Precise biosynthesis of β-1,2-glucan from cellulosic materials by in vitro metabolic engineering

The biosynthesis of polysaccharides with precisely defined structures, such as tunable degree of polymerization (DP) and low polydispersity index (PDI), remains a significant challenge in microbial cell factories due to their intricate endogenous metabolic networks. In vitro metabolic engineering (ivME) has emerged as a promising alternative, offering simplified pathway design and easy process optimization. In this study, ivME was utilized for the precise synthesis of β-1,2-glucans from β-1,4-linked cellobiose. A four-enzyme system comprising cellobiose phosphorylase, β-1,2-oligoglucan phosphorylase, glucose oxidase, and catalase operated under pH self-neutralized conditions, efficiently producing β-1,2-glucan at a concentration of 31.9 ± 0.4 g/L with a high molar yield (93.3 ± 1.3 %) and a rapid productivity of 4.0 ± 0.1 g/L/h. β-1,2-Glucans with tunable DPs (75-531) and narrow molecular weight distributions (PDI as low as 1.2) were synthesized by adjusting primer concentration, enzyme loadings, and reaction time. The low PDI values of β-1,2-glucans were attributed to the smart pathway design, the careful selection of β-1,2-oligoglucan phosphorylase, and the use of sophorose as the primer. The DP values were mainly influenced by the concentration and type of primers with sophorose outperforming glucose. This strategy of direct glycosidic bond rearrangement from β-1,4 to β-1,2 linkages without coenzymes (e.g., CoA, NAD, ATP, UTP) or external energy input provided a new route for lignocellulosic biomass utilization and significantly enhanced the capabilities of ivME for the production of tailored polysaccharides.