Cellulose degradation in Gastrophysa viridula (Coleoptera: Chrysomelidae): functional characterization of two CAZymes belonging to glycoside hydrolase family 45 reveals a novel enzymatic activity

Abstract

Cellulose is a major component of the primary and secondary cell walls in plants. Cellulose is considered to be the most abundant biopolymer on Earth and represents a large potential source of metabolic energy. Yet, cellulose degradation is rare and mostly restricted to cellulolytic microorganisms. Recently, various metazoans, including leaf beetles, have been found to encode their own cellulases, giving them the ability to degrade cellulose independently of cellulolytic symbionts. Here, we analyzed the cellulosic capacity of the leaf beetle Gastrophysa viridula, which typically feeds on Rumex plants. We identified three putative cellulases member of two glycoside hydrolase (GH) families, namely GH45 and GH9. Using heterologous expression and functional assays, we demonstrated that both GH45 proteins are active enzymes, in contrast to the GH9 protein. One GH45 protein acted on amorphous cellulose as an endo-β-1,4-glucanase, whereas the other evolved to become an endo-β-1,4-xyloglucanase. We successfully knocked down the expression of both GH45 genes using RNAi, but no changes in weight gain or mortality were observed compared to control insects. Our data indicated that the breakdown of these polysaccharides in G. viridula may facilitate access to plant cell content, which is rich in nitrogen and simple sugars.

Pectin digestion in herbivorous beetles: Impact of pseudoenzymes exceeds that of their active counterparts

“…Thus, the observed phenotype cannot be correlated with lower cellulase activity with total certainty. Moreover, the knockdown of GH45 cellulases in the leaf beetle Gastrophysa viridula had no effect on larval fitness [56], ), indicating that the impact of cellulase activity in herbivorous insects depends on both species and context, such as the diet provided for feeding assays..…”

“…Thus, although PG activity in the 28a treatment is impaired, the interaction time of PGs and pectin in the gut might be enough for sufficient enzyme function. As "Phytophaga" beetles also possess gene families encoding active cellulases and hemicellulases [25,56,[66][67][68][69], it is unclear whether the lack of a specific PCWDE activity, such as that of PGs, is costly or can be compensated for by the concurrent action of other enzymatic functions.…”

Functional analyses of the horizontally acquired Phytophaga glycoside hydrolase family 45 (GH45) proteins reveal distinct functional characteristics

“…Interestingly, all Chrysomelid GH45 xyloglucanases (except LDE5 and DVI6), including G. viridula GVI1 from our previous study, (Busch, et al 2018), displayed a substitution from aspartate to glutamate at the stabilizing site (Asp114) (Fig. 2).…”

“…The first GH45 that was functionally characterized in a beetle originated from Apriona germari (Chrysomeloidea: Cerambycidae: Lamiinae), which had the ability to degrade amorphous cellulose (Lee, et al 2004). Until recently, GH45s in beetles have been functionally characterized in only a few Chrysomeloidea species, mostly Cerambycidae (Chang, et al 2012; Pauchet, Kirsch, et al 2014; Mei, et al 2015) and Diabrotica virgifera virgifera (Chrysomelidae: Galerucinae) (Valencia, et al 2013), and another two from our previous study in Gastrophysa viridula (Chrysomelidae: Chrysomelinae) (Busch, et al 2018). Although GH45 sequences have been identified in Curculionoidea beetles (Pauchet, et al 2010; Keeling, et al 2013; Vega, et al 2015), to date none has ever been functionally characterized.…”

Analyzing the Substrate Specificity of a Class of Long‐Horned‐Beetle‐Derived Xylanases by Using Synthetic Arabinoxylan Oligo‐ and Polysaccharides

“…Such substrates would be helpful to have for analyzing other classes of PCWDEs, such as those acting on substituted polysaccharides like xyloglucan or galactomannan/galactoglucomannan. The synthesis of xyloglucan oligosaccharides by glycan‐automated assembly has been developed, and such substrates could be used to functionally characterize insect‐derived xyloglucanases such as the GH45 proteins described from several chrysomelid and curculionid species …”