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Lycopene cyclase gene in sphingomonas sp. and use thereof

A technology of lycopene and less active sheath, which is applied in the fields of application, genetic engineering, plant genetic improvement, etc.

Inactive Publication Date: 2013-04-10
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there is no gene sequence of sphingomonas paucimobilis lycopene cyclase and the use of gene knockout technology to knock out the lycopene cyclase gene of sphingomonas paucimobilis to co-produce tomato by gellan gum fermentation red pigment report

Method used

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  • Lycopene cyclase gene in sphingomonas sp. and use thereof
  • Lycopene cyclase gene in sphingomonas sp. and use thereof
  • Lycopene cyclase gene in sphingomonas sp. and use thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0020] Embodiment 1: Obtaining of DNA sequence of crtY gene of Sphingomonas paucimobilis

[0021] According to the partial sequence of Sphingomonas paucimobilis ATCC 31461 crtY (NCBI accession number: HQ202920), specific primers for upstream genes were designed, and the complete sequence including crtY gene and the sequence with NCBI accession number HQ202920 were obtained by SiteFinding-PCR technology Part of the flanking sequence required for gene knockout was obtained by splicing. The sequences of the upstream gene-specific primers, SiteFinder, and SFP primers used are as follows:

[0022] Upstream sequence-specific primers:

[0023] UP1: 5′-GCTGTAATAGGTGTCCTCGACGA-3′

[0024] UP2: 5′-TGAACGGCAGGCAATAGACGAAG-3′

[0025] SiteFinder:

[0026] SiteFl:

[0027] 5′-CACGACACGCTACTCAACACACCACCACGCACAGCGTCCTCAANNNNNNCATGG-3′

[0028] SiteF2:

[0029] 5′-CACGACACGCTACTCAACACACCACCACGCACAGCGTCCTCAANNNNNNCATGC-3′

[0030] SiteF3:

[0031] 5′-CACGACACGCTACTCAACACACCACCACGCACAG...

Embodiment 2

[0038] Example 2: Construction of a recombinant strain of Sphingomonas paucimobilis deficient in lycopene cyclase

[0039] 1. Construction of gene knockout vector pLO3-△Y

[0040] Primers were designed based on the obtained DNA sequences. Genomic DNA (using the AxyPrep Bacterial Genomic DNA Miniprep Kit) was extracted from Sphingonmonas paucimobilis ATCC 31461 (purchased from ATCC) as a PCR template. Y1 and Y2 are primers Taq enzyme (Takara) to amplify upstream homologous sequences. The PCR reaction program is 95°C pre-denaturation for 5 minutes to enter the cycle process; Extend at 72°C for 10 min. Y3 and Y4 are the primers Taq enzyme (Takara) to amplify downstream homologous sequences. The PCR reaction program is: 95°C pre-denaturation for 5 minutes to enter the cycle process; 94°C denaturation for 30 sec, 63°C annealing for 30 sec, 72°C extension for 40 s, 30 cycles, Finally, it was extended at 72°C for 10 min. The above primer sequences are as follows:

[0041] Y1: 5′-A...

Embodiment 3

[0064] Example 3: Determination of Gel Production Ability of Sphingomonas paucimobilis Strains Deleted in crtY Gene

[0065] 1. Wild-type strain (Sphingomonas paucimobilis ATCC 31461) and crtY gene-deleted Sphingomonas paucimobilis (△Y1~△Y7, where △Y1~△Y4 are derived from Sphingomonas paucimobilis ATCC 31461, And △Y5~△Y7 are derived from Sphingomonas paucimobilis DSM 6314) inoculated on the slant of YM medium, cultured at 30°C for 72h;

[0066] 2. First-level seed culture: respectively insert slant seeds into 50ml first-level seed medium (filled in a 250ml Erlenmeyer flask), and culture at 30°C and 200rpm for 24 hours, which is the first-level seed liquid;

[0067] 3. Secondary seed culture: Inoculate the primary seed solution with 5% volume ratio into 100ml secondary seed culture medium (filled in a 500mL triangular flask), shake and cultivate at 30°C and 200rpm for 12h, which is the secondary seed solution. seed liquid;

[0068] 4. Fermentation: Put the secondary seed liqu...

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Abstract

The invention provides the DNA sequence of a lycopene cyclase gene (crtY) in sphingomonas sp., a recombinant strain lacking lycopene cyclase and use thereof. The nucleotide sequence of the crtY gene is represented by SEQ ID NO.1. The DNA sequence of the lycopene cyclase gene (crtY) in sphingomonas sp., which is provided by the invention, lays a foundation for the genetic modification of a sphingomonas sp. carotenoid biological synthesis means. In addition, compared with a wild strain, the sphingomonas sp. recombinant strain lacking the lycopene cyclase gene, which is constructed by a gene knockout method, has the advantages that: the gellengum yield is basically unchanged; and lycopene can be produced in the production of gellengum by fermentation. And the strain can be used for further gene knockout or metabolic engineering modification.

Description

(1) Technical field [0001] The invention relates to the lycopene cyclase gene (crtY) of sphingomonas paucimobilis, the recombinant sphingomonas paucimobilis and the application thereof. (2) Background technology [0002] Gellan gum is linked and polymerized by β-1,3-D-glucose, β-1,4-D-glucuronic acid, β-1,3-D-glucose, α-1,4-L-rhamnose Composition of microbial exopolysaccharides. Compared with similar products, it has many superior properties, such as less dosage; adjustable freezing point, melting point, etc.; acid and alkali resistance, high thermal stability; good water solubility, taste, moisture retention, film forming, Sustained release, etc. In recent years, gellan gum has been widely used in food, medicine, chemical and other industrial fields as a new type of emulsifier, suspending agent, thickener, stabilizer, gelling agent, slow-release agent, and film-forming material. In addition, gellan gum can be used in combination with other food gums, so that the food can...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/60C12N1/20C12P19/04C12P23/00C12R1/01
Inventor 吴雪昌朱亮李欧
Owner ZHEJIANG UNIV
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