Synergism of Glycoside Hydrolase Secretomes from Two Thermophilic Bacteria Cocultivated on Lignocellulose

Zhang, Kundi; Chen, Xiaohua; Schwarz, Wolfgang H.; Li, Fu‐Li

doi:10.1128/aem.00295-14

Cited by 25 publications

(31 citation statements)

References 60 publications

(59 reference statements)

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“…All chromatography procedures were performed on an ÄKTA purifier system (GE Healthcare). The purified proteins were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis (36). All putative brown alga degradation-related genes except dp0100 were selected for heterogeneous expression by following the method of Zhang et al (36).…”

Section: Methodsmentioning

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: Methodsmentioning

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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“…The cross-linking and cleavage products were determined by SDS-PAGE. The bands were cut from the gel, digested in gel, and analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) with a Surveyor Plus ion trap mass spectrometer (LTQ XL; Thermo Fisher, San Jose, CA) equipped with a nanospray source (39).…”

Section: Methodsmentioning

confidence: 99%

Distinct Roles for Carbohydrate-Binding Modules of Glycoside Hydrolase 10 (GH10) and GH11 Xylanases from Caldicellulosiruptor sp. Strain F32 in Thermostability and Catalytic Efficiency

Meng

Chen

et al. 2015

Appl Environ Microbiol

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cXylanases are crucial for lignocellulosic biomass deconstruction and generally contain noncatalytic carbohydrate-binding modules (CBMs) accessing recalcitrant polymers. Understanding how multimodular enzymes assemble can benefit protein engineering by aiming at accommodating various environmental conditions. Two multimodular xylanases, XynA and XynB, which belong to glycoside hydrolase families 11 (GH11) and GH10, respectively, have been identified from Caldicellulosiruptor sp. strain F32. In this study, both xylanases and their truncated mutants were overexpressed in Escherichia coli, purified, and characterized. GH11 XynATM1 lacking CBM exhibited a considerable improvement in specific activity (215.8 U nmol ؊1 versus 94.7 U nmol ؊1 ) and thermal stability (half-life of 48 h versus 5.5 h at 75°C) compared with those of XynA. However, GH10 XynB showed higher enzyme activity and thermostability than its truncated mutant without CBM. Site-directed mutagenesis of N-terminal amino acids resulted in a mutant, XynATM1-M, with 50% residual activity improvement at 75°C for 48 h, revealing that the disordered region influenced protein thermostability negatively. The thermal stability of both xylanases and their truncated mutants were consistent with their melting temperature (T m ), which was determined by using differential scanning calorimetry. Through homology modeling and cross-linking analysis, we demonstrated that for XynB, the resistance against thermoinactivation generally was enhanced through improving both domain properties and interdomain interactions, whereas for XynA, no interdomain interactions were observed. Optimized intramolecular interactions can accelerate thermostability, which provided microbes a powerful evolutionary strategy to assemble catalysts that are adapted to various ecological conditions. X ylanases (endo-1,4-␤-xylanase; EC 3.2.1.8) hydrolyze the ␤-1,4 bond in the xylan backbone of hemicellulose, which yields short xylooligosaccharides or xylose. They are distributed in several glycoside hydrolase families (GH), such as GH5, GH7, GH8, GH10, GH11, and GH43, and most of them belong to GH10 and GH11 according to the CAZy (Carbohydrate Active Enzymes) database (1). Xylanase families differ in their physicochemical properties (molecular mass and isoelectric point [pI]), three-dimensional structures, and catalytic mechanisms (2). Enzymes from GH family 11 have a relatively low molecular weight and a high pI, and they possess a ␤-jelly roll secondary structure, a double displacement catalytic mechanism, and two glutamates acting as catalytic residues (3). In contrast, members of GH10 typically have a high molecular mass and a low pI and display an (␣/␤) 8 barrel fold. Xylanases have been found in many organisms, such as bacteria, fungi, algae, and protozoa (4). Xylanases from fungi or mesophilic bacteria generally share a low optimum temperature and poor thermal stability (5). Thermostable xylanases have been characterized from some thermophilic microorganisms, such as Clostridium thermocellum (6...

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“…(Supplementary Table S1). All of our MAGs meet the medium-quality requirement (> 50% completeness and < 10% contamination) of the MIMAG standard [29] (with one exception: ZC4RG07 had 10.87% contamination); 34 MAGs meet the high-quality requirement (> 90% completeness and < 5% contamination). The average number of contigs in these MAGs is 363.75, with a minimum of 15 and a maximum of 1,655.…”

Section: Mags Recovered From Compostingmentioning

confidence: 88%

Temporal dynamics and ecophysiology of thermophilic composting analyzed through metagenome-assembled genomes

Setúbal¹,

Braga²,

Pereira³

et al. 2020

Preprint

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Background: Thermophilic composting is a semi-engineered process carried out by diverse microbial communities. Composting is an environment friendly way of degrading biomass; its study may help uncover important biomass-degrading organisms and key enzymes. DNA sequence-based previous studies have presented a general description of the microbial-molecular features of composting, but they have lacked more specific information on the key organisms that are active during the process and their genomes. Methods: We present an analysis of metagenome-assembled genomes (MAGs) obtained from time-series samples of a thermophilic composting process in the São Paulo Zoological Park (Brazil). Our results are based on a careful analysis of MAG gene content and on metabolic modeling of their interactions. Results: We recovered 60 MAGs from sequencing datasets from two separate composting cells. Phylogenetic analysis shows that 47 of these MAGs represent novel taxa at the genus or higher levels. We have analyzed the gene repertoire of these MAGs in terms of lignocellulose degradation, secondary metabolite production, antibiotic resistance genes, denitrification genes, sulfur metabolism, hydrogen metabolism, and oxygen metabolism. For one of the composting cells we also had metatranscriptome data, which allowed a deeper analysis of 49 MAGs. This analysis showed the presence of three distinct clusters of MAGs with varying activity during the 99-day composting process. The interaction model pointed to Sphaerobacter thermophilus and Thermobispora bispora as key players in the process, as well as other bacteria that are novel. Our results also show the importance of coadjuvant bacteria and of microbial functions related to efficient bioenergetic processes during biomass conversion, such as N2O reduction and hydrogenases. A novel acidobacteria MAG encodes N2O reductase hallmark genes (nosZD). Strong metabolic dependencies predicted between MAGs revealed that cross-feeding in composting can be determined by complementary functions found in the genomes of producers and consumers, supporting the Black Queen hypothesis for co-evolutionary interactions. Conclusions: This study reveals for the first time the key bacterial players in thermophilic composting and provides a model of their dynamic metabolic interactions. These findings pave the way for more rational composting procedures and provide information that could help the development of novel biomass-degrading technologies.

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Synergism of Glycoside Hydrolase Secretomes from Two Thermophilic Bacteria Cocultivated on Lignocellulose

Cited by 25 publications

References 60 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

Distinct Roles for Carbohydrate-Binding Modules of Glycoside Hydrolase 10 (GH10) and GH11 Xylanases from Caldicellulosiruptor sp. Strain F32 in Thermostability and Catalytic Efficiency

Temporal dynamics and ecophysiology of thermophilic composting analyzed through metagenome-assembled genomes

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