Role of a cell-wall glucan-degrading enzyme in mating of Schizophyllum commune

Mycelial enzyme extracts of Schizophyllum commune were prepared during vegetative growth matings leading to common-A and common-B heterokaryons and the dikaryon, and were examined for hydrolytic activity against an alkaliinsoluble cell-wall glucan (R-glucan) isolated from this mushroom. In extracts from several individual homokaryotic mycelia the R-glucanase activity was low and did not increase when the cultures exhausted glucose in the medium. In common-A matings, a 30-fold increase in specific activity of intracellular R-glucanase was found even in the presence of glucose in the broth. An increase of this magnitude was not observed in the common-B mating nor in the fully compatible cross leading to the dikaryon. Extracts of the dikaryon did show elevated R-glucanase activity after exogenous glucose disappearance and subsequent fruiting. In none of these situations was an enzyme activity detected towards an alkali-soluble cell-wall glucan (S-glucan) prepared from S. commune. Changes in R-glucanase were not parallelled by identical changes in laminarinase, pustulanase, cellobiase, and p-nitrophenyl-beta-d-glucosidase, but comparable increases in specific activities were found for hydrolysis of glycogen and maltose. After interaction of the various mycelia in mating combinations, the S-glucan/R-glucan ratio of the cell wall of the dikaryon was found to be similar to that of the homokaryons, but increased in the common-B interaction and was elevated almost threefold in the common-A heterokaryon.     

Biochemistry of Sexual Morphogenesis in Schizophyllum commune: Effect of Mutations Affecting the Incompatibility System on Cell-Wall Metabolism

A number of homokaryons of Schizophyllum commune, which carry various mutations affecting the incompatibility system, and a wild-type homokaryon were grown and examined for differences in the net synthesis of the cell-wall polysaccharides S-glucan, R-glucan, and chitin and in the activity of an enzyme hydrolyzing R-glucan (R-glucanase) in mycelial extracts and culture media. Only slight differences were observed for the accumulation of S-glucan and chitin. In comparison with the wild-type homokaryon, a very high S-glucan/R-glucan ratio was found in a primary B-factor mutant strain. Essentially, wild-type S-glucan/R-glucan ratios were restored in two strains in which additional mutations restored normal morphology: a strain carrying a secondary B-factor mutation and a strain carrying a modifier mutation in addition to the primary B-factor mutation. The S-glucan/R-glucan ratios in three A-factor mutants were intermediate between those of the wild-type homokaryon and the primary B-factor mutant. In young, growing cultures of the various homokaryons, except for the A-factor mutants, a correlation was found between the S-glucan/R-glucan ratios in the cell wall and the activities of R-glucanase in mycelial extracts. A certain specificity of the effect of the studied mutations on enzyme activities was indicated by the fact that, in young cultures, changes in R-glucanase activities were not paralleled by similar changes in the activities of laminarinase and maltase. The results can be correlated with particular morphological features of the homokaryons and, together with earlier results obtained with heterokaryons, indicate the activity of R-glucanase as an integral component of sexual morphogenesis regulated by the incompatibility factors.     

Direct glucose production from lignocellulose using <Emphasis Type="Italic">Clostridium thermocellum</Emphasis> cultures supplemented with a thermostable β-glucosidase

Background

Cellulases continue to be one of the major costs associated with the lignocellulose hydrolysis process. Clostridium thermocellum is an anaerobic, thermophilic, cellulolytic bacterium that produces cellulosomes capable of efficiently degrading plant cell walls. The end-product cellobiose, however, inhibits degradation. To maximize the cellulolytic ability of C. thermocellum, it is important to eliminate this end-product inhibition.

Results

This work describes a system for biological saccharification that leads to glucose production following hydrolysis of lignocellulosic biomass. C. thermocellum cultures supplemented with thermostable beta-glucosidases make up this system. This approach does not require any supplementation with cellulases and hemicellulases. When C. thermocellum strain S14 was cultured with a Thermoanaerobacter brockii beta-glucosidase (CglT with activity 30 U/g cellulose) in medium containing 100 g/L cellulose (617 mM initial glucose equivalents), we observed not only high degradation of cellulose, but also accumulation of 426 mM glucose in the culture broth. In contrast, cultures without CglT, or with less thermostable beta-glucosidases, did not efficiently hydrolyze cellulose and accumulated high levels of glucose. Glucose production required a cellulose load of over 10 g/L. When alkali-pretreated rice straw containing 100 g/L glucan was used as the lignocellulosic biomass, approximately 72% of the glucan was saccharified, and glucose accumulated to 446 mM in the culture broth. The hydrolysate slurry containing glucose was directly fermented to 694 mM ethanol by addition of Saccharomyces cerevisiae, giving an 85% theoretical yield without any inhibition.

Conclusions

Our process is the first instance of biological saccharification with exclusive production and accumulation of glucose from lignocellulosic biomass. The key to its success was the use of C. thermocellum supplemented with a thermostable beta-glucosidase and cultured under a high cellulose load. We named this approach biological simultaneous enzyme production and saccharification (BSES). BSES may resolve a significant barrier to economical production by providing a platform for production of fermentable sugars with reduced enzyme amounts.

     

Two new <Emphasis Type="Italic">Entoloma</Emphasis> s.L. species with serrulatum-type lamellar edge from Changbai Mountains,Northeast China

Two species of Entoloma with serrulatum-type lamellar edge, E. cyanostipitum and E. subcaesiocinctum from the Changbai Mountains in northeast China, are described as new. E. cyanostipitum is distinguished by a brownish orange pileus with a central depression, black-blue lamellar edge, deep blue stipe, and cylindrical cheilocystidia with intense blue, intracellular pigment. E. subcaesiocinctum is characterized by a depressed pileus covered with brownish squamules, blackish blue lamellar edge, and cylindrical to clavate cheilocystidia with intense blue intracellular pigment. The two new species are further confirmed based on the ITS and nLSU sequences.     

Three new species of <Emphasis Type="Italic">Russula</Emphasis> from South China

Russula lotus, R. nivalis, and R. purpureoverrucosa are proposed here as new taxa based on morphological and molecular evidences. The new species are described with illustration photographs and line drawings, and compared against related species. Morphologically, R. lotus (subgenus Heterophyllidia, section Heterophyllae, subsection Cyanoxanthinae) is characterized by a medium-sized basidioma with a pale pink to purplish pink pileus whose center area is yellowish white, white to cream white lamellae occasionally forked with lamellulae, a cream white smooth stipe, broadly ellipsoid to ellipsoid basidiospores ornamented with disconnected amyloid warts and inamyloid suprahilar plage, dispersed sulphovanillin (SV)-negative pileocystidia, and the pileipellis with suprapellis cells claw-assembled. Russula nivalis (subgenus Russula, section Russula, subsection Russula) is characterized by a very small pure white basidioma with a pileus 7–12 mm in diameter, abundant clavate pileocystidia and caulocystidia changing purplish red in SV, and broadly ellipsoidal to ellipsoid basidiospores ornamented with strongly amyloid warts and ridges interconnected by fine lines in an uncompleted or completed reticulum, mostly with inamyloid suprahilar plage. Russula purpureoverrucosa (subgenus Incrustatula, section Lilaceinae, subsection Lilaceinae) is characterized by a medium-sized basidioma with a red to grayish magenta pileus slightly areolate in small irregular warts from center to margin, a stipe with the same color and warts as the pileus, white to cream white occasionally forked lamellae with lamellulae, broadly ellipsoidal to ellipsoid basidiospores ornamented with amyloid warts and ridges rarely connected, abundant clavate pleurocystidia covered with thick yellowish incrustations, and the pileipellis with suprapellis cells a typical trichoderm, some of which covered with yellowish incrustations, pileocystidia absent. Phylogenetic analysis of nucleotide sequences of the internal transcribed spacer (ITS) region provided further evidence that the described species belong to the subsections above respectively, and represent new taxa.     

Isolation and properties of fungal β-glucosidases

Using chromatography on different matrixes, three β-glucosidases (120, 116, and 70 kDa) were isolated from enzymatic complexes of the mycelial fungi Aspergillus japonicus, Penicillium verruculosum, and Trichoderma reesei, respectively. The enzymes were identified by MALDI-TOF mass-spectrometry. Substrate specificity, kinetic parameters for hydrolysis of specific substrates, ability to catalyze the transglucosidation reaction, dependence of the enzymatic activity on pH and temperature, stability of the enzymes at different temperatures, adsorption ability on insoluble cellulose, and the influence of glucose on catalytic properties of the enzymes were investigated. According to the substrate specificity, the enzymes were shown to belong to two groups: i) β-glucosidase of A. japonicus exhibiting high specific activity to the low molecular weight substrates cellobiose and pNPG (the specific activity towards cellobiose was higher than towards pNPG) and low activity towards polysaccharide substrates (β-glucan from barley and laminarin); ii) β-glucosidases from P. verruculosum and T. reesei exhibiting relatively high activity to polysaccharide substrates and lower activity to low molecular weight substrates (activity to cellobiose was lower than to pNPG).     

Xylan extraction from pretreated sugarcane bagasse using alkaline and enzymatic approaches

Background

New biorefinery concepts are necessary to drive industrial use of lignocellulose biomass components. Xylan recovery before enzymatic hydrolysis of the glucan component is a way to add value to the hemicellulose fraction, which can be used in papermaking, pharmaceutical, and food industries. Hemicellulose removal can also facilitate subsequent cellulolytic glucan hydrolysis.

Results

Sugarcane bagasse was pretreated with an alkaline-sulfite chemithermomechanical process to facilitate subsequent extraction of xylan by enzymatic or alkaline procedures. Alkaline extraction methods yielded 53% (w/w) xylan recovery. The enzymatic approach provided a limited yield of 22% (w/w) but produced the xylan with the lowest contamination with lignin and glucan components. All extracted xylans presented arabinosyl side groups and absence of acetylation. 2D-NMR data suggested the presence of O-methyl-glucuronic acid and p-coumarates only in enzymatically extracted xylan. Xylans isolated using the enzymatic approach resulted in products with molecular weights (Mw) lower than 6 kDa. Higher Mw values were detected in the alkali-isolated xylans. Alkaline extraction of xylan provided a glucan-enriched solid readily hydrolysable with low cellulase loads, generating hydrolysates with a high glucose/xylose ratio.

Conclusions

Hemicellulose removal before enzymatic hydrolysis of the cellulosic fraction proved to be an efficient manner to add value to sugarcane bagasse biorefining. Xylans with varied yield, purity, and structure can be obtained according to the extraction method. Enzymatic extraction procedures produce high-purity xylans at low yield, whereas alkaline extraction methods provided higher xylan yields with more lignin and glucan contamination. When xylan extraction is performed with alkaline methods, the residual glucan-enriched solid seems suitable for glucose production employing low cellulase loadings.

     

New species of <Emphasis Type="Italic">Austroboletus</Emphasis> (Boletaceae) in Australia

Three new species of Austroboletus are described from Australia. Austroboletus austrovirens has been collected repeatedly in northern Queensland and the Northern Territory, and is primarily distinguished by its dry, green pileus and stipe ornamentation, and boletoid spores with meandering ridges and pits. Austroboletus viscidoviridis is known from southeastern coastal Queensland and Cape Tribulation; it is recognized by a glutinous-viscid, olive-green pileus, brown reticulum on the stipe, and amygdaliform spores with nodulose-tuberculate ornamentation. Austroboletus roseialbus has only been collected in eastern New South Wales and is characterized by a white pileus with subtle rosy tints, and flattened, white, lanose patches; the spores are amygdaliform with irregular alveolate-reticulate ornamentation.     

Trithorax-group protein ATX5 mediates the glucose response via impacting the HY1-ABI4 signaling module

Key message

Trithorax-group Protein ARABIDOPSIS TRITHORAX5 modulates the glucose response.

Abstract

Glucose is an evolutionarily conserved modulator from unicellular microorganisms to multicellular animals and plants. Extensive studies have shown that the Trithorax-group proteins (TrxGs) play essential roles in different biological processes by affecting histone modifications and chromatin structures. However, whether TrxGs function in the glucose response and how they achieve the control of target genes in response to glucose signaling in plants remain unknown. Here, we show that the Trithorax-group Protein ARABIDOPSIS TRITHORAX5 (ATX5) affects the glucose response and signaling. atx5 loss-of-function mutants display glucose-oversensitive phenotypes compared to the wild-type (WT). Genome-wide RNA-sequencing analyses have revealed that ATX5 impacts the expression of a subset of glucose signaling responsive genes. Intriguingly, we have established that ATX5 directly controls the expression of HY1 by trimethylating H3 lysine 4 of the Arabidopsis Heme Oxygenase1 (HY1) locus. Glucose signaling causes the suppression of ATX5 activity and subsequently reduces the H3K4me3 levels at the HY1 locus, thereby leading to the increased expression of ABSCISIC ACID-INSENSITIVE4 (ABI4). This result suggests that an important ATX5-HY1-ABI4 regulatory module governs the glucose response. This idea is further supported by genetic evidence showing that an atx5 hy1-100 abi4 triple mutant showed a similar glucose-insensitive phenotype as compared to that of the abi4 single mutant. Our findings show that a novel ATX5-HY1-ABI4 module controls the glucose response in Arabidopsis thaliana.

     

Role of bacterial γ-glutamyltranspeptidase as a novel virulence factor in bone-resorbing pathogenesis

Mammalian γ-glutamyltranspeptidase (GGT) has been identified as a bone-resorbing factor. Since GGT of Bacillus subtilis exhibits similarity in their primary structure and enzymatic characteristics with mammalian GGTs, the bone-resorbing activity of bacterial GGT was examined in this study. Osteoclastogenesis was performed in a co-culture system of mouse calvaria-derived osteoblasts and bone marrow cells. A conditioned medium from GGT-overproducing B. subtilis culture showed significantly higher activity of osteoclast formation than a conditioned medium from wild-type B. subtilis culture. Recombinant GGT (rGGT) of wild-type B. subtilis and an enzymatic activity-defected rGGT of B. subtilis 2288 mutant were expressed in Escherichia coli and purified using His tag. Both purified rGGTs induced similar levels of osteoclastogenesis, suggesting that B. subtilis GGT possesses virulent bone-resorbing activity and its activity is probably independent of its enzymatic activity. Furthermore, a recombinant protein of B. subtilis GGT heavy subunit (Bs rGGT/H) showed strong activity of osteoclastogenesis while the light subunit failed to show strong activity, suggesting that the bone-resorbing activity is mainly located at the heavy subunit. More importantly, the GGT enzymatic activity may not be required for this virulence activity since the light subunit contains the catalytic pocket. In addition, B. subtilis rGGT stimulated mRNA expressions of receptor activator of nuclear factor kappa-B ligand (RANKL) and cyclooxygenase-2 (COX-2), while an osteoprotegerin inhibited the osteoclast formation induced by Bs rGGT/H. This is the first demonstration that bacterial GGT itself is sufficient to act as a bone-resorbing virulence factor via RANKL-dependent pathway. Therefore, it can be hypothesized that GGT of periodontopathic bacteria may play an important role as a virulence factor in bone destruction.     

Co-expression of Beta-Glucosidase and Laccase in <Emphasis Type="Italic">Trichoderma reesei</Emphasis> by Random Insertion with Enhanced Filter Paper Activity

Trichoderma reesei strain Rut-C30 was modified with enhanced beta-glycosidase (BGL) activity to balance the cellulase system and generated laccase (LAC) protein for lignin degradation. Initially, the binary plasmid p1300-w1 was constructed to express T. reesei bgl2 under the control of promoter P _pki and T-nos terminator. Random insertion was performed via Agrobacterium tumefaciens-mediated transformation. A total of 353 mutants were obtained, and 34PTrb2 was exceptionally stable with increased FPA and BGL activity after screening for extracellular enzyme activity. Subsequently, 34PTrb2 was used as parent strain via the same method to insert the lac gene from Fomes lignosus, with promoter P _gpd , followed by cbh1 signal peptide trss and T-nos as terminator. Several mutants successfully expressed enzyme LAC with stable activity of approximately 0.13 U/mL. The mutant 15Gsslac increased activity by 40.4% FPA compared with that of the host Rut-C30.     

The Auxin-Deficient <Emphasis Type="Italic">Defective Kernel18 (dek18)</Emphasis> Mutation Alters the Expression of Seed-Specific Biosynthetic Genes in Maize

The dek18 mutant of maize was previously classified as a collapsed kernel mutant named cp*-931A, which has a decreased auxin content in kernels. Molecular and functional characterization of this mutant line offers the possibility to better understand auxin biology during maize seed development. Seeds of the dek18 mutants are smaller compared to wild-type seeds and the vegetative development of dek18 is delayed. Here we analyzed the expression of several auxin-related genes in dek18 homozygous seeds and normal-sized seeds (Dek18/-) segregating on the same ear. Three genes related to auxin biosynthesis ZmAlliinase/Tar3, ZmTar1, and ZmYuc1 were highly downregulated in the mutant compared to the wild type. Sequence analysis of these genes revealed that no nucleotide difference is present in dek18 homozygous seeds compared to Dek18/-, except for ZmYuc1. Two different ZmYuc1 cDNAs sequences are produced: a normal-sized sequence of 1197 bp and a shorter coding sequence lacking the third exon. Ectopic expression of ZmYuc1 cDNAs in Arabidopsis indicates that (i) the ZmYuc1 gene is functional in Arabidopsis and (ii) the third exon is required for the enzymatic activity of the YUCCA1 protein. Because ZmYuc1, ZmTar1, and ZmAlliinase are barely expressed in dek18 homozygous seeds, it is proposed that the mutation responsible for the dek18 phenotype alters the upstream regulation of the auxin biosynthetic pathway.     

The role of microorgansisms in maintaining the chitin balance in the Barents Sea

In this study, we investigated chitin hydrolysis by the bacteria inhabiting the ground of the Barents Sea. Four microbial cultures isolated from the ground were described as the genera of Rhodococcus sp., Bacillus sp., Pseudomonas sp., and Acinetobacter sp. Protein complexes with endochitinase and exochitinase activities were purified from the culture liquid. These microorganisms can participate in chitin degradation in sea water. The average molecular weight of the protein fraction with the chitinolytic activity constituted 92–135 kDa. The ratio of the endo-/exochitinase activities of the enzymatic systems was increased in the order Pseudomonas sp. < Bacillus sp. < Acinetobacter sp. < Rhodococcus sp.     

Polymorphism of <Emphasis Type="Italic">yellow</Emphasis> locus in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> mutants from natural populations

We have studied the molecular characteristics of the yellow locus (y; 1–0.0), which determines the body color of phenotypically wild-type and mutant alleles isolated in different years from geographically distant populations of Drosophila melanogaster. According to the Southern blot, data restriction maps of the yellow locus of all examined strains differ from one another, as well as from Oregon stock. FISH analysis shows that, in the neighborhood of the yellow locus in the X chromosome, neither P nor hobo elements are found in y^1–775 stock, while only hobo is found in these region in y^1–859 and y^1–866 stocks, only the P element is found in y⁺sn⁸⁴⁹ stock, and both elements are found in y^1–719 stock. Thus, all yellow mutants studied are of independent origin. Locus yellow located on the end of X chromosome (region 1A5–8 on the cytologic map) carries significantly more transposon than retrotransposon induced mutations compared to the white locus (region 3C2). It is possible that, at the ends of Drosophila melanogaster chromosomes, transposons are more active than retrotransposons.     

<Emphasis Type="Italic">R</Emphasis>-acetoin accumulation and dissimilation in <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis>

Klebsiella pneumoniae is a 2,3-butanediol producer, and R-acetoin is an intermediate of 2,3-butanediol production. R-acetoin accumulation and dissimilation in K. pneumoniae was studied here. A budC mutant, which has lost 2,3-butanediol dehydrogenase activity, accumulated high levels of R-acetoin in culture broth. However, after glucose was exhausted, the accumulated R-acetoin could be reused by the cells as a carbon source. Acetoin dehydrogenase enzyme system, encoded by acoABCD, was responsible for R-acetoin dissimilation. acoABCD mutants lost the ability to grow on acetoin as the sole carbon source, and the acetoin accumulated could not be dissimilated. However, in the presence of another carbon source, the acetoin accumulated in broth of acoABCD mutants was converted to 2,3-butanediol. Parameters of R-acetoin production by budC mutants were optimized in batch culture. Aerobic culture and mildly acidic conditions (pH 6–6.5) favored R-acetoin accumulation. At the optimized conditions, in fed-batch fermentation, 62.3 g/L R-acetoin was produced by budC and acoABCD double mutant in 57 h culture, with an optical purity of 98.0 %, and a substrate conversion ratio of 28.7 %.