Molecular identification of unsaturated uronate reductase prerequisite for alginate metabolism in Sphingomonas sp. A1

Takase, Ryuichi; Ochiai, Akihito; Mikami, Bunzo; Hashimoto, Wataru; Murata, Kousaku

doi:10.1016/j.bbapap.2010.05.010

Cited by 66 publications

(71 citation statements)

References 32 publications

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“…Dp1759 was predicted to be a DEH reductase and shared 38% amino acid identity with an NADH-dependent reductase of Sphingomonas sp. strain A1 (47). A small gene cluster including dp1703-dp1705 was involved in the conversion of KDG to pyruvate (Fig.…”

Section: Survey and Isolation Of The Communitymentioning

confidence: 99%

Defluviitalea phaphyphila sp. nov., a Novel Thermophilic Bacterium That Degrades Brown Algae

Wang

Lü

et al. 2016

Appl Environ Microbiol

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Brown algae are one of the largest groups of oceanic primary producers for CO 2 removal and carbon sinks for coastal regions. However, the mechanism for brown alga assimilation remains largely unknown in thermophilic microorganisms. In this work, a thermophilic alginolytic community was enriched from coastal sediment, from which an obligate anaerobic and thermophilic bacterial strain, designated Alg1, was isolated. Alg1 shared a 16S rRNA gene identity of 94.6% with Defluviitalea saccharophila LIND6LT2 T . Phenotypic, chemotaxonomic, and phylogenetic studies suggested strain Alg1 represented a novel species of the genus Defluviitalea, for which the name Defluviitalea phaphyphila sp. nov. is proposed. Alg1 exhibited an intriguing ability to convert carbohydrates of brown algae, including alginate, laminarin, and mannitol, to ethanol and acetic acid. Three gene clusters participating in this process were predicted to be in the genome, and candidate enzymes were successfully expressed, purified, and characterized. Six alginate lyases were demonstrated to synergistically deconstruct alginate into unsaturated monosaccharide, followed by one uronic acid reductase and two 2-keto-3-deoxy-D-gluconate (KDG) kinases to produce pyruvate. A nonclassical mannitol 1-phosphate dehydrogenase, catalyzing D-mannitol 1-phosphate to fructose 1-phosphate in the presence of NAD ؉ , and one laminarase also were disclosed. This work revealed that a thermophilic brown alga-decomposing system containing numerous novel thermophilic alginate lyases and a unique mannitol 1-phosphate dehydrogenase was adopted by the natural ethanologenic strain Alg1 during the process of evolution in hostile habitats. Brown algae (Phaeophyceae) are a large group of marine vegetation that have abundant amounts of the yellow-brown pigment fucoxanthin (1). Their huge biomass makes them one of the largest oceanic primary producers for CO 2 removal and carbon storage for coastal regions (2). Moreover, as an emerging feedstock for liquid biofuel production, recently the bioconversion of brown algae has been reported frequently (1, 3-6). They have a complex sugar composition, mainly including alginate, mannitol, and laminarin (6). Alginate is a unique structural polysaccharide in brown algae, and it is abundant in the cell wall for mechanical protection (1). It is a linear polysaccharide consisting of two uronic acids, ␣-L-guluronate (G) and ␤-D-mannuronate (M) (7). The content of alginate varies by species from 20% to 40% dry cell weight (8, 9). Mannitol is a sugar alcohol form of mannose. Laminarin, a storage polysaccharide in many brown algae, is a linear polysaccharide of ␤-1,3-linked glucose with small amounts of ␤-1,6-linkages (10). Mannitol and laminarin are mostly accumulated in the summer, and their content could reach as high as 25% and 30% in the species Laminaria hyperborean (11).The microbial degradation of brown algae involves the decomposition of the structural polysaccharides (mainly alginate and laminarin) and then the catabolism of the resulting mon...

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Section: Survey and Isolation Of The Communitymentioning

confidence: 99%

Defluviitalea phaphyphila sp. nov., a Novel Thermophilic Bacterium That Degrades Brown Algae

Wang

Lü

et al. 2016

Appl Environ Microbiol

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“…For instance, Laminaria hyperborean and Saccharina latissima contain laminarin at up to 32% of dry weight (6), and 84% of total carbohydrates are laminarin in Fucus vesiculosus (7). Unlike other marine polysaccharides, such as alginate and agar, from which monomeric sugars are not fermentable by terrestrial microorganisms (8,9), the final hydrolysis product of laminarin is glucose, which can be directly fermented by microorganisms. Therefore, laminarin is an ideal substrate for the production of biofuels and biologically based products.…”

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confidence: 99%

A Novel Glycoside Hydrolase Family 5 β-1,3-1,6-Endoglucanase from Saccharophagus degradans 2-40 ^T and Its Transglycosylase Activity

Wang

Kim

Seo

et al. 2016

Appl Environ Microbiol

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In this study, we characterized Gly5M, originating from a marine bacterium, as a novel ␤-1,3-1,6-endoglucanase in glycoside hydrolase family 5 (GH5) in the Carbohydrate-Active enZyme database. The gly5M gene encodes Gly5M, a newly characterized enzyme from GH5 subfamily 47 (GH5_47) in Saccharophagus degradans 2-40 T . The gly5M gene was cloned and overexpressed in Escherichia coli. Through analysis of the enzymatic reaction products by thin-layer chromatography, high-performance liquid chromatography, and matrix-assisted laser desorption ionization-tandem time of flight mass spectrometry, Gly5M was identified as a novel ␤-1,3-endoglucanase (EC 3.2.1.39) and bacterial ␤-1,6-glucanase (EC 3.2.1.75) in GH5. The ␤-1,3-endoglucanase and ␤-1,6-endoglucanase activities were detected by using laminarin (a ␤-1,3-glucan with ␤-1,6-glycosidic linkages derived from brown macroalgae) and pustulan (a ␤-1,6-glucan derived from fungal cell walls) as the substrates, respectively. This enzyme also showed transglycosylase activity toward ␤-1,3-oligosaccharides when laminarioligosaccharides were used as the substrates. Since laminarin is the major form of glucan storage in brown macroalgae, Gly5M could be used to produce glucose and laminarioligosaccharides, using brown macroalgae, for industrial purposes. IMPORTANCEIn this study, we have discovered a novel ␤-1,3-1,6-endoglucanase with a unique transglycosylase activity, namely, Gly5M, from a marine bacterium, Saccharophagus degradans 2-40 T . Gly5M was identified as the newly found ␤-1,3-endoglucanase and bacterial ␤-1,6-glucanase in GH5. Gly5M is capable of cleaving glycosidic linkages of both ␤-1,3-glucans and ␤-1,6-glucans. Gly5M also possesses a transglycosylase activity toward ␤-1,3-oligosacchrides. Due to the broad specificity of Gly5M, this enzyme can be used to produce glucose or high-value ␤-1,3-and/or ␤-1,6-oligosaccharides. Marine macroalgae have been considered as potential sources of carbohydrates for the production of biofuels and biologically based products (1). Among the marine macroalgae, over 70 million tons of brown macroalgae are produced annually (2). The major carbon storage compounds of brown algae are alginate, laminarin, fucoidan, and mannitol (3). Laminarin is a linear polysaccharide composed of D-glucose with ␤-1,3-glycosidic linkages in the main backbone and degrees of polymerization (DPs) of 20 to 25 (4), with some 6-O-branching from D-glucose in the main chain and some ␤-1,6-glycosidic linkages as branches; the ␤-1,3-glucan/␤-1,6-glucan ratio is approximately 3:1, depending on the species of brown macroalgae (5). Among brown macroalgae, the main sources of laminarin are Laminaria and Saccharina spp. For instance, Laminaria hyperborean and Saccharina latissima contain laminarin at up to 32% of dry weight (6), and 84% of total carbohydrates are laminarin in Fucus vesiculosus (7). Unlike other marine polysaccharides, such as alginate and agar, from which monomeric sugars are not fermentable by terrestrial microorganisms (8, 9), the final hydrolys...

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“…3 DEH is reduced to 2-keto-3-deoxy-D-gluconate by NADPH-dependent reductase, A1-R (ID: SPH3227), and 2-keto-3-deoxy-D-gluconate is catabolized to glyceraldehyde 3-phosphate and pyruvate through subsequent reactions by 2-keto-3-deoxy-D-gluconate kinase and aldolase, respectively ( Fig. 1) (43).…”

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“…Proteins on the gel were stained with Coomassie Brilliant Blue R-250. DEH was prepared from sodium alginate by using exotype alginate lyase, Atu3025 (63), and purified using Centriprep centrifugal filter and Bio-Gel P-2 size-exclusion column as described previously (43). Thiobarbituric acid method (64) was used for determination of DEH concentration.…”

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confidence: 99%

Structure-based Conversion of the Coenzyme Requirement of a Short-chain Dehydrogenase/Reductase Involved in Bacterial Alginate Metabolism

Takase¹,

Mikami

Kawai³

et al. 2014

Journal of Biological Chemistry

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Background: NADPH-dependent ␣-keto acid reductase, belonging to the short-chain dehydrogenase/reductase family, is involved in bacterial alginate metabolism. Results: A novel NADH-dependent ␣-keto acid reductase was identified, and its tertiary structure was determined by x-ray crystallography. Conclusion: Two short and long loops are structural determinants for coenzyme specificity. Significance: A method for structure-based conversion of a coenzyme requirement was established.

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Molecular identification of unsaturated uronate reductase prerequisite for alginate metabolism in Sphingomonas sp. A1

Cited by 66 publications

References 32 publications

Defluviitalea phaphyphila sp. nov., a Novel Thermophilic Bacterium That Degrades Brown Algae

Defluviitalea phaphyphila sp. nov., a Novel Thermophilic Bacterium That Degrades Brown Algae

A Novel Glycoside Hydrolase Family 5 β-1,3-1,6-Endoglucanase from Saccharophagus degradans 2-40 ^T and Its Transglycosylase Activity

Structure-based Conversion of the Coenzyme Requirement of a Short-chain Dehydrogenase/Reductase Involved in Bacterial Alginate Metabolism

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Molecular identification of unsaturated uronate reductase prerequisite for alginate metabolism in Sphingomonas sp. A1

Cited by 66 publications

References 32 publications

Defluviitalea phaphyphila sp. nov., a Novel Thermophilic Bacterium That Degrades Brown Algae

Defluviitalea phaphyphila sp. nov., a Novel Thermophilic Bacterium That Degrades Brown Algae

A Novel Glycoside Hydrolase Family 5 β-1,3-1,6-Endoglucanase from Saccharophagus degradans 2-40 T and Its Transglycosylase Activity

Structure-based Conversion of the Coenzyme Requirement of a Short-chain Dehydrogenase/Reductase Involved in Bacterial Alginate Metabolism

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A Novel Glycoside Hydrolase Family 5 β-1,3-1,6-Endoglucanase from Saccharophagus degradans 2-40 ^T and Its Transglycosylase Activity