Self-Sustaining Cell-Free Systems

自我维持的无细胞系统

• 批准号: MR/V027107/1
• 负责人: Nadanai Laohakunakorn
• 金额: $ 191.81万
• 依托单位: University of Edinburgh
• 依托单位国家: 英国
• 项目类别: Fellowship
• 财政年份: 2022
• 资助国家: 英国
• 起止时间: 2022 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=MR%2FV027107%2F1
• 关键词: Self; Sustaining; Cell; Free; Systems

Cell-free gene expression systems have recently emerged as a powerful platform for synthetic biology applications. Using cell lysates or purified enzymes, complex biological processes are harnessed in controlled biochemical reactions, which allows sophisticated synthetic genetic and metabolic programmes to be implemented free from the constraints of living cells. By decoupling protein production from growth, resources can be efficiently channelled to produce proteins of interest; the open reaction allows the composition and conditions of the reaction to be defined and manipulated in real time; and the non-living nature of the reaction enables production and detection of compounds otherwise lethal to engineered cells. These benefits have led to breakthrough applications in portable, on-demand bioproduction of therapeutic proteins, paper-based environmental and pathogenic biosensors, and industrial-scale production of high-value compounds. A major limitation of existing cell-free systems is that unlike living cells, they are not able to regenerate their enzymatic components, which degrade over time. This sets a fundamental limit on the reaction lifetime as well as the protein synthesis capacity of cell-free systems. This fellowship proposes to develop cell-free systems which can completely regenerate their enzymatic components from a supply of energy and substrates. This involves designing systems to efficiently direct resources to self-regeneration processes. Refreshing the enzymes in this way improves the reaction performance in a positive feedback loop known as autocatalysis, a process routinely achieved by living cells. Achieving autocatalysis in cell-free systems will be transformative, enabling entirely new classes of applications as well as elucidating a fundamental property of life. To achieve this challenging and ambitious goal, I propose a research programme divided into three major objectives: 1) Self-sustaining cell-free metabolism will be achieved by engineering novel metabolic systems to maintain homeostasis of energy and metabolites, extending the lifetime of cell-free reactions beyond the state-of-the-art. 2) Self-regeneration of cell-free enzymes will be achieved by directing the protein synthesis reaction to continuously regenerate its enzymatic components, improving reaction lifetime as well as productivity. 3) Metabolism-aware cell-free genetic programs will be used to direct the system's self-production of its components. These will be designed using a novel integrated computational framework.The success of this goal promises to yield a new paradigm of operation for cell-free systems, where the reaction itself is used to bootstrap the regeneration of its own components. The eventual outcome is a fully autocatalytic, self-sustaining cell-free reaction which will have profound consequences for the economics of cell-free bioproduction, the biocomputational power available for cell-free sensing and diagnostic applications, and the future development of artificial cells.

无细胞基因表达系统最近已成为合成生物学应用的强大平台。使用细胞裂解物或纯化的酶，在受控的生化反应中利用复杂的生物过程，这使得复杂的合成遗传和代谢程序能够在不受活细胞限制的情况下实施。通过将蛋白质生产与生长分离，可以有效地引导资源以生产感兴趣的蛋白质;开放反应允许在真实的时间内定义和操纵反应的组成和条件;并且反应的非生命性质使得能够生产和检测否则对工程化细胞致命的化合物。这些优点导致了便携式、按需生物生产治疗性蛋白质、纸基环境和病原生物传感器以及工业规模生产高价值化合物等方面的突破性应用。现有无细胞系统的一个主要限制是，与活细胞不同，它们不能再生其酶组分，这些酶组分会随着时间的推移而降解。这对反应寿命以及无细胞系统的蛋白质合成能力设定了基本限制。该奖学金计划开发无细胞系统，该系统可以从能量和底物的供应中完全再生其酶组分。这涉及设计系统，以有效地引导资源自我再生过程。以这种方式更新酶可以改善被称为自催化的正反馈回路中的反应性能，这是活细胞常规实现的过程。在无细胞系统中实现自催化将是变革性的，使全新的应用类别成为可能，并阐明了生命的基本性质。为了实现这一具有挑战性和雄心勃勃的目标，我提出了一个分为三个主要目标的研究计划：1）自我维持的无细胞代谢将通过设计新的代谢系统来实现，以维持能量和代谢物的稳态，延长了无细胞反应的寿命，超过了现有技术的水平。2）细胞的自我再生，通过引导蛋白质合成反应以连续再生其酶组分、提高反应寿命以及生产率，将获得游离酶。3)代谢意识无细胞遗传程序将用于指导系统的自我生产其组件。这一目标的成功有望为无细胞系统提供一种新的操作模式，其中反应本身用于引导其自身组件的再生。最终的结果是一个完全自催化，自我维持的无细胞反应，这将对无细胞生物生产的经济学，可用于无细胞传感和诊断应用的生物计算能力，以及人工细胞的未来发展产生深远的影响。

项目成果

Self-Assembling Protein Surfaces for In Situ Capture of Cell-Free-Synthesized Proteins.

• DOI: 10.3389/fbioe.2022.915035
• 发表时间: 2022
• 期刊: FRONTIERS IN BIOENGINEERING AND BIOTECHNOLOGY
• 影响因子: 5.7
• 作者: Thornton, Ella Lucille;Paterson, Sarah Maria;Gidden, Zoe;Horrocks, Mathew H.;Laohakunakorn, Nadanai;Regan, Lynne
• 通讯作者: Regan, Lynne