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

Huber, M. L. B.; Paschal, Jonathan W.; Leeds, James P.; Kirst, Herbert A.; Wind, Julie A.; Miller, F. D.; Turner, J. R.

doi:10.1128/aac.34.8.1535

Cited by 25 publications

(12 citation statements)

References 24 publications

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“…nov. (tetraketide and pentaketide) and the corresponding triketide in a nonproducing mutant (Kinoshita et al, 1988). Tetra-and pentaketides were observed in nonproducing mutants of the tylosin producer Streptomyces fradiae (Huber et al, 1990). These observations, together with our results, suggest that the formation of noncyclized polyketide intermediates is perhaps a general phenomenon during either the normal or impaired catalysis by type I PKS.…”

Section: Discussionsupporting

confidence: 67%

Intermediates of rifamycin polyketide synthase produced by an Amycolatopsis mediterranei mutant with inactivated rifF gene

et al. 1999

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Rifamycin B biosynthesis in Amycolatopsis mediterranei N/813 was inactivated by introducing a small deletion in the rifF gene situated directly downstream of the rifamycin polyketide synthase (PKS) gene cluster. The corresponding mutant strain produced a series of linear intermediates of rifamycin B biosynthesis that are most probably generated by obstruction of the normal release of the end product of the rifamycin PKS. This result provides evidence that the rifF gene product catalyses the release of the completed linear polyketide from module 10 of the PKS and the intramolecular macrocyclic ring closure by formation of an amide bond, as indicated by sequence similarity of this protein to amide synthases. The chemical structures of the new rifamycin polyketide synthase intermediates released from modules 4 to 10 were determined by spectroscopic methods (UV, IR, NMR and MS) and gave insight into the reaction steps of rifamycin ansa chain biosynthesis and the timing of the formation of the naphthoquinone ring. The intermediates released from modules 6 and 8 were isolated as lactones formed by the terminal carboxyl group ; proton NMR double resonance and ROESY(rotated frame nuclear Overhauser enhancement spectroscopy) experiments enabled the deduction of the relative configurations in the linear chain which correspond to the known absolute stereochemistry of rifamycin B.Keywords : antibiotic biosynthesis, ansamycins, amide synthase, gene replacement, pathway engineering INTRODUCTIONRifamycins are clinically important ansamycin antibiotics, composed of a naphthalenic chromophore spanned by a long aliphatic ansa chain. The rifamycins and the semisynthetic drugs derived from them exert their antibiotic activity by specific inhibition of bacterial DNA-dependent RNA polymerase (Wehrli, 1977). At higher concentrations, these antibiotics also inhibit the RNA-dependent DNA polymerase of retroviruses (Szabo et al., 1976 bacterium leprae, causative agents of tuberculosis and leprosy, respectively, they are also active against a variety of other organisms, including bacteria and viruses (Szabo et al., 1976 ;Oppenheim et al., 1986 ;Barakett et al., 1993 ;Bachs et al., 1992). Furthermore, it was shown recently that rifampicin-containing regimens are able to cure staphylococcal implant-related infections (Zimmerli et al., 1998).The identification and sequencing of the rifamycin polyketide synthase (PKS) gene cluster by our group (Schupp et al., 1998) and by August et al. (1998) Our interest in further studying rifamycin B biosynthesis led us to undertake the inactivation of the rifF gene, situated directly downstram of the PKS genes, and to analyse the effect of this mutation on rifamycin biosynthesis. The rifF gene product has been characterized as rifamycin amide synthase by sequence homologies to different arylamine N-acetyltransferases (August et al., 1998) and the putative function of the RifF protein in rifamycin biosynthesis was suggested to be a cyclase, catalysing the formation of an intramolecular amide bond between ...

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Section: Discussionsupporting

confidence: 67%

Intermediates of rifamycin polyketide synthase produced by an Amycolatopsis mediterranei mutant with inactivated rifF gene

et al. 1999

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“…This was done by reverse phase HPLC (as described by Huber et al, 1990) using a 3·9 × 300 mm C18 gBondapak column protected by a C18 gBondapak guard column (Waters Associates). Chloroform extracts were applied to the column in 04 % (w/v) ammonium formate (pH 4·0) containing 50 % (v/v) methanol and products were eluted using a similar buffer with a linear concentration gradient (50—80 %) of methanol at a flow rate of 1·75 ml min −1 .…”

Section: Methodsmentioning

confidence: 99%

Stimulation of polyketide metabolism in Streptomyces fradiae by tylosin and its glycosylated precursors

Fish¹,

Cundliffe

1997

Microbiology

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Three glycosyltransferases are involved in tylosin biosynthesis in Streptomyces fradiae. The first sugar to be added to the polyketide aglycone (tylactone) is mycaminose and the gene encoding mycaminosyltransferase is oH2* (tyhW2). However, targeted disruption of od2* did not lead to the accumulation of tylactone under conditions that normally favour tylosin production; instead, the synthesis of tylactone was virtually abolished. This may, in part, have resulted from a polar effect on the expression of genes downstream of oH2*, particularly od4* (ccr) which encodes crotonyl-CoA reductase, an enzyme that supplies 4-carbon extender units for polyketide metabolism. However, that cannot be the entire explanation, since tylosin production was restored a t about 10% of the wild-type level when oH2* was re-introduced into the disrupted strain. When glycosylated precursors of tylosin were fed to the disrupted strain, they were converted to tylosin, confirming that two of the three glycosyltransferase activities associated with tylosin biosynthesis were still intact. Interestingly, however, tylactone also accumulated under such conditions and, to a much lesser extent, when tylosin was added to similar fermentations. It is concluded that glycosylated macrolides exert a pronounced positive effect on polyketide metabolism in S. fradiae. ;ity OfKeywords : tylosin production, glycosyltransferase, polyketide metabolism, Streptomyces fradiae INTRODUCTIONThe macrolide antibiotic, tylosin (Fig. l ) , is produced by Streptomyces fradiae via a combination of polyketide and 6-deoxyhexose metabolism. Glycosylation of tylactone, the cyclized polyketide product, always begins with the addition of mycaminose followed, in a preferred but not obligatory order, by deoxyallose and then mycarose to generate demethyl-macrocin. Stepwise bis 0-methylation then converts the deoxyallose moiety to mycinose as macrocin is produced and converted to tylosin (Baltz et al., 1983). Characterization of the tylosin biosynthetic pathway (Fig. 2) was facilitated by the availability of non-producing mutants of S. fradiae, generated using NTG (Baltz & Seno, 1981). Collectively, these exhibited nine distinct phenotypes in cross-feeding Abbreviations: OMT, 0-mycaminosyl-tylonolide; DMT, demycinosyltyl osi n. experiments and allowed 13 genetic loci (tylA-M) to be mapped (Fig. 3) following complementation studies using cloned fragments of tyl DNA (Beckmann et al.,

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“…Working on the mycinamicin producer, Micromonospora griseorubida, Kinoshita et al (29) isolated from a nonproducing mutant a triketide containing the starter and first two chain-extension units and, from the wild type, the corresponding tetraketide and the decarboxylation product of the pentaketide. Tetra-and decarboxylated pentaketides were isolated from nonproducing mutants of the tylosin producer Streptomyces fradiae (30). However, noncyclized polyketide intermediates have not been observed during extensive work with 6-deoxyerythronolide B synthase (31) Because the ketides isolated from the rifF(Ϫ) mutant also are found in the wild-type extract, although their presence is masked by large amounts of rifamycin, their formation is not the result of the mutation to eliminate the off-loading mechanism.…”

Section: Discussionmentioning

confidence: 99%

Direct evidence that the rifamycin polyketide synthase assembles polyketide chains processively

Shen²,

Doi-Katayama³

et al. 1999

Proc. Natl. Acad. Sci. U.S.A.

133

130

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The assembly of the polyketide backbone of rifamycin B on the type I rifamycin polyketide synthase (PKS), encoded by the rifA-rifE genes, is terminated by the product of the rifF gene, an amide synthase that releases the completed undecaketide as its macrocyclic lactam. Inactivation of rifF gives a rifamycin B nonproducing mutant that still accumulates a series of linear polyketides ranging from the tetra-to a decaketide, also detected in the wild type, demonstrating that the PKS operates in a processive manner. Disruptions of the rifD module 8 and rifE module 9 and module 10 genes also result in accumulation of such linear polyketides as a consequence of premature termination of polyketide assembly. Whereas the tetraketide carries an unmodified aromatic chromophore, the penta-through decaketides have undergone oxidative cyclization to the naphthoquinone, suggesting that this modification occurs during, not after, PKS assembly. The structure of one of the accumulated compounds together with 18 O experiments suggests that this oxidative cyclization produces an 8-hydroxy-7,8-dihydronaphthoquinone structure that, after the stage of proansamycin X, is dehydrogenated to an 8-hydroxynaphthoquinone.

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

Cited by 25 publications

References 24 publications

Intermediates of rifamycin polyketide synthase produced by an Amycolatopsis mediterranei mutant with inactivated rifF gene

Intermediates of rifamycin polyketide synthase produced by an Amycolatopsis mediterranei mutant with inactivated rifF gene

Stimulation of polyketide metabolism in Streptomyces fradiae by tylosin and its glycosylated precursors

Direct evidence that the rifamycin polyketide synthase assembles polyketide chains processively

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