Quantitative iTRAQ Secretome Analysis of Aspergillus niger Reveals Novel Hydrolytic Enzymes

Abstract

The natural lifestyle of Aspergillus niger made them more effective secretors of hydrolytic proteins and becomes critical when this species were exploited as hosts for the commercial secretion of heterologous proteins. The protein secretion profile of A. niger and its mutant at different pH was explored using iTRAQ-based quantitative proteomics approach coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). This study characterized 102 highly confident unique proteins in the secretome with zero false discovery rate based on decoy strategy. The iTRAQ technique identified and relatively quantified many hydrolyzing enzymes such as cellulases, hemicellulases, glycoside hydrolases, proteases, peroxidases, and protein translocating transporter proteins during fermentation. The enzymes have potential application in lignocellulosic biomass hydrolysis for biofuel production, for example, the cellulolytic and hemicellulolytic enzymes glucan 1,4-alpha-glucosidase, alpha-glucosidase C, endoglucanase, alpha l-arabinofuranosidase, beta-mannosidase, glycosyl hydrolase; proteases such as tripeptidyl-peptidase, aspergillopepsin, and other enzymes including cytochrome c oxidase, cytochrome c oxidase, glucose oxidase were highly expressed in A. niger and its mutant secretion. In addition, specific enzyme production can be stimulated by controlling pH of the culture medium. Our results showed comprehensive unique secretory protein profile of A. niger, its regulation at different pH, and the potential application of iTRAQ-based quantitative proteomics for the microbial secretome analysis.

Proteomic Analysis of the Vibrio cholerae Type II Secretome Reveals New Proteins, Including Three Related Serine Proteases

“…This proteomic approach allows for simultaneous comparison of complex protein mixtures and was recently applied to profile bacterial, fungal, and eukaryotic secretomes (22,(63)(64)(65). To provide additional controls for these experiments, we used two isogenic eps mutants (N⌬eps and N⌬epsD) in addition to the wild-type strain (58).…”

Biochemical characterization of Aspergillus niger CfcI, a glycoside hydrolase family 18 chitinase that releases monomers during substrate hydrolysis

“…This suggests the function of CfcI might be different from currently characterized GH18 family members. Expression of CfcI has been identified by proteome and transcriptome studies during the late-exponential or stationary growth phase and under nutrient limitation, but (with one exception) not during exponential growth (Adav et al, 2010;Ferreira de Oliveira et al, 2011;Jørgensen et al, 2010;Lu et al, 2010;Martens-Uzunova & Schaap, 2009;van den Berg et al, 2010;Yuan et al, 2008). These expression conditions indicate that the physiological function of CfcI is important in processes that take place during the late stages of the aspergillus life cycle, such as autolysis or sporulation.…”

“…Analysis of expression conditions showed that CfcI of A. niger N402 is detected after the exponential growth phase, during nutrient limitation (Adav et al, 2010;Jørgensen et al, 2010;Lu et al, 2010;Martens-Uzunova & Schaap, 2009;van den Berg et al, 2010;Yuan et al, 2008). It is therefore an interesting candidate for a chitinase involved in sporulation or autolysis, the process where the ageing mycelium degrades part of its own hyphae (White et al, 2002).…”

“…The large proteins (up to 200 kDa) often contain multiple carbohydrate-binding modules (CBMs) of family 18 (CBM18) and lysine motif (LysM) modules (Gruber et al, 2011). CBM18 modules consist of around 40 amino acids, organized in a structure of small a-helices, b-sheets and mostly coil regions, for which binding to chitin and chitin oligosaccharides has been shown (Boraston et al, 2004).Analysis of expression conditions showed that CfcI of A. niger N402 is detected after the exponential growth phase, during nutrient limitation (Adav et al, 2010;Jørgensen et al, 2010;Lu et al, 2010;Martens-Uzunova & Schaap, 2009;van den Berg et al, 2010;Yuan et al, 2008). It is therefore an interesting candidate for a chitinase involved in sporulation or autolysis, the process where the ageing mycelium degrades part of its own hyphae (White et al, 2002).…”

Crop Residue Degradation by Autochthonous Fungi Isolated from Cropping System Management Scenarios

“…Species of Aspergilli are known to produce all three enzymatic activities of the cellulose complex and exhibit strong hydrolytic activity towards cellulose. A. niger (Sharma et al 2001;Adav et al 2010), A. terreus (Emtiazi et al 2001), A. c a n d i d u s (Mila la e t a l . 2 0 0 9 ) and A. flavus (Saritha and Maruthi 2010) are known to be effective in the biodegradation of lignocellulosic biomass by producing oxidative and hydrolytic enzymes.…”