Search

Fucoidan, a sulfated polysaccharide found in algae, plays a central role in marine carbon sequestration and exhibits a wide array of bioactivities. However, the molecular diversity and structural complexity of fucoidan hinder precise structure-function studies. To address this, we present an automated method for generating well-defined linear and branched α-fucan oligosaccharides. Our syntheses include oligosaccharides with up to 20 cis -glycosidic linkages, diverse branching patterns, and 11 sulfate monoesters. In this study, we demonstrate the utility of these oligosaccharides by (i) characterizing two endo -acting fucoidan glycoside hydrolases (GH107), (ii) utilizing them as standards for NMR studies to confirm suggested structures of algal fucoidans, and (iii) developing a fucoidan microarray. This microarray enabled the screening of the molecular specificity of four monoclonal antibodies (mAb) targeting fucoidan. It was found that mAb BAM4 has cross-reactivity to β-glucans, while mAb BAM2 has reactivity to fucoidans with 4- O -sulfate esters. Knowledge of the mAb BAM2 epitope specificity provided evidence that a globally abundant marine diatom, Thalassiosira weissflogii , synthesizes a fucoidan with structural homology to those found in brown algae. Automated glycan assembly provides access to fucoidan oligosaccharides. These oligosaccharides provide the basis for molecular level investigations into fucoidan's roles in medicine and carbon sequestration.

Marine algae drive the marine carbon cycle, converting carbon dioxide into organic material. A major component of this produced biomass is a variety of glycans. Marine α-glucans include a range of storage glycans from red and green algae, bacteria, fungi, and animals. Although these compounds are likely to account for a high amount of the carbon stored in the oceans they have not been quantified in marine samples so far. Here we present a method to extract and quantify α-glucans (and compare it with the β-glucan laminarin) in particulate organic matter from algal cultures and environmental samples using sequential physicochemical extraction and enzymes as α-glucan-specific probes. This enzymatic assay is more specific and less susceptible to side reactions than chemical hydrolysis. Using HPAEC-PAD to detect the hydrolysis products allows for a glycan quantification in particulate marine samples down to a concentration of ≈2 µg/L. We measured glucans in three cultured microalgae as well as in marine particulate organic matter from the North Sea and western North Atlantic Ocean. While the β-glucan laminarin from diatoms and brown algae is an essential component of marine carbon turnover, our results further indicate the significant contribution of starch-like α-glucans to marine particulate organic matter. Henceforth, the combination of glycan-linkage-specific enzymes and chromatographic hydrolysis product detection can provide a powerful tool in the exploration of marine glycans and their role in the global carbon cycle., (© 2022 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

$\text{L} $ -Fucose is the most widely distributed $\text{L} $-hexose in marine and terrestrial environments and presents a variety of functional roles. $\text{L} $-Fucose is the major monosaccharide in the polysaccharide fucoidan from cell walls of brown algae and is found in human milk oligosaccharides (HMOs) and the Lewis blood group system, where it is important in cell signaling and immune response stimulation. Removal of fucose from these biomolecules is catalyzed by fucosidases belonging to different carbohydrate-active enzyme (CAZy) families. Fucosidases of glycoside hydrolase family 29 (GH29) release α-$\text{L} $-fucose from non-reducing ends of glycans and display activities targeting different substrate compositions and linkage types. While several GH29 fucosidases from terrestrial environments have been characterized, much less is known about marine members of GH29 and their substrate specificities, as only four marine GH29 enzymes were previously characterized. Here, five GH29 fucosidases originating from an uncultured fucoidan-degrading marine bacterium (Paraglaciecola sp.) were cloned and produced recombinantly in Escherichia coli. All five enzymes (Fp231, Fp239, Fp240, Fp251 and Fp284) hydrolyzed the synthetic substrate CNP-α-$\text{L} $-fucose. Assayed against up to 17 fucose-containing oligosaccharides, Fp239 showed activity against the Lewis Y antigen, 2'- and 3-fucosyllactose, while Fp284 degraded 2'-fucosyllactose and Fuc(α1,6)GlcNAc. Furthermore, Fp231 displayed strict specificity against Fuc(α1,4)GlcNAc, a previously unreported specificity in GH29. Fp231 is a monomeric enzyme with pH and temperature optima at pH 5.6-6.0 and 25°C, hydrolyzing Fuc(α1,4)GlcNAc with kcat = 1.3 s-1 and Km = 660 μM. Altogether, the findings extend our knowledge about GH29 family members from the marine environment, which are so far largely unexplored., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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 strain A3 T 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 A3 T 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 genomic 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 has been 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 A3 T is an example of how the gain of gene(s) can lead to the evolutionary expansion of agar-specific polysaccharide utilization loci (PUL). C. echini A3 T 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 A3 T . The addition of α-agarases to the agarolytic enzyme repertoire might allow marine agarolytic bacteria to increase competitive abilities through metabolic versatility.

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 κ-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 β- and κ-carrageenan motifs. We show that furcellaranases degrade furcellaran into neocarratetraose-43- O -monosulfate [DA-(α1,3)-G4S-(β1,4)-DA-(α1,3)-G], and we propose a molecular model of furcellaranases and compare the active site architectures of furcellaranases, κ-carrageenases, β-agarases, and β-porphyranases. Furthermore, C. echini A3 T was shown to encode κ-carrageenases, ι- carrageenases, and members of a new class of enzymes, active only on hybrid β/κ-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, κ-carrageenan, and ι- carrageenan. IMPORTANCE Here, we report that a recently described bacterium, Colwellia echini , harbors a large number of enzymes enabling the bacterium to grow on κ-carrageenan and agar. The genes are organized in two clusters that encode enzymes for the total degradation of κ-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 β/κ-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 κ-carrageenases, β-agarases, and β-porphyranases. Furthermore, we describe a new class of enzymes distantly related to GH42 and GH160 β-galactosidases and show that this new class of enzymes is active only on hybrid β/κ-carrageenan oligosaccharides. Finally, we propose a new model for how the carrageenolytic enzyme repertoire enables C. echini to metabolize β/κ-, κ-, and ι- carrageenan., (Copyright © 2020 Christiansen et al.)

Here, we report the genome sequences of two Arthrobacter sp. strains isolated from potato and capable of degrading the toxic potato-derived glycoalkaloids (GAs) α-chaconine and α-solanine. Information from the genome sequences will provide insight into the genetic mechanism of GA degradation by these isolates., (Copyright © 2019 Hennessy et al.)

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., (© 2018 Federation of European Biochemical Societies.)

Marine microbes are a rich source of enzymes for the degradation of diverse polysaccharides. Paraglaciecola hydrolytica S66 T 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 S66 T 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 S66 T . 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.

A novel bacterial strain, A3 T , was isolated from the intestines of the sea urchin Strongylocentrotus droebachiensis collected in Øresund, Denmark. The strain was Gram-reaction-negative, rod-shaped and facultatively anaerobic, and displayed growth at 5-25 °C (optimum 20 °C), pH 7-9 (optimum at pH 7) and 1-6 % (w/v) NaCl (optimum 3 %). Furthermore, strain A3 T grew on agar, agarose, κ-carrageenan, alginate and laminarin as sole carbon source. Complete liquefaction of agar and κ-carrageenan was observed on solid plate media as a result of enzymatic activities. Major fatty acids were summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c) and C16 : 0. The respiratory quinones were determined to be ubiquinones Q-8 (92 %) and Q-7 (8 %), and polar lipids were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. The DNA G+C content was 36.9 mol%. Phylogenetical analyses based on the 16S rRNA gene showed that the bacterium was affiliated with the genus Colwellia within the Alteromonadaceae of the Gammaproteobacteria. The level of 16S rRNA gene sequence similarity between strain A3 T and its closest relatives in the genus Colwellia (C. psychrerythraea ATCC 27364 T and C. asteriadis KMD 002 T ) was 97.5 %. The average nucleotide identity between strain A3 T and other members of Colwellia was 78.6-80.5 %, and DNA-DNA hybridization prediction revealed values of less than 23 % relatedness between strain A3 T and other Colwellia species. The phenotypic, phylogenetic and genomic analyses support the hypothesis that strain A3 T represents a novel species of the genus Colwellia, for which the name Colwellia echini sp. nov. is proposed. The type strain is A3 T (=LMG 30125 T =NCIMB 15095 T ).

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).

A novel agarolytic gammaproteobacterium, ITALIC! Paraglaciecolasp. S66, was isolated from marine samples of eelgrass ( ITALIC! Zosterasp.) and sequenced. The draft genome contains a large number of enzyme-encoding genes with predicted function against several complex polysaccharides found in the cell walls of algae., (Copyright © 2016 Schultz-Johansen et al.)