PDF(Pubmed)
Abstract：
Cooperation between catabolism and anabolism is crucial for maintaining homeostasis in living cells. The most fundamental systems for catabolism and anabolism are the glycolysis of sugars and the transcription-translation (TX-TL) of DNA, respectively. Despite their importance in living cells, the in vitro reconstitution of their cooperation through purified factors has not been achieved, which hinders the elucidation of the design principle in living cells. Here, we reconstituted glycolysis using sugars and integrated it with the PURE system, a commercial in vitro TX-TL kit composed of purified factors. By optimizing key parameters, such as glucokinase and initial phosphate concentrations, we determined suitable conditions for their cooperation. The optimized system showed protein synthesis at up to 33% of that of the original PURE system. We observed that ATP consumption in upstream glycolysis inhibits TX-TL and that this inhibition can be alleviated by the co-addition of glycolytic intermediates, such as glyceraldehyde 3-phosphate, with glucose. Moreover, the system developed here simultaneously synthesizes a subset of its own enzymes, that is, glycolytic enzymes, in a single test tube, which is a necessary step toward self-replication. As glycolysis and TX-TL provide building blocks for constructing cells, the integrated system can be a fundamental material for reconstituting living cells from purified factors.
摘要：
分解代谢和合成代谢之间的合作对于维持活细胞的稳态至关重要。分解代谢和合成代谢的最基本系统是糖的糖酵解和DNA的转录翻译（TX-TL），分别。尽管它们在活细胞中很重要，尚未实现通过纯化因子进行合作的体外重建，这阻碍了活细胞中设计原理的阐明。这里,我们使用糖重建糖酵解，并将其与PURE系统集成，由纯化因子组成的商业体外TX-TL试剂盒。通过优化关键参数，如葡萄糖激酶和初始磷酸盐浓度，我们为他们的合作确定了合适的条件。优化的系统显示蛋白质合成高达原始PURE系统的33%。我们观察到上游糖酵解中的ATP消耗会抑制TX-TL，并且这种抑制作用可以通过糖酵解中间体的共同添加来缓解。如甘油醛3-磷酸，与葡萄糖。此外，这里开发的系统同时合成自己的酶的一个子集，也就是说,糖酵解酶,在一个试管里，这是迈向自我复制的必要步骤。糖酵解和TX-TL为构建细胞提供了基础，集成系统可以是从纯化因子中重建活细胞的基本材料。
