To examine as randomly as possible the role of the ␤-ketoacyl and acyl carrier protein (ACP) components of bacterial type II polyketide synthases (PKSs), homologs of the chain-length-factor (CLF) genes were cloned from the environmental community of microorganisms. With PCR primers derived from conserved regions of known ketosynthase (KS ␣ ) and ACP genes specifying the formation of 16-to 24-carbon polyketides, two CLF (KS ␤ ) genes were cloned from unclassified streptomycetes isolated from the soil, and two were cloned from soil DNA without the prior isolation of the parent microorganism. The sequence and deduced product of each gene were distinct from those of known KS ␤ genes and, by phylogenetic analysis, belonged to antibiotic-producing PKS gene clusters. Hybrid PKS gene cassettes were constructed with each novel KS ␤ gene substituted for the actI-ORF2 or tcmL KS ␤ subunit genes, along with the respective actI-ORF1 or tcmK KS ␣ , tcmM ACP, and tcmN cyclase genes, and were found to produce an octaketide or decaketide product characteristic of the ones known to be made by the heterologous KS ␣ gene partner. Since substantially less than 1% of the microorganisms present in soil are thought to be cultivatable by standard methods, this work demonstrates a potential way to gain access to a more extensive range of microbial molecular diversity and to biosynthetic pathways whose products can be tested for biological applications.The polyketide metabolites are a large group of structurally complex and diverse natural products, many of which are clinically valuable antibiotics or chemotherapeutic agents or have other useful pharmacological activities (48). Polyketides are synthesized by polyketide synthases (PKSs) which, like the related fatty acid synthases, are multifunctional enzyme assemblies that catalyze repeated decarboxylative condensations between enzyme-bound acylthioesters (52). The structural diversity of polyketides is a result of the different numbers and types of acyl units involved, coupled with various possibilities for enzyme-mediated reduction, dehydration, cyclization, and aromatization of the initial ␤-ketoacyl condensation products (52). Manipulation of the sequence or specificity of the PKS enzymes can lead to the production of novel secondary metabolites (28,29,33). The key to further exploitation of this approach to molecular diversity lies in an increased understanding of the genetics and biochemistry of the various PKS systems.Recent progress in the cloning and sequencing of microbial PKS genes has led to the identification of at least two architecturally different types of PKSs (28, 33). Modular type I PKSs, represented by erythromycin A (11, 17), avermectin (41), rapamycin (58), and soraphen (57), are discrete multifunctional enzymes sporting numerous unique active site domains. The domains are grouped into distinct modules that in turn control the sequential addition of acylthioester units to the growing polyketide chain and the subsequent modification of the respective condensation prod...

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