A second generation snp-derived Escherichia coli–Streptomyces shuttle expression vector that is generally transferable by conjugation

• Published in: Plasmid Vol. 56; no. 3; pp. 223 - 227
• Main Authors: Nikodinovic, Jasmina, Priestley, Nigel D.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.11.2006
• Subjects: Base Sequence; Blotting, Western; Chromosome Mapping; Conjugation; Conjugation, Genetic - genetics; DNA Primers; Escherichia; Escherichia coli; Escherichia coli - genetics; Gene Transfer Techniques; Genes, Bacterial - genetics; Genetic Vectors - genetics; Heterologous expression; Molecular Sequence Data; oriT; RK2; Sequence Analysis, DNA; Shuttle vector; snpA promoter; Streptomyces; Streptomyces - genetics; Streptomyces griseus; Streptomyces; Shuttle vector; RK2; oriT; Conjugation; snpA promoter; Heterologous expression
• ISSN: 0147-619X; 1095-9890
• DOI: 10.1016/j.plasmid.2006.05.002

An Escherichia coli– Streptomyces shuttle vector (pJN100) was constructed, by inserting an origin of transfer ( oriT), derived from the E. coli broad host range plasmid RK2, into pANT1202, a high-copy-number vector for gene expression in Streptomyces. The resulting conjugably transferable vector contains the pANT1202-derived SnpR (LysR-like protein) activated snpA promoter that drives strong heterologous expression of proteins. We initially demonstrated that plasmid pJN100 was transferred with high frequency (10 −5–7 exconjugants per recipient) into several Streptomyces strains that were refractory to transformation by other means. Plasmid pJN100 was also shown to be stable in E. coli and Streptomyces. We confirmed functional protein expression by using a pJN100 derivative to complement a mutant of Streptomyces griseus with a disrupted chromosomal copy of the gene nonM, a gene encoding an essential reductase in the nonactin biosynthesis gene cluster. High levels of protein expression were confirmed using Western blotting to assess the production of the serine esterase NonR, an enzyme responsible for nonactin resistance in the nonactin producer S. griseus.