Discovery and Biotechnological Exploitation of Glycoside-Phosphorylases

Abstract

Among carbohydrate active enzymes, glycoside phosphorylases (GPs) are valuable catalysts for white biotechnologies, due to their exquisite capacity to efficiently re-modulate oligo- and poly-saccharides, without the need for costly activated sugars as substrates. The reversibility of the phosphorolysis reaction, indeed, makes them attractive tools for glycodiversification. However, discovery of new GP functions is hindered by the difficulty in identifying them in sequence databases, and, rather, relies on extensive and tedious biochemical characterization studies. Nevertheless, recent advances in automated tools have led to major improvements in GP mining, activity predictions, and functional screening. Implementation of GPs into innovative in vitro and in cellulo bioproduction strategies has also made substantial advances. Herein, we propose to discuss the latest developments in the strategies employed to efficiently discover GPs and make the best use of their exceptional catalytic properties for glycoside bioproduction.

Keywords: biotechnology; carbohydrate-active enzymes; functional metagenomics; glycochemistry; glycoside phosphorylases; glycosides; screening; sequence similarity networks.

Figure 1

Inverting GPs and GHs catalytic mechanisms and family classification. The carbohydrates indicated for each family correspond to the glycosyl donors used by GPs of this family.

Figure 2

Retaining GPs catalytic mechanisms and family classification. The carbohydrates indicated for each family correspond to the glycosyl donors used by GPs of this family.

Figure 3

Structural representatives of the phosphorylase families with their main substrate (glycoside donnor, or acceptor, in green, or inorganic phosphate in red, when available). (A) GH3 β-glucoside phosphorylase BglX bound to 2FGlc (5VQE); (B) GH13_18 sucrose phosphorylase E232Q mutant from Bifidobacterium adolescentis in complex with sucrose (2GDU); (C) GH65 kojibiose phosphorylase from Caldicellulosiruptor saccharolyticus DSM 8903 in complex with kojibiose (3WIQ); (D) GH94 chitobiose phosphorylase from Vibrio proteolyticus in complex with GlcNAc (1V7W); (E) GH112 galacto-N-biose phosphorylase from Bifidobacterium longum subsp. longum JCM 1217 in complex with GalNAc (2ZUT). (F) GH130 MannosylGlucose phosphorylase from Bacteroides fragilis NCTC 9343 in complex with mannosyl-glucose and phosphate (3WAS); (G) GH149 β-1,3-oligoglucan phosphorylase in complex with laminaribiose (6HQ8); (H) GT35 maltodextrin phosphorylase from Escherichia coli str. K-12 substr. MG1655 in complex with maltose (1AHP); (I) GT108 Dual-Activity Glycosyltransferase-Phosphorylase from Leishmania mexicana MHOM/GT/2001/U1103 in complex with inorganic phosphate (6Q50).

Figure 4

Biotechnological applications of GPs: the different strategies employed for improving glycoside production yields are marked in red.

Figure 5

Sequence- and activity-based approaches for GP discovery from (meta)genomes and functional characterization.