Comparative secretome analyses of two Trichoderma reesei RUT-C30 and CL847 hypersecretory strains

Abstract

Background: Due to its capacity to produce large amounts of cellulases, Trichoderma reesei is increasingly been researched in various fields of white biotechnology, especially in biofuel production from lignocellulosic biomass. The commercial enzyme mixtures produced at industrial scales are not well characterized, and their proteinaceous components are poorly identified and quantified. The development of proteomic methods has made it possible to comprehensively overview the enzymes involved in lignocellulosic biomass degradation which are secreted under various environmental conditions.

Results: The protein composition of the secretome produced by industrial T. reesei (strain CL847) grown on a medium promoting the production of both cellulases and hemicellulases was explored using two-dimensional electrophoresis and MALDI-TOF or LC-MS/MS protein identification. A total of 22 protein species were identified. As expected, most of them are potentially involved in biomass degradation. The 2D map obtained was then used to compare the secretomes produced by CL847 and another efficient cellulolytic T. reesei strain, Rut-C30, the reference cellulase-overproducing strain using lactose as carbon source and inducer of cellulases.

Conclusion: This study provides the most complete mapping of the proteins secreted by T. reesei to date. We report on the first use of proteomics to compare secretome composition between two cellulase-overproducing strains Rut-C30 and CL847 grown under similar conditions. Comparison of protein patterns in both strains highlighted many unexpected differences between cellulase cocktails. The results demonstrate that 2D electrophoresis is a promising tool for studying cellulase production profiles, whether for industrial characterization of an entire secretome or for a more fundamental study on cellulase expression at genome-wide scale.

Figure 1

Coomassie blue-stained 2DE gel of secreted proteins from T. reesei CL847 cultivated on xylose–lactose medium. The protein spots identified are labeled by the protein abbreviations given in Tables 1 and 2. Spots names with asterisks refer to degraded form of proteins.

Figure 2

Coomassie blue-stained 2DE gel of secreted proteins from T. reesei CL847 (A) and Rut-C30 (B) cultivated on lactose medium. The protein spots identified are labeled by the protein abbreviations given in Tables 1 and 2.

Figure 3

Spot volume distribution of Rut-C30 and CL847 secretomes. Standard deviations are calculated from three replicates. Differences between the two strains can be related to both small spots, which are more abundant in CL847, and isoforms, equally more abundant in this strain.

Figure 4

Comparative Analysis of Rut-C30 (white histograms) and CL847 (grey histograms) secretomes. Values are expressed in %Volume except for B where it is a %Cel7A/%Cel7B ratio. A: Volume of total cellobiohudrolases; B: Cel7A-to-Cel6A ratio; C: Secondary endoglucanases; D: β-glucosidase; E: Non-xylolytic hemicellulases and F: Xylanases.