Cloning and expression in Escherichia coli of thermophilic Clostridium sp. F1 genes related to cellulose hydrolysis.

Abstract

Eleven distinct fragments of Clostridium sp. stain Fl (which was isolated and identified in this laboratory) DNAhave been cloned in E. coli and shown to express enzymatic activities related to cellulose hydrolysis. Six of the ll E. coli clones showed endoglucanase activity, 2 showed cellobiohydrolase activity as well as endoglycanase activity, one showed /?-glucosidase activity and the 2 others showedexoglucanase activity as well as xylanase activity. On comparingthe restriction mapsof the plasmids constructed in this study with those of C. thermocellum genes, it was concluded that 7 of the 1 1 clones carried hitherto unidentified cellulase genes, i.e., 4 endoglucanase genes, l/?-glucosidase gene and 2 xylanase/exoglucanase genes, and that the 4 other clones carried the same genes as those found in C. thermocellum.

Cloning, DNA sequencing, and expression of the gene encoding Clostridium thermocellum cellulase CelJ, the largest catalytic component of the cellulosome

“…Previously, we cloned eight distinct endoglucanase genes from C. thermocellum F1 and found that four of them were hitherto unidentified genes (38). Although a recombinant plasmid, pKS304, containing a new endoglucanase gene, celJ, conferred endoglucanase activity on E. coli, DNA sequencing of this gene revealed that the open reading frame (ORF) of celJ extended beyond the 3Ј end of the genomic DNA insert.…”

“…1). The construction of pKS304 was described previously (38). Cloning of a DNA fragment encoding the C-terminal end of CelJ by PCR.…”

“…Cloning of a DNA fragment encoding the C-terminal end of CelJ by PCR. C. thermocellum genomic DNA was isolated as described previously (38). Targeted gene walking PCR (36) involved (i) digestion of C. thermocellum genomic DNA with BamHI, (ii) ligation of the genomic fragments to pBluescript II SK(Ϫ), and (iii) PCR from the chimeric plasmid library with an internal primer specific for celJ and the universal T7 primer.…”

“…We constructed a gene bank of C. thermocellum F1, which was isolated from a compost heap, and cloned eight endoglucanase genes, two xylanase genes, and a ␤-glucosidase gene (38). On comparing the restriction maps of the plasmids constructed from C. thermocellum F1 genomic DNA and those of C. thermocellum NCIB 10682 genes, we found that four of eight endoglucanase genes cloned from strain F1 are homologous with the genes from the type strain, i.e., celC, celF, celH, and an uncharacterized endoglucanase gene (38).…”

“…On comparing the restriction maps of the plasmids constructed from C. thermocellum F1 genomic DNA and those of C. thermocellum NCIB 10682 genes, we found that four of eight endoglucanase genes cloned from strain F1 are homologous with the genes from the type strain, i.e., celC, celF, celH, and an uncharacterized endoglucanase gene (38). Comparison of the nucleotide sequences of the homologous celC genes showed only six substitutions in the coding region, resulting in three amino acid changes (40).…”