Wood extractives, solvent-soluble fractions of woody biomass, are considered to be a factor imped... more Wood extractives, solvent-soluble fractions of woody biomass, are considered to be a factor impeding or excluding fungal colonization on the freshly harvested conifers. Among wood decay fungi, the basidiomycete Phlebiopsis gigantea has evolved a unique enzyme system to efficiently transform or degrade conifer extractives but little is known about the mechanism(s). In this study, to clarify the mechanism(s) of softwood degradation, we examined the transcriptome, proteome, and metabolome of P. gigantea when grown on defined media containing microcrystalline cellulose and pine sapwood extractives. Beyond the conventional enzymes often associated with cellulose, hemicellulose and lignin degradation, an array of enzymes implicated in the metabolism of softwood lipophilic extractives such as fatty and resin acids, steroids and glycerides was significantly up-regulated. Among these, a highly expressed and inducible lipase is likely responsible for lipophilic extractive degradation, based o...
Developing an efficient deconstruction step of woody biomass for biorefinery has been drawing con... more Developing an efficient deconstruction step of woody biomass for biorefinery has been drawing considerable attention since its xylem cell walls display highly recalcitrance nature. Here, we explored transcriptional factors (TFs) that reduce wood recalcitrance and improve saccharification efficiency in Populus species. First, 33 TF genes up-regulated during poplar wood formation were selected as potential regulators of xylem cell wall structure. The transgenic hybrid aspens (Populus tremula × Populus tremuloides) overexpressing each selected TF gene were screened for in vitro enzymatic saccharification. Of these, four transgenic seedlings overexpressing previously uncharacterized TF genes increased total glucan hydrolysis on average compared to control. The best performing lines overexpressing Pt × tERF123 and Pt × tZHD14 were further grown to form mature xylem in the greenhouse. Notably, the xylem cell walls exhibited significantly increased total xylan hydrolysis as well as initial...
Recent studies show the potential impacts of insect symbiont microbes on biofuel application with... more Recent studies show the potential impacts of insect symbiont microbes on biofuel application with regard to their degradation capability of a recalcitrant plant cell wall. In this study, we describe a novel fungal isolate, D. decipiens oita, as a single symbiotic fungus from the Xiphydria woodwasp found in the northern forests of Japan. Our detailed secretome analyses of D. decipiens oita, together with activity measurements, reveal that this insect-associated fungus exhibits high and broad activities for plant cell wall material degradation, suggesting potential applications within the biomass conversion industry for plant mass degradation.
Highlights d The Striga genome reflects a three-phase model of parasitic plant genome evolution d... more Highlights d The Striga genome reflects a three-phase model of parasitic plant genome evolution d A family of strigolactone receptors has undergone a striking expansion in Striga d Genes in lateral root development are coordinately induced in a parasitic organ d Host genes and retrotransposons are horizontally transferred into Striga
Watanabe et al. Genomes of Sulfur-Oxidizing Chemolithoautotrophs and Sterolibacteriaceae were obs... more Watanabe et al. Genomes of Sulfur-Oxidizing Chemolithoautotrophs and Sterolibacteriaceae were observed to harbor genes encoding ArxAB, a type of arsenite oxidase that has been identified in a limited number of bacteria. These findings were made with the newly obtained genomes, including those from 6 genera from which no genome sequence of an isolated organism was previously available. These genomes will serve as valuable references to interpret nucleotide sequences.
Biological polymer synthetic systems, which utilize no template molecules, normally synthesize ra... more Biological polymer synthetic systems, which utilize no template molecules, normally synthesize random copolymers. We report an exception, a synthesis of block polyhydroxyalkanoates (PHAs) in an engineered Escherichia coli. Using an engineered PHA synthase, block copolymers poly[(R)-2-hydroxybutyrate(2HB)-b-(R)-3-hydroxybutyrate(3HB)] were produced in E. coli. The covalent linkage between P(2HB) and P(3HB) segments was verified with solvent fractionation and microphase separation. Notably, the block sequence was generated under the simultaneous consumption of two monomer precursors, indicating the existence of a rapid monomer switching mechanism during polymerization. Based on in vivo metabolic intermediate analysis and the relevant in vitro enzymatic activities, we propose a model in which the rapid intracellular 3HB-CoA fluctuation during polymer synthesis is a major factor in generating block sequences. The dynamic change of intracellular monomer levels is a novel regulatory principle of monomer sequences of biopolymers.
Journal of the Science of Food and Agriculture, 2018
BACKGROUND Mushrooms have been widely considered as health foods as their extracts have anti-hype... more BACKGROUND Mushrooms have been widely considered as health foods as their extracts have anti-hypertensive and anti-tumor activities. After a thorough literature survey, we hypothesized that enzymes in mushroom extracts play an important role in synthesizing functional molecules. Therefore, in the present study, proteins extracted from Reishi mushroom (Ganoderma lucidum), which is used in oriental medicine, were identified by the proteomic approach, and appropriate extraction methods for improving angiotensin-converting enzyme (ACE) inhibitory activities were investigated. RESULTS Various glycoside hydrolases (GHs), such as β-N-acetylhexaminidase (GH family 20), α-1,2-mannosidase (GH family 47), endo-β-1,3-glucanase (GH family 128) and β-1,3-glucanase (GH152), that degrade glycans in the fruiting body were identified. The residual glucanase activities generated -oligosaccharides. Additionally, the glutamic acid protease of the peptidase G1 family was determined as the major protein in the extract, and the residual peptidase activity of the extracts was found to improve ACE This article is protected by copyright. All rights reserved. Accepted Article inhibitory activities. Finally, it was observed that extraction at 50°C is suitable for yielding functional molecules with high ACE inhibitory activities. CONCLUSION Water extraction is generally believed to extract only functional macromolecules that exist in mushroom fruiting bodies. The present study proposed a new concept that describes how functional molecules are produced by enzymes, including proteases and GHs, during extraction.
Applied and environmental microbiology, Jan 10, 2018
Fungi play a key role cycling nutrients in forest ecosystems but the mechanisms remain uncertain.... more Fungi play a key role cycling nutrients in forest ecosystems but the mechanisms remain uncertain. To clarify the enzymatic processes involved in wood decomposition, metatranscriptomics and metaproteomics of extensively decayed lodgepole pine were examined by RNAseq and LC-MS/MS, respectively. Following metatranscriptome assembly, 52,011 contigs were searched for functional domains and homology to database entries. Contigs similar to to basidiomycete transcripts dominated and many of these were most closely related to ligninolytic white rot fungi or cellulolytic brown rot fungi. A diverse array of carbohydrate active enzymes (CAzymes) representing a total of 132 families or subfamilies were identified. Among these were 672 glycoside hydrolases including highly expressed cellulases or hemicellulases. The CAzymes also included 162 genes encoding redox enzymes classified within Auxiliary Activity (AA) families. Eighteen of these were manganese peroxidases, key components of ligninolytic...
D-Lactate (LA)-based oligomers (D-LAOs) are unusual oligoesters consisting of D-LA and D-3-hydrox... more D-Lactate (LA)-based oligomers (D-LAOs) are unusual oligoesters consisting of D-LA and D-3-hydroxybutyrate that are produced and secreted by engineered Escherichia coli grown on glucose. The cells heterologously express LApolymerizing polyhydroxyalkanoate synthase and monomer-supplying enzymes. In this study, we attempted to identify the D-LAO secretion route in E. coli, which is thought to be mediated by intrinsic membrane proteins. To this end, a loss-of-function screening of D-LAO secretion was carried out using 209 single-gene membrane protein deletants, which are involved in the transport of organic compounds. Among the deletants of the outer membrane-associated proteins,
Arabinogalactan-proteins (AGPs) are highly diverse plant proteoglycans found on the plant cell su... more Arabinogalactan-proteins (AGPs) are highly diverse plant proteoglycans found on the plant cell surface. AGPs have large arabinogalactan (AG) moieties attached to a core-protein rich in hydroxyproline (Hyp). The AG undergoes hydrolysis by various glycoside hydrolases, most of which have been identified, whereas the core-proteins is presumably degraded by unknown proteases/peptidases secreted from fungi and bacteria in nature. Although several enzymes hydrolyzing other Hyp-rich proteins are known, the enzymes acting on the core-proteins of AGPs remain to be identified. The present study describes the detection of protease/peptidase activity toward AGP core-proteins in the culture medium of winter mushroom (Flammulina velutipes) and partial purification of the enzyme by several conventional chromatography steps. The enzyme showed higher activity toward Hyp residues than toward proline and alanine residues and acted on core-proteins prepared from gum arabic. Since the activity was inhib...
Subfamily 2 of SNF1-related protein kinase (SnRK2) plays important roles in plant abiotic stress ... more Subfamily 2 of SNF1-related protein kinase (SnRK2) plays important roles in plant abiotic stress responses as a global positive regulator of abscisic acid signaling. In the genome of the model tree Populus trichocarpa, 12 SnRK2 genes have been identified, and some are upregulated by abiotic stresses. In this study, we heterologously overexpressed the PtSnRK2 genes in Arabidopsis thaliana and found that overexpression of PtSnRK2.5 and PtSnRK2.7 genes enhanced stress tolerance. In the PtSnRK2.5 and PtSnRK2.7 overexpressors, chlorophyll content, and root elongation were maintained under salt stress conditions, leading to higher survival rates under salt stress compared with those in the wild type. Transcriptomic analysis revealed that PtSnRK2.7 overexpression affected stress-related metabolic genes, including lipid metabolism and flavonoid metabolism, even under normal growth conditions. However, the stress response genes reported to be upregulated in Arabidopsis SRK2C/SnRK2.6 and whea...
Efficient and complete degradation of woody plant cell walls requires the concerted action of hyd... more Efficient and complete degradation of woody plant cell walls requires the concerted action of hydrolytic and oxidative systems possessed by a relatively small group of filamentous basidiomycetous fungi. Among these wood decay species, Phanerochaete chrysosporium was the first to be sequenced (Martinez et al. 2004). In the intervening 10 years, over 100 related saprophytes have been sequenced. There genomes have revealed impressive sequence diversity. and recent functional analyses are providing a deeper understanding of their roles in the deconstruction of plant cell walls and the transformation of xenobiotics. Wood cell walls are primarily composed of cellulose, hemicellulose and lignin. Many microbes are capable of hydrolyzing the linkages in cellulose and hemicel lulose, even though crystalline regions within cellulose can be rather challenging substrates (reviewed in Baldrian and Lopez-Mondejar 2014; van den Brink and de Vries 2011). In contrast, few microbes possess the oxidative enzymes required to efficiently degrade the recalcitrant lignin, a complex, amorphous, and insoluble phenylpropanoid polymer (Higuchi 1990; Ralph et al. 2004). These unusual wood decay fungi secrete extracellular peroxidases with impressive oxidative potential. Potential applications have focused primarily on lignocellulosic bioconversions to
Collectively classified as white-rot fungi, certain basidiomycetes efficiently degrade the major ... more Collectively classified as white-rot fungi, certain basidiomycetes efficiently degrade the major structural polymers of wood cell walls. A small subset of these Agaricomycetes, exemplified by Phlebiopsis gigantea, is capable of colonizing freshly exposed conifer sapwood despite its high content of extractives, which retards the establishment of other fungal species. The mechanism(s) by which P. gigantea tolerates and metabolizes resinous compounds have not been explored. Here, we report the annotated P. gigantea genome and compare profiles of its transcriptome and secretome when cultured on freshcut versus solvent-extracted loblolly pine wood. The P. gigantea genome contains a conventional repertoire of hydrolase genes involved in cellulose/hemicellulose degradation, whose patterns of expression were relatively unperturbed by the absence of extractives. The expression of genes typically ascribed to lignin degradation was also largely unaffected. In contrast, genes likely involved in the transformation and detoxification of wood extractives were highly induced in its presence. Their products included an ABC transporter, lipases, cytochrome P450s, glutathione S-transferase and aldehyde dehydrogenase. Other regulated genes of unknown function and several constitutively expressed genes are also likely involved in P. gigantea's extractives metabolism. These results contribute to our fundamental understanding of pioneer colonization of conifer wood and provide insight into the diverse chemistries employed by fungi in carbon cycling processes.
With the aim of developing a new easy-to-use method for rescuing flood-damaged paper, the effect ... more With the aim of developing a new easy-to-use method for rescuing flood-damaged paper, the effect of saltwater on the inhibition of fungal growth on paper was investigated. This procedure could be used instead of, or assisted by freeze drying. Cellulose-digesting Trichoderma reesei, three types of fungi representative of fungi growing on paper (T. reesei, Aspergillus terreus, and Aureobasidium pullulans) and various naturally airborne fungi were cultured on cellulosic materials in liquid media containing artificial seawater with different salt concentrations. The addition of salts successfully inhibited the growth of T. reesei on microcrystalline cellulose at the concentration of 3.2% (m/m) or higher. The critical salt concentration, 3.2%, is within the general range of salt content in seawater. Other solutions of salts similar to sodium chloride also inhibited fungal growth. Although the observed growth-inhibiting effect was attributed to the high osmotic pressure of the salt solution, physiological effects depending on ion species used were also considered to be possible. The growth of all three types of fungi on copy paper was inhibited effectively when the salt concentration was increased. The growth of various fungi on pure cellulose, with enough oxygen supplied to pores, was completely inhibited (as assessed by visual examination) for 24 days at salt concentrations of 3.5% (m/m) or greater. The fact that the effect of saltwater on cellulosic materials was observed even under optimum medium conditions implies that fungi would be considerably inhibited on flood-damaged paper immersed in saltwater. This method is a promising first aid measure when circumstances do not allow for flood-damaged paper to be dried immediately.
Dating Wood Rot Specific lineages within the basidiomycete fungi, white rot species, have evolved... more Dating Wood Rot Specific lineages within the basidiomycete fungi, white rot species, have evolved the ability to break up a major structural component of woody plants, lignin, relative to their non–lignin-decaying brown rot relatives. Through the deep phylogenetic sampling of fungal genomes, Floudas et al. (p. 1715 ; see the Perspective by Hittinger ) mapped the detailed evolution of wood-degrading enzymes. A key peroxidase and other enzymes involved in lignin decay were present in the common ancestor of the Agaricomycetes. These genes then expanded through gene duplications in parallel, giving rise to white rot lineages.
To degrade the polysaccharides, wooddecay fungi secrete a variety of glycoside hydrolases (GHs) a... more To degrade the polysaccharides, wooddecay fungi secrete a variety of glycoside hydrolases (GHs) and carbohydrate esterases (CEs) classified into various sequence-based families of carbohydrateactive enzymes (CAZys) and their appended carbohydrate-binding modules (CBM). Oxidative enzymes, such as cellobiose dehydrogenase (CDH) and lytic polysaccharide monooxygenase (LPMO, formerly GH61), also have been implicated in cellulose degradation. To examine polysaccharide-degrading potential between white-and brown-rot fungi, we performed genomewide analysis of CAZys and these oxidative enzymes in 11 Polyporales, including recently sequenced monokaryotic strains of Bjerkandera adusta, Ganoderma sp. and Phlebia brevispora. Furthermore, we conducted comparative secretome analysis of seven Polyporales grown on wood culture. As a result, it was found that genes encoding cellulases belonging to families GH6, GH7, GH9 and carbohydrate-binding module family CBM1 are lacking in genomes of brown-rot polyporales. In addition, the presence of CDH and the expansion of LPMO were observed only in white-rot genomes. Indeed, GH6, GH7, CDH and LPMO peptides were identified only in white-rot polypores. Genes encoding aldose 1-epimerase (ALE), previously detected with CDH and cellulases in the culture filtrates, also were identified in white-rot genomes, suggesting a physiological connection between ALE, CDH, cellulase and possibly LPMO. For hemicellulose degradation, genes and peptides corresponding to GH74 xyloglucanase, GH10 endo-xylanase, GH79 b-glucuronidase, CE1 acetyl xylan esterase and CE15 glucuronoyl methylesterase were significantly increased in white-rot genomes compared to brown-rot genomes. Overall, relative to brown-rot Polyporales, white-rot Polyporales maintain greater enzymatic diversity supporting lignocellulose attack.
Proceedings of the National Academy of Sciences of the United States of America, Jan 3, 2012
Efficient lignin depolymerization is unique to the wood decay basidiomycetes, collectively referr... more Efficient lignin depolymerization is unique to the wood decay basidiomycetes, collectively referred to as white rot fungi. Phanerochaete chrysosporium simultaneously degrades lignin and cellulose, whereas the closely related species, Ceriporiopsis subvermispora, also depolymerizes lignin but may do so with relatively little cellulose degradation. To investigate the basis for selective ligninolysis, we conducted comparative genome analysis of C. subvermispora and P. chrysosporium. Genes encoding manganese peroxidase numbered 13 and five in C. subvermispora and P. chrysosporium, respectively. In addition, the C. subvermispora genome contains at least seven genes predicted to encode laccases, whereas the P. chrysosporium genome contains none. We also observed expansion of the number of C. subvermispora desaturase-encoding genes putatively involved in lipid metabolism. Microarray-based transcriptome analysis showed substantial up-regulation of several desaturase and MnP genes in wood-co...
Wood extractives, solvent-soluble fractions of woody biomass, are considered to be a factor imped... more Wood extractives, solvent-soluble fractions of woody biomass, are considered to be a factor impeding or excluding fungal colonization on the freshly harvested conifers. Among wood decay fungi, the basidiomycete Phlebiopsis gigantea has evolved a unique enzyme system to efficiently transform or degrade conifer extractives but little is known about the mechanism(s). In this study, to clarify the mechanism(s) of softwood degradation, we examined the transcriptome, proteome, and metabolome of P. gigantea when grown on defined media containing microcrystalline cellulose and pine sapwood extractives. Beyond the conventional enzymes often associated with cellulose, hemicellulose and lignin degradation, an array of enzymes implicated in the metabolism of softwood lipophilic extractives such as fatty and resin acids, steroids and glycerides was significantly up-regulated. Among these, a highly expressed and inducible lipase is likely responsible for lipophilic extractive degradation, based o...
Developing an efficient deconstruction step of woody biomass for biorefinery has been drawing con... more Developing an efficient deconstruction step of woody biomass for biorefinery has been drawing considerable attention since its xylem cell walls display highly recalcitrance nature. Here, we explored transcriptional factors (TFs) that reduce wood recalcitrance and improve saccharification efficiency in Populus species. First, 33 TF genes up-regulated during poplar wood formation were selected as potential regulators of xylem cell wall structure. The transgenic hybrid aspens (Populus tremula × Populus tremuloides) overexpressing each selected TF gene were screened for in vitro enzymatic saccharification. Of these, four transgenic seedlings overexpressing previously uncharacterized TF genes increased total glucan hydrolysis on average compared to control. The best performing lines overexpressing Pt × tERF123 and Pt × tZHD14 were further grown to form mature xylem in the greenhouse. Notably, the xylem cell walls exhibited significantly increased total xylan hydrolysis as well as initial...
Recent studies show the potential impacts of insect symbiont microbes on biofuel application with... more Recent studies show the potential impacts of insect symbiont microbes on biofuel application with regard to their degradation capability of a recalcitrant plant cell wall. In this study, we describe a novel fungal isolate, D. decipiens oita, as a single symbiotic fungus from the Xiphydria woodwasp found in the northern forests of Japan. Our detailed secretome analyses of D. decipiens oita, together with activity measurements, reveal that this insect-associated fungus exhibits high and broad activities for plant cell wall material degradation, suggesting potential applications within the biomass conversion industry for plant mass degradation.
Highlights d The Striga genome reflects a three-phase model of parasitic plant genome evolution d... more Highlights d The Striga genome reflects a three-phase model of parasitic plant genome evolution d A family of strigolactone receptors has undergone a striking expansion in Striga d Genes in lateral root development are coordinately induced in a parasitic organ d Host genes and retrotransposons are horizontally transferred into Striga
Watanabe et al. Genomes of Sulfur-Oxidizing Chemolithoautotrophs and Sterolibacteriaceae were obs... more Watanabe et al. Genomes of Sulfur-Oxidizing Chemolithoautotrophs and Sterolibacteriaceae were observed to harbor genes encoding ArxAB, a type of arsenite oxidase that has been identified in a limited number of bacteria. These findings were made with the newly obtained genomes, including those from 6 genera from which no genome sequence of an isolated organism was previously available. These genomes will serve as valuable references to interpret nucleotide sequences.
Biological polymer synthetic systems, which utilize no template molecules, normally synthesize ra... more Biological polymer synthetic systems, which utilize no template molecules, normally synthesize random copolymers. We report an exception, a synthesis of block polyhydroxyalkanoates (PHAs) in an engineered Escherichia coli. Using an engineered PHA synthase, block copolymers poly[(R)-2-hydroxybutyrate(2HB)-b-(R)-3-hydroxybutyrate(3HB)] were produced in E. coli. The covalent linkage between P(2HB) and P(3HB) segments was verified with solvent fractionation and microphase separation. Notably, the block sequence was generated under the simultaneous consumption of two monomer precursors, indicating the existence of a rapid monomer switching mechanism during polymerization. Based on in vivo metabolic intermediate analysis and the relevant in vitro enzymatic activities, we propose a model in which the rapid intracellular 3HB-CoA fluctuation during polymer synthesis is a major factor in generating block sequences. The dynamic change of intracellular monomer levels is a novel regulatory principle of monomer sequences of biopolymers.
Journal of the Science of Food and Agriculture, 2018
BACKGROUND Mushrooms have been widely considered as health foods as their extracts have anti-hype... more BACKGROUND Mushrooms have been widely considered as health foods as their extracts have anti-hypertensive and anti-tumor activities. After a thorough literature survey, we hypothesized that enzymes in mushroom extracts play an important role in synthesizing functional molecules. Therefore, in the present study, proteins extracted from Reishi mushroom (Ganoderma lucidum), which is used in oriental medicine, were identified by the proteomic approach, and appropriate extraction methods for improving angiotensin-converting enzyme (ACE) inhibitory activities were investigated. RESULTS Various glycoside hydrolases (GHs), such as β-N-acetylhexaminidase (GH family 20), α-1,2-mannosidase (GH family 47), endo-β-1,3-glucanase (GH family 128) and β-1,3-glucanase (GH152), that degrade glycans in the fruiting body were identified. The residual glucanase activities generated -oligosaccharides. Additionally, the glutamic acid protease of the peptidase G1 family was determined as the major protein in the extract, and the residual peptidase activity of the extracts was found to improve ACE This article is protected by copyright. All rights reserved. Accepted Article inhibitory activities. Finally, it was observed that extraction at 50°C is suitable for yielding functional molecules with high ACE inhibitory activities. CONCLUSION Water extraction is generally believed to extract only functional macromolecules that exist in mushroom fruiting bodies. The present study proposed a new concept that describes how functional molecules are produced by enzymes, including proteases and GHs, during extraction.
Applied and environmental microbiology, Jan 10, 2018
Fungi play a key role cycling nutrients in forest ecosystems but the mechanisms remain uncertain.... more Fungi play a key role cycling nutrients in forest ecosystems but the mechanisms remain uncertain. To clarify the enzymatic processes involved in wood decomposition, metatranscriptomics and metaproteomics of extensively decayed lodgepole pine were examined by RNAseq and LC-MS/MS, respectively. Following metatranscriptome assembly, 52,011 contigs were searched for functional domains and homology to database entries. Contigs similar to to basidiomycete transcripts dominated and many of these were most closely related to ligninolytic white rot fungi or cellulolytic brown rot fungi. A diverse array of carbohydrate active enzymes (CAzymes) representing a total of 132 families or subfamilies were identified. Among these were 672 glycoside hydrolases including highly expressed cellulases or hemicellulases. The CAzymes also included 162 genes encoding redox enzymes classified within Auxiliary Activity (AA) families. Eighteen of these were manganese peroxidases, key components of ligninolytic...
D-Lactate (LA)-based oligomers (D-LAOs) are unusual oligoesters consisting of D-LA and D-3-hydrox... more D-Lactate (LA)-based oligomers (D-LAOs) are unusual oligoesters consisting of D-LA and D-3-hydroxybutyrate that are produced and secreted by engineered Escherichia coli grown on glucose. The cells heterologously express LApolymerizing polyhydroxyalkanoate synthase and monomer-supplying enzymes. In this study, we attempted to identify the D-LAO secretion route in E. coli, which is thought to be mediated by intrinsic membrane proteins. To this end, a loss-of-function screening of D-LAO secretion was carried out using 209 single-gene membrane protein deletants, which are involved in the transport of organic compounds. Among the deletants of the outer membrane-associated proteins,
Arabinogalactan-proteins (AGPs) are highly diverse plant proteoglycans found on the plant cell su... more Arabinogalactan-proteins (AGPs) are highly diverse plant proteoglycans found on the plant cell surface. AGPs have large arabinogalactan (AG) moieties attached to a core-protein rich in hydroxyproline (Hyp). The AG undergoes hydrolysis by various glycoside hydrolases, most of which have been identified, whereas the core-proteins is presumably degraded by unknown proteases/peptidases secreted from fungi and bacteria in nature. Although several enzymes hydrolyzing other Hyp-rich proteins are known, the enzymes acting on the core-proteins of AGPs remain to be identified. The present study describes the detection of protease/peptidase activity toward AGP core-proteins in the culture medium of winter mushroom (Flammulina velutipes) and partial purification of the enzyme by several conventional chromatography steps. The enzyme showed higher activity toward Hyp residues than toward proline and alanine residues and acted on core-proteins prepared from gum arabic. Since the activity was inhib...
Subfamily 2 of SNF1-related protein kinase (SnRK2) plays important roles in plant abiotic stress ... more Subfamily 2 of SNF1-related protein kinase (SnRK2) plays important roles in plant abiotic stress responses as a global positive regulator of abscisic acid signaling. In the genome of the model tree Populus trichocarpa, 12 SnRK2 genes have been identified, and some are upregulated by abiotic stresses. In this study, we heterologously overexpressed the PtSnRK2 genes in Arabidopsis thaliana and found that overexpression of PtSnRK2.5 and PtSnRK2.7 genes enhanced stress tolerance. In the PtSnRK2.5 and PtSnRK2.7 overexpressors, chlorophyll content, and root elongation were maintained under salt stress conditions, leading to higher survival rates under salt stress compared with those in the wild type. Transcriptomic analysis revealed that PtSnRK2.7 overexpression affected stress-related metabolic genes, including lipid metabolism and flavonoid metabolism, even under normal growth conditions. However, the stress response genes reported to be upregulated in Arabidopsis SRK2C/SnRK2.6 and whea...
Efficient and complete degradation of woody plant cell walls requires the concerted action of hyd... more Efficient and complete degradation of woody plant cell walls requires the concerted action of hydrolytic and oxidative systems possessed by a relatively small group of filamentous basidiomycetous fungi. Among these wood decay species, Phanerochaete chrysosporium was the first to be sequenced (Martinez et al. 2004). In the intervening 10 years, over 100 related saprophytes have been sequenced. There genomes have revealed impressive sequence diversity. and recent functional analyses are providing a deeper understanding of their roles in the deconstruction of plant cell walls and the transformation of xenobiotics. Wood cell walls are primarily composed of cellulose, hemicellulose and lignin. Many microbes are capable of hydrolyzing the linkages in cellulose and hemicel lulose, even though crystalline regions within cellulose can be rather challenging substrates (reviewed in Baldrian and Lopez-Mondejar 2014; van den Brink and de Vries 2011). In contrast, few microbes possess the oxidative enzymes required to efficiently degrade the recalcitrant lignin, a complex, amorphous, and insoluble phenylpropanoid polymer (Higuchi 1990; Ralph et al. 2004). These unusual wood decay fungi secrete extracellular peroxidases with impressive oxidative potential. Potential applications have focused primarily on lignocellulosic bioconversions to
Collectively classified as white-rot fungi, certain basidiomycetes efficiently degrade the major ... more Collectively classified as white-rot fungi, certain basidiomycetes efficiently degrade the major structural polymers of wood cell walls. A small subset of these Agaricomycetes, exemplified by Phlebiopsis gigantea, is capable of colonizing freshly exposed conifer sapwood despite its high content of extractives, which retards the establishment of other fungal species. The mechanism(s) by which P. gigantea tolerates and metabolizes resinous compounds have not been explored. Here, we report the annotated P. gigantea genome and compare profiles of its transcriptome and secretome when cultured on freshcut versus solvent-extracted loblolly pine wood. The P. gigantea genome contains a conventional repertoire of hydrolase genes involved in cellulose/hemicellulose degradation, whose patterns of expression were relatively unperturbed by the absence of extractives. The expression of genes typically ascribed to lignin degradation was also largely unaffected. In contrast, genes likely involved in the transformation and detoxification of wood extractives were highly induced in its presence. Their products included an ABC transporter, lipases, cytochrome P450s, glutathione S-transferase and aldehyde dehydrogenase. Other regulated genes of unknown function and several constitutively expressed genes are also likely involved in P. gigantea's extractives metabolism. These results contribute to our fundamental understanding of pioneer colonization of conifer wood and provide insight into the diverse chemistries employed by fungi in carbon cycling processes.
With the aim of developing a new easy-to-use method for rescuing flood-damaged paper, the effect ... more With the aim of developing a new easy-to-use method for rescuing flood-damaged paper, the effect of saltwater on the inhibition of fungal growth on paper was investigated. This procedure could be used instead of, or assisted by freeze drying. Cellulose-digesting Trichoderma reesei, three types of fungi representative of fungi growing on paper (T. reesei, Aspergillus terreus, and Aureobasidium pullulans) and various naturally airborne fungi were cultured on cellulosic materials in liquid media containing artificial seawater with different salt concentrations. The addition of salts successfully inhibited the growth of T. reesei on microcrystalline cellulose at the concentration of 3.2% (m/m) or higher. The critical salt concentration, 3.2%, is within the general range of salt content in seawater. Other solutions of salts similar to sodium chloride also inhibited fungal growth. Although the observed growth-inhibiting effect was attributed to the high osmotic pressure of the salt solution, physiological effects depending on ion species used were also considered to be possible. The growth of all three types of fungi on copy paper was inhibited effectively when the salt concentration was increased. The growth of various fungi on pure cellulose, with enough oxygen supplied to pores, was completely inhibited (as assessed by visual examination) for 24 days at salt concentrations of 3.5% (m/m) or greater. The fact that the effect of saltwater on cellulosic materials was observed even under optimum medium conditions implies that fungi would be considerably inhibited on flood-damaged paper immersed in saltwater. This method is a promising first aid measure when circumstances do not allow for flood-damaged paper to be dried immediately.
Dating Wood Rot Specific lineages within the basidiomycete fungi, white rot species, have evolved... more Dating Wood Rot Specific lineages within the basidiomycete fungi, white rot species, have evolved the ability to break up a major structural component of woody plants, lignin, relative to their non–lignin-decaying brown rot relatives. Through the deep phylogenetic sampling of fungal genomes, Floudas et al. (p. 1715 ; see the Perspective by Hittinger ) mapped the detailed evolution of wood-degrading enzymes. A key peroxidase and other enzymes involved in lignin decay were present in the common ancestor of the Agaricomycetes. These genes then expanded through gene duplications in parallel, giving rise to white rot lineages.
To degrade the polysaccharides, wooddecay fungi secrete a variety of glycoside hydrolases (GHs) a... more To degrade the polysaccharides, wooddecay fungi secrete a variety of glycoside hydrolases (GHs) and carbohydrate esterases (CEs) classified into various sequence-based families of carbohydrateactive enzymes (CAZys) and their appended carbohydrate-binding modules (CBM). Oxidative enzymes, such as cellobiose dehydrogenase (CDH) and lytic polysaccharide monooxygenase (LPMO, formerly GH61), also have been implicated in cellulose degradation. To examine polysaccharide-degrading potential between white-and brown-rot fungi, we performed genomewide analysis of CAZys and these oxidative enzymes in 11 Polyporales, including recently sequenced monokaryotic strains of Bjerkandera adusta, Ganoderma sp. and Phlebia brevispora. Furthermore, we conducted comparative secretome analysis of seven Polyporales grown on wood culture. As a result, it was found that genes encoding cellulases belonging to families GH6, GH7, GH9 and carbohydrate-binding module family CBM1 are lacking in genomes of brown-rot polyporales. In addition, the presence of CDH and the expansion of LPMO were observed only in white-rot genomes. Indeed, GH6, GH7, CDH and LPMO peptides were identified only in white-rot polypores. Genes encoding aldose 1-epimerase (ALE), previously detected with CDH and cellulases in the culture filtrates, also were identified in white-rot genomes, suggesting a physiological connection between ALE, CDH, cellulase and possibly LPMO. For hemicellulose degradation, genes and peptides corresponding to GH74 xyloglucanase, GH10 endo-xylanase, GH79 b-glucuronidase, CE1 acetyl xylan esterase and CE15 glucuronoyl methylesterase were significantly increased in white-rot genomes compared to brown-rot genomes. Overall, relative to brown-rot Polyporales, white-rot Polyporales maintain greater enzymatic diversity supporting lignocellulose attack.
Proceedings of the National Academy of Sciences of the United States of America, Jan 3, 2012
Efficient lignin depolymerization is unique to the wood decay basidiomycetes, collectively referr... more Efficient lignin depolymerization is unique to the wood decay basidiomycetes, collectively referred to as white rot fungi. Phanerochaete chrysosporium simultaneously degrades lignin and cellulose, whereas the closely related species, Ceriporiopsis subvermispora, also depolymerizes lignin but may do so with relatively little cellulose degradation. To investigate the basis for selective ligninolysis, we conducted comparative genome analysis of C. subvermispora and P. chrysosporium. Genes encoding manganese peroxidase numbered 13 and five in C. subvermispora and P. chrysosporium, respectively. In addition, the C. subvermispora genome contains at least seven genes predicted to encode laccases, whereas the P. chrysosporium genome contains none. We also observed expansion of the number of C. subvermispora desaturase-encoding genes putatively involved in lipid metabolism. Microarray-based transcriptome analysis showed substantial up-regulation of several desaturase and MnP genes in wood-co...
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Papers by Chiaki Hori