Identification of glucanases and phosphorylases involved in hypoxic paramylon degradation in Euglena gracilis

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The phytoflagellate Euglena gracilis accumulates a large amount of paramylon , an aggregation of linear β-1,3-glucan chains forming a granule , as a storage polysaccharide . E. gracilis rapidly catabolizes paramylon and converts it into wax esters to produce ATP under low-oxygen conditions. Previously, three dominant β-1,3-glucanases were identified in E. gracilis , but it remains unclear if these enzymes are substantially responsible for the paramylon degradation process . In this study, we first demonstrated that these known β-1,3-glucanases are not required for hypoxic paramylon degradation, and then performed a combination of functional proteomics and reverse genetic analyses to identify enzymes responsible for paramylon degradation. The proteomics analysis of the proteins extracted from the isolated paramylon identified two endo-glucanases (EgENG1A and EgENG2) and one laminaridextrin phosphorylase (EgLDP1) as candidate paramylon-degrading enzymes. Furthermore, the bioinformatics-based identification of proteins homologous to these enzymes suggested the involvement of one more glucanase (EgENG1B) and two more phosphorylases (EgLDP2 and EgP1) in paramylon degradation. Simultaneous knockdown (KD) of any two to three endo-glucanase genes showed a significant, but partial, retardation of paramylon breakdown under hypoxic conditions. A simultaneous KD cell line dKD- ldp1 / p1 for two genes encoding laminaridextrin phosphorylases showed more severe phenotypes on both paramylon synthesis and degradation than did the respective single KDs, indicating that EgLDP1 and EgP1 act additively to regulate paramylon metabolism. These results clearly indicate that the glucanases and phosphorylases identified in this study play a role in substantial paramylon degradation under low-oxygen conditions. • Substantial enzymes in hypoxic paramylon degradation were identified in Euglena . • Three of each glucanase and glucan phosphorylase were selected by proteomics and in silico analyses. • Gene silencing analysis demonstrated the importance of the enzymes in paramylon degradation.