ATCC 824 utilizes glucose, xylose, and lichenan, but not laminarin or cellulose, for growth and solvent production. Lichenan has a structure that resembles the β-glucans that are abundant in plant cell walls of cereals such as barley, rye, and wheat (25
). This polymer is a substrate for several different enzymes, including lichenanases (EC 22.214.171.124) and 1,3-1,4-β-glucanases (EC 126.96.36.199), and is often degraded by cellulases of both the exo- and the endo- type (1
). Laminarin is degraded mainly by laminarinases (EC 188.8.131.52), lichenanases, and 1,3-1,4-β-glucanases. All of these enzymes belong to family 16 of the glycoside hydrolases, but each class contains important unique sequences, especially in the region surrounding the strictly conserved catalytic residues (25
). The genome of C. acetobutylicum
ATCC 824 contains a single gene (CAC2807) that encodes a glycoside hydrolase from family 16 which does not belong to the cellulosome gene cluster and lacks dockerin modules. The protein encoded by this gene shows high homology to LicB from Clostridium thermocellum
(54% identity and 70% similarity), which seems to be a cellulosomal component. When produced by E. coli
, LicB showed a high level of activity on lichenan and no activity on laminarin (31
). The absence of a gene coding for a canonical laminarinase from family 16 in the genome of C. acetobutylicum
could explain the low laminarinase activity (Table ) produced and the bacterium's inability to utilize laminarin.
Although C. acetobutylicum
produced CMCase activity under certain conditions (Table ), it did not grow on CMC, either because the hydrolysis products obtained could not be further converted due to the presence of the carboxymethyl groups or because the degradation rate was not fast enough to support growth. A similar inability to grow on CMC, even when the medium was supplemented with Celluclast 1.5L, was observed previously for Clostridium beijerinckii
For lichenan-grown cultures, all of the enzymatic activities measured were the highest observed for all cultures, and a low but significant avicelase activity could be detected, suggesting an induced expression of cellulolytic enzymes by lichenan (Table ). In a previous study, members of our laboratory could not detect extracellular avicelase activity in similarly grown C. acetobutylicum
). In the experiments described here, however, the protein concentrations were substantially higher, as the extracellular proteins were concentrated approximately 60-fold. In xylose-grown cultures, the activities were the second highest (Table ) of those observed, and activity on MUC was detectable, although it was lower than that in lichenan-grown cultures (Fig. ). Lee et al. (12
) reported the production of endoglucanase (CMCase) and cellobiosidase (pNPCase) activities by C. acetobutylicum
grown on various carbon sources (glucose, cellobiose, xylose, and mannose) in a chemostat.
A conserved feature between cellulosomes produced by clostridial species is the presence of a family 48 glycoside hydrolase enzyme with exoglucanase activity that seems to play an essential role in cellulosomal function. The only gene that possibly encodes a glycoside hydrolase from family 48 in the genome of C. acetobutylicum
, was overexpressed in E. coli
. The resulting protein, CelF, was produced in inclusion bodies, always appearing in the insoluble fraction of the cell lysates, and therefore its enzymatic activity (CMCase or avicelase) could not be determined. Other clostridial cellulases from family 48, such as ExgS from C. cellulovorans
) and CelF from C. cellulolyticum
), have been produced by E. coli
, for which denaturation-renaturation methods or optimized production conditions allowed solubilization of at least part of the corresponding protein. However, similar methods were unsuccessful in the case of CelF from C. acetobutylicum
. Recently, it was shown that the fusion of a cellulose-binding domain to the catalytic domains increased the solubilities of cellulosomal and noncellulosomal cellulases of C. cellulovorans
upon expression in E. coli
), a strategy that looks promising for the solubilization of recombinant CelF.
Polyclonal antibodies raised against the E. coli
-produced CelF protein were used to detect the production of this protein by C. acetobutylicum
ATCC 824 grown on different substrates. On all substrates, CelF was specifically detected in the extracellular medium as a single band of the expected size (Fig. ). CelF was found to be constitutively produced by C. acetobutylicum
but was induced during growth on lichenan, xylose, or Avicel. Our results are consistent with those in which the expression of CelF by C. acetobutylicum
grown on cellobiose was confirmed by N-terminal sequencing of the extracellular protein (29
). In C. cellulovorans
, the major cellulosomal components are constitutively expressed, and depending on the growth substrate, specific enzymes are induced and the compositions of the cellulosomes vary (10
). In C. thermocellum
, transcriptional regulation of the cellulosomal cel48A
gene (encoding a cellulosomal subunit analogous to CelF in C. acetobutylicum
) appears to be dependent not only on the growth substrate, but also on the growth rate (3
), but whether this is the case in C. acetobutylicum
still needs to be investigated.
In the genome of C. acetobutylicum, there is a gene encoding a CcpA-like regulator, CAC3037, and several CRE sites that are putative targets for CcpA. A search for CREs in the genome was performed by the use of well-characterized consensus sequences, and various hits were found both in the chromosome and in the megaplasmid. CRE sequences are present in many genes, including some that encode extracellular polymer-degrading enzymes (Table ). No CRE sites were found in the promoter or in the coding regions of genes encoding putative cellulosomal subunits.
Despite the presence of genes involved in cellulose degradation in its genome and the production of cellulolytic enzymes, C. acetobutylicum ATCC 824 is not able to utilize cellulose for growth. In this study, we showed that CelF, a protein encoded by one of the genes present in the cellulosomal gene cluster, is produced and exported to the extracellular medium during growth on glucose, cellobiose, xylose, lichenan, or Avicel. On the last three substrates, there were larger amounts of CelF in the extracellular medium, which correlated with increased cellulolytic activities. However, at this stage, these activities cannot be assigned to CelF only, since other genes coding for different glycoside hydrolases are present in the genome. A functional study of the genes encoding proteins involved in (hemi)cellulose degradation, in particular the putative cellulosomal genes, will be an essential step toward understanding the reason for the lack of true cellulolytic properties by C. acetobutylicum ATCC 824 and the enhancement of the utilization of lignocellulosic substrates for the production of acetone, butanol, and ethanol by this strain.