Biosynthesis of the macrolactone ring of FK506 involves 10 elongation cycles that mechanistically resemble the steps in fatty acid synthesis. Sequencing of a 40-kb DNA segment of the FK506 gene cluster from Streptomyces sp. MA6548 has revealed two additional polyketide synthases (PKS) genes fkbB and fkbC which lie upstream of fkbA, a PKS gene recently shown to be responsible for the last four condensation steps of the FK506 biosynthesis [Motamedi, H., Cai, S. J., Shafiee, A. & Elliston, K. O. (1997) Eur. J. Biochem. 244, 74Ϫ80]. fkbB and fkbC are contiguous and encode respectively, the first (790 129 Da) and the second (374 438 Da) components of the FK506 polyketide synthase, a complex of three multidomain polypeptides. The predicted domain structures of FkbB and FkbC are analogous to that of FkbA and comprise 30 fatty-acid-synthase(FAS)-like domains arranged in 6 modules. Each module performs a specific extension cycle in the assembly of the carbon skeleton of the FK506 macrolactone ring. The component activities for the initiation of the polyketide chain consisting of a dihydrocyclohexenylcarbonyl coenzyme A (CoA) synthetase and a dihydrocyclohexenylcarbonyl CoA reductase required for the formation of the dihydrocyclohexylcarbonyl CoA starter unit and an acyl-carrier-protein to which the starter unit is anchored and translocated to the appropriate site on the PKS multienzyme are located at the N-terminal region of the FkbB polypeptide. A third gene, fkbL, lies at one end of the cluster and encodes lysine cyclodeaminase which catalyzes A-deamination and cyclization of the lysine into pipecolate. A fourth gene fkbP located at the other end of the sequence reported here encodes a peptide synthetase required for the activation and incorporation of the pipecolate moiety into the completed acyl chain. Finally the cluster carries a gene, fkbO, whose product is presumed to carry out a post-polyketide oxidation step of the FK506 marocycle.

Key information about the biosynthesis of polyketide metabolites has been uncovered by sequence analysis of the tetracenomycin C polyketide synthase genes (tcml) from Streptomyces glaucescens GLA.0. The sequence data revealed the presence of three complete open reading frames (ORFs). ORF1 and ORF2 appear to be translationally coupled and would encode proteins containing 426 and 405 amino acids, respectively. The two deduced proteins are homologous to known beta‐ketoacyl synthases. ORF3 begins 70 nucleotides after the stop codon of ORF2 and would code for an 83 amino acid protein with a strong resemblance to known bacterial, animal and plant acyl‐carrier proteins (ACP). The presence of an ACP gene within the tcm gene cluster suggests that different ACPs are used in fatty acid and polyketide biosynthesis in Streptomyces. We conclude from these data and earlier information that polyketide biosynthesis in S. glaucescens, and most likely in other bacteria, involves a multienzyme complex consisting of at least five types of enzymes: acylCoA transferases that load the acyl and 2‐carboxyacyl precursors onto the ACP; a beta‐ketoacyl synthase that, along with the acylated ACP, forms the poly‐beta‐ketoacyl intermediates; a poly‐beta‐ketone cyclase that forms carbocyclic structures from the latter intermediates; a beta‐ketoacyl oxidoreductase that forms beta‐hydroxyacyl intermediates or reduces ketone groups in fully formed polyketides; and a thioesterase that releases the assembled polyketide from the enzyme.

Mutations in the tcmll-temlV region of the Streptomyces glaucescens chromosome block the C-3 and C-8 0-methylations of the polyketide antibiotic tetracenomycin C (Tcm C). The nucleotide sequence of this region reveals the presence of two genes, tcmN and temO, whose deduced protein products display similarity to the hydroxyindole 0-methyl transferase of the bovine pineal gland, an enzyme that catalyzes a phenolic 0-methylation analogous to those required for the biosynthesis of Tcm C. The deduced product of the tcmN gene also has an N-terminal domain that shows similarity to the putative ActVH and WhiE ORFVI proteins of Streptomyces coelicolor. The tcmN N-terminal domain can be separated from the remainder of the tcmN gene product, and when coupled on a plasmid with the Tcm C polyketide synthase genes (tcmKLM), this domain enables high-level production of an early, partially cyclized intermediate of Tcm C in a Tcm C-null mutant or in a heterologous host (Streptomyces lividans). By analogy to fatty acid biosynthesis, the tcmKLM polyketide synthase gene products are probably sufficient to produce the linear decaketide precursor of Tcm C; thus, the tcmN N-terminal domain is most likely responsible for one or more of the early cyclizations and, perhaps, the attendant dehydrations that lead to the partially cyclized intermediate. The temN gene therefore appears to encode a multifunctional cyclase-dehydratase-3-0-methyl transferase. The tcmO gene encodes the 8-0-methyl transferase.Polyketide metabolites are a structurally diverse family of compounds that encompasses both aromatic and aliphatic members (Fig. 1A). These molecules are commonly synthesized via secondary metabolic pathways in bacteria, fungi, and plants (10). Importantly, many of these compounds have found clinical utility as antibiotics and chemotherapeutic agents (10).The feature that binds the seemingly disparate polyketide family together is the mechanism of biosynthesis. The carbon backbone of a polyketide is synthesized in a manner that is similar to long-chain fatty acid biosynthesis: small fatty acid units (acetate, propionate, butyrate, etc.) are sequentially condensed to yield extended linear precursors. A notable difference distinguishes polyketide biosynthesis from fatty acid biosynthesis, however. The condensation reactions of polyketide chain growth are not always followed by the cycles of reduction and dehydration that characterize the synthesis of fatty acids. As a result, the linear intermediate of polyketide biosynthesis is peppered with reactive carbonyl groups that form the basis for subsequent chemical elaborations.Tetracenomycin C (Tcm C) is a relatively simple aromatic polyketide antibiotic that is produced by Streptomyces glaucescens (25). The tetracyclic backbone of this molecule is most likely formed by cyclization of a 20-carbon linear decaketide (Fig. 1B). Previously, we cloned the genes required for the biosynthesis of Tcm C from S. (14) and reported the DNA sequence of the polyketide synthase (PKS) genes from the tcmIa region t...

A second open reading frame, fkbD, was found upstream of fkbM in all three aforementioned species and was predicted to encode a protein of 388 residues that showed a strong resemblance to cytochrome P-450 hydroxylases. Disruption of fkbD had a polar effect on the synthesis of the downstream fkbM gene product and resulted in the formation of 9-deoxo-31-O-demethyl-FK506. This established the product of fkbD as the cytochrome P-450 9-deoxo-FK506 hydroxylase, which is responsible for hydroxylation at position C-9 of the FK506 and FK520 macrolactone ring.The polyketide, immunosuppressant compound FK506 ( Fig. 1) (13) is a 23-membered macrolide with potent antifungal activity produced by several Streptomyces species. FK506 is approximately 100-fold more potent than the structurally unrelated immunosuppressive compound cyclosporin A. Both drugs are important therapeutic agents for the prevention of graft rejection following organ and bone marrow transplantations and for the treatment of autoimmune diseases (22). FK520 (also known as immunomycin and ascomycin) is another immunosuppressive compound similar to FK506 (Fig. 1) in which the allyl group is replaced by an ethyl group at position C-21 of the macrolactone ring (9). Both the antifungal and the immunosuppressive activities of FK520 are approximately one-half of those exhibited by FK506 (9).Through precursor incorporation experiments, Byrne et al. (3) demonstrated that the polyketide portion of FK506 and FK520 is derived, for the most part, from acetate and propionate. Those authors also established the origin of the pipecolate and the cyclohexyl rings to be lysine and shikimic acid, respectively, and demonstrated that the source of the methyl portion of the methoxyl groups at C-13, C-15, and C-31 of FK520 (Fig. 1) is L-methionine.The enzymology of FK506 biosynthesis has also been explored to some extent. The pipecolate-activating enzyme which presumably incorporates pipecolate into the completed polyketide chain has been characterized previously (19). Both 31-O-demethyl-FK520 methyltransferase and 31-O-demethyl-FK506 methyltransferase (FKMT) have been isolated from the producing strains (3, 27). These two enzymes can use each other's substrate interchangeably and methylate the C-31 OH and not the C-13 or C-15 OH group (27).Here, we report the isolation and molecular characterization of two genes involved in the biosynthesis of FK506. One gene, fkbM, encodes FKMT, and the other, fkbD, encodes a cytochrome P-450 9-deoxo-FK506 hydroxylase that catalyzes hydroxylation at C-9. MATERIALS AND METHODSStandard recombinant DNA techniques were performed as described by Sambrook et al. (24).Probe design. N-terminal amino acid sequencing of FKMT from Streptomyces sp. strain MA6858 (27) gave a 39-mer with the sequence SDVVETLRLPNGA TVAHVNAGEAQFLYREIFTDRCYLRH. This peptide sequence was then used to design two nonoverlapping degenerate oligonucleotide probes, P1 and P2, in which inosine was incorporated at the third position of highly degenerate codons (2). P1 corresponded to ...

Many important antibiotics such as tetracyclines, erythromycin, adriamycin, monensin, rifamycin and avermectins are polyketides. In their biosynthesis, multifunctional synthases catalyse iterated condensation of thio-esters derived from acetate, propionate or butyrate to yield aliphatic chains of varying length and carrying different alkyl substituents. Subsequent modifications, including aromatic or macrolide ring closure or specific methylations or glycosylations, generate further chemical diversity. It has been suggested that, if different polyketide synthases had a common evolutionary origin, cloned DNA coding for one synthase might be used as a hybridization probe for the isolation of others. We show here that this is indeed possible. Study of a range of such synthase genes and their products should help to elucidate what determines the choice and order of condensation of different residues in polyketide assembly, and might yield, by in vitro recombination or mutagenesis, synthase genes capable of producing novel antibiotics. Moreover, because genes for entire antibiotic pathways are usually clustered in Streptomyces, cloned polyketide synthase genes are valuable in giving access to groups of linked biosynthetic genes.