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Gene engineering strain and application thereof

A genetically engineered bacteria and gene technology, applied in the fields of genetic engineering and biology, can solve the problems of little yield, difficult separation and purification of arabitol, poor specificity of xylose reductase, etc., and achieve the effect of improving enzyme activity

Inactive Publication Date: 2013-07-17
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Although the microbial method has these advantages, in the existing genetically engineered bacteria, the specificity of xylose reductase is relatively poor, and arabinose is reduced to arabinitol while catalyzing the reduction of xylose into xylitol, while xylose Alcohol and arabinitol are epimers of each other, and their physical and chemical properties are very similar, and the hemicellulose hydrolyzate contains a large amount of arabinose, and the generated arabinitol brings difficulties to separation and purification
[0007] In recent years, researchers have continuously improved the specificity of xylose reductase in microorganisms to xylose through screening, optimization, genetic engineering and other means to increase the yield of xylitol and reduce production costs, but little has been achieved.

Method used

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  • Gene engineering strain and application thereof
  • Gene engineering strain and application thereof
  • Gene engineering strain and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1 Construction of genetically engineered bacteria E.coli BL21 (pET30a-xr)

[0059] 1 Obtaining the xylose reductase gene

[0060] (1) According to the xylose reductase gene (xr) sequence (Gene ID: 2593996254, SEQ ID No.1) published on NCBI and the characteristics of the multiple cloning site on the expression vector pET30a(+), design using bioinformatics software Synthesize degenerate primers, the upstream and downstream primers are:

[0061] P1: 5'-GA AGATCT GATGGTACCAGGTCTAAGGCTCAACTC-3' (the underline is the BglⅡ restriction site) (SEQ ID No.4);

[0062] P2: 5'-CG GAATTC CTATGCGAAAAACCAGAGGTTCTC-3'; (contains EcoR Ⅰ restriction site) (SEQ ID No.5);

[0063] (2) Culture Neurospora crassa and extract its total DNA;

[0064] (3) Using the total DNA as a template and using P1 and P2 as primers, carry out PCR amplification;

[0065] The PCR reaction system is: 25 μL DNA PrimerStarMix, 21 μL H 2 O, 1.5 μL P1, 1.5 μL P2, 1 μL DNA template.

[0066] The PCR ...

Embodiment 2

[0084] Example 2 Construction of genetically engineered bacteria E.coli BL21 (pET30a-xr+pCDFDuet-1-gnd) 1 to obtain gnd gene

[0085] (1) According to the 6-phosphogluconate dehydrogenase gene (gnd) sequence (GeneID: 388478084, SEQ ID No.2) published on NCBI and the characteristics of the multiple cloning site on the expression vector pCDFDuet-1, using bioinformatics software Design and synthesize degenerate primers, the upstream and downstream primers are:

[0086] P3: 5'-TCC GAATTC GATGTCAAAGCAACAGATCGG-3' (containing EcoR Ⅰ restriction site) (SEQ ID No.6);

[0087] P4: 5'-GCTT GTC GAC TTAATCCAGCCATTCGGTATG-3' (containing Sal Ⅰ restriction site) (SEQ ID No.7).

[0088] (2) Using E.coli K-12 genomic DNA as a template, PCR amplification was performed using primers P3 and P4;

[0089]The PCR reaction system is: 25 μL DNA PrimerStarMix, 21 μL H 2 O, 1.5 μL P3, 1.5 μL P4, 1 μL DNA template.

[0090] The PCR reaction conditions were: pre-denaturation at 95°C for 2 min; den...

Embodiment 3

[0108] Example 3 Construction of engineering bacteria E.coli BL21 (pET30a-xr+pCDFDuet-1-gnd-zwf)

[0109] 1 Get the zwf gene

[0110] (1) According to the glucose 6-phosphate dehydrogenase gene (zwf) sequence (GeneID: 388477926, SEQ ID No.3) published on NCBI and the characteristics of the multiple cloning site on the expression vector pCDFDuet-1, the bioinformatics software was used to design Synthesize degenerate primers, the upstream and downstream primers are:

[0111] P5: 5'-TGGC AGATCT CATGGCGGTAACGCAAACA-3' (containing Bgl Ⅱ restriction site) (SEQ ID No.8);

[0112] P6: 5'-CAGA CTCGAG TTACTCAAACTCATTCCAGG-3' (containing Xho Ⅰ restriction site) (SEQ ID No.9);

[0113] (2) Using E.coli K-12 genomic DNA as a template, PCR amplification was performed using primers P5 and P6;

[0114] The PCR reaction system is: 25 μL DNA Primer StarMix, 21 μL H 2 O, 1.5 μL P5, 1.5 μL P6, 1 μL DNA template.

[0115] The PCR reaction conditions were: pre-denaturation at 95°C for 2 min...

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Abstract

The invention discloses a gene engineering strain and an application thereof. The gene engineering strain comprises host cells and target genes transferred into the host cells, wherein the target genes include xylose reductase genes and NADPH (Nicotinamide Adenine Dinucleotide Phosphate) regenerate enzyme genes. The gene engineering strain is applied to the preparation of xylitol which is prepared through the following steps of: inoculating the engineering strain to a culture solution to culture until the OD600 is equal to 0.6-0.8, adding an inducer for inducible expression, and separating and purifying the culture solution after the inducible expression is finished. Compared with the prior art, the activity of the xylose reductase in the engineering strain is greatly improved and is 5 times as high as that of a neurospora crassa; and the gene engineering strain disclosed by the invention also contains an NADPH regeneration system, so that the xylose conversion ratio of the gene engineering strain is as high as 80.32%.

Description

technical field [0001] The invention belongs to the field of genetic engineering and biotechnology, and in particular relates to a genetically engineered bacterium and its application. Background technique [0002] Xylitol is a five-carbon sugar alcohol, its chemical name is 1,2,3,4,5-pentol, and its molecular formula is C 5 h 12 o 5 , is a white crystal or crystalline powder, its solubility, solution density and refractive index and other physical and chemical properties are basically the same as sucrose, and can be used as a substitute for sucrose. The US FAD has regarded xylitol as a "recognized safe substance" since 1963, allowing it to be used in food, beverage, medicine and cosmetics. [0003] Xylitol has attracted more and more attention, such as in the field of pharmacy, as an auxiliary material of pharmaceuticals, xylitol surfactant; in the field of medicine, as a high-quality infusion carrier, and can be combined with antibacterial drugs, cardiovascular Drugs, ...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12P7/18C12R1/19
Inventor 吴绵斌焦静雨林建平杨立荣
Owner ZHEJIANG UNIV
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