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

A technology of genetic recombination and butanediol, applied in the direction of microorganism-based methods, applications, genetic engineering, etc., can solve problems such as difficult large-scale production, high cost, and low product productivity

Inactive Publication Date: 2009-10-28
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0026] In the above 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

[0079] Example 1: Construction of recombinant genetic engineering strain E.coli BL21 (pETDuet-ydjLnox) CCTCC NO: M 208259

[0080] 1. Cloning of 2R, 3R-BDH gene (ydjL)

[0081] The genomic DNA of bacterial strain Bacillus subtilis 168 is prepared by a conventional method, and the process can refer to the method for small-scale preparation of bacterial genomes in the "Guide to Molecular Biology" published by Science Press to extract the genomic DNA of Bacillus subtilis 168; Butanediol dehydrogenase gene ydjL was amplified by PCR with primers from the genomic DNA of Bacillus subtilis 168;

[0082] Bacillus subtilis 168 was used as the source of the ydjL gene. According to the sequenced genome sequence of the bacterium, primers were designed: the upstream primer 5′-TCACCATGGGCATGAAGGCAGCAAGA-3′, carrying an NcoI site; the downstream primer 5′-GGCGTCGACTTAGTTAGGTCTAACA-3′, carrying A SalI site.

[0083] 2. Cloning of NADH oxidase gene (nox)

[0084]The genomic DNA of bacterial ...

Embodiment 2

[0092] Example 2: Preparation of Whole Cell Catalyst

[0093] (1) Plate culture: Streak the recombinant Escherichia coli E.coli BL21 (pETDuet-ydjLnox) CCTCC NO: M 208259 strain on an ampicillin LB plate containing 1.5% agar with a mass volume ratio of 100 μg / mL , 37°C for 12 hours.

[0094] (2) First-class seeds: under sterile conditions, use a sterile toothpick to pick a single colony on the plate in step (1), and then inoculate it into 5 mL of liquid medium containing 100 μg / mL ampicillin, 37 Cultivate on a shaker for 12 hours.

[0095] (3) Secondary seeds: under aseptic conditions, take the culture solution cultivated in step (2) with an inoculum volume of 2% by volume, and inoculate 30 mL to 500 mL of LB liquid medium containing 100 μg / mL of ampicillin Incubate at 37°C for 10 hours with shaking.

[0096] (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 ...

Embodiment 3

[0102] Example 3: Preparation of Whole Cell Catalyst

[0103] (1) Plate culture: Streak the recombinant Escherichia coli E.coli BL21 (pETDuet-ydjLnox) CCTCC NO: M 208259 strain on an ampicillin LB plate containing 1.5% agar with a mass volume ratio of 100 μg / mL , 37°C for 12 hours.

[0104] (2) First-class seeds: under sterile conditions, use a sterile toothpick to pick a single colony on the plate in step (1), and then inoculate it into 5 mL of liquid medium containing 100 μg / mL ampicillin, 37 Cultivate on a shaker for 12 hours.

[0105] (3) Secondary seeds: under aseptic conditions, take the culture solution cultivated in step (2) with an inoculum volume of 2% by volume, and inoculate 30 mL to 500 mL of LB liquid medium containing 100 μg / mL of ampicillin Incubate at 37°C for 10 hours with shaking.

[0106] (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 ...

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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 by catalyzing 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 application thereof, specifically, relates to a recombination capable of combining 2R, 3R-butanediol dehydrogenase (2R, 3R-BDH) gene (ydjL) and NADH oxidase gene ( nox) co-expressed Escherichia coli, and use this engineering bacteria to efficiently transform meso-2, 3-butanediol (meso-BD) to produce S-acetoin (S-AC), 2R, 3R-butanediol (2R, 3R-BD) the method for producing R-acetoin (R-AC) and resolution 2,3-butanediol (2,3-BD) mixture produces 2S, 3S-butanediol (2S, 3S -BD) approach. 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 food, fra...

Claims

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Application Information

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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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