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导过程。除了它们的天然功能外，蛋白质还广泛用于化学，生物技术和医学-例如作为胰岛素等治疗剂或作为洗衣剂中使用的生物催化剂或分解塑料。尽管蛋白质具有显著的结构和功能多样性，但绝大多数蛋白质仅由20种标准构建块（标准氨基酸）组成。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。为了解决这一基本限制，已经开发了一种强大的蛋白质工程技术，称为遗传密码扩展（GCE），以允许从>20个氨基酸的构建块产生蛋白质。使用这种技术，数百种具有新功能侧链的非经典氨基酸（ncAA）现在可以选择性地引入蛋白质中，从而导致新一代生物催化剂，先进材料和新生物治疗剂的开发。然而，尽管取得了巨大的进展和巨大的商业潜力，GCE从学术实验室转化为商业蛋白质产品受到现有技术限制的阻碍。目前的翻译障碍包括含ncAA的蛋白质的低产量和需要大量过量的ncAA，这最终导致高得令人望而却步的生产成本。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。汇集来自学术界和工业界的多学科研究人员，我们将开发能够生物合成新功能ncAA的工程菌株，并将其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。为了提高我们的技术，我们将与我们的工业合作伙伴AstraZeneca，GSK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治疗剂的大规模生产。展望未来，在该提案中开发的通用GCE平台和工程生物学工具将使多样化功能化蛋白质的可规模化生产成为可能，以满足新兴的社会需求。

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.