The enediynes exemplify nature's ingenuity. We have cloned and characterized the biosynthetic locus coding for perhaps the most notorious member of the nonchromoprotein enediyne family, calicheamicin. This gene cluster contains an unusual polyketide synthase (PKS) that is demonstrated to be essential for enediyne biosynthesis. Comparison of the calicheamicin locus with the locus encoding the chromoprotein enediyne C-1027 reveals that the enediyne PKS is highly conserved among these distinct enediyne families. Contrary to previous hypotheses, this suggests that the chromoprotein and nonchromoprotein enediynes are generated by similar biosynthetic pathways.

The drrC gene, cloned from the daunorubicin (DNR)-and doxorubicin-producing strain of Streptomyces peucetius ATCC 29050, encodes a 764-amino-acid protein with a strong sequence similarity to the Escherichia coli and Micrococcus luteus UvrA proteins involved in excision repair of DNA. Expression of drrC was correlated with the timing of DNR production in the growth medium tested and was not dependent on the presence of DNR. Since introduction of drrC into Streptomyces lividans imparted a DNR resistance phenotype, this gene is believed to be a DNR resistance gene. The drrC gene could be disrupted in the non-DNR-producing S. peucetius dnrJ mutant but not in the wild-type strain, and the resulting dnrJ drrC double mutant was significantly more sensitive to DNR in efficiency-of-plating experiments. Expression of drrC in an E. coli uvrA strain conferred significant DNR resistance to this highly DNR-sensitive mutant. However, the DrrC protein did not complement the uvrA mutation to protect the mutant from the lethal effects of UV or mitomycin even though it enhanced the UV resistance of a uvrA ؉ strain. We speculate that the DrrC protein mediates a novel type of DNR resistance, possibly different from the mechanism of DNR resistance governed by the S. peucetius drrAB genes, which are believed to encode a DNR antiporter.Microorganisms require one or more self-resistance determinants to produce antibiotics, except in cases in which they are insensitive to the antibiotic's effect(s). The resistance genes usually are clustered with the structural (biosynthetic) and regulatory genes and encode proteins that either inactivate the antibiotic, facilitate its export, or modify the host to render it insensitive to the antibiotic (9). Multiple rather than single resistance mechanisms are often found; in this case, it is not known whether any one resistance mode is sufficient to ensure survival or antibiotic production. We address this question here.Streptomyces peucetius, which produces the important antitumor drugs daunorubicin (DNR) (4, 10) and doxorubicin (DXR) (2), contains the drrAB (17) and ric2 (8) resistance genes, which are assumed to provide self-resistance to these two antibiotics because they confer DNR and DXR resistance when introduced into Streptomyces lividans. The DrrA protein strongly resembles bacterial proteins that transport compounds by an ATP-dependent process (20), as well as the Mdr1 P glycoprotein responsible for DNR-DXR resistance of human cancer cells and known to act as an ATP-dependent transporter (19). DrrA thus is a candidate for a DNR-DXR binding and transport protein, whereas the hydrophobic DrrB protein could be responsible for binding DrrA to the bacterial membrane. The ric2 locus, which unlike drrAB is not part of the cluster of DXR production genes (8), may also be important for self-resistance. A third gene, drrC, in the same cluster of genes as is drrAB, encodes the DrrC protein described here. S. lividans drrC ϩ transformants display a DNR resistance phenotype similar to that of drrAB tran...

Doxorubicin-overproducing strains of Streptomyces peucetius ATCC 29050 can be obtained through manipulation of the genes in the region of the doxorubicin (DXR) gene cluster that containsdpsH, the dpsG polyketide synthase gene, the putative dnrU ketoreductase gene, dnrV, and thedoxA cytochrome P-450 gene. These five genes were characterized by sequence analysis, and the effects of replacingdnrU, dnrV, doxA, ordpsH with mutant alleles and of doxAoverexpression on the production of the principal anthracycline metabolites of S. peucetius were studied. The exact roles of dpsH and dnrV could not be established, although dnrV is implicated in the enzymatic reactions catalyzed by DoxA, but dnrU appears to encode a ketoreductase specific for the C-13 carbonyl of daunorubicin (DNR) and DXR or their biosynthetic precursors. The highest DXR titers were obtained in a dnrX dnrU (N. Lomovskaya, Y. Doi-Katayama, S. Filippini, C. Nastro, L. Fonstein, M. Gallo, A. L. Colombo, and C. R. Hutchinson, J. Bacteriol. 180:2379–2386, 1998) double mutant and a dnrX dnrU dnrH (C. Scotti and C. R. Hutchinson, J. Bacteriol. 178:7316–7321, 1996) triple mutant. Overexpression of doxA in adoxA::aphII mutant resulted in the accumulation of DXR precursors instead of in a notable increase in DXR production. In contrast, overexpression of dnrV and doxAjointly in the dnrX dnrU double mutant or the dnrX dnrU dnrH triple mutant increased the DXR titer 36 to 86%.

The high-yielding glycosylation of the aromatic polyketide epsilon-rhodomycinone using plasmid-borne deoxysugar biosynthesis genes proves that the minimal information for L-daunosamine biosynthesis and attachment in the heterologous host is encoded by the dnmLMJVUTS genes. This is a general approach to making both known and new glycosides of anthracyclines, several of which have medically important antitumor activity.

The plasmid-based reconstruction of the avr deoxysugar genes for expression in a heterologous system combined with biotransformation has led to new information about the mechanism of 2,6-deoxysugar biosynthesis. The structures of the di-demethyldeoxysugar avermectins accumulated indicate that in the oleandrose pathway the stereochemistry at C-3 is ultimately determined by the 3-O-methyltransferase and not by the 3-ketoreductase or a possible 3,5-epimerase. The AvrF protein is therefore a 5-epimerase and not a 3,5-epimerase. The ability of the AvrB (mono-)glycosyltransferase to accommodate different deoxysugar intermediates is evident from the structures of the novel avermectins produced.

The Streptomyces peucetius dpsY and dnrXgenes govern early and late steps in the biosynthesis of the clinically valuable antitumor drugs daunorubicin (DNR) and doxorubicin (DXR). Although their deduced products resemble those of genes thought to be involved in antibiotic production in several other bacteria, this information could not be used to identify the functions ofdpsY and dnrX. Replacement of dpsYwith a mutant form disrupted by insertion of the aphIIneomycin-kanamycin resistance gene resulted in the accumulation of UWM5, the C-19 ethyl homolog of SEK43, a known shunt product of iterative polyketide synthases involved in the biosynthesis of aromatic polyketides. Hence, DpsY must act along with the other components of the DNR-DXR polyketide synthase to form 12-deoxyaklanonic acid, the earliest known intermediate of the DXR pathway. Mutation ofdnrX in the same way resulted in a threefold increase in DXR production and the disappearance of two acid-sensitive, unknown compounds from culture extracts. These results suggest thatdnrX, analogous to the role of the S. peucetius dnrH gene (C. Scotti and C. R. Hutchinson, J. Bacteriol. 178:7316–7321, 1996), may be involved in the metabolism of DNR and/or DXR to acid-sensitive compounds, possibly related to the baumycins found in many DNR-producing bacteria.

A system for gene disruption and replacement based on a streptomycete temperate phage vector was developed to introduce DNA in the rapamycinproducing Streptomyces hygroscopicus strain ATCC 29253. This will be useful in attempts to produce, through genetic manipulation, novel forms of the therapeutically important immunosuppressive drug rapamycin. Recombinant phages were constructed from the qK31 phage derivative KC515 (c+ attP) carrying a thiostrepton or viomycin resistance gene along with segments of the S. hygroscopicus chromosome. Each of the cloned segments also contained the aphll neomycin/kanamycin resistance gene to enable gene replacement by loss of the phage-derived DNA. Specific deletion of the entire polyketide synthase (PKS) believed to govern rapamycin biosynthesis resulted in the loss of rapamycin production. In contrast, disruption or deletion of a region predicted to encode four PKS open reading frames, or another region predicted to encode another PKS plus a cytochrome P450 hydroxylase and ferredoxin, had no effect on the production of rapamycin or nigericin, a polyether antibiotic also produced b y S. hygroscopicus. Therefore, 5. hygroscopicus may have the capacity to produce polyketides additional to rapamycin and nigericin.

No abstract