Converting Bulk Sugars into Functional Fibers: Discovery and Application of a Thermostable ß-1,3-Oligoglucan Phosphorylase.

Despite their broad application potential, the widespread use of ß-1,3-glucans has been hampered by the high cost and heterogeneity associated with current production methods. To address this challenge, scalable and economically viable processes are needed for the production of ß-1,3-glucans with tailorable molecular mass distributions. Glycoside phosphorylases have shown to be promising catalysts for the bottom-up synthesis of ß-1,3-(oligo)glucans since they combine strict regioselectivity with a cheap donor substrate (i.e., α-glucose 1-phosphate). However, the need for an expensive priming substrate (e.g., laminaribiose) and the tendency to produce shorter oligosaccharides still form major bottlenecks. Here, we report the discovery and application of a thermostable ß-1,3-oligoglucan phosphorylase originating from Anaerolinea thermophila (AtßOGP). This enzyme combines a superior catalytic efficiency toward glucose as a priming substrate, high thermostability, and the ability to synthesize high molecular mass ß-1,3-glucans up to DP 75. Coupling of AtßOGP with a thermostable variant of Bifidobacterium adolescentis sucrose phosphorylase enabled the efficient production of tailorable ß-1,3-(oligo)glucans from sucrose, with a near-complete conversion of >99 mol %. This cost-efficient process for the conversion of renewable bulk sugar into ß-1,3-(oligo)glucans should facilitate the widespread application of these versatile functional fibers across various industries.

Development and Application of a High-Throughput Functional Metagenomic Screen for Glycoside Phosphorylases.

Glycoside phosphorylases (GPs) catalyze the reversible phosphorolysis of glycosidic bonds, releasing sugar 1-phosphates. To identify a greater range of these under-appreciated enzymes, we have developed a high-throughput functional screening method based on molybdenum blue formation. In a proof-of-principle screen focused on cellulose-degrading GPs we interrogated ∼23,000 large insert (fosmid) clones sourced from microbial communities inhabiting two separate environments and identified seven novel GPs from carbohydrate active enzyme family GH94 and one from GH149. Characterization identified cellobiose phosphorylases, cellodextrin phosphorylases, laminaribiose phosphorylases, and a ß-1,3-glucan phosphorylase. To demonstrate the versatility of the screening method, varying substrate combinations were used to identify GP activity from families GH13, GH65, GH112, and GH130 in addition to GH94 and GH149. These pilot screen and substrate versatility results provide a screening paradigm platform for recovering diverse GPs from uncultivated microbial communities acting on different substrates with considerable potential to unravel previously unknown degradative pathways within microbiomes.