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crtz gene and crtg gene of Sphingomonas paucimobilis and their application

A sphingomonas, less motile technology, applied in application, genetic engineering, plant genetic improvement and other directions

Inactive Publication Date: 2011-12-21
ZHEJIANG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, there is no sequence of β-carotene hydroxylase gene and 2,2′-β hydroxylase gene of Sphingomonas paucimobilis and knockout of this gene in Sphingomonas paucimobilis. Report of two genes leading to the co-production of carotenoids required for gellan gum fermentation

Method used

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  • crtz gene and crtg gene of Sphingomonas paucimobilis and their application
  • crtz gene and crtg gene of Sphingomonas paucimobilis and their application
  • crtz gene and crtg gene of Sphingomonas paucimobilis and their application

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Embodiment 1

[0028] Embodiment 1: Obtaining of DNA sequence of Sphingomonas paucimobilis crtZ gene

[0029]Eight species of Erythrobacter gaetbuli, Erythrobacter litoralis HTCC 2594, Erythrobacter sp.NAP1, Brevundimonas sp.SD 212, Brevundimonas bacteroides, Brevundimonas vesicularis, Aurantimonas manganoxydans SI85-2trohobact from Sphingomonas paucimobilis belonging to the order Sphingomonadales The β-carotene hydroxylase protein sequence of bacteria (data from NCBI protein database) was analyzed using Block Maker (http: / / blocks.fhcrc.org / blocks / make_blocks.html), and multiple CodeHop degenerate primers were designed. Among them, the following 1 pair of primers can amplify bands with good consistency and strong specificity.

[0030] Upstream primers:

[0031] crtZ sense: 5′-GCCTGGTCGATGCACAAGTAYRTNATGCAYG-3′

[0032] Downstream primers:

[0033] crtZ anti: 5′-CGGCGTGGTGCAGCYKRTGNGCYTG-3′

[0034] Genomic DNA (using the AxyPrep Bacterial Genomic DNA Miniprep Kit) extracted from Sphmgomo...

Embodiment 2

[0056] Embodiment 2: Obtaining of Sphingomonas paucimobilis crtG gene DNA sequence

[0057] 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 the crtG gene and the genes required for gene knockout were obtained by several SiteFinding-PCR methods. part of the flanking sequence. The gene-specific primers used were designed according to known sequences, and the sequences of SiteFinder and SFP primers were the same as above.

[0058] After splicing the resulting sequences, a sequence including the crtG gene was obtained, as shown in SEQ ID No.4, wherein the full length of the crtG gene (SEQ ID No.2) contained was 798bp (position 1255 of SEQ ID No.4 base to the 2052nd base).

Embodiment 3

[0059] Example 3: Recombinant strains of Sphingomonas paucimobilis with deletion of β-carotene hydroxylase and 2,2'-β-hydroxylase and 2,2'-β-hydroxylase deficiency Construction of Recombinant Strains of Sphingomonas

[0060] 1. Construction of gene knockout vectors pLO3-ΔZ and pLO3-ΔG

[0061] 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. Z1 and Z2 are primers Taq enzyme (Takara) to amplify the upstream homologous sequence of the crtZ gene. 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 45 sec, 30 cycles , and finally extended at 72 ° C for 10 min. Z3 and Z4 are primers Taq enzyme (Takara) to amplify the downstream homologous sequence of the crtZ gene. The PCR reaction program is:...

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Abstract

The invention provides the DNA sequences of a beta-carotene hydroxylase gene (crtZ) and a 2,2'-betahydroxylase gene (crtG) in sphingomonas sp., recombinant strains lacking beta-carotene hydroxylase and 2,2'-betahydroxylase and the use of the two strains. The nucleotide sequence of the crtZ gene is represented by SEQ ID NO.1; and the nucleotide sequence of the crtG gene is represented by SEQ ID NO.2. The DNA sequences of the beta-carotene hydroxylase gene and the 2,2'-betahydroxylase gene in the sphingomonas sp., which are provided by the invention, lay a foundation for the genetic modification of a sphingomonas sp. carotenoid biological synthesis means. In addition, compared with wild strains, the recombinant strains of the sphingomonas sp., which are constructed by a gene knockout method, have the advantages that: gellengum yield is basically unchanged; and beta-carotene or zeaxanthin can be produced in the production of gellengum by fermentation. And the strains can be used in further gene knockout or metabolic engineering modification.

Description

(1) Technical field [0001] The present invention relates to β-carotene hydroxylase gene (crtZ) and 2,2'-β hydroxylase gene (crtG) of sphingomonas kinesis, gene knockout recombinant bacterial strain and application. (2) Background technology [0002] Gellan gum is a novel microbial exopolysaccharide produced by Sphingomonas paucimobilis. Compared with similar products, gellan gum has many superior properties, such as less dosage; high transparency of the formed gel; adjustable freezing point, melting point, elasticity, etc.; acid resistance, alkali resistance, high temperature resistance; good water solubility, taste properties, moisture retention, etc. Many of its excellent characteristics determine its broad market prospects. At present, gellan gum has been widely used as emulsifier, suspending agent, thickener, stabilizer, gelling agent, slow-release agent, film-forming material, etc. in the food and pharmaceutical industries. In addition, gellan gum can also be compoun...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N1/20C12P19/04C12P23/00C12R1/01
Inventor 吴雪昌朱亮李欧
Owner ZHEJIANG UNIV
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