A cocktail of protein engineering strategies: Breaking the enzyme bottleneck one by one for high UTP production in vitro

Abstract

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.

Multivariate Modular Metabolic Engineering for High Titer Uridine Triphosphate Production in Escherichia coli

“…55,56 For example, the ppk from Thermus thermophiles and Sulfurovum lithotrophicum were selected for ATP regeneration to provide energy supply for cytidine triphosphate and UTP synthesis, respectively. 8,57 However, two weaknesses emerged: (1) the accumulation of free phosphate ions during polyphosphate utilization, and (2) the relatively low utilization rate of phosphate residue in polyphosphate. 58 To address these limitations, we focused on ETC, which tightly couples oxidative phosphorylation with ATP synthase to facilitate ATP generation.…”

“…An ATP-sensing switch was employed to regulate ETC to ensure a stable intracellular ATP supply and drive increased UTP titer and cell density. Previous studies have established the inextricable connection between pyrimidine derivatives accumulation and ATP levels. ,, To increase ATP abundance, conventional approaches primarily utilized polyphosphate and polyphosphate kinases (PPK) for ATP regeneration. , For example, the ppk from Thermus thermophiles and Sulfurovum lithotrophicum were selected for ATP regeneration to provide energy supply for cytidine triphosphate and UTP synthesis, respectively. , However, two weaknesses emerged: (1) the accumulation of free phosphate ions during polyphosphate utilization, and (2) the relatively low utilization rate of phosphate residue in polyphosphate . To address these limitations, we focused on ETC, which tightly couples oxidative phosphorylation with ATP synthase to facilitate ATP generation.…”

“…26 The determination methods of nucleoside diphosphate kinase (NDK) were improved according to the previous research. 8 The reaction was carried out in a 0.5 mL reaction mixture containing 6 mM UDP, 6 mM ATP, and 30 mM MgCl 2 (to ensure an excess of Mg 2+ , required for enzyme activity) in 50 mM Tris−HCl (pH 7.0) and an appropriate amount of enzyme. Routine assays were performed at 37 °C with a 4 min incubation time and immediately terminated by adding an equal volume of 0.8 M HCl on ice.…”

“…Currently, UTP production involves two main methods: chemical and enzymatic synthesis. 8,9 However, the chemical method usually requires the use of numerous organic solvents and complex steps, such as the reaction of salicyl phosphorochoridite and tributylammonium pyrophosphate, to generate a phosphitylating reagent that selectively phosphitylates the nonprotected nucleosides to produce 5′phosphite. Then, this cyclic phosphite is oxidized by iodine to generate the 5′-cyclic triphosphate, followed by hydrolysis to afford the UTP.…”

“…Uridine triphosphate (UTP), an important member of pyrimidine derivatives, not only participates in nucleic acid and glycogen synthesis but also plays crucial roles in immune regulation, myocardial repair, and intestinal development promotion in living bodies. , It has also been reported that UTP can act as a signaling molecule and the donor of energy transfer to regulate metabolic processes. , In addition, UTP has been used in clinical practice to inhibit platelet aggregation, treat myocardial infarction, and improve the anti-injury ability of nerve cells. Currently, UTP production involves two main methods: chemical and enzymatic synthesis. , However, the chemical method usually requires the use of numerous organic solvents and complex steps, such as the reaction of salicyl phosphorochoridite and tributylammonium pyrophosphate, to generate a phosphitylating reagent that selectively phosphitylates the nonprotected nucleosides to produce 5′-phosphite. Then, this cyclic phosphite is oxidized by iodine to generate the 5′-cyclic triphosphate, followed by hydrolysis to afford the UTP .…”