Construction of new vectors for high-level expression in actinomycetes.
A new integrative vector (pCJR24) was constructed for use in the erythromycin producer Saccharopolyspora erythraea and in other actinomycetes. It includes the pathway-specific activator gene actII-ORF4 from the actinorhodin biosynthetic gene cluster of Streptomyces coelicolor. The actI promoter and the associated ribosome binding site are located upstream of an NdeI site (5'-CATATG-3') which encompasses the actI start codon allowing protein(s) to be produced at high levels in response to nutritional signals if these signals are faithfully mediated by the ActII-ORF4 activator. Several polyketide synthase genes were cloned in pCJR24 and overexpressed in S. erythraea after integration of the vector into the chromosome by homologous recombination, indicating the possibility that the S. coelicolor promoter/activator functions appropriately in S. erythraea. pCJR24-mediated recombination was also used to place the entire gene set for the erythromycin-producing polyketide synthase under the control of the actI promoter. The resulting strain produced copious quantities of erythromycins and precursor macrolides when compared with wild-type S. erythraea. The use of this system provides the means for rational strain improvement of antibiotic-producing actinomycetes.
Cambridge Centre for Molecular Recognition and Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, UK.
C J
CJ
Rowe
J
J
Cortés
S
S
Gaisser
J
J
Staunton
P F
PF
Leadlay
D000192:Actinomycetales / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism; D000900:Anti-Bacterial Agents / Q000378:metabolism; D004274:DNA, Recombinant; D004917:Erythromycin / Q000031:analogs & derivatives / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial / Q000235:genetics; D005818:Genetic Engineering; D005822:Genetic Vectors / Q000235:genetics*; D009097:Multienzyme Complexes / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics; D016930:Saccharopolyspora / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D013869:Thiolester Hydrolases / Q000235:genetics
D000900:Anti-Bacterial Agents; D004274:DNA, Recombinant; D009097:Multienzyme Complexes; D011993:Recombinant Fusion Proteins; C034277:6-deoxyerythronolide B; D004917:Erythromycin; D013869:Thiolester Hydrolases