Dataset Information

Transcriptome analysis of Aspergillus niger grown on sugarcane bagasse.

ABSTRACT: BACKGROUND:Considering that the costs of cellulases and hemicellulases contribute substantially to the price of bioethanol, new studies aimed at understanding and improving cellulase efficiency and productivity are of paramount importance. Aspergillus niger has been shown to produce a wide spectrum of polysaccharide hydrolytic enzymes. To understand how to improve enzymatic cocktails that can hydrolyze pretreated sugarcane bagasse, we used a genomics approach to investigate which genes and pathways are transcriptionally modulated during growth of A. niger on steam-exploded sugarcane bagasse (SEB). RESULTS:Herein we report the main cellulase- and hemicellulase-encoding genes with increased expression during growth on SEB. We also sought to determine whether the mRNA accumulation of several SEB-induced genes encoding putative transporters is induced by xylose and dependent on glucose. We identified 18 (58% of A. niger predicted cellulases) and 21 (58% of A. niger predicted hemicellulases) cellulase- and hemicellulase-encoding genes, respectively, that were highly expressed during growth on SEB. CONCLUSIONS:Degradation of sugarcane bagasse requires production of many different enzymes which are regulated by the type and complexity of the available substrate. Our presently reported work opens new possibilities for understanding sugarcane biomass saccharification by A. niger hydrolases and for the construction of more efficient enzymatic cocktails for second-generation bioethanol.

SUBMITTER: de Souza WR

PROVIDER: S-EPMC3219568 | biostudies-literature | 2011 Oct

REPOSITORIES: biostudies-literature

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Transcriptome analysis of Aspergillus niger grown on sugarcane bagasse.

de Souza Wagner R WR de Gouvea Paula F PF Savoldi Marcela M Malavazi Iran I de Souza Bernardes Luciano A LA Goldman Maria Helena S MHS de Vries Ronald P RP de Castro Oliveira Juliana V JV Goldman Gustavo H GH

Biotechnology for biofuels 20111018

<h4>Background</h4>Considering that the costs of cellulases and hemicellulases contribute substantially to the price of bioethanol, new studies aimed at understanding and improving cellulase efficiency and productivity are of paramount importance. Aspergillus niger has been shown to produce a wide spectrum of polysaccharide hydrolytic enzymes. To understand how to improve enzymatic cocktails that can hydrolyze pretreated sugarcane bagasse, we used a genomics approach to investigate which genes a ...[more]

PMID: 22008461

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Project description:Second generation (2G) ethanol is produced by breaking down lignocellulosic biomass into fermentable sugars. In Brazil, sugarcane bagasse has been proposed as the lignocellulosic residue for this biofuel production. The enzymatic cocktails for the degradation of biomass-derived polysaccharides are mostly produced by fungi, such as Aspergillus niger and Trichoderma reesei. However, it is not yet fully understood how these microorganisms degrade plant biomass. In order to identify transcriptomic changes during steam-exploded bagasse (SEB) breakdown, we conducted a RNA-seq comparative transcriptome profiling of both fungi growing on SEB as carbon source.Particular attention was focused on CAZymes, sugar transporters, transcription factors (TFs) and other proteins related to lignocellulose degradation. Although genes coding for the main enzymes involved in biomass deconstruction were expressed by both fungal strains since the beginning of the growth in SEB, significant differences were found in their expression profiles. The expression of these enzymes is mainly regulated at the transcription level, and A. niger and T. reesei also showed differences in TFs content and in their expression. Several sugar transporters that were induced in both fungal strains could be new players on biomass degradation besides their role in sugar uptake. Interestingly, our findings revealed that in both strains several genes that code for proteins of unknown function and pro-oxidant, antioxidant, and detoxification enzymes were induced during growth in SEB as carbon source, but their specific roles on lignocellulose degradation remain to be elucidated.This is the first report of a time-course experiment monitoring the degradation of pretreated bagasse by two important fungi using the RNA-seq technology. It was possible to identify a set of genes that might be applied in several biotechnology fields. The data suggest that these two microorganisms employ different strategies for biomass breakdown. This knowledge can be exploited for the rational design of enzymatic cocktails and 2G ethanol production improvement.

Project description:The production of bioethanol from non-food agricultural residues represents an alternative energy source to fossil fuels for incorporation into the world's economy. Within the context of bioconversion of plant biomass into renewable energy using improved enzymatic cocktails, Illumina RNA-seq transcriptome profiling was conducted on a strain of Aspergillus tamarii, efficient in biomass polysaccharide degradation, in order to identify genes encoding proteins involved in plant biomass saccharification. Enzyme production and gene expression was compared following growth in liquid and semi-solid culture with steam-exploded sugarcane bagasse (SB) (1% w/v) and glucose (1% w/v) employed as contrasting sole carbon sources. Enzyme production following growth in liquid minimum medium supplemented with SB resulted in 0.626 and 0.711 UI.mL-1 xylanases after 24 and 48 h incubation, respectively. Transcriptome profiling revealed expression of over 7120 genes, with groups of genes modulated according to solid or semi-solid culture, as well as according to carbon source. Gene ontology analysis of genes expressed following SB hydrolysis revealed enrichment in xyloglucan metabolic process and xylan, pectin and glucan catabolic process, indicating up-regulation of genes involved in xylanase secretion. According to carbohydrate-active enzyme (CAZy) classification, 209 CAZyme-encoding genes were identified with significant differential expression on liquid or semi-solid SB, in comparison to equivalent growth on glucose as carbon source. Up-regulated CAZyme-encoding genes related to cellulases (CelA, CelB, CelC, CelD) and hemicellulases (XynG1, XynG2, XynF1, XylA, AxeA, arabinofuranosidase) showed up to a 10-fold log2FoldChange in expression levels. Five genes from the AA9 (GH61) family, related to lytic polysaccharide monooxygenase (LPMO), were also identified with significant expression up-regulation. The transcription factor gene XlnR, involved in induction of hemicellulases, showed up-regulation on liquid and semi-solid SB culture. Similarly, the gene ClrA, responsible for regulation of cellulases, showed increased expression on liquid SB culture. Over 150 potential transporter genes were also identified with increased expression on liquid and semi-solid SB culture. This first comprehensive analysis of the transcriptome of A. tamarii contributes to our understanding of genes and regulatory systems involved in cellulose and hemicellulose degradation in this fungus, offering potential for application in improved enzymatic cocktail development for plant biomass degradation in biorefinery applications.

Project description:BACKGROUND:Sugarcane bagasse has been proposed as a lignocellulosic residue for second-generation ethanol (2G) produced by breaking down biomass into fermentable sugars. The enzymatic cocktails for biomass degradation are mostly produced by fungi, but low cost and high efficiency can consolidate 2G technologies. A. fumigatus plays an important role in plant biomass degradation capabilities and recycling. To gain more insight into the divergence in gene expression during steam-exploded bagasse (SEB) breakdown, this study profiled the transcriptome of A. fumigatus by RNA sequencing to compare transcriptional profiles of A. fumigatus grown on media containing SEB or fructose as the sole carbon source. Secretome analysis was also performed using SDS-PAGE and LC-MS/MS. RESULTS:The maximum activities of cellulases (0.032 U mL-1), endo-1,4-β--xylanase (10.82 U mL-1) and endo-1,3-β glucanases (0.77 U mL-1) showed that functional CAZymes (carbohydrate-active enzymes) were secreted in the SEB culture conditions. Correlations between transcriptome and secretome data identified several CAZymes in A. fumigatus. Particular attention was given to CAZymes related to lignocellulose degradation and sugar transporters. Genes encoding glycoside hydrolase classes commonly expressed during the breakdown of cellulose, such as GH-5, 6, 7, 43, 45, and hemicellulose, such as GH-2, 10, 11, 30, 43, were found to be highly expressed in SEB conditions. Lytic polysaccharide monooxygenases (LPMO) classified as auxiliary activity families AA9 (GH61), CE (1, 4, 8, 15, 16), PL (1, 3, 4, 20) and GT (1, 2, 4, 8, 20, 35, 48) were also differentially expressed in this condition. Similarly, the most important enzymes related to biomass degradation, including endoxylanases, xyloglucanases, β-xylosidases, LPMOs, α-arabinofuranosidases, cellobiohydrolases, endoglucanases and β-glucosidases, were also identified in the secretome. CONCLUSIONS:This is the first report of a transcriptome and secretome experiment of Aspergillus fumigatus in the degradation of pretreated sugarcane bagasse. The results suggest that this strain employs important strategies for this complex degradation process. It was possible to identify a set of genes and proteins that might be applied in several biotechnology fields. This knowledge can be exploited for the improvement of 2G ethanol production by the rational design of enzymatic cocktails.

Project description:Brazil is the world’s second largest producer of ethanol. Increasing demand for this biofuel has called for investment in new technologies. One example of such technologies is the second-generation (2G) ethanol production. Lignocellulose comprises mainly cellulose and hemicellulose, which consist of high-energy sugars that can be converted into ethanol. Aspergillus sp play an important role in the recycling of lignocellulosic biomass. These species have been investigated as a cell factory to produce industrial enzymes. To improve our understanding of the mechanisms used by Aspergillus sp during degradation of plant biomass and to identify the hydrolytic enzymes secreted by these fungi, we have analyzed the transcriptome and secretome of an Aspergillus species grown on sugarcane bagasse (SEB). A. fumigatus was cultivated in the presence of fructose or SEB. Its cellulolytic and xylanolytic activities depended on time. The maximum activities of endoglucanase and xylanase from the fungus grown on SEB were 0.0029 and 10.82 U mL-1, respectively. Characterization of the transcriptome by RNAseq technology helped to identify genes that participated in the degradation of the biomass demonstrating potential application in the process of enzymatic hydrolysis. The RNAseq data revealed that 2287 genes were differentially expressed in the fungus grown on SEB; 1181 and 1046 of these genes were up- and down-regulated, respectively. There were several CAZymes among the up-regulated genes. Most of these enzymes belonged to the GH family, which includes important hydrolytic and accessory enzymes involved in the degradation of lignocellulose. Similarly, proteomic studies showed that a total of 130 proteins existed in the fungus grown on SEB. These proteins were classified into several groups of secreted extracellular enzymes, and their functional classification demonstrated that 59% of the proteins were CAZymes such as GH45-endoglucanases, GH6 and GH7-cellobiohydrolases, GH3-β-glucosidases, GH10-xylanases, and AA9-LPMO. Data obtained by transcriptome and secretome analyses indicated that A. fumigatus produces a considerable number of CAZymes that participate in the hydrolysis of biomass. These CAZymes are valuable for the lignocellulosic bioenergy industry, provide information for future studies on the degradation of biomass, and improve understanding of the role genes and enzymes play in the degradation process.

Project description:BackgroundOur dependence on fossil fuel sources and concern about the environment has generated a worldwide interest in establishing new sources of fuel and energy. Thus, the use of ethanol as a fuel is advantageous because it is an inexhaustible energy source and has minimal environmental impact. Currently, Brazil is the world's second largest producer of ethanol, which is produced from sugarcane juice fermentation. However, several studies suggest that Brazil could double its production per hectare by using sugarcane bagasse and straw, known as second-generation (2G) bioethanol. Nevertheless, the use of this biomass presents a challenge because the plant cell wall structure, which is composed of complex sugars (cellulose and hemicelluloses), must be broken down into fermentable sugar, such as glucose and xylose. To achieve this goal, several types of hydrolytic enzymes are necessary, and these enzymes represent the majority of the cost associated with 2G bioethanol processing. Reducing the cost of the saccharification process can be achieved via a comprehensive understanding of the hydrolytic mechanisms and enzyme secretion of polysaccharide-hydrolyzing microorganisms. In many natural habitats, several microorganisms degrade lignocellulosic biomass through a set of enzymes that act synergistically. In this study, two fungal species, Aspergillus niger and Trichoderma reesei, were grown on sugarcane biomass with two levels of cell wall complexity, culm in natura and pretreated bagasse. The production of enzymes related to biomass degradation was monitored using secretome analyses after 6, 12 and 24 hours. Concurrently, we analyzed the sugars in the supernatant.ResultsAnalyzing the concentration of monosaccharides in the supernatant, we observed that both species are able to disassemble the polysaccharides of sugarcane cell walls since 6 hours post-inoculation. The sugars from the polysaccharides such as arabinoxylan and ?-glucan (that compose the most external part of the cell wall in sugarcane) are likely the first to be released and assimilated by both species of fungi. At all time points tested, A. niger produced more enzymes (quantitatively and qualitatively) than T. reesei. However, the most important enzymes related to biomass degradation, including cellobiohydrolases, endoglucanases, ?-glucosidases, ?-xylosidases, endoxylanases, xyloglucanases, and ?-arabinofuranosidases, were identified in both secretomes. We also noticed that the both fungi produce more enzymes when grown in culm as a single carbon source.ConclusionOur work provides a detailed qualitative and semi-quantitative secretome analysis of A. niger and T. reesei grown on sugarcane biomass. Our data indicate that a combination of enzymes from both fungi is an interesting option to increase saccharification efficiency. In other words, these two fungal species might be combined for their usage in industrial processes.

Project description:BACKGROUND:Enzymatic plant biomass degradation by fungi is a highly complex process and one of the leading challenges in developing a biobased economy. Some industrial fungi (e.g. Aspergillus niger) have a long history of use with respect to plant biomass degradation and for that reason have become 'model' species for this topic. A. niger is a major industrial enzyme producer that has a broad ability to degrade plant based polysaccharides. A. niger wild-type, the (hemi-)cellulolytic regulator (xlnR) and xylulokinase (xkiA1) mutant strains were grown on a monocot (corn stover, CS) and dicot (soybean hulls, SBH) substrate. The xkiA1 mutant is unable to utilize the pentoses D-xylose and L-arabinose and the polysaccharide xylan, and was previously shown to accumulate inducers for the (hemi-)cellulolytic transcriptional activator XlnR and the arabinanolytic transcriptional activator AraR in the presence of pentoses, resulting in overexpression of their target genes. The xlnR mutant has reduced growth on xylan and down-regulation of its target genes. The mutants therefore have a similar phenotype on xylan, but an opposite transcriptional effect. D-xylose and L-arabinose are the most abundant monosaccharides after D-glucose in nearly all plant-derived biomass materials. In this study we evaluated the effect of the xlnR and xkiA1 mutation during growth on two pentose-rich substrates by transcriptome analysis. RESULTS:Particular attention was given to CAZymes, metabolic pathways and transcription factors related to the plant biomass degradation. Genes coding for the main enzymes involved in plant biomass degradation were down-regulated at the beginning of the growth on CS and SBH. However, at a later time point, significant differences were found in the expression profiles of both mutants on CS compared to SBH. CONCLUSION:This study demonstrates the high complexity of the plant biomass degradation process by fungi, by showing that mutant strains with fairly straightforward phenotypes on pure mono- and polysaccharides, have much less clear-cut phenotypes and transcriptomes on crude plant biomass.

Dataset Information

Transcriptome analysis of Aspergillus niger grown on sugarcane bagasse.

Publications

Transcriptome analysis of Aspergillus niger grown on sugarcane bagasse.

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