Cloning and analysis of the spinosad biosynthetic gene cluster of Saccharopolyspora spinosa.

BACKGROUND: Spinosad is a mixture of novel macrolide secondary metabolites produced by Saccharopolyspora spinosa. It is used in agriculture as a potent insect control agent with exceptional safety to non-target organisms. The cloning of the spinosyn biosynthetic gene cluster provides the starting materials for the molecular genetic manipulation of spinosad yields, and for the production of novel derivatives containing alterations in the polyketide core or in the attached sugars. RESULTS: We cloned the spinosad biosynthetic genes by molecular probing, complementation of blocked mutants, and cosmid walking, and sequenced an 80 kb region. We carried out gene disruptions of some of the genes and analyzed the mutants for product formation and for the bioconversion of intermediates in the spinosyn pathway. The spinosyn gene cluster contains five large open reading frames that encode a multifunctional, multi-subunit type I polyketide synthase (PKS). The PKS cluster is flanked on one side by genes involved in the biosynthesis of the amino sugar forosamine, in O-methylations of rhamnose, in sugar attachment to the polyketide, and in polyketide cross-bridging. Genes involved in the early common steps in the biosynthesis of forosamine and rhamnose, and genes dedicated to rhamnose biosynthesis, were not located in the 80 kb cluster. CONCLUSIONS: Most of the S. spinosa genes involved in spinosyn biosynthesis are found in one 74 kb cluster, though it does not contain all of the genes required for the essential deoxysugars. Characterization of the clustered genes suggests that the spinosyns are synthesized largely by mechanisms similar to those used to assemble complex macrolides in other actinomycetes. However, there are several unusual genes in the spinosyn cluster that could encode enzymes that generate the most striking structural feature of these compounds, a tetracyclic polyketide aglycone nucleus.

Genes for the biosynthesis of spinosyns: applications for yield improvement in Saccharopolyspora spinosa.

Spinosyns A and D are the active ingredients in an insect control agent produced by fermentation of Saccharopolyspora spinosa. Spinosyns are macrolides with a 21-carbon, tetracyclic lactone backbone to which the deoxysugars forosamine and tri-O-methylrhamnose are attached. The spinosyn biosynthesis genes, except for the rhamnose genes, are located in a cluster that spans 74 kb of the S. spinosa genome. DNA sequence analysis, targeted gene disruptions and bioconversion studies identified five large genes encoding type I polyketide synthase subunits, and 14 genes involved in sugar biosynthesis, sugar attachment to the polyketide or cross-bridging of the polyketide. Four rhamnose biosynthetic genes, two of which are also necessary for forosamine biosynthesis, are located outside the spinosyn gene cluster. Duplication of the spinosyn genes linked to the polyketide synthase genes stimulated the final step in the biosynthesis--the conversion of the forosamine-less pseudoaglycones to endproducts. Duplication of genes involved in the early steps of deoxysugar biosynthesis increased spinosyn yield significantly.

A cluster of genes for the biosynthesis of spinosyns, novel macrolide insect control agents produced by Saccharopolyspora spinosa.

Spinosyns A and D are the active ingredients in a family of insect control agents produced by fermentation of Saccharopolyspora spinosa. Spinosyns are 21-carbon tetracyclic lactones to which are attached two deoxysugars. Most of the genes involved in spinosyn biosynthesis are clustered in an 74 kb region of the S. spinosa genome. This region has been characterized by DNA sequence analysis and by targeted gene disruptions. The spinosyn biosynthetic gene cluster contains five large genes encoding a type I polyketide synthase, and 14 genes involved in modification of the macrolactone, or in the synthesis, modification and attachment of the deoxysugars. Four genes required for rhamnose biosynthesis (two of which are also required for forosamine biosynthesis) are not present in the cluster. A pathway for the biosynthesis of spinosyns is proposed.

Conjugal transfer of cosmid DNA from Escherichia coli to Saccharopolyspora spinosa: effects of chromosomal insertions on macrolide A83543 production.

Cosmid pOJ436, containing large inserts of Saccharopolyspora spinosa (Ss) DNA, was transferred by conjugation from Escherichia coli to Ss an integrated into the chromosome, apparently by homologous recombination, at high frequencies (10(-5) to 10(-4) per recipient). Transfer was mediated by the plasmid RP4 (RK2) transfer functions in E. coli, and the RK2 oriT function located on pOJ436 [Bierman et al., Gene 116 (1992) 43-49]. pOJ436 lacking Ss DNA, or containing a small insert (approx. 2 kb) of Ss DNA, conjugated from E. coli and integrated at either of two bacteriophage phi C31 attB sites at low frequency (approx. 10(-7) per recipient). Exconjugants containing homologous inserts or inserts at the phi C31 attB sites were stable in the absence of antibiotic selection, and most produced control levels of tetracyclic macrolide A83543 factors. Some exconjugants contained similar kinds of large deletions and were defective in macrolide production.

Pneumocystis carinii dihydrofolate reductase used to screen potential antipneumocystis drugs.

Pneumocystis carinii was obtained in high yield from the lungs of immunosuppressed rats by rupturing mammalian host cells, washing away the soluble mammalian dihydrofolate reductase, and harvesting intact organisms in association with the mammalian plasma membranes. P. carinii dihydrofolate reductase, measured in the 100,000 x g supernatant from sonicated organisms, was obtained in yields ranging up to 62 IU per rat. The enzyme prepared in the presence of protease inhibitors was stable when frozen in liquid nitrogen. P. carinii dihydrofolate reductase differed from the mammalian enzyme in that the former was slightly inhibited by 150 mM KCl, whereas the latter was stimulated over twofold by 150 mM KCl. The standard assay for P. carinii dihydrofolate reductase contained 0.12 mM NADPH and 92 microM dihydrofolic acid. Under these conditions, the 50% inhibitory concentrations of the known inhibitors trimethoprim, trimetrexate, and pyrimethamine were 12 microM, 42 nM, and 3.8 microM, respectively. These standard compounds were also tested against dihydrofolate reductase from rat liver to allow an assessment of the selectivity of the drugs. Although it was the least potent, trimethoprim was the most selective. Pyrimethamine was more potent but was nonselective. Trimetrexate was extremely potent but was selective for mammalian dihydrofolate reductase. A series of experimental compounds was obtained from the National Cancer Institute and other sources through the Developmental Therapeutics Branch of the Division of AIDS at the National Institute of Allergy and Infectious Diseases. Among the first 87 compounds tested, 11 had 50% inhibitory concentrations below that of trimetrexate and 3 were more selective than trimethoprim. The most promising compounds in this original group were chemically related to methotrexate.

Polyene resistance in ergosterol producing strains of Candida albicans.

Following nitrous acid mutagenesis, one nystatin- (nyl) and two amphotericin B (AB)-resistant (ab1 and ab2) mutants of Candida albicans were isolated and characterized. The three mutants plus a previously described cytochrome P450-deficient mutant (D10) of this organism were analyzed for polyene cross resistance. Cross resistance was noted for ny1 and D10 but not for ab1 and ab2. Sterol analysis indicated that ny1 was a delta 8-delta 7 isomerase mutant while ab1 and ab2 showed wild type sterol profiles. Fatty acid analysis showed no significant differences for ab1, ab2, and ny1 compared to wild type while D10 showed more pronounced differences. AB- and Triton X-100-induced potassium leakage studies indicated that ab1 and ab2 are resistant to low AB levels and ny1 is resistant to higher AB levels. In contrast, ab1 and ab2 were more resistant to detergent-induced potassium leakage than the wild type or mutants ny1 and D10. Significant differences in growth rate, ethanol sensitivity, and response to Tergitol were also noted among the resistant strains. The data indicate a different mechanism of action for the two polyenes and indicate a resistance mechanism for ab1 and ab2 based on subtle alterations of membrane structure rather than sterol substitution.

Azole susceptibility and hyphal formation in a cytochrome P-450-deficient mutant of Candida albicans.

A cytochrome P-450-deficient mutant of Candida albicans, strain D10, was employed to study the mode of action of imidazole antifungal agents. This mutant accumulates exclusively 14-alpha-methylsterols, resulting in a sterol profile which mimics that of azole-treated wild-type strains. Since the widely accepted primary effect of imidazoles is the inhibition of cytochrome P-450-mediated demethylation of the ergosterol precursor lanosterol, strain D10 and its wild-type revertant, strain D10R, were grown in the presence of concentrations of clotrimazole, miconazole, and ketoconazole known to inhibit demethylation. The growth of strain D10 was unaffected by these antifungal agents, while that of strain D10R was significantly reduced. At higher azole concentrations (which are known to exert a direct, disruptive action on the cell membrane), the growth of both strains was immediately and completely inhibited by clotrimazole and miconazole. Ketoconazole was membrane disruptive only for strain D10; this is the first report of a direct membrane effect for this drug. Because hyphal formation has been implicated in the pathogenesis of C. albicans and because it has been shown to be inhibited by azoles, the hypha-forming capability of strain D10 was examined. Strain D10 was shown to be seriously defective in hyphal formation, suggesting that this function may be dependent on the 14-alpha-demethylation of lanosterol. The results of this study suggest that inhibition of lanosterol demethylation per se is neither fungicidal nor fungistatic, although the growth rate is reduced. In addition, the substitution of 14-alpha-methylsterols for ergosterol results in defective hyphal formation and in a cell that is more susceptible to membrane-active agents such as ketoconazole.

Membrane fluidity alterations in a cytochrome P-450-deficient mutant of Candida albicans.

A cytochrome P450-deficient mutant of the pathogenic fungus, Candida albicans, which accumulates exclusively 14 alpha-methylsterols in place of the normal end product sterol, ergosterol, was examined for alterations in membrane fluidity by electron paramagnetic resonance. The results using four nitroxyl spin labels indicated that exponential phase cultures of the mutant strain, D10, had a uniformly more rigid membrane than similarly grown wild type. Since D10 shows a sterol spectrum similar to that of wild type cells treated with imidazole and triazole antifungal agents, many of the physiological effects reported as the result of azole application may be the result of alterations in membrane fluidity.