Self-Sustaining Cell-Free Systems

无细胞基因表达系统最近成为合成生物学应用的一个强大平台。利用细胞裂解物或纯化酶，复杂的生物过程在受控的生化反应中被利用，这使得复杂的合成遗传和代谢程序可以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，资源可以有效地用于生产感兴趣的蛋白质；开放反应允许实时定义和操纵反应的组成和条件；该反应的无生命性质使其能够产生和检测对工程细胞致命的化合物。这些优点已经在便携式、按需生物生产治疗蛋白、纸质环境和致病生物传感器以及工业规模生产高价值化合物方面取得了突破性的应用。现有无细胞系统的一个主要限制是，与活细胞不同，它们不能再生其酶成分，酶成分会随着时间的推移而降解。这就限制了无细胞系统的反应寿命和蛋白质合成能力。该研究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成分。这包括设计系统来有效地引导资源进行自我再生过程。以这种方式更新酶可以提高反应性能，这是一种被称为自催化的正反馈回路，是活细胞通常实现的过程。在无细胞系统中实现自催化将具有变革性，使全新的应用类别成为可能，并阐明了生命的基本属性。为了实现这一具有挑战性和雄心勃勃的目标，我提出了一个研究计划，分为三个主要目标：1)通过设计新的代谢系统来维持能量和代谢物的稳态，将无细胞反应的寿命延长到最先进的水平，从而实现自我维持的无细胞代谢。2)通过指导蛋白质合成反应不断地再生其酶组分，提高反应寿命和生产率，实现无细胞酶的自我再生。3)代谢感知的无细胞遗传程序将用于指导系统的自我生产其组成部分。这些将使用一个新的集成计算框架来设计。这一目标的成功有望为无细胞系统产生一种新的操作范式，在这种系统中，反应本身被用来引导其自身成分的再生。最终的结果是一种完全自催化、自我维持的无细胞反应，这将对无细胞生物生产的经济性、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。

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.