Identificação e caracterização do secretoma e celulossoma de um novo isolado de Clostridium thermocellum (B8) para a sacarificação de biomassas lignocelulósicas

Dissertação (mestrado)—Universidade de Brasília, Instituto de Ciências Biológicas, Departamento de Biologia Celular, Programa de Pós-Graduação em Biologia Molecular, 2015.O presente trabalho tem como propósito a purificação de celulossomas, sua caracterização e do secretoma de um novo isolado de Clostridium thermocellum B8, obtido do rúmen de caprino. Para obtenção das amostras proteicas C. thermocellum foi cultivada em meio líquido contendo celulose como fonte de carbono. O secretoma que constitui a amostra de Proteínas Ligadas á Celulose (PLC) foi eluído a partir da celulose residual da cultura. As frações cromatográficas do principal pico de proteína resultante do fracionamento da amostra PLC em uma cromatografia de exclusão molecular (Superdex S-200) compõe a amostra de Celulossomas Parcialmente Purificados (CPP). Ambas as amostras apresentaram atividades enzimáticas celulolíticas e xilanolíticas. As análises de SDS-PAGE, DLS e espectrometria de massa confirmaram que a amostra CPP se trata de um complexo celulossomal, com uma massa molecular estimada de 11,3 MDa ± 4,4 MDa. As proteínas constituintes de CPP foram identificadas por espectrometria de massa LC-MS/MS, levando a identificação de: proteína estrutural (CipA), glicosil hidrolases das famílias 5, 8, 9, 10 e 48 e transportadores do tipo ABC. Na amostra PLC foram identificadas principalmente proteínas envolvidas no catabolismo de polissacarídeos, além de proteínas relacionadas com o mecanismo de transporte de moléculas e processos metabólicos. Dentre as proteínas que têm a função no catabolismo de polissacarídeos, foram identificados glicosil hidrolases das famílias 5, 8, 9, 10, 11, 26, 30, 43 e 53, com atividades de endoglucanase, celobiohidrolases, endoxilanases, endoxilanases com esterase feruloil e acetil com função de xilana esterase, xiloglucanases, arabinoxilanase, glucoxilana xilanahidrolase, arabinofuranosidase, exogalactanase, endogalactanase (arabinogalactana), quitinase, mananase. CPP e PLC apresentaram atividade máxima no intervalo de temperatura entre 60°C-70°C, pH 5.0, e estabilidade térmica a 50, 60 e 70°C, principalmente, no que diz respeito a amostra de PLC. As atividades holocelulolíticas de PLC foram inibidas por compostos fenólicos, enquanto que CPP apresentou um aumento nas atividades de CMCase e xilanase na presença de vários fenóis. A partir dos resultados da sacarificação dos substratos de celulose, palha de cana e piolho de algodão por PLC e CPP, foi observado maiores quantidades de açúcares liberados principalmente a 50°C, em comparação com os dados de 60°C, após 10 dias de incubação. Entretanto, foi observado que após o segundo dia de sacarificação, que os produtos obtidos pela desconstrução de materiais lignocelulósicos, acabam por interferir na velocidade de hidrólise das enzimas de C. thermocellum B8. Em conclusão, este estudo demonstrou potencial para utilização de ambas as amostras (CPP e PLC) de C. thermocellum B8 para hidrolisar biomassas lignocelulósicas, destacando-se o aumento da atividade celulolítica e xilanolítica de CPP na presença de compostos fenólicos, além da termoestabilidade de PLC.The present work aimed the purification and characterization of the secretome produced by Clostridium thermocellum B8, a novel isolate obtained from goat rumen, after growth on liquid medium containing cellulose as carbon source. The secretome were eluted from the residual substrate, constituting the Protein Linked on Cellulose (PLC) sample. The main protein peak of PLC fractionation onto a molecular exclusion (Superdex S-200) composes the Cellulosomes Partially Purified (CPP) sample. Both samples presented enzymatic activities of cellulases and xylanases. It was elucidated through SDS-PAGE, DLS and mass spectrometry that CPP sample is a cellulosome complex, with an estimated mass of 11,3 MDa ± 4,4 MDa. CPP’s constituting proteins were identified by mass spectrometry LC-MS/MS leading the identification of: scaffolding protein (CipA), glycoside hydrolase proteins classified on the families 5, 8, 9, 10 and 48 and ABC transporter substrate-binding protein. In the PLC sample were identified mostly proteins involved in the catabolism of polysaccharides, besides proteins related to the transport mechanism molecules and metabolic processes. Among the proteins which have catabolism of polysaccharides function, were identified glycosyl hydrolase families 5, 8, 9, 10, 11, 26, 30, 43 and 53 with endoglucanase activity, cellobiohydrolases, endoxylanases, endoxylanases with feruloil esterase and acetyl xylan esterase function, xyloglucanases, arabinoxylanase, glucoxylana xylanhydrolase, arabinofuranosidase, exogalactanase, endogalactanase (arabinogalactan), chitinase, mannanase. CPP and PLC presented maximal activity in the range of 60° to 70°C and pH 5.0, and also those samples have a high thermostability at 50, 60 and 70°C mainly for PLC sample. PLC holocelullolytic activities were inhibited by phenolic compounds, while CPP showed improvement or was less inhibited on its xylanase and CMCase activity in phenols presence. Saccharification results of cellulose, sugarcane straw and cotton gin waste by PLC and CPP, showed highest amounts of sugar released mostly at 50°C in comparison to 60°C and after 10 days of incubation. In summary, this research demonstrated the potential of using CPP and PLC samples of C. thermocellum B8 to hydrolyze lignocellulosic biomasses, with the ability of CPP increase its holocelullolytic activities in the presence of phenolic compounds, and the interesting thermostability of PLC sample, both being valuable for second generation production of biofuels

Used but not Sensed - The Paradox of D-xylose Metabolism in Saccharomyces cerevisiae

The realization that the extraction and combustion of fossil fuels is having serious effects on the environment and the climate, together with the ever-growing need for fuels, has led to the development of the concept of the biorefinery. Biorefineries are refineries in which fossil resources, such as oil, are replaced by renewable biomaterials to produce biofuels and biochemicals. Non-edible biomass is used in a lignocellulose-based refinery, which avoids the conflict between fuel and food production, but a number of inherent technical challenges must be overcome. The robust and genetic engineering-friendly yeast Saccharomyces cerevisiae (baker’s yeast) is a promising platform organism for biomass fermentation, but it lacks functional assimilatory pathways to utilise D-xylose, the second most abundant sugar in a wide range of lignocellulosic materials. During the past two decades, recombinant forms of S. cerevisiae have been developed able to efficiently convert D-xylose to ethanol. However, the rate of conversion is slow, and D-xylose appears not to be recognised by S. cerevisiae as a fermentable sugar.This thesis is focused on investigating the role of the sugar sensing and signalling routes in the unusual behaviour of S. cerevisiae on D-xylose. A panel of in vivo biosensors coupled to D-glucose signalling routes was used under different physiological conditions and in the presence of different genetic modifications. The green fluorescent protein gene (yEGFP3) was coupled to different endogenous yeast promoters known to be regulated by at least one of the three main sugar pathways: Snf3p/Rgt2p, cAMP/PKA and SNF1/Mig1p.The signallome investigation revealed that a recombinant strain of S. cerevisiae able to assimilate D-xylose could sense high concentrations of D-xylose, but the signal was similar to that observed with low levels of D-glucose: inducing SUC2p (SNF1/Mig1p pathway) and HXT2p (Snf3p/Rgt2p pathway) but repressing HXT1p (Snf3p/Rgt2p and cAMP/PKA pathway). Strains unable to metabolise D-xylose provided no clear signal in the presence of D-xylose due to heterogeneity in the population of the biosensor strains. However, in strains that were able to assimilate D-xylose, the signalling induction pattern was completely opposite to the signal obtained when protein kinase A (PKA) was activated by high levels of D-glucose. It was therefore hypothesized that the signal triggered by a high D-xylose level similar to a low D-glucose signal was due to a low PKA activity.Further validation of the role of sugar signalling was obtained by using targeted deletants known to improve the D-xylose consumption rate without being directly associated with D-xylose catabolic routes. Notably, it was found that the signalling response on D-xylose changed from a low D-glucose signal in the background strain, to simultaneous signalling of high and low D-glucose in the best strain (ira2Δisu1Δ). Since IRA2 is a repressor of PKA activity, this finding supported the hypothesis of the malfunction of PKA activity on D-xylose due to poor sensing through this route.This study also focused on understanding whether the sensing signal observed in the presence of high concentrations of D-xylose could be linked to a metabolite acting as a pathway regulator. Using strains in which PGI1, which encodes an isomerase enabling the reversible conversion of glucose-6-phosphate and fructose-6-phosphate, had been deleted, it was possible to link changes in signalling to disturbances in the levels of glycolytic intermediates. The findings presented in this thesis support the hypothesis of a dysfunctional sugar signalling mechanism on D-xylose, and show that the phenotype is a result of the lack of membrane sensing in connection with alterations in intracellular signalling

Assessing the effect of d-xylose on the sugar signaling pathways of Saccharomyces cerevisiae in strains engineered for xylose transport and assimilation

One of the challenges of establishing an industrially competitive process to ferment lignocellulose to value-added products using Saccharomyces cerevisiae is to get efficient mixed sugar fermentations. Despite successful metabolic engineering strategies, the xylose assimilation rates of recombinant S. cerevisiae remain significantly lower than for the preferred carbon source, glucose. Previously, we established a panel of in vivo biosensor strains (TMB371X) where different promoters (HXT1/2/4p; SUC2p, CAT8p; TPS1p/2p, TEF4p) from the main sugar signaling pathways were coupled with the yEGFP3 gene, and observed that wild-type S. cerevisiae cannot sense extracellular xylose. Here, we expand upon these strains by adding a mutated galactose transporter (GAL2-N376F) with improved xylose affinity (TMB372X), and both the transporter and an oxidoreductase xylose pathway (TMB375X). On xylose, the TMB372X strains displayed population heterogeneities, which disappeared when carbon starvation was relieved by the addition of the xylose assimilation pathway (TMB375X). Furthermore, the signal in the TMB375X strains on high xylose (50 g/L) was very similar to the signal recorded on low glucose (≤5 g/L). This suggests that intracellular xylose triggers a similar signal to carbon limitation in cells that are actively metabolizing xylose, in turn causing the low assimilation rates

D-xylose sensing in saccharomyces cerevisiae : Insights from D-glucose signaling and native D-xylose utilizers

Extension of the substrate range is among one of the metabolic engineering goals for microorganisms used in biotechnological processes because it enables the use of a wide range of raw materials as substrates. One of the most prominent examples is the engineering of baker’s yeast Saccharomyces cerevisiae for the utilization of D-xylose, a five-carbon sugar found in high abundance in lignocellulosic biomass and a key substrate to achieve good process economy in chemical production from renewable and non-edible plant feedstocks. Despite many excellent engineering strategies that have allowed recombinant S. cerevisiae to ferment D-xylose to ethanol at high yields, the consumption rate of D-xylose is still significantly lower than that of its preferred sugar D-glucose. In mixed D-glucose/D-xylose cultivations, D-xylose is only utilized after D-glucose depletion, which leads to prolonged process times and added costs. Due to this limitation, the response on D-xylose in the native sugar signaling pathways has emerged as a promising next-level engineering target. Here we review the current status of the knowledge of the response of S. cerevisiae signaling pathways to D-xylose. To do this, we first summarize the response of the native sensing and signaling pathways in S. cerevisiae to D-glucose (the preferred sugar of the yeast). Using the Dglucose case as a point of reference, we then proceed to discuss the known signaling response to Dxylose in S. cerevisiae and current attempts of improving the response by signaling engineering using native targets and synthetic (non-native) regulatory circuits