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Gene engineering strain streptomyces tsukubaensis L20 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, cost reduction, and stable genetic traits

Active Publication Date: 2015-12-16
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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  • Gene engineering strain streptomyces tsukubaensis L20 and application thereof
  • Gene engineering strain streptomyces tsukubaensis L20 and application thereof
  • Gene engineering strain streptomyces tsukubaensis L20 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] The acquisition of embodiment 1 Streptomyces tsukuba L20

[0025] The DNA sequence of SEQIDNO1 was amplified by polymerase chain reaction (PCR), and the amplified DNA fragments were digested by DNA restriction endonuclease and inserted into plasmid pIJ8660 respectively, 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 plasmids into 40ml LB liquid medium (containing corresponding antibiotics) and culture at 37°C until OD 600 0.6, collect the bacteria by centrifugation, wash once with 10ml LB liquid medium, and resuspend with 0.5ml 2×YT liquid medium; add 20 μl of the screened Streptomyces tsukuba spores into 0.5ml 2×YT liquid medium Resuspend, heat shock at 45°C for 10 minutes, then cool in an ice bath for 2 minutes; mix the spores of the above-mentioned Escherichia...

Embodiment 2

[0027] The thalline morphology characteristic of embodiment 2 Streptomyces tsukuba L20

[0028] Streptomyces tsukuba L20 was cultured on ISP4 solid medium for 7 days, the hyphae and aerial hyphae in the base were well developed, the spore filaments were flexible or curved, the number of mature spore chain spores was more than 10, and the spores were short rods (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 L20

[0031] Inoculate 20 μl of Streptomyces tsukuba L20 spores into the seed medium at 250 rpm and incubate at 28°C for 24 hours; inoculate the mycelium in the seed medium at 10% inoculum into the fermentation medium at 250 rpm at 28°C After culturing for 120 hours, samples were taken after culturing for 24, 48, 72, 96 and 120 hours to measure the production of tacrolimus.

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Abstract

The invention provides a gene engineering strain streptomyces tsukubaensis L20 and application thereof. The gene engineering strain streptomyces tsukubaensis L20 is preserved in China General Microbiological Culture Collection Center; the preservation date is August 19, 2015; the preservation number is CGMCC No. 11252. The gene engineering strain streptomyces tsukubaensis L20 is relatively stable in inheritable character and fermentation unit, the culture and fermentation conditions are suitable for industrialized production, and the fermentation unit reaches a high level; the gene engineering strain streptomyces tsukubaensis L20 is utilized for improving the expression amount of a rate-limiting enzyme in a tacrolimus biosynthetic pathway through gene engineering, and serves as the rate-limiting enzyme during gene engineering of the tacrolimus biosynthetic pathway to improve the yield of tacrolimus, so that the cost of an industrialized production process can be reduced, and the fermentation unit of tacrolimus can be improved by 30%.

Description

technical field [0001] The invention belongs to the field of bioengineering, and relates to a gene engineering bacterial strain Streptomyces tsukuba L20 in the field of microbial pharmacy and an application 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 Japan and the...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12P17/18C12R1/465
Inventor 李永泉王月月江辉张小晟骆红豆
Owner ZHEJIANG UNIV
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