Comprehensive functional characterization of the glycoside hydrolase family 3 enzymes from Cellvibrio japonicus reveals unique metabolic roles in biomass saccharification

Abstract

Lignocellulose degradation is central to the carbon cycle and renewable biotechnologies. The xyloglucan (XyG), β(1→3)/β(1→4) mixed-linkage glucan (MLG) and β(1→3) glucan components of lignocellulose represent significant carbohydrate energy sources for saprophytic microorganisms. The bacterium Cellvibrio japonicus has a robust capacity for plant polysaccharide degradation, due to a genome encoding a large contingent of Carbohydrate-Active enZymes (CAZymes), many of whose specific functions remain unknown. Using a comprehensive genetic and biochemical approach, we have delineated the physiological roles of the four C. japonicus glycoside hydrolase family 3 (GH3) members on diverse β-glucans. Despite high protein sequence similarity and partially overlapping activity profiles on disaccharides, these β-glucosidases are not functionally equivalent. Bgl3A has a major role in MLG and sophorose utilization, and supports β(1→3) glucan utilization, while Bgl3B underpins cellulose utilization and supports MLG utilization. Bgl3C drives β(1→3) glucan utilization. Finally, Bgl3D is the crucial β-glucosidase for XyG utilization. This study not only sheds the light on the metabolic machinery of C. japonicus, but also expands the repertoire of characterized CAZymes for future deployment in biotechnological applications. In particular, the precise functional analysis provided here serves as a reference for informed bioinformatics on the genomes of other Cellvibrio and related species.

Figure 1. Representative structure of xyloglucan (XyG) and mixed linkage β-glucan (MLG)

(A) Dicot (fucogalacto)xyloglucan indicating variable sidechain substitution. (B) General Poales MLG structure comprised of β(1→4)-linked cellotetraose and cellotriose units connected by β(1→3) linkages. Representations of monosaccharide residues are according to (Varki et al., 2009): glucose, blue circles; xylose, orange stars; galactose, yellow circles; L-fucose, red triangles.

Figure 2. Growth of E. coli strains expressing individual C. japonicus GH3 genes using mono- and disaccharides

(A) glucose, (B) sophorose, (C) gentiobiose, (D) laminaribiose. E. coli harboring the empty pBBRMCS-5 vector (pVOC) was included as a negative control. Experiments were performed in biological triplicate with the error bars representing the standard deviation. Growth rates and maximum optical density are summarized in Table S1.

Figure 3. Growth of C. japonicus wild-type and GH3 gene deletion mutants on the β(1→2) linked dissacharide sophorose

(A) single, (B) double, (C) triple and quadruple mutants versus wild type. Experiments were performed in biological triplicate with the error bars representing the standard deviation. Growth rates and maximum optical density are summarized in Table S1. All mutants grew as wild type on glucose, as shown previously (Nelson et al., 2017).

Figure 4. Growth of C. japonicus wild-type and GH3 gene deletion mutants on the β(1→3) linkage-containing substrates

(A–C) laminaribiose, (D–F) curdlan, (G–I) MLG. Experiments were performed in biological triplicate with the error bars representing the standard deviation. Growth rates and maximum optical density are summarized in Table S1. All mutants grew as wild type on glucose, as shown previously (Nelson et al., 2017).

Figure 5. Growth of C. japonicus wild-type and GH3 gene deletion mutants on xyloglucan and xylogluco-oliogsaccharides

(A) single, (B) double, (C) triple and quadruple mutants versus wild type on XyG. (D) Growth of select strains on XyGOs. Experiments were performed in biological triplicate with the error bars representing the standard deviation. Growth rates and maximum optical density are summarized in Table S1. All mutants grew as wild type on glucose, as shown previously (Nelson et al., 2017).

Figure 6. Updated model of xyloglucan utilization by C. japonicus

XyG hydrolysis is initiated outside of the cell by endo-xyloglucanases (e.g. CjGH74 (Attia et al., 2016)), followed by XyGO transport into the periplasm by a TonB-dependent transporter (TBDT). The C. japonicus α-L-fucosidase Afc95A removes terminal fucosyl residues, enabling full access of the β-galactosidase Bgl35A to both pendant galactosyl residues (Larsbrink et al., 2014a) Activity of the α-xylosidase Xyl31A is restricted to terminal non-reducing-end xylosyl residues (Larsbrink et al., 2014a), such that cycling between the α-xylosidase and the primary XyGO-specific β-glucosidase Bgl3D is required for complete saccharification to monosaccharides for primary metabolism (see Fig S3).