Abstract 2377 Thermophilic E1 endoglucanase from Acidothermus cellulolyticus undergoes extensive hydroxyproline-O-glycosylation when expressed in planta

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Thermophilic E1 enzyme derived from Acidothermus cellulolyticus is a well characterized endo-1,4-β-glucanase that has been frequently expressed in planta to reconstruct the plant cell wall to overcome biomass recalcitrance. However, the holoenzyme expressed in planta and targeted to apoplast exhibited a larger molecular size (∼100 kDa) than the theoretical one (57 kDa). This potentially resulted from the posttranslational modification occurring in the recombinant enzyme within plant cells. The objective of this study is to investigate the glycosylation of E1 holoenzyme expressed in tobacco plants and determine how the glycosylation affected the enzyme activity and biomass digestibility. The E1 holoenzyme, E1 active domain (E1cd) and linker region (E1Lk) were each expressed in tobacco plants and suspension cells. The accumulation of holoenzyme was 2.0- to 2.3- times higher than that of E1cd. The proline-rich E1Lk region was extensively hydroxyproline-O-glycosylated with arabinogalactan polysaccharides with sugar accounting for 41.3% of the holoenzyme. Compared with E1cd, the holoenzyme with a Hyp-O-glycosylated E1Lk displayed a broader optimal temperature range (70 to 85 ºC). When grown in greenhouse, the expression of E1 holoenzyme induced notable phenotypic changes in plants, including delayed flowering and leaf variegation post-flowering. Due to the synergetic action of the E1Lk and cellulose binding domain in reducing cell wall recalcitrance, plant biomass engineering with E1 demonstrated markedly higher saccharification efficiency than the E1cd and wild type plants. This study revealed the post-translational modifications occurring in the E1 holoenzyme when expressed in planta and elucidated their effects on both enzyme activity and the enzymatic digestibility of biomass. This work was supported by the Agriculture and Food Research Initiative Grant no. 2014-04034 from the USDA National Institute of Food and Agriculture and the Arkansas Biosciences Institute, the major research component of the Arkansas Tobacco Settlement Proceeds Act of 2000.