Rapamycin biosynthesis: elucidation of gene product function

Gregory, Matthew A.; Hong, Hui; Lill, Rachel E.; Gaisser, Sibylle; Petković, Hrvoje; Low, Lindsey; Sheehan, Lesley S.; Carletti, Isabelle; Ready, Sarah J.; Ward, Michael J.; Kaja, Andrew L.; Weston, Alison J.; Challis, Iain R.; Leadlay, Peter F.; Martin, Christine; Wilkinson, Barrie; Sheridan, Rose

doi:10.1039/b608813a

Cited by 48 publications

(51 citation statements)

References 23 publications

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“…BIOT-4010 was grown in a Braun 22 L bioreactor (×2) and subsequent processing was performed as described previously (51). The resulting material (13.0 g) was suspended in a methanol∶water mix (80∶20; 300 mL) and extracted twice with hexane (300 mL).…”

Section: Methodsmentioning

confidence: 99%

“…pGP9 is an E. coli-actinomycete shuttle plasmid shown to be effective for gene expression in S. hygroscopicus (52). Fermentation was performed in 50 mL Falcon tubes (8 mL working volume) or Braun 22 L Bioreactors (15 L working volume) as described previously (51). Feeding experiments were performed routinely in 50 mL Falcon tubes, with the required carboxylic acid feed being added after 24 h growth in production media.…”

Section: Methodsmentioning

confidence: 99%

“…Plasmid DNA was introduced into S. hygroscopicus by conjugal transfer as described previously (51). pGP9 is an E. coli-actinomycete shuttle plasmid shown to be effective for gene expression in S. hygroscopicus (52).…”

Section: Methodsmentioning

confidence: 99%

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Biosynthesis of the immunosuppressants FK506, FK520, and rapamycin involves a previously undescribed family of enzymes acting on chorismate

Andexer

Kendrew²,

Nur‐e‐Alam³

et al. 2011

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

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The macrocyclic polyketides FK506, FK520, and rapamycin are potent immunosuppressants that prevent T-cell proliferation through initial binding to the immunophilin FKBP12. Analogs of these molecules are of considerable interest as therapeutics in both metastatic and inflammatory disease. For these polyketides the starter unit for chain assembly is (4 R ,5 R )-4,5-dihydroxycyclohex-1-enecarboxylic acid derived from the shikimate pathway. We show here that the first committed step in its formation is hydrolysis of chorismate to form (4 R ,5 R )-4,5-dihydroxycyclohexa-1,5-dienecarboxylic acid. This chorismatase activity is encoded by fkbO in the FK506 and FK520 biosynthetic gene clusters, and by rapK in the rapamycin gene cluster of Streptomyces hygroscopicus . Purified recombinant FkbO (from FK520) efficiently catalyzed the chorismatase reaction in vitro, as judged by HPLC-MS and NMR analysis. Complementation using fkbO from either the FK506 or the FK520 gene cluster of a strain of S. hygroscopicus specifically deleted in rapK (BIOT-4010) restored rapamycin production, as did supplementation with (4 R ,5 R )-4,5-dihydroxycyclohexa-1,5-dienecarboxylic acid. Although BIOT-4010 produced no rapamycin, it did produce low levels of BC325, a rapamycin analog containing a 3-hydroxybenzoate starter unit. This led us to identify the rapK homolog hyg5 as encoding a chorismatase/3-hydroxybenzoate synthase. Similar enzymes in other bacteria include the product of the bra8 gene from the pathway to the terpenoid natural product brasilicardin. Expression of either hyg5 or bra8 in BIOT-4010 led to increased levels of BC325. Also, purified Hyg5 catalyzed the predicted conversion of chorismate into 3-hydroxybenzoate. FkbO, RapK, Hyg5, and Bra8 are thus founder members of a previously unrecognized family of enzymes acting on chorismate.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Biosynthesis of the immunosuppressants FK506, FK520, and rapamycin involves a previously undescribed family of enzymes acting on chorismate

Andexer

Kendrew²,

Nur‐e‐Alam³

et al. 2011

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

Self Cite

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“…Recently, the Sheridan group identified the functions of several gene products involved in post-PKS tailoring by biotransformation and gene complementation. 52 The predicted functions of the post-PKS tailoring enzymes corresponding to the conversion of prerapamycin into rapamycin are shown in Figure 3. RapJ and RapN encode cytochrome P450 monooxygenases that catalyze the specific oxidation step in C9 and C27, respectively, of the pre-rapamycin macrolide.…”

Section: Biological Activities Of Rapamycin and Its Analogsmentioning

confidence: 99%

“…The deduced gene products of rapM, rapQ and rapI are similar to S-adenosylmethionine-dependent O-methyltransferases, which carry out methylation at the hydroxyl groups of C16, C27 and C39, respectively, in the rapamycin molecule. 15,52 REGULATION OF RAPAMYCIN BIOSYNTHESIS Despite accumulated information on the rapamycin biosynthetic pathway, most of the nutritional control and regulatory factors remain unknown. Demain's group examined the effect of carbon 53 and nitrogen sources 54 on the biosynthesis of rapamycin in S. hygroscopicus.…”

Section: Biological Activities Of Rapamycin and Its Analogsmentioning

confidence: 99%

Biosynthesis of rapamycin and its regulation: past achievements and recent progress

et al. 2010

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Rapamycin and its analogs are clinically important macrolide compounds produced by Streptomyces hygroscopicus. They exhibit antifungal, immunosuppressive, antitumor, neuroprotective and antiaging activities. The core macrolactone ring of rapamycin is biosynthesized by hybrid type I modular polyketide synthase (PKS)/nonribosomal peptide synthetase systems primed with 4,5-dihydrocyclohex-1-ene-carboxylic acid. The linear polyketide chain is condensed with pipecolate by peptide synthetase, followed by cyclization to form the macrolide ring and modified by a series of post-PKS tailoring steps. The aim of this review was to outline past and recent advances in the biosynthesis and regulation of rapamycin, with an emphasis on the distinguished contributions of Professor Demain to the study of rapamycin. In addition, this article describes the biological activities as well as mechanism of action of rapamycin and its derivatives. Recent attempts to improve the productivity of rapamycin and generate diverse rapamycin analogs through mutasynthesis and mutagenesis are also introduced, along with some future perspectives.

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Natural Products as Cytotoxic Agents

Pullman,

León,

Cutler

2021

Burger's Medicinal Chemistry and Drug Discovery

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Nature has proved to be a wellspring of important antineoplastic agents. In recent years, there has been a resurgence in the study of natural products with an emphasis on the discovery of novel therapeutic agents. New technologies and approaches such as high‐throughput screening, parallel synthesis, and other programs has facilitated this increased interest in natural products research. The scientific literature indicates that natural sources not previously considered as bioactive have in fact, become potential valuable treasure troves of novel chemical structures. For example, marine products from diverse sources such as thermal vents, sponges, terrestrial thermal vents, glacial cryogenic organisms, and xerophytic and endophytic organisms now give rise to a vast assortment of microorganisms. Additionally, the study of the biosynthetic machineries and biochemical process in microorganism has enhanced the research of cryptic natural products, fueling the search for natural products in the treatment of cancer. It comes as no surprise that nature biosynthesizes chemical compounds that chemists have not considered even in their imaginations. Although this article provides a few of these examples, it is certain that more will be present in the literature as the intellectual property in these discoveries are protected.

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Rapamycin biosynthesis: elucidation of gene product function

Abstract: The function of gene products involved in the biosynthesis of the clinically important polyketide rapamycin were elucidated by biotransformation and gene complementation.

Cited by 48 publications

References 23 publications

Biosynthesis of the immunosuppressants FK506, FK520, and rapamycin involves a previously undescribed family of enzymes acting on chorismate

Biosynthesis of the immunosuppressants FK506, FK520, and rapamycin involves a previously undescribed family of enzymes acting on chorismate

Biosynthesis of rapamycin and its regulation: past achievements and recent progress

Natural Products as Cytotoxic Agents

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