Construction and characterization of multicopy expression-vectors in Streptomyces spp

Abstract

A strong transcriptional signal previously cloned from the Streptomyces griseus genome in S. lividans was subcloned and its nucleotide sequence was determined. Upstream of the transcriptional start point which was determined by high-resolution S1 nuclease mapping, -35 (5'-TTGCCG-3') and -10 (5'-TAGCGT-3') sequences, separated by 18 nucleotides, were present. By replacing the tet promoter of pBR322 with the Streptomyces promoter, no expression of the tet gene was observed in Escherichia coli cells. The result suggests that notwithstanding a similarity to the E. coli -35 and -10 sequences, the Streptomyces promoter is not functional in E. coli. The strong promoter was inserted in multi-copy and wide host range plasmids pIJ702 and pKS11, resulting in the pSEV series of expression-vectors with several unique restriction endonuclease cleavage sites downstream of the promoter for cloning of foreign genes. The extremely heat-stable malate dehydrogenase of Thermus flavus, when its coding sequence with a ribosome-binding site was located downstream of the strong promoter in pSEV2, was produced in large quantities in S. lividans throughout growth. When an extracellular cellulase from Bacillus subtilis was expressed in a cellulase-negative S. lividans strain, virtually all of the cellulase activity was found in the culture supernatant.