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.