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Culture medium for preparing epothilone through fermenting myxococcus Xanthus for heterologously expressing epothilone gene

A technology of Myxococcus aureus and Epothilone, which is applied in the directions of fermentation, microorganism-based methods, biochemical equipment and methods, etc., to achieve the effect of improving the level of heterologous expression, easy preparation and good water solubility

Active Publication Date: 2015-12-30
SHANDONG UNIV
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  • Abstract
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  • Claims
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Problems solved by technology

At present, there are few relevant literature reports on the medium composition and optimization of epothilones fermented by Myxococcus xanthus. Therefore, how to obtain a yellow viscose that can effectively improve the fermentation yield and efficiency and is suitable for heterologous expression of epothilones The medium for producing epothilone by cocci fermentation has become an urgent problem to be solved

Method used

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  • Culture medium for preparing epothilone through fermenting myxococcus Xanthus for heterologously expressing epothilone gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] (1) Selection of fermentation strains: Myxococcus xanthus DZ strain among the myxobacteria strains.

[0045] (2) Activation of strains: the above strains were inoculated on a solid plate medium, cultured at 30° C. for 3 days, then inoculated into a liquid medium, and cultured at 30° C. at 200 rpm for 2 days. Then, collect the bacterial solution for future use.

[0046] in,

[0047] The composition of the solid plate culture medium is: casein peptone 1%, MgSO 4 ·7H 2 O8mM, Tris HCl (pH7.6) 10mM, KH 2 PO 4 / K 2 HPO 4 (pH7.6) 1 mM, pH7.6. Add 1.5% agar to the solid medium, and sterilize with damp heat at 121°C for 30 minutes.

[0048] The composition of the liquid medium is: casein peptone 1%, yeast extract 0.5%, MgSO 4 ·7H 2 O4mM, MOPS10mM; pH7.6. Sterilize with damp heat at 121°C for 30min.

[0049] (3) Fermentation culture method: Take 3 mL of the bacterial liquid prepared in step (2) and inoculate them into the counter liquid medium (OCM medium), and ferment...

Embodiment 2

[0063] (1) Selection of fermentation strains: Myxococcus xanthus DZ strain among the myxobacteria strains.

[0064] (2) Activation of strains: the above strains were inoculated on a solid plate medium, cultured at 30° C. for 3 days, then inoculated into a liquid medium, and cultured at 30° C. at 200 rpm for 2 days. Then, collect the bacterial solution for future use.

[0065] in,

[0066] The composition of the solid plate culture medium is: casein peptone 1%, MgSO 4 ·7H 2 O8mM, Tris HCl (pH7.6) 10mM, KH 2 PO 4 / K 2 HPO 4 (pH7.6) 1 mM, pH7.6. Add 1.5% agar to the solid medium, and sterilize with damp heat at 121°C for 30 minutes.

[0067] The composition of the liquid medium is: casein peptone 1%, yeast extract 0.5%, MgSO 4 ·7H 2 O4mM, MOPS10mM; pH7.6. Sterilize with damp heat at 121°C for 30min.

[0068] (3) Fermentation culture method: Take 3 mL of the bacterial solution prepared in step (2) and inoculate them into the counter liquid medium (OCM medium), and ferme...

Embodiment 3

[0082] (1) Selection of fermentation strains: Myxococcus xanthus DZ strain among the myxobacteria strains.

[0083](2) Activation of strains: the above strains were inoculated on a solid plate medium, cultured at 30° C. for 3 days, then inoculated into a liquid medium, and cultured at 30° C. at 200 rpm for 2 days. Then, collect the bacterial solution for future use.

[0084] in,

[0085] The composition of the solid plate culture medium is: casein peptone 1%, MgSO 4 ·7H 2 O8mM, Tris HCl (pH7.6) 10mM, KH 2 PO 4 / K 2 HPO 4 (pH7.6) 1 mM, pH7.6. Add 1.5% agar to the solid medium, and sterilize with damp heat at 121°C for 30 minutes.

[0086] The composition of the liquid medium is: casein peptone 1%, yeast extract 0.5%, MgSO 4 ·7H 2 O4mM, MOPS10mM; pH7.6. Sterilize with damp heat at 121°C for 30min.

[0087] (3) Fermentation culture method: Take 3 mL of the bacterial solution prepared in step (2) and inoculate them into the counter liquid medium (OCM medium), and fermen...

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Abstract

The invention discloses a culture medium for preparing epothilone through fermenting myxococcus Xanthus for heterologously expressing an epothilone gene. The culture medium comprises the following components with the contents: 10g / L of casein peptone, 1.0g / L of MgSO4.7H2O, 2.07g / L of 3-(N-morpholinyl)propanesulfonic acid sodium salt, 5-15ml / L of cis-9-octadecenoic acid methyl ester, 0.8-1.6g / L of threonine, 0.5-1.2g / L of methionine, 1.0-3.0g / L of potassium propionate, 1mL / L of a microelement solution and 20mL / L of macroporous resin XAD-16. The culture medium has the remarkable characteristic that cis-9-octadecenoic acid methyl ester, L-threonine, methionine and potassium propionate are added. Experiments prove that the fermentation level and efficiency of an epothilone compound are remarkably improved through combination and optimization, and the culture medium has a favorable application prospect in production of the epothilone through industrial fermentation of myxococcus Xanthus for heterologously expressing the epothilone gene.

Description

technical field [0001] The invention provides a special fermentation medium, in particular to a medium for producing epothilones by fermenting Myxococcus xanthus expressing epothilones genes heterologously, and belongs to the technical field of fermentation mediums for preparing antitumor drugs. Background technique [0002] Epothilones are a class of antitumor drugs with great application prospects. Epothilone compounds belong to polyketide macrolide compounds, which have a strong stabilizing effect on polymeric microtubules. The mechanism of action is similar to paclitaxel, and it is superior to paclitaxel in terms of water solubility, cytotoxicity and drug resistance. , is considered to be the replacement product of paclitaxel, and is a better anticancer drug. [0003] At present, the preparation methods of epothilones mainly include chemical synthesis and biosynthesis. The chemical total synthesis method has many steps and low yield, and has no cost advantage compared ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P17/16C12R1/01
Inventor 胡玮李越中郑连帅
Owner SHANDONG UNIV
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