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Cladosporium fulvum genetically engineered bacterium and application of cladosporium fulvum genetically engineered bacterium in production of hypericin and sperm

A technology of genetically engineered bacteria and xanthosporium, applied in genetic engineering, microorganism-based methods, applications, etc., to achieve the effect of fermentation production and efficient preparation

Pending Publication Date: 2022-05-06
NANJING UNIVERSITY OF TRADITIONAL CHINESE MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But so far, there is no literature report to produce hypericin (hypericin) and skyrin (skyrin) by microbial fermentation.

Method used

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  • Cladosporium fulvum genetically engineered bacterium and application of cladosporium fulvum genetically engineered bacterium in production of hypericin and sperm
  • Cladosporium fulvum genetically engineered bacterium and application of cladosporium fulvum genetically engineered bacterium in production of hypericin and sperm
  • Cladosporium fulvum genetically engineered bacterium and application of cladosporium fulvum genetically engineered bacterium in production of hypericin and sperm

Examples

Experimental program
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Effect test

Embodiment 1

[0025] Example 1: Construction of Xanthosporium ΔclaC-rugG genetically engineered bacteria

[0026] This embodiment illustrates the construction method of Xanthosporium ΔclaC-rugG genetically engineered bacteria, including the following steps:

[0027] (1) Using the pCAMBIA1391Xa plasmid (disclosed in P.Hajdukiewicz, Z.Svab, P.Maliga, PlantMol.Biol.1994,25,989–994) and the resistance gene hygromycin as a template, design primers hph-F / hph-R Amplify the hygromycin fragment, recombine it with the pCAMBIA1391Xa plasmid in vitro to construct the pCAMBIA1391Xa-hph plasmid containing the hygromycin resistance gene, and design primers pCAMBIA1391Xa-F / pCAMBIA1391Xa-R to amplify the pCAMBIA1391Xa-containing plasmid from the pCAMBIA1391Xa-hph plasmid The hygromycin resistance gene fragment of the homology arm.

[0028] (2) Using the genome of Cladosporium fulvum (Cladosporium fulvum) ACCC 37291 (purchased from China Agricultural Microorganism Culture Collection Center (ACCC)) as a temp...

Embodiment 2

[0057] Example 2 Fermentation production of hypericin and Tianjing by the gene engineering strain ΔclaC-rugG of Xanthosporium sp.

[0058] The Xanthosporium genetically engineered strain ΔclaC-rugG constructed in Example 1 was cultivated in a 3L fermenter at 22°C with PDB medium, the stirring speed was 100r / min, and the ventilation rate was 1vvm. After cultivating for 4 days, maltose was added to Quality final concentration is 4% in the fermented liquid, continues to ferment to 8 days ( image 3 ). It was determined that the yield of protohypericin reached 35.2 mg / L, and that of skyrin reached 305 mg / L. The fermentation broth was filtered, and the supernatant and mycelia were extracted three times with ethyl acetate respectively. After the extract was irradiated with visible light for 24 hours, the original hypericin was spontaneously transformed into hypericin under the action of visible light, and concentrated by rotary steaming at 35°C. Measured by high performance liquid...

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Abstract

The invention provides a cladosporium fulvum genetically engineered bacterium and application of the cladosporium fulvum genetically engineered bacterium in production of hypericin and sperm, and belongs to the technical field of biological pharmacy. The cladosporium fulvum genetically engineered bacterium is obtained by replacing a trihydroxy naphthol reductase gene in cladosporium fulvum with a cytochrome P450 gene. The invention also provides a method for producing the hypericin and the eriosperm by adopting the cladosporium fulvum genetically engineered bacterium, and the method comprises the step of carrying out fermentation culture by adopting the cladosporium fulvum genetically engineered bacterium to obtain the hypericin and the eriosperm. The cladosporium fulvum genetically engineered bacterium disclosed by the invention can be used for preparing hypericin and lysozyme through a fermentation method, and is efficient and environment-friendly.

Description

technical field [0001] The invention belongs to the technical field of biopharmaceuticals, and in particular relates to a Xanthosporium genetically engineered bacterium and its application for producing hypericin and Tianjing. Background technique [0002] Diseases such as tumors, immunity and infection have a high incidence in our country. At present, there is still a lack of ideal clinical treatment drugs, which brings a heavy burden to families and society, and seriously affects the health of the people and the construction of a healthy China. Hypericin has significant antitumor and antiviral activities, making it a potential clinical drug source molecule. [0003] Hypericin is mainly a dianthrone compound isolated from the plant Hypericum perforatum of Garciniaceae, and its molecular formula is C 30 h 16 o 8 , the molecular weight is 504.44. However, the low content of hypericin in plants (0.01%-0.47%) leads to insufficient extraction supply (J.Chinese Pharm.Sci.2012...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/15C12N15/80C12N15/90C12N15/53C12P7/26C12R1/645
CPCC12N15/80C12N15/902C12N9/0006C12N9/0081C12P7/26C12Y114/15006
Inventor 吴薛明关秋艳韩云宾谭仁祥
Owner NANJING UNIVERSITY OF TRADITIONAL CHINESE MEDICINE
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