Xylan Utilization Research Articles

A synergistic fermentation system of probiotics with low-cost and high butyric acid production: Lactiplantibacillus plantarum and Clostridium tyrobutyricum

Butyric acid plays a significant role in maintaining body health, and thus the research on cost-effective and efficient biological conversion strategies for butyric acid production holds great importance. This study utilized the mutual interactions between prebiotics and multiple probiotic strains to target increasing the butyric acid yield in a co-fermentation system. Briefly, xylan, an inexpensive biomass material, was used as a prebiotic to screen lactic acid bacteria that capable of being promoted for proliferation and metabolism in its presence. Subsequently, the interaction between the screened lactic acid bacteria and a butyric acid-producing strain (Clostridium tyrobutyricum) was utilized to enhance butyric acid production. The results showed that xylan significantly improved the proliferation and lactic acid metabolizing ability of Lactiplantibacillus plantarum C0502. Whole-genome analysis revealed that L. plantarum carries at least four distinct enzyme genes related to the hydrolysis and utilization of xylan. Co-cultivation of L. plantarum with C. tyrobutyricum demonstrated that the C. tyrobutyricum was able to effectively convert lactic and acetic acids into butyric acid. Furthermore, the addition of xylan into this co-culture system yielded a remarkable butyric acid production of up to 5.01 ± 0.04 g/L, which is 4.58 times higher than the yield obtained from the monoculture of C. tyrobutyricum in reinforced Clostridium medium. The results of this study suggested that the co-culture system of xylan, L. plantarum, and C. tyrobutyricum has the potential to significantly enhance butyric acid production while markedly reduce the production cost. This has immense prospects for practical applications.

Verrucomicrobia of the Family Chthoniobacteraceae Participate in Xylan Degradation in Boreal Peat Soils.

The phylum Verrucomicrobiota is one of the main groups of soil prokaryotes, which remains poorly represented by cultivated organisms. The major recognized role of Verrucomicrobiota in soils is the degradation of plant-derived organic matter. These bacteria are particularly abundant in peatlands, where xylan-type hemicelluloses represent one of the most actively decomposed peat constituents. The aim of this work was to characterize the microorganisms capable of hydrolyzing xylan under the anoxic conditions typical of peatland soils. The laboratory incubation of peat samples with xylan resulted in the pronounced enrichment of several phylotypes affiliated with the Verrucomicrobiota, Firmicutes, and Alphaproteobacteria. Sequencing of the metagenome of the enrichment culture allowed us to recover high-quality metagenome-assembled genomes (MAGs) assigned to the genera Caproiciproducens, Clostridium, Bacillus (Firmicutes), and Rhizomicrobium (Alphaproteobacteria), Cellulomonas (Actinobacteriota) and the uncultured genus-level lineage of the family Chthoniobacteraceae (Verrucomicrobiota). The latter bacterium, designated "Candidatus Chthoniomicrobium xylanophilum" SH-KS-3, dominated in the metagenome and its MAG was assembled as a complete closed chromosome. An analysis of the SH-KS-3 genome revealed potential endo-1,4-beta-xylanases, as well as xylan beta-1,4-xylosidases and other enzymes involved in xylan utilization. A genome analysis revealed the absence of aerobic respiration and predicted chemoheterotrophic metabolism with the capacity to utilize various carbohydrates, including cellulose, and to perform fermentation or nitrate reduction. An analysis of other MAGs suggested that Clostridium and Rhizomicrobium could play the role of primary xylan degraders while other community members probably took advantage of the availability of xylo-oligosaccharides and xylose or utilized low molecular weight organics.

Efficient conversion of hemicellulose into high-value product and electric power by enzyme-engineered bacterial consortia

As an abundant agricultural and forestry biomass resource, hemicelluloses are hard to be effectively degraded and utilized by microorganisms due to the constraints of membrane and metabolic regulations. Herein, we report a synthetic extracellular metabolic pathway with hemicellulose-degrading-enzymes controllably displayed on Escherichia coli surface as engineered bacterial consortia members for efficient utilization of xylan, the most abundant component in hemicellulose. Further, we develop a hemicellulose/O2 microbial fuel cell (MFC) configuring of enzyme-engineered bacterial consortia based bioanode and bacterial-displayed laccase based biocathode. The optimized MFC exhibited an open-circuit voltage of 0.71 V and a maximum power density (Pmax) of 174.33 ± 4.56 µW cm−2. Meanwhile, 46.6% (w/w) α-ketoglutarate was produced in this hemicellulose fed-MFC. Besides, the MFC retained over 95% of the Pmax during 6 days’ operation. Therefore, this work establishes an effective and sustainable one-pot process for catalyzing renewable biomass into high-value products and electricity in an environmentally-friendly way.

Intracellular removal of acetyl, feruloyl and p-coumaroyl decorations on arabinoxylo-oligosaccharides imported from lignocellulosic biomass degradation by Ruminiclostridium cellulolyticum

BackgroundXylans are polysaccharides that are naturally abundant in agricultural by-products, such as cereal brans and straws. Microbial degradation of arabinoxylan is facilitated by extracellular esterases that remove acetyl, feruloyl, and p-coumaroyl decorations. The bacterium Ruminiclostridium cellulolyticum possesses the Xua (xylan utilization associated) system, which is responsible for importing and intracellularly degrading arabinoxylodextrins. This system includes an arabinoxylodextrins importer, four intracellular glycosyl hydrolases, and two intracellular esterases, XuaH and XuaJ which are encoded at the end of the gene cluster.ResultsGenetic studies demonstrate that the genes xuaH and xuaJ are part of the xua operon, which covers xuaABCDD’EFGHIJ. This operon forms a functional unit regulated by the two-component system XuaSR. The esterases encoded at the end of the cluster have been further characterized: XuaJ is an acetyl esterase active on model substrates, while XuaH is a xylan feruloyl- and p-coumaryl-esterase. This latter is active on oligosaccharides derived from wheat bran and wheat straw. Modelling studies indicate that XuaH has the potential to interact with arabinoxylobiose acylated with mono- or diferulate. The intracellular esterases XuaH and XuaJ are believed to allow the cell to fully utilize the complex acylated arabinoxylo-dextrins imported into the cytoplasm during growth on wheat bran or straw.ConclusionsThis study reports for the first time that a cytosolic feruloyl esterase is part of an intracellular arabinoxylo-dextrin import and degradation system, completing its cytosolic enzymatic arsenal. This system represents a new pathway for processing highly-decorated arabinoxylo-dextrins, which could provide a competitive advantage to the cell and may have interesting biotechnological applications.

Marine Bacteroidetes enzymatically digest xylans from terrestrial plants.

Marine Bacteroidetes that degrade polysaccharides contribute to carbon cycling in the ocean. Organic matter, including glycans from terrestrial plants, might enter the oceans through rivers. Whether marine bacteria degrade structurally related glycans from diverse sources including terrestrial plants and marine algae was previously unknown. We show that the marine bacterium Flavimarina sp. Hel_I_48 encodes two polysaccharide utilization loci (PULs) which degrade xylans from terrestrial plants and marine algae. Biochemical experiments revealed activity and specificity of the encoded xylanases and associated enzymes of these PULs. Proteomics indicated that these genomic regions respond to glucuronoxylans and arabinoxylans. Substrate specificities of key enzymes suggest dedicated metabolic pathways for xylan utilization. Some of the xylanases were active on different xylans with the conserved β-1,4-linked xylose main chain. Enzyme activity was consistent with growth curves showing Flavimarina sp. Hel_I_48 uses structurally different xylans. The observed abundance of related xylan-degrading enzyme repertoires in genomes of other marine Bacteroidetes indicates similar activities are common in the ocean. The here presented data show that certain marine bacteria are genetically and biochemically variable enough to access parts of structurally diverse xylans from terrestrial plants as well as from marine algal sources.

Impact of fermentable fiber, xylo-oligosaccharides and xylanase on laying hen productive performance and nutrient utilization

This study evaluated the impact of feeding xylo-oligosaccharides (XOS), fermentable fiber in the form of wheat bran (WB), and xylanase (XYL) on laying hen productive performance and nutrient digestibility. The hypothesis was that the WB would provide the microbiota in the hindgut with fermentable dietary xylan, and the XOS and XYL would further upregulate xylan fermentation pathways, resulting in improved nutrient utilization. Isa Brown hens (n=96) were obtained at 39 wk of age. They were fed 12 dietary treatments, 8 hens per treatment, for 56 d. A commercial laying hen ration was fed, and for half of the treatments 10% of this ration was directly replaced with WB. The diets were then supplemented with either 1) no supplements; 2) XOS 50 g/t; 3) XOS 2000 g/t; 4) XYL (16,000 BXU/kg); 5) XYL+XOS 50 g/t, or 6) XYL+XOS 2,000 g/t. Hen performance and egg quality were measured every 14 d. On d56, ileum digesta samples were collected for determination of starch, nonstarch polysaccharide (NSP), XOS, protein, energy, and starch digestibility. Ceca digesta samples were also collected for analysis of XOS, short chain fatty acid (SCFA), xylanase and cellulase activity and microbial counts. Feeding 2,000 g/t XOS increased ileal protein digestibility. Combined 2,000 g/t XOS and XYL increased cecal Bifidobacteria concentration. This combination also increased cecal xylanase activity in birds fed the control diet. Cecal cellulase activity was improved by feeding WB, XYL, and 2,000 g/t XOS. XYL increased cecal lactate production. Feeding 2,000 g/t XOS with WB increased insoluble NSP degradability and shell breaking strength at d56. In summary, supplementing laying hen diets with fermentable fiber, XYL and XOS increases utilization of dietary xylan, improving nutrient utilization, performance, and gastrointestinal health.

Genetic Determinants of Xylan Utilization in Humisphaera borealis M1803T, a Planctomycete of the Class Phycisphaerae

—Planctomycetes of the class Phycisphaerae are aerobic and anaerobic heterotrophic bacteria that colonize a wide range of marine and terrestrial habitats. Their functional roles in the environment, however, are still poorly understood. Humisphaera borealis M1803T is one of the very few characterized planctomycetes of this class. It is also the first described representative of the previously uncultured group WD2101, which is commonly detected in soils and peatlands. This work analyzed the genetic determinants that define the ability of Humisphaera borealis M1803T to grow on xylan, one of the plant cell wall polymers. The whole genome sequence analysis of this planctomycete resulted in identification of five genes encoding the proteins homologous to previously described endo-β-xylanases. For two of these proteins, evolutionarily closer experimentally characterized homologs with other substrate specificities were found. In a member of the GH10 family of glycoside hydrolases, the active center of the enzyme was destroyed. We consider two proteins from GH62 and GH141 families as the most likely candidates for the role of β-xylanase responsible for xylan utilization. Phylogenetic analysis of proteins of GH10, GH62, and GH141 families was carried out. The role of lateral transfers in the evolution of the genes for glycoside hydrolases and their close homologs is discussed.

Surface tethered xylosidase activity improved xylan conversion in engineered strains of Saccharomyces cerevisiae

AbstractBACKGROUNDSecond‐generation biofuel production strategies require utilization of the largest possible fraction of the available sugars in renewable biomass. Xylose can make up as much as 35% of plant dry cell weight (DCW), primarily in the form of the hemicellulose polymer xylan. Xylo‐oligosaccharides (XOS) are breakdown products of xylan released during pre‐treatment and hydrolysis that may inhibit the fermentation process. To enable growth on xylan and the removal of inhibitory XOS, CRISPR‐Cas9 was used to confer xylanase and GH43 xylosidase activity to xylose‐assimilating Saccharomyces cerevisiae strains. Xylosidase activity was engineered to be cell free or tethered to the yeast cell surface.RESULTSGene‐integration and expression of both xylanolytic enzyme‐encoding genes was successful. Secretion yielded higher overall xylosidase activity when strains were cultivated on glucose, but cell‐associated activity was higher when strains were cultivated in the presence of xylose. Growth trials on both xylan and XOS showed that a combination of both enzymes, along with cell‐associated xylosidase production, resulted in the best growth on both substrates. The increased growth on xylan also translated to substantial improvements in ethanol production from polymeric xylan as sole carbon source.CONCLUSIONIncreased enzyme production, growth capabilities, and hemicellulosic substrate conversion due to cell‐tethered activity over secreted activity were shown for the first time. The use of a GH43 xylosidase to help avoid problematic transglycosylation for this purpose is also novel. The development of S. cerevisiae strains capable of xylan utilization and fermentation brings the industry a step closer to the ideal of utilizing the full range of sugars in biomass feedstocks for large scale ethanol production. © 2022 Society of Chemical Industry (SCI).

Xylan alleviates dietary fiber deprivation-induced dysbiosis by selectively promoting Bifidobacterium pseudocatenulatum in pigs

BackgroundLow dietary fiber intake has been shown to disturb the gut microbiome community, damage the mucus barrier, and promote pathogen susceptibility. However, little is known about the temporal response of the gut microbiome to dietary fiber deprivation and the recovery induced by dietary fiber inclusion in pigs.ObjectiveIn the present study, temporal responses of ileal and fecal microbiota to dietary fiber deprivation were profiled using an ileum cannulated growing pig model. In addition, the potential of dietary-resistant starch, β-glucan, and xylan to alleviate gut dysbiosis throughout the gastrointestinal tract, as well as its possible mechanisms were investigated.MethodsSix cannulated growing pigs were fed a fiber deprivation diet for 35 days. Ileal digesta and feces were collected at days 0, 7, 21, and 35 for 16S rRNA sequencing and short-chain fatty acid (SCFA) determination. Another twenty-four healthy growing pigs were assigned to one of four dietary treatments including (1) fiber-free diet, (2) resistant starch diet, (3) β-glucan diet, and (4) xylan diet. These twenty-four pigs were fed a corresponding diet for 35 days and slaughtered. Gut microbiome and SCFA concentration were profiled along the gastrointestinal tract.ResultsDietary fiber deprivation-induced consistent microbiota extinction, mainly Bifidobacterium and Lactobacillus, and decreased SCFA concentrations in both ileum and feces. The community structure partially recovered at day 35 compared with baseline while SCFA concentrations remained low. Xylan supplementation alleviated gut dysbiosis by selectively promoting Bifidobacterium pseudocatenulatum within the large intestine. SCFA concentration increased significantly after xylan supplementation and exhibited a positive association with B. pseudocatenulatum abundance. An elevated abundance of xylan degradation-related enzyme genes was also observed in the gut microbiome after xylan supplementation. In vitro growth assay further verified the xylan utilization capacity of B. pseudocatenulatum.ConclusionsDietary fiber deprivation could induce probiotic extinction and loss of the SCFA production while potential pathogen was promoted. Xylan intervention could partially restore dietary fiber deprivation-induced gut dysbiosis through selectively promoting B. pseudocatenulatum and therefore normalizing the gut environment. These findings collectively provide evidence that dietary fiber-driven microbiota metabolism bridges the interplay between microbiome and gut health.DsGU9NsKuPME7_kAmChQecVideo abstract

Bacterial xylan utilization regulons: systems for coupling depolymerization of methylglucuronoxylans with assimilation and metabolism.

Bioconversion of lignocellulosic resources offers an economically promising path to renewable energy. Technological challenges to achieving bioconversion include the development of cost-effective processes that render the cellulose and hemicellulose components of these resources to fermentable hexoses and pentoses. Natural bioprocessing of the hemicellulose fraction of lignocellulosic biomass requires depolymerization of methylglucuronoxylans. This requires secretion of endoxylanases that release xylooligosaccharides and aldouronates. Physiological, biochemical, and genetic studies with selected bacteria support a process in which a cell-anchored multimodular GH10 endoxylanase catalyzes release of the hydrolysis products, aldotetrauronate, xylotriose, and xylobiose, which are directly assimilated and metabolized. Gene clusters encoding intracellular enzymes, including α-glucuronidase, endoxylanase, β-xylosidase, ABC transporter proteins, and transcriptional regulators, are coordinately responsive to substrate induction or repression. The rapid rates of glucuronoxylan utilization and microbial growth, along with the absence of detectable products of depolymerization in the medium, indicate that assimilation and depolymerization are coupled processes. Genomic comparisons provide evidence that such systems occur in xylanolytic species in several genera, including Clostridium, Geobacillus, Paenibacillus, and Thermotoga. These systems offer promise, either in their native configurations or through gene transfer to other organisms, to develop biocatalysts for efficient production of fuels and chemicals from the hemicellulose fractions of lignocellulosic resources.

Endo-xylanases from Cohnella sp. AR92 aimed at xylan and arabinoxylan conversion into value-added products.

The genus Cohnella belongs to a group of Gram-positive endospore-forming bacteria within the Paenibacillaceae family. Although most species were described as xylanolytic bacteria, the literature still lacks some key information regarding their repertoire of xylan-degrading enzymes. The whole genome sequence of an isolated xylan-degrading bacterium Cohnella sp. strain AR92 was found to contain five genes encoding putative endo-1,4-β-xylanases, of which four were cloned, expressed, and characterized to better understand the contribution of the individual endo-xylanases to the overall xylanolytic properties of strain AR92. Three of the enzymes, CoXyn10A, CoXyn10C, and CoXyn11A, were shown to be effective at hydrolyzing xylans-derived from agro-industrial, producing oligosaccharides with substrate conversion values of 32.5%, 24.7%, and 10.6%, respectively, using sugarcane bagasse glucuronoarabinoxylan and of 29.9%, 19.1%, and 8.0%, respectively, using wheat bran-derived arabinoxylan. The main reaction products from GH10 enzymes were xylobiose and xylotriose, whereas CoXyn11A produced mostly xylooligosaccharides (XOS) with 2 to 5 units of xylose, often substituted, resulting in potentially prebiotic arabinoxylooligosaccharides (AXOS). The endo-xylanases assay displayed operational features (temperature optima from 49.9 to 50.4°C and pH optima from 6.01 to 6.31) fitting simultaneous xylan utilization. Homology modeling confirmed the typical folds of the GH10 and GH11 enzymes, substrate docking studies allowed the prediction of subsites (- 2 to + 1 in GH10 and - 3 to + 1 in GH11) and identification of residues involved in ligand interactions, supporting the experimental data. Overall, the Cohnella sp. AR92 endo-xylanases presented significant potential for enzymatic conversion of agro-industrial by-products into high-value products.Key points• Cohnella sp. AR92 genome encoded five potential endo-xylanases.• Cohnella sp. AR92 enzymes produced xylooligosaccharides from xylan, with high yields.• GH10 enzymes from Cohnella sp. AR92 are responsible for the production of X2 and X3 oligosaccharides.• GH11 from Cohnella sp. AR92 contributes to the overall xylan degradation by producing substituted oligosaccharides.

CAZyme prediction in ascomycetous yeast genomes guides discovery of novel xylanolytic species with diverse capacities for hemicellulose hydrolysis

BackgroundAscomycetous yeasts from the kingdom fungi inhabit every biome in nature. While filamentous fungi have been studied extensively regarding their enzymatic degradation of the complex polymers comprising lignocellulose, yeasts have been largely overlooked. As yeasts are key organisms used in industry, understanding their enzymatic strategies for biomass conversion is an important factor in developing new and more efficient cell factories. The aim of this study was to identify polysaccharide-degrading yeasts by mining CAZymes in 332 yeast genomes from the phylum Ascomycota. Selected CAZyme-rich yeasts were then characterized in more detail through growth and enzymatic activity assays.ResultsThe CAZyme analysis revealed a large spread in the number of CAZyme-encoding genes in the ascomycetous yeast genomes. We identified a total of 217 predicted CAZyme families, including several CAZymes likely involved in degradation of plant polysaccharides. Growth characterization of 40 CAZyme-rich yeasts revealed no cellulolytic yeasts, but several species from the Trichomonascaceae and CUG-Ser1 clades were able to grow on xylan, mixed-linkage β-glucan and xyloglucan. Blastobotrys mokoenaii, Sugiyamaella lignohabitans, Spencermartinsiella europaea and several Scheffersomyces species displayed superior growth on xylan and well as high enzymatic activities. These species possess genes for several putative xylanolytic enzymes, including ones from the well-studied xylanase-containing glycoside hydrolase families GH10 and GH30, which appear to be attached to the cell surface. B. mokoenaii was the only species containing a GH11 xylanase, which was shown to be secreted. Surprisingly, no known xylanases were predicted in the xylanolytic species Wickerhamomyces canadensis, suggesting that this yeast possesses novel xylanases. In addition, by examining non-sequenced yeasts closely related to the xylanolytic yeasts, we were able to identify novel species with high xylanolytic capacities.ConclusionsOur approach of combining high-throughput bioinformatic CAZyme-prediction with growth and enzyme characterization proved to be a powerful pipeline for discovery of novel xylan-degrading yeasts and enzymes. The identified yeasts display diverse profiles in terms of growth, enzymatic activities and xylan substrate preferences, pointing towards different strategies for degradation and utilization of xylan. Together, the results provide novel insights into how yeast degrade xylan, which can be used to improve cell factory design and industrial bioconversion processes.

The Role of Clt1-Regulated Xylan Metabolism in Melanin and Toxin Formation for the Pathogenicity of Curvularia lunata in Maize.

We previously reported that the BTB (brica-brac, tramtrack, and broad) domain-containing protein Clt1 regulates melanin and toxin synthesis, conidiation, and pathogenicity in Curvularia lunata, but the interacting proteins and regulative mechanism of Clt1 are unclear. In this research, we identified two proteins, which respectively correspond to xylanase (Clxyn24) and acetyl xylan esterase (Claxe43) from C. lunata, that were regulated by Clt1. Yeast two-hybrid (Y2H) and bimolecular fluorescence complementation assays were conducted to verify the interaction of Clt1 with full-length Clxyn24 and Claxe43. Furthermore, the Y2H assay revealed that Clt1 physically interacted with Clxyn24 and Claxe43 through its BTB domain to degrade xylan, which was used as a carbon source for C. lunata growth. The utilization of xylan provides acetyl-CoA for the synthesis of melanin and toxin as well as energy and other intermediate metabolites for conidiation. Furthermore, transcriptome analysis revealed that PKS18 and its 13 flanking genes found clustered in a region spanning 57.89 kb on scaffold 9 of the C. lunata CX-3 genome were down-regulated in toxin production-deficient mutant T806, and this cluster is possibly responsible for toxin biosynthesis of C. lunata.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Cloning and Heterologous Expression of a Novel Xylanase Gene TAX1 from Trichoderma atroviride and Its Application in the Deconstruction of Corn Stover.

Xylanase plays a vital role in the efficient utilization of xylan, which accounts for up to 30% of plant dry matter. However, the production cost of xylanase remains high, and the enzymatic characteristics of xylanases of most microorganisms are not suitable for industrial production. Therefore, it is of great significance to discover and develop new and efficient xylanases. In this study, the xylanase gene TAX1 (672 bp cDNA) was cloned from Trichoderma atroviride 3.3013 and expressed in Pichia pastoris. The TAX1 gene encoded a 223-amino acid protein (TAX1) with a molecular weight of 24.2 kDa which showed high similarity to glycoside hydrolase family 11. Enzyme activity assay verified that the recombinant xylanase TAX1 had optimal activity (215.3 IU/mL) at 50°C and pH 6.0. Stable working conditions were measured as pH 4.0-7.0 and 40-60°C. By adding Zn2+, the relative enzymatic activity of recombinant TAX1 was enhanced by 26%. The recombinant xylanase showed high activity toward birchwood xylan and corn stover. The Km and Kcat for xylan and corn stover were 0.36 mg/mL and 0.204 S-1 and 0.48 mg/mL and 0.149 S-1, respectively. The enzymatic activity of the TAX1 produced by P. pastoris was about 2.4-4 times higher that directly isolated from T. atroviride, so engineered P. pastoris for xylanase production could be an ideal candidate for industrial enzyme production.

Reclassification of the genus Natronolimnobius: proposal of two new genera, Natronolimnohabitans gen. nov. to accommodate Natronolimnobius innermongolicus and Natrarchaeobaculum gen. nov. to accommodate Natronolimnobius aegyptiacus and Natronolimnobius sulfurireducens.

The genus Natronolimnobius, currently including four species, is a member of the order Natrialbales, class Halobacteria, and consists of obligately alkaliphilic and extremely halophilic members found exclusively in highly alkaline hypersaline soda lakes. The species were classified into this genus mostly based on phylogenetic analysis of the 16S rRNA gene. However, a more advanced phylogenomic reconstruction based on 122 conserved single-copy archaeal protein markers clearly indicates a polyphyletic origin of the species included into this genus, thus warranting its reclassification into three separate genera. We therefore propose to transfer Nlb. innermongolicus (type strain N-1311) to a new genus Natronolimnohabitans as Nlh. innermongolicus comb. nov. and to transfer Nlb. aegyptiacus (type strain JW/NM-HA 15) and Nlb. sulfurireducens (type strain AArc1) to a new genus Natrarchaeobaculum as Nbl. aegyptiacum comb. nov. and Nbl. sulfurireducens comb. nov. The phylogenomic differentiation of these four species is also supported by the ANI/AAI distances and unique phenotypes. The most important physiological differences includes a previously unreported ability for cellulose and xylan utilization in Nlb. baerhuensis, thermophily in Nbl. aegyptiacus and anaerobic sulfur respiration in Nbl. sulfurireducens. We further present an emended description of Natronolimnobius baerhuensis.

Dynamic consolidated bioprocessing for direct production of xylonate and shikimate from xylan by Escherichia coli

Numerous value-added chemicals can be produced using xylan as a feedstock. However, the product yields are limited by low xylan utilization efficiency, as well as by carbon flux competition between biomass production and biosynthesis. Herein, a dynamic consolidated bioprocessing strategy was developed, which coupled xylan utilization and yield optimization modules. Specifically, we achieved the efficient conversion of xylan to valuable chemicals in a fully consolidated manner by optimizing the expression level of xylanases and xylose transporter in the xylan utilization module. Moreover, a cell density-dependent, and Cre-triggered dynamic system that enabled the dynamic decoupling of biosynthesis and biomass production was constructed in the yield optimization module. The final shake flask-scale titers of xylonate, produced through an exogenous pathway, and shikimate, produced through an endogenous pathway, reached 16.85 and 3.2 g L−1, respectively. This study not only provides an efficient microbial platform for the utilization of xylan, but also opens up the possibility for the large-scale production of high value-added chemicals from renewable feedstocks.

Characterization and diversity of the complete set of GH family 3 enzymes from Rhodothermus marinus DSM 4253

The genome of Rhodothermus marinus DSM 4253 encodes six glycoside hydrolases (GH) classified under GH family 3 (GH3): RmBgl3A, RmBgl3B, RmBgl3C, RmXyl3A, RmXyl3B and RmNag3. The biochemical function, modelled 3D-structure, gene cluster and evolutionary relationships of each of these enzymes were studied. The six enzymes were clustered into three major evolutionary lineages of GH3: β-N-acetyl-glucosaminidases, β-1,4-glucosidases/β-xylosidases and macrolide β-glucosidases. The RmNag3 with additional β-lactamase domain clustered with the deepest rooted GH3-lineage of β-N-acetyl-glucosaminidases and was active on acetyl-chitooligosaccharides. RmBgl3B displayed β-1,4-glucosidase activity and was the only representative of the lineage clustered with macrolide β-glucosidases from Actinomycetes. The β-xylosidases, RmXyl3A and RmXyl3B, and the β-glucosidases RmBgl3A and RmBgl3C clustered within the major β-glucosidases/β-xylosidases evolutionary lineage. RmXyl3A and RmXyl3B showed β-xylosidase activity with different specificities for para-nitrophenyl (pNP)-linked substrates and xylooligosaccharides. RmBgl3A displayed β-1,4-glucosidase/β-xylosidase activity while RmBgl3C was active on pNP-β-Glc and β-1,3-1,4-linked glucosyl disaccharides. Putative polysaccharide utilization gene clusters were also investigated for both R. marinus DSM 4253 and DSM 4252T (homolog strain). The analysis showed that in the homolog strain DSM 4252TRmar_1080 (RmXyl3A) and Rmar_1081 (RmXyl3B) are parts of a putative polysaccharide utilization locus (PUL) for xylan utilization.

Consolidated bioprocessing performance of bacterial consortium EMSD5 on hemicellulose for isopropanol production

Consolidated bioprocessing (CBP) of lignocellulose by bacterial consortium for isopropanol production is considered as the most promising strategy. However, low utilization of xylan caused by the complex sidechain structure remains inhibit the conversion of full-biomass. In this study, isopropanol production from different lignocelluloses by the consortium EMSD5 through CBP was performed. A total of 7.00 g/L of isopropanol was obtained from corncob by optimizing fermentation conditions. Isopropanol production by EMSD5 was mainly based on utilizing xylan in corncob and isopropanol titer was increased by 47.71% and reached up to 8.39 g/L using arabinoxylan compared with linear xylan. The analysis of community structures and the isolation of key strain confirmed the enrichment of the isopropanol producer, Clostridium beijierinckii, in the bacterial community when it was incubated with corn glucuronoarabinoxylan and the cooperation between C. beijerinckii and lignocellulose degraders. The unique features of EMSD5 could lead to large-scale isopropanol production using lignocellulose.

A novel fungal GH30 xylanase with xylobiohydrolase auxiliary activity

BackgroundThe main representatives of hemicellulose are xylans, usually decorated β-1,4-linked d-xylose polymers, which are hydrolyzed by xylanases. The efficient utilization and complete hydrolysis of xylans necessitate the understanding of the mode of action of xylan degrading enzymes. The glycoside hydrolase family 30 (GH30) xylanases comprise a less studied group of such enzymes, and differences regarding the substrate recognition have been reported between fungal and bacterial GH30 xylanases. Besides their role in the utilization of lignocellulosic biomass for bioenergy, such enzymes could be used for the tailored production of prebiotic xylooligosaccharides (XOS) due to their substrate specificity.ResultsThe expression of a putative GH30_7 xylanase from the fungus Thermothelomyces thermophila (synonyms Myceliophthora thermophila, Sporotrichum thermophile) in Pichia pastoris resulted in the production and isolation of a novel xylanase with unique catalytic properties. The novel enzyme designated TtXyn30A, exhibited an endo- mode of action similar to that of bacterial GH30 xylanases that require 4-O-methyl-d-glucuronic acid (MeGlcA) decorations, in contrast to most characterized fungal ones. However, TtXyn30A also exhibited an exo-acting catalytic behavior by releasing the disaccharide xylobiose from the non-reducing end of XOS. The hydrolysis products from beechwood glucuronoxylan were MeGlcA substituted XOS, and xylobiose. The major uronic XOS (UXOS) were the aldotriuronic and aldotetrauronic acid after longer incubation indicating the ability of TtXyn30A to cleave linear parts of xylan and UXOS as well.ConclusionsHereby, we reported the heterologous production and biochemical characterization of a novel fungal GH30 xylanase exhibiting endo- and exo-xylanase activity. To date, considering its novel catalytic properties, TtXyn30A shows differences with most characterized fungal and bacterial GH30 xylanases. The discovered xylobiohydrolase mode of action offers new insights into fungal enzymatic systems that are employed for the utilization of lignocellulosic biomass. The recombinant xylanase could be used for the production of X2 and UXOS from glucuronoxylan, which in turn would be utilized as prebiotics carrying manifold health benefits.

A novel bifunctional xylanase/cellulase TcXyn10A from Thermoascus crustaceus JCM12803

Efficient utilization of cellulose and xylan is of importance in the bioethanol industry. In this study, a novel bifunctional xylanase/cellulase gene, Tcxyn10a, was cloned from Thermoascus crustaceus JCM12803, and the gene product was successfully overexpressed in Pichia pastoris GS115. The recombinant protein was then purified and characterized. The pH and temperature optima of TcXyn10A were determined to be 5.0 and 65-70 °C, respectively. The enzyme retained stable under acid to alkaline conditions (pH 3.0-11.0) or after 1-h treatment at 60 °C. The specific activities of TcXyn10A towards beechwood xylan, wheat arabinoxylan, sodium carboxymethyl cellulose and lichenan were (1 480±26) U/mg, (2 055±28) U/mg, (7.4±0.2) U/mg and (10.9±0.4) U/mg, respectively. Homologous modeling and molecular docking analyses indicated that the bifunctional TcXyn10A has a single catalytic domain, in which the substrate xylan and cellulose shared the same binding cleft. This study provides a valuable material for the study of structure and function relationship of bifunctional enzymes.