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Genetic engineering strain Streptomyces tsukubaensis L21 and application thereof

A technology of genetically engineered strains, Streptomyces tsukuba, applied in the field of bioengineering, can solve the problem of low fermentation units of strains, and achieve the effects of stable fermentation units, high fermentation units, and stable genetic traits

Active Publication Date: 2015-12-23
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] It is now found that there are many Streptomyces producing tacrolimus, some genetic traits and fermentation units are relatively stable, and the culture and fermentation conditions are suitable for industrial production, but the fermentation units of the strains are relatively low

Method used

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  • Genetic engineering strain Streptomyces tsukubaensis L21 and application thereof
  • Genetic engineering strain Streptomyces tsukubaensis L21 and application thereof
  • Genetic engineering strain Streptomyces tsukubaensis L21 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] The acquisition of embodiment 1 Streptomyces tsukuba L21

[0025] The DNA sequence of SEQ ID NO.1 was amplified by polymerase chain reaction (PCR), and the amplified DNA fragment was digested with a restriction endonuclease and inserted into the plasmid pIJ8660, and then the plasmid containing the above DNA sequence was transformed into the large intestine Bacillus ET12567 (which already contains plasmid pUZ8002).

[0026] Inoculate Escherichia coli ET12567 (already containing plasmid pUZ8002) containing the above recombinant plasmid into 50mL LB liquid medium (containing corresponding antibiotics), and cultivate to OD at 37°C 600 was 0.4, the cells were collected by centrifugation, washed once with 20 mL LB liquid medium, and then washed once with 10 mL LB. Then centrifuge the supernatant and resuspend with 0.5mL LB liquid medium; add 20μL of the screened Streptomyces tsukuba spores to resuspend in 0.5mL 2×YT liquid medium, heat shock at 45°C for 10min, and then cool ...

Embodiment 2

[0027] The thalline morphology characteristic of embodiment 2 Streptomyces tsukuba L21

[0028] Streptomyces tsukuba L21 was cultured in ISP4 solid medium for 7 days, observed under the electron microscope, it can be seen that the hyphae and aerial hyphae in the base are well developed, the spore filaments are flexible or curved, and the number of mature spore chainspores is less than 10 Above, the spores are short rod-shaped (see figure 1 ). The apparent characteristics in ISP4 solid medium, the results are shown in Table 1.

[0029]

Embodiment 3

[0030] The fermentation verification of embodiment 3 Streptomyces tsukuba L21

[0031] Inoculate 20 μL of Streptomyces tsukuba L21 spores into the seed medium at a rotational speed of 250 rpm and incubate at 28°C for 24 hours; inoculate the mycelium in the seed medium into the fermentation medium at a rotational speed of 250 rpm at 28°C at 10% of the inoculum amount At 120 hours, samples were taken after 24, 48, 72, 96, and 120 hours of culture to measure the production of tacrolimus.

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Abstract

The invention provides genetic engineering strain Streptomyces tsukubaensis L21. The fungus preservation unit is CGMCC (China General Microbiological Culture Collection Center), the preservation date is August 19, 2015, and the preservation number is CGMCC No. 11253. According to the genetic engineering strain Streptomyces tsukubaensis L21, the strain genetic character and the fermentation unit are stable, culture and fermentation conditions are suitable for industrial production, and the fermentation unit is high. The copy number of specific controlling genes in the Tacrolimus creature biosynthetic pathway is increased through genetic engineering, and the genes improve rate-limiting enzyme in the Tacrolimus creature biosynthetic pathway so as to increase the Tacrolimus yield, reduce the cost in the industrial production process and improves the Tacrolimus fermentation unit to 945 mg / L.

Description

technical field [0001] The invention belongs to the field of bioengineering, and relates to a genetically engineered bacterial strain Streptomyces tsukuba L21 in the field of microbial pharmacy and a cultivation method thereof. Background technique [0002] Tacrolimus, also known as FK506, is derived from the genus Streptomyces ( streptomyces ), the chemical structure of the fermentation product isolated in ) belongs to 23-membered macrolide antibiotics. It has strong immunosuppressant activity and antifungal activity, and is mainly used clinically for the treatment of immune rejection after organ transplantation and the treatment of skin inflammation. As a powerful new immunosuppressant, it mainly inhibits the release of interleukin-2 (L-2) and comprehensively inhibits the effect of T lymphocytes, which is 100 times stronger than cyclosporine (CsA). In recent years, as a first-line drug for liver and kidney transplantation, it has been marketed in 14 countries including J...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12P17/18C12R1/465
Inventor 李永泉张小晟江辉骆红豆王月月
Owner ZHEJIANG UNIV
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