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Streptomyces calculus genetically engineered bacterium and constructing method and application thereof

A technology of Streptomyces albicans and genetically engineered bacteria, applied in the field of genetic engineering, can solve the problem of few actinomycetes, and achieve the effects of reducing the obstruction of intracellular enzymes, improving utilization, and having good industrial application prospects.

Active Publication Date: 2016-03-30
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The research on ammonium ion transporters mainly focuses on Escherichia coli and yeast, but there are few related reports on actinomycetes

Method used

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  • Streptomyces calculus genetically engineered bacterium and constructing method and application thereof
  • Streptomyces calculus genetically engineered bacterium and constructing method and application thereof
  • Streptomyces calculus genetically engineered bacterium and constructing method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1: Cloning of ammonium transporter gene (amtB gene) and construction of genetically engineered bacteria.

[0026] 1.1 PCR amplification of the ammonium transporter gene (amtB gene).

[0027] The genomic DNA of S. albulusPD-1 in logarithmic growth phase was extracted with GenomicDNA Purification Kit (Takara, Dalian), and the obtained genomic DNA was detected by 2% (20 g / L) agarose gel electrophoresis.

[0028] Use VectorNTI software to design the following two primers:

[0029] Primer 1: 5′-CCATATGG CATATG GTGAACCTCTCAGGTTCCGATGA-3′

[0030] (The underline is the NdeI restriction site)

[0031] Primer 2: 5′-CTCTAGAG AGATCT CTACTTCTGCCGCTTGTAGAAGG-3′

[0032] (The underline is the XbaI restriction site)

[0033] The obtained genomic DNA of S. albulusPD-1 was used as a template to amplify the target gene fragment.

[0034] PCR system: 2 μL of genomic DNA, 1 μL each of primer 1 and primer 2, 2 μL of dNTP, 10× exTaq buffer (containing Mg 2+ ) 2.5 μL, exTaq ...

Embodiment 2

[0052] Example 2: Verification of high-efficiency nitrogen source using recombinant bacteria shake flask fermentation.

[0053] Prepare seed liquid 100mL, culture medium is M3G liquid culture medium (Glucose50g / L, YeastExtract5g / L, (NH 4 ) 2 SO 4 10g / L, K 2 HPO 4 0.8g / L, KH 2 PO 4 1.36g / L, MgSO 4 ·7H 2 O0.5g / L, FeSO 4 ·7H 2 O0.03g / L, ZnSO 4 ·7H 2 (00.04g / L, pH6.8), put into 500mL wide-mouth Erlenmeyer flask after being sterilized by high pressure at 121℃ for 15min. Insert a loop of genetically engineered bacteria Streptomycesalbulus PD-4 into the seed solution with an inoculation loop, and place it on a shaker at 30°C at a speed of 200rpm for overnight culture for 3 days. Fermentation results such as figure 2 As shown, the yield of shake flask fermentation reached ε-PL1.38g / L for three days, the dry weight (DCW) of bacteria per unit volume could reach 7.2g / L, the S.albulusPD-1 control could reach 1.23g / L, and DCW could up to 6.5g / L. Compared with the control, ε...

Embodiment 3

[0054] Example 3: Two-stage fermentation optimization of C / N in shake flasks using recombinant bacteria as a high-efficiency nitrogen source.

[0055] After culturing the engineering bacteria S.albulusPD-4 and the control bacteria S.albulusPD-130°C at 200rpm for one day, centrifuge at 6000rpm for 5min, collect the bacteria, wash and transfer to cultures with a glucose concentration of 10g / L and different ammonium sulfate concentrations. In base I, control C / N at 2.36:1, 3:1, 4.71:1, 9.17:1, 11.5:1, 16.5:1, and 23:1 to continue fermentation for 7 days, and take samples to measure the ε-PL output at the end of fermentation. At the end of the fermentation, it was found that, as shown in Table 1, the ε-PL of the original strain reached the maximum when the optimal C / N was 3:1 in the two-stage fermentation process of the shake flask, and the maximum value was 1.236g / L, which was supplied under the original feeding process The C / N supply of S.albulusPD-1 with a certain concentration...

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Abstract

The invention discloses a streptomyces calculus genetically engineered bacterium PD-4. An ammonium transporter gene amtB from an S.albulus PD-1 genome is subjected to overexpression, and polylysine (epsilon-poly-L-lysine, epsilon-PL) synthesis ability higher than that of streptomyces albus S.albulus PD-1 is achieved. The sequence of the ammonium transporter gene is shown as SEQ ID No:1. The invention further discloses a construction method and fermentation verification of the recombinant strain. By means of efficient expression of amtB, limitations caused by defects of ammonium transporters are eliminated, and the rate of a nitrogen source in fermentation liquid utilized by S.albulus PD-4 is increased, so that the yield of epsilon-PL is increased, production cost is reduced, and huge economic benefits are brought.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a polylysine fermentation strain and a construction method and application of the strain. Background technique [0002] ε-PL is a new polyamino group first discovered by Dr. S.Shima and H.Sakai in Japan in 1977 when they screened a large number of alkaloids. ε-PL is composed of α-carboxyl and ε of L-lysine. - A polycation formed by the polymerization of amide bonds formed between amino groups. As a natural polyamino acid, ε-PL has excellent biocompatibility and biodegradability, and its molecular side chain contains a large number of hydrophilic free amino groups. It is convenient for its functional transformation, so ε-PL has broad development and application prospects in the fields of health food, gene carrier, drug sustained release, biomedical materials and other fields. Biological synthesis of polylysine uses renewable resources as raw materials, and...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/76C12P13/02C12R1/465
Inventor 徐虹曹长虹冯小海许召贤许宗奇徐铮徐得磊
Owner NANJING UNIV OF TECH
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