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Genome and transcriptome analyses of the thermophilic zygomycete fungus Rhizomucor miehei

ABSTRACT: The zygomycete fungi-like Rhizomucor miehei have been extensively exploited for the production of various enzymes. As a thermophilic fungus, R. miehei is capable of growing at temperatures that approach the upper limits for all eukaryotes. In order to study the thermophilic mechanism, the transcriptional profiles of R. miehei CAU432 grown at two different temperatures (at 30°C and 50°C) were investigated by RNA-seq analysis. Approximately 35 million high-quality reads were generated from each library, and 62% reads were uniquely mapped to the genome. A high percentage of reads (67.1%) were mapped to predicted protein-coding genes, while 3.96% reads were distributed in splice junctions, 3.49% reads in antisense transcripts, 2.27% in introns, and 21.9% in other genomic regions. The frequency of reads which mapped to different genes ranged from one to over 300,000. More than 90% of predicted genes (9,680, 93% at 30°C; 9,618, 93.6% at 50°C) were detected with at least one read, while 128 genes and 190 genes were uniquely expressed at 50°C and 30°C, respectively. The results show that 2,117 genes were differently expressed (P<0.001) by the fungus with more than two-fold changes. These genes include 849 up-regulated and 1,268 down-regulated genes in mycelia grown at 50°C. These significantly differently expressed genes include many genes, putatively involved in thermophilic process, such as HSP, chaperones and proteasome.

ORGANISM(S): Rhizomucor miehei

PROVIDER: GSE31580 | GEO | 2013/08/31

SECONDARY ACCESSION(S): PRJNA145613

REPOSITORIES: GEO

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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.

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Genome and transcriptome analyses of the thermophilic zygomycete fungus Rhizomucor miehei

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