Construction of a De Novo Nucleotide Biosynthesis Pathway in Artificial Cells for RNA Transcription

The reconstitution of the de novo nucleotide synthesis pathway from inorganic compounds for RNA transcription is essential for an autonomous artificial cell. Here, a de novo uridine triphosphate (UTP) synthesis pathway is successfully constructed starting from NH₄HCO₃, involving eight enzymes of carbamoyl phosphate synthetase (CPS), aspartate transcarbamoylase (ATC), dihydroorotase (DHO), dihydroorotate dehydrogenase (DHODH), ribose-phosphate pyrophosphokinase (RPPK), uridine 5'-monophosphate synthase (UMPs), uridylate kinase (UK), and nucleoside diphosphate kinase (NDK). ATP regeneration system composed of creatine kinase (CK) and creatine phosphate is incorporated to drive this pathway. 0.85 mM UTP is produced within 180 min under the optimized conditions, which is further used for RNA transcription along with preadded CTP, GTP, regenerated ATP, and T7 RNA polymerase. The de novo UTP synthesis pathway and RNA transcription components are encapsulated into giant unilamellar vesicles to build artificial cells capable of nucleotide de novo production for subsequent RNA transcription. The successful RNA transcription inside artificial cells is visualized using pyronin Y. The successful reconstitution of the de novo UTP synthesis pathway for RNA transcription inside artificial cells lays a solid foundation for the reproduction of an autonomous artificial cell.