Project description:Microarrays have become established tools for describing microbial systems, however the assessment of expression profiles for environmental microbial communities still presents unique challenges. Notably, the concentration of particular transcripts are likely very dilute relative to the pool of total RNA, and PCR-based amplification strategies are vulnerable to amplification biases and the appropriate primer selection. Thus, we apply a signal amplification approach, rather than template amplification, to analyze the expression of selected lignin-degrading enzymes in soil. Controls in the form of known amplicons and cDNA from Phanerochaete chrysosporium were included and mixed with the soil cDNA both before and after the signal amplification in order to assess the dynamic range of the microarray. We demonstrate that restored prairie soil expresses a diverse range of lignin-degrading enzymes following incubation with lignin substrate, while farmed agricultural soil does not. The mixed additions of control cDNA with soil cDNA indicate that the mixed biomass in the soil does interfere with low abundance transcript changes, nevertheless our microarray approach consistently reports the most robust signals. Keywords: comparative analysis, microbial ecology, soil microbial communities We used lignin degradation as a model process to demonstrate the use of an oligonucleotide microarray for directly detecting gene expression in soil communities using signal amplification instead of template amplification to avoid the introduction of PCR bias. In the current study, we analyzed mRNA isolated from two distinct soil microbial communities and demonstrate our ability to detect the expression of a small subset of lignin degrading genes following exposure to a lignitic substrate. We also included purified control amplicons and mixed target experiments with pure P. chrysosporium genomic cDNA to determine the level of interference from soil biomass on target hybridization.
Project description:Lignin is a biopolymer found in plant cell walls that accounts for 30% of the organic carbon in the biosphere. White-rot fungi (WRF) are considered the most efficient organisms at degrading lignin in Nature. While lignin depolymerization by WRF has been exhaustively studied, the possibility that WRF are able to utilize lignin as a carbon source is still a matter of controversy. Here we employ 13C-labeling and systems biology approaches to demonstrate that two WRF, Trametes versicolor and Gelatoporia subvermispora, funnel lignin-derived aromatic compounds into central carbon metabolism via intracellular catabolic pathways. These results provide insights into global carbon cycling in soil ecosystems, and furthermore establishes a foundation for employing WRF in simultaneous lignin depolymerization and bioconversion to bioproducts – a key step towards enabling a sustainable bioeconomy.
Project description:Wood-degrading fungi play a critical role in global carbon cycling, and their varied mechanisms for deconstruction offer pathways for industrial bioconversion. In this study, we used comparative genomics to isolate upregulation patterns among fungi with brown rot (carbohydrate-selective) or white rot (lignin-degrading) nutritional modes. Specifically, we used whole-transcriptome profiling to compare early, middle, and late decay stages on wood wafers, matching differentially-expressed gene (DEG) patterns with fungal growth and enzyme activities. This approach highlighted 34 genes uniquely upregulated in early brown rot stages, with notable candidates involved in generating reactive oxygen species (ROS) as a pretreatment mechanism during brown rot. This approach further isolated 18 genes in late brown rot stages that may be adapted to handle oxidatively-reacted lignocellulose components. By summing gene expression levels in functional classes, we also identified a broad and reliable distinction in glycoside hydrolase (GH) versus lignocellulose oxidative (LOX) transcript counts that may reflect the energy investment burden of lignin-degrading machinery among white rot fungi.
Project description:The Trametes versicolor genome is predicted to encode many enzymes that can effectively degrade lignin, making it a has potentially useful application intool for biopulping and biobleaching. Poplar is an important and widely cultivated species of tree species, which isand extensively applied used in the pulping industry. However, the wood degradation mechanism of T. versicolor from transcriptomic level is not clear. To reveal identify the enzymes that contributeing to lignocellulose degraredauction and its degradation mechanisms, we evaluated transcriptomic how study theof T. versicolor transcriptome was changes during evaluated growthing on the poplar wood relative to growth on glucose medium. 853 genes were differentially expressed;, 360 genes were up-regulated on poplar wood, and 493 genes were down-regulated on poplar wood. Notably, most genes relative involved into lignin degradation were up-regulated, including eight lignin peroxidase (LiP) genes, and two manganese peroxidase (MnP) genes etc. Genes encoding cellulose and hemicelluloses degrading-enzymesation were mostly down-regulated, including six endo-β-1, 4-glucanase genes, three cellobiohydrolase I genes, and one cellobiohydrolase II gene, etc. MeanwhileAdditionally, expression of more significant expansion of P450s in T. versicolor genome, along with differences in carbohydrate- and lignin-degrading enzymes, could bewere correlated withto poplar wood degradation. Our results revealed transcriptomic characterizeation transcriptomic changes related toof lignocellulose degradation. Therefore, our results cwould be benuseful for the development ofefit T. versicolor as a tool to improve the efficiency of lignin degradation, and provide a theoretical foundation for a new paper pulp manufacturing processe 1,T.versicolor groewn on PDA medium. 2, T. versicolor growing on the a glucose carbon medium of glucose. 3, T. versicolor growing on poplar medium
Project description:Microarrays have become established tools for describing microbial systems, however the assessment of expression profiles for environmental microbial communities still presents unique challenges. Notably, the concentration of particular transcripts are likely very dilute relative to the pool of total RNA, and PCR-based amplification strategies are vulnerable to amplification biases and the appropriate primer selection. Thus, we apply a signal amplification approach, rather than template amplification, to analyze the expression of selected lignin-degrading enzymes in soil. Controls in the form of known amplicons and cDNA from Phanerochaete chrysosporium were included and mixed with the soil cDNA both before and after the signal amplification in order to assess the dynamic range of the microarray. We demonstrate that restored prairie soil expresses a diverse range of lignin-degrading enzymes following incubation with lignin substrate, while farmed agricultural soil does not. The mixed additions of control cDNA with soil cDNA indicate that the mixed biomass in the soil does interfere with low abundance transcript changes, nevertheless our microarray approach consistently reports the most robust signals. Keywords: comparative analysis, microbial ecology, soil microbial communities
Project description:The Trametes versicolor genome is predicted to encode many enzymes that can effectively degrade lignin, making it a has potentially useful application intool for biopulping and biobleaching. Poplar is an important and widely cultivated species of tree species, which isand extensively applied used in the pulping industry. However, the wood degradation mechanism of T. versicolor from transcriptomic level is not clear. To reveal identify the enzymes that contributeing to lignocellulose degraredauction and its degradation mechanisms, we evaluated transcriptomic how study theof T. versicolor transcriptome was changes during evaluated growthing on the poplar wood relative to growth on glucose medium. 853 genes were differentially expressed;, 360 genes were up-regulated on poplar wood, and 493 genes were down-regulated on poplar wood. Notably, most genes relative involved into lignin degradation were up-regulated, including eight lignin peroxidase (LiP) genes, and two manganese peroxidase (MnP) genes etc. Genes encoding cellulose and hemicelluloses degrading-enzymesation were mostly down-regulated, including six endo-β-1, 4-glucanase genes, three cellobiohydrolase I genes, and one cellobiohydrolase II gene, etc. MeanwhileAdditionally, expression of more significant expansion of P450s in T. versicolor genome, along with differences in carbohydrate- and lignin-degrading enzymes, could bewere correlated withto poplar wood degradation. Our results revealed transcriptomic characterizeation transcriptomic changes related toof lignocellulose degradation. Therefore, our results cwould be benuseful for the development ofefit T. versicolor as a tool to improve the efficiency of lignin degradation, and provide a theoretical foundation for a new paper pulp manufacturing processe