“Integrative genomic analysis of the bioprospection of regulators and accessory enzymes associated with cellulose degradation in a filamentous fungus (Trichoderma harzianum)”

- Integrative genomic analysis of the.
- In total, 1676 genes were annotated in the genomic regions analyzed.
- harzianum IOC3844.
- The genes induced/repressed under cellulose conditions included those important for plant biomass degradation, including CIP2 of the CE15 family and a copper-dependent LPMO of the AA9 family..
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- Full list of author information is available at the end of the article Ferreira Filho et al.
- Thus, increasing the number of studies related to the biotech- nological potential of T.
- Different strains of a specific fungal species have dif- ferent potentials for the degradation of plant biomass, and these differences may be associated with differences in the genome and regulation of CAZyme enzymes [17]..
- The three main groups involved in the hydrolysis of CEL are cellobiohydrolases, endo-β-1,4-glucanases and β-glucosidases.
- One of the great challenges in understanding the mo- lecular mechanism of biomass degradation is to capture how the transcription factors (TFs) act.
- in the regulation of plant biomass [25].
- This number has expanded rapidly in recent years, mainly due to the de- velopment of whole genome sequencing technologies as- sociated with the exponential increase in the number of bioinformatics analysis tools that produced massive amounts of information and increased the numbers of identified genes [25, 26]..
- We sequenced a massive amount of DNA and used the information obtained to integrate genomic data (genomic regions containing CAZymes), expression patterns (transcriptome under degradation conditions), proteins (secretome by mass spectrometry) and systems biology (with gene regulatory networks) to obtain a broad and precise overview of the CEL degradation pathways.
- Our study characterized the main genes, accessory enzymes and regions involved in the degrad- ation and regulation of hydrolytic enzymes.
- Screening for genes of interest resulted in a total of 62 regions that contained CAZyme genes related to the degradation of plant biomass in the ThIOC3844 genome.
- Sequencing of these regions generated 5 Mb total of the estimated 40 Mb genome (Additional file 1: Supplementary Table S2 and S3).
- 1 Distribution of the T.
- harzianum IOC3844 genes on the 1 Mb intervals of the seven chromosomes of T.
- For this analysis, we compared the genomic regions of ThIOC3844 against the entire genomes of different strains and species of the genus Trichoderma.
- Genomic comparison of the sequenced regions of ThIOC3844 with two other strains of the same species (T.
- For the T.
- virens Gv29–8 (TvGv29–8), the similarity was 86.55%, and for T.
- 2 Functional annotation of the genes predicted in the genomic regions of T.
- Expression determined by RNA-Seq and secreted proteins All genes predicted in the genomic regions were analyzed according to the expression data by RNA-Seq (under CEL.
- harzianum IOC3844 and those of other species of the genus Trichoderma spp.
- 3 CAZy classification of genes annotated in the genomic regions of T.
- among them, 51 were classified as CAZymes, such as beta-glucosidase of GH1 family (1.8-fold change - FC), LPMOs of the AA9 family (FC 5.0) and a hypothetical protein with CBM1 do- main (FC 3.7).
- Among the genes annotated as CAZymes in ThIOC3844, 31 were found in the secretome of ThIOC3844 under CEL conditions, and the main.
- In this analysis, we also used the expression levels of the secreted proteins.
- The gene with the highest transcripts per million (TPM) index (1567.4 TPM) was a cellobiohydrolase (EC 3.2.1.91) of the GH6 fam- ily.
- Phylogenetic analysis of the CLR2 factor showed a clear separation of this TF in relation to Basidiomycetes and Ascomycetes (Fig.
- However, even within these groups, con- siderable phylogenetic diversity was observed among the Table 1 Proteins identified in genomic and in the T.
- Structural modeling analysis of the CLR2 protein of ThIOC3844 was performed using T.
- For ThIOC3844, 59% of the residues were modeled, while T.
- A coregulation network of genes directly related to the CLR2 regulator was constructed, to search for insights.
- about other important proteins in the process of cellu- lases expression.
- Using the gene expression data of the secreted proteins, a Bayesian network of induced/repressed genes was con- structed based on the CEL growth conditions for T.
- 5 Molecular phylogeny of the CLR2 transcription factor in Ascomycota and Basidiomycota (a).
- In the present study, an integrative multiomics approach was used to mine CAZyme-rich regions of ThIOC3884..
- harzianum for the degradation of plant biomass.
- This is the first study that integrates results from different bio- technological approaches and focuses on the prediction of the most important enzymes and TFs used by T.
- reesei, the major CAZy families related to CEL degradation were identified in the genome (GH1, GH3, GH6, GH7, GH12, GH45 and AA9) [34], and the three-dimensional structures of cellu- lases have already been solved.
- Despite that, many key proteins in the degradation process still not well known, as well as transporters and TFs related to the regulation of related enzymes..
- The study of genomic regions is an important tool that provides a global view of the important genes and regu- latory regions of a genome [27, 35].
- However, se- quencing several regions of the T.
- involved in the degradation of CEL and hemicellulose, beyond the study of these genes’ clusters..
- In the used approach, we chose to select regions of interest instead of sequencing the complete T.
- A large number of fungal genomes have already been used as a platform to search for new genes related to the degradation of biomass.
- Our study results with the genomic re- gions of ThIOC3844 showed a large number of enzymes classified as CAZymes as well as TFs and transporters in clusters in the genome, important information for future studies on genetic modification of this lineage..
- however, a direct relationship does not always exist between the gene being highly expressed and the proteins that are important/active in the extracellular medium.
- Thus, in this work, in addition to studying the genes most expressed in the genomic re- gions, we also searched for those with a confirmed pres- ence in the fungal secretome under CEL degradation conditions.
- harzianum (IOC3844, B97 and T6776), which indicates that differences in enzyme production and efficiency may be related more to gene regulation mechanisms than to differences in the se- quence itself.
- In addition, synteny analysis showed a greater difference in relation to the T.
- 7 Molecular scheme of the enzymatic model in the degradation of cellulose in Trichoderma spp.
- harzianum strains are highly similar, studies from our group showed that stains differences can be observed in terms of regulation of CEL degradation-related genes and in the enzyme activity profiles [17, 18].
- harzianum strains (such as SNPs in introns and gene regions) can play an important role in the efficiency of strains..
- how- ever, CLR2 functional role in fungi of the genus Tricho- derma, including T.
- In the ThIOC3844 genome, we found a cluster with the CLR2 TF in association with other putative TFs, CAZymes, transporters and MFS permease.
- In this way, we analyzed the coregulation net- work of the CLR2 regulator.
- The present study illuminates unclear areas of the genomic organization, expression, and putative regulation of CLR2 in T..
- these genes may be important in the regulatory process of this factor, which is linked to the expression of cellulases in other filamentous fungi.
- We found several TFs, accessory enzymes, and transporters in the genomic re- gions of ThIOC3844 that may be important for the ex- pression/secretion of CAZyme genes.
- harzianum IOC3844 (ThIOC3844) was obtained from Institute Oswaldo Cruz (IOC, Rio de Janeiro, Brazil) and deposited on the Brazilian Collection of Environment and Industrial Microorganisms (CBMAI, Campinas, Brazil).
- In this way, three selections were made to find the positive clone, searches in the plate pool, in the column pool and in the positive column, to obtain the coordinates of the positive clones.
- An- notations of the ontologies were performed with Blas- t2GO [53].
- harzianum IOC3844 using global alignment through Nucmer (−maxmatch), which is part of the software package MUMmmer 3.23 [55].
- Delta-filter (−q), show- coords (−rcl), and DNADIFF (standard parameters) were used for filtering, obtaining the mapping coordinates and generating the statistical report in the alignment, re- spectively.
- In silico modeling of the CLR2 domain was performed using RaptorX protein structure prediction software (http://raptorx.uchicago.edu/) [61]..
- The reads from the RNA-Seq library were mapped against those of the ThIOC3844 genes using the CLC Genomics Workbench (QIAGEN, Aarhus, Denmark) [62].
- Secreted pro- teins were analyzed using a Blastp search of the annotated proteins in ThIOC3844 against a local protein database (Additional file 7 ) generated by a previous study that deter- mined the proteins secreted by the fungus under CEL and GLU conditions using a liquid chromatography tandem mass spectrometry (LC-MS/MS) technique [18]..
- The induction and repression networks were constructed based on the expres- sion data of a set of genes that were identified in the secre- tome of the CEL growth condition by the Bayesian inference method [63].
- If the secreted protein was present in the condition, it was assigned a value of one.
- The treatment conditions were considered regulators of the network to detect the direct relationships between the con- ditions and the genes.
- Cytoscape software v https://cytoscape.org/) was used for data ana- lysis and construction of the CLR2 subnetwork..
- Screening genes of interest in the genomic library of T.
- Distribution of the main GO terms of the annotated genes in T.
- Description of the species used for the phylogenetic analysis of the transcription factor CLR2.
- Description of the genes found in the coregulation networks..
- Description of the genomic regions sequenced in T.
- harzianum IOC3844..
- Description of the EC codes for T.
- Description of the CAZyme genes in T.
- Expression levels of the genes annotated in T.
- harzianum IOC3844 under cellulose conditions developed in the work of Horta et al., 2018..
- We also thank David Kudrna (Arizona Genomics Institute – The University of Arizona, USA), who assisted with the PacBio BAC sequencing.
- The funding bodies played no role in the design of the study, analysis and interpretation of the data or writing of the manuscript..
- The raw data of the genomic regions (PacBio reads) can be found by the accession number PRJNA647392.
- The raw data of the RNA-Seq (Illumina reads) can be found by the accession number PRJNA336221.
- The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest..
- Crystal structure and biochemical characterization of the recombinant ThBgl, a GH1 β -glucosidase overexpressed in Trichoderma harzianum under biomass degradation conditions.
- Cellulases and beyond: the first 70 years of the enzyme producer Trichoderma reesei.
- Genome sequencing and analysis of the biomass-degrading fungus Trichoderma reesei (syn..
- analysis of the secretome of Trichoderma reesei grown on unconventional carbon source.
- Re- annotation of the CAZy genes of Trichoderma reesei and transcription in the presence of lignocellulosic substrates.
- Comparative analysis of the Trichoderma reesei transcriptome during growth on the cellulase inducing substrates wheat straw and lactose.
- Draft whole-genome sequence of the biocontrol agent Trichoderma harzianum T6776.
- Evolution and comparative genomics of the most common Trichoderma species.
- Gene duplication in the sugarcane genome: a case study of allele interactions and evolutionary patterns in two genic regions.
- Genomic clustering and co-regulation of transcriptional networks in the pathogenic fungus Fusarium graminearum

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