Engineering a cyclic enzymatic cascade for efficient trehalose biosynthesis from maltodextrin substrates

Trehalose, a nonreducing disaccharide widely distributed in nature, is commonly used in pharmaceuticals, food, and cosmetics because of its stability and protein-preserving properties. Currently, industrial trehalose production relies on a two-step enzymatic cascade catalyzed by maltooligosyltrehalose synthase (TreY) and maltooligosyltrehalose trehalohydrolase (TreZ). However, the overall conversion rate remains suboptimal, primarily because of limited enzyme performance and the accumulation of short-chain maltooligosaccharides as nonconvertible by-products. In this study, a computer-aided enzyme engineering strategy was employed to enhance the thermostability and catalytic efficiency of TreY and TreZ from Arthrobacter ramosus. The TreZ mutant T212P exhibited a 32.3 % increase in thermal stability, whereas the TreY mutant M1 exhibited 2.97-fold and 0.63-fold improvements in thermostability and catalytic activity, respectively. To further improve substrate utilization, a highly efficient 4-α-glucanotransferase (CSMalQ) from Corallococcus sp. EGB was identified via computational screening. CSMalQ catalyzes the disproportionation of short-chain maltooligosaccharides, converting them into longer chains that can be recycled into the trehalose biosynthetic pathway. By integrating CSMalQ into the enzymatic system, a cyclic multienzyme cascade was established using low-cost maltodextrin as the substrate, resulting in a trehalose conversion rate of 84.31 %, which is the highest level reported to date. This study presents a promising strategy for the biosynthetic production of trehalose.