Background
Deposition of new genetic sequences in online databases is expanding at an unprecedented rate. As a result, sequence identification continues to outpace functional characterization of carbohydrate active enzymes (CAZymes). In this paradigm, the discovery of enzymes with novel functions is often hindered by high volumes of uncharacterized sequences particularly when the enzyme sequence belongs to a family that exhibits diverse functional specificities (i.e., polyspecificity). Therefore, to direct sequence-based discovery and characterization of new enzyme activities we have developed an automated in silico pipeline entitled: Sequence Analysis and Clustering of CarboHydrate Active enzymes for Rapid Informed prediction of Specificity (SACCHARIS). This pipeline streamlines the selection of uncharacterized sequences for discovery of new CAZyme or CBM specificity from families currently maintained on the CAZy website or within user-defined datasets.
Conclusions
Establishing sequence-function and sequence-structure relationships in polyspecific CAZyme families are promising approaches for streamlining enzyme discovery. SACCHARIS facilitates this process by embedding CAZyme and CBM family trees generated from biochemically to structurally characterized sequences, with protein sequences that have unknown functions. In addition, these trees can be integrated with user-defined datasets (e.g., genomics, metagenomics, and transcriptomics) to inform experimental characterization of new CAZymes or CBMs not currently curated, and for researchers to compare differential sequence patterns between entire CAZomes. In this light, SACCHARIS provides an in silico tool that can be tailored for enzyme bioprospecting in datasets of increasing complexity and for diverse applications in glycobiotechnology.
Results
SACCHARIS was used to generate a phylogenetic tree of a GH43, a CAZyme family with defined subfamily designations. This analysis confirmed that large datasets can be organized into sequence clusters of manageable sizes that possess related functions. Seeding this tree with a GH43 sequence from Bacteroides dorei DSM 17855 (BdGH43b, revealed it partitioned as a single sequence within the tree. This pattern was consistent with it possessing a unique enzyme activity for GH43 as BdGH43b is the first described α-glucanase described for this family. The capacity of SACCHARIS to extract and cluster characterized carbohydrate binding module sequences was demonstrated using family 6 CBMs (i.e., CBM6s). This CBM family displays a polyspecific ligand binding profile and contains many structurally determined members. Using SACCHARIS to identify a cluster of divergent sequences, a CBM6 sequence from a unique clade was demonstrated to bind yeast mannan, which represents the first description of an α-mannan binding CBM. Additionally, we have performed a CAZome analysis of an in-house sequenced bacterial genome and a comparative analysis of B. thetaiotaomicron VPI-5482 and B. thetaiotaomicron 7330, to demonstrate that SACCHARIS can generate "CAZome fingerprints", which differentiate between the saccharolytic potential of two related strains in silico. Conclusions: Establishing sequence-function and sequence-structure relationships in polyspecific CAZyme families are promising approaches for streamlining enzyme discovery. SACCHARIS facilitates this process by embedding CAZyme and CBM family trees generated from biochemically to structurally characterized sequences, with protein sequences that have unknown functions. In addition, these trees can be integrated with user-defined datasets (e.g., genomics, metagenomics, and transcriptomics) to inform experimental characterization of new CAZymes or CBMs not currently curated, and for researchers to compare differential sequence patterns between entire CAZomes. In this light, SACCHARIS provides an in silico tool that can be tailored for enzyme bioprospecting in datasets of increasing complexity and for diverse applications in glycobiotechnology.