Jan R. Turner

Cloning and analysis of the spinosad biosynthetic gene cluster of Saccharopolyspora spinosa1

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.

PRODUCTION OF A NOVEL POLYKETIDE THROUGH THE CONSTRUCTION OF A HYBRID POLYKETIDE SYNTHASE

The lactone rings of the polyketides platenolide and tylactone are synthesized by condensation of acetate-, proprionate-, and butyrate-derived precursors. A hybrid tylactone/platenolide synthase was constructed to determine if the choice of substrate is programmed by the polyketide synthase and to ascertain if a substrate different than that normally used in the first step of platenolide synthesis could be incorporated into the final polyketide. In this work, we report the successful incorporation of a propionate in place of the acetate normally used in the first step of platenolide synthesis. This result demonstrates that polyketide synthases choose a particular substrate at defined steps and provides strong evidence that substrate choice is programmed by the acyl transferase domain of a large, multifunctional polyketide synthase.

Research PaperCloning and analysis of the spinosad biosynthetic gene cluster of Saccharopolyspora spinosa1

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.

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.

Characterization of a novel regulatory gene governing the expression of a polyketide synthase gene in Streptomyces ambofaciens

A key step in the biosynthesis of macrolide antibiotics is the assembly of a large macrocyclic lactone ring by a multienzyme protein complex called the polyketide synthase. In the species Streptomyces ambofaciens, the polyketide synthase for the assembly of the 16‐membered ring of the macrolide antibiotic spiramycin is encoded by the biosynthetic gene srmG. Here we show that the accumulation of transcripts from the srmG promoter is governed by the regulatory gene srmR, whose predicted product, a 65 kDa polypeptide, is not significantly similar in its deduced amino acid sequence to that of previously reported proteins in the protein databases. The srmR gene product is also required for the accumulation of transcripts from srmX, an additional gene in the vicinity of srmR, but not for the accumulation of transcripts from srmR itself. Interestingly, mutations in srmR prevent the accumulation of transcripts from the spiramycin resistance gene srmB, but this is an indirect consequence of the failure of srmR mutants to produce spiramycin, which is an inducer of its own resistance gene. The possibility that srmR is the prototype for a new class of regulatory genes governing early events in the biosynthesis of macrolide antibiotics is discussed.

Production of a hybrid macrolide antibiotic in Streptomyces ambofaciens and Streptomyces lividans by introduction of a cloned carbomycin biosynthetic gene from Streptomyces thermotolerans

The structurally related macrolide antibiotics carbomycin (Cb) and spiramycin (Sp) are produced by Streptomyces thermotolerans and Streptomyces ambofaciens, respectively. Both antibiotics contain 16-membered lactone rings to which deoxysugars are attached. There are three sugars in Sp (forosamine, mycaminose and mycarose) and two sugars in Cb (mycaminose and a derivative of mycarose containing an isovaleryl group at position 4). We have identified the gene from S. thermotolerans (designated carE), which appears to encode an enzyme that acylates this mycarose sugar, and have shown that recombinant strains containing carE can use Sp as a substrate and convert it to the hybrid antibiotic, isovaleryl Sp (ivSp). Expression of carE was demonstrated in two heterologous hosts: in S. ambofaciens, where endogenously synthesized Sp was converted to ivSp, and in Streptomyces lividans where exogenously added Sp was converted to ivSp. The carE gene was isolated on a cosmid that also encodes genes required for Cb-lactone formation. These genes reside on a DNA segment of about 70 kb and are part of a Cb biosynthetic gene cluster that is flanked by two Cb-resistance genes, carA and carB. Mapping studies and nucleotide sequence analysis revealed that carE is located at one end of this gene cluster, immediately adjacent to the carB gene. Genes carB and carE are transcribed convergently and may share a common transcriptional terminator sequence.

Rapid detection of ampicillin-resistant Haemophilus influenzae and their susceptibility to sixteen antibiotics.

Ampicillin-resistant and -susceptible strains of Haemophilus influenzae were tested for susceptibility to 16 antibiotics. Chloramphenicol and a new cephalosporin, cefamandole, were most active with minimal inhibitory concentrations (MICs) for all bacteria tested between 0.5 to 2.0 μg/ml. All but two organisms were susceptible to tetracycline. Ampicillin-resistant strains of H. influenzae were less susceptible (MIC, 4 to 32 μg/ml) to carbenicillin and ticarcillin than ampicillin-susceptible organisms (MIC, 0.25 to 1.0 μg/ml). A rapid assay for β-lactamase, utilizing a chromogenic cephalosporin substrate, detected enzyme production in all 17 ampicillin-resistant strains of H. influenzae. Images

Branched-chain fatty acids produced by mutants of Streptomyces fradiae, putative precursors of the lactone ring of tylosin.

Three branched-chain fatty acids (7-hydroxy-4,6-dimethylnona-2,4-dienoic acid [compound 1], its 7-epimer [compound 2], and 7-keto-4,6-dimethylnona-2,4-dienoic acid [compound 3]) and a ketone (9-hydroxy-6,8-dimethylundeca-4,6-dien-3-one [compound 4]) were isolated from the culture broth of mutants of Streptomyces fradiae which were blocked in the biosynthesis of the macrolide antibiotic tylosin. Two phenotypic classes of mutants of this organism which were blocked in the addition of mycaminose to tylactone (compound 6) accumulated these compounds. These compounds were not produced by mutants which were blocked in lactone synthesis, in steps beyond mycaminose addition, or by the wild-type strain. Synthesis of these compounds, like synthesis of tylosin, was inhibited by the addition of cerulenin. Compounds 1, 2, and 3 were partially interconvertible by these mutants; but they were not produced from the degradation of tylactone and they were not directly incorporated into tylosin by intact cells. The structures of compounds 1 and 2 were equivalent to that of a predicted intermediate (S. Yue, J. S. Duncan, Y. Yamamoto, and C. R. Hutchinson, J. Am. Chem. Soc. 109:1253-1255, 1987) in the biosynthesis of tylactone. The ketone (compound 4) reported previously (N. D. Jones, M. O. Chaney, H. A. Kirst, G. M. Wild, R. H. Baltz, R. L. Hamill, and J. W. Paschal, J. Antibiot. 35:420-425, 1982) appears to be the decarboxylation product of the intermediate following that represented by compound 1. This represents the first report of the isolation of putative precursors of tylactone from tylosin-producing organisms.

Delineation of the Relative Antibacterial Activity of Cefamandole and Cefamandole Nafate

By conventional laboratory evaluation procedures, the in vitro antibacterial activities of cefamandole and its O-formyl ester, cefamandole nafate, appear virtually identical. When the activities of these two compounds were examined for their ability to lyse log-phase cultures of susceptible bacteria, however, cefamandole was found to be about 10 times more active than cefamandole nafate. Cefamandole nafate was shown to be rapidly converted to cefamandole in bacteriological media, with a half-life of less than 1 h at a pH of 7.0 or above. At pH 6.0, in log-phase inhibition experiments, however, cefamandole nafate is more stable, allowing delineation of the activity between cefamandole and cefamandole nafate. The efficacy of cefamandole was identical to that of cefamandole nafate in treating experimental animal infections, indicating that rapid conversion of cefamandole nafate to cefamandole occurs in vivo.