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

• Published in: Biotechnology and bioengineering 2022-06, Vol.119 (6), p.1405-1415
• Main Authors: Li, Zonglin, Sun, Chuanqi, Lou, Longwei, Li, Zhimin
• Format: Article
• Language: English
• Subjects: computational strategy; conformational dynamics; Enzymes; Kinases; Metabolic engineering; Metabolic pathways; Metabolism; Microorganisms; multienzyme catalysis; Nucleoside-diphosphate kinase; Nucleosides; Polyphosphate kinase; Protein engineering; Proteins; semirational design; Substrates; Thermal stability; Uridine; Uridylate kinase; UTP
• ISSN: 0006-3592; 1097-0290
• DOI: 10.1002/bit.28061

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 four‐enzyme coupled in vitro production system for uridine triphosphate (UTP) was constructed. To overcome multiple enzymatic bottlenecks, the authors used a variety of protein engineering strategies including semirational design, high‐throughput screening, conformational analysis, computational strategies, and iterative conformational dynamics methods. The limit‐breaking system resulted in a 6‐fold increase in UTP yield compared to the original case.