The application of molecular cloning to antibiotic-producing microorganisms should lead to enhanced antibiotic productivity and to the biosynthesis of novel antibiotics by in vitro interspecific recombination. To allow such approaches, the genes for antibiotic synthesis must be isolated, analysed and perhaps modified. Certain Streptomyces species produce nearly two-thirds of the known natural antibiotics; the recent development of cloning systems in the genus makes it possible to isolate and analyse Streptomyces genes. However, antibiotics are metabolites which require sets of several enzymes for their synthesis and attempts to isolate the corresponding genes have so far yielded clones carrying either individual genes of the set, or only incomplete gene sets. We describe here the isolation of a large continuous segment of Streptomyces coelicolor DNA which apparently carries the complete genetic information required for synthesis of an antibiotic, actinorhodin , from simple primary metabolites. Not only can the cloned DNA 'complement' all available classes of actinorhodin non-producing mutants of S. coelicolor but, on introduction into a different host, Streptomyces parvulus , it directs the synthesis of the antibiotic. The tendency for the genes for antibiotic synthesis to be clustered together on the chromosomes of Streptomyces species and the availability of plasmid vectors which can carry stable inserts of DNA larger than 30 kilobase pairs (kb) and which can be introduced efficiently into Streptomyces protoplasts, suggest that the experiments described have general significance for this area of biotechnology.

Hybrid antibiotics mederrhodins A and B were produced by a recombinant strain consisting of the medermycin-producing Streptomyces sp. strain AM7161 containing part of the gene clusters for actinorhodin biosynthesis of Streptomyces coelicolor A3(2). Mederrhodin A has a hydroxyl group at the C-6 position of the medermycin molecule, and mederrhodin B is dihydromederrhodin A. The antimicrobial activity of mederrhodin A resembled that of medermycin. Mederrhodin B was almost devoid of antimicrobial activity.

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Complementation of defined actinorhodin j8-ketoacyl synthase (KS) mutants by various other KS genes suggested that the ORFl-encoded KS may be relatively generalized in function, whereas the ORF2-encoded KS component may provide specificity in polyketide chain construction. Evidence for differential temporal-spatial expression of the actinorhodin and spore pigment KSs in Streptomyces coelicolor was obtained.Significant recent progress in the cloning and analysis of polyketide synthase (PKS) genes from members of the genus Streptomyces and related bacteria (11, 12) has been made. This includes type I (multifunctional) and type II (multicomponent) PKS systems. The type I PKSs specify the construction of macrolide antibiotics and can utilize a range of different precursor starter and extender unit carboxyl coenzyme A (CoA) esters for carbon chain assembly (4,7,12). In contrast, the type II PKS systems so far investigated specify the construction of a diverse class of aromatic metabolites, which are derived from a more limited range of starter units (usually acetyl-CoA) and seven, eight, or nine malonyl-CoA extender units (2,3,5,8,11,16,23). Molecular genetic analysis of several type II PKS systems has shown that the earliest biosynthetic steps (i.e., carbon chain construction) are catalyzed by three gene products, named actI-ORF1, actI-ORF2, and actI-ORF3 in the actinorhodin model system of Streptomyces coelicolor A3(2) (Fig. 1). One of these (the ORF1 product) resembles closely the Escherichia coli fabB KS, including an appropriately positioned cysteine active site residue (13). Another related gene product (that of ORF2) appears to be translationally coupled in most type II PKSs and also resembles the E. colifabB KS, but the active-site Cys has been replaced by another amino acid (glutamine). It has been suggested that a heterodimeric KS consisting of the ORF1 and ORF2 products is required for polyketide biosynthesis in Streptomyces spp. (2, 23). An acyl carrier protein (ACP), the product of ORF3, is also required for a functional type II PKS. This protein also closely resembles its E. coli fatty acid synthase (FAS) counterpart, in addition to a variety of plant type II FAS ACPs (11).In a previous study, we showed that it is possible to complement, in trans, PKS mutations corresponding to actI-ORF1 and actI-ORF2, as well as mutations in actIII (ORF5) and actVII (ORF4), which encode the polyketide ketoreductase and cyclase, respectively, by the corresponding homologous genes from the gra cluster (Fig. 1) Transformation of act KS mutants by low-copy plasmids containing PKS KS genes. In order to determine the ability of the otcY-ORF1, otcY-ORF2, whiE-ORFIII, and whiE-ORFIV genes to complement actI-ORF1 or actI-ORF2 mutations, individual genes were cloned as described in Table 1. In each case, a DNA fragment containing the gene of interest (Fig. 1) was inserted into the single-copy SCP2*-derived Streptomyces vector pIJ941 (17) and introduced into Streptomyces lividans by transformation. The cloning sites and fragment ...

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