Exoproteome analysis of Clostridium cellulovorans in natural soft-biomass degradation

Abstract

Clostridium cellulovorans is an anaerobic, cellulolytic bacterium, capable of effectively degrading various types of soft biomass. Its excellent capacity for degradation results from optimization of the composition of the protein complex (cellulosome) and production of non-cellulosomal proteins according to the type of substrates. In this study, we performed a quantitative proteome analysis to determine changes in the extracellular proteins produced by C. cellulovorans for degradation of several types of natural soft biomass. C. cellulovorans was cultured in media containing bagasse, corn germ, rice straw (natural soft biomass), or cellobiose (control). Using an isobaric tag method and a liquid chromatograph equipped with a long monolithic silica capillary column/mass spectrometer, we identified 372 proteins in the culture supernatant. Of these, we focused on 77 saccharification-related proteins of both cellulosomal and non-cellulosomal origins. Statistical analysis showed that 18 of the proteins were specifically produced during degradation of types of natural soft biomass. Interestingly, the protein Clocel_3197 was found and commonly involved in the degradation of every natural soft biomass studied. This protein may perform functions, in addition to its known metabolic functions, that contribute to effective degradation of natural soft biomass.Electronic supplementary materialThe online version of this article (doi:10.1186/s13568-014-0089-9) contains supplementary material, which is available to authorized users.

Elucidation of the recognition mechanisms for hemicellulose and pectin in Clostridium cellulovorans using intracellular quantitative proteome analysis

“…First, we focused on profiles of metabolism-related proteins, such as enzymes involved in substrate degradation and metabolism, and other characteristic metabolic pathway. C. cellulovorans is known to change production of carbohydrate-related enzymes secreted into media from exoproteome analyses (Esaka et al 2015 ; Matsui et al 2013 ) and alternation of production of different metabolic pathways depending on which substrates are available in culture is also predicted.…”

“…Similarly annotated genes are part of mannan metabolic pathways in other bacteria (Senoura et al 2011 ), and we observed increased levels of proteins in this pathway (Clocel_3198, 3205) in the presence of galactomannan. However, Clocel_3197, which is known to play an important role in the mannan metabolic pathway (Esaka et al 2015 ), was only identified in our qualitative proteome analysis (Additional file 2 : Table S2). This protein has been also detected with “exoproteome” analysis (Esaka et al 2015 ).…”

“…Clostridium cellulovorans is a gram-positive, mesophilic, and cellulosome-producing anaerobe of the Family 1 (Cluster I) of Clostridia that can degrade and directly metabolize various kinds of carbohydrates such as cellulose, hemicellulose (xylan and mannan), and pectin (Petitdemange et al 1984 ; Tamaru et al 2010 ). Furthermore, C. cellulovorans alters its production of secreted enzymes, depending on which substrates are available (Morisaka et al 2012 ; Matsui et al 2013 ; Esaka et al 2015 ). Thus, C. cellulovorans can distinguish among varied substrates and adapt to survive in different environments.…”

Direct ethanol fermentation of the algal storage polysaccharide laminarin with an optimized combination of engineered yeasts

“…The crude solution was centrifuged at 12,000 × g for 10 min at 4°C, and the supernatant was obtained. After reduction, alkylation, and digestion by trypsin of each supernatant, the tryptic digests were subjected to nano liquid chromatography (LC)-mass spectrometry (MS)/MS analysis (Esaka et al, 2015).…”

Xylanase B fromClostridium cellulovorans743B: overexpression, purification, crystallization and X-ray diffraction analysis

“…The xynA and xynB genes have been cloned and confirmed to have xylanase activity (Kosugi et al, 2002;Han et al, 2004). Additionally, proteome analysis verified that C. cellulovorans expresses XynA and XynB proteins (Morisaka et al, 2012;Matsui et al, 2013;Esaka et al, 2015;Aburaya et al, 2015). We believe that these two xylanases play an important role in the hydrolysis of the xylan contained in lignocellulose.…”