A rapid in vitro enzymatic biosynthesis system has been developed as a biological manufacturing platform with potential industrial uses. Cytidine 5′-monophosphate (5′-CMP) is a key intermediate in the preparation of several nucleotide derivatives and is widely used in food and pharmaceutical industries. In this study, a highly efficient biosynthesis system was constructed for manufacturing 5′-CMP in vitro. Cytidine kinase (CK) was used for the biotransformation of cytidine to 5′-CMP, while polyphosphate kinase (PPK) was coupled for adenosine triphosphate regeneration. Both CK and PPK were selected from extremophiles, possessing great potential for biocatalytic synthesis. The effects of temperature, substrate concentration, and enzyme ratios were investigated to enhance the titer and yield of 5′-CMP. After optimization, 96 mM 5′-CMP was produced within 6 h, and the yield reached nearly 100%. This work highlights the ease of 5′-CMP production by an in vitro biomanufacturing platform and provides a green and efficient approach for the industrial synthesis of 5′-CMP.

The pyrimidine metabolic pathway is tightly regulated in microorganisms, allowing limited success in metabolic engineering for the production of pathway‐related substances. Here, we constructed a four‐enzyme coupled system for the in vitro production of uridine triphosphate (UTP). The enzymes used include nucleoside kinase, uridylate kinase, nucleoside diphosphate kinase, and polyphosphate kinase for energy regeneration. All these enzymes are derived from extremophiles. To increase the total and unit time yield of the product, three enzymes other than polyphosphate kinase were modified separately by multiple protein engineering strategies. A nucleoside kinase variant with increased specific activity from 2.7 to 36.5 U/mg, a uridylate kinase variant (specific activity of 37.1 U/mg) with a 5.2‐fold increase in thermostability, and a nucleoside diphosphate kinase variant with a 2‐fold increase in a specific activity to over 900 U/mg were obtained, respectively. The reaction conditions of the coupled system were further optimized, and a two‐stage method was taken to avoid the problem of enzymatic pH adaptation mismatch. Under optimal conditions, this system can produce more than 65 mM UTP (31.5 g/L) in 3.0 h. The substrate conversion rate exceeded 98% and the maximum UTP productivity reached 40 mM/h.

A novel in vitro multi-enzymatic system was constructed for the conversion of CO2 into optically pure N-carbamoyl-L-aspartate and orotate. Four cascade enzymes, carbamoyl phosphate synthase (CPS), aspartate carbamoyl-transferase (ATCase), dihydroorotase...

Increasing yields while reducing costs is one of the ultimate pursuits of industrial production. To achieve this goal in the enzymatic production of multiple nucleotides, in this study, a co-immobilized polyphosphate kinase–nucleoside kinase hybridized nanoflower system (PPK@NK) was constructed. To improve the productivity, the nucleoside kinase (NK) used was rationally designed, and a variant with significantly increased activity compared to the wild type was obtained. The polyphosphate kinase (PPK) and NK could be sequentially adsorbed on the surface of hybrid nanoflowers at room temperature (25 °C) through the interaction of Cu2+ and proteins without any other chemical pretreatment. The optimal preparation conditions and reaction parameters of PPK@NK hybrid nanoflowers were investigated. Under optimal reaction conditions, the newly prepared co-immobilization system could catalyze the conversion of 100 mM uridine, cytidine, and inosine to the corresponding nucleotides completely within 4 h and could be reused at least six times. The storage stability of the co-immobilized system was more than 2-fold higher than that of the free enzyme, and there was no significant difference in thermostability. PPK@NK hybridized nanoflowers have properties such as easy preparation and storage and low cost, indicating their suitability for the efficient production of nucleotides.