ABSTRACT: Transcriptional profiling of the A. niger WT (N402) strain treated with xylan (1%, w/v) for 6, 12 and 24 h. The main objective was to identify genes related to cellulases and hemicellulases after treatment with the polysaccharide xylan. The experiment was further validated by enzymatic assays. Three-condition experiment: WT-Xylan for 6, 12 and 24 h at 30oC in batch culture. Firstly, the WT (N402) strain was grown in minimal medium with fructose as carbon source (control), and then transferred to 1% (w/v) xylan as carbon source. 2 biological replicates per time point.
Project description:Transcriptional profiling of the A. niger WT (N402) strain treated with xylan (1%, w/v) for 6, 12 and 24 h. The main objective was to identify genes related to cellulases and hemicellulases after treatment with the polysaccharide xylan. The experiment was further validated by enzymatic assays. Overall design: Three-condition experiment: WT-Xylan for 6, 12 and 24 h at 30oC in batch culture. Firstly, the WT (N402) strain was grown in minimal medium with fructose as carbon source (control), and then transferred to 1% (w/v) xylan as carbon source. 2 biological replicates per time point.
Project description:Hemicellulose, the second most abundant plant biomass fraction after cellulose, is widely viewed as a potential feedstock for the production of liquid fuels and other value-added materials. Degradation of hemicellulose by filamentous fungi requires production of many different enzymes, which are induced by biopolymers or its derivatives and regulated mainly at the transcriptional level through transcription factors (TFs). Neurospora crassa has been shown to express and secrete plant cell wall associated enzymes. To better understand genes specifically associated with degradation of hemicellulose, we identified 353 genes by transcriptome analysis of N. crassa wild type strain grown on beechwood xylan. Exposure to xylan induces 9 of the 19 predicted hemicellulase genes. The xylanolytic phenotype of strains with deletions in genes identified from the secretome and transcriptome analysis of wild type showed that none were essential for growth on beechwood xylan. The transcription factor XlnR/Xyr1 in Aspergillus and Trichoderma species is considered to be the major transcriptional regulator of genes encoding both cellulases and hemicellulases. We identified a xlnR/xyr1 homolog in N. crassa, NCU06971, termed xlr-1 (xylanase regulator 1). Deletion of xlr-1 in N. crassa abolishes the growth on xylan and xylose, but growth on cellulose was indistinguishable from wild type. To determine regulatory mechanisms associated with hemicellulose degradation, we explored the transcriptional regulon of XLR-1 under xylose and xylanolytic versus cellulolytic conditions. XLR-1 regulated only some predicted hemicellulase genes in N. crassa and was required for a full induction of several cellulase genes. Hemicellulase gene expression was induced by a combination of release from carbon catabolite repression (CCR) and induction. However, in N. crassa, xlr-1 is subject to non-CRE-1 mediated CCR. This systematic analysis provides the similarities and differences of hemicellulose degradation and regulation mechanisms used by N. crassa in comparison to other filamentous fungi. Four-condition experiments (minimal medium, xylan medium,xylose and Avicel medium) of mutant strain(xlr-1) compared to wild type strain; Cy3 and Cy5 dye swap
Project description:Transcriptional profiling of A. niger comparing WT strain vs. ΔXlnR strain treated with steam-exploded sugarcane bagasse (SESB) for 6, 12 and 24 h. The main objective was to identifiy genes related to cellulases and hemicellulases, comparing the differences between WT strain and the strain with the disrupted xylanolytic transcriptional activator gene, XlnR, after treatment with steam-exploded sugarcane. The experiment was further validated by real-time PCR, mass spectrometry of secreted proteins and enzymatic assays. Three-condition experiment : WT-SESB or ΔXlnR-SESB for 6, 12 and 24 h at 30 oC in batch culture. Firstly, WT and ΔXlnR strains were grown in minimal medium with fructose as carbon source (control), and then transferred to SESB as carbon source.
Project description:Investigation of whole genome gene expression level changes in Streptomyces sp. SirexAA-E (ActE) when grown on different carbon sources. The results of this study demonstrate that ActE upregulates a small number of genes specific for the utilization of the avaliable carbon source. Cellulolytic Streptomyces sp. SirexAA-E (ActE), isolated from the pinewood-boring wasp Sirex noctilio, has a genome enriched for biomass utilization. The secreted proteomes obtained from growth on pure polysaccharides catalyzed hydrolysis of cellulose, mannan, and xylan with specific activities comparable to Spezyme CP, a commercial cellulase preparation. During reaction of an ActE secretome with cellulose, reducing sugar release was markedly stimulated in the presence of O2. ActE also expresses and secretes an expanded repertoire of enzymes during growth on natural and pre-treated biomass. These results indicate a new microbial contribution to biomass utilization that is widely distributed in natural environments by insects Streptomyces sp. ActE was grown in minimal medium supplimented with 0.5% carbon source (glucose, sigmacell-20, xylan, chitin, cellobiose, or AFEX). Cells were grown for 7 days and total RNA was extracted from the cell pellet. At least 3 biological replicates were performed for each carbon source (glucose, 3; sigmacell, 3; xylan, 5; chitin, 3; cellobiose 3; AFEX 3). Each biological replicate contained 3 technical replicates. The complete dataset were RMA Background Corrected, quantile normalized, the RMA algorithm was utilized by DNAStar ArrayStar.
Project description:Transcriptional profiling of A. niger comparing mutant strains with the disrupted xylanolytic transcriptional activator gene, XlnR, the arabinolytic transcriptional activator gene, AraR, and the double mutant (ΔXlnR, ΔaraR and ΔaraRΔXlnR, repsectively) treated with steam-exploded sugarcane bagasse (SEB) for 12 and 24 h. The main objective was to identify genes related to cellulases and hemicellulases in mutant strains grown on SEB, with indirect comparisons with the WT strain grown on SEB [the (WT/SEB) data deposited in GSE24798]. The experiment was further validated by real-time PCR and enzymatic assay. Two-condition experiment : A. niger mutant strains on SEB for 12 and 24 h at 30 oC in batch culture. Firstly, the strains were pre-grown in minimal medium with fructose as carbon source (control), and then transferred to SEB as carbon source.
Project description:Transcriptional profiling of A. niger comparing different strains (WT, ΔXlnR, ΔaraR and ΔaraRΔXlnR) treated with 25mM xylose + 25mM arabinose for 2 and 8h. The main objective was to identifiy genes related to primary metabolism and carbohydrate metabolism in general, for instance, cellulases and hemicellulases. The study will compare the differences between WT strain and the strains with the disrupted xylanolytic transcriptional activator gene, XlnR, the arabinolytic transcriptional activator gene, AraR, and the double mutant,after treatment with xylose+arabinose (XA treatment). The experiment was further validated by real-time PCR and enzymatic assays. Two-condition experiment : A. niger mutant strains on XA for 2 and 8 h at 30 oC in batch culture. Firstly, the strains were pre-grown in minimal medium with fructose as carbon source (control), and then transferred to xylose+arabinose (25 mM each) as carbon source.
Project description:Transcriptome of A. nidulans TNO2a3 and ∆ptpB strains when grown on minimal media plus casaminoacids and transferred to minimal media plus glucose as a sole carbon source for 4 hours Three conditions minimal media plus casaminoacids during 24 hours (reference) and minimal media plus glucose for 4 hours. Three strains TNO2a3 and ∆ptpB. Three biological repetitions of each timepoint of TNO2a3 / ∆ptpB
Project description:High throughput “omics technologies” such as transcriptomics and proteomics provide insights into the metabolic potential of an organism and have been used to understand the genetic and the central carbon metabolism mechanisms for the production of desired end products in various cellulolytic clostridia cultured on different substrates In this study, C. termitidis was cultured on lignocellulose derived simple and complex sugars: cellobiose, xylose, xylan and α–cellulose as sole carbon sources. 2D HPLC-MS/MS quantitative Proteomic profiles and RNA seq transcriptome profiles (next generation sequencing to identify and quantify RNA in biological samples) were analyzed to identify the genes involved in substrate degradation, cellodextrin transport and end product synthesis related genes Identification of these genes is important in understanding the metabolic networks of C. termitidis and could be valuable engineering targets for improving biomass to biofuel production. Closridium termitidis was cultured on 2g/L each of α-cellulose, xylan, cellobiose and xylose. Samples were collected from the exponential phase. 2 replicate experiments were conducted under each substrate condition
Project description:Filamentous fungi are powerful producers of hydrolytic enzymes for the deconstruction of plant cell wall polysaccharides. However, the central question of how these sugars are perceived in the context of the complex cell wall matrix remains largely elusive. To address this question in a systematic fashion we performed an extensive comparative systems analysis of how the model filamentous fungus Neurospora crassa responds to the three main cell wall polysaccharides: pectin, hemicellulose and cellulose. We found the pectic response to be largely independent of the cellulolytic one with some overlap to hemicellulose, and in its extent surprisingly high, suggesting advantages for the fungus beyond being a mere carbon source. Our approach furthermore allowed us to identify carbon source-specific adaptations, such as the induction of the unfolded protein response on cellulose, and a commonly induced set of 29 genes likely involved in carbon scouting. Moreover, by hierarchical clustering we generated a co-expression matrix useful for the discovery of new components involved in polysaccharide utilization. This is exemplified by the identification of lat-1, which we demonstrate to encode for the physiologically relevant arabinose transporter in Neurospora. The analyses presented here are an important step towards understanding fungal degradation processes of complex biomass. Our data are based on carbon source transfer experiments. For this, N. crassa pregrown in sucrose for 16 hrs was washed in media without carbon and then transferred to either pectin, orange peel powder (OPP), xylan, Avicel (cellulose), sucrose, or media without carbon (NoC) for another 4 hrs. Biological triplicates (pectin, Avicel, sucrose, NoC) or duplicates (OPP) were used to identify differentially expressed genes in WT. For xylan only a single library was prepared, which was found to correlate well with previously published microarray data (Sun et al. 2012; doi: 10.1128/EC.05327-11)
Project description:Various saprotrophic microorganisms, especially filamentous fungi, can efficiently degrade lignocellulose that is one of the most abundant natural material on earth. It consists of complex carbohydrates and aromatic polymers found in plant cell wall and thus in plant debris. Aspergillus fumigatus Z5 was isolated from compost heaps and showed highly efficient plant biomass-degradation capability.Genome analysis revealed an impressive array of genes encoding cellulases, hemicellulases, and pectinases involved in lignocellulosic biomass degradation. We sequenced the transcriptomes of Aspergillus fumigatus Z5 induced by sucrose, xylan, cellulose and rice straw, respectively. There were 444, 1711 and 1386 significantly differently (q-value ≤ 0.0001 and |log2 of the ratio of the RPM values| ≥ 2) expressed genes in xylan, cellulose and rice straw,respectively, relative to sucrose control. After incubation at 45 ℃, 145rpm for 20 hours with sucrose as the carbon source, mycelia were induced for 16 hours using xylan, cellulose and rice straw, respectively. Transcriptome induced by sucrose was used as the control when comparing the differences between other three transcriptomes (induced by xylan, cellulose and rice straw, respectively).