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Method for fermenting to produce acetoin by utilizing bacillus licheniformis engineered strain

A technology of Bacillus licheniformis and engineering strain, which is applied in the field of fermentation and production of acetoin to achieve the effects of high production intensity, low probability of contaminating miscellaneous bacteria and high yield

Active Publication Date: 2019-03-19
山东大学深圳研究院 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, by knocking out the key enzyme genes (gdh and budC) of the by-product 2,3-butanediol synthesis pathway, and replacing the Bacillus licheniformis lactate dehydrogenase gene with the NADH oxidase gene (nox) from Thermococcusprofundus DT5432 strain (ldh) reduce the production mode of by-product lactic acid to construct bacillus licheniformis engineered bacterial strain, to realize the method for the efficient production of acetoin has not yet been reported

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1: Construction of an engineering bacterium Bacillus licheniformisΔgdhΔbudCΔldh::nox that produces acetoin

[0028] The starting strain is Bacillus licheniformis10-1-A, which was deposited in the General Microorganism Center of China Committee for the Collection of Microorganisms on November 14, 2011, with the preservation number: CGMCC NO.5461.

[0029] (1) Knockout of glycerol dehydrogenase gdh gene

[0030] The sequence length of the glycerol dehydrogenase gene gdh is 1104 bases, and its nucleotide sequence is shown in SEQ ID NO.1.

[0031]Genomic DNA of B. licheniformis 10-1-A was prepared by a conventional method. The process referred to the method for small-scale preparation of bacterial genomes in the "Guide to Molecular Biology" published by Science Press to extract Bacillus licheniformis 10- Genomic DNA of 1-A; primers "gdh1-f and gdh1-r" and "gdh2-f and gdh2-r" were used to PCR amplify the upstream and downstream homology arms of the gdh-encoding gene...

Embodiment 2

[0063] Example 2: Rotational speed optimization of Bacillus licheniformisΔgdhΔbudCΔldh::nox strain in a 1L fermenter for the production of acetoin

[0064] Referring to Example 1, using the wild-type Bacillus licheniformis 10-1-A as the starting strain, the glycerol dehydrogenase gdh gene and the 2,3-butanediol dehydrogenase gene budC were knocked out to express NADH oxidation in Thermococcus profundus DT5432 Enzyme gene nox to replace lactate dehydrogenase ldh, named Bacillus licheniformisΔgdhΔbudCΔldh::nox.

[0065] (1) Streak the recombinant Bacillus licheniformisΔgdhΔbudCΔldh::nox onto an LB medium plate containing 1.5-1.8% agar in a mass-volume ratio, and culture it on a shaker at 50±1°C for 12±1 hours;

[0066](2) Under sterile conditions, use a sterile toothpick to pick a single colony on the plate in step (1), then inoculate it into 5 mL of LB medium, and culture it on a shaker at 50±1°C for 12±1 hours , obtaining first-class seeds of recombinant Bacillus licheniformi...

Embodiment 3

[0073] Example 3: Optimization of the ventilation rate of Bacillus licheniformisΔgdhΔbudCΔldh::nox strain in a 1L fermenter for the production of acetoin

[0074] Referring to Example 1, using the wild-type Bacillus licheniformis 10-1-A as the starting strain, the glycerol dehydrogenase gdh gene and the 2,3-butanediol dehydrogenase gene budC were knocked out to express NADH oxidation in Thermococcus profundus DT5432 Enzyme gene nox to replace lactate dehydrogenase ldh, named Bacillus licheniformisΔgdhΔbudCΔldh::nox.

[0075] (1) Streak the recombinant Bacillus licheniformisΔgdhΔbudCΔldh::nox onto an LB medium plate containing 1.5-1.8% agar in a mass-volume ratio, and culture it on a shaker at 50±1°C for 12±1 hours;

[0076] (2) Under sterile conditions, use a sterile toothpick to pick a single colony on the plate in step (1), then inoculate it into 5 mL of LB medium, and culture it on a shaker at 50±1°C for 12±1 hours , obtaining first-class seeds of recombinant Bacillus lich...

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Abstract

The invention discloses a method for fermenting to produce acetoin by utilizing a bacillus licheniformis engineered strain. The method comprises the following steps: knocking out glycerol dehydrogenase genes gdh and 2,3-butanediol dehydrogenase genes budC by taking bacillus licheniformis 10-1-A as an original strain, replacing lactic dehydrogenase genes ldh with NADH oxidase genes nox in Thermococcus profundus DT5432, and constructing a bacillus licheniformis engineered strain capable of being used for producing acetoin; taking glucose as a substrate, biologically fermenting the strain, and preparing the acetoin from fermentation liquor. Experiments prove that the engineered strain disclosed by the invention is capable of producing 82.14g / L of acetoin, and the production efficiency reaches2.28g / L per hour. Moreover, the cost in the production process is low, the yield is high, and the method has excellent application value and considerable economic benefits.

Description

technical field [0001] The invention relates to a method for fermenting and producing acetoin, in particular to a method for fermenting and producing acetoin by using Bacillus licheniformis engineering strains. Background technique [0002] Acetoin (3-hydroxy-2-butanone) is a volatile compound, which is widely used in food additives, plant growth promotion and biological pest control. In addition, acetoin can also be used as a precursor for chemical synthesis of a series of compounds, such as diacetyl and alkyl-containing pyrazines, the latter including 2,3,5,6-tetramethylpyrazine. In view of the wide use of acetoin and the potential of realizing industrial production, it is listed as one of the 30 priority platform compounds by the US Department of Energy. [0003] At present, the main source of commercial acetoin is the chemical synthesis method based on non-renewable resources. The process steps are complex, the reaction conditions are harsh, and the pollution is serious...

Claims

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Application Information

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IPC IPC(8): C12P7/26C12N1/21C12R1/10
CPCC12N9/0004C12N9/0006C12P7/26C12Y101/01004C12Y101/01006C12Y101/01076C12Y106/00
Inventor 高超马翠卿严金鑫刘秋媛张一鹏
Owner 山东大学深圳研究院
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