Thesis (M.Sc. (Microbiology))--Mahidol University, 1984.

Made available in DSpace on 2017-07-10T14:38:30Z (GMT). No. of bitstreams: 1 DISSERTAcaoO-MESTRADO-SANDRA-VERSaO-FINAL.pdf: 1113857 bytes, checksum: e8421d0365b2d79f28487d63317ac109 (MD5) Previous issue date: 2016-02-26 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The production of xylanolitic enzymes have been widely explored in the past few years by several species of filamentous fungi. The objective of this study was to evaluate the production of xylanases by Penicillium brefeldianum an isolated filamentous fungus of Parana Atlantic Forest using brewers spent grain and evaluate its potential in the agro-waste saccharification. The enzyme extract was obtained from liquid culture using 1% (w / v) brewers spent grain in modified under stationary conditions at 28 ° for 9 days. The fungus had large capacity in the production of xylanase (830 U mL-1), pectinase (295 U mL-1), β-xylosidase (3,46 U mL-1), FPase (0,60 U mL-1), avicelase (13, 71 U ml-1), cellulase (1.89 U mL-1) and β -glycosidase (29,45 U mL-1). The optimum pH of 4.5 was found for xylanase and stability in 98% range of 2.5 to 8.0 for 96 hours. The optimum temperature was 55 °C with a half life of 90 minutes. The zymogram of the crude extract of P. brefeldianum exhibited three bands of xylanase activity with molecular masses of 60 and 97kDa. P. brefeldianum crude enzymatic extract was used for saccharification of various waste (sugarcane bagasse, brewers spent grain, rice straw, corn stover, wheat straw, sorghum biomass and sorghum low-lignin) untreated and previously treated with NaOH 30°C for 12h (NaOH30) and at 121°C for 30 minutes (NaOH121). The best results were obtained with waste treated under high pressure and temperature (121°C). Among waste, wheat straw showed higher amount of total sugar (63%) released after the enzymatic hydrolysis, followed brewers spent grain (60%), corn straw (58%), sorghum biomass (54%) and sorghum low-lignin (35%). The pentose released after saccharification showed that sorghum low-lignin treated at high temperature and pressure was the best substrate releasing 7,35 times more pentose compared to sorghum low-lignin untreated, followed by straw rice (5,52x) and sorghum biomass (4,39x). Thus, the filamentous fungus P. brefeldianum isolated from the West of Paraná Atlantic Forest was able to use the brewers spent grain as inductor of various degrading enzymes the cell wall, and furthermore, showed potential for saccharification of lignocellulosic biomass from agro-waste. / A produção de enzimas xilanolíticas tem sido amplamente explorada nos últimos anos por diversas espécies de fungos filamentosos. O objetivo deste trabalho foi avaliar a produção de xilanases por Penicillium brefeldianum, um fungo filamentoso isolado da Mata Atlântica do Paraná, utilizando bagaço de cervejaria para avaliar seu potencial na sacarificação de resíduos agroindustriais. O extrato enzimático foi obtido a partir de cultivo líquido contendo 1% de bagaço de cervejaria (p/v) e cultivado em condições estacionárias a 28° por 9 dias. O fungo apresentou versatilidade em produzir diversas enzimas tais como: xilanase (830 U mL-1), pectinase (295 U mL-1), β -glicosidase (29,45 U mL-1), avicelase (13,71 U mL-1) β-xilosidase (3,46 U mL-1), celulase (1,89 U mL-1) e Fpase (0,60 U mL-1). A xilanase produzida por esse fungo apresentou pH ótimo de atividade enzimática de 4,5 e estabilidade de 98% na faixa de pH 2,5 a 8,0 por até 96 horas. A temperatura ótima de atividade da xilanase foi 55°C com meia vida de 90 minutos. No processo de sacarificação enzimática de resíduos (bagaço de cana, bagaço de cervejaria, palha de arroz, palha de milho, palha de trigo, sorgo biomassa) com extrato bruto de P. brefeldianum foi obtido que o bagaço de cervejaria apresentou melhor resultado sem qualquer tipo de pré-tratamento, exibindo 35% de sacarificação. Porém, o pré-tratamento dos resíduos com NaOH 1% a 30°C por 12 horas ou a 121°C e pressão de 1 atm por 30 minutos resultaram em melhores rendimentos de açúcares redutores totais. E dentre os resíduos sacarificados, o bagaço de cervejaria (60%) liberou maior quantidade de açucares totais, seguido de palha de trigo (58,73%), palha de milho (58%) sorgo biomassa (54%) e sorgo com baixa lignina (35%). Entretanto, as pentoses produzidas após sacarificação mostraram que o tratado com NaOH (121ºC e pressão de 1 atm) apresentou 7,35 vezes mais pentoses quando comparado ao in natura, seguido de casca de arroz (5,52 vezes) e sorgo biomassa (4,39 vezes). Dessa forma, o fungo filamentoso P. brefeldianum isolado da Mata Atlântica do Oeste do Paraná foi capaz de utilizar o bagaço de cervejaria como indutor de hemicelulases, celulases e pectinases, além disso, exibiu potencial de sacarificação de resíduos agroindustriais para produção de açucares redutores.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2016-10-06T10:55:44Z No. of bitstreams: 2 Dissertação - Aline Rodrigues Gama - 2016.pdf: 2281415 bytes, checksum: a79af0203dd2a1cfd381c59e3e9d6d7b (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2016-10-06T11:22:35Z (GMT) No. of bitstreams: 2 Dissertação - Aline Rodrigues Gama - 2016.pdf: 2281415 bytes, checksum: a79af0203dd2a1cfd381c59e3e9d6d7b (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2016-10-06T11:22:35Z (GMT). No. of bitstreams: 2 Dissertação - Aline Rodrigues Gama - 2016.pdf: 2281415 bytes, checksum: a79af0203dd2a1cfd381c59e3e9d6d7b (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016-09-14 / The use of carbohydrates derived from agricultural waste is important in the industry of food, textiles, paper, detergents, animal feed and in the production of bioethanol. Some filamentous bacteria are used in the enzymatic degradation processes, such as Streptomyces thermocerradoensis I3, which was isolated from soil and this work was selected for the production of cellulases and xylanases by growing in semi-solid fermentation (SSF) in medium supplemented with wheat bran (WB) as carbon source. S. thermocerradoensis I3 it was maintained for 4 days at 37 ° C and showed a higher production of Endoglucanase (2,92 U/mL) and Xylanase (12,34 U/mL) after 72 hours of cultivation and β-glucosidase (0,023 U/mL) and FPase (2,82 U/mL) after 96 hours of cultivation. The 96-hour extract was concentrated. The enzyme present in the concentrated extract presented molecular mass of 17 kDa. It showed activity for cellulase and xylanases confirmed by zimograms and enzymatic activities. The crude extract (EB) and the concentrated extract (EC) were analyzed for pH and temperature optima for activity of cellulases and xylanases. The results showed that the highest activity of the total cellulase (FPase) was at pH 6.0 at 60 °C (EB) and pH in the range 3.0 to 6.0 at 80 °C (EC); the endoglucanase has higher activity at pH 6.0 at 55 °C (EB and EC); xylanase showed higher activity at pH 8.0 at 70 °C (EB and EC). The xylanase activity of EB and EC showed thermostability 60% after 2 hours of incubation at 60 °C and the endoglucanase activity of EB and EC remained above 50% after 4 hours of incubation at 50 °C, 60 °C and 70 °C. Qualitative analysis observed by TLC (Thin layer chromatography) showed the production of oligosaccharides and xilooligômeros from the hydrolysis of different substrates. S. thermocerradoensis I3 successfully used the WB in SSF producing enzymes that have the characteristics necessary for their industrial application. / A utilização de carboidratos oriundos dos resíduos agrícolas faz-se importante nas indústrias de alimentos, tecidos, papeis, detergentes, ração animal e ainda na produção de bioetanol. Algumas bactérias filamentosas são utilizadas nos processos de hidrólise enzimática, como Streptomyces thermocerradoensis I3, o qual foi isolado do solo e, neste trabalho, selecionado para produção de celulases e xilanases através de cultivo em fermentação semi-sólida (FSS) em Meio Mínimo suplementado com farelo de trigo (FT) como fonte de carbono. S. thermocerradoensis I3 foi cultivado por 4 dias à 37 °C e apresentou maior produção de Endoglucanase (2,92 U/mL) e Xilanase (12,34 U/mL) após 72 horas de cultivo e de β-glicosidase (0,023 U/mL) e FPase (2,82 U/mL) após 96 horas de cultivo. O extrato de 96 horas foi concentrado e a enzima presente no extrato concentrado apresentou Massa Molecular de 17 kDa. Ela apresentou atividade de celulase e xilanase, as quais foram confirmadas por zimogramas e determinação das atividades enzimáticas. O extrato bruto (EB) e o extrato concentrado (EC) foram analisados quanto ao pH e temperatura ótimos para a atividade de celulases e xilanase. Os resultados obtidos demonstraram que a maior atividade de celulases totais (FPase) foi em pH 6,0 à 60 °C (EB) e pH na faixa de 3,0 e 6,0 à 80 °C (EC); a enduglucanase apresentou maior atividade na faixa de pH 6,0 à 55 °C (EB e EC); a xilanase apresentou maior atividade em pH 8,0 à 70 °C (EB e EC). A atividade de xilanase do EB e da EC apresentou termoestabilidade de 60% após 2 horas de incubação à 60 °C e a atividade de endoglucanase do EB e do EC permaneceu acima de 50% após 4 horas de incubação à 50, 60 e 70 °C. As análises qualitativas observadas por TLC (Thin Layer Chromatography) revelaram a liberação de oligossacarídeos e xilooligômeros a partir da hidrólise de diferentes substratos. S. thermocerradoensis I3 utilizou com sucesso o FT em FSS produzindo enzimas que apresentam as características necessárias para sua aplicação industrial.

Cette thèse a été réalisée dans le cadre du projet Futurol, un projet national français qui vise à produire du bioéthanol à partir de biomasses végétales telles que le bois ou la paille de céréale. Pour cela, la biomasse doit être prétraitée puis digérée enzymatiquement pour libérer des sucres fermentescibles. Ma contribution dans ce projet a été de découvrir des enzymes originales pour l’hydrolyse de l’hémicellulose, un hétéropolysaccharide, constituant majeur de la paroi cellulaire des cellules végétales. Afin de rechercher de nouveaux biocatalyseurs, une approche de métagénomique a été adoptée afin de sonder les intestins de deux espèces de termites : N. corniger, un termite xylophage, et T. hispaniolae un termite humivore / xylophage. 30 000 clones métagénomiques ont été criblés sur 10 substrats cellulosiques et hémicellulosique, et 660 hits ont été obtenus. La comparaison phénotypique a montré une différence claire entre ces deux banques, probablement liée au régime alimentaire des deux espèces de termite. Le séquençage de 45 clones N. corniger a révélé 120 séquences codant pour des enzymes originales, de nombreuses étant multimodulaires et / ou organisées en cluster de gènes. Dans un second temps, une approche à haut-débit a été adoptée pour le clonage, l’expression et la caractérisation légère de 104 enzymes entières ou formes tronquées. 45 protéines recombinantes ont été produites de manière soluble, et les activités de 19 enzymes et de 12 modules enzymatiques ont été montrées, permettant la mise au point d’une boite à outil hemicellulolytique. Dans certains cas, l’activité de modules classés « Inconnus » a pu être déterminée. Cette approche a été particulièrement pertinente dans le cas de Pm69, une enzyme multimodulaire GH3-UNK-CBM48-CE1 montrant les 3 activités glucosidase, xylosidase and estérase. Cette étude a permis de poser les bases d’un brevet sur cette enzyme. D’un autre côté, les enzymes ayant montré une activité xylanase ou féruloyle-estérase se sont révélées complémentaires d’un cocktail cellulolytique durant la dégradation de paille de blé prétraitée. Enfin, dans une troisième partie, nous avons étudié un fragment d’ADN provenant la banque P. militaris, codant pour 19 ORFs et appartenant à une espèce du genre Bacteroides. La caractérisation biochimique d’Abn43A, Abn43B, Abf51A et Abf51B-trunc a montré que ces 4 enzymes portent des actions complémentaires sur l’hydrolyse de l’arabinane, et qu’elles peuvent agir de manière synergique pour la dégradation de ce polymère pectique. Enfin, l’étude détaillée des 19 ORFs codées sur ce fragment d’ADN nous a permis de proposer un schéma global de détection, d’hydrolyse et de métabolisation de l’arabinane par cette espèce du genre Bacteroides. / This thesis was performed in the context of the Futurol project, a French national project that aims at producing bioethanol from plant biomass such as wood and cereal straw. To reach that goal, the biomass must be pretreated, and enzymatically degraded to release fermentable simple sugar. My implication in that project was to discover original enzymes that can hydrolyze the hemicellulose, a major heteropolysaccharide found in plant cell wall.To mine for new biocatalysts, the gut microbial communities of two species of termite were investigated by a metagenomic approach : Nasutitermes corniger, a wood-feeder termite, and Termes hispaniolae supposed to be a soil-wood feeder. 30 000 metagenomic clones were screened on an array of 10 cellulosic and hemicellulosic substrates and 660 hits were obtained. Phenotypic comparison showed clear differences between both environments, probably related to the diet of the termite. The sequence of 45 N. corniger metagenomic inserts revealed 120 original sequences encoding for putative enzymes of interest. Original sequences encoding for multimodular enzymes were revealed and many ORFs were organized in clusters, suggesting that these enzymes are encoded on Polysaccharides Utilization Locus. In a second part, a high-throughput approach was used for the cloning, the expression and the slight characterization of 104 full-size and truncated enzymes. Forty five recombinant proteins were produced soluble, and their investigation revealed the activity of 19 enzymes and of 12 enzymatic modules, representing a hemicellulolytic tool-box for endo- and exo-type activities. In some cases, the implication of “Unkown” domains in the activity of multimodular enzymes was demonstrated. This approach was particularly efficient for the study of the GH3-UNKCBM48-CE1 Pm69, and this study triggered the patent process for this multiactive glucosidase, xylosidase and esterase. The xylanases and the feruloyl esterases were shown to be particularly efficient to supplement cellulolytic cocktails on pretreated wheat straw. In a third part, we investigated a DNA fragment belonging to a species of the genus Bacteroides and that encoded 19 ORFs. The biochemical characterization of Abn43A, Abn43B, Abf51A and Abf51B-trunc showed that these four enzymes harbored complementary actions for the hydrolysis of the arabinan, and that they can act synergistically for the hydrolysis of this pectic polymer. We also revealed that Abn43B had an original mode of action that we classified as exo-arabinanase. Finally, the in-depth study of the 19 ORFs allowed us to propose the entire scheme for arabinan detection, hydrolysis and utilization by the Bacteroides species carrying this DNA sequence

Les co-produits agricoles, son et paille de blé, sont riches en arabinoxylanes (AX) qui après hydrolyse peuvent fournir des molécules pour des usages variés: xylo-oligosides (prébiotiques), xylose (xylitol), acide férulique (vanilline, antioxydant). L’étude a visé à mettre en place un procédé intégré et éco-compatible, depuis l'hydrolyse enzymatique des AX du son de blé jusqu'à la purification de l'acide férulique. Des cocktails hémicellulasiques produits par culture de la bactérie Thermobacillus xylanilyticus sur paille ou son de blé et mis en œuvre dans des conditions variées ont permis de libérer des glucides (mono- et oligomères) et de l’acide férulique. Afin d’augmenter la monomérisation, deux nouvelles β-xylosidases ont été produites à partir de T. xylanilyticus et caractérisées. Elles ont été testées pour complémenter des cocktails hémicellulasiques complexes issus de T. xylanilyticus et étudiées en mélange avec une xylanase et une arabinosidase pures. Le choix d’une résine anionique faible sous forme base libre pour séparer les fractions glucidique et phénolique et purifier l’acide férulique s’est également inscrit dans une démarche d’éco-conception : la résine Amberlyst A21 a montré une bonne affinité pour l’acide férulique et sa régénération a libéré une fraction très concentrée. La déminéralisation préalable de l’hydrolysat par électrodialyse a permis d’accroître la capacité de la résine pour l’acide et la pureté de la fraction récupérée, potentiellement cristallisable. / Agriculture by-products (wheat bran and straw) are rich in arabinoxylans (AX). These polymers composed of a main chain of β-(1,4) linked xylose ramified by arabinose and ferulic acid, are sources of molecules for various applications: xylooligosaccharides as prebiotics, xylose to synthesize xylitol, a non-cariogenic sweetener, or ferulic acid as a precursor of vanillin or an antioxidant molecule for packaging applications. The aim of this work was to set up an eco-friendly process ranging from wheat bran AX hydrolysis to ferulic acid purification.Hemicellulasic cocktails obtained by growing Thermobacillus xylanilyticus on wheat straw or wheat bran were implemented in various conditions. They released a carbohydrate fraction (mono- and oligosaccharides) and ferulic acid. In order to increase the monomerization, two new β-xylosidases were grown from T. xylanilyticus and characterized. They were tested to supplement the complex hemicellulasic cocktails from T. xylanilyticus and studied in mixture with a pure xylanase and a pure arabinosidase.The choice of a weak anionic resin under free base form to separate the glucidic and the phenolic fractions and to purify ferulic acid was also driven by environnement purposes: Amberlyst A21 resin showed a good affinity for ferulic acid and regeneration allowed a concentrated fraction of ferulate to be obtained. Prior demineralization by electrodialysis increased the capacity of the resin for ferulic acid and the purity of the recovered fraction, potentially allowing crystallization.

Palm oil empty fruit bunches (EFB) is an abundant and cheap lignocellulose material in South East Asia. Its use as the sole medium for producing lignocellulose-hydrolyzing enzymes would increase its commercial value. A newly isolated Aspergillus niger DSM 26641 was investigated for its capability of producing hemicellulases in EFB hydrolysate obtained by treatment with pressurized hot water (1-20%, w/v) at 120-180◦C in a 1 L Parr reactor for 10-60 min. The optimal hydrolysate for the fungal growth and endoxylanase production was obtained when 10% (w/v) of empty fruit bunch was treated at 120◦C or 150◦C for 10 min, giving an endoxylanase activity of 24.5 mU ml-1 on RBB-Xylan and a saccharification activity of 5 U ml-1 on xylan (DNS assay). When the hydrolysates were produced at higher temperatures, longer treatment times or higher biomass contents, only less than 20% of the above maximal endoxylanase activity was detected, possibly due to the higher carbohydrate concentrations in the medium. Transcriptome analysis showed that 3 endoxylanases (expression levels 59-100%, the highest level was set as 100%), 2 b-xylosidases (4%), 4 side chain-cleaving arabinofuranosidases (1-95%), 1 acetyl xylan esterase (9%) and 2 ferulic acid esterases (0.3-9%) were produced together.

O mundo se depara atualmente com a perspectiva de um significativo aumento na demanda por etanol combustível. O bagaço de cana está entre os maiores subprodutos agroindustriais no Brasil, sendo uma das alternativas na utilização para a produção do etanol de segunda geração. A degradação do bagaço de cana requer a ação de muitas enzimas diferentes que são reguladas transcripcionalmente. Considerando-se que o custo de celulases e hemicelulases contribuem substancialmente no preço do bioetanol, novos estudos visando o entendimento da eficiência e produtividade de celulases são de grande importância. Para entender como melhorar coquetéis de enzimas que podem hidrolizar o bagaço de cana-de-açúcar pré-tratado, uitlizou-se um experimento de genômica para investigar-se quais genes e vias são transcripcionalmente moduladas durante o crescimento de A. niger em bagaço de cana-de-açúcar explodido. Neste trabalho foram identificados genes que codificam celulases e hemicelulases com aumento da expresão durante o crescimento em bagaço de cana-de-açúcar explodido. Foi também realizada a determinação do acúmulo de mRNA de diversos genes que codificam transportadores para verificar se estes eram induzidos por xilose e por depedência de glicose. Foram identificados 18 genes que corresponde a 58% de celulases preditas em A. niger e 21 genes que correponde a 58% de hemicelulases preditas em A. niger os quias foram altamente expressos durante o crescimento em bagaço de cana-de-açúcar explodido. Foi investigado também o papel central realizado pelas proteínas quinases e fosfatases não essenciais (NPKs e NPPs, respectivamente) quando em presença de celulose como fonte de carbono, no sensoriamento do estado energético e na subsequente via de sinalização no fungo filamentoso modelo Aspergillus nidulans. O estudo com A. nidulans identificou 11 quinases e 7 fosfatases não essências, NPKs e NPPs, respectivamente, envolvidas na produção de celulases e em alguns casos, na produção também de hemicelulases. O envolvimento destas NPKs identificadas na resposta induzida por avicel e na desrepressão foram acessados pela análise do transcriptoma da cepa selvagem e por microscopia de fluorescência através da cepa de fusão CreA::GFP expressa no selvagem e no background dos mutantes de NPKs. A ausência das quinases snfA e schA reduziu dramaticamente a resposta transcricional induzida por celulose incluindo a expressão de enzimas hidrolíticas e transportadores, enquanto que a ausência de snfA resultou em uma quase completa modulação gênica induzida por celulose. O mecanismo pelo qual essas duas quinases controlam a transcrição gênica foi identificado, onde os dois mutantes de quinases foram capazes de desbloquear o CreA mediante a repressão catabólica do carbono (CCR), sob condições de desrepressão, como em baixa presença de carbono ou crescimento em celulose. Desta forma, este trabalho abriu novas possibilidades para o entendimento da sacarificação do bagaço de cana-de-açúcar por hidrolases de A. niger e para a construção de coquetéis de enzimas mais eficientes para a obtenção do etanol de segunda geração. Também possibilitou a identificação de muitas quinases e fosfatases envolvidas no sensoriamento do carbono e do estado energético, as quais demonstraram papéis sobrespostos e distintos de snfA e schA na regulação da desrepressão de CreA e na produção de enzimas hidrolíticas em A. nidulans. / The world today is faced with the prospect of a significant increase in demand for fuel ethanol. Sugarcane bagasse is among the largest agro-industrial by-products in Brazil, one of the alternatives in use for the production of second generation ethanol. Degradation of sugarcane bagasse requires the action of many different enzymes which are transcriptionally regulated. Considering that the costs of cellulases and hemicellulases contribute substantially to the price of bioethanol, new studies aimed at understanding and improving cellulase efficiency and productivity are of paramount importance. To understand how to improve enzymatic cocktails that can hydrolyze pretreated sugarcane bagasse, we used a genomics approach to investigate which genes and pathways are transcriptionally modulated during growth of A. niger on steam-exploded sugarcane bagasse. We also sought to determine whether the mRNA accumulation of several steam-exploded sugarcane bagasseinduced genes encoding putative transporters is induced by xylose and dependent on glucose. We identified 18 genes that corresponds to 58% of A. niger predicted cellulases and 21 genes that correspond to 58% of A. niger predicted hemicellulases, that were highly expressed during growth on sugarcane bagasse. The central role performed by nonessential protein kinases (NPK) and phosphatases (NPP) when grown on cellulose as a sole carbon source, in the sensing energetic status and the subsequent signalling pathways was assessed in the model filamentous fungus Aspergillus nidulans. This study identified multiple kinases and phosphatases (NPKs and NPPs, respectively) involved in the sensing of carbon or energetic status, while demonstrating the overlapping and distinct roles of snfA and schA in the regulation of CreA derepression and hydrolytic enzyme production in A.nidulans. The involvement of the identified NPKs in avicel-induced responses and CreA derepression was assessed by genome-wide transcriptomics and fluorescent microscopy of a CreA::GFP fusion proteinexpressed in the wild-type and NPK-deficient mutant backgrounds. The absence of either the schA or snfA kinase dramatically reduced cellulose-induced transcriptional responses including the expression of hydrolytic enzymes and transporters, while the absence snfA resulted in a near complete loss of wild-typecellulose-induced gene modulation. The mechanism by which these two NPKs controlled gene transcription was identified, as neither of NPK-deficient mutants were able to unlock CreA-mediated carbon catabolite repression, under derepressing conditions, such as carbon starvation or growth on cellulose. Our presently reported work opens new possibilities for understanding sugarcane biomass saccharification by A. niger hydrolases and for the construction of more efficient enzymatic cocktails for second-generation bioethanol. This work also enable the identification of multiple kinases and phosphatases involved in the sensing of carbon or energetic status, while demonstrating the overlapping and distinct roles of snfA and schA in the regulation of CreA derepression and hydrolytic enzyme production in A.nidulans.

O mundo se depara atualmente com a perspectiva de um significativo aumento na demanda por etanol combustível. O bagaço de cana está entre os maiores subprodutos agroindustriais no Brasil, sendo uma das alternativas na utilização para a produção do etanol de segunda geração. A degradação do bagaço de cana requer a ação de muitas enzimas diferentes que são reguladas transcripcionalmente. Considerando-se que o custo de celulases e hemicelulases contribuem substancialmente no preço do bioetanol, novos estudos visando o entendimento da eficiência e produtividade de celulases são de grande importância. Para entender como melhorar coquetéis de enzimas que podem hidrolizar o bagaço de cana-de-açúcar pré-tratado, uitlizou-se um experimento de genômica para investigar-se quais genes e vias são transcripcionalmente moduladas durante o crescimento de A. niger em bagaço de cana-de-açúcar explodido. Neste trabalho foram identificados genes que codificam celulases e hemicelulases com aumento da expresão durante o crescimento em bagaço de cana-de-açúcar explodido. Foi também realizada a determinação do acúmulo de mRNA de diversos genes que codificam transportadores para verificar se estes eram induzidos por xilose e por depedência de glicose. Foram identificados 18 genes que corresponde a 58% de celulases preditas em A. niger e 21 genes que correponde a 58% de hemicelulases preditas em A. niger os quias foram altamente expressos durante o crescimento em bagaço de cana-de-açúcar explodido. Foi investigado também o papel central realizado pelas proteínas quinases e fosfatases não essenciais (NPKs e NPPs, respectivamente) quando em presença de celulose como fonte de carbono, no sensoriamento do estado energético e na subsequente via de sinalização no fungo filamentoso modelo Aspergillus nidulans. O estudo com A. nidulans identificou 11 quinases e 7 fosfatases não essências, NPKs e NPPs, respectivamente, envolvidas na produção de celulases e em alguns casos, na produção também de hemicelulases. O envolvimento destas NPKs identificadas na resposta induzida por avicel e na desrepressão foram acessados pela análise do transcriptoma da cepa selvagem e por microscopia de fluorescência através da cepa de fusão CreA::GFP expressa no selvagem e no background dos mutantes de NPKs. A ausência das quinases snfA e schA reduziu dramaticamente a resposta transcricional induzida por celulose incluindo a expressão de enzimas hidrolíticas e transportadores, enquanto que a ausência de snfA resultou em uma quase completa modulação gênica induzida por celulose. O mecanismo pelo qual essas duas quinases controlam a transcrição gênica foi identificado, onde os dois mutantes de quinases foram capazes de desbloquear o CreA mediante a repressão catabólica do carbono (CCR), sob condições de desrepressão, como em baixa presença de carbono ou crescimento em celulose. Desta forma, este trabalho abriu novas possibilidades para o entendimento da sacarificação do bagaço de cana-de-açúcar por hidrolases de A. niger e para a construção de coquetéis de enzimas mais eficientes para a obtenção do etanol de segunda geração. Também possibilitou a identificação de muitas quinases e fosfatases envolvidas no sensoriamento do carbono e do estado energético, as quais demonstraram papéis sobrespostos e distintos de snfA e schA na regulação da desrepressão de CreA e na produção de enzimas hidrolíticas em A. nidulans. / The world today is faced with the prospect of a significant increase in demand for fuel ethanol. Sugarcane bagasse is among the largest agro-industrial by-products in Brazil, one of the alternatives in use for the production of second generation ethanol. Degradation of sugarcane bagasse requires the action of many different enzymes which are transcriptionally regulated. Considering that the costs of cellulases and hemicellulases contribute substantially to the price of bioethanol, new studies aimed at understanding and improving cellulase efficiency and productivity are of paramount importance. To understand how to improve enzymatic cocktails that can hydrolyze pretreated sugarcane bagasse, we used a genomics approach to investigate which genes and pathways are transcriptionally modulated during growth of A. niger on steam-exploded sugarcane bagasse. We also sought to determine whether the mRNA accumulation of several steam-exploded sugarcane bagasseinduced genes encoding putative transporters is induced by xylose and dependent on glucose. We identified 18 genes that corresponds to 58% of A. niger predicted cellulases and 21 genes that correspond to 58% of A. niger predicted hemicellulases, that were highly expressed during growth on sugarcane bagasse. The central role performed by nonessential protein kinases (NPK) and phosphatases (NPP) when grown on cellulose as a sole carbon source, in the sensing energetic status and the subsequent signalling pathways was assessed in the model filamentous fungus Aspergillus nidulans. This study identified multiple kinases and phosphatases (NPKs and NPPs, respectively) involved in the sensing of carbon or energetic status, while demonstrating the overlapping and distinct roles of snfA and schA in the regulation of CreA derepression and hydrolytic enzyme production in A.nidulans. The involvement of the identified NPKs in avicel-induced responses and CreA derepression was assessed by genome-wide transcriptomics and fluorescent microscopy of a CreA::GFP fusion proteinexpressed in the wild-type and NPK-deficient mutant backgrounds. The absence of either the schA or snfA kinase dramatically reduced cellulose-induced transcriptional responses including the expression of hydrolytic enzymes and transporters, while the absence snfA resulted in a near complete loss of wild-typecellulose-induced gene modulation. The mechanism by which these two NPKs controlled gene transcription was identified, as neither of NPK-deficient mutants were able to unlock CreA-mediated carbon catabolite repression, under derepressing conditions, such as carbon starvation or growth on cellulose. Our presently reported work opens new possibilities for understanding sugarcane biomass saccharification by A. niger hydrolases and for the construction of more efficient enzymatic cocktails for second-generation bioethanol. This work also enable the identification of multiple kinases and phosphatases involved in the sensing of carbon or energetic status, while demonstrating the overlapping and distinct roles of snfA and schA in the regulation of CreA derepression and hydrolytic enzyme production in A.nidulans.