Computationally assisted semirational engineering of Nicotiana attenuata α-glucan phosphorylase enhances cellobiose-to-amylose cascade synthesis

A library of 37 Nicα GP mutants was built by computational engineering. • L226Y/V437L/F751H showed 2.10 fold higher activity than WT Nicα GP. • The triple mutant improved thermostability and kinetics. • Cascade productivity increased by 26.5 % at a cellobiose/G4 ratio of 1:2. • Structural analysis linked activity gains to regulatory layer optimization. Converting non-food cellulosic sugars into programmable amylose offers a route to higher-value carbohydrate materials. α -Glucan phosphorylase ( α GP) controls glucose-1-phosphate (G-1-P)-dependent chain elongation in cellobiose-to-amylose cascades. Here, Nicotiana attenuata α GP ( Nicα GP) was engineered by integrating AlphaFold3-based structural modeling, ProteinMPNN, EVcouplings, structural priors, and DynaMut2 filtering. Of 916 candidate substitutions, 37 were selected for experimental validation, and combinatorial screening identified L226Y/V437L/F751H as the optimal triple mutant, with 2.10-fold higher relative activity than the same-batch wild-type (WT) control and clear positive epistasis. The mutant further shifted the optimal temperature from 40 to 45 °C, increased thermal transition midpoint from 56.2 to 58.8 °C, and enhanced catalytic efficiency toward both maltotetraose and G-1-P. In the Clostridium thermocellum cellobiose phosphorylase ( Ct CBP)– Nicα GP cascade, L226Y/V437L/F751H reached 38.86 ± 2.06 % conversion at 72 h versus the previously reported WT value of 35.84 ± 1.04 % at 84 h, increasing apparent productivity by 26.5 %. Docking, CAVER, and molecular dynamics (MD) analyses suggested that these gains may be associated with optimization of structural regions surrounding the conserved catalytic core. This work provides an engineered α GP for amylose synthesis from cellobiose and supports peripheral regulatory engineering as a practical strategy for improving phosphorylase-based biocatalytic cascades. Download: Download high-res image (230KB) Download: Download full-size image