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Gene recombination bacterium and application thereof in preparing chiral pure acetoin and 2,3-butanediol

A gene recombination, butanediol technology, applied in microorganism-based methods, applications, genetic engineering, etc.

Inactive Publication Date: 2010-10-13
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0027] In view of the above-mentioned existing methods, the productivity of the product is low, the cost is high, and it is difficult to produce on a large scale.

Method used

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  • Gene recombination bacterium and application thereof in preparing chiral pure acetoin and 2,3-butanediol
  • Gene recombination bacterium and application thereof in preparing chiral pure acetoin and 2,3-butanediol
  • Gene recombination bacterium and application thereof in preparing chiral pure acetoin and 2,3-butanediol

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0084] 2. Cloning of NADH oxidase gene (nox)

[0085] The genomic DNA of bacterial strain Lactobacillus brevis is prepared by a conventional method, and this process can be referred to the method for the small-scale preparation of bacterial genome in "Guide to Molecular Biology" published by Science Press to extract the genomic DNA of Lactobacillus brevis bacterial strain; Primers PCR amplified the NADH oxidase gene nox from the genomic DNA of Lactobacillus brevis strain;

[0086] Lactobacillus brevis is used as the source bacterium of the nox gene, and primers are designed according to the sequenced genome sequence of the bacterium and the sequence of the gene reported:

[0087] The upstream primer 5'-GCGAGATCTCATGAAAGTCACAGTTG-3' carries a Bgl II site;

[0088] The downstream primer, 5'-GATCTCGAGTTAAGCGTTAACTGAT-3', carries an XhoI site.

[0089] 3. Link the fragment amplified by step (1) to pEasy-T to obtain the recombinant plasmid pEasy-TydjL, link the fragment amplified b...

Embodiment 2

[0097] (4) Fermentation tank culture: under sterile conditions, take the culture solution obtained in step (3) and inoculate the improved M9 liquid medium of 2L to 10L with a volume ratio of 8% inoculum, cultivate at 37°C for 10 hours, add IPTG with a final concentration of 1 mM was induced at 20°C for 12 hours, so that the transformation ability of the cells reached 15 U / ml.

[0098] Wherein: the formula of the LB medium described in the above steps (1)-(3) is: peptone 10g / L; yeast powder 5g / L; NaCl 10g / L, pH 7.0; sterilized at 115°C for 15 minutes.

[0099] The improved M9 liquid culture medium formula described in the above-mentioned steps (4) is: peptone 10g / L; Yeast powder 5g / L; Na 2 HPO 4 ·7H 2 O 12.8g / L; KH 2 PO 4 3g / L; NaCl 0.5g / L; NH 4 C1 1g / L; CaCl 2 0.011g / L; Glucose 4g / L; Sterilize at 115°C for 15 minutes.

[0100] The method for measuring the transformation ability of the above cells is: suspend the cultured cells in 200mM pH7.8 phosphate buffer containin...

Embodiment 3

[0107] (4) Fermentation tank culture: under sterile conditions, take the culture solution obtained in step (3) and inoculate the improved M9 liquid medium of 2L to 10L with a volume ratio of 8% inoculum, cultivate at 37°C for 14 hours, add IPTG with a final concentration of 1 mM was induced at 10°C for 20 hours to make the transformation ability of the cells reach 20 U / ml.

[0108] Wherein: the formula of the LB medium described in the above steps (1)-(3) is: peptone 10g / L; yeast powder 5g / L; NaCl 10g / L, pH 7.0; sterilized at 115°C for 15 minutes.

[0109] The improved M9 liquid culture medium formula described in the above-mentioned steps (4) is: peptone 10g / L; Yeast powder 5g / L; Na 2 HPO 4 ·7H 2 O 12.8g / L; KH 2 PO 4 3g / L; NaCl 0.5g / L; NH 4 Cl 1g / L; CaCl 2 0.011g / L; Glucose 4g / L; Sterilize at 115°C for 15 minutes.

[0110] The method for measuring the transformation ability of the above cells is: suspend the cultured cells in 200mM pH7.8 phosphate buffer containing 1...

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Abstract

The invention discloses an E. coli BL21(pETDuet-ydjLnox) containing a 2R,3R-butanediol dehydrogenase gene ydjL and an NADH oxidase gene nox, wherein the strain is preserved in the 'China Center for Type Culture Collection' on December 23 in 2008, and the preservation number is CCTCC NO: M 208259. The invention also discloses application of a gene recombination bacterium in producing chiral S-AC bycatalyzing meso-BD, producing chiral R-AC by catalyzing 2R,3R-BD and producing chiral pure 2S,3S-BD by splitting a 2,3-BD mixture. The concentration of a chiral AC prepared from the recombinant E. coli can reach over 6 grams per liter (the ee value is more than or equal to 96 percent); and the ee value of the chiral pure 2S,3S-BD is more than or equal to 98 percent, and the regeneration of a cofactor is achieved, thus the gene recombination bacterium has great industrial application prospect.

Description

technical field [0001] The present invention relates to a gene recombination bacterium and its application, specifically, to a recombination capable of combining 2R, 3R-2, 3-butanediol dehydrogenase (2R, 3R-BDH) gene (ydjL) and NADH Escherichia coli co-expressing the oxidase (NOX) gene (nox), and using this engineering bacteria to efficiently transform meso-2,3-butanediol (meso-BD) to produce S-acetoin (S-AC), 2R, 3R-2, 3-butanediol (2R, 3R-BD) production method of R-acetoin (R-AC) and resolution of 2,3-butanediol (2,3-BD) mixture production 2S,3S-2,3-Butanediol (2S,3S-BD) method. Background technique [0002] Acetoin (AC) is usually pale yellow liquid or crystal, has two isomers (R-AC and S-AC), naturally exists in wine, honey, cocoa, butter, coffee, strawberry and Rainbow currant and other substances. The national standard GB2760-86 stipulates that it is a food spice that is allowed to be used, and the FEMA safety number is 2008. AC also has very important uses in the...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/53C12P41/00C12P7/18C12P7/26C12R1/19
Inventor 马翠卿吕传娟肖梓军秦加阳许平
Owner SHANDONG UNIV
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