Although the Actinomycetales produce more than half of the known antibiotics having valuable clinical and other applications as secondary metabolites and, thus, are recognized as a key target for application of gene manipulation techniques, many problems remain to be overcome before specific useful genes are successfully identified and cloned ["Molecular Breeding and Genetics of Applied Microorganisms", Sakaguchi and Okanishi, eds., Academic Press (New York) Kodansha Ltd. (Toyko) 1980, pgs. 130-131]. Until the present work, cloning of a .beta.-galactosidase gene from a Streptomyces species onto a suitable vector followed by introduction and expression of such vector has not been reported. Prior work has concerned development of other cloning systems or vectors for Streptomycetes [Bibb et al. (1978), Nature 274: 398-400; Hayakawa et al. (1979), J. Antibiot. XXXII(12): 1348-1350; Okanishi et al. (1980), J. Antibiot. XXXIII(1): 88-91; Bibb et al. (1980), Nature 284: 526-531; Thompson et al. (1980), Nature 286: 525-527; Suarez et al. (1980), Nature 286: 527-529; Bibb et al. (1981), Mol. Gen. Genet. 184: 230-240]; [Bibb (1981), "Microbiology-1981", Schlessinger, ed., American Society for Microbiology, (Washington, D.C.) 1981, pgs. 367-370 and Hopwood et al. (1981), "Microbiology-1981", supra. pgs. 376-379], cloning and expression in Streptomyces sp. of genes derived from Escherichia coli [Schottel et al. (1981), J. Bacteriol. 146: 360-368] and cloning of genes from Streptomycetes in Escherichia coli ["Molecular Breeding and Genetics of Applied Microorganisms", supra; pgs. 130-137]. Chater et al. (1982), Current Topics in Microbiol. and Immunol. 96: 69-95, review gene cloning in Streptomyces and is incorporated by reference herein as though fully set forth.
Work with various .beta.-galactosidase genes, their expression and application of such expression as an assay or detection method has been reported by Rose et al. (1981), Proc. Natl. Acad. Sci. USA 78(4): 2460-2464, for expression in yeast of yeast genes fused to .beta.-galactosidase genes from Escherichia coli; by Casadaban et al. (1980), J. Mol. Biol. 138: 179-207, for fusion of .beta.-galactosidase genes to promoters in Escherichia coli and assay following transformation; and by Talmadge et al. (1981), Nature 294: 176-178, for construction of Escherichia coli containing a plasmid encoding a .beta.-galactosidase-preproinsulin fusion protein.
Collinge et al., U.S. Pat. No. 3,816,259, disclose that a Streptomyces coelicolor preparation had a .beta.-galactosidase activity.
In general, the activity of promoters can be assayed by measuring the amount of gene product which is formed as a consequence of transcription starting from a specific promoter. The amount of gene product formed is determined by using a specific property of that gene product, such as enzymatic activity. In studying gene expression or in constructing high expression vectors which rely on highly efficient promoters, the gene which is naturally expressed from such a promoter is replaced by the structural gene whose product can be more easily monitored. The lacZ gene from Escherichia coli [Casadaban et al. (1980), supra.] is frequently used for this purpose.
A variety of chromogenic substrates, such as 5-bromo-4-chloro-3-indolyl-.beta.-D-galactosidase (referred to as "X-gal") or o-nitrophenyl-.beta.-D-galactoside (referred to as "ONPG") can be used to monitor enzymatic activity as described by Miller (1972), "Experiments in Molecular Genetics", Cold Spring Harbor Laboratories (Cold Spring Harbor, N.Y.). These substrates are advantageous since the efficiency of a promoter fused to a gene coding for an enzyme which can react with the substrate, such as the lacZ gene, can be monitored by growing the organism on a solid agar medium containing the substrate and observing for enzyme-substrate reaction. In this manner, several hundred individual colonies can be scored at one time for their ability to express the gene. Thus, relatively rare events such as the occurrence of a highly efficient promoter can be detected. .beta.-Galactosidase expression can be used in such a procedure to assay gene transcription and to detect and isolate mutants which over-produce a particularly desired protein, such as an enzyme involved in antibiotic production.
In order to effectively use such a powerful approach as described above, it is crucial that the chosen substrate has the opportunity to react with the enzyme. If, as in the case of .beta.-galactosidase produced by Escherichia coli, the enzyme is intracellular, the substrate must enter the cell in order for the enzyme-substrate reaction to occur. With Streptomyces lividans, however, the commonly used substrates, X-gal and ONPG, enter the cell only poorly as verified by comparing the intracellular .beta.-galactosidase activity of a suitable organism with dye formation on plates. For example, we have found that although intracellular activity as measured with cell extracts and with ONPG as substrate was very high (300 nmoles/mg protein/min), no significant color reaction with whole cells and with either ONPG or X-gal was found. Furthermore, Actinomycetes differ from many other microorganisms by the formation of an aerial mycelium which separates the cells physically from the substrate, thus further restricting access of the substrate to the cells [Kalakoutskii et al. (1976), Bacteriol. Rev. 40(2): 469-524].