Global View of the Clostridium thermocellum Cellulosome Revealed by Quantitative Proteomic Analysis

Abstract

A metabolic isotope-labeling strategy was used in conjunction with nano-liquid chromatography-electrospray ionization mass spectrometry peptide sequencing to assess quantitative alterations in the expression patterns of subunits within cellulosomes of the cellulolytic bacterium Clostridium thermocellum, grown on either cellulose or cellobiose. In total, 41 cellulosomal proteins were detected, including 36 type I dockerin-containing proteins, which count among them all but three of the known docking components and 16 new subunits. All differential expression data were normalized to the scaffoldin CipA such that protein per cellulosome was compared for growth between the two substrates. Proteins that exhibited higher expression in cellulosomes from cellulose-grown cells than in cellobiose-grown cells were the cell surface anchor protein OlpB, exoglucanases CelS and CelK, and the glycoside hydrolase family 9 (GH9) endoglucanase CelJ. Conversely, lower expression in cellulosomes from cells grown on cellulose than on cellobiose was observed for the GH8 endoglucanase CelA; GH5 endoglucanases CelB, CelE, CelG; and hemicellulases XynA, XynC, XynZ, and XghA. GH9 cellulases were the most abundant group of enzymes per CipA when cells were grown on cellulose, while hemicellulases were the most abundant group on cellobiose. The results support the existing theory that expression of scaffoldin-related proteins is coordinately regulated by a catabolite repression type of mechanism, as well as the prior observation that xylanase expression is subject to a growth rate-independent type of regulation. However, concerning transcriptional control of cellulases, which had also been previously shown to be subject to catabolite repression, a novel distinction was observed with respect to endoglucanases.Clostridium thermocellum, a thermophilic, strictly anaerobic gram-positive bacterium, has the highest rate of cellulose utilization of any bacterium, and for this reason it is deemed of great significance to the pursuit of biofuel production from the cellulosic materials in plant biomass (3,6,20,32). The organism achieves hydrolysis of crystalline cellulose by virtue of a large cell surface-bound protein complex known as the cellulosome, the structure of which consists of a central noncatalytic scaffoldin protein (CipA) bearing up to nine catalytic subunits (44). The attachment of a given subunit is mediated by the interaction of its type I dockerin (Doc1) domain with one of the nine cohesin type I domains of CipA (26). CipA is, in turn, bound to the cell surface by virtue of the interaction of its type II dockerin domain with the type II cohesin domain of one of three S-layer anchor proteins, SdbA, Orf2p, or OlpB (6). CipA also contains a type III cellulose-binding module for attachment of the complex to cellulose (13).Previous studies have shown that cellulolytic activity in C. thermocellum is regulated by either carbon source or growth rate (or both) and that changes with respect to one or the other are reflected in overall cellulase ...

Metaproteomics of cellulose methanisation under thermophilic conditions reveals a surprisingly high proteolytic activity

“…The identified proteins included some major cellulosomal structural and catalytic components. In particular, CelS and CelJ proteins are among the most upregulated enzymes for C. thermocellum Avicel-grown cells compared with cellobiose-grown cells (Gold and Martin, 2007). Hence, even if a limited number of the over 30 cellulosomal proteins from C. thermocellum (Gold and Martin, 2007) were detected, the information was sufficient to confirm the cellulolytic activity of strains closely related to C. thermocellum.…”

Mutations from a family-shuffling-library reveal amino acid residues responsible for the thermostability of endoglucanase CelA from Clostridium thermocellum

“…This domain is known to anchor CelA to a scaffolding protein in cellulosome (Gold and Martin 2007) and is devoid of catalytic activity (Alzari et al 1996). The exchange of this domain appeared have no significant effect on the thermostability of CelA.…”

“…It is the major endoglucanase in the cellulosome as demonstrated by both early studies (Petre et al 1981) and recent proteomic analysis (Gold and Martin 2007;Zverlov et al 2005). It is optimally active at 75°C and is resistant to the denaturing effects of detergents and organic solvents (Schwarz et al 1986).…”

Innovative Biological Solutions to Challenges in Sustainable Biofuels Production

“…Biobutanol is an attractive fuel as it possesses better energy properties than ethanol, including higher energy content per volume, lower water absorption, and better blending ability. Additionally, C. thermocellum appears to be a cellulose-utilizing specialist (Freier et al, 1988;Demain et al, 2005;Tripathi et al, 2010) and produces cellulosomes, a multienzyme cellulose-solubilizing complex (Bayer et al, 1985;Bayer et al, 2004;Gold & Martin, 2007;Tripathi et al, 2010). Because of the exemplary capacity of C. thermocellum to convert cellulosic biomass without the addition of purified cellulose or hemicellulase enzymes, the CBP platform using C. thermocellum provides a promising means for low-cost production of renewable biofuels.…”