Data for iTRAQ secretomic analysis of Aspergillus fumigatus in response to different carbon sources.

Here, we provide data related to the research article entitled "Quantitative proteomics study of Aspergillus fumigatus secretome revealed deamidation of secretory enzymes" by Adav et al. (J. Proteomics (2015) [1]). Aspergillus sp. plays an important role in lignocellulosic biomass recycling. To explore biomass hydrolyzing enzymes of A. fumigatus, we profiled secretome under different carbon sources such as glucose, cellulose, xylan and starch by high throughput quantitative proteomics using isobaric tags for relative and absolute quantification (iTRAQ). The data presented here represents the detailed comparative abundances of diverse groups of biomass hydrolyzing enzymes including cellulases, hemicellulases, lignin degrading enzymes, and peptidases and proteases; and their post translational modification like deamidation.

Quantitative proteomic study of Aspergillus Fumigatus secretome revealed deamidation of secretory enzymes.

Aspergillus sp. plays an essential role in lignocellulosic biomass recycling and is also exploited as cell factories for the production of industrial enzymes. This study profiled the secretome of Aspergillus fumigatus when grown with cellulose, xylan and starch by high throughput quantitative proteomics using isobaric tags for relative and absolute quantification (iTRAQ). Post translational modifications (PTMs) of proteins play a critical role in protein functions. However, our understanding of the PTMs in secretory proteins is limited. Here, we present the identification of PTMs such as deamidation of secreted proteins of A. fumigatus. This study quantified diverse groups of extracellular secreted enzymes and their functional classification revealed cellulases and glycoside hydrolases (32.9%), amylases (0.9%), hemicellulases (16.2%), lignin degrading enzymes (8.1%), peptidases and proteases (11.7%), chitinases, lipases and phosphatases (7.6%), and proteins with unknown function (22.5%). The comparison of quantitative iTRAQ results revealed that cellulose and xylan stimulates expression of specific cellulases and hemicellulases, and their abundance level as a function of substrate. In-depth data analysis revealed deamidation as a major PTM of key cellulose hydrolyzing enzymes like endoglucanases, cellobiohydrolases and glucosidases. Hemicellulose degrading endo-1,4-beta-xylanase, monosidases, xylosidases, lignin degrading laccase, isoamyl alcohol oxidase and oxidoreductases were also found to be deamidated. BIOLOGICAL SIGNIFICANCE: The filamentous fungi play an essential role in lignocellulosic biomass recycling and fungal strains belonging to Aspergillus were also exploited as cell factories for the production of organic acids, pharmaceuticals, and industrially important enzymes. In this study, extracellular proteins secreted by thermophilic A. fumigatus when grown with cellulose, xylan and starch were profiled using isobaric tags for relative and absolute quantification (iTRAQ) by adopting liquid chromatography tandem mass spectrometry. The comparison of quantitative iTRAQ results revealed that cellulose and xylan stimulate expression of specific cellulases and hemicellulases, and expression level as a function of substrate. Post translational modifications revealed deamidation of key cellulases including endoglucanases, cellobiohydrolases and glucosidases; and hemicellulases and lignin degrading enzymes. The knowledge on deamidated enzymes along with specific sites of modifications could be crucial information for further functional studies of these enzymes of A. fumigatus.

Effects of vegetation type on microbial biomass carbon and nitrogen in subalpine mountain forest soils.

BACKGROUND/PURPOSE: Microbial biomass plays an important role in nutrient transformation and conservation of forest and grassland ecosystems. The objective of this study was to determine the microbial biomass among three vegetation types in subalpine mountain forest soils of Taiwan. METHODS: Tatachia is a typical high-altitude subalpine temperate forest ecosystem in Taiwan with an elevation of 1800-3952 m and consists of three vegetation types: spruce, hemlock, and grassland. Three plots were selected in each vegetation type. Soil samples were collected from the organic layer, topsoil, and subsoil. Microbial biomass carbon (Cmic) was determined by the chloroform fumigation-extraction method, and microbial biomass nitrogen (Nmic) was determined from the total nitrogen (Ntot) released during fumigation-extraction. Bacteria, actinomycetes, fungi, cellulolytic microbes, phosphate-solubilizing microbes, and nitrogen-fixing microbes were also counted. RESULTS: The Cmic and Nmic were highest in the surface soil and declined with the soil depth. These were also highest in spruce soils, followed by in hemlock soils, and were lowest in grassland soils. Cmic and Nmic had the highest values in the spring season and the lowest values in the winter season. Cmic and Nmic had significantly positive correlations with total organic carbon (Corg) and Ntot. Contributions of Cmic and Nmic, respectively, to Corg and Ntot indicated that the microbial biomass was immobilized more in spruce and hemlock soils than in grassland soils. Microbial populations of the tested vegetation types decreased with increasing soil depth. CONCLUSION: Cmic and Nmic were high in the organic layer and decreased with the depth of layers. These values were higher for spruce and hemlock soils than for grassland soils. Positive correlations were observed between Cmic and Nmic and between Corg and Ntot.

Study of Phanerochaete chrysosporium secretome revealed protein glycosylation as a substrate-dependent post-translational modification.

Lignocellulosic biomass is a potential sustainable resource of mixed sugars that can be exploited for biofuel and other biomaterials. Phanerochaete chrysosporium (P. chrysosporium) produce an arsenal of extracellular enzymes, the secretome, for efficiently degrading lignocellulosic biomass. Post-translational modifications (PTMs) of these biomass-degrading enzymes generate remarkable diversity, complexity, heterogeneity and also alter physiological behavior, function, and activities. Identification of PTMs and the sites of modifications of these secreted proteins remain as an essential but unexploited step to understand the biomass degradation mechanism. Therefore, this study applied electrostatic repulsion hydrophilic interaction chromatography (ERLIC) for glycopeptides enrichment and coupled with tandem mass spectrometry (LC-MS/MS) analysis for glycosylated secreted enzymes of P. chrysosporium during glucose, cellulose, and lignin degradation. Varied groups of enzymes, including cellulases, glycoside hydrolases, hemicellulases, lignin-degrading enzymes, were glycosylated. The comparisons of the glycosylated secreted enzymes of P. chrysosporium in glucose, cellulose, and lignin culture conditions revealed glycosylation as substrate-dependent PTMs.

Proteomic analysis of temperature dependent extracellular proteins from Aspergillus fumigatus grown under solid-state culture condition.

Fungal species of the genus Aspergillus are filamentous ubiquitous saprophytes that play a major role in lignocellulosic biomass recycling and also are considered as cell factories for the production of organic acids, pharmaceuticals, and industrially important enzymes. Analysis of extracellular secreted biomass degrading enzymes using complex lignocellulosic biomass as a substrate by solid-state fermentation could be a more practical approach to evaluate application of the enzymes for lignocellulosic biorefinery. This study isolated a fungal strain from compost, identified as Aspergillus fumigatus, and further analyzed it for lignocellulolytic enzymes at different temperatures using label free quantitative proteomics. The profile of secretome composition discovered cellulases, hemicellulases, lignin degrading proteins, peptidases and proteases, and transport and hypothetical proteins; while protein abundances and further their hierarchical clustering analysis revealed temperature dependent expression of these enzymes during solid-state fermentation of sawdust. The enzyme activities and protein abundances as determined by exponentially modified protein abundance index (emPAI) indicated the maximum activities at the range of 40-50 °C, demonstrating the thermophilic nature of the isolate A. fumigatus LF9. Characterization of the thermostability of secretome suggested the potential of the isolated fungal strain in the production of thermophilic biomass degrading enzymes for industrial application.

Quantitative proteomic analysis of secretome of microbial consortium during saw dust utilization.

Proteomics analysis of lignocellulolytic proteins by lignocellulosic biomass degrading microbes and compatible microbial consortium is a promising approach that offers a new means to enzyme discovery. The abundance of proteins in complex secretome by microbial communities would highlight key lignocellulolytic proteins for lignocellulosic biorefinery. In this study, lignocellulolytic enzymes of potent lignin degrading basidiomycota and effective cellulolytic ascomycota fungal strains, and their co-cultures were analyzed using high throughput isobaric tag for relative and absolute quantitation (iTRAQ) technique using liquid chromatography tandem mass spectrometry. Protein abundances in the iTRAQ-multiplexed samples were determined by integrating relative quantitation and exponentially modified protein abundance index (emPAI). The functional classification of the secretory proteins by individual culture and co-culture demonstrated 36.77% cellulolytic proteins, 13.06% hemicellulases, 14.09% ligninolytic proteins, 19.59% proteolytic enzymes. 7.22% hypothetical proteins and 6.87% cell morphogenesis proteins. The abundance of the proteins by individual cultures and co-cultured fungal consortium revealed that co-culturing of Phanerochaete chrysosporium with Trichoderma reesei QM6a and Trichoderma reesei Rut C30 induced the production of cellulolytic proteins and stimulated expression of hemicellulolytic enzymes. The hierarchical clustering of proteins in secretome of fungal strains and their co-cultures elucidated differential expressions of lignocellulolytic proteins by the microbial consortium.

Label free quantitative proteomic analysis of secretome by Thermobifida fusca on different lignocellulosic biomass.

Solid state fermentation of lignocellulosic biomass by filamentous microorganisms to induced enzyme production has been recognized as an attractive and cost effective technology. The secretion profile of lignocellulolytic enzymes by thermostable filamentous Thermobifida fusca (T. fusca) in solid state fermentation of different lignocellulosic biomasses, such as corn stover, hay; saw dust; sugarcane bagasse; wood chips; and un-dried green plant were explored using label-free exponentially modified protein abundance index (emPAI) based quantitative proteomics. Comparative analyses of T. fusca secretion profiles between cellulose and the various lignocellulosic biomasses showed induced expression of cellulolytic proteins by cellulose, and expression of hemicellulose, pectin and lignin degrading enzymes were induced by lignocellulosic biomasses. The solid state fermentation by T. fusca on lignocellulosic biomasses also revealed increased expressions of various transport proteins and hypothetical proteins. The Bray-Curtis similarity indices, clustering, and multidimensional scaling plot explicated differential protein expressions by T. fusca on different lignocellulosic biomasses, indicating that protein secretion by T. fusca is reliant on substrate complexity.

Quantitative proteomic analysis of lignocellulolytic enzymes by Phanerochaete chrysosporium on different lignocellulosic biomass.

Lignocellulosic biomass from agricultural crop residues and forest waste represents an abundant renewable resource for bioenergy and future biofuel. The current bottleneck of lignocellulosic biofuel production is the hydrolysis of biomass to sugar. To understand the enzymatic hydrolysis of complex biomasses, in this report, lignocellulolytic enzymes secretion by Phanerochaete chrysosporium cultivated in different natural lignocellulosic biomass such as corn stover, hay, sawdust, sugarcane baggase, wheat bran and wood chips were quantitatively analyzed with the iTRAQ technique using LC-MS/MS. A diverse groups of enzymes, including cellulases, glycoside hydrolases, hemicellulases, lignin degrading enzymes, peroxidases, esterases, lipases, chitinases, peptidases, protein translocating transporter and hypothetical proteins were quantified, of which several were novel lignocellulosic biomass hydrolyzing enzymes. The quantitative expression and regulation of lignocellulolytic enzymes by P. chrysosporium were dependent on the nature and complexity of lignocellulosic biomass as well as physical size of the biomass. The iTRAQ data revealed oxidative and hydrolytic lignin degrading mechanism of P. chrysosporium. Numerous proteins presumed to be involved in natural lignocellulosic biomass transformation and degradation were expressed and produced in variable quantities in response to different agricultural and forest wastes.

Proteomic analysis of pH and strains dependent protein secretion of Trichoderma reesei.

Bioenergy, particularly biofuel, from lignocellulosic biomass has been considered as one of the most promising renewable and sustainable energies. The industrial productivity and efficiency of microbial lignocellulolytic enzymes for cellulosic biofuel applications are significantly affected by pH of culture condition. This study established and compared hydrolytic protein expression profiles of Trichoderma reesei QM6a, QM9414, RUT C30 and QM9414MG5 strains at different pH in cellulosic culture media. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of secretome of T. reesei cultured from pH 3.0-9.0 revealed significantly higher hydrolytic protein expressions at acidic pH. The Bray-Curtis similarity indices, clustering, and Shannon diversity index elucidated differences in protein secretion at different pHs in individuals and among the strains. This study demonstrated a comparative lignocellulolytic enzyme secretion profile of T. reesei and its mutants at different pHs and provides pH sensitive and resistance enzyme targets for industrial lignocellulose hydrolysis.

Seasonal variation of microbial populations and biomass in Tatachia grassland soils of Taiwan.

To investigate the seasonal variations of microbial ecology in grassland of Tatachia forest, soil properties, microbial populations, microbial biomass, and 16S rDNA clone library analysis were determined. The soil had temperatures 6.6-18.4 degrees C, pH 3.6-5.1, total organic carbon 1.11-10.68%, total nitrogen 0.18-0.78%, and C/N ratios 3.46-20.55. Each gram of dry soil contained bacteria, actinomycetes, fungi, cellulolytic, phosphate-solubilizing microbes, and nitrogen-fixing microbes 4.54 x 10(4) to 3.79 x 10(7), 3.43 x 10(2) to 2.17 x 10(5), 5.74 x 10(3) to 3.76 x 10(6), 1.97 x 10(3) to 1.34 x 10(6), 8.49 x 10(2) to 5.59 x 10(5), and 3.86 x 10(2) to 3.75 x 10(5) CFU, respectively. Each gram of soil contained 117-2,482 microg of microbial biomass carbon, 23-216 microg of microbial biomass nitrogen and 9-29 microg of DNA. The microbial populations, microbial biomass, and DNA decreased stepwise with the depth of soil, and they had low values in winter seasons. The microbial populations, microbial biomass carbon, microbial biomass nitrogen, and DNA at the BW2 horizon were 8.42-17.84, 19.26-64.40, 16.84-61.11, and 31.03-46.26% of those at the O horizon, respectively. When analyzing 16S rDNA library, members of Proteobacteria, Acidobacteria, Actinobacteria, Bacteroidetes, Chloroflexi, Firmicutes, candidate division TMI, candidate division TM7, Gammatimonadetes, and Verrucomicrobia were identified. Members of Proteobacteria (44.4%) and Acidobacteria (33.3%) dominated the clone libraries. Within the phylum Proteobacteria, alpha-, beta-, and gamma-Proteobacteria were most numerous, followed by delta-Proteobacteria.