Cloning, Sequencing, and Characterization of Two Clustered Cellulase-Encoding Genes, celS1 and celS2, from Streptomyces viridosporus T7A and their Expression in Escherichia coli.

Abstract

A recombinant clone, pBLP, containing a 4.1kb fragment of Streptomyces viridosporus T7A chromosomal DNA was shown to confer endoglucanase activity in Escherichia coli cells. Further subcloning and sequence analysis revealed two co-transcribed Open Reading Frames (ORF) with high sequence similarities to other Streptomyces endoglucanase-encoding genes. A signal peptide, a catalytic domain and cellulose binding domain were identified within ORF1 (celS1). The amino acid sequence of ORF2 (celS2) showed similarity with cellulose binding protein (p40) of Streptomyces halstedii. The hydrophobic analysis of CelSl revealed that it belongs to the family H cellu-lase catalytic domain. The rare TTA codon encoding leucine was found in the signal sequence of both the cellu-lase ORFs, indicating translational dependence on the bldA gene product, a cognate tRNA responsible for translating the TTA codons. The presence of 14 bp inverted repeats in the 5’-end of these genes, was consistent with the highly conserved positive regulatory structure found in other endoglucanase genes.