Analysis of Trichoderma harzianum transcriptome for bioprospecting of hydrolytic enzymes

Orientadores: Anete Pereira de Souza, Sindélia Freitas AzzoniTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: Buscando contribuir com o desenvolvimento da tecnologia de produção do etanol de segunda geração, o presente estudo analisa o transcriptoma de T. harzianum IOC-3844 utilizando técnicas de sequenciamento high-thoughput. O principal objetivo dessas análises foi identificar, caracterizar e catalogar os transcritos expressos por T. harzianum relacionados com a degradação de substratos complexos, como o bagaço de cana de açúcar, revelando o conjunto de genes envolvidos na degradação da biomassa. A análise do transcriptoma do fungo Trichoderma harzianum sob condições que induzem a degradação da biomassa permitiu a identificação de sequências de genes potencialmente eficazes no processo de biodegradação, uma etapa essencial à compreensão do processo de hidrólise enzimática. O sequenciamento resultou em 246 milhões de sequências com 72 pb, o que corresponde a 14,7 GPB analisados. Após a montagem , 32.494 contigs foram gerados, submetidos à identificação e classificados de acordo com sua identidade. Todas as sequências de contigs foram comparados com o banco de dados do NCBI, Gene Ontology (GO terms), Enciclopédia de Genes Kyoto (KEGG), Carbohydrate Active-Enzymes (CAZYmes). Foram identificados 487 CAZymes no transcriptoma, inclusive aquelas ligadas as reações químicas de despolimerização de celulose e hemicelulose. As sequências classificadas como atividade catalítica (6.975) e atividade reguladora (143) podem estar envolvidas com esse tipo de reação.A análise permitiu definir o principal conjunto de genes envolvidos na degradação da celulose e de hemicelulose do T. harzianum , e genes acessórios relativos à despolimerização de biomassa. Uma análise dos níveis de expressão permitiu determinar os conjuntos de genes diferencialmente expressos em diferentes condições de cultivo. Os resultados obtidos acrescentam conhecimento sobre a constituição do genoma, as atividades de expressão gênica do fungo Trichoderma harzianum e fornece informações importantes a respeito dos mecanismos genéticos de degradação de biomassa que o fungo utiliza. As informações obtidas poderão ser utilizadas para outras espécies de fungos filamentosos com potencial para a biodegradaçãoAbstract: In order to contribute to the development of second-generation ethanol technology, this study analyzes the transcriptome of T. harzianum IOC-3844 using high-thoughput sequencing techniques. The main objective of this analysis was to identify, characterize and catalog the transcripts expressed by T. harzianum related to the degradation of complex substrates such as sugar cane bagasse, revealing the set of genes involved in the degradation of biomass. The analysis of the transcriptome of the fungus Trichoderma harzianum under conditions that induce the degradation of biomass allowed the identification of genes potentially effective in the biodegradation process, an essential step for understanding the enzymatic process. Sequencing resulted in 246 million sequences with 72 bp, which corresponds to 14.7 GBP analyzed. After assembly, 32,494 contigs were generated, identified and classified according to their identity. All sequence contigs were compared with NCBI database, Gene Ontology (GO terms), Kyoto Encyclopedia of Genes (KEGG), Carbohydrate Active-Enzymes (CAZYmes). 487 CAZymes were identified in the transcriptome, including those related to reactions of cellulose and hemicellulose depolymerization. Sequences classified as catalytic activity (6,975) and regulatory activity (143) may be involved with this type of reaction. This analysis define the set of genes involved in the degradation of cellulose and hemicellulose of T. harzianum, and accessories genes related to depolymerization of the biomass. An analysis of expression levels was used to calculate the set of differentially expressed genes in different culture conditions. The results add to knowledge about the composition of the genome and gene expression activity of the fungus Trichoderma harzianum, and provides important information regarding the genetic mechanisms of biomass degradation that the fungus uses. The information obtained may be used for other species of filamentous fungi with potential for biodegradationDoutoradoGenetica Vegetal e MelhoramentoDoutora em Genética e Biologia Molecula

Transcriptome Profile of Trichoderma harzianum IOC-3844 Induced by Sugarcane Bagasse

Profiling the transcriptome that underlies biomass degradation by the fungus Trichoderma harzianum allows the identification of gene sequences with potential application in enzymatic hydrolysis processing. in the present study, the transcriptome of T. harzianum IOC-3844 was analyzed using RNA-seq technology. the sequencing generated 14.7 Gbp for downstream analyses. de novo assembly resulted in 32,396 contigs, which were submitted for identification and classified according to their identities. This analysis allowed us to define a principal set of T. harzianum genes that are involved in the degradation of cellulose and hemicellulose and the accessory genes that are involved in the depolymerization of biomass. An additional analysis of expression levels identified a set of carbohydrate-active enzymes that are upregulated under different conditions. the present study provides valuable information for future studies on biomass degradation and contributes to a better understanding of the role of the genes that are involved in this process.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Univ Campinas UNICAMP, CBMEG, Campinas, SP, BrazilBrazilian Ctr Res Energy & Mat CNPEM, Brazilian Bioethanol Sci & Technol Lab CTBE, Campinas, SP, BrazilUniv São Paulo, Phys Inst Sao Carlos, Sao Carlos, SP, BrazilFed Univ São Paulo UNIFESP, Inst Sci & Technol, Sao Jose Dos Campos, SP, BrazilUniv Campinas UNICAMP, Dept Plant Biol, Inst Biol, Campinas, SP, BrazilFed Univ São Paulo UNIFESP, Inst Sci & Technol, Sao Jose Dos Campos, SP, BrazilWeb of Scienc

Examination of Genome-Wide Ortholog variation in clinical and environmental isolates of the fungal pathogen Aspergillus fumigatus

Aspergillus fumigatus is a cosmopolitan species of fungus responsible for thousands of cases of invasive disease annually. Clinical and environmental isolates of A. fumigatus exhibit extensive phenotypic differences, including differences related to virulence and antifungal drug resistance. Aspergillus fumigatus is both an environmental saprobe and an opportunistic human fungal pathogen. Knowledge of genomic variation across A. fumigatus isolates is essential for understanding the evolution of pathogenicity, virulence, and resistance to antifungal drugs. Here, we investigated 206 A. fumigatus isolates (133 clinical and 73 environmental isolates), aiming to identify genes with variable presence across isolates and test whether this variation was related to the clinical or environmental origin of isolates. The PanOrtho genome of A. fumigatus consists of 13,085 ortholog groups, of which 7,773 (59.4\%) are shared by all isolates (core groups) and 5,312 (40.6\%) vary in their gene presence across isolates (accessory groups plus singletons). Despite differences in the distribution of orthologs across all isolates, no significant differences were observed among clinical versus environmental isolates when phylogeny was accounted for. Orthologs that differ in their distribution across isolates tend to occur at low frequency and/or be restricted to specific isolates; thus, the degree of genomic conservation between orthologs of A. fumigatus is high. These results suggest that differences in the distribution of orthologs within A. fumigatus cannot be associated with the clinical or environmental origin of isolates. IMPORTANCE Aspergillus fumigatus is a cosmopolitan species of fungus responsible for thousands of cases of invasive disease annually. Clinical and environmental isolates of A. fumigatus exhibit extensive phenotypic differences, including differences related to virulence and antifungal drug resistance. A comprehensive survey of the genomic diversity present in A. fumigatus and its relationship to the clinical or environmental origin of isolates can contribute to the prediction of the mechanisms of evolution and infection of the species. Our results suggest that there is no significant variation in ortholog distribution between clinical and environmental isolates when accounting for evolutionary history. The work supports the hypothesis that environmental and clinical isolates of A. fumigatus do not differ in their gene contents.We thank Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) 2020/10536-9 (M.A.C.H.) and 2016/07870-9 (G.H.G.) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) 301058/2019-9 and 404735/2018-5 (G.H.G.), both in Brazil, and National Institutes of Health/National Institute of Allergy and Infectious Diseases (R01AI153356) (A.R. and G.H.G.), in the United States.Peer ReviewedPostprint (published version

Studies on Fe3+ reduction by aqueous extracts recovered from cultures of Ceriporiopsis subvermispora on wood and its correlation with lipid peroxidation

O presente trabalho está inserido dentro de um estudo que pretende entender os mecanismos químicos e bioquímicos envolvidos na biodegradação da madeira por Ceriporiopsis subvermispora. Particularmente avaliou-se a relação entre a atividade redutora de Fe3+ presente em extratos aquosos de cultivos de Ceriporiopsis subvermispora sobre madeira com a iniciação de reações de peroxidação de ácido linoleico. A madeira foi biotratada por períodos de 7, 14 e 28 dias. Após a biodegradação, os cavacos foram extraídos com água e os extratos gerados foram caracterizados quanto a atividade redutora de Fe3+ e a capacidade de peroxidar ácido linoleico in vitro. As reações de peroxidação foram avaliadas a partir do monitoramento do consumo de O2 em um oxímetro. A partir desses ensaios foi possível determinar que tanto a oxidação de íons Fe2+ adicionados diretamente ao meio reacional ou a adição de íons Fe3+ e extratos com atividade redutora foram capazes de gerar radicais hidroperoxila que podem iniciar a peroxidação de ácido linoleico. As reações controle indicaram que a peroxidação não ocorre na presença de Fe3+. A adição de metanol às reações contendo Fe2+ não proporcionou menor capacidade de peroxidação de lipídeos, indicando que a reação não deve ser iniciada por OH radical. Tanto a atividade redutora de Fe+3 como a capacidade de iniciar a peroxidação de ácido linoleico dos extratos aumentaram em função do tempo de cultivo. A ultrafiltação dos extratos através de membranas seletivas associada com estudos de cromatografia de permeação em gel (CPG) indicaram que a maior parte da atividade redutora e da capacidade peroxidativa pode ser atribuída a compostos menores que 5kDa. Entre os compostos orgânicos identificados nas frações menores que 5 kDa, foram identificados, por cromatografia gasosa acoplada à espectrometria de massas (CG/EM), alguns ácidos graxos como o palmítico e o ácido esteárico, além de glicerol e ácido oxálico e compostos aromáticos como a vanilina, e os ácidos vanílico, siríngico e gálico. A vanilina, detectada em todas as amostras em estudo, foi usada como padrão para um estudo comparativo das atividades redutoras de Fe3+ detectadas nos extratos. As concentrações estimadas de vanilina nos extratos foram de 5 a 22 ? moles/L de extrato ultrafiltrado. Soluções padrão de vanilina foram então preparadas em concentrações de 5 a 30 ? M e avaliadas quanto a capacidade de reduzir Fe3+ na presença de ferrozina. Essas misturas levaram à formação de Fe2+ nas concentrações de 0,5 a 0,7 ? M de Fe2+ , após 10 min de reação. A comparação desses dados com a capacidade redutora de Fe3+ encontrada nos ultrafiltrados da amostra de madeira biotratada por 14 dias, 43 ?M de Fe2+ em 10 min, sugeriu que a maior parte da capacidade redutora dos extratos não pode ser atribuída aos compostos detectados por CG/EM e sim deve ser atribuída a compostos que apresentam massa molar próxima àquela obtida pela calibração do sistema de CPG, entre 3 e 5 kDa. Isso sugere que a atividade redutora não estaria presente em compostos de baixa massa molar compatíveis com estruturas contendo 1 ou, no máximo, 2 anéis aromáticos e sim em derivados fenólicos maiores, oriundos da biodegradação da lignina.The present work is inserted in a broad study aiming to understand the chemical and biochemical mechanisms involved in wood biodegradation by Ceriporiopsis subvermispora. Particularly, the relationship between the Fe3+-reducing activity present in aqueous extracts recovered from Ceriporiopsis subvermispora cultures on wood with the initiation of linoleic acid peroxidation reactions was evaluated. Wood chips were biotreated for periods varying from 7 to 28 days. After biotreatment, the wood chips were extracted with water and the resulting extracts were characterized according to their Fe3+-reducing activity, as well as their capacity to initiate linoleic acid peroxidation reactions in vitro. The peroxidation reactions were monitored through O2 consumption by using an appropriated Oximeter. Based on these assays, it was possible to show that Fe2+ ions added directly to the reaction media or the addition of Fe3+ ions plus aqueous extracts were able to generate hidroperoxil radicals that initiated linoleic acid peroxidation. The control reactions indicated that the peroxidation did not occur in the presence of Fe3+ ions alone. Addition of methanol to the reaction media containing Fe2+ did not diminish the lipid peroxidation extent, suggesting that the reaction should not depend on the formation of hydroxyl radical. The Fe3+-reducing activity and the capacity to initiate linoleic acid peroxidation increased in the aqueous wood extracts as a function of culturing time. The ultrafiltration of the extracts associated with gel permeation chromatography (GPC) studies indicated that most of the reducing activity and the peroxidation capacity present in the extracts were assigned to compounds presenting molecular mass lower than 5 kDa. In the fraction lower than 5 kDa some organic compounds were identified by using gas chromatography coupled to mass spectrometry (GC/MS). Palmitic and estearic acids, glycerol, oxalic acid and some aromatic compounds such as vanillin, vanillic, gallic and syringic acids were identified. Vanillin, detected in all the studied samples, was used as a standard for comparative evaluation of the Fe3+-reducing activities detected in the extracts. The estimated concentrations of vanillin in the extracts varied from 5 to 22 μmol/L of ultrafiltrated extract. Therefore, 5 to 30 μM standard solutions of va nillin were assayed for their Fe3+-reducing activity. These solutions reduced 0.5 to 0.7?M of Fe3+ after 10 min reaction. The comparison of these data with the Fe3+-reducing capacity found in the ultrafiltrated aqueous extract from the 14-day biotreated sample, 43 ? M of Fe3+ reduced after 10 min reaction, suggested that most of the reducing capacity in the extracts could not be attributed to compounds detect by GC/MS. Conversely, the Fe3+-reducing capacity could be assigned to compounds with higher molar mass as already observed in the GPC studies (3 to 5 kDa). These data suggest that the reducing activity detected in the aqueous extracts would not be compatible with low molar mass compounds containing 1 or, at least 2, aromatic rings. Instead, the Fe3+-reducing capacity should be assigned to high molar mass phenolic derivatives produced during the lignin biodegradation

Studies on Fe3+ reduction by aqueous extracts recovered from cultures of Ceriporiopsis subvermispora on wood and its correlation with lipid peroxidation

O presente trabalho está inserido dentro de um estudo que pretende entender os mecanismos químicos e bioquímicos envolvidos na biodegradação da madeira por Ceriporiopsis subvermispora. Particularmente avaliou-se a relação entre a atividade redutora de Fe3+ presente em extratos aquosos de cultivos de Ceriporiopsis subvermispora sobre madeira com a iniciação de reações de peroxidação de ácido linoleico. A madeira foi biotratada por períodos de 7, 14 e 28 dias. Após a biodegradação, os cavacos foram extraídos com água e os extratos gerados foram caracterizados quanto a atividade redutora de Fe3+ e a capacidade de peroxidar ácido linoleico in vitro. As reações de peroxidação foram avaliadas a partir do monitoramento do consumo de O2 em um oxímetro. A partir desses ensaios foi possível determinar que tanto a oxidação de íons Fe2+ adicionados diretamente ao meio reacional ou a adição de íons Fe3+ e extratos com atividade redutora foram capazes de gerar radicais hidroperoxila que podem iniciar a peroxidação de ácido linoleico. As reações controle indicaram que a peroxidação não ocorre na presença de Fe3+. A adição de metanol às reações contendo Fe2+ não proporcionou menor capacidade de peroxidação de lipídeos, indicando que a reação não deve ser iniciada por OH radical. Tanto a atividade redutora de Fe+3 como a capacidade de iniciar a peroxidação de ácido linoleico dos extratos aumentaram em função do tempo de cultivo. A ultrafiltação dos extratos através de membranas seletivas associada com estudos de cromatografia de permeação em gel (CPG) indicaram que a maior parte da atividade redutora e da capacidade peroxidativa pode ser atribuída a compostos menores que 5kDa. Entre os compostos orgânicos identificados nas frações menores que 5 kDa, foram identificados, por cromatografia gasosa acoplada à espectrometria de massas (CG/EM), alguns ácidos graxos como o palmítico e o ácido esteárico, além de glicerol e ácido oxálico e compostos aromáticos como a vanilina, e os ácidos vanílico, siríngico e gálico. A vanilina, detectada em todas as amostras em estudo, foi usada como padrão para um estudo comparativo das atividades redutoras de Fe3+ detectadas nos extratos. As concentrações estimadas de vanilina nos extratos foram de 5 a 22 ? moles/L de extrato ultrafiltrado. Soluções padrão de vanilina foram então preparadas em concentrações de 5 a 30 ? M e avaliadas quanto a capacidade de reduzir Fe3+ na presença de ferrozina. Essas misturas levaram à formação de Fe2+ nas concentrações de 0,5 a 0,7 ? M de Fe2+ , após 10 min de reação. A comparação desses dados com a capacidade redutora de Fe3+ encontrada nos ultrafiltrados da amostra de madeira biotratada por 14 dias, 43 ?M de Fe2+ em 10 min, sugeriu que a maior parte da capacidade redutora dos extratos não pode ser atribuída aos compostos detectados por CG/EM e sim deve ser atribuída a compostos que apresentam massa molar próxima àquela obtida pela calibração do sistema de CPG, entre 3 e 5 kDa. Isso sugere que a atividade redutora não estaria presente em compostos de baixa massa molar compatíveis com estruturas contendo 1 ou, no máximo, 2 anéis aromáticos e sim em derivados fenólicos maiores, oriundos da biodegradação da lignina.The present work is inserted in a broad study aiming to understand the chemical and biochemical mechanisms involved in wood biodegradation by Ceriporiopsis subvermispora. Particularly, the relationship between the Fe3+-reducing activity present in aqueous extracts recovered from Ceriporiopsis subvermispora cultures on wood with the initiation of linoleic acid peroxidation reactions was evaluated. Wood chips were biotreated for periods varying from 7 to 28 days. After biotreatment, the wood chips were extracted with water and the resulting extracts were characterized according to their Fe3+-reducing activity, as well as their capacity to initiate linoleic acid peroxidation reactions in vitro. The peroxidation reactions were monitored through O2 consumption by using an appropriated Oximeter. Based on these assays, it was possible to show that Fe2+ ions added directly to the reaction media or the addition of Fe3+ ions plus aqueous extracts were able to generate hidroperoxil radicals that initiated linoleic acid peroxidation. The control reactions indicated that the peroxidation did not occur in the presence of Fe3+ ions alone. Addition of methanol to the reaction media containing Fe2+ did not diminish the lipid peroxidation extent, suggesting that the reaction should not depend on the formation of hydroxyl radical. The Fe3+-reducing activity and the capacity to initiate linoleic acid peroxidation increased in the aqueous wood extracts as a function of culturing time. The ultrafiltration of the extracts associated with gel permeation chromatography (GPC) studies indicated that most of the reducing activity and the peroxidation capacity present in the extracts were assigned to compounds presenting molecular mass lower than 5 kDa. In the fraction lower than 5 kDa some organic compounds were identified by using gas chromatography coupled to mass spectrometry (GC/MS). Palmitic and estearic acids, glycerol, oxalic acid and some aromatic compounds such as vanillin, vanillic, gallic and syringic acids were identified. Vanillin, detected in all the studied samples, was used as a standard for comparative evaluation of the Fe3+-reducing activities detected in the extracts. The estimated concentrations of vanillin in the extracts varied from 5 to 22 μmol/L of ultrafiltrated extract. Therefore, 5 to 30 μM standard solutions of va nillin were assayed for their Fe3+-reducing activity. These solutions reduced 0.5 to 0.7?M of Fe3+ after 10 min reaction. The comparison of these data with the Fe3+-reducing capacity found in the ultrafiltrated aqueous extract from the 14-day biotreated sample, 43 ? M of Fe3+ reduced after 10 min reaction, suggested that most of the reducing capacity in the extracts could not be attributed to compounds detect by GC/MS. Conversely, the Fe3+-reducing capacity could be assigned to compounds with higher molar mass as already observed in the GPC studies (3 to 5 kDa). These data suggest that the reducing activity detected in the aqueous extracts would not be compatible with low molar mass compounds containing 1 or, at least 2, aromatic rings. Instead, the Fe3+-reducing capacity should be assigned to high molar mass phenolic derivatives produced during the lignin biodegradation

Degradative Capacity of Two Strains of Rhodonia placenta : From Phenotype to Genotype

Brown rot fungi, such as Rhodonia placenta (previously Postia placenta), occur naturally in northern coniferous forest ecosystems and are known to be the most destructive group of decay fungi, degrading wood faster and more effectively than other wood-degrading organisms. It has been shown that brown rot fungi not only rely on enzymatic degradation of lignocellulose, but also use low molecular weight oxidative agents in a non-enzymatic degradation step prior to the enzymatic degradation. R. placenta is used in standardized decay tests in both Europe and North America. However, two different strains are employed (FPRL280 and MAD-698, respectively) for which differences in colonization-rate, mass loss, as well as in gene expression have been observed, limiting the comparability of results. To elucidate the divergence between both strains, we investigated the phenotypes in more detail and compared their genomes. Significant phenotypic differences were found between the two strains, and no fusion was possible. MAD-698 degraded scots pine more aggressively, had a more constant growth rate and produced mycelia faster than FPRL280. After sequencing the genome of FPRL280 and comparing it with the published MAD-698 genome we found 660,566 SNPs, resulting in 98.4% genome identity. Specific analysis of the carbohydrate-active enzymes, encoded by the genome (CAZome) identified differences in many families related to plant biomass degradation, including SNPs, indels, gaps or insertions within structural domains. Four genes belonging to the AA3_2 family could not be found in or amplified from FPRL280 gDNA, suggesting the absence of these genes. Differences in other CAZy encoding genes that could potentially affect the lignocellulolytic activity of the strains were also predicted by comparison of genome assemblies (e.g., GH2, GH3, GH5, GH10, GH16, GH78, GT2, GT15, and CBM13). Overall, these mutations help to explain the phenotypic differences observed between both strains as they could interfere with the enzymatic activities, substrate binding ability or protein folding. The investigation of the molecular reasons that make these two strains distinct contributes to the understanding of the development of this important brown rot reference species and will help to put the data obtained from standardized decay tests across the globe into a better biological context. © Copyright © 2020 Kölle, Horta, Nowrousian, Ohm, Benz and Pilgård.Funding details: Deutsche Forschungsgemeinschaft, DFG; Funding details: Svenska ForskningsrÃ¥det Formas, NO407/7-1, 942-2015-530; Funding details: VetenskapsrÃ¥det, VR; Funding text 1: MK and AP gratefully acknowledge financial support from The Swedish Research Council and MN from the DFG. We gratefully acknowledge excellent technical assistance by Petra Arnold (TUM). Thanks also to Dan Cullen for providing us a sample of the Rhodonia placenta MAD-SB12 strain for laboratory tests. Funding. This work was supported by the Swedish Research Council Formas 942-2015-530 to AP; DFG project NO407/7-1 to MN.</p

Degradative Capacity of Two Strains of Rhodonia placenta: From Phenotype to Genotype

Brown rot fungi, such as Rhodonia placenta (previously Postia placenta), occur naturally in northern coniferous forest ecosystems and are known to be the most destructive group of decay fungi, degrading wood faster and more effectively than other wood-degrading organisms. It has been shown that brown rot fungi not only rely on enzymatic degradation of lignocellulose, but also use low molecular weight oxidative agents in a non-enzymatic degradation step prior to the enzymatic degradation. R. placenta is used in standardized decay tests in both Europe and North America. However, two different strains are employed (FPRL280 and MAD-698, respectively) for which differences in colonization-rate, mass loss, as well as in gene expression have been observed, limiting the comparability of results. To elucidate the divergence between both strains, we investigated the phenotypes in more detail and compared their genomes. Significant phenotypic differences were found between the two strains, and no fusion was possible. MAD-698 degraded scots pine more aggressively, had a more constant growth rate and produced mycelia faster than FPRL280. After sequencing the genome of FPRL280 and comparing it with the published MAD-698 genome we found 660,566 SNPs, resulting in 98.4% genome identity. Specific analysis of the carbohydrate-active enzymes, encoded by the genome (CAZome) identified differences in many families related to plant biomass degradation, including SNPs, indels, gaps or insertions within structural domains. Four genes belonging to the AA3_2 family could not be found in or amplified from FPRL280 gDNA, suggesting the absence of these genes. Differences in other CAZy encoding genes that could potentially affect the lignocellulolytic activity of the strains were also predicted by comparison of genome assemblies (e.g., GH2, GH3, GH5, GH10, GH16, GH78, GT2, GT15, and CBM13). Overall, these mutations help to explain the phenotypic differences observed between both strains as they could interfere with the enzymatic activities, substrate binding ability or protein folding. The investigation of the molecular reasons that make these two strains distinct contributes to the understanding of the development of this important brown rot reference species and will help to put the data obtained from standardized decay tests across the globe into a better biological context

Carbohydrate-active enzymes in Trichoderma harzianum: a bioinformatic analysis bioprospecting for key enzymes for the biofuels industry

Abstract Background Trichoderma harzianum is used in biotechnology applications due to its ability to produce powerful enzymes for the conversion of lignocellulosic substrates into soluble sugars. Active enzymes involved in carbohydrate metabolism are defined as carbohydrate-active enzymes (CAZymes), and the most abundant family in the CAZy database is the glycoside hydrolases. The enzymes of this family play a fundamental role in the decomposition of plant biomass. Results In this study, the CAZymes of T. harzianum were identified and classified using bioinformatic approaches after which the expression profiles of all annotated CAZymes were assessed via RNA-Seq, and a phylogenetic analysis was performed. A total of 430 CAZymes (3.7% of the total proteins for this organism) were annotated in T. harzianum, including 259 glycoside hydrolases (GHs), 101 glycosyl transferases (GTs), 6 polysaccharide lyases (PLs), 22 carbohydrate esterases (CEs), 42 auxiliary activities (AAs) and 46 carbohydrate-binding modules (CBMs). Among the identified T. harzianum CAZymes, 47% were predicted to harbor a signal peptide sequence and were therefore classified as secreted proteins. The GH families were the CAZyme class with the greatest number of expressed genes, including GH18 (23 genes), GH3 (17 genes), GH16 (16 genes), GH2 (13 genes) and GH5 (12 genes). A phylogenetic analysis of the proteins in the AA9/GH61, CE5 and GH55 families showed high functional variation among the proteins. Conclusions Identifying the main proteins used by T. harzianum for biomass degradation can ensure new advances in the biofuel production field. Herein, we annotated and characterized the expression levels of all of the CAZymes from T. harzianum, which may contribute to future studies focusing on the functional and structural characterization of the identified proteins

Degradative Capacity of Two Strains of Rhodonia placenta: From Phenotype to Genotype

Brown rot fungi, such as Rhodonia placenta (previously Postia placenta), occur naturally in northern coniferous forest ecosystems and are known to be the most destructive group of decay fungi, degrading wood faster and more effectively than other wood-degrading organisms. It has been shown that brown rot fungi not only rely on enzymatic degradation of lignocellulose, but also use low molecular weight oxidative agents in a non-enzymatic degradation step prior to the enzymatic degradation. R. placenta is used in standardized decay tests in both Europe and North America. However, two different strains are employed (FPRL280 and MAD-698, respectively) for which differences in colonization-rate, mass loss, as well as in gene expression have been observed, limiting the comparability of results. To elucidate the divergence between both strains, we investigated the phenotypes in more detail and compared their genomes. Significant phenotypic differences were found between the two strains, and no fusion was possible. MAD-698 degraded scots pine more aggressively, had a more constant growth rate and produced mycelia faster than FPRL280. After sequencing the genome of FPRL280 and comparing it with the published MAD-698 genome we found 660,566 SNPs, resulting in 98.4% genome identity. Specific analysis of the carbohydrate-active enzymes, encoded by the genome (CAZome) identified differences in many families related to plant biomass degradation, including SNPs, indels, gaps or insertions within structural domains. Four genes belonging to the AA3_2 family could not be found in or amplified from FPRL280 gDNA, suggesting the absence of these genes. Differences in other CAZy encoding genes that could potentially affect the lignocellulolytic activity of the strains were also predicted by comparison of genome assemblies (e.g., GH2, GH3, GH5, GH10, GH16, GH78, GT2, GT15, and CBM13). Overall, these mutations help to explain the phenotypic differences observed between both strains as they could interfere with the enzymatic activities, substrate binding ability or protein folding. The investigation of the molecular reasons that make these two strains distinct contributes to the understanding of the development of this important brown rot reference species and will help to put the data obtained from standardized decay tests across the globe into a better biological context

Contribution to the taxonomy and ecology of the Azorean benthic marine algae

Evolution and Conservation in the North Atlantic Islands (Madeira, Canaries, Azores). Manchester, Reino Unido, 4-6 de Setembro de 1990