Genomic studies of global gene expression of filamentous fungus Trichoderma reesei grown in bagasse and culm of sugarcane

Orientadores: Gustavo Henrique Goldman, Juliana Velasco de Castro OliveiraDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: A parede celular vegetal é uma estrutura recalcitrante, composta por polissacarídeos complexos que podem ser quebrados em açúcares fermentáveis. A desconstrução desse material complexo pode ser feita por diversos tipos de enzimas hidrolíticas, que são produzidas naturalmente por uma variedade de microrganismos. Entre eles, o fungo Trichoderma reesei se destaca pela capacidade de produzir e secretar estas enzimas em grandes quantidades. Embora alguns trabalhos utilizando abordagens de proteômica e transcriptômica já tenham sido realizados com esse fungo, ainda não são conhecidos em detalhes os mecanismos moleculares responsáveis pela degradação da parede e a regulação gênica envolvida nesse sistema lignocelulolítico. O presente trabalho tem como objetivo principal a análise da expressão gênica global de T. reesei, crescido por 6, 12 e 24 horas em bagaço e colmo de cana-de-açúcar como fontes únicas de carbono, pela técnica de sequenciamento high-throughput de RNA (RNA-Seq). No transcriptoma de T. reesei foram identificadas sendo hiper-expressas as principais celulases, hemicelulases e proteínas acessórias relacionadas direta ou indiretamente com a desconstrução da parede vegetal. De modo geral, as celulases e hemicelulases apresentaram uma expressão maior do que outras enzimas, e o nível dos seus transcritos foi crescente ao longo do tempo tanto em colmo quanto no bagaço. A grande maioria dos genes de CAZymes e proteínas acessórias hiper-expressos foram compartilhados pelos dois substratos, o que demonstra que a estratégia usada por T. reesei para degradar a parede celular do colmo e do bagaço é similar. Adicionalmente, vários fatores de transcrição, proteínas de função desconhecida e transportadores supostamente envolvidos na assimilação dos açúcares liberados também foram hiper-expressos nas condições amostradas. Para validação do RNA-Seq, foi realizado PCR em tempo real de diversos genes hiper-expressos que codificam para enzimas hidrolíticas, reguladores transcricionais, proteínas acessórias e genes ainda não caracterizados. Para isso, a análise temporal foi ampliada para 30 minutos, 2, 4, 6, 12 e 24 horas de crescimento após o inóculo, o que permitiu uma análise mais detalhada da expressão desses genes. Como objetivo secundário, foi analisado o secretoma deste fungo e os açúcares concomitantemente liberados no sobrenadante. Estas análises indicaram que a desconstrução da parede celular já se inicia dentro de 6 horas pós inoculo, com a liberação de monômeros (principalmente xilose e glicose) dos polissacarídeos e secreção de diversas CAZymes. Ensaios enzimáticos também foram realizados, mostrando atividades celulo e hemicelulolíticas. Assim, descrevemos pela primeira vez o arsenal de enzimas transcritas e secretadas por T. reesei RUT C30, desde pontos inicias de crescimento, em bagaço explodido e colmo de cana-de-açúcar. Por fim, este trabalho também permitiu a identificação de vários genes, com função predita ou não, que podem abrir caminho para a descoberta de novos atuantes na resposta do fungo ao substrato lignocelulósicoAbstract: Plant cell wall is a recalcitrant structure composed of complex polysaccharides which can be broken down into fermentable sugars. The deconstruction of this complex material can be made by a variety of hydrolytic enzymes which are naturally produced by a variety of microorganisms. Among them, stands out the fungus Trichoderma reesei, able to produce and secrete those enzymes in large quantities. Although some studies using transcriptomics and proteomics approaches have been performed with this fungus, the molecular mechanisms responsible for the degradation of the cell wall and gene regulation involved in this lignocellulosic system are not well known. This work has as main objective the analysis of global gene expression of T. reesei grown at 6, 12 and 24 hours in sugarcane bagasse and culm as sole carbon sources by high-throughput RNA sequencing technology (RNA-Seq). In the T. reesei transcriptome, it was identified the major cellulases, hemicellulases and accessory proteins directly or indirectly related to the deconstruction of plant cell wall. In general, cellulases and hemicellulases exhibited higher expression than other enzymes, and the level of their transcripts was increased over the time in both culm and bagasse cultures. Most of up-regulated CAZymes and accessory proteins were shared between the two substrates, which demonstrates the strategy used by T. reesei to degrade the bagasse and culm cell wall is similar. Furthermore, several transcription factors, proteins of unknown function and transporters supposedly involved in the assimilation of sugars were also up-regulated in the sampled conditions. To validate the RNA-Seq data, real-time PCR of several up-regulated genes encoding hydrolytic enzymes, transcriptional regulators, accessory proteins and proteins not yet characterized was carried out. The time points was extended to 30 min, 2, 4, 6, 12 and 24 hours of growth after inoculation, allowing a more detailed analysis of the expression of these genes. As a secondary objective, T. reesei secretome and the sugars released in the supernatant were analyzed. It was shown that the sugarcane cell wall deconstruction begins within the first 6 hours post inoculation, releasing sugar monomers (mainly xylose and glucose) from polysaccharides due to the secretion of several hydrolytic enzymes. Enzymatic assays were also performed, showing cellulosic and hemicellulosic activities. Finally, this is the first study showing the arsenal of enzymes transcribed and secreted by T. reesei grown on steam exploded sugarcane bagasse and culm, at early time points. It was possible identify several genes, with predicted function or not, that can open new paths to discover novel players on the fungus response to lignocellulosic substrateMestradoMicrobiologiaMestre em Genética e Biologia Molecula

Gene Co-expression Network Reveals Potential New Genes Related to Sugarcane Bagasse Degradation in Trichoderma reesei RUT-30

The biomass-degrading fungus Trichoderma reesei has been considered a model for cellulose degradation, and it is the primary source of the industrial enzymatic cocktails used in second-generation (2G) ethanol production. However, although various studies and advances have been conducted to understand the cellulolytic system and the transcriptional regulation of T. reesei, the whole set of genes related to lignocellulose degradation has not been completely elucidated. In this study, we inferred a weighted gene co-expression network analysis based on the transcriptome dataset of the T. reesei RUT-C30 strain aiming to identify new target genes involved in sugarcane bagasse breakdown. In total, ~70% of all the differentially expressed genes were found in 28 highly connected gene modules. Several cellulases, sugar transporters, and hypothetical proteins coding genes upregulated in bagasse were grouped into the same modules. Among them, a single module contained the most representative core of cellulolytic enzymes (cellobiohydrolase, endoglucanase, β-glucosidase, and lytic polysaccharide monooxygenase). In addition, functional analysis using Gene Ontology (GO) revealed various classes of hydrolytic activity, cellulase activity, carbohydrate binding and cation:sugar symporter activity enriched in these modules. Several modules also showed GO enrichment for transcription factor activity, indicating the presence of transcriptional regulators along with the genes involved in cellulose breakdown and sugar transport as well as other genes encoding proteins with unknown functions. Highly connected genes (hubs) were also identified within each module, such as predicted transcription factors and genes encoding hypothetical proteins. In addition, various hubs contained at least one DNA binding site for the master activator Xyr1 according to our in silico analysis. The prediction of Xyr1 binding sites and the co-expression with genes encoding carbohydrate active enzymes and sugar transporters suggest a putative role of these hubs in bagasse cell wall deconstruction. Our results demonstrate a vast range of new promising targets that merit additional studies to improve the cellulolytic potential of T. reesei strains and to decrease the production costs of 2G ethanol

Advances in Recombinant Lipases: Production, Engineering, Immobilization and Application in the Pharmaceutical Industry

Lipases are one of the most used enzymes in the pharmaceutical industry due to their efficiency in organic syntheses, mainly in the production of enantiopure drugs. From an industrial viewpoint, the selection of an efficient expression system and host for recombinant lipase production is highly important. The most used hosts are Escherichia coli and Komagataella phaffii (previously known as Pichia pastoris) and less often reported Bacillus and Aspergillus strains. The use of efficient expression systems to overproduce homologous or heterologous lipases often require the use of strong promoters and the co-expression of chaperones. Protein engineering techniques, including rational design and directed evolution, are the most reported strategies for improving lipase characteristics. Additionally, lipases can be immobilized in different supports that enable improved properties and enzyme reuse. Here, we review approaches for strain and protein engineering, immobilization and the application of lipases in the pharmaceutical industry

Insights into the plant polysaccharide degradation potential of the xylanolytic yeast Pseudozyma brasiliensis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)In second-generation (2G) bioethanol production, plant cell-wall polysaccharides are broken down to release fermentable sugars. The enzymes of this process are classified as carbohydrate-active enzymes (CAZymes) and contribute substantially to the cost of biofuel production. A novel basidiomycete yeast species, Pseudozyma brasiliensis, was recently discovered. It produces an endo-beta-1,4-xylanase with a higher specific activity than other xylanases. This enzyme is essential for the hydrolysis of biomass-derived xylan and has an important role in 2G bioethanol production. In spite of the P. brasiliensis biotechnological potential, there is no information about how it breaks down polysaccharides. For the first time, we characterized the secretome of P. brasiliensis grown on different carbon sources (xylose, xylan, cellobiose and glucose) and also under starvation conditions. The growth and consumption of each carbohydrate and the activity of the CAZymes of culture supernatants were analyzed. The CAZymes found in its secretomes, validated by enzymatic assays, have the potential to hydrolyze xylan, mannan, cellobiose and other polysaccharides. The data show that this yeast is a potential source of hydrolases, which can be used for biomass saccharification.In second-generation (2G) bioethanol production, plant cell-wall polysaccharides are broken down to release fermentable sugars. The enzymes of this process are classified as carbohydrate-active enzymes (CAZymes) and contribute substantially to the cost of162112FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCAPES - COORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIORFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)2014/11766-72015/08222-82013/18910-3SEM INFORMAÇÃOThe authors would like to thank Dr. Marina Camara Mattos Martins, Ms. Lucia Wolff and Dr Maria Teresa Borges Pimenta Barbosa for assistance with HPAEC-PAD analysis. We gratefully acknowledge the CNPEM facility MAS at LNBi

Comparative transcriptome analysis reveals different strategies for degradation of steam-exploded sugarcane bagasse by Aspergillus niger and Trichoderma reesei

Abstract Background 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. Results 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. Conclusions 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

A comparison of the abundance of secreted enzymes by <i>A</i>. <i>niger</i> and <i>T</i>. <i>reesei</i>.

<p>A semi-quantitative analysis of the amount of enzyme secreted by both fungi after 6, 12 and 24 hours (h) growth on sugarcane culm (C) and bagasse (B).</p

Graphical representation of secreted CAZYmes.

<p>Heat-map of the number of enzymes of each CAZY family secreted by <i>A</i>. <i>niger</i> and <i>T</i>. <i>reesei</i> after 6, 12 and 24 hours (h) growth on sugarcane culm and bagasse. This map includes only enzymes/proteins related to biomass degradation (Tables <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129275#pone.0129275.t001" target="_blank">1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129275#pone.0129275.t002" target="_blank">2</a>).</p