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Phytoene desaturase gene of sphingomonas sp. and application thereof

A technology of phytoene and monas, which is applied in the fields of application, genetic engineering, plant genetic improvement, etc.

Inactive Publication Date: 2013-05-01
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, there are no reports on the sequence of the phytoene dehydrogenase gene of S. paucimobilis and the knockout of the phytoene dehydrogenase gene of S. paucimobilis by gene knockout technology.

Method used

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  • Phytoene desaturase gene of sphingomonas sp. and application thereof
  • Phytoene desaturase gene of sphingomonas sp. and application thereof
  • Phytoene desaturase gene of sphingomonas sp. and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Embodiment 1: Obtaining of DNA sequence of Sphingomonas paucimobilis crtI gene

[0023] The phytoene dehydrogenase protein sequences of 4 species of Erythrobacterlitoralis HTCC2594, Erythrobacter longus, Erythrobacter sp.NAP1, and Erythrobacter sp.SD-21 from the order sphingomonadales belonging to Sphingomonas paucimobilis (data from NCBI protein Database) was compared with ClustalW using the software MEGA4, and it was found that there were protein conservative sequences TFDA(G)GPT, VGAGTHPGAG. According to the above conserved sequence and codon preference query (data from the Japanese kazusa Codon Usage Database), the following degenerate primers were designed to amplify the partial sequence of the crtI gene.

[0024] Upstream primers:

[0025] crtIsense: 5′-ACSTTYGAYGCNGGBCCSACS-3′

[0026] Downstream primers:

[0027] crtIanti: 5′-VCCNGCVCCSGGRTGSGTVCCNGGCVCCNAC-3′

[0028] Genomic DNA extracted from Sphmgomonas paucimobilis ATCC31461 (purchased from ATCC) (using...

Embodiment 2

[0050] Example 2: Construction of a recombinant strain of Sphingomonas paucimobilis lacking in phytoene dehydrogenase

[0051] 1. Construction of gene knockout vector pLO3-ΔI

[0052] Primers were designed based on the obtained DNA sequences. Genomic DNA (using the AxyPrep Bacterial Genomic DNA Miniprep Kit) was extracted from Sphingomonas paucimobilis ATCC31461 (purchased from ATCC) as a template for PCR. I1 and I2 are primers for amplifying the upstream sequence of the crtI gene. The PCR reaction program is: pre-denaturation at 95°C for 5 minutes to enter the cycle process; denaturation at 94°C for 30 sec, annealing at 63°C for 30 sec, extension at 72°C for 1 min, 30 cycles, and finally extension at 72°C 10min. I3 and I4 are primers for amplifying the downstream sequence of the crtI gene. The PCR reaction program is: pre-denaturation at 95°C for 5 minutes to enter the cycle process; denaturation at 94°C for 30 sec, annealing at 64°C for 30 sec, extension at 72°C for 1 min,...

Embodiment 3

[0076] Example 3: Determination of Gel Production Ability of Sphingomonas paucimobilis Strains Deleted in the crtI Gene

[0077] 1. Wild-type strain (Sphingomonas paucimobilis ATCC 31461) and crtI gene-deleted Sphingomonas paucimobilis (ΔI1~ΔI7) were inoculated on the slant of YM medium, and cultured at 30°C for 72 hours;

[0078] 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;

[0079] 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;

[0080] 4. Fermentation: Put the secondary seed liquid into 100mL grade fermentation medium (filled in a 500mL Erlenmeyer flask) with an inoculation amount of 5% volume ...

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Abstract

The invention provides a DNA sequence of a phytoene desaturase gene of sphingomonas sp., a recombinant strain with phytoene desaturase lost and application thereof. The phytoene desaturase gene (crtI) of the sphingomonas sp. has a nucleotide sequence shown by SEQ ID No.1. The DNA sequence of the phytoene desaturase gene (crtI) of the sphingomonas sp. provided by the invention lays a foundation for the genetic transformation of a biological synthesis manner of sphingomonas sp. carotene. Moreover, compared with the wild strains, the sphingomonas sp. recombinant strain with the phytoene desaturase lost, which is constructed by a gene knockout method, has gellan glue yield unchanged, generates no uranidin, reduces the amount of ethanol or isopropanol which is used during gellan glue extraction and therefore reduces the production cost of the gellan glue; meanwhile, the strain can also be applied to the further gene knockout or metabolic engineering transformation.

Description

(1) Technical field [0001] The invention relates to the phytoene dehydrogenase gene (crtI) of Sphingomonas paucimobilis and its application. (2) Background technology [0002] Gellan gum is a new type of microbial exopolysaccharide, which is composed of β-1,3-D-glucose, β-1,4-D-glucuronic acid and α-1,4-L-rhamnose in molar ratio 2:1:1 composition of polymers. Compared with similar products, it has many superior properties, such as less dosage; adjustable freezing point, melting point, elasticity, hardness, etc.; the formed gel has high transparency and strength; good taste performance and high thermal stability. Due to its good properties, gellan gum is widely used in food and pharmaceutical industries as emulsifier, suspending agent, thickener, stabilizer, gelling agent, sustained release agent, film-forming material, etc. In addition, gellan gum can also be compounded with other colloids to give the product a unique taste and flavor, and has broad application prospects. ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/53C12N1/20C12P19/04C12R1/01
Inventor 吴雪昌朱亮李欧
Owner ZHEJIANG UNIV
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