Primer Unit Specificity in Rifamycin Biosynthesis Principally Resides in the Later Stages of the Biosynthetic Pathway

Hunziker, Daniel; Yu, Tianyi; Hutchinson, C. Richard; Floss, and Heinz G.; Khosla, Chaitan

doi:10.1021/ja9736368

Cited by 42 publications

(41 citation statements)

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“…Feeding of AHBA to this mutant restored full rifamycin B production, but feeding of two analogs gave copious amounts of the corresponding tetraketides ( Figure 9). This result, confirmed subsequently by in vitro studies (Admiraal et al, 2001), showed that the loading module of the rif PKS is rather promiscuous, but that a down-stream step, apparently after the third chain elongation, discriminates against the structurally altered intermediates (Hunziker et al, 1998). An explanation for the latter phenomenon was revealed when rifF, the gene encoding the downloading enzyme, was inactivated.…”

Section: Halogenationsmentioning

confidence: 65%

“…The rifamycin biosynthetic gene cluster from Amycolatopsis mediterranei and the pathway of rifamycin B biosynthesis (August et al, 1998) The ansamitocin biosynthetic gene cluster from Actinosynnema pretiosum and the structure and biosynthesis of ansamitocin P-3 . Production of tetraketides upon feeding of AHBA analogs to an AHBA(−) mutant of the rifamycin producer, Amycolatopsis mediterranei (Hunziker et al, 1998). Ketides isolated from mutants of the rifamycin producer, Amycolatopsis mediterranei, carrying an inactivated rifF gene (Yu et al, 1999;Stratmann et al, 1999).…”

Section: Halogenationsmentioning

confidence: 99%

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Combinatorial biosynthesis—Potential and problems

Floss

2006

Journal of Biotechnology

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Because of their ecological functions, natural products have been optimized in evolution for interaction with biological systems and receptors. However, they have not necessarily been optimized for other desirable drug properties and thus can often be improved by structural modification. Using examples from the literature, this paper reviews the opportunities for increasing structural diversity among natural products by combinatorial biosynthesis, i.e., the genetic manipulation of biosynthetic pathways. It distinguishes between combinatorial biosynthesis in a narrower sense to generate libraries of modified structures, and metabolic engineering for the targeted formation of specific structural analogs. Some of the problems and limitations encountered with these approaches are also discussed. Work from the author's laboratory on ansamycin antibiotics is presented which illustrates some of the opportunities and limitations. KeywordsCombinatorial biosynthesis; metabolic engineering; natural products; rifamycin; maytansinoids; polyketides Combinatorial biosynthesis can be defined as the application of genetic engineering to modify biosynthetic pathways to natural products in order to produce new and altered structures using nature's biosynthetic machinery. The feasibility of this approach was first demonstrated in work by David Hopwood and colleagues (Hopwood et al., 1985). Following the cloning of the biosynthetic genes for the antibiotic actinorhodin from Streptomyces coelicolor (Malpartida & Hopwood, 1984), Hopwood and coworkers cloned some or all of these genes into the producers of the antibiotics medermycin (Takano et al., 1976) and dihydrogranaticin (Corbaz et al., 1957), respectively. The transformant of the medermycin producer, Streptomyces sp. AM-7161 produced, in addition to medermycin, large amounts of a new compound, mederrhodin A, which carried an additional hydroxy group characteristic of actinorhodin ( Figure 1). The transformant of the dihydrogranaticin producer, Streptomyces violaceoruber Tü 22 produced a new compound, dihydrogranatirhodin, which has the actinorhodin configuration at one and the dihydrogranaticin configuration at the other stereocenter of the isochromane quinone system. This work represents the first, albeit modest, implementation of the concept of combinatorial biosynthesis of natural products. Since then, a large number of examples encompassing a wide range of natural product classes have appeared in the literature, and this approach has been accepted as a useful tool to increase the chemical diversity of natural products (Staunton and Wilkinson, 2001;Walsh, 2002;Rix et al., 2002;Reeves, 2003). Following the author's primary interests, this article focuses on the application of the combinatorial biosynthesis approach to microbial natural products and within these to Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript ...

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Section: Halogenationsmentioning

confidence: 65%

Section: Halogenationsmentioning

confidence: 99%

Combinatorial biosynthesis—Potential and problems

Floss

2006

Journal of Biotechnology

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114

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“…Thus, the enzyme(s) catalyzing it either must be part of the PKS or must dock to the PKS, and module 4 of RifB may only chain-extend a (dihydro)naphthoquinoid, i.e., cyclized tetraketide. Downstream recognition of modifications in the starter unit had been demonstrated earlier by the observation that 3-hydroxy-and 3,5-dihydroxybenzoic acid were used as starter units but resulted only in the formation of the desamino or 3-hydroxy analogs of 3, respectively (32). The cumulative evidence points to module 4 as the site of this discrimination.…”

Section: Discussionmentioning

confidence: 91%

Direct evidence that the rifamycin polyketide synthase assembles polyketide chains processively

Shen²,

Doi-Katayama³

et al. 1999

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

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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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“…It seems that the transition from module 3 to module 4 is a critical step during the rifamycin B synthesis. This is strengthened by results from Hunziker et al (1998), who showed that feeding of altered starter units, with a missing amino group at C-3 or an additional missing hydroxyl group at C-5, to a mutant in AHBA synthesis results in a termination of the rifamycin synthesis after module 3. In those feeding experiments, the corresponding derivatives of the well-known tetraketide shunt product P8\1-OG were detected (Hunziker et al, 1998).…”

Section: Discussionmentioning

confidence: 94%

“…This is strengthened by results from Hunziker et al (1998), who showed that feeding of altered starter units, with a missing amino group at C-3 or an additional missing hydroxyl group at C-5, to a mutant in AHBA synthesis results in a termination of the rifamycin synthesis after module 3. In those feeding experiments, the corresponding derivatives of the well-known tetraketide shunt product P8\1-OG were detected (Hunziker et al, 1998). Different rifamycin molecules have been postulated as the earliest macrocyclic intermediates of rifamycin biosynthesis : proansamycin A or B deduced from the early intermediate protorifamycin I and structure comparison with streptovaricin precursors (Ghisalba et al, 1978(Ghisalba et al, , 1979 and proansamycin X, based on hypothetical intermediates of the rifamycin PKS gene cluster (August et al, 1998 ;Tang et al, 1998).…”

Section: Discussionmentioning

confidence: 94%

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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Primer Unit Specificity in Rifamycin Biosynthesis Principally Resides in the Later Stages of the Biosynthetic Pathway

Cited by 42 publications

References 10 publications

Combinatorial biosynthesis—Potential and problems

Combinatorial biosynthesis—Potential and problems

Direct evidence that the rifamycin polyketide synthase assembles polyketide chains processively

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

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