Daptomycin-producing Streptomyces strain and use thereof

Abstract

The present invention provides a novel daptomycin-producing Streptomyces strain and use thereof, the strain is a subspecies of streptomyces griseus, named Streptomyces griseus L340, and the preservation number is CGMCC17921. The genetic character and fermentation unit of the strain provided by the present invention are relatively stable, the culture and fermentation conditions are suitable for industrial production of daptomycin, and the interference of pigment in later separation and purification is eliminated. The secondary metabolite daptomycin is obtained through fermentation of the Streptomyces provided by the present invention, which has the advantages of no interference of pigment accumulation, cleaner metabolic spectrum and thus is obviously superior in later separation and purification.

Claims

1. A novel daptomycin-producing Streptomyces strain, wherein the Streptomyces strain is classified and named as Streptomyces griseus L340 preserved in China General Microbiological Culture Collection Center with a preservation date of Jun. 12, 2019 and a preservation number of CGMCC No. 17921, and the strain is kept in a media with a buffer to maintain the pH 5-11.

2. A method for culturing the Streptomyces strain according to claim 1, comprising the following steps: (1) solid culture: inoculating the strain on an agarslantculture-medium, and culturing in an incubator at 30 C. for 5-8 days; and (2) liquid culture: cutting spores from the agarslantculture-medium and inoculating into a seed broth, culturing on a shaker with a rotation speed of 250 rpm at 30 C. for 36 hours, then inoculating a transfer amount of 4% into a fermentation medium, and culturing on a shaker with a rotation speed of 250 rpm at 30 C. for 4-6 days.

3. The method according to claim 2, wherein the agarslantculture-medium in the step (1) is a R5 medium, each liter of medium which contains 103 g of sucrose, 10 g of glucose, 5 g of yeast extract, 0.1 g of casein hydrolysate, 0.25 g of K.sub.2SO.sub.4, 10.12 g of MgCl.sub.2.Math.6H.sub.2O, 2 mL of a trace element solution, 5.73 g of TES buffer, and 0.3 g of proline, which are diluted by water to a volume of 1 L, and 22 g of agar is added to a solid medium; in the step (2), the seed broth is a Trypticase Soy Broth medium: TSB 2%, PEG 6000 5%, which are diluted by water to a volume of 1 L, wherein the percentages are percentages by mass; and the fermentation medium in the step (2) is a yeast malt medium: 0.3% yeast extract, 0.3% malt extract, 0.5% tryptone, and 4% glucose, which are diluted to a volume of 1 L.

4. A production method of daptomycin using the Streptomyces griseus L340 according to claim 1, comprising the following steps: (1) cutting spores from an agarslantculture-medium, inoculating the spores into a seed broth, and culturing on a shaker with a rotation speed of 250 rpm at 30 C. for 36 hours until a bacterial liquid is sticky and granular; (2) harvesting 4% by volume of the bacterial liquid from the seed broth, inoculating into the fermentation medium, and culturing on a shaker with a rotation speed of 250 rpm at 30 C.; and after 48 hours, feeding decanoic acid in 0.1% by volume of the fermentation medium every 12 hours; (3) after 144 hours, treating the fermentation broth with methanol of equal volume, and subjecting a supernatant obtained after centrifugation to subsequent separation and purification to obtain the daptomycin.

5. The production method according to claim 4, wherein, definitions of the agarslantculture-medium, the seed broth and the fermentation medium are the same as in claim 3.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1: a monoclonal morphology of Streptomyces colonies cultured on a R5 plate medium for 7 days, wherein, the monoclonal irregularly round as a whole, with a slightly convex surface and radial cracks; the spores are gray-white and easy to fall off, which are concentrated at the edge of the monoclonal.

(2) FIG. 2: the morphology of bacteria in Streptomyces fermentation broth provided by the present invention.

(3) FIG. 3: comparison of fermentation broth morphology between the Streptomyces and the traditional daptomycin-producing strain, i.e., Streptomyces roseosporus, in a traditional daptomycin-producing medium, a YEME(4%) liquid medium; wherein, FIG. 3-A shows the broth morphology after 144 h of fermentation, red representing Streptomyces roseosporus, and white representing Streptomyces of the present application, the right being the same; FIG. 3-B shows the supernatant and mycelial morphology of the fermentation liquid after centrifugation. It can be seen that a large amount of red pigment is produced in the fermentation process of Streptomyces roseosporus, while the present invention declares that no pigment substance is produced in either the mycelium or the supernatant of Streptomyces (the light yellow color of the supernatant is the background color of the medium).

(4) FIG. 4: the phylogenetic tree of the 16s rDNA sequence shows that the homology with streptomyces griseus is 99.41%, and the homology with Streptomyces coelicolor is 96.9%, which indicates that the present Streptomyces has similar genetic relationship with streptomyces griseus.

(5) FIG. 5: the metabolic spectrum of the fermentation broth of the Streptomyces of the present application in the YEME(4%) fermentation experiment after 144 hours detected by HPLC.

DESCRIPTION OF EMBODIMENTS

(6) The present invention will be further explained with reference to drawings and examples.

(7) The experimental methods in the following examples are all conventional methods, unless otherwise specified.

(8) The medium used in the examples:

(9) A slant R5 agar medium: each liter of the medium contains 103 g of sucrose, 10 g of glucose, 5 g of yeast extract, 0.1 g of casein hydrolysate, 0.25 g of K.sub.2SO.sub.4, 10.12 g of MgCl.sub.2.Math.6H.sub.2O, 2 mL of a trace element solution, 5.73 g of TES buffer, 0.3 g of L-proline, which are diluted by water to a volume of 1 L (22 g of agar is added to a solid medium), which is sterilized at 115 C. for 25 min. After sterilization, the following solutions are added in turn: 10 mL of 5% KH.sub.2PO.sub.4, 4 mL of 5M CaCl.sub.2.2H.sub.2O, 700 L of 10M NaOH, which are shaken well, poured onto the plate and solidified for later use. A trace element storage solution: 40 mg of ZnCl.sub.2, 200 mg of FeCl.sub.3.Math.6H.sub.2O, 10 mg of CuCl.sub.2.Math.2H.sub.2O, 10 mg of MnCl.4H.sub.2O, 10 mg of Na.sub.2B.sub.4O.sub.7.Math.10H.sub.2O and 10 mg of (NH.sub.4) Mo.sub.7O.sub.24.Math.4H.sub.2O, to which water is added to a volume of 1 L for later use.

(10) A seed broth (TSB): TSB 2%, PEG 6000 5%, which are diluted by water to a volume of 1 L; wherein, the percentages are by mass. The medium is sterilized at 115 C. for 25 min for later use.

(11) A fermentation medium (YEME): 0.3% yeast extract, 0.3% malt extract, 0.5% tryptone, and 4% glucose, which are diluted by water to a volume of 1 L; wherein, the percentages are by mass. The medium is sterilized at 115 C. for 25 min for later use.

EXAMPLE 1

(12) The present invention provides a novel daptomycin-producing Streptomyces strain. Compare with the traditional daptomycin-producing bacteria Streptomyces roseosporus, the novel daptomycin-producing strain provided by the invention has the great advantage that pigment substances are not produced in the process of subculture and fermentation, thereby reducing the difficulty for subsequent separation and purification operations. The strain provided by the present invention is preserved in China General Microbiological Culture Collection Center with the preservation date of Jun. 12, 2019, and the preservation number of CGMCC17921.

(13) The apparent characteristics in different media are shown in Table 1, in which the colony morphology on a R5 medium is shown in FIG. 1, and the mycelium morphology on a YEME medium is shown in FIG. 2. See Table 2 for physiological and biochemical characteristics of L10.

(14) Compared with other 17 Streptomyces strains (see FIG. 4 for the phylogenetic tree), the sequencing results of 16srDNA of Streptomyces declared by the present invention show that the homology with streptomyces griseus is 99.41%, and the homology with Streptomyces coelicolor is 96.9%, indicating that Streptomyces has similar genetic relationship with streptomyces griseus.

(15) TABLE-US-00001 TABLE 1 Cultural characteristics of strains: Color of the back Soluble Aerial side of the Medium pigment mycelium medium YEME none white yellowish ISP4 none white yellowish MM grey white white R5 none white yellowish

(16) TABLE-US-00002 TABLE 2 Physiological and biochemical characteristics of strains: Characteristics Results pH range for growth pH < 4 pH = 5-11 + pH = 12 W NaCl tolerance 3% NaCl + 5% NaCl W 7% NaCl Growth temperature 30 C. + 37 C. Melanin production Milk coagulation + Starch hydrolysis + Gelatin liquefaction + Used carbon source D-glucose + D-fructose + Maltose + Sucrose + Glycerol + Inositol + D-mannitol + L-arabinose D-xylose L-rhamnose Sodium acetate Raffinose Sorbitol Note: W indicates a weak positive result, + indicates a positive result and indicates a negative result

EXAMPLE 2

(17) The method for culturing the daptomycin-producing Streptomyces strain is as below:

(18) (1) solid culture: inoculating the strain on a agarslantculture-medium, and culturing the strain in an incubator at 30 C. for 5-8 days;

(19) (2) liquid culture: inoculating the strain into a seed broth, culturing the strain on a shaker with a rotation speed of 250 rpm at 30 C. for 36 hours, then inoculating the strain into a fermentation medium based on a transfer amount of 4%, and culturing the strain on a shaker with a rotation speed of 250 rpm at 30 C. for 4-6 days; feeding 0.1% by volume of decanoic acid every 12 hours after 48 hours.

EXAMPLE 3

(20) Shake flask fermentation was carried out according to Example 2, and samples were taken after 144 h hours. The color of the bacterial liquid at this time is shown in FIG. 3, which shows that compared with traditional daptomycin-producing strains, pigment is completely eliminated, which is beneficial to subsequent separation. Methanol was added in a volume ratio of 1:1, and the supernatant was identified by HPLC (high performance liquid chromatography) after shaking centrifugation. As shown in FIG. 5, daptomycin could be detected before and after 21 min, and the yield calculated by the standard curve of daptomycin was 31.5 mg/L.

(21) The HPLC detection method is: HPLC chromatographic column: XDB-C18

(22) Phase A: H.sub.2O+0.05% formic acid; phase B: acetonitrile +0.05% formic acid; Gradient of phase B is 5%-100%, 35 min.

(23) The absorption peak of daptomycin can be seen at 21 min.