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:This study aims to reveal genes related to lignin production in Cenchrus purpureus through RNA-Seq. This species is widely used as forrage for cattle, and for the last years, due to its high biomass yield, has been considered as source to lignocellulosic ethanol. Given those two importances, lignin is a molecule related to low digestibility in cattle and recalcitrance in biofuel production. Eight samples were chosen from previous lignin production and forage quality data; four samples had low lignin production, and four had high lignin production. The highest nodes were collected for RNA extraction using TRIzol reagent, following Jordon-Thaden et a;. (2015) protocol. The cDNA library preparation was generated according to Illumina TruSeq Stranded mRNA Sample Prep kit protocol, and RNA sequencing was performed using HiSeq 2500 sequencer. Quality control was measured by FastQC software v 0.11.8. The sequenced reads were aligned to Cenchrus purpureus genome through STAR software v. 2.5.2b (Dobin et al., 2012). After that, the transcriptome was assembled using Stringtie v 2.0.4 software (Pertea et al., 2015). Salmon v 0.7.2 software (Patro et al., 2017) was used to quantify the sequenced reads. The DEG was identified using DESeq2 package, and genes functions were annotated through Trinotate software (Bryant et al., 2017). In total, approximately 130 million reads were sequenced. The final assembled transcriptome was formed by 101,169 transcripts. The differential expressed genes analysis revealed 52 significatively genes. Here, we highlighted genes related to sterol, L-serine and terpene biosynthetic process.
Project description:Transcriptomic analysis was performed on the main inflorescence stems of wild-type and lignin-modified lines growing under the same conditions.
Project description:Lignin is an aromatic plant cell wall polymer that facilitates water transport through the vasculature of plants. Although lignin’s ability to reduce bacterial growth been previously reported, it’s hydrophobicity complicates the ability to examine its biological effects on living cells in aqueous growth media. We recently described the ability to solvate lignin in Good’s buffers with neutral pH, a breakthrough that has allowed examination of lignin’s antimicrobial effects against the human pathogen Staphylococcus aureus. We previously showed that lignin damages the S. aureus cell membrane, causes increased cell clustering, and inhibits growth synergistically with tunicamycin, a teichoic acid synthesis inhibitor. In this current study, additional experiments were performed to better understand the physiological and transcriptomic responses of S. aureus to lignin. Intriguingly, lignin restored the susceptibility of genetically resistant S. aureus isolates to β-lactam antibiotics, dysregulated intracellular pH, and impaired normal cell division. Additionally, RNAseq analysis of lignin-treated cultures revealed a number of gene expression changes related to cell envelope, cell wall physiology, fatty acid metabolism and stress resistance. Altogether, these results represent the first comprehensive analysis of lignin’s antibacterial activity against S. aureus that provide clarity in deciphering the mechanisms of lignin’s antibacterial activity, while supporting the notion that lignin has potential to be repurposed for biomedical applications.
Project description:This study aims to reveal genes related to lignin production in Urochloa humidicola through RNA-Seq. This species is widely used as forrage for cattle, being some species of Urochloa responsible for 85% of pastures in Brazil. Given this importance, lignin is a molecule directly related to low digestibility in cattle. Eight samples were chosen from previous lignin production and forage quality data; four samples had low lignin production, and four had high lignin production. The second extended leaves were collected for RNA extraction using RNeasy® Plant Mini Kit. The cDNA library preparation was generated according to Illumina TruSeq Stranded mRNA Sample Prep kit protocol, and RNA sequencing was performed using HiSeq 2500 sequencer. Quality control was measured by FastQC software v 0.11.8. As Urochloa humidicola does not have a sequenced genome, a transcriptome assembly was built by two approaches: through Trinity v 2.8.4 (Grabherr et al., 2011), and Stringtie v 2.0.4 software (Pertea et al., 2015), using Urochloa ruziziensis as reference genome. Then, the final transcriptome was assembled using those two de novo assembles by PASA v 2.2 (Haas et al., 2003). Salmon v 0.7.2 software (Patro et al., 2017) was used to quantify the sequenced reads. The DEG was identified using DESeq2 package, and genes functions were annotated through Trinotate software (Bryant et al., 2017). In total, approximately 123 million reads were sequenced. The final assembled transcriptome was formed by 48,695 transcripts. The differential expressed genes analysis revealed 258 significatively genes. Here, we highlighted genes related to flavonoid biosynthetic process, regulation of phenylpropanoid metabolic process, and Myb-like DNA-binding domain.
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.