Marine microbes are a rich source of enzymes for the degradation of diverse polysaccharides. Paraglaciecola hydrolytica S66T is a marine bacterium capable of hydrolyzing polysaccharides found in the cell wall of red macroalgae. In this study, we applied an approach combining genomic mining with functional analysis to uncover the potential of this bacterium to produce enzymes for the hydrolysis of complex marine polysaccharides. A special feature of P. hydrolytica S66T is the presence of a large genomic region harboring an array of carbohydrate-active enzymes (CAZymes) notably agarases and carrageenases. Based on a first functional characterization combined with a comparative sequence analysis, we confirmed the enzymatic activities of several enzymes required for red algal polysaccharide degradation by the bacterium. In particular, we report for the first time, the discovery of novel enzyme activities targeting furcellaran, a hybrid carrageenan containing both β-carrageenan and κ/β-carrageenan motifs. Some of these enzymes represent a new subfamily within the CAZy classification. From the combined analyses, we propose models for the complete degradation of agar and κ/β-type carrageenan by P. hydrolytica S66T. The novel enzymes described here may find value in new bio-based industries and advance our understanding of the mechanisms responsible for recycling of red algal polysaccharides in marine ecosystems.

Algal cell wall polysaccharides constitute a large fraction in the biomass of marine primary producers and are thus important in nutrient transfer between trophic levels in the marine ecosystem. In order for this transfer to take place, polysaccharides must be degraded into smaller mono-and disaccharide units, which are subsequently metabolized, and key components in this degradation are bacterial enzymes. The marine bacterium Colwellia echini A3 T is a potent enzyme producer since it completely hydrolyzes agar and -carrageenan. Here, we report that the genome of C. echini A3 T harbors two large gene clusters for the degradation of carrageenan and agar, respectively. Phylogenetical and functional studies combined with transcriptomics and in silico structural modeling revealed that the carrageenolytic cluster encodes furcellaranases, a new class of glycoside hydrolase family 16 (GH16) enzymes that are key enzymes for hydrolysis of furcellaran, a hybrid carrageenan containing both ␤and -carrageenan motifs. We show that furcellaranases degrade furcellaran into neocarratetraose-43-O-monosulfate [DA-(␣1,3)-G4S-(␤1,4)-DA-(␣1,3)-G], and we propose a molecular model of furcellaranases and compare the active site architectures of furcellaranases, -carrageenases, ␤-agarases, and ␤-porphyranases. Furthermore, C. echini A3 T was shown to encode -carrageenases, -carrageenases, and members of a new class of enzymes, active only on hybrid ␤/-carrageenan tetrasaccharides. On the basis of our genomic, transcriptomic, and functional analyses of the carrageenolytic enzyme repertoire, we propose a new model for how C. echini A3 T degrades complex sulfated marine polysaccharides such as furcellaran, -carrageenan, and -carrageenan. IMPORTANCE Here, we report that a recently described bacterium, Colwellia echini, harbors a large number of enzymes enabling the bacterium to grow on -carrageenan and agar. The genes are organized in two clusters that encode enzymes for the total degradation of -carrageenan and agar, respectively. As the first, we report on the structure/ function relationship of a new class of enzymes that hydrolyze furcellaran, a partially sulfated ␤/-carrageenan. Using an in silico model, we hypothesize a molecular structure of furcellaranases and compare structural features and active site architectures of furcellaranases with those of other GH16 polysaccharide hydrolases, such as -carrageenases, ␤-agarases, and ␤-porphyranases. Furthermore, we describe a new class of enzymes distantly related to GH42 and GH160 ␤-galactosidases and show that this new class of enzymes is active only on hybrid ␤/-carrageenan oligosaccharides. Finally, we propose a new model for how the carrageenolytic enzyme repertoire enables C. echini to metabolize ␤/-, -, and -carrageenan.

Sulfated fucans, often denoted as fucoidans, are highly variable cell wall polysaccharides of brown algae, which possess a wide range of bioactive properties with potential pharmaceutical applications. Due to their complex architecture, the structures of algal fucans have until now only been partly determined. Enzymes capable of hydrolyzing sulfated fucans may allow specific release of defined bioactive oligosaccharides and may serve as a tool for structural elucidation of algal walls. Currently, such enzymes include only a few hydrolases belonging to the glycoside hydrolase family 107 (GH107), and little is known about their mechanistics and the substrates they degrade. In this study, we report the identification and recombinant production of three novel GH107 family proteins derived from a marine metagenome. Activity screening against a large substrate collection showed that all three enzymes degraded sulfated fucans from brown algae in the order Fucales. This is in accordance with a hydrolytic activity against α-1,4-fucosidic linkages in sulfated fucans as reported for previous GH107 members. Also, the activity screening gave new indications about the structural differences in brown algal cell walls. Finally, sequence analyses allowed identification of the proposed catalytic residues of the GH107 family. The findings presented here form a new basis for understanding the GH107 family of enzymes and investigating the complex sulfated fucans from brown algae. DATABASE: The assembled metagenome and raw sequence data is available at EMBL-EBI (Study number: PRJEB28480). Sequences of the GH107 fucanases (Fp273, Fp277, and Fp279) have been deposited in GenBank under accessions MH755451-MH755453.

Marine microorganisms encode a complex repertoire of carbohydrate-active enzymes (CAZymes) for the catabolism of algal cell wall polysaccharides. While the core enzyme cascade for degrading agar is conserved across agarolytic marine bacteria, gain of novel metabolic functions can lead to the evolutionary expansion of the gene repertoire. Here, we describe how two less abundant GH96 α-agarases harbored in the agar specific polysaccharide utilization locus (PUL) of Colwellia echini A3T facilitate the versatility of the agarolytic pathway. The cellular and molecular functions of the α-agarases examined by genomic, transcriptomic, and biochemical analyses revealed that α-agarases of C. echini A3T create a novel auxiliary pathway. α-Agarases convert even-numbered neoagarooligosaccharides to odd-numbered agaro- and neoagarooligosaccharides, providing an alternative route for the depolymerization process in the agarolytic pathway. Comparative genome analysis of agarolytic bacteria implied that the agarolytic gene repertoire in marine bacteria has been diversified during evolution while the essential core agarolytic gene set was conserved. The expansion of the agarolytic gene repertoire and novel hydrolytic functions, including the elucidated molecular functionality of α-agarase, promote metabolic versatility by channeling agar metabolism through different routes. Importance Colwellia echini A3T is an example of how the gene gain can lead to the evolutionary expansion of agar specific polysaccharide utilization loci (PUL). C. echini A3T encodes two α-agarases in addition to the core β-agarolytic enzymes in its agarolytic PUL. Among the agar-degrading CAZymes identified so far, only a few α-agarases have been biochemically characterized. The molecular and biological functions of two α-agarases revealed that their unique hydrolytic pattern leads to the emergence of auxiliary agarolytic pathways. Through the combination of transcriptomic, genomic, and biochemical evidence, we elucidate the complete α-agarolytic pathway in C. echini A3T. The addition of α-agarases to the agarolytic enzyme repertoire might allow marine agarolytic bacteria to increase competitive abilities through metabolic versatility.

A novel bacterial strain, S66T, was isolated from eelgrass collected on the coastline of Zealand, Denmark. Polyphasic analyses involving phenotypic, phylogenetic and genomic methods were used to characterize strain S66T. The strain was Gram-reaction-negative, rod-shaped, aerobic, and displayed growth at 10-25 °C (optimum 20-25 °C) and at pH 7-9 (optimum pH 7.5). Furthermore, strain S66T grew on seaweed polysaccharides agar, agarose, porphyran, κ-carrageenan, alginate and laminarin as sole carbon sources. Major fatty acids were C16 : 0, C16 : 1ω7c and C18 : 1ω7c. The respiratory quinone was determined to be Q-8, and major polar lipids were phosphatidylethanolamine and phosphatidylglycerol. The DNA G+C content was determined to be 42.2 mol%. Phylogenetic analyses based on the 16S rRNA gene and GyrB sequence comparisons showed that the bacterium was affiliated with the genus Paraglaciecola within the family Alteromonadaceae of the class Gammaproteobacteria. The percentage similarity between the 16S rRNA gene and GyrB sequences of strain S66T and other members of the genus Paraglaciecola were 94-95 % and 84-85 %, respectively. Based on the genome sequence of S66T, the average nucleotide identity (ANI) between strain S66T and other members of the genus Paraglaciecola was 77-80 %, and DNA-DNA hybridization prediction showed values of less than 24 % relatedness, respectively, between S66T and other species of the genus Paraglaciecola. The phenotypic, phylogenetic and genomic analyses support the hypothesis that strain S66T represents a novel species of the genus Paraglaciecola, for which the name Paraglaciecola hydrolytica sp. nov. is proposed. The type strain is S66T (=LMG 29457T=NCIMB 15060T=DSM 102834T).