Cloning and DNA sequence of the gene coding for Clostridium thermocellum cellulase Ss (CelS), a major cellulosome component

Abstract

Clostridium thermocellum ATCC 27405 produces an extracellular cellulase system capable of hydrolyzing crystalline cellulose. The enzyme system involves a multicomponent protein aggregate (the cellulosome) with a total molecular weight in the millions, impeding mechanistic studies. However, two major components of the aggregate, SS (M(r) = 82,000) and SL (M(r) = 250,000), which act synergistically to hydrolyze crystalline cellulose, have been identified (J. H. D. Wu, W. H. Orme-Johnson, and A. L. Demain, Biochemistry 27:1703-1709, 1988). To further study this synergism, we cloned and sequenced the gene (celS) coding for the SS (CelS) protein by using a degenerate, inosine-containing oligonucleotide probe whose sequence was derived from the N-terminal amino acid sequence of the CelS protein. The open reading frame of celS consisted of 2,241 bp encoding 741 amino acid residues. It encoded the N-terminal amino acid sequence and two internal peptide sequences determined for the native CelS protein. A putative ribosome binding site was identified at the 5' end of the gene. A putative signal peptide of 27 amino acid residues was adjacent to the N terminus of the CelS protein. The predicted molecular weight of the secreted protein was 80,670. The celS gene contained a conserved reiterated sequence encoding 24 amino acid residues found in proteins encoded by many other clostridial cel or xyn genes. A palindromic structure was found downstream from the open reading frame. The celS gene is unique among the known cel genes of C. thermocellum. However, it is highly homologous to the partial open reading frame found in C. cellulolyticum and in Caldocellum saccharolyticum, indicating that these genes belong to a new family of cel genes.

Cellulase, Clostridia, and Ethanol

“…It is an exoglucanase (168-170, 231, 234). Sequencing of celS revealed an open reading frame of 2,241 bp encoding 741 amino acid residues with a predicted molecular weight of 80,670 (341,343). The sequence revealed that CelS belonged to a new cellulase family (341).…”

“…Of particular interest was our gene sequencing of S L (96,277) and the gene sequencing of S S (341).…”

A diverse set of family 48 bacterial glycoside hydrolase cellulases created by structure‐guided recombination

“…Three extensively characterized Cel48 cellulases were chosen as parents for construction of the SCHEMA recombination library: CelF [10] from the mesophile Clostridium cellulolyticum ATCC 35319, CelY [18] from the thermophile Clostridium stercorarium, and CelS (also known as CelA) [19] from the thermophile Clostridium thermocellum ATCC 27405.…”

Structure of the Clostridium stercorarium gene celY encoding the exo-1,4-�-glucanase Avicelase II

“…Recently, CelS, the most abundant catalytic subunit in cellulosomes of the cellulolytic thermophile Clostridiurn thermocellurn, was characterized as an exo-1,4-P-glucanase (Morag et al, 1991;Wang et al, 1993;Kruus et a/., 1995). Furthermore, CbhA and CbhB of Cellulomonas fimi Shen et al, 1995) and presumably CelF of Clostridium cellulolyticum were shown to represent this type of cellulolytic enzyme in mesophilic bacteria.…”