Bacillus licheniformis engineered bacterium capable of high production of poly-Gamma-glutamic acid

A technology of Bacillus licheniformis and glutamic acid, which is applied in the field of genetic engineering and microbial metabolic engineering, can solve the problem that no one has studied the fermentation yield of poly-γ-glutamic acid, achieve broad application prospects, and increase yield

Active Publication Date: 2017-03-15
HUBEI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the relationship between Zwf expression level and

Method used

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  • Bacillus licheniformis engineered bacterium capable of high production of poly-Gamma-glutamic acid
  • Bacillus licheniformis engineered bacterium capable of high production of poly-Gamma-glutamic acid
  • Bacillus licheniformis engineered bacterium capable of high production of poly-Gamma-glutamic acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] This example provides Bacillus licheniformis WX-02 / pHY-zwf and its construction method.

[0028] 1 Cloning of expression elements in recombinant vectors

[0029] Using the total DNA of Bacillus subtilis 168 as a template, according to the genome sequence of Bacillus subtilis 168 published by NCBI, the promoter P43 sequence SEQ ID NO: 1 was amplified by PCR method, and the primers were P43-F SEQ ID NO :2, P43-R SEQ ID NO: 3; using the total DNA of Bacillus licheniformis WX-02 as a template, according to the genome sequence of Bacillus licheniformis WX-02 published on NCBI, the method for amplifying 6-glucose phosphate by PCR Dehydrogenase gene zwf sequence SEQ ID NO: 4, primers are zwf-F SEQ ID NO: 5, zwf-R SEQ ID NO: 6; using the total DNA of Bacillus licheniformis WX-02 as a template, according to the lichen published on NCBI For the genome sequence of Bacillus sp. WX-02, the amylase gene amyL terminator sequence SEQ ID NO: 7 was amplified by PCR method, and the prime...

Embodiment 2

[0039] This example provides a method for producing poly-γ-glutamic acid by Bacillus licheniformis WX-02 / pHY-zwf. Fermentation experiments were carried out under different fermentation conditions by Bacillus licheniformis WX-02 / pHY-zwf to obtain high-yield poly-γ-glutamic acid. Glutamate method.

[0040] The poly-γ-glutamic acid that described fermentation experiment obtains all adopts following measuring method and quantitative method:

[0041] Add 2 times the volume of absolute ethanol to 2.0 mL of fermentation broth, shake and mix, and centrifuge at 8000 r / min for 10 min. Discard the supernatant, and dissolve the centrifuged pellet with 2.0 mL deionized water, and shake evenly. Take 1.0mL of the solution in a colorimetric tube, add 1mL of concentrated hydrochloric acid, seal it and hydrolyze it at 100°C for 24h. After hydrolysis, dilute to 10 mL with 1 mol / L NaOH, and then filter through a 0.22 μm microporous membrane. Lichrospher C18 chromatographic column (25cm×4.6mm) ...

Embodiment 3

[0068] This example provides a method for Bacillus licheniformis WX-02 / pHY-zwf to produce poly-γ-glutamic acid in an optimized medium, and the specific steps include:

[0069] Pick the colony WX-02 / pHY-zwf and inoculate it in 5 mL of LB medium, and cultivate overnight at 37°C with shaking at 240r / min. Then transfer 1% inoculum to 50mL fresh LB medium until OD600 is 1.0, inoculate 1% inoculum into the pre-prepared optimized medium, 40°C, 240r / min, fermentation time 36h.

[0070] The optimized medium: 100g / L glucose; 12g / L NH 4 Cl; 5g / L sodium citrate; 10g / L sodium glutamate; 1.5g / LK 2 HPO 4 ·3H 2 O; 1.5g / LMgSO 4 ·7H 2 O; 1.5g / LZnSO 4 ·7H 2 O; 1.5g / LCaCl 2 ;0.15g / LMnSO 4 ·H 2 O; pH 8.0.

[0071] The obtained fermentation result is basically consistent with that of Example 2, and the yield of poly-γ-glutamic acid is significantly improved compared with the original strain.

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Abstract

The invention provides a Bacillus licheniformis engineered bacterium capable of high production of poly-Gamma-glutamic acid, collected under CCTCC No: M2016439 and named as Bacillus licheniformis (WX-02)/pHY-zwf, and further provides a construction method of the Bacillus licheniformis (WX-02)/pHY-zwf and a method for high production of poly-Gamma-glutamic acid. The synthetic level of intracellular NADPH is increased through genetic engineering modification, and the yield of Gamma-PGA. By constructing glucose-6-phosphate dehydrogenase overexpression support pHY-zwf and converting to Gamma-PGA-producing Bacillus licheniformis WX-02 pre-stored in a lab, the Bacillus licheniformis (WX-02)/pHY-zwf capable of producing Gamma-PGA is re-constructed. The bacterium may provide significantly synthesis of Gamma-PGA under optimized media and culture conditions. The yield of Gamma-PGA is up to 51.13 g/L which is 49% significantly higher than the Gamma-PGA yield of an original bacterium.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering and microbial metabolism engineering, in particular to a bacillus licheniformis engineering bacterium capable of high-yielding poly-γ-glutamic acid. Background technique [0002] Poly-γ-glutamic acid is a widely used anionic polymer with many characteristics: easy to dissolve in water; good water retention capacity; adsorption of metal ions; slow release and biodegradability. Because of the special biological properties of poly-γ-glutamic acid, it is widely used in agriculture, medicine, food, cosmetics and other fields. [0003] Related literature reports that the producing bacteria of poly-γ-glutamic acid are mainly Bacillus subtilis, Bacillus amyloliquefaciens and Bacillus licheniformis. Generally speaking, the synthesis of poly-γ-glutamic acid mainly consists of two ways: endogenous and exogenous. The exogenous pathway is mainly to add glutamic acid in the medium, and the glutami...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12P13/02C12R1/10
CPCC12N9/0006C12P13/02C12Y101/01049
Inventor 陈守文蔡冬波陆兴澄王勤陈杨阳何鹏辉
Owner HUBEI UNIV
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