Abstract
BackgroundXyloglucan (XyG) is a ubiquitous and fundamental polysaccharide of plant cell walls. Due to its structural complexity, XyG requires a combination of backbone-cleaving and sidechain-debranching enzymes for complete deconstruction into its component monosaccharides. The soil saprophyte Cellvibrio japonicus has emerged as a genetically tractable model system to study biomass saccharification, in part due to its innate capacity to utilize a wide range of plant polysaccharides for growth. Whereas the downstream debranching enzymes of the xyloglucan utilization system of C. japonicus have been functionally characterized, the requisite backbone-cleaving endo-xyloglucanases were unresolved.ResultsCombined bioinformatic and transcriptomic analyses implicated three glycoside hydrolase family 5 subfamily 4 (GH5_4) members, with distinct modular organization, as potential keystone endo-xyloglucanases in C. japonicus. Detailed biochemical and enzymatic characterization of the GH5_4 modules of all three recombinant proteins confirmed particularly high specificities for the XyG polysaccharide versus a panel of other cell wall glycans, including mixed-linkage beta-glucan and cellulose. Moreover, product analysis demonstrated that all three enzymes generated XyG oligosaccharides required for subsequent saccharification by known exo-glycosidases. Crystallographic analysis of GH5D, which was the only GH5_4 member specifically and highly upregulated during growth on XyG, in free, product-complex, and active-site affinity-labelled forms revealed the molecular basis for the exquisite XyG specificity among these GH5_4 enzymes. Strikingly, exhaustive reverse-genetic analysis of all three GH5_4 members and a previously biochemically characterized GH74 member failed to reveal a growth defect, thereby indicating functional compensation in vivo, both among members of this cohort and by other, yet unidentified, xyloglucanases in C. japonicus. Our systems-based analysis indicates distinct substrate-sensing (GH74, GH5E, GH5F) and attack-mounting (GH5D) functions for the endo-xyloglucanases characterized here.ConclusionsThrough a multi-faceted, molecular systems-based approach, this study provides a new insight into the saccharification pathway of xyloglucan utilization system of C. japonicus. The detailed structural–functional characterization of three distinct GH5_4 endo-xyloglucanases will inform future bioinformatic predictions across species, and provides new CAZymes with defined specificity that may be harnessed in industrial and other biotechnological applications.
Highlights
Xyloglucan (XyG) is a ubiquitous and fundamental polysaccharide of plant cell walls
Transcriptomic analysis reveals a potential keystone endo‐xyloglucanase from Glycoside Hydrolase (GH) family 5, subfamily 4 We previously showed via quantitative PCR that the C. japonicus gene cluster containing xyl31A (CJA_2706), bgl35A (CJA_2707), CJA_2709, and afc95A (CJA_2710) (Fig. 1b), was up-regulated during growth on xyloglucan-containing medium [13]
Samples were collected from both exponentially growing and stationary phase cells grown on glucose or xyloglucan as the sole carbon source to allow for analyses of gene expression based on early-stage substrate detection (Additional file 1: Figure S1), late-stage substrate detection (Additional file 1: Figure S2A), or growth rate (Additional file 1: Figure S2B)
Summary
Xyloglucan (XyG) is a ubiquitous and fundamental polysaccharide of plant cell walls. Due to its structural complexity, XyG requires a combination of backbone-cleaving and sidechain-debranching enzymes for complete deconstruction into its component monosaccharides. The soil saprophyte Cellvibrio japonicus has emerged as a genetically tractable model system to study biomass saccharification, in part due to its innate capacity to utilize a wide range of plant polysaccharides for growth. Chemically and structurally complex in nature and require harsh thermo-chemical treatment to yield fermentable sugars. Such processes often generate undesirable by-products that inhibit subsequent microbial conversion [3]. In light of their ability to catalyze the degradation of recalcitrant plant cell walls under ambient conditions, enzymes from saprophytic microorganisms constitute an attractive palette of biocatalysts for improved biomass saccharification [4]. The recent development of genome editing techniques for C. japonicus has further advanced the biology and bioengineering of this bacterium in biomass conversion [9,10,11,12,13]
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