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. © 2022 Wiley Periodicals LLC.