Production of xylooligosaccharides from enzymatic hydrolysis of xylan by the white-rot fungi Pleurotus

The white-rot fungi basidiomycetes Pleurotus sp. BCCB068 and Pleurotus tailandia were used to degrade oat-spelt xylan under submerged fermentation over a period of 40 days. Activities of endo-1,4-β-xylanase and β-xylosidase and xylan degradation products were determined. Xylan degradation by Pleurotus sp. BCCB068 and P. tailandia reached 75.1% and 73.4%, respectively. The formation of xylooligosaccharides and the simple sugars xylose, arabinose, cellobiose, mannose, and maltose were observed for both strains. The xylan degradation exhibited by these Pleurotus strains indicates they have potential for use in biotechnological processes related to degradation of hemicellulose sources.     

Modulation of cellulosome composition in Clostridium cellulolyticum: Adaptation to the polysaccharide environment revealed by proteomic and carbohydrate‐active enzyme analyses

Clostridium cellulolyticum is a model mesophilic anaerobic bacterium that efficiently degrades plant cell walls. The recent genome release offers the opportunity to analyse its complete degradation system. A total of 148 putative carbohydrate‐active enzymes were identified, and their modular structures and activities were predicted. Among them, 62 dockerin‐containing proteins bear catalytic modules from numerous carbohydrate‐active enzymes' families and whose diversity reflects the chemical and structural complexity of the plant carbohydrate. The composition of the cellulosomes produced by C. cellulolyticum upon growth on different substrates (cellulose, xylan, and wheat straw) was investigated by LC MS/MS. The majority of the proteins encoded by the cip‐cel operon, essential for cellulose degradation, were detected in all cellulosome preparations. In the presence of wheat straw, the natural and most complex of the substrates studied, additional proteins predicted to be involved in hemicellulose degradation were produced. A 32‐kb gene cluster encodes the majority of these proteins, all harbouring carbohydrate‐binding module 6 or carbohydrate‐binding module 22 xylan‐binding modules along dockerins. This newly identified xyl‐doc gene cluster, specialised in hemicellulose degradation, comes in addition of the cip‐cel operon for plant cell wall degradation. Hydrolysis efficiencies determined on the different substrates corroborates the finding that cellulosome composition is adapted to the growth substrate.     

Review: Microbial synthesis of agrochemical metabolites

The synthesis of agrochemical metabolite reference standards by microbial cultures can serve as a useful alternative to conventional chemical synthesis, particularly when the chemical synthesis is difficult. Microbially generated metabolites of agrochemicals can also be useful for predicting degradative pathways in animals, plants and soils prior to conducting animal, plant and soil metabolism studies which are required by regulatory agencies to support agrochemical registrations. Examples from the literature are used to illustrate the utility of synthesizing metabolites of agrochemicals by common microbes. Received 17 January 1997/ Accepted in revised form 20 March 1997     

Glucuronoyl esterases: diversity,properties and biotechnological potential. A review

Glucuronoyl esterases (GEs) belonging to the carbohydrate esterase family 15 (CE15) are involved in microbial degradation of lignocellulosic plant materials. GEs are capable of degrading complex polymers of lignin and hemicellulose cleaving ester bonds between glucuronic acid residues in xylan and lignin alcohols. GEs promote separation of lignin, hemicellulose and cellulose which is crucial for efficient utilization of biomass as an energy source and feedstock for further processing into products or chemicals. Genes encoding GEs are found in both fungi and bacteria, but, so far, bacterial GEs are essentially unexplored, and despite being discovered >10?years ago, only a limited number of GEs have been characterized. The first laboratory scale example of improved xylose and glucuronic acid release by the synergistic action of GE with cellulolytic enzymes was only reported recently (improved C5 sugar and glucuronic acid yields) and, until now, not much is known about their biotechnology potential. In this review, we discuss the diversity, structure and properties of microbial GEs and consider the status of their action on natural substrates and in biological systems in relation to their future industrial use.     

Relative fibrolytic activities of anaerobic rumen fungi on untreated and sodium hydroxide treated barley straw in in vitro culture

The fibrolytic activities of rumen fungi were studied in terms of dry matter loss, plant cell wall degradation and enzyme (cellulase and xylanase) activities, when grown in vitro on either untreated or sodium hydroxide treated stems of barley straw over a 12 day period. Changes in fungal growth, development and overall biomass were followed using chitin assay and scanning electron microscopy. Treatment with sodium hydroxide resulted in a decrease in the NDF content together with the disruption of cuticle and the loosening and separation of the plant cells within the straw fragments. The enzyme activities of the anaerobic fungi have a high positive correlation (R(2)=0.99) with their biomass concentration assessed by chitin assay indicating that chitin is a valuable index for the estimation of the fungal biomass in vitro. The anaerobic fungi produced very extensive rhizoidal systems in these in vitro cultures. After incubation with rumen fungi, dry matter losses were, respectively, 35% and 38% for the untreated and treated straw samples and the overall fungal biomass, determined by chitin assay, was significantly higher in the treated samples. In vitro degradation of cellulose and hemicellulose was also higher in the treated than that of untreated cultures. Although, comparatively, xylanase activity was higher than that of cellulase, the cellulose fraction of the straw was degraded more than hemicellulose in both treated and untreated straw.     

Mapping the polysaccharide degradation potential of Aspergillus niger

ABSTRACT: BACKGROUND: The degradation of plant materials by enzymes is an industry of increasing importance. For sustainable production of second generation biofuels and other products of industrial biotechnology, efficient degradation of non-edible plant polysaccharides such as hemicellulose is required. For each type of hemicellulose, a complex mixture of enzymes is required for complete conversion to fermentable monosaccharides. In plant-biomass degrading fungi, these enzymes are regulated and released by complex regulatory structures. In this study, we present a methodology for evaluating the potential of a given fungus for polysaccharide degradation. RESULTS: Through the compilation of information from 203 articles, we have systematized knowledge on the structure and degradation of 16 major types of plant polysaccharides to form a graphical overview. As a case example, we have combined this with a list of 188 genes coding for carbohydrate-active enzymes from Aspergillus niger, thus forming an analysis framework, which can be queried. Combination of this information network with gene expression analysis on mono- and polysaccharide substrates has allowed elucidation of concerted gene expression from this organism. One such example is the identification of a full set of extracellular polysaccharide-acting genes for the degradation of oat spelt xylan. CONCLUSIONS: The mapping of plant polysaccharide structures along with the corresponding enzymatic activities is a powerful framework for expression analysis of carbohydrate-active enzymes. Applying this network-based approach, we provide the first genome-scale characterization of all genes coding for carbohydrate-active enzymes identified in A. niger.     

Patterns of functional enzyme activity in fungus farming ambrosia beetles

ABSTRACT: INTRODUCTION: In wood-dwelling fungus-farming weevils, the so-called ambrosia beetles, wood in the excavated tunnels is used as a medium for cultivating fungi by the combined action of digging larvae (which create more space for the fungi to grow) and of adults sowing and pruning the fungus. The beetles are obligately dependent on the fungus that provides essential vitamins, amino acids and sterols. However, to what extent microbial enzymes support fungus farming in ambrosia beetles is unknown. Here we measure (i) 13 plant cell-wall degrading enzymes in the fungus garden microbial consortium of the ambrosia beetle Xyleborinus saxesenii, including its primary fungal symbionts, in three compartments of laboratory maintained nests, at different time points after gallery foundation and (ii) four specific enzymes that may be either insect or microbially derived in X. saxesenii adult and larval individuals. RESULTS: We discovered that the activity of cellulases in ambrosia fungus gardens is relatively small compared to the activities of other cellulolytic enzymes. Enzyme activity in all compartments of the garden was mainly directed towards hemicellulose carbohydrates such as xylan, glucomannan and callose. Hemicellulolytic enzyme activity within the brood chamber increased with gallery age, whereas irrespective of the age of the gallery, the highest overall enzyme activity were detected in the gallery dump material expelled by the beetles. Interestingly endo-beta-1,3(4)-glucanase activity capable of callose degradation was identified in whole-body extracts of both larvae and adult X. saxesenii, whereas endo-beta-1,4-xylanase activity was exclusively detected in larvae. CONCLUSION: Similar to closely related fungi associated with bark beetles in phloem, the microbial symbionts of ambrosia beetles do not degrade cellulose. Instead, their enzyme activity is directed mainly towards comparatively more easily accessible hemicellulose components of the ray-parenchyma cells in the wood xylem. Furthermore, the detection of xylanolytic enzymes exclusively in larvae and not in adults indicates that larvae (pre-) digest plant cell wall structures exclusively in larvae (which feed on fungus colonized wood) and not in adults (which feed only on fungi). This implies that in X. saxesenii and likely also in many other ambrosia beetles, adults and larvae do not compete for the same food within their nests - in contrast, larvae increase colony fitness by facilitating enzymatic wood degradation and fungus cultivation.     

Screening for distinct xylan degrading enzymes in complex shake flask fermentation supernatants

The efficient degradation of complex xylans needs collaboration of many xylan degrading enzymes. Assays for xylan degrading activities based on reducing sugars or PNP substrates are not indicative for the presence of enzymes able to degrade complex xylans: They do not provide insight into the possible presence of xylanase-accessory enzymes within enzyme mixtures. A new screening method is described, by which specific xylan modifying enzymes can be detected.Fermentation supernatants of 78 different fungal soil isolates grown on wheat straw were analyzed by HPLC and MS. This strategy is powerful in recognizing xylanases, arabinoxylan hydrolases, acetyl xylan esterases and glucuronidases.No fungus produced all enzymes necessary to totally degrade the substrates tested. Some fungi produce high levels of xylanase active against linear xylan, but are unable to degrade complex xylans. Other fungi producing relative low levels of xylanase secrete many useful accessory enzyme component(s).     

Diversity,role in decomposition,and succession of zoosporic fungi and straminipiles on submerged decaying leaves in a woodland stream

Leaf litter is a very important primary source of energy in woodland streams. Decomposition of leaf litter is a process mediated by many groups of microorganisms which release extracellular enzymes for the degradation of complex macromolecules. In this process, true fungi and straminipiles are considered to be among the most active groups, more active than the bacteria, at least during the early stages of the process. Colonization increases the quality of the leaves as a food resource for detritivores. In this way, matter and energy enter detritus-based food chains. Previously, aquatic hyphomycetes were considered to be the major fungal group responsible for leaf litter decomposition. Although zoosporic fungi and straminipiles are known to colonize and decompose plant tissues in various environments, there is scant information on their roles in leaf decomposition. This study focuses on the communities of zoosporic fungi and straminipiles in a stream which are involved in the decomposition of leaves of two plant species, Ligustrum lucidum and Pouteria salicifolia, in the presence of other groups of fungi. A characteristic community dominated by Nowakowskiella elegans, Phytophthora sp., and Pythium sp. was found. Changes in the fungal community structure over time (succession) was observed: terrestrial mitosporic fungi appeared during the early stages, zoosporic fungi, straminipiles, and aquatic Hyphomycetes in early-to-intermediate stages, while representatives of the phylum Zygomycota were found at early and latest stages of the decomposition. These observations highlight the importance of zoosporic fungi and straminipiles in aquatic ecosystems.     

Monitoring of in planta gene expression for xylan degradation and assimilation in the maize pathogen Bipolaris maydis

Swift and efficient onset of feeding on host tissue by phytopathogenic fungi is a requisite event for their successful infection and propagation. Necrotrophic fungi colonizing host cell walls appear to obtain carbon and energy sources from plant wall degradants, but what they actually utilize for nutrition after host invasion remains unclear. Here we focus on plant wall xylan, the major hemicellulosic polysaccharide in cereal plants, and study its participation in post-invasion nutrition of the maize necrotrophic pathogen Bipolaris maydis (syn: Cochliobolus heterostrophus). Using a fluorescence reporter assay, we demonstrated that a B. maydis β-xylosidase gene, BmXyp1, is strongly upregulated at the beginning of infection, specifically within invading hyphae. Additionally, our time-course measurements of mRNA expression during maize infection revealed that xylan degradation and assimilation are concomitantly induced during an early infection stage. These findings suggest that this fungus can access xylan degradants as an early in planta nutrient source after host penetration; however, mutant strains deficient in xylan-assimilation ability still retained virulence, although the lesion size was decreased as compared with the wild-type strain. Overall, we conclude that xylan degradation and assimilation by B. maydis are initial post-invasion events but do not play an essential role in fungal nutrient acquisition.     

The α-glucuronidase Agu1 from <Emphasis Type="Italic">Schizophyllum commune</Emphasis> is a member of a novel glycoside hydrolase family (GH115)

Schizophyllum commune produces an α-glucuronidase that is active on polymeric xylan, while the ascomycete α-glucuronidases are only active on xylan oligomers. In this study, we have identified the gene (agu1) encoding this enzyme and confirmed the functionality by overexpression of the gene in S. commune and degradation of aldopentauronic acids, (MeGlcA)³-Xyl₄, in the cultivation medium of the transformants. Expression analysis demonstrated that agu1 is not co-regulated with the predominant xylanase-encoding gene (xynA) of S. commune. The detailed sequence analysis of Agu1 demonstrated that this gene belongs to a novel glycoside hydrolase family (GH115) that also contains candidate genes from ascomycete fungi and bacteria. Phylogenetic analysis showed that the fungal GH115 α-glucuronidases are distinctly separate from the prokaryotic clade and distributed over three branches. The identification of putative genes encoding this enzyme in industrial fungi, such as Aspergillus oryzae and Hypocrea jecorina, will provide a starting point for further analysis of the importance of this enzyme for the hydrolysis of plant biomass.     

Production of Polysaccharidases in Different Carbon Sources by Leucoagaricus gongylophorus Möller (Singer), the Symbiotic Fungus of the Leaf-Cutting Ant Atta sexdens Linnaeus

Leucoagaricus gongylophorus, the fungus cultured by the leaf-cutting ant Atta sexdens, produces polysaccharidases that degrade leaf components by generating nutrients believed to be essential for ant nutrition. We evaluated pectinase, amylase, xylanase, and cellulase production by L. gongylophorus in laboratory cultures and found that polysaccharidases are produced during fungal growth on pectin, starch, cellulose, xylan, or glucose but not cellulase, whose production is inhibited during fungal growth on xylan. Pectin was the carbon source that best stimulated the production of enzymes, which showed that pectinase had the highest production activity of all of the carbon sources tested, indicating that the presence of pectin and the production of pectinase are key features for symbiotic nutrition on plant material. During growth on starch and cellulose, polysaccharidase production level was intermediate, although during growth on xylan and glucose, enzyme production was very low. We propose a possible profile of polysaccharide degradation inside the nest, where the fungus is cultured on the foliar substrate.     

A comparative systems analysis of polysaccharide‐elicited responses in Neurospora crassa reveals carbon source‐specific cellular adaptations

Filamentous fungi are powerful producers of hydrolytic enzymes for the deconstruction of plant cell wall polysaccharides. However, the central question of how these sugars are perceived in the context of the complex cell wall matrix remains largely elusive. To address this question in a systematic fashion we performed an extensive comparative systems analysis of how the model filamentous fungus Neurospora crassa responds to the three main cell wall polysaccharides: pectin, hemicellulose and cellulose. We found the pectic response to be largely independent of the cellulolytic one with some overlap to hemicellulose, and in its extent surprisingly high, suggesting advantages for the fungus beyond being a mere carbon source. Our approach furthermore allowed us to identify carbon source‐specific adaptations, such as the induction of the unfolded protein response on cellulose, and a commonly induced set of 29 genes likely involved in carbon scouting. Moreover, by hierarchical clustering we generated a coexpression matrix useful for the discovery of new components involved in polysaccharide utilization. This is exemplified by the identification of lat‐1, which we demonstrate to encode for the physiologically relevant arabinose transporter in Neurospora. The analyses presented here are an important step towards understanding fungal degradation processes of complex biomass.     

Biochemical analyses of multiple endoxylanases from the rumen bacterium Ruminococcus albus 8 and their synergistic activities with accessory hemicellulose-degrading enzymes

Ruminococcus albus 8 is a ruminal bacterium capable of metabolizing hemicellulose and cellulose, the major components of the plant cell wall. The enzymes that allow this bacterium to capture energy from the two polysaccharides, therefore, have potential application in plant cell wall depolymerization, a process critical to biofuel production. For this purpose, a partial genome sequence of R. albus 8 was generated. The genomic data depicted a bacterium endowed with multiple forms of plant cell wall-degrading enzymes. The endoxylanases of R. albus 8 exhibited diverse modular architectures, including incorporation of a catalytic module, a carbohydrate binding module, and a carbohydrate esterase module in a single polypeptide. The accessory enzymes of xylan degradation were a β-xylosidase, an α-l-arabinofuranosidase, and an α-glucuronidase. We hypothesized that due to the chemical complexity of the hemicellulose encountered in the rumen, the bacterium uses multiple endoxylanases, with subtle differences in substrate specificities, to attack the substrate, while the accessory enzymes hydrolyze the products to simple sugars for metabolism. To test this hypothesis, the genes encoding the predicted endoxylanases were expressed, and the proteins were biochemically characterized either alone or in combination with accessory enzymes. The different endoxylanase families exhibited different patterns of product release, with the family 11 endoxylanases releasing more products in synergy with the accessory enzymes from the more complex substrates. Aside from the insights into hemicellulose degradation by R. albus 8, this report should enhance our knowledge on designing effective enzyme cocktails for release of fermentable sugars in the biofuel industry.     

Enhanced production of polyhydroxyalkanoates (PHAs) from beechwood xylan by recombinant Escherichia coli

Microbial conversion of plant biomass to value-added products is an attractive option to address the impacts of petroleum dependency. In this study, a bacterial system was developed that can hydrolyze xylan and utilize xylan-derived xylose for growth and production of polyhydroxyalkanoates (PHAs). A β-xylosidase and an endoxylanase were engineered into a P(LA-co-3HB)-producing Escherichia coli strain to obtain a xylanolytic strain. Although PHA production yields using xylan as sole carbon source were minimal, when the xylan-based media was supplemented with a single sugar (xylose or arabinose) to permit the accumulation of xylan-derived xylose in the media, PHA production yields increased up to 18-fold when compared to xylan-based production, and increased by 37 % when compared to production from single sugar sources alone. ¹H-Nuclear magnetic resonance (NMR) analysis shows higher accumulation of xylan-derived xylose in the media when xylan was supplemented with arabinose to prevent xylose uptake by catabolite repression. ¹H-NMR, gel permeation chromatography, and differential scanning calorimetry analyses corroborate that the polymers maintain physical properties regardless of the carbon source. This study demonstrates that accumulation of biomass-derived sugars in the media prior to their uptake by microbes is an important aspect to enhance PHA production when using plant biomass as feedstock.     

Comparison of glycoside hydrolase family 3 β-xylosidases from basidiomycetes and ascomycetes reveals evolutionarily distinct xylan degradation systems

Xylan is the most common hemicellulose in plant cell walls, though the structure of xylan polymers differs between plant species. Here, to gain a better understanding of fungal xylan degradation systems, which can enhance enzymatic saccharification of plant cell walls in industrial processes, we conducted a comparative study of two glycoside hydrolase family 3 (GH3) β-xylosidases (Bxls), one from the basidiomycete Phanerochaete chrysosporium (PcBxl3), and the other from the ascomycete Trichoderma reesei (TrXyl3A). A comparison of the crystal structures of the two enzymes, both with saccharide bound at the catalytic center, provided insight into the basis of substrate binding at each subsite. PcBxl3 has a substrate-binding pocket at subsite -1, while TrXyl3A has an extra loop that contains additional binding subsites. Furthermore, kinetic experiments revealed that PcBxl3 degraded xylooligosaccharides faster than TrXyl3A, while the K_M values of TrXyl3A were lower than those of PcBxl3. The relationship between substrate specificity and degree of polymerization of substrates suggested that PcBxl3 preferentially degrades xylobiose (X₂), while TrXyl3A degrades longer xylooligosaccharides. Moreover, docking simulation supported the existence of extended positive subsites of TrXyl3A in the extra loop located at the N-terminus of the protein. Finally, phylogenetic analysis suggests that wood-decaying basidiomycetes use Bxls such as PcBxl3 that act efficiently on xylan structures from woody plants, whereas molds use instead Bxls that efficiently degrade xylan from grass. Our results provide added insights into fungal efficient xylan degradation systems.     

Modification of the carbohydrate composition of sulfite pulp by purified and characterized beta-xylanase and beta-xylosidase of Aureobasidium pullulans

Both beta-xylanase and beta-xylosidase were purified to homogeneity from a xylose-grown culture of Aureobasidium pullulans. Cellular distribution studies of enzyme activities revealed that beta-xylanase was an extracellular enzyme, during both the exponential and stationary phases, whereas beta-xylosidase was mostly periplasmic associated. The beta-xylanase exhibited very high specificity for xylan extracted from Eucalyptus grandis dissolving pulp, whereas the beta-xylosidase was only active on p-nitrophenyl xyloside and xylobiose. Comparison of kcat/Km ratios showed that the beta-xylanase hydrolyzed xylan from dissolving pulp 1.3, 2.1, and 2. 3 times more efficiently than Eucalyptus hemicellulose B, Eucalyptus hemicellulose A, and larchwood xylan, respectively. The beta-xylosidase exhibited a transxylosylation reaction during the hydrolysis of xylobiose. When applied on acid sulfite pulp, both enzymes released xylose and hydrolyzed xylan to a different extent. Although beta-xylosidase (0.4 U/g pulp) liberated more xylose from pulp than beta-xylanase (4.7 U/g pulp), it was responsible for only 3% of xylan solubilization. Treatment of pulp with beta-xylanase liberated 51.7 microgram of xylose/g and hydrolyzed 10% of xylan. The two enzymes acted additively on pulp and removed 12% of pulp xylan. A synergistic effect in terms of release of xylose from pulp was observed when the enzyme mixture of beta-xylanase and beta-xylosidase was supplemented with beta-mannanase. However, this did not result in further enzymatic degradation of pulp xylan. Both beta-xylanase and beta-xylosidase altered the carbohydrate composition of sulfite pulp by increasing the relative cellulose content at the expense of reduced hemicellulose content of pulp.