The polyketide-derived macrolactone of the antibiotic erythromycin is made through successive condensation and processing of seven three-carbon units. The fourth cycle involves complete processing of the newly formed 13-keto group (.3keto reduction, dehydration, and enoyl reduction) to yield the methylene that will appear at C-7 of the lactone ring. Synthesis of this molecule in Saccharopolyspora erythraea is determined by the three large eryA genes, organized in six modules, each governing one condensation cycle. Two amino acid substitutions were introduced in the putative NAD(P)H binding motif in the proposed enoyl reductase domain encoded by eryAMi. The metabolite produced by the resulting strain was identified as A6"7-anhydroerythromycin C resulting from failure ofenoyl reduction during the fourth cycle ofsynthesis ofthe macrolactone. This result demonstrates the involvement of at least the enoyl reductase from the fourth module in the fourth cycle and indicates that a virtually complete macrolide can be produced through reprogramming of polyketide synthesis.A wide variety of natural compounds, exhibiting antibacterial, antihelminthic, antitumor, and immunosuppressive activities, contain a polyketide-derived skeleton. Biosynthesis of polyketides is mechanistically equivalent to formation of long-chain fatty acids (1), where the fatty acid synthase (FAS) condenses the extender unit malonate with the starter unit acetate and the resulting (3-keto group undergoes three processing steps, 3-keto reduction, dehydration, and enoyl reduction, to yield a fully saturated butyryl unit. The C4 chain is elongated through repeated addition of two carbon atoms (derived from malonate) and fully processed at each cycle, until the proper length of a symmetrical chain has been reached. Many polyketides, in contrast, retain ketone, hydroxyl, or olefinic functions and contain methyl or ethyl side groups interspersed along an acyl chain of length comparable to that of common fatty acids. This asymmetry in structure implies that the polyketide synthase (PKS), the enzyme system responsible for formation of these molecules, although mechanistically equivalent to FAS, must somehow be programmed to produce the correct molecular structure.The current model (2) for biosynthesis of complex polyketides (defmed as compounds whose synthesis requires each FAS-like cycle to be usually different from the previous one) is exemplified, for the erythromycin aglycone DEB (Fig. 1) teins-EryAl, EryAII, and EryAIII-encoded by eryA (Fig. 2). Thus, the noniterative processive synthesis ofasymmetric acyl chains found in complex polyketides is accomplished through the use of a programmed protein template, where the nature of the chemical reactions occurring at each point is determined by the specificities of the domains contained in each SU.The involvement of a distinct enzymatic activity in each synthesis step implies that a modification affecting a single activity should perturb only the corresponding step. Such modifications offer the potent...

The starter unit used in the biosynthesis of daunorubicin is propionyl coenzyme A (CoA) rather than acetyl-CoA, which is used in the production of most of the bacterial aromatic polyketides studied to date. In the daunorubicin biosynthesis gene cluster ofStreptomyces peucetius, directly downstream of the genes encoding the β-ketoacyl:acyl carrier protein synthase subunits, are two genes, dpsC and dpsD, encoding proteins that are believed to function as the starter unit-specifying enzymes. Recombinant strains containing plasmids carrying dpsC anddpsD, in addition to other daunorubicin polyketide synthase (PKS) genes, incorporate the correct starter unit into polyketides made by these genes, suggesting that, contrary to earlier reports, the enzymes encoded by dpsC and dpsD play a crucial role in starter unit specification. Additionally, the results of a cell-free synthesis of 21-carbon polyketides from propionyl-CoA and malonyl-CoA that used the protein extracts of recombinant strains carrying other daunorubicin PKS genes to which purified DpsC was added suggest that this enzyme has the primary role in starter unit discrimination for daunorubicin biosynthesis.

In an effort to characterize the diversity of mechanisms involved in cellular self-protection against the antitumor antibiotic mitomycin C (MC), DNA fragments from the producing organism (Streptomyces lavendulae) were introduced into Streptomyces lividans and transformants were selected for resistance to the drug. Subcloning of a 4.0-kb BclI fragment revealed the presence of an MC resistance determinant, mrd. Nucleotide sequence analysis identified an open reading frame consisting of 130 amino acids with a predicted molecular weight of 14,364. Transcriptional analysis revealed that mrd is expressed constitutively, with increased transcription in the presence of MC. Expression of mrd in Escherichia coli resulted in the synthesis of a soluble protein with an M r of 14,400 that conferred high-level cellular resistance to MC and a series of structurally related natural products. Purified MRD was shown to function as a drug-binding protein that provides protection against cross-linking of DNA by preventing reductive activation of MC.Streptomyces species are gram-positive soil bacteria known for their ability to produce a wide range of biologically active metabolites. In addition, many resistance genes have been cloned from these bacteria. Mechanisms of cellular self-protection include drug inactivation, target site modification, reduction of intracellular concentration via efflux, and drug binding (8). The presence of multiple modes of self-protection toward a single antibiotic is well documented (9), often with one or more resistance determinants located adjacent to the corresponding biosynthetic genes.Mitomycin C (MC) was identified in 1956 as an antibiotic produced by Streptomyces lavendulae (18) and subsequently established as an important antitumor agent (19,21). MC functions as a prodrug and requires enzymatic or chemical reduction to become a highly reactive alkylating agent (19,40). The intracellular activation of MC is specified by endogenous flavoreductases (34) and proceeds by single electron reduction to the MC semiquinone radical. The relatively long-lived semiquinone species either rearranges to an alkylating intermediate (by further reduction) or transfers an electron to molecular oxygen to generate superoxide (32). Therefore, the ability of MC to inhibit bacterial and mammalian cell growth involves the combined action of DNA alkylation and the formation of reactive oxygen species. Other naturally occurring compounds within this class include bleomycin (37), enediynes (10), and the more recently discovered dihydrobenzoxazines (25,42).Recently, a locus, mcr, that confers high-level MC resistance in S. lavendulae has been reported (2, 3). The resistance gene, mcrA, encodes a flavoenzyme (MCRA) that reoxidizes reductively activated MC (23). In another example involving a DNA damaging agent, bleomycin self-resistance has been determined by drug modification (Bat) and binding (BLMA) proteins in Streptomyces verticillus (38). Beyond these examples, little is known about bacterial resistance to the growing cl...

Sequence analysis of Streptomyces lavendulae NRRL 2564 chromosomal DNA adjacent to the mitomycin resistance locusmrd (encoding a previously described mitomycin-binding protein [P. Sheldon, D. A. Johnson, P. R. August, H.-W. Liu, and D. H. Sherman, J. Bacteriol. 179:1796–1804, 1997]) revealed a putative mitomycin C (MC) transport gene (mct) encoding a hydrophobic polypeptide that has significant amino acid sequence similarity with several actinomycete antibiotic export proteins. Disruption of mct by insertional inactivation resulted in an S. lavendulae mutant strain that was considerably more sensitive to MC. Expression of mct in Escherichia coli conferred a fivefold increase in cellular resistance to MC, led to the synthesis of a membrane-associated protein, and correlated with reduced intracellular accumulation of the drug. Coexpression ofmct and mrd in E. coli resulted in a 150-fold increase in resistance, as well as reduced intracellular accumulation of MC. Taken together, these data provide evidence that MRD and Mct function as components of a novel drug export system specific to the mitomycins.

The dorrigocins are newsecondary metabolites produced by submerged fermentation of a streptomycete which was isolated from a soil sample collected in Australia. The dorrigocins show moderate antifungal activity and reverse the morphology of ray-transformed NIH/3T3cells from a transformed phenotype to a normal one. The producing culture was identified as Streptomyces platensis subsp. rosaceus strain AB198 1F-75.The dorrigocins are novel glutarimide antifungal antibiotics discovered in the fermentation broth and myceliumof Streptomyces platensis subsp. rosaceus strain AB198 1F-75. This paper describes the taxonomy of the producing strain and the fermentation, antifungal and antitumor activity of the dorrigocins. The isolation, structural elucidation, biological properties and mechanism of action of these compounds are described in accompanying publication1>2). Materials and Methods Micro organismsStrain AB1981F-75 was isolated from soil collected on the Dorrigo plateau in NewSouth Wales, Australia. A subculture of the microorganism was deposited at

Self-protection in the mitomycin C (MC)-producing microorganism Streptomyces lavendulae includes MRD, a protein that binds MC in the presence of NADH and functions as a component of a unique drug binding-export system. Characterization of MRD revealed that it reductively transforms MC into 1,2- cis -1-hydroxy-2,7-diaminomitosene, a compound that is produced in the reductive MC activation cascade. However, the reductive reaction catalyzed by native MRD is slow, and both MC and the reduced product are bound to MRD for a relatively prolonged period. Gene shuffling experiments generated a mutant protein (MRD E55G ) that conferred a 2-fold increase in MC resistance when expressed in Escherichia coli . Purified MRD E55G reduces MC twice as fast as native MRD, generating three compounds that are identical to those produced in the reductive activation of MC. Detailed amino acid sequence analysis revealed that the region around E55 in MRD strongly resembles the second active site of prokaryotic catalase-peroxidases. However, native MRD has an aspartic acid (D52) and a glutamic acid (E55) residue at the positions corresponding to the catalytic histidine and a nearby glycine residue in the catalase-peroxidases. Mutational analysis demonstrated that MRD D52H and MRD D52H/E55G conferred only marginal resistance to MC in E. coli . These findings suggest that MRD has descended from a previously unidentified quinone reductase, and mutations at the active site of MRD have greatly attenuated its catalytic activity while preserving substrate-binding capability. This presumed evolutionary process might have switched MRD from a potential drug-activating enzyme into the drug-binding component of the MC export system.