Profile of native cellulosomal proteins of Clostridium cellulovorans adapted to various carbon sources

The development of white biotechnology requires degradation of biomass using biocatalysts. The cellulosome, which is produced by many cellulolytic gram-positive anaerobic bacteria such as Clostridium, efficiently degrades plant cell wall polysaccharides. However, the molecular mechanism of cellulosome formation has not been characterized. We sequenced the entire genome of Clostridium cellulovorans (Tamaru et al. [2010a]) and identified all genes, including those that encode proteins of known and unknown functions, related to cellulosome composition (Tamaru et al. [2010a]). Genome analysis of C. cellulovorans indicated the presence of 57 cellulosomal enzymes-encoding genes including 4 scaffold proteins-encoding genes and 53 cellulosomal enzymes-encoding genes with dockerin domains (Doi and Tamaru [2001]). The major scaffold protein, CbpA, is comprised of 9 cohesin domains that bind to various cellulosomal enzymes via cohesin-dockerin interactions. Most cellulosomal enzymes are glycoside hydrolases, but they possess an interesting property. The cellulosome consists not only of glycoside hydrolases but of other proteins such as proteases, protease inhibitors, and unknown proteins. This characteristic may be important in the degradation of various resources. However, we have only general subcellular proteinous information regarding the proteins that actively degrade biomass, although genome analysis has provided many interesting insights into the characteristics of C. cellulovorans. The next step was to directly analyze cellulosomal proteins in the culture media.

Although proteome analysis has been advanced by the development of instruments such as the mass spectrometer (Link et al. [1999]), but difficulties remain. Ionization suppression in mass spectrometry is a significant problem that needs to be overcome for mass spectrometry to be used as a detector in proteome analysis. Results can be improved by complete pre-separation; construction of an efficient separation system is indispensable to obtain satisfactory measurement. The monolithic silica column is a novel separation medium of liquid chromatography that exhibits lower column back pressure owning by its high permeability. This property enables use of the long column that is impossible by conventional particle-packed columns and shows higher performance in comparison to conventional columns (Minakuchi et al. [1996], Minakuchi et al. [1998]). In addition, monolithic silica can be prepared in a long-fused silica capillary (300 cm) because of the attachment of the silica monolithic skeletons to the tube wall (Motokawa et al. [2002]). Proteome analysis methods could be improved by using long ultra-performance monolithic columns to overcome ionization suppression.

In this study, a novel analysis using the long monolithic column was applied to a proteome analysis focused on the cellulosome of C. cellulovorans to characterize the molecular mechanism that underlies efficient degradation of various biomasses.

The quality of the mass spectrum is very important in proteome analysis. The number of identified proteins is dependent on the number of detected peptides, which is dependent on the efficiency of the separation prior to ionization. Thus, the separation performance of the liquid chromatography method directly influences the quality of the proteome analysis. De Godoy et al ([2008]) reported that complete pre-separation contributed quality of proteome analysis such as the number of identified proteins. We examined the validity of a proteome analysis system using a long monolithic column (300 cm) which has higher resolution versus a conventional packed column (15 cm). The long monolithic column showed good separation and decreased ionization suppression. In the proteome analysis with the conventional system, protein purification was required because the number of detectable peptides was reduced by ionization suppression. With the new system, proteome analysis of cellulosomal proteins could be performed directly from crude extracted samples thanks to the ultra-performance separation by the long monolithic column. By this benefit, the analysis of non-cellulosomal enzyme of C. cellulovorans became possible simultaneously. The ratio of cellulosomal / non-cellulosomal genes encoding glycosyl hydrolases and polysaccharide lyases of C. cellulovorans was 0.48 (29/61) while that of C. thermocellum which is other cellulosome-producing clostridia was 3.31(53/16) (Tamaru et al. [2010b]). From proteome analysis of supernatants, total 59 proteins annotated glycosyl hydrolases and polysaccharide lyases were identified and its ratio of cellulosomal / non-cellulosomal was 0.69 (24/35). It was confirmed by proteome analysis that C. cellulovorans has much non-cellulosomal enzyme as compared with other clostridia. The degradation of various resources by C. cellulovorans is highly dependent on not only cellulosomal enzymes but also these secretory proteins.

To analyze the mechanism of cellulosome formation, our proteome analysis focused on the cellulosomes of C. cellulovorans. Although 57 cellulosomal genes including 4 scaffold proteins and 53 cellulosomal enzymes were identified by a genome analysis of C. cellulovorans (Tamaru et al. [2010b]), what kinds of cellulosomal proteins have been produced in various carbon sources has not been completely confirmed (Oded and Doi [1990]).

From our experiments, the number and members of cellulosomal enzymes were found to be changeable altered, caused by the benefit of good separation using the better performance system equipped with a long monolithic column (300 cm). This suggests that C. cellulovorans may change cellulosomal components to match its carbon sources. In addition, a set of 11 enzymes described as closed circles in Table1 including 5-assingned cellulases, 5-assingned hemicellulases, and 1-assingned other protein identified in all culture conditions were the same enzymes produced by cultivation with cellobiose (disaccharide made from units of glucose), which is the simplest substrate. We hypothesized that a basic cellulosome could be constructed by this set of 11 enzymes. Cellulosomes have been formed by adding other cellulosomal enzymes described as open circles in Table1 necessary for degradation of polysaccharides such as avicel (polysaccharides made from units of glucose) and xylan (polysaccharides made from units of xylose) (Figure2). It is interesting to note that these adding functions of enzymes included many other proteins (Table2), whose functions in saccharification, have not been clarified. The functions of these proteins categorized into ‘other proteins’ have been not made clear in the previous reports on the analyses of natural cellulosomal proteins, although cellulosomal protease/peptidase inhibitors (named cyspins) of C. cellulovorans classified into other proteins showed inhibition activities by heterologous gene expression systems (Meguro et al [2011]). These other proteins might play some an important role in the efficient degradation of each biomass. We will further investigate the stoichiometric ratio of cellulosomal enzymes and proteins in different cultivation media. From these results, we hope to develop a model for the formation of cellulosomes by the change of carbon sources and to apply it to utilize the various waste biomasses.