Sun Chuanqi,Li Zonglin,Ning Xiao,Xu Wentian,Li Zhimin
Adenosine triphosphate (ATP) acts as a crucial energy currency in vivo , and it is a widely used energy and/or phosphate donor for enzyme-catalyzed reactions in vitro. In this study, we established an in vitro multi-enzyme cascade system for ATP production. Using adenosine and inorganic polyphosphate (polyP) as key substrates, we combined adenosine kinase and two functionally distinct polyphosphate kinases (PPKs) in a one-pot reaction to achieve chain-like ATP regeneration and production. Several sources of PPK were screened and characterized, and two suitable PPKs were selected to achieve high rates of ATP production. Among these, Sulfurovum lithotrophicum PPK (SlPPK) exhibited excellent activity over a wide pH range (pH 4.0–9.0) and synthesized ATP from ADP using short-chain polyP. Furthermore, it had a half-life > 155.6 h at 45 °C. After optimizing the reaction conditions, we finally carried out the coupling-catalyzed reaction with different initial adenosine concentrations of 10, 20, and 30 mM. The highest yields of ATP were 76.0, 70.5, and 61.3%, respectively. Graphical Abstract
Boyu Yang,Chao Li,Yongshuo Ren,Weichen Wang,Xiangxiang Zhang,Xiaojun Han
The bottom-up construction of artificial cells is beneficial for understanding cell working mechanisms. The glycolysis metabolism mimicry inside artificial cells is challenging. Herein, the glycolytic pathway (Entner–Doudoroff pathway in archaea) is reconstituted inside artificial cells. The glycolytic pathway comprising glucose dehydrogenase (GDH), gluconate dehydratase (GAD), and 2-keto-3-deoxygluconate aldolase (KDGA) converts glucose molecules to pyruvate molecules. Inside artificial cells, pyruvate molecules are further converted into alanine with the help of alanine dehydrogenase (AlaDH) to build a metabolic pathway for synthesizing amino acid. On the other hand, the pyruvate molecules from glycolysis stimulate the living mitochondria to produce ATP inside artificial cells, which further trigger actin monomers to polymerize to form actin filaments. With the addition of methylcellulose inside the artificial cell, the actin filaments form adjacent to the inner lipid bilayer, deforming the artificial cell from a spherical shape to a spindle shape. The spindle-shaped artificial cell reverses to a spherical shape by depolymerizing the actin filament upon laser irradiation. The glycolytic pathway and its further extension to produce amino acids (or ATP) inside artificial cells pave the path to build functional artificial cells with more complicated metabolic pathways.
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