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 T1/2 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.