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Whole-aircraft coupled aerodynamics, flight dynamics, and structural dynamics models are commonly employed during the aircraft design process, generally increasing in fidelity, and associatively accuracy, as the design matures. While literature acknowledges the impact of explicitly representing the propulsion system in such models, it does not address the impact of structural idealizations on key metrics that inform early conceptual design. This paper investigates the impact of a range of propulsion system structural modeling strategies and their attendant idealizations on the accuracy with which key structural performance metrics may be acquired. A conventional tube-and-wing airframe with under-wing-mounted, high-bypass-ratio, turbofan propulsion systems is employed as a verification case. The lowest-fidelity modeling strategies defined herein are capable of acquiring airframe loads and vertical power plant center-of-mass inertial loads within [Formula: see text]. Higher-fidelity, predominantly 2D strategies are necessary for acquisition of propulsion system displacements, deformations, and power plant swing frequency within [Formula: see text] of a global finite element model. However, sufficient data must be available to allow the construction of higher-fidelity strategies during early design stages.

The brown rot fungus Fomitopsis pinicola efficiently depolymerizes wood cellulose via the combined activities of oxidative and hydrolytic enzymes. Mass spectrometric analyses of culture filtrates identified specific proteins, many of which were differentially regulated in response to substrate composition.

Wood-decaying fungi tend to have characteristic substrate ranges that partly define their ecological niche.

Wood-decaying fungi tend to have characteristic substrate ranges that partly define their ecological niche. Fomitopsis pinicola is a brown rot species of Polyporales that is reported on 82 species of softwoods and 42 species of hardwoods. We analyzed gene expression levels of F. pinicola from submerged cultures with ground wood powder (sampled at 5 days) or solid wood wafers (sampled at 10 and 30 days), using aspen, pine, and spruce substrates (aspen was used only in submerged cultures). Fomitopsis pinicola expressed similar sets of wood-degrading enzymes typical of brown rot fungi across all culture conditions and time points. Nevertheless, differential gene expression was observed across all pairwise comparisons of substrates and time points. Genes exhibiting differential expression encode diverse enzymes with known or potential function in brown rot decay, including laccase, benzoquinone reductase, aryl alcohol oxidase, cytochrome P450s, and various glycoside hydrolases. Comparing transcriptomes from submerged cultures and wood wafers, we found that culture conditions had a greater impact on global expression profiles than substrate wood species. These findings highlight the need for standardization of culture conditions in studies of gene expression in wood-decaying fungi. IMPORTANCE All species of wood-decaying fungi occur on a characteristic range of substrates (host plants), which may be broad or narrow. Understanding the mechanisms that allow fungi to grow on particular substrates is important for both fungal ecology and applied uses of different feedstocks in industrial processes. We grew the wood-decaying polypore Fomitopsis pinicola on three different wood species—aspen, pine, and spruce—under various culture conditions. We found that F. pinicola is able to modify gene expression (transcription levels) across different substrate species and culture conditions. Many of the genes involved encode enzymes with known or predicted functions in wood decay. This study provides clues to how wood-decaying fungi may adjust their arsenal of decay enzymes to accommodate different host substrates.

Fungi that decay wood have characteristic associations with certain tree species, but the mechanistic bases for these associations are poorly understood. We studied substrate-specific gene expression and RNA editing in six species of wood-decaying fungi from the 'Antrodia clade' (Polyporales, Agaricomycetes) on three different wood substrates (pine, spruce, and aspen) in submerged cultures. We identified dozens to hundreds of substrate-biased genes (i.e., genes that are significantly upregulated in one substrate relative to the other two substrates) in each species, and these biased genes are correlated with their host ranges. Evolution of substrate-biased genes is associated with gene family expansion, gain and loss of genes, and variation in cis- and trans- regulatory elements, rather than changes in protein coding sequences. We also demonstrated widespread RNA editing events in the Antrodia clade, which differ from those observed in the Ascomycota in their distribution, substitution types, and the genomic environment. Moreover, we found that substrates could affect editing positions and frequency, including editing events occurring in mRNA transcribed from wood-decay-related genes. This work shows the extent to which gene expression and RNA editing differ among species and substrates, and provides clues into mechanisms by which wood-decaying fungi may adapt to different hosts.

Wood-decaying fungi tend to have characteristic substrate ranges that partly define their ecological niche. Fomitopsis pinicola is a brown rot species of Polyporales that is reported on 82 species of softwoods and 42 species of hardwoods. We analyzed the gene expression levels and RNA editing profiles of F. pinicola from submerged cultures with ground wood powder (sampled at 5 days) or solid wood wafers (sampled at 10 and 30 days), using aspen, pine, and spruce substrates (aspen was used only in submerged cultures). Fomitopsis pinicola expressed similar sets of wood-degrading enzymes typical of brown rot fungi across all culture conditions and time points. Nevertheless, differential gene expression and RNA editing were observed across all pairwise comparisons of substrates and time points. Genes exhibiting differential expression and RNA editing encode diverse enzymes with known or potential function in brown rot decay, including laccase, benzoquinone reductase, aryl alcohol oxidase, cytochrome P450s, and various glycoside hydrolases. There was no overlap between differentially expressed and differentially edited genes, suggesting that these may provide F. pinicola with independent mechanisms for responding to different conditions. Comparing transcriptomes from submerged cultures and wood wafers, we found that culture conditions had a greater impact on global expression profiles than substrate wood species. In contrast, the suites of genes subject to RNA editing were much less affected by culture conditions. These findings highlight the need for standardization of culture conditions in studies of gene expression in wood-decaying fungi.IMPORTANCE All species of wood-decaying fungi occur on a characteristic range of substrates (host plants), which may be broad or narrow. Understanding the mechanisms that enable fungi to grow on particular substrates is important for both fungal ecology and applied uses of different feedstocks in industrial processes. We grew the wood-decaying polypore Fomitopsis pinicola on three different wood species, aspen, pine, and spruce, under various culture conditions. We examined both gene expression (transcription levels) and RNA editing (posttranscriptional modification of RNA, which can potentially yield different proteins from the same gene). We found that F. pinicola is able to modify both gene expression and RNA editing profiles across different substrate species and culture conditions. Many of the genes involved encode enzymes with known or predicted functions in wood decay. This work provides clues to how wood-decaying fungi may adjust their arsenal of decay enzymes to accommodate different host substrates.

Office of Science of the U.S. Department of Energy (DE-AC02-05CH11231) ; NSF (1457695 ; 1457721) ; JSPS (16K18727) ; French National Research Agency (ANR-11-LABX 0002 01); Fungi play a key role cycling nutrients in forest ecosystems, but the mechanisms remain uncertain. To clarify the enzymatic processes involved in wood decomposition, the metatranscriptomics and metaproteomics of extensively decayed lodgepole pine were examined by RNA sequencing (RNA-seq) and liquid chromatography-tandem mass spectrometry (LC-MS/MS), respectively. Following de novo metatranscriptome assembly, 52,011 contigs were searched for functional domains and homology to database entries. Contigs similar 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 predicted redox enzymes classified within auxiliary activity (AA) families. Eighteen of these were manganese peroxidases, which are key components of ligninolytic white rot fungi. The expression of other redox enzymes supported the working of hydroquinone reduction cycles capable of generating reactive hydroxyl radicals. These have been implicated as diffusible oxidants responsible for cellulose depolymerization by brown rot fungi. Thus, enzyme diversity and the coexistence of brown and white rot fungi suggest complex interactions of fungal species and degradative strategies during the decay of lodgepole pine.IMPORTANCE The deconstruction of recalcitrant woody substrates is a central component of carbon cycling and forest health. Laboratory investigations have contributed substantially toward understanding the mechanisms employed by model wood decay fungi, but few studies have examined the physiological processes in natural environments. Herein, we identify the functional genes present in field samples of extensively decayed lodgepole pine (Pinus contorta), a major species distributed throughout the North American Rocky Mountains. The classified transcripts and proteins revealed a diverse array of oxidative and hydrolytic enzymes involved in the degradation of lignocellulose. The evidence also strongly supports simultaneous attack by fungal species employing different enzymatic strategies.

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-containing medium. MS identified MnP proteins in C. subvermispora culture filtrates, but none in P. chrysosporium cultures. These results support the importance of MnP and a lignin degradation mechanism whereby cleavage of the dominant nonphenolic structures is mediated by lipid peroxidation products. Two C. subvermispora genes were predicted to encode peroxidases structurally similar to P. chrysosporium lignin peroxidase and, following heterologous expression in Escherichia coli , the enzymes were shown to oxidize high redox potential substrates, but not Mn 2+ . Apart from oxidative lignin degradation, we also examined cellulolytic and hemicellulolytic systems in both fungi. In summary, the C. subvermispora genetic inventory and expression patterns exhibit increased oxidoreductase potential and diminished cellulolytic capability relative to P. chrysosporium .

3 p.-2 tab. Gaskell, Jill et al., We report the genome of Postia (Rhodonia) placenta MAD-SB12, a homokaryotic wood decay fungus (Basidiomycota, Polyporales). Intensively studied as a representative brown rot decayer, the gene complement is consistent with the rapid depolymerization of cellulose but not lignin., The work conducted by the U.S. Department of Energy Joint Genome Institute, a DOE Office of Science User Facility, was supported by the Office of Science of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231. BH was funded by the AMIDEX foundation (MicrobioE project, grant number ANR-11-IDEX-0001-02).

The white-rot basidiomycetes efficiently degrade all wood cell wall polymers. Generally, these fungi simultaneously degrade cellulose and lignin, but certain organisms, such as Ceriporiopsis subvermispora , selectively remove lignin in advance of cellulose degradation. However, relatively little is known about the mechanism of selective ligninolysis. To address this issue, C. subvermispora was grown in liquid medium containing ball-milled aspen, and nano-liquid chromatography-tandem mass spectrometry was used to identify and estimate extracellular protein abundance over time. Several manganese peroxidases and an aryl alcohol oxidase, both associated with lignin degradation, were identified after 3 days of incubation. A glycoside hydrolase (GH) family 51 arabinofuranosidase was also identified after 3 days but then successively decreased in later samples. Several enzymes related to cellulose and xylan degradation, such as GH10 endoxylanase, GH5_5 endoglucanase, and GH7 cellobiohydrolase, were detected after 5 days. Peptides corresponding to potential cellulose-degrading enzymes GH12, GH45, lytic polysaccharide monooxygenase, and cellobiose dehydrogenase were most abundant after 7 days. This sequential production of enzymes provides a mechanism consistent with selective ligninolysis by C. subvermispora .

Furylglycolic acid (FA), a pseudoaromatic hydroxy-acid suitable for copolymerization with lactic acid, can be produced from glucose via enzymatically derived cortalcerone using a combination of Bronsted and Lewis acid catalysts. Cortalcerone is first converted to furylglyoxal hydrate (FH) over a Bronsted acid site (HCl or Al-containing beta-zeolite), and FH is subsequently converted to FA over a Lewis acid site (Sn-beta zeolite). Selectivity for conversion of FH to FA is as high as 80% at 12% conversion using tetrahydrofuran (THF) as a solvent at 358 K. Higher conversion of FH leads to FA-catalyzed degradation of FH and subsequent deactivation of the catalyst by the deposition of carbonaceous residues. The deactivated catalyst can be regenerated by calcination. Cortalcerone can be produced from 10% glucose solution using recombinant Escherichia coli strains expressing pyranose 2-oxidase and aldos-2-ulose dehydratase from the wood-decay fungus Phanerochaete chrysosporium BKM-F-1767. This enzymatically deri...

Certain wood decay basidiomycetes, collectively referred to as brown rot fungi, rapidly depolymerize cellulose while leaving behind the bulk of cell wall lignin as a modified residue. The mechanism(s) employed is unclear, but considerable evidence implicates the involvement of diffusible oxidants generated via Fenton-like chemistry. Toward a better understanding of this process, we have examined the transcriptome and secretome of Wolfiporia cocos when cultivated on media containing glucose, purified crystalline cellulose, aspen ( Populus grandidentata ), or lodgepole pine ( Pinus contorta ) as the sole carbon source. Compared to the results obtained with glucose, 30, 183, and 207 genes exhibited 4-fold increases in transcript levels in cellulose, aspen, and lodgepole pine, respectively. Mass spectrometry identified peptides corresponding to 64 glycoside hydrolase (GH) proteins, and of these, 17 corresponded to transcripts upregulated on one or both woody substrates. Most of these genes were broadly categorized as hemicellulases or chitinases. Consistent with an important role for hydroxyl radical in cellulose depolymerization, high transcript levels and upregulation were observed for genes involved in iron homeostasis, iron reduction, and extracellular peroxide generation. These patterns of regulation differ markedly from those of the closely related brown rot fungus Postia placenta and expand the number of enzymes potentially involved in the oxidative depolymerization of cellulose. IMPORTANCE The decomposition of wood is an essential component of nutrient cycling in forest ecosystems. Few microbes have the capacity to efficiently degrade woody substrates, and the mechanism(s) is poorly understood. Toward a better understanding of these processes, we show that when grown on wood as a sole carbon source the brown rot fungus W. cocos expresses a unique repertoire of genes involved in oxidative and hydrolytic conversions of cell walls.

Identification of specific genes and enzymes involved in conversion of lignocellulosics from an expanding number of potential feedstocks is of growing interest to bioenergy process development. The basidiomycetous wood decay fungi Phanerochaete chrysosporium and Postia placenta are promising in this regard because they are able to utilize a wide range of simple and complex carbon compounds. However, systematic comparative studies with different woody substrates have not been reported. To address this issue, we examined gene expression of these fungi colonizing aspen ( Populus grandidentata ) and pine ( Pinus strobus ). Transcript levels of genes encoding extracellular glycoside hydrolases, thought to be important for hydrolytic cleavage of hemicelluloses and cellulose, showed little difference for P. placenta colonizing pine versus aspen as the sole carbon source. However, 164 genes exhibited significant differences in transcript accumulation for these substrates. Among these, 15 cytochrome P450s were upregulated in pine relative to aspen. Of 72 P. placenta extracellular proteins identified unambiguously by mass spectrometry, 52 were detected while colonizing both substrates and 10 were identified in pine but not aspen cultures. Most of the 178 P. chrysosporium glycoside hydrolase genes showed similar transcript levels on both substrates, but 13 accumulated >2-fold higher levels on aspen than on pine. Of 118 confidently identified proteins, 31 were identified in both substrates and 57 were identified in pine but not aspen cultures. Thus, P. placenta and P. chrysosporium gene expression patterns are influenced substantially by wood species. Such adaptations to the carbon source may also reflect fundamental differences in the mechanisms by which these fungi attack plant cell walls.

The wood decay basidiomycete Phanerochaete chrysosporium was grown under standard ligninolytic or cellulolytic conditions and subjected to whole-genome expression microarray analysis and liquid chromatography-tandem mass spectrometry of extracellular proteins. A total of 545 genes were flagged on the basis of significant changes in transcript accumulation and/or peptide sequences of the secreted proteins. Under nitrogen or carbon limitation, lignin and manganese peroxidase expression increased relative to nutrient replete medium. Various extracellular oxidases were also secreted in these media, supporting a physiological connection based on peroxide generation. Numerous genes presumed to be involved in mobilizing and recycling nitrogen were expressed under nitrogen limitation, and among these were several secreted glutamic acid proteases not previously observed. In medium containing microcrystalline cellulose as the sole carbon source, numerous genes encoding carbohydrate-active enzymes were upregulated. Among these were six members of the glycoside hydrolase family 61, as well as several polysaccharide lyases and carbohydrate esterases. Presenting a daunting challenge for future research, more than 190 upregulated genes are predicted to encode proteins of unknown function. Of these hypothetical proteins, approximately one-third featured predicted secretion signals, and 54 encoded proteins detected in extracellular filtrates. Our results affirm the importance of certain oxidative enzymes and, underscoring the complexity of lignocellulose degradation, also support an important role for many new proteins of unknown function.

20 p.-3 tab.-9 fig., 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., The major portions of this work were performed under US Department of Agriculture Cooperative State, Research, Education, and Extension Service Grant 2007-35504-18257 (to DC and RAB). The US Department of Energy Joint Genome Institute is supported by the Office of Science of the US Department of Energy under Contract DE-AC02-05CH11231. This work was also supported by the HIPOP (BIO2011-26694) project of the Spanish Ministry of Economy and Competitiveness (MINECO) (to FJRD), the PEROXICATS (KBBE-2010-4-265397) and INDOX (KBBE-2013-.3.3-04-613549) European projects (to ATM), and the Chilean National Fund for Scientific and Technological Development Grant 1131030 (to LFL).

The white rot basidiomycete, Phanerochaete chrysosporium, employs an array of extracellular enzymes to completely degrade the major polymers of wood: cellulose, hemicellulose and lignin. Towards the identification of participating enzymes, 268 likely secreted proteins were predicted using SignalP and TargetP algorithms. To assess the reliability of secretome predictions and to evaluate the usefulness of the current database, we performed shotgun LC-MS/MS on cultures grown on standard cellulose-containing medium. A total of 182 unique peptide sequences were matched to 50 specific genes, of which 24 were among the secretome subset. Underscoring the rich genetic diversity of P. chrysosporium, identifications included 32 glycosyl hydrolases. Functionally interconnected enzyme groups were recognized. For example, the multiple endoglucanases and processive exocellobiohydrolases observed quite probably attack cellulose in a synergistic manner. In addition, a hemicellulolytic system included endoxylanases, alpha-galactosidase, acetyl xylan esterase, and alpha-l-arabinofuranosidase. Glucose and cellobiose metabolism likely involves cellobiose dehydrogenase, glucose oxidase, and various inverting glycoside hydrolases, all perhaps enhanced by an epimerase. To evaluate the completeness of the current database, mass spectroscopy analysis was performed on a larger and more inclusive dataset containing all possible ORFs. This allowed identification of a previously undetected hypothetical protein and a putative acid phosphatase. The expression of several genes was supported by RT-PCR amplification of their cDNAs.

Pyranose 2-oxidase (POX) was recovered fromPhanerochaete chrysosporiumBKM-F-1767 solid substrate culture using mild extraction conditions and was purified.13C-nuclear magnetic resonance confirmed production ofd-arabino-hexos-2-ulose (glucosone) fromd-glucose with the oxidase. Peptide fingerprints generated by liquid chromatography-tandem mass spectrometry of tryptic digests and analysis of the corresponding cDNA revealed a structurally unusual sequence for theP. chrysosporiumPOX. Relatively high levels ofpoxtranscript were detected under carbon-starved culture conditions but not under nutrient sufficiency. This regulation pattern is similar to that observed for lignin peroxidases, manganese peroxidases, and glyoxal oxidase ofP. chrysosporium, supporting evidence that POX has a role in lignocellulose degradation.

We examined gene expression patterns in the lignin-degrading fungus Phanerochaete chrysosporium when it colonizes hybrid poplar ( Populus alba × tremula ) and syringyl (S)-rich transgenic derivatives. A combination of microarrays and liquid chromatography-tandem mass spectrometry (LC-MS/MS) allowed detection of a total of 9,959 transcripts and 793 proteins. Comparisons of P. chrysosporium transcript abundance in medium containing poplar or glucose as a sole carbon source showed 113 regulated genes, 11 of which were significantly higher (>2-fold, P < 0.05) in transgenic line 64 relative to the parental line. Possibly related to the very large amounts of syringyl (S) units in this transgenic tree (94 mol% S), several oxidoreductases were among the upregulated genes. Peptides corresponding to a total of 18 oxidoreductases were identified in medium consisting of biomass from line 64 or 82 (85 mol% S) but not in the parental clone (65 mol% S). These results demonstrate that P. chrysosporium gene expression patterns are substantially influenced by lignin composition.

To degrade the polysaccharides, wood-decay fungi secrete a variety of glycoside hydrolases (GHs) and carbohydrate esterases (CEs) classified into various sequence-based families of carbohydrate-active 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 β-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.

Lignin peroxidase-like genes were PCR amplified from Phanerochaete sordida and Ceriporiopsis subvermispora, fungi lacking lignin peroxidase (LiP) activity. Amplification products were highly similar to previously described LiP genes. Using reverse transcription-coupled PCR a LiP-like cDNA clone was amplified from P. sordida RNA. In contrast, no evidence was obtained for transcription of C. subvermispora LiP genes.

A 1747-bp insertion within a lignin peroxidase allele of Phanerochaete chrysosporium BKM-F-1767 is described. Pce1, the element, lies immediately adjacent to the fourth intron of lip12. Southern blots reveal the presence of Pce1-homologous sequences in other P. chrysosporium strains. Transposon-like features include inverted terminal repeats and a dinucleotide (TA) target duplication. Atypical of transposons, Pce1 is present at very low copy numbers (one to five copies), and conserved transposase motifs are lacking. The mutation transcriptionally inactivates lip12 and is inherited in a 1:1 Mendelian fashion among haploid progeny. Thus, Pce1 is a transposon-like element that may play a significant role in generating ligninolytic variation in certain P. chrysosporium strains.

5 páginas, 1 figura, 1 tabla, 22 figuras suplementarias, 16 tablas suplementarias -- PAGS nros. 1715-1719 et al., Wood is a major pool of organic carbon that is highly resistant to decay, owing largely to the presence of lignin. The only organisms capable of substantial lignin decay are white rot fungi in the Agaricomycetes, which also contains non–lignin-degrading brown rot and ectomycorrhizal species. Comparative analyses of 31 fungal genomes (12 generated for this study) suggest that lignin-degrading peroxidases expanded in the lineage leading to the ancestor of the Agaricomycetes, which is reconstructed as a white rot species, and then contracted in parallel lineages leading to brown rot and mycorrhizal species. Molecular clock analyses suggest that the origin of lignin degradation might have coincided with the sharp decrease in the rate of organic carbon burial around the end of the Carboniferous period, The work conducted by the U.S. Department of Energy Joint Genome Institute was supported by the Office of Science of the U.S. Department of Energy under contract DE-AC02-05CH11231. Also supported by the Assembling the Fungal Tree of Life (AFTOL) project under NSF awards DEB-0732968 (D.S.H.), DEB-0732993 (J.W.S.), and DEB-0732550 (D.J.M.).

Determining linkage is problematic for genes lacking easily identifiable phenotypes and for organisms without well-defined genetic recombination systems. Phanerochaete chrysosporium with its lignin peroxidase (LiP) gene family typifies these difficulties. We describe an experimental approach whereby the segregation of specific alleles is directly monitored during sexual fruiting. The method establishes linkage relationships among genes for which there are no mutations, and it is applicable to a wide range of genes, gene families and organisms. Using this approach, five P. chrysosporium linkage groups were identified. Ten LiP genes were distributed among three of these groups. One co-segregating group contained eight closely linked LiP genes. Another LiP gene was linked to a cellobiohydrolase gene cluster. These genetic linkages were consistent with physical mapping by pulsed field gel electrophoresis. Based on the identification of allelic relationships, a uniform nomenclature for LiP genes is also described.

Lignin depolymerization is catalyzed by extracellular peroxidases of white-rot basidiomycetes such as Phanerochaete chrysosporium . In submerged culture, production of multiple lignin peroxidase (LiP) isozymes is derepressed under carbon, nitrogen, or sulfur limitation. The roles of the individual isozymes in lignin degradation and their genetic regulation are poorly understood (reviewed by Kirk and Farrell, 1987).

Cellulose degradation by brown rot fungi, such as Postia placenta , is poorly understood relative to the phylogenetically related white rot basidiomycete, Phanerochaete chrysosporium . To elucidate the number, structure, and regulation of genes involved in lignocellulosic cell wall attack, secretome and transcriptome analyses were performed on both wood decay fungi cultured for 5 days in media containing ball-milled aspen or glucose as the sole carbon source. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), a total of 67 and 79 proteins were identified in the extracellular fluids of P. placenta and P. chrysosporium cultures, respectively. Viewed together with transcript profiles, P. chrysosporium employs an array of extracellular glycosyl hydrolases to simultaneously attack cellulose and hemicelluloses. In contrast, under these same conditions, P. placenta secretes an array of hemicellulases but few potential cellulases. The two species display distinct expression patterns for oxidoreductase-encoding genes. In P. placenta , these patterns are consistent with an extracellular Fenton system and include the upregulation of genes involved in iron acquisition, in the synthesis of low-molecular-weight quinones, and possibly in redox cycling reactions.

Lignin peroxidases (LiP) of Phanerochaete chrysosporium are encoded by a family of six closely related genes. Five LiP genes have been localized to the same dimorphic chromosome. In this investigation, relative transcript levels of the LiP genes were determined. Transcripts of the LiPA, LiPB, and O282 genes were at similar levels in both carbon- and nitrogen-limited cultures. In contrast, transcription of the GLG5, V4, and GLG4 genes was dramatically altered by culture conditions. Under carbon-limited conditions, GLG4 transcripts were, by far, the most abundant. Southern blot analyses of clamped homogeneous field gels were used to map the GLG4 gene to a dimorphic chromosome separate from the other LiP genes.

Brown-rot fungi such as Postia placenta are common inhabitants of forest ecosystems and are also largely responsible for the destructive decay of wooden structures. Rapid depolymerization of cellulose is a distinguishing feature of brown-rot, but the biochemical mechanisms and underlying genetics are poorly understood. Systematic examination of the P. placenta genome, transcriptome, and secretome revealed unique extracellular enzyme systems, including an unusual repertoire of extracellular glycoside hydrolases. Genes encoding exocellobiohydrolases and cellulose-binding domains, typical of cellulolytic microbes, are absent in this efficient cellulose-degrading fungus. When P. placenta was grown in medium containing cellulose as sole carbon source, transcripts corresponding to many hemicellulases and to a single putative β -1–4 endoglucanase were expressed at high levels relative to glucose-grown cultures. These transcript profiles were confirmed by direct identification of peptides by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Also upregulated during growth on cellulose medium were putative iron reductases, quinone reductase, and structurally divergent oxidases potentially involved in extracellular generation of Fe(II) and H2O2. These observations are consistent with a biodegradative role for Fenton chemistry in which Fe(II) and H2O2 react to form hydroxyl radicals, highly reactive oxidants capable of depolymerizing cellulose. The P. placenta genome resources provide unparalleled opportunities for investigating such unusual mechanisms of cellulose conversion. More broadly, the genome offers insight into the diversification of lignocellulose degrading mechanisms in fungi. Comparisons with the closely related white-rot fungus Phanerochaete chrysosporium support an evolutionary shift from white-rot to brown-rot during which the capacity for efficient depolymerization of lignin was lost. This work was supported by the U.S. Department of Energy’s Office of Science, Biological and Environmental Research Program, and University of California, Lawrence Berkeley National Laboratory Contract DE-AC02–05CH11231; Lawrence Livermore National Laboratory Contract DE-AC52–07NA27344; Los Alamos National Laboratory Contract DE-AC02–06NA25396; University of Wisconsin Grant DE-FG02–87ER13712; Forest Products Laboratory, U.S. Department of Agriculture, Cooperative State Research, Education, and Extension Services Grant 2007–35504-18257; National Institutes of Health Grant GM060201 (to University of New Mexico); Centro de Investigaciones Biológicas (Madrid) EUproject NMP2–2006-026456; Ministry of Education Czech Republic Grant LC06066.

The white-rot basidiomycete Phanerochaete chrysosporium employs extracellular enzymes to completely degrade the major polymers of wood: cellulose, hemicellulose, and lignin. Analysis of a total of 10,048 v2.1 gene models predicts 769 secreted proteins, a substantial increase over the 268 models identiWed in the earlier database (v1.0). Within the v2.1 ‘computational secretome,’ 43% showed no signiWcant similarity to known proteins, but were structurally related to other hypothetical protein sequences. In contrast, 53% showed signiWcant similarity to known protein sequences including 87 models assigned to 33 glycoside hydrolase families and 52 sequences distributed among 13 peptidase families. When grown under standard ligninolytic conditions, peptides corresponding to 11 peptidase genes were identiWed in culture Wltrates by mass spectrometry (LS–MS/MS). Five peptidases were members of a large family of aspartyl proteases, many of which were localized to gene clusters. Consistent with a role in dephosphorylation of lignin peroxidase, a mannose-6-phosphatase (M6Pase) was also identiWed in carbon-starved cultures. Beyond proteases and M6Pase, 28 speciWc gene products were identiWed including several representatives of gene families. These included 4 lignin peroxidases, 3 lipases, 2 carboxylesterases, and 8 glycosyl hydrolases. The results underscore the rich genetic diversity and complexity of P. chrysosporium’s extracellular enzyme systems. Published by Elsevier Inc.

Indexación: Scopus. Genetic analyses of the lignin-degrading fungus Ceriporiopsis subvermispora is complicated by a dikaryotic nuclear condition and the absence of spore forms. Previous investigations had identified a family of closely related sequences encoding manganese peroxidase (MnP), but the relationship between genes and allelic variants could not be experimentally established. Addressing this issue, homokaryotic derivatives of C. subvermipora strain FP105752 were isolated from regenerated protoplasts. Designated CsA and CsB, their homokaryotic nature was established by polymerase chain reaction amplification and sequence analysis of the allelic variants of three MnP genes. Isoelectrofocusing revealed fewer MnP isoenzymes in filtrates of homokaryon cultures relative to the parental strain. The homokaryotic strains will simplify genetic analyses, particularly the identification of new genes. https://academic-oup-com.recursosbiblioteca.unab.cl/femsle/article/199/1/91/553870

Analysis of complex gene families in the lignin-degrading basidiomycete Phanerochaete chrysosporium has been hampered by the dikaryotic nuclear condition. To facilitate genetic investigations in P. chrysosporium strain BKM-F-1767, we isolated a homokaryon from regenerated protoplasts. The nuclear condition was established by PCR amplification of five unlinked genes followed by probing with allele-specific oligonucleotides. Under standard nitrogen-limited culture conditions, lignin peroxidase, manganese peroxidase, and glyoxal oxidase activities of the homokaryon were equivalent to those of the parental dikaryon. We used the homokaryon to determine the genomic organization and to assess transcriptional effects of a family of repetitive elements. Previous studies had identified an insertional mutation, Pce1 , within lignin peroxidase allele lipI2 . The element resembled nonautonomous class II transposons and was present in multiple copies in strain BKM-F-1767. In the present study, three additional copies of the Pce1 -like element were cloned and sequenced. The distribution of elements was nonrandom; all localized to the same 3.7-Mb chromosome, as assessed by segregation analysis and Southern blot analysis of the homokaryon. Reverse transcription-PCR (RT-PCR) showed that Pce1 was not spliced from the lipI2 transcript in either the homokaryon or the parental dikaryon. However, both strains had equivalent lignin peroxidase activity, suggesting that some lip genes may be redundant.

The transcripts of structurally related cellobiohydrolase genes in Phanerochaete chrysosporium -colonized wood chips were quantified. The transcript patterns obtained were dramatically different from the transcript patterns obtained previously in defined media. Cellobiose dehydrogenase transcripts were also detected, which is consistent with the hypothesis that such transcripts play an important role in cellulose degradation.

A method to identify and differentiate allelic variants of the gene encoding lignin peroxidase isozyme H8 is presented. The strategy involves amplifying a variable region of the gene's carboxy terminus by use of the polymerase chain reaction and then probing with allele-specific oligonucleotides.

Three lignin peroxidase (LiP) genes from the basidiomycete Phanerochaete chrysosporium were cloned on a single 30 kb cosmid insert. One gene, GLG5, is the genomic equivalent of a previously reported cDNA clone, CLG5. The other two LiP genes are transcriptionally convergent and map to a position approximately 15 kb downstream of GLG5. The translational stop codons of these genes are separated by 1.3 kb. Analysis of homokaryons established allelic relationships to previously described LiP clones. Using clamped homogeneous electrical field electrophoresis (CHEF), seven chromosomal bands were resolved from P. chrysosporium genomic DNA. On CHEF gel Southern blots, the LiP gene family was localized to a single, dimorphic chromosome.

Promoter sequences from the Aspergillus niger glucoamylase-encoding gene (glaA) were linked to the bacterial hygromycin (Hy) phosphotransferase-encoding gene (hph) and this chimeric marker was used to select Hy-resistant (HyR) Ustilago maydis transformants. This is an example of an Ascomycete promoter functioning in a Basidiomycete. HyR transformants varied with respect to copy number of integrated vector, mitotic stability, and tolerance to Hy. Only 216 bp of glaA promoter sequence is required for expression in U. maydis but this promoter is not induced by starch as it is in Aspergillus spp. The transcriptional start points are the same in U. maydis and A. niger.

Cultures of Phanerochaete chrysosporium were examined for the presence of bacteria as previously described (F. Seigle-Murandi, P. Guiraud, C. Falsen, and K.-E. Eriksson, Appl. Environ. Microbiol. 62:2477-2481, 1996). Under no conditions could bacteria be isolated from cultures of P. chrysosporium. With PCR primers corresponding to small-subunit rRNA genes, no bacterium-like product could be amplified from cultures of the widely used P. chrysosporium strain BKM-F-1767. Thus, we could find no evidence of bacteria in association with P. chrysosporium BKM-F-1767.

The genomic clones encoding lignin peroxidase isozyme H8 and two closely related genes were isolated from Phanerochaete chrysosporium BKM-1767, and their nucleotide sequences were determined. The positions and approximate lengths of introns were found to be highly conserved in all three clones. Analysis of homokaryotic derivatives indicated that the three clones are not alleles of the same gene(s).