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Borrelidin-producing polyketide synthase and its use

A spirovoxel and nucleotide sequence technology, which is applied in the field of materials for preparing polyketide compounds, can solve the problems of colon adenocarcinoma cells, such as the reduced ability to metastasize and form

Inactive Publication Date: 2006-02-08
BIOTICA TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Similarly, using the colon 38 spleen-liver model, treatment with subtoxic doses of this borrelidin derivative significantly reduced the metastasis-forming ability of mouse colon adenocarcinoma cells transplanted into the mouse spleen

Method used

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  • Borrelidin-producing polyketide synthase and its use
  • Borrelidin-producing polyketide synthase and its use
  • Borrelidin-producing polyketide synthase and its use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0222] Example 1: Fermentation of Streptomyces minor strains

[0223] The following methods can generally be used to grow Streptomyces parvum to produce borrelidin and / or borrelidin analogs:

[0224] Seed flasks containing NYG medium (30ml in a 250ml Erlenmeyer flask) were inoculated from the working stock (0.5ml). NYG medium contained (in deionized water): beef extract (0.3%), bactopeptone (0.5%), glucose (1%) and yeast extract (0.5%). After shaking for 2 days in a rotating incubator (2-inch eccentricity; 30° C.; 250 rpm), the resulting paste culture was used to inoculate PYDG production medium (30 ml in a 250 ml Erlenmeyer flask; 10% inoculum). PYDG medium contained per liter of deionized water: peptonized milk nutrients (1.5%), yeast autolysate (0.15%), dextrin (4.5%) and glucose (0.5%), adjusted to pH 7.0. After 5 days of shaking on a rotating incubator (after 2-inch eccentricity; 30°C; 250 rpm), cultures were harvested for analysis as described in Example 4, or for isol...

Embodiment 2

[0227] Embodiment 2: the cryopreservation of Streptomyces minor strain

[0228] working stock

[0229] A working stock of vegetative mycelium was prepared by mixing a 2-day old seed culture grown in NGY medium (0.5 ml) with cryopreservation solution (0.5 ml). The cryopreservation solution consisted of 20% glycerol and 10% lactose in deionized water.

[0230] Spore stock

[0231] Strains of Streptomyces minor were incubated on HA agar plates at 30°C. After 14 days, the resulting spores were harvested from individual plates and suspended in cryopreservation solution (1 ml). HA agar contained in deionized water: 0.4% yeast extract, 1% malt extract, 0.4% dextrose and 1.5% agar, adjusted to pH 7.3.

Embodiment 3

[0232] Example 3: Cloning of Borrelidin Biosynthesis Gene Cluster and Disruption of borA2 & borA3

[0233] Cosmid library generation

[0234] The Gigapack_III Gold Packaging Extract kit was used according to the manufacturer's manual (Stratagene). Cosmid libraries were constructed in the pWE15 cosmid vector. Chromosomal DNA was extracted from Streptomyces minor Tü4055 and treated with Sau3AI and cloned into pWE15 according to standard protocols (Kieser et al., 2000). A number of resulting E. coli transformants (3300) were picked and transferred to 96-well microtiter plates containing Luria broth (LB) medium (0.1 ml / well) and ampicillin (100 μg / ml). The resulting clones were replicated on Luria agar (LA) plates containing ampicillin (100 μg / ml). After overnight incubation at 37°C, colonies were transferred to nylon filters for in situ colony hybridization analysis according to published protocols (Sambrook et al., 1989).

[0235] library screening

[0236] Cosmid libraries...

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Abstract

The present invention relates to the biosynthesis of polyketides and derives from the cloning of nucleic acids encoding a polyketide synthase and other associated proteins involved in the synthesis of the polyketide borrelidin. Materials and methods including enzyme systems, nucleic acids, vectors and cells are provided for the preparation of polyketides including borrelidin and analogues and derivatives thereof. Novel polyketide molecules are also provided.

Description

field of invention [0001] The present invention relates to materials and methods for the preparation of polyketides. Enzyme systems, nucleic acids, vectors and cells for the preparation of polyketides, especially the polyketide macrolide borrelidin, are provided. Background of the invention [0002] Polyketides are natural products produced by various organisms, especially microorganisms. Polyketides have many important pharmaceutical, veterinary and agricultural uses. Polyketides include a large range of chemical structural space and have various related biological activities. Polyketides used in medicine include antibiotics, immunosuppressants, antineoplastic agents, other chemotherapeutic agents, and other compounds with a wide range of therapeutic and biological properties. The Gram-positive bacterium Streptomyces and its closely related genera are excellent producers of polyketides, and the genetics and biochemistry of polyketide biosynthesis in these organisms are r...

Claims

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Application Information

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IPC IPC(8): C12N15/52C07D313/00C12P17/08C12R1/465
CPCC12P17/08C07D313/00C12N15/52A61P17/06A61P19/02A61P31/04A61P33/06A61P35/00A61P9/10
Inventor J·A·萨拉斯C·门德斯C·奥拉诺C·桑切斯A·F·布拉尼亚B·威尔金森C·J·马丁S·莫斯P·F·利德雷M·奥利尼克
Owner BIOTICA TECH
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