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A strain of Streptomyces parvus genetic engineering bacteria and its construction method and application

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

Active Publication Date: 2019-01-18
NANJING TECH UNIV
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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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  • A strain of Streptomyces parvus genetic engineering bacteria and its construction method and application
  • A strain of Streptomyces parvus genetic engineering bacteria and its construction method and application
  • A strain of Streptomyces parvus genetic engineering bacteria and its construction method and application

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

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

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

[0030] (The underline is the Nde I restriction site)

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

[0032] (The underline is the Xba I restriction site)

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

[0034] PCR system: Genomic DNA 2 μL, primer 1 and primer 2 1 μL each, dNTP 2 μL, 10× exTaq buffer (containing Mg 2+ ) 2.5 μL, exTaq enzyme 0....

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 (Glucose 50g / L, Yeast Extract 5g / 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 O 0.5g / L, FeSO 4 ·7H 2 O0.03g / L, ZnSO 4 ·7H 2 O 0.04g / L, pH 6.8), after being sterilized by high pressure at 121°C for 15 minutes, put it into a 500mL wide-mouth Erlenmeyer flask. Inject a loop of genetically engineered bacteria Streptomyces albulus 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 in three days, and the dry weight (DCW) of bacteria per unit volume could reach 7.2g / L, and the S.albulus PD-1 control could reach 1.23g / L, DCW Up to 6.5g / L. Compared ...

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.albulus PD-4 and the control bacteria S.albulus PD-130°C at 200rpm for one day, centrifuge at 6000rpm for 5min, collect the bacteria, wash and transfer to a glucose concentration of 10g / L, with different ammonium sulfate concentrations In medium I, control C / N at 2.36:1, 3:1, 4.71:1, 9.17:1, 11.5:1, 16.5:1, 23:1 and continue to ferment for 7 days, and take samples to measure ε-PL at the end of fermentation Yield, found at the end of fermentation, as shown in Table 1, 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. The original feeding process Under the condition of supplying S.albulus PD-1 with a certain concentration of glucose and ammonium sulfate, the C / N supply can be b...

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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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Patent Type & Authority Patents(China)
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 TECH UNIV
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