Integrative vectors for heterologous gene expression in Streptomyces spp.
Integrative expression vectors for heterologous expression of the genes in Streptomyces were developed. The vectors are comprised of a strong constitutive promoter, PE, a synthetic ribosome-binding site, ATG start codon, multiple cloning site, transcription terminator and hygromycin-resistance-encoding gene. The vectors also contain a ColE1 replicon for propagation in Escherichia coli and a wide-host-range Streptomyces integration element, the mini-circle, to direct the insertion of the vectors into the Streptomyces genome at the mini-circle attachment site. HyR transformants are stable in the absence of drug selection. Conjugative derivatives were also constructed by incorporating oriT, the origin of transfer of the IncP plasmid RK2, into these vectors, and conjugal transfer was demonstrated from an appropriate E. coli donor to Steptomyces lividans (Sl). Derivatives of these vectors potentially useful for gene disruption, as well as complementation, are also described. Replicative forms of the constructed mini-circle-based vectors in Sl, that co-exist with the integrated copy of the vector, were also present without any apparent instability problems. The utility of the vectors was demonstrated by expression of the gene encoding 31-O-methyltransferase, which is involved in methylation at position 31 of the immunosuppressive drug FK506, in Sl.
Motamedi
H
H
Merck Research Laboratories, Rahway, NJ 07065, USA.
Shafiee
A
A
Cai
S J
SJ
D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular / Q000379:methods; D003062:Codon; D003227:Conjugation, Genetic; D017931:DNA Primers; D004926:Escherichia coli; D015870:Gene Expression; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D015183:Restriction Mapping; D012270:Ribosomes / Q000378:metabolism; D013302:Streptomyces / Q000235:genetics* / Q000378:metabolism*; D013728:Terminator Regions, Genetic; D014158:Transcription, Genetic
D003062:Codon; D017931:DNA Primers