Carbomycin resistance-conferring gene, designated cara, for use in streptomyces and other organisms

#The present invention comprises a novel carbomycin resistance-conferring gene, designated carA, a method for using the carA gene, recombinant DNA cloning vectors that comprise the novel gene, and transformants containing the carbomycin resistance-conferring vectors. Streptomyces thermotolerans (ATCC 11416) produces carbomycin, a macrolide antibiotic consisting of a 16-member cyclic lactone and two sugar residues. The antibiotic activity of carbomycin, like that of other macrolides, is due to inhibition of protein synthesis by a mechanism that involves the binding of carbomycin to the ribosome.

#The present invention provides carbomycin resistance-conferring cloning vectors for use in Streptomyces and other host cells. The development and exploitation of recombinant DNA technology in Streptomyces depends upon the availability of selectable genetic markers on suitable cloning vectors. This development has been somewhat retarded by the low number of selectable markers presently available for use in Streptomyces. The present invention is useful and especially important in that it expands the number of selectable markers suitable for such use.

#The vectors of the present invention are particularly useful, because the vectors are small, versatile, and can be transformed and selected in a variety of carbomycin-sensitive Streptomyces cells. Streptomyces provides over half of the clinically important antibiotics and thus is a commercially significant group. The present invention provides new and useful cloning systems and vectors for this industrially important group and allows for the cloning of genes both for increasing the yields of known antibiotics and also for producing new antibiotics and antibiotic derivatives.

#The figures described below are drawn to scale; however, observed restriction fragment size may vary somewhat from calculated size based on map distances. For some restriction enzymes, such as SauIIIAl, only the significant cut sites are shown.

#FIG. 1 is a restriction site and function map of plasmid pOJ158.

#FIG. 2 is a restriction site and function map of plasmid pOJ197.

#The present invention concerns a novel carbomycin resistance-conferring gene, designated carA, that is useful as a genetic marker in many organisms. The carA gene is useful in a method for selecting a recombinant DNA-containing host cell that comprises transforming a carbomycin-sensitive host cell with a recombinant DNA vector that codes for expression of the carA gene product and culturing the transformed cell under conditions suitable for selection of carbomycin-resistant transformants. The carA gene can be isolated from plasmid pOJ158 on an .about.4 kb PstI-EcoRI restriction fragment; plasmid pOJ158 can be isolated from Streptomyces griseofuscus C581/pOJ158, a strain deposited and made part of the permanent culture collection of the Agricultural Research Service, Northern Regional Research Center (NRRL), Peoria, Ill. 61604, under the accession number NRRL 18089. A restriction site and function map of plasmid pOJ158 is presented in FIG. 1 of the accompanying drawings. Plasmid pOJ158 can be isolated from S. griseofuscus C581/pOJ158 in substantial accordance with the procedure described in Example 1.

#Plasmid pOJ158 serves as useful starting material for the construction of other vectors that confer carA-mediated carbomycin resistance. For example, the .about.4 kb PstI-EcoRI, carbomycin resistance-conferring restriction fragment of plasmid pOJ158 was isolated and inserted into EcoRI-PstI-digested plasmid pOJ160 (NRRL B-18088) to yield plasmid pOJ197. The construction protocol for plasmid pOJ197 is given in Example 2; a restriction site and function map of plasmid pOJ197 is presented in FIG. 2 of the accompanying drawings.

#The carA gene was isolated from a carbomycinproducing strain of Streptomyces thermotolerans (ATCC 11416). Thus, genomic DNA of S. thermotolerans was partially digested with restriction enzyme MboI, and the resulting DNA was inserted into BglII-digested plasmid pIJ702 (ATCC 39155, Katz et al., 1983, J. Gen. Microbiol. 129:2703) to yield a number of carA-containing plasmids, including plasmid pOJ158. Because the carA gene was isolated from S. thermotolerans, the carA gene functions in S. thermotolerans, but the carA gene with its natural promoter also functions in other organisms.