Construction and characterization of multicopy expression-vectors in Streptomyces spp

Area of Science:

• Molecular Biology
• Microbial Genetics
• Gene Expression Systems

Background:

• Characterization of a strong transcriptional signal from Streptomyces griseus.
• Investigation of promoter function across different bacterial species.

Purpose of the Study:

• To determine the nucleotide sequence of a strong Streptomyces promoter.
• To assess the heterologous expression capability of the Streptomyces promoter in E. coli and S. lividans.
• To develop novel expression vectors for S. lividans.

Main Methods:

• Subcloning and nucleotide sequencing of the Streptomyces promoter.
• High-resolution S1 nuclease mapping to identify the transcriptional start point.
• Construction of expression vectors (pSEV series) by inserting the promoter into pIJ702 and pKS11 plasmids.
• Heterologous gene expression studies in E. coli and S. lividans.

Main Results:

• The Streptomyces promoter sequence was determined, revealing characteristic -35 and -10 regions.
• The promoter showed no activity in Escherichia coli, despite sequence similarities to E. coli promoters.
• High-level production of Thermus flavus malate dehydrogenase and Bacillus subtilis cellulase was achieved in S. lividans using the developed pSEV expression vectors.

Conclusions:

• The identified Streptomyces promoter is functional in S. lividans but not in E. coli.
• The pSEV expression vectors are effective tools for high-level foreign protein production in S. lividans.
• This work facilitates advanced genetic engineering applications in Streptomyces species.