Project description:Using whole genome microarrays based on the annotated genomes of Phanerochaete chrysosporium, we monitored the changes in its transcriptomes relevant to cell wall degradation during growth on three chemically distinct Populus trichocarpa (poplar) wood substrates. Results of this study are sumbitted for review in Biotechnology for Biofuels From a data set of 10004 unique gene models, each NimbleGen (Madison, WI) array targeted 9959 genes and featured 12 unique 60mers per gene, all in triplicate. RNA and protein were obtained from P. chrysosporium strain RP-78 (USDA Forest Mycology Center, Forest Products Laboratory, Madison WI) grown in malt extract agar for 10 days prior to inoculation with wood wafers. Three poplar wood substrates with distinct cell wall chemical properties were selected from several hundred 4-year old Populus trichocarpa trees grown in a common garden field trial at the University of British Columbia (Canada). We selected three poplar genotypes based on cell wall chemical traits. Substrate A corresponds to a genotype with a higher than average lignin content and lower that average glucose content; Substrate B, a lower than average lignin content and higher that average glucose content; Substrate C lignin and glucose contents are near the population average. Poplar wood stems were cut into 0.5 mm wafers on a microtome, sterilized for 20 min at 121°C, dried at 50°C overnight, and cooled to room temperature. The specimens were then inoculated in Petri dishes with actively growing mycelia. Approximately 5 g of wood wafers were placed in each Petri dish (exact weights were recorded), sealed and incubated at 22°C and 70 ± 5% relative humidity for 10, 20 or 30 days. For RNA, the degraded wafers were removed from the Petri dishes, immediately snap-frozen in liquid nitrogen and stored at -80°C for later use. Total RNA was converted to Cy3 labelled cDNA, hybridized to microarrays and scanned as previously described by Vanden Wymelenberg et al 2010 (Appl Enviro Microbiol 76:3599-3610).The 24 arrays per fungal species were scanned and data extracted using NimbleScan v.2.4. The raw data was loaded into GeneSpring, where the intensities were converted to log2 and quantile normalized, and all probes per gene averaged. This data was then exported and further analyzed in R. For substrates “A” and “B”, three replicates were used for each wood substrate/fungus and incubation period combination. For substrate “C” only 2 biological replicates were employed.

Project description:The biodegradation of lignocellulose requires the disruption of its lignin, which shields the metabolically assimilable polysaccharides in this recalcitrant natural composite. Although a variety of microorganisms can attack lignocellulose, white rot basidiomycetes are uniquely efficient at this process, cleaving the recalcitrant intermonomer linkages of lignin via extracellular oxidative mechanisms and mineralizing many of the resulting fragments to carbon dioxide via intracellular processes. Considerable progress has been made in understanding this process in the model white rot fungus Phanerochaete chrysosporium, which expresses important components of its ligninolytic system in response to nutrient limitation, as part of its secondary metabolism. Biochemical and genetic evidence point to an important role in P. chrysosporium for secreted lignin peroxidases (LiPs), manganese peroxidases (MnPs), and H2O2-producing oxidases, which are thought to work together to cleave lignin into low molecular weight fragments. However, many aspects of ligninolysis by P. chrysosporium remain poorly understood. Although a definitive picture of the entire ligninolytic system in P. chrysosporium is not yet attainable, transcriptome analyses of the fungus grown on wood can provide useful clues. With the advent of the initial genome assembly and annotations (v1.0 and v2.1), microarray-based transcriptome analysis allowed examination of transcript levels of P. chrysosporium genes when grown in ball-milled wood and in defined growth media. This approach provided useful insights but was limited to 10048 v2.1 targets and complicated by the unpredictable manner in which the fungus responds to unnatural carbon sources in submerged basal salts media. A complete, fully coordinated ligninolytic system is likely not expressed by P. chrysosporium on ball-milled wood, because a potential route for regulatory feedback has been eliminated: the cellulose and hemicellulose in this substrate is readily accessible to enzymes, and thus ligninolysis is not essential for growth. An alternative approach is to compare levels of gene expression just before and after the onset of secondary metabolism and extracellular substrate oxidation by P. chrysosporium as it utilizes solid wood as its carbon source. If this can be done, and decay of the substrate is also confirmed, then the genes undergoing marked changes in expression during the metabolic transition can be identified with greater confidence. Although not all such genes are expected to have roles in biodegradation, this strategy may identify interesting candidates for future investigation. Here we report RNAseq-based transcriptomes to characterize changes in gene expression that occur during the transition to ligninolytic metabolism.

Project description:The biodegradation of lignocellulose requires the disruption of its lignin, which shields the metabolically assimilable polysaccharides in this recalcitrant natural composite. Although a variety of microorganisms can attack lignocellulose, white rot basidiomycetes are uniquely efficient at this process, cleaving the recalcitrant intermonomer linkages of lignin via extracellular oxidative mechanisms and mineralizing many of the resulting fragments to carbon dioxide via intracellular processes. Considerable progress has been made in understanding this process in the model white rot fungus Phanerochaete chrysosporium, which expresses important components of its ligninolytic system in response to nutrient limitation, as part of its secondary metabolism. Biochemical and genetic evidence point to an important role in P. chrysosporium for secreted lignin peroxidases (LiPs), manganese peroxidases (MnPs), and H2O2-producing oxidases, which are thought to work together to cleave lignin into low molecular weight fragments. However, many aspects of ligninolysis by P. chrysosporium remain poorly understood. Although a definitive picture of the entire ligninolytic system in P. chrysosporium is not yet attainable, transcriptome analyses of the fungus grown on wood can provide useful clues. With the advent of the initial genome assembly and annotations (v1.0 and v2.1), microarray-based transcriptome analysis allowed examination of transcript levels of P. chrysosporium genes when grown in ball-milled wood and in defined growth media. This approach provided useful insights but was limited to 10048 v2.1 targets and complicated by the unpredictable manner in which the fungus responds to unnatural carbon sources in submerged basal salts media. A complete, fully coordinated ligninolytic system is likely not expressed by P. chrysosporium on ball-milled wood, because a potential route for regulatory feedback has been eliminated: the cellulose and hemicellulose in this substrate is readily accessible to enzymes, and thus ligninolysis is not essential for growth. An alternative approach is to compare levels of gene expression just before and after the onset of secondary metabolism and extracellular substrate oxidation by P. chrysosporium as it utilizes solid wood as its carbon source. If this can be done, and decay of the substrate is also confirmed, then the genes undergoing marked changes in expression during the metabolic transition can be identified with greater confidence. Although not all such genes are expected to have roles in biodegradation, this strategy may identify interesting candidates for future investigation. Here we report RNAseq-based transcriptomes to characterize changes in gene expression that occur during the transition to ligninolytic metabolism. Phanerochaete chrysosporium was inoculated onto thin sections of wood. RNA was purified from colonized material after 40 and 96 hours. Single read 100 bp Illumina runs were performed.

Project description:BackgroundCellobiose dehydrogenase from Phanerochaete chrysosporium (PcCDH) is a key enzyme in lignocellulose depolymerization, biosensors and biofuel cells. For these applications, it should retain important molecular and catalytic properties when recombinantly expressed. While homologous expression is time-consuming and the prokaryote Escherichia coli is not suitable for expression of the two-domain flavocytochrome, the yeast Pichia pastoris is hyperglycosylating the enzyme. Fungal expression hosts like Aspergillus niger and Trichoderma reesei were successfully used to express CDH from the ascomycete Corynascus thermophilus. This study describes the expression of basidiomycetes PcCDH in T. reesei (PcCDHTr) and the detailed comparison of its molecular, catalytic and electrochemical properties in comparison with PcCDH expressed by P. chrysosporium and P. pastoris (PcCDHPp).ResultsPcCDHTr was recombinantly produced with a yield of 600 U L-1 after 4 days, which is fast compared to the secretion of the enzyme by P. chrysosporium. PcCDHTr and PcCDH were purified to homogeneity by two chromatographic steps. Both enzymes were comparatively characterized in terms of molecular and catalytic properties. The pH optima for electron acceptors are identical for PcCDHTr and PcCDH. The determined FAD cofactor occupancy of 70% for PcCDHTr is higher than for other recombinantly produced CDHs and its catalytic constants are in good accordance with those of PcCDH. Mass spectrometry showed high mannose-type N-glycans on PcCDH, but only single N-acetyl-D-glucosamine additions at the six potential N-glycosylation sites of PcCDHTr, which indicates the presence of an endo-N-acetyl-?-D-glucosaminidase in the supernatant.ConclusionsHeterologous production of PcCDHTr is faster and the yield higher than secretion by P. chrysosporium. It also does not need a cellulose-based medium that impedes efficient production and purification of CDH by binding to the polysaccharide. The obtained high uniformity of PcCDHTr glycoforms will be very useful to investigate electron transfer characteristics in biosensors and biofuel cells, which are depending on the spatial restrictions inflicted by high-mannose N-glycan trees. The determined catalytic and electrochemical properties of PcCDHTr are very similar to those of PcCDH and the FAD cofactor occupancy is good, which advocates T. reesei as expression host for engineered PcCDH for biosensors and biofuel cells.

Dataset Information

Quantitation of fungal mRNAs in complex substrates by reverse transcription PCR and its application to Phanerochaete chrysosporium-colonized soil.

Publications

Quantitation of fungal mRNAs in complex substrates by reverse transcription PCR and its application to Phanerochaete chrysosporium-colonized soil.

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