Dataset Information

Combined genome and transcriptome sequencing to investigate the plant cell wall degrading enzyme system in the thermophilic fungus Malbranchea cinnamomea.

ABSTRACT: Background:Genome and transcriptome sequencing has greatly facilitated the understanding of biomass-degrading mechanisms in a number of fungal species. The information obtained enables the investigation and discovery of genes encoding proteins involved in plant cell wall degradation, which are crucial for saccharification of lignocellulosic biomass in second-generation biorefinery applications. The thermophilic fungus Malbranchea cinnamomea is an efficient producer of many industrially relevant enzymes and a detailed analysis of its genomic content will considerably enhance our understanding of its lignocellulolytic system and promote the discovery of novel proteins. Results:The 25-million-base-pair genome of M. cinnamomea FCH 10.5 was sequenced with 225× coverage. A total of 9437 protein-coding genes were predicted and annotated, among which 301 carbohydrate-active enzyme (CAZyme) domains were found. The putative CAZymes of M. cinnamomea cover cellulases, hemicellulases, chitinases and pectinases, equipping the fungus with the ability to grow on a wide variety of biomass types. Upregulation of 438 and 150 genes during growth on wheat bran and xylan, respectively, in comparison to growth on glucose was revealed. Among the most highly upregulated CAZymes on xylan were glycoside hydrolase family GH10 and GH11 xylanases, as well as a putative glucuronoyl esterase and a putative lytic polysaccharide monooxygenase (LPMO). AA9-domain-containing proteins were also found to be upregulated on wheat bran, as well as a putative cutinase and a protein harbouring a CBM9 domain. Several genes encoding secreted proteins of unknown function were also more abundant on wheat bran and xylan than on glucose. Conclusions:The comprehensive combined genome and transcriptome analysis of M. cinnamomea provides a detailed insight into its response to growth on different types of biomass. In addition, the study facilitates the further exploration and exploitation of the repertoire of industrially relevant lignocellulolytic enzymes of this fungus.

SUBMITTER: Huttner S

PROVIDER: S-EPMC5683368 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Combined genome and transcriptome sequencing to investigate the plant cell wall degrading enzyme system in the thermophilic fungus <i>Malbranchea cinnamomea</i>.

Hüttner Silvia S Nguyen Thanh Thuy TT Granchi Zoraide Z Chin-A-Woeng Thomas T Ahrén Dag D Larsbrink Johan J Thanh Vu Nguyen VN Olsson Lisbeth L

Biotechnology for biofuels 20171113

<h4>Background</h4>Genome and transcriptome sequencing has greatly facilitated the understanding of biomass-degrading mechanisms in a number of fungal species. The information obtained enables the investigation and discovery of genes encoding proteins involved in plant cell wall degradation, which are crucial for saccharification of lignocellulosic biomass in second-generation biorefinery applications. The thermophilic fungus <i>Malbranchea cinnamomea</i> is an efficient producer of many industr ...[more]

PMID: 29158777

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Project description:The thermophilic fungus Malbranchea cinnamomea belongs to the order of Onygenales and is a promising source of thermostable, industrially relevant biocatalysts, as it can grow at temperatures of more than 50°C and is able to utilise many different types of plant biomass. Enzymes from M. cinnamomea that have been characterised so far include an α-amylase, an α-glucosidase, xylanases and an alkaline β-1,3-1,4-glucanase (lichenase), all of which have been reported to have temperature optima between 50°C and 80°C. With this study, we complement the knowledge of the enzymatic repertoire of M. cinnamomea with a transcriptomic analysis of strain FCH 10.5 to provide a more comprehensive view of its lignocellulolytic enzyme system. Genes differentially expressed during growth on two different polymeric substrates, beechwood xylan and wheat bran, point to differences in the fungal response to the deconstruction of a hardwood hemicellulose (beechwood xylan) and a cereal hemicellulose (wheat bran). The data presented here will form the basis for a systematic exploration of the full potential of this fungus as a source of thermostable enzymes. We sequenced the genome of M. cinnamomea FCH 10.5, which was isolated from the compost of a waste treatment plant in Hanoi, Vietnam (PMC5604768, https://www.ncbi.nlm.nih.gov/nuccore/FQSS02000000). For RNAseq, the fungus was grown on three different carbon sources (glucose, wheat bran, beechwood xylan) at 50°C. Mycelium was harvested after 4h and 48h and RNA was extracted. For RNAseq analysis, the RNA of 4h and 48h samples was mixed 1:1, to get information about both early- and late-response genes during growth on the different carbon sources. Two independent duplicate experiments were done for each substrate. Total RNA was extracted using TRIzol (Invitrogen) and chloroform, and further purified with the RNeasy Plant RNA Kit with on-column DNAse digestion (QIAGEN). The quality of the purified RNA was verified by agarose gel electrophoresis, Nanodrop (Thermo Scientific) and Qubit (Life Technologies). The NEBNext Ultra Directional RNA Library Prep Kit for Illumina (New England Biolabs) was used to process the samples according to the manufacturer’s instructions. Briefly, mRNA was isolated from total RNA using oligo-dT magnetic beads and used to synthesise cDNA. The cDNA was ligated with sequencing adapters and PCR amplified. The quality and yield after sample preparation were determined with the Fragment Analyzer (Advanced Analytical). The size of the resulting products was consistent with the expected size distribution (a broad peak between 300-500 bp). Standard Illumina primers for Illumina cBot and HiSeq 2500, and the HiSeq control software HCS v2.2.58 were used according to manufacturer’s protocols for clustering and DNA sequencing with a concentration of 16.0 pM. The Illumina data analysis pipelines RTA v1.18.64 and Bcl2fastq v2.17 were used for image analysis, base calling, and quality check. Sequencing was performed on an Illumina HiSeq 2500 sequencer. The assembled genome from the DNA sequencing was used as a reference to map the reads using the packages Tophat (v2.0.14. Linux_x86_64) and Bowtie (v2-2.1.0) with a default mismatch rate of 2%. The frequency with which a read was mapped on a transcript was determined based on the mapped locations from the alignment. To normalise for transcript length, fpkm (fragments per kilobase of transcript per million mapped reads) were calculated. For differential expression analysis, the read counts were loaded into the DESeq package v 1.10.1. Genes were considered differentially expressed if they showed a log2 fold change ≥ 1 and the adjusted p-value was < 0.05.

Project description:The thermophilic biomass-degrader Malbranchea cinnamomea exhibits poor growth on cellulose but excellent growth on hemicelluloses as the sole carbon source. This is surprising considering that its genome encodes eight lytic polysaccharide monooxygenases (LPMOs) from auxiliary activity family 9 (AA9), enzymes known for their high potential in accelerating cellulose depolymerization. We characterized four of the eight (M. cinnamomea AA9s) McAA9s, namely, McAA9A, McAA9B, McAA9F, and McAA9H, to gain a deeper understanding about their roles in the fungus. The characterized McAA9s were active on hemicelluloses, including xylan, glucomannan, and xyloglucan, and furthermore, in accordance with transcriptomics data, differed in substrate specificity. Of the McAA9s, McAA9H is unique, as it preferentially cleaves residual xylan in phosphoric acid-swollen cellulose (PASC). Moreover, when exposed to cellulose-xylan blends, McAA9H shows a preference for xylan and for releasing (oxidized) xylooligosaccharides. The cellulose dependence of the xylan activity suggests that a flat conformation, with rigidity similar to that of cellulose microfibrils, is a prerequisite for productive interaction between xylan and the catalytic surface of the LPMO. McAA9H showed a similar trend on xyloglucan, underpinning the suggestion that LPMO activity on hemicelluloses strongly depends on the polymers' physicochemical context and conformation. Our results support the notion that LPMO multiplicity in fungal genomes relates to the large variety of copolymeric polysaccharide arrangements occurring in the plant cell wall.IMPORTANCE The Malbranchea cinnamomea LPMOs (McAA9s) showed activity on a broad range of soluble and insoluble substrates, suggesting their involvement in various steps of biomass degradation besides cellulose decomposition. Our results indicate that the fungal AA9 family is more diverse than originally thought and able to degrade almost any kind of plant cell wall polysaccharide. The discovery of an AA9 that preferentially cleaves xylan enhances our understanding of the physiological roles of LPMOs and enables the use of xylan-specific LPMOs in future applications.

Project description:BackgroundEnzymatic hydrolysis is a key step in the conversion of lignocellulosic polysaccharides to fermentable sugars for the production of biofuels and high-value chemicals. However, current enzyme preparations from mesophilic fungi are deficient in their thermostability and biomass-hydrolyzing efficiency at high temperatures. Thermophilic fungi represent promising sources of thermostable and highly active enzymes for improving the biomass-to-sugar conversion process. Here we present a comprehensive study on the lignocellulosic biomass-degrading ability and enzyme system of thermophilic fungus Malbranchea cinnamomea N12 and the application of its enzymes in the synergistic hydrolysis of lignocellulosic biomass.ResultsMalbranchea cinnamomea N12 was capable of utilizing untreated wheat straw to produce high levels of xylanases and efficiently degrading lignocellulose under thermophilic conditions. Temporal analysis of the wheat straw-induced secretome revealed that M. cinnamomea N12 successively degraded the lignocellulosic polysaccharides through sequential secretion of enzymes targeting xylan and cellulose. Xylanase-enriched cocktail from M. cinnamomea N12 was more active on native and alkali‑pretreated wheat straw than the commercial xylanases from Trichoderma reesei over temperatures ranging from 40 to 75 °C. Integration of M. cinnamomea N12 enzymes with the commercial cellulase preparation increased the glucose and xylose yields of alkali‑pretreated wheat straw by 32 and 166%, respectively, with pronounced effects at elevated temperature.ConclusionsThis study demonstrated the remarkable xylanase-producing ability and strategy of sequential lignocellulose breakdown of M. cinnamomea N12. A new process for the hydrolysis of lignocellulosic biomass was proposed, comprising thermophilic enzymolysis by enzymes of M. cinnamomea N12 followed with mesophilic enzymolysis by commercial cellulases. Developing M. cinnamomea N12 as platforms for thermophilic enzyme mixture production will provide new perspectives for improved conversion yields for current biomass saccharification schemes.

Dataset Information

Combined genome and transcriptome sequencing to investigate the plant cell wall degrading enzyme system in the thermophilic fungus Malbranchea cinnamomea.

Publications

Combined genome and transcriptome sequencing to investigate the plant cell wall degrading enzyme system in the thermophilic fungus <i>Malbranchea cinnamomea</i>.

Similar Datasets

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