restricts our ability to develop proteins with new and improved functions. To address this fundamental limitation, a powerful protein engineering technique called genetic code expansion (GCE) has been developed to allow proteins to be produced from >20 amino acid building blocks. Using this technique, hundreds of non-canonical amino acids (ncAAs) with new functional side chains can now be selectively introduced into proteins, leading to the development of new generations of biocatalysts, advanced materials and new biotherapeutics. However, despite great progress and its enormous commercial potential, the translation of GCE from academic labs into commercial protein products has been hindered by existing limitations of the technology. Current barriers to translation include the low yields of ncAA-containing proteins and the requirement for large excesses of ncAAs, which ultimately result in prohibitively high production costs. In this proposal we will develop a fully integrated engineering biology platform to translate GCE into commercial applications. Bringing together multidisciplinary researchers from across academia and industry, we will develop engineered strains capable of biosynthesizing new functional ncAAs and efficiently introduce them into proteins to deliver precisely functionalized proteins at substantially lower costs than existing platforms. To exemplify our technology, we will work in collaboration with our industrial partners AstraZeneca, GSK and Prozomix, to apply our engineered strains to the large-scale production of next generation biocatalysts and protein therapeutics. Moving forward, the versatile GCE platform and engineering biology tools developed within this proposal will enable the scalable production of diverse functionalized proteins in response to emerging societal needs.