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Construction method and application of genetically engineering strain capable of producing indigo pigment

A technology of genetically engineered strains and indigo pigments, applied in the field of food additive manufacturing, can solve problems such as difficult separation and purification, achieve efficient production and improve the effect of indigo pigment synthesis

Inactive Publication Date: 2016-07-27
BEIJING TECHNOLOGY AND BUSINESS UNIVERSITY
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AI Technical Summary

Problems solved by technology

One way is that under the catalysis of monooxygenase and isomerase, indole is oxidized to oxindole, and oxindole is polymerized into indigo molecules; the other way is under the action of dioxygenase and isomerase Under the action of catalysis, indole is oxidized into oxindole and 2-oxindole, oxindole polymerizes into indigo molecules, oxindole and 2-oxindole polymerize into indirubin molecules, and the final products are indigo and indigo Red mixture, difficult to separate and purify

Method used

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  • Construction method and application of genetically engineering strain capable of producing indigo pigment
  • Construction method and application of genetically engineering strain capable of producing indigo pigment
  • Construction method and application of genetically engineering strain capable of producing indigo pigment

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Embodiment Construction

[0024] 1. Cloning of styAB gene

[0025] 1.1 Extraction of PseudomonasputidaB4 Genomic DNA

[0026] The freshly activated Pseudomonasputida B4 strain was inoculated into LB liquid medium, placed in a shaker at 30°C, and shaken at 220rpm for overnight cultivation.

[0027] (1) Take 1 mL of bacterial liquid into a 1.5 mL centrifuge tube, centrifuge at 12,000 rpm for 1 min at room temperature, and discard the supernatant;

[0028] (2) Bacterial genomic DNA extraction kit (Tiangen Biochemical Technology (Beijing) Co., Ltd.) was used to extract genomic DNA.

[0029] 1.2 Cloning of styAB gene

[0030] Specific primers were designed, and the styAB gene fragment was amplified by PCR method using PseudomonasputidaB4 genomic DNA as a template.

[0031] styAB gene primer sequence:

[0032] Upstream primer: 5'-CGGGGTACCAGCAACCCACAACAAAAACAAG-3'

[0033] Downstream primer: 5'-CCCAAGCTTGTGATCGGCACAGAAAGG-3'

[0034] PCR reaction system

[0035]

[0036] KpnI and HindIII restrictio...

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Abstract

The invention relates to a recombinant genetically engineering strain Pseudomonas putida and a preparation method thereof. The engineering strain carries an expression vector with overexpressed endogenous styrene monooxygenase genes styAB. The preparation method of the engineering strain comprises the following steps: 1) endogenous styrene monooxygenase genes styAB fragments are amplified in a Pseudomonas putida genome with a PCR (polymerase chain reaction) method; 2) a specific restriction enzyme digestion site is designed and constructed onto a Pseudomonas plasmid vector pBBR1MCS-2; 3) a recombinant plasmid is transformed into Pseudomonas putida with an electroporation method. The recombinant genetically engineering strain Pseudomonas putida prepared with the method can remarkably increase the synthetic amount of the natural pigment indigo and can be applied to industries such as food, medicine, daily chemicals and the like.

Description

technical field [0001] The invention relates to a construction method and application of an indigo pigment-producing genetically engineered bacterium, which belongs to the technical field of food additive manufacturing. The prepared indigo pigment has the same chemical structure as plant indigo, and is a natural food pigment. The pigment product can be applied to food, Health products, daily chemical and pharmaceutical industries. Background technique [0002] Food coloring is an important food additive, widely used in food, medicine, cosmetics, printing and dyeing and other industrial fields. According to the principle of color technology, red, yellow, and blue are the basic three primary colors, and the three primary colors can be formulated according to different concentration combinations to produce various shades except white. The global annual demand for dyes is 800,000 tons, of which blue pigments exceed 80,000 tons, which are currently mainly produced through chemic...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/78C12N1/21C12N15/11C12R1/40
CPCC12N15/78C12N9/0071C12N15/11C12N2310/10C12N2800/108C12N2800/60C12Y114/14011
Inventor 王成涛尹胜郝帅程雷孙宝国
Owner BEIJING TECHNOLOGY AND BUSINESS UNIVERSITY
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