Genes for the biosynthesis of epothilones

A biosynthetic, 0.5mnapo4ph7.0 technology, used in plant genetic improvement, genetic engineering, biochemical equipment and methods, etc., can solve the problems of complex genes and unisolated

Inactive Publication Date: 2008-04-16
NOVARTIS AG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the gene encoding the polypeptide responsible for epothilone biosynthesis has not been isolated so far
Moreover, the epothilone-producing strain, So ce90, also produces at least one other polyketide spirangien, possibly complicating the isolation of genes specifically responsible for epothilone biosynthesis

Method used

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  • Genes for the biosynthesis of epothilones
  • Genes for the biosynthesis of epothilones
  • Genes for the biosynthesis of epothilones

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0114] Example 1: Cultivation of S. cellulosus strains producing epothilone

[0115] Cellulose bacteria strain 90 (DSM 6773, Deutsche Sammlung vonMikroorganismen und Zellkulturen, Braunschweig) in SolE medium (0.35% glucose, 0.05% tryptone, 0.15% MgSO 4 ·7H 2 O, 0.05% ammonium sulfate, 0.1% CaCl 2 , 0.006%K 2 HPO 4 , 0.01% sodium dithionite, 0.0008% Fe-EDTA, 1.2% HEPES, 3.5% [vol / vo / ] sterilized supernatant of a culture of S. cellulosus in stationary phase) pH adjusted to 7.4 on an agar plate Streak and incubate at 30°C. Pick from 1cm 2 The cells were inoculated into 5ml G51t liquid medium (0.2% glucose, 0.5% starch, 0.2% tryptone, 0.1% probion S, 0.05% CaCl 2 2H 2 O, 0.05% MgSO 4 ·7H 2 O, 1.2% HEPES, pH adjusted to 7.4), and cultured at 30° C. with shaking at 225 rpm. After 4 days, the culture was transferred to 50 ml G51t and grown for 5 days as described above. This culture was used to inoculate 500 ml of G51t and cultured for 6 days as described above. The cult...

Embodiment 2

[0116] Example 2: Construction of Bacterial Artificial Chromosome Library

[0117] To construct the Bac library, S. cellulosus cells cultured as described in Example 1 were embedded in agarose blocks, lysed, and the released genomic DNA was partially digested with the restriction enzyme HindIII. Digested DNA was separated by pulse electrophoresis on an agarose gel. Large fragments of DNA (approximately 90-150 kb) were isolated from agarose gels and ligated into the vector pBelobacII. pBelobacII contains the gene encoding chloramphenicol resistance, a multiple cloning site within the lacZ gene providing blue-white selection on appropriate media, and genes required for replication and maintenance of one or two copies of the plasmid per cell. Using conventional electroporation techniques, the ligation mixture was transformed into E. coli DH10B electrocompetent cells. Chloramphenicol-resistant recombinant (white spot, lacZ mutation) colonies were transferred to positively charge...

Embodiment 3

[0118] Example 3: Screening of related sequences of the first class of polyketide synthases in the Bac library of S. cellulosus 90

[0119] Bac library filters were probed using a conventional Southern hybridization procedure. The probes used encode the beta-ketoacyl synthase domains of the first and second modules of the rifamycin polyketide synthase (Schupp et al., FEMS Microbiology Letters 159:210-207 (1998)). Probe DNA was generated by PCR using primers flanking each ketosynthase domain using plasmid pNE95 (pNE95 is cosmid 2 described in Schupp et al. (1998)) as a template. 25 ng of PCR-amplified DNA was isolated from a 0.5% agarose gel and labeled with a random primer kit (Gibco-BRL, Methesda, MD, USA) according to the supplier's instructions. 32 P-dCTP labeling. Hybridization was performed at 65°C for 36 hours, and the membrane was washed under highly stringent conditions (0.1xSSC and 0.5% SDS at 65°C for 20 minutes three times). The labeled spots were exposed to a phos...

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Abstract

Nucleic acid molecules are isolated from Sorangium cellulosum that encode polypeptides necessary for the biosynthesis of epothilone. Disclosed are methods for the production of epothilone in recombinant hosts transformed with the genes of the invention. In this manner, epothilone can be produced in quantities large enough to enable their purification and use in pharmaceutical formulations such as those for the treatment of cancer.

Description

field of invention [0001] The present invention generally relates to polyketides and the genes used to synthesize them. In particular, the present invention relates to the isolation and identification of novel polyketide synthase and nonribosomal peptide synthase genes essential for the biosynthesis of epothilone A and B from Sorangium cellulosum. Background of the invention [0002] Polyketides are compounds synthesized from two-carbon structural units, the β-carbon of which always carries a ketone group, hence the name polyketides. These compounds include many important antibiotics, immunosuppressants, cancer chemotherapeutics, and other compounds with a wide range of biological properties. The enormous structural diversity arises from the different lengths of the polyketide chains, the different side chains introduced (either as part of the two-carbon building block or after formation of the polyketide backbone), and the stereochemistry of these groups. Keto groups can ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/52C12N15/62C12N15/63C12P21/02C12P9/00C12N15/31C07K14/195
Inventor T·斯彻普J·M·利根I·莫尔纳R·泽克尔J·戈拉彻D·西尔
Owner NOVARTIS AG
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