Engineering the Thermostability of Sucrose Synthase by Reshaping the Subunit Interaction Contributes to Efficient UDP-Glucose Production

Abstract

The restricted availability of UDP-glucose, an essential precursor that targets oligo/polysaccharide and glycoside synthesis, makes its practical application difficult. Sucrose synthase (Susy), which catalyzes one-step UDP-glucose synthesis, is a promising candidate. However, due to poor thermostability of Susy, mesophilic conditions are required for synthesis, which slow down the process, limit productivity, and prevent scaled and efficient UDP-glucose preparation. Here, we obtained an engineered thermostable Susy (mutant M4) from Nitrosospira multiformis through automated prediction and greedy accumulation of beneficial mutations. The mutant improved the T 1/2 value at 55 °C by 27-fold, resulting in UDP-glucose synthesis at 37 g/L/h of space-time yield that met industrial biotransformation standards. Furthermore, global interaction between mutant M4 subunits was reconstructed by newly formed interfaces according to molecular dynamics simulations, with residue Trp162 playing an important role in strengthening the interface interaction. This work enabled effective, time-saving UDP-glucose production and paved the way for rational thermostability engineering of oligomeric enzymes.

Highly Efficient Production of UDP-Glucose from Sucrose via the Semirational Engineering of Sucrose Synthase and a Cascade Route Design

“…The primers used are listed in the Supporting Information, Table S1. Detailed gene cloning, culture, and purification were performed as previously described . The target protein was eluted at an imidazole concentration of 200 mM (for SuSy) or 300 mM (for MIase).…”

“…Detailed gene cloning, culture, and purification were performed as previously described. 18 The target protein was eluted at an imidazole concentration of 200 mM (for SuSy) or 300 mM (for MIase). The amount of purified protein was determined using the Bradford method.…”

“…UDP-Glc was monitored through high-performance liquid chromatography (HPLC) as previously described. 18 Briefly, the HPLC system contained a Dionex CarboPac PA10 (Thermo Scientific, Waltham, MA, USA). The mobile phase contained 2 mM NaOH and 1 M NaAc.…”

“…The UDP-Glc yield of WT SuSy was only 12% at the optimum temperature of 55 °C, whereas enhancing its thermostability significantly improved the yield. 18 The half-life time of the enzyme activity of the mutant in this study was also increased by ∼10-fold (Supporting Information, Figure S2). Enhanced thermostability ensured more activated enzymes for catalysis.…”

Multiscale QM/MM Simulations Identify the Roles of Asp239 and 1-OH···Nucleophile in Transition State Stabilization in Arabidopsis thaliana Cell-Wall Invertase 1

“…In nature, sucrose is predominantly derived from photosynthetically active leaves and is translocated through the phloem to sink organs. Once this disaccharide is delivered to sinks, its cleavage is triggered by a chemical reaction catalyzed by either sucrose synthase (EC 2.4.1.13) or invertase (EC 3.2.1.26). , Sucrose synthase hydrolyzes sucrose into uridine-diphosphate-glucose and fructose, whereas invertase irreversibly cleaves sucrose into glucose and fructose. Sucrose and its hydrolyzed products serve as carbon skeleton for specialized metabolites, and sucrose is thus of high importance for cellular metabolism and plant growth and development .…”