Hybrid Computationally Guided Strategies for Engineering a Laminaribiose Phosphorylase to Facilitate the Industrial Production of Laminaribiose In Vitro

Efficient enzymatic synthetic pathways for biomanufacturing chemicals often require addressing bottlenecks by enhancing the catalytic efficiency or thermostability of unsatisfied enzymes. Laminaribiose, a valuable commodity disaccharide, can be biosynthesized from starch via an in vitro four-enzyme cascade previously described, but a thermolabile laminaribiose phosphorylase from Paenibacillus sp. YM1 (PsLBP) is a potential bottleneck. Here, through several computationally guided approaches, the thermostability of PsLBP was successfully enhanced, with the best mutant, M8, exhibiting a 15.3 C-degrees higher T m value, a 71.1-fold longer half-life, and a 2.4-fold increased specific activity at 60 C-degrees than those of the wild-type enzyme. Structure analysis and molecular dynamics simulations revealed that the formation of new disulfide bonds and salt bridges and the changes to hydrogen bonds optimized the overall structure and improved the thermostability of M8. Subsequently, by employing an industrial procedure of heat-permeabilized whole cells, the in vitro biosystem incorporating PsLBP-M8 in a volume of 30 L converted 100.0 g/L starch to similar to 77.0 g/L laminaribiose with a molar yield of similar to 75% at 60 C-degrees. These findings highlight the utility of hybrid computationally guided strategies for engineering large proteins such as laminaribiose phosphorylase and demonstrate the power of adapting incompatible enzymes in in vitro biosystems to enhance industrial performance.