CONSTRUCTION OF NEW VECTORS FOR HIGH-LEVEL EXPRESSION IN ACTINOMYCETES

A new integrative vector (pCJR24) was constructed for use in the eryth romycin producer Saccharopolyspora erythraea and in other actinomycete s. It includes the pathway-specific activator gene actII-ORF4 from the actinorhodin biosynthetic gene cluster of Streptomyces coelicolor. Th e actI promoter and the associated ribosome binding site are located u pstream of an NdeI site (5'-CATATG-3') which encompasses the actI star t 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 i n pCJR24 and overexpressed in S, erythraea after integration of the ve ctor into the chromosome by homologous recombination, indicating the p ossibility that the S. coelicolor promoter/activator functions appropr iately in S. erythraea, pCJR24-mediated recombination was also used to place the entire gene set for the erythromycin-producing polyketide s ynthase under the control of the actI promoter. The resulting strain p roduced copious quantities of erythromycins and precursor macrolides w hen compared with wild-type S. erythraea. The use of this system provi des the means for rational strain improvement of antibiotic-producing actinomycetes. (C) 1998 Elsevier Science B.V. All rights reserved.