A new strain of Streptomyces producing daptomycin and its application

A technology of daptomycin and streptomyces, applied in the field of microorganisms, can solve the problems of complex secondary metabolites, difficulty in separating and purifying daptomycin, accumulation of by-product pigments, etc., achieve good growth conditions, convenient and direct microscopic examination , The effect of reducing the consumption level of carbon and nitrogen sources

Active Publication Date: 2020-08-11
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] For a long time, the strain used in the fermentative production of daptomycin is Streptomyces roseospora, its secondary metabolites are complex, and the by-product pigments accumulate more, which brings difficulties to the later separation and purification of daptomycin

Method used

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  • A new strain of Streptomyces producing daptomycin and its application
  • A new strain of Streptomyces producing daptomycin and its application
  • A new strain of Streptomyces producing daptomycin and its application

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Embodiment 1

[0037] The present invention provides a new daptomycin-producing Streptomyces strain, compared with the traditional daptomycin-producing strain Streptomyces roseospora, the biggest advantage is that it will not produce pigment substances during subculture and fermentation, so that It reduces the difficulty for subsequent separation and purification operations. The bacterial strain provided by the present invention is preserved in the China General Microorganism Culture Collection and Management Center, the preservation date: 2019.6.12, and the preservation number: CGMCC17921.

[0038] The appearance characteristics in various mediums, the results are shown in Table 1, and the colony morphology on the R5 medium is shown in figure 1 , see the mycelium morphology in YEME medium figure 2 ; The physiological and biochemical characteristics of L10 are shown in Table 2.

[0039] The Streptomyces 16s rDNA sequencing result (see attachment) declared by the present invention is compa...

Embodiment 2

[0046] The culture method of the Streptomyces bacterial strain producing daptomycin is realized through the following steps:

[0047] (1) Solid culture: inoculate the bacterial strain on a slant medium, and culture it in a 30° C. incubator for 5 to 7 days; wait for it to produce spores, and store it for future use.

[0048] (2) Liquid culture: the bacteria were inoculated in the seed medium TSB, cultivated for 36 h on a shaker with a rotating speed of 250 rpm at a temperature of 30° C., and inoculated in the fermentation medium YEME (4%) according to a 4% transfer amount. Cultivate for 4-6 days at a temperature of 30° C. on a shaker with a rotational speed of 250 rpm. From 48h, add 0.1% capric acid solution by volume every 12h.

Embodiment 3

[0050] Carry out shake bottle fermentation by embodiment 2, 144h sampling, this moment, bacterial liquid color sees image 3 , it can be seen that compared with the traditional daptomycin-producing bacteria, the pigment is completely eliminated, which is beneficial to subsequent isolation. Add methanol according to the volume 1:1, shake and centrifuge and carry out HPLC (high performance liquid chromatography) identification on the supernatant, as attached Figure 5 As shown, daptomycin can be detected around 21 min, and the output calculated by daptomycin standard curve is 31.5 mg / L.

[0051] HPLC detection method: HPLC column: XDB-C18

[0052] Phase A: H 2 O+0.05% formic acid; B phase: acetonitrile+0.05% formic acid; B phase gradient 5%-100%, 35min.

[0053] The absorption peak of daptomycin can be seen at 21min.

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Abstract

The invention provides a new strain of daptomycin-producing Streptomyces and its application, which is a subspecies of Streptomyces griseus, which is named Streptomyces griseus L340 (Streptomyces griseus), and its preservation number is CGMCC17921. The genetic character and fermentation unit of the strain provided by the invention are relatively stable, the culture and fermentation conditions are suitable for industrial production of daptomycin, and the pigment interference in the later separation and purification is eliminated. The Streptomyces provided by the present invention obtains the secondary metabolite daptomycin through fermentation, which has the advantages of no interference from pigment accumulation, cleaner metabolic profile, and obvious advantages in later stage separation and purification.

Description

technical field [0001] The invention belongs to the field of microorganisms, and relates to a new daptomycin-producing streptomyces strain and application thereof. Background technique [0002] Daptomycin is a cyclolipopeptide antibiotic with a new structure extracted from the fermentation broth of S. reseosporus. It was discovered by Eli Lilly and Company in the 1980s and developed by Cubist Pharmaceuticals in 1997. Successful, it not only has a novel chemical structure, but also its mode of action is different from any approved antibiotic: it hinders the biosynthesis of bacterial cell wall peptidoglycan by disrupting the transport of amino acids by the cell membrane, and changes the cytoplasmic membrane. It can destroy the function of bacterial cell membrane in many ways, and quickly kill Gram-positive bacteria. In addition to its ability to act on most clinically relevant Gram-positive bacteria, daptomycin is more important for isolates that have shown resistance to meth...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12P21/04C12R1/545
CPCC12N1/20C12P21/02C07K7/08C12R2001/545C12N1/205
Inventor 李永泉方教乐毛旭明陈新爱
Owner ZHEJIANG UNIV
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