CN108384819B

CN108384819B - A kind of culture medium and fermentation method for fermenting tacrolimus

Info

Publication number: CN108384819B
Application number: CN201710063681.XA
Authority: CN
Inventors: 胡海峰; 边佳昕; 陈昌发; 闵涛玲
Original assignee: Shanghai Institute of Pharmaceutical Industry; China State Institute of Pharmaceutical Industry
Current assignee: Shanghai Institute of Pharmaceutical Industry; China State Institute of Pharmaceutical Industry
Priority date: 2017-02-03
Filing date: 2017-02-03
Publication date: 2021-06-25
Anticipated expiration: 2037-02-03
Also published as: CN108384819A

Abstract

The invention discloses a culture medium for fermenting tacrolimus and a fermentation method. The culture medium comprises a carbon source and a nitrogen source, wherein the carbon source comprises 30-70g/L of soluble starch, 5-20g/L of fructose and 5-20g/L of lactose, and the nitrogen source comprises 1-40g/L of fish meal peptone and 10-30g/L of yeast extract. The culture medium or the fermentation method of the invention has high yield of the fermented tacrolimus, and the titer is 3 times of the fermentation titer of the existing culture medium and reaches more than 1000 mg/L.

Description

Culture medium for fermenting tacrolimus and fermentation method

Technical Field

The invention belongs to the technical field of industrial microorganisms, and particularly relates to a culture medium for fermenting tacrolimus and a fermentation method.

Background

Tacrolimus is a fermentation product separated from streptomyces, is a powerful immunosuppressant and is 100 times stronger than cyclosporine. Clinical tests prove that the traditional Chinese medicine composition has good curative effect in transplantation of heart, lung, intestine, bone marrow and the like, and plays an active role in various immune diseases in the middle and old years.

Tacrolimus is currently obtained mainly by fermentation of bacterial microorganisms (or actinomycete fermentation). The fermentation yield of the tacrolimus is improved mainly by strain modification and improvement of culture medium components.

Wherein, the modification of the strain mainly comprises the modes of amplifying gene segments such as acetyl CoA synthetase, carboxylase and the like, and improving the tolerance of the strain to a target product (Jung S, Moon S, Lee K, et al.

With regard to the improvement of the medium composition, the prior art has predominantly used the addition of precursor amino acids (Singh B P, Behera B K. Regulation of microorganisms production by alteration of primary source of carbohydrates and amino acids [ J]Letters in Applied Microbiology,2009,49(2): 254-9), pipecolic acid (II) (III)

J,Gajzlerska W,Klimaszewska M.Enhancement of tacrolimus productivity in Streptomyces tsukubaensis by the use of novel precursors for biosynthesis[J].Enzyme&Microbiological Technology,2012,51(6-7):388-95), methyl oleate (Mo S J, Ban Y H, Park J W, et al].Journal of Industrial Microbiology&Biotechnology,2009,36(12):1473-].Journal of Microbiology&Biotechnology,2007,17(10): 1638-1644): improving lipase activity), composite carbon source (mixture ratio of dextrin and soluble starch) (Wanya, Huanghe, Bijiaxin, etc.. Tacrolimus engineering bacteria construction and preliminary fermentation process optimization [ J]2016,47(2):152-,

J.Use of three-carbon chain compounds as biosynthesis precursors to enhance tacrolimus production in Streptomyces tsukubaensis[J]new Biotechnology,2015,32(1):32-39)) to increase the yield of tacrolimus.

At present, the components of the culture medium are improved, and most of the components need to be directed to specific tacrolimus production strains; in other words, different Tacrolimus producing strains, such as the wild strain of the Tacrolimus producing strain Streptomyces tsukubaensis No.9993 (CGMCC No.0083) and the genetically engineered strain thereof, need to improve different components in the culture medium to obtain higher yield of Tacrolimus (construction of Tacrolimus engineered strain and optimization of the primary fermentation process [ J ] J.China J.Med.Med.industrials, 2016,47(2): 152-157). The culture medium has weak universality, and the process of constructing the strain is complicated, large in investment and long in time consumption; in addition, the existing culture medium has complex formula and high cost, and inorganic salt containing heavy metal ions is mostly adopted. The problems are not beneficial to the industrial production of tacrolimus.

Disclosure of Invention

Therefore, the technical problem to be solved by the present invention is to solve the above-mentioned drawbacks of the prior art, and provide a novel culture medium and fermentation method for fermenting tacrolimus, which can simplify the formulation, reduce metal pollution, and is suitable for different streptomyces tsubaensis strains producing tacrolimus, improve the fermentation unit of tacrolimus, and improve the production efficiency, so as to be suitable for industrial production.

The technical scheme for solving the technical problems is as follows:

one of the technical solutions adopted to solve the above technical problems of the present invention is: a culture medium for fermenting tacrolimus comprises a carbon source and a nitrogen source, wherein the carbon source comprises 30-70g/L of soluble starch, 5-20g/L of fructose and 5-20g/L of lactose; the nitrogen source comprises 1-40g/L of fish meal peptone and 10-30g/L of yeast extract.

Preferably, the carbon source is 30-70g/L of soluble starch, 5-20g/L of fructose and 5-20g/L of lactose, and/or the nitrogen source is 1-40g/L of fish meal peptone and 10-30g/L of yeast extract.

Preferably, the culture medium further comprises amino acid, and the content of the amino acid is preferably 1-10 g/L. The amino acid is conventional in the art, and is preferably L-arginine.

More preferably, the culture medium further comprises an inorganic salt, wherein the inorganic salt is preferably (NH)₄)₂SO₄And anhydrous magnesium sulfate; said (NH)₄)₂SO₄And the content of anhydrous magnesium sulfate is preferably 1 to 20g/L and 1 to 10g/L, respectively.

Even more preferably, the culture medium further comprises inorganic salt CaCO₃Said CaCO₃Has excellent content ofIs selected to be 0.5-1.5 g/L.

Most preferably, the medium further comprises natural sources of natural plant extracts; the content of the natural killer is preferably 1 to 20 g/L.

In the present invention, the pH of the medium is usually, naturally or by regulation, and preferably pH 6.2-6.8.

The second technical scheme adopted by the invention for solving the technical problems is as follows: a method for the fermentation of tacrolimus comprising the steps of:

(1) inoculating a seed solution of the streptomyces tsukubaensis producing tacrolimus into the fermentation culture medium for liquid fermentation culture to obtain a fermentation liquid;

(2) isolating tacrolimus from the fermentation broth.

In the invention, the Streptomyces tsukubaensis can be various existing Streptomyces tsukubaensis producing tacrolimus, and is preferably Streptomyces tsukubaensis (S.tsukubaensis) No.9993, namely CGMCC No. 0083.

The preparation and inoculation method of the seed liquid for fermentation culture is conventional in the field, preferably, the culture time of the first-stage seeds is 48-72h (h), the culture time of the second-stage seeds is 24-48h, the inoculation amount of the second-stage seeds is 1-2%, and the inoculation amount of the fermentation liquid is 5-15%; the percentage is volume percentage.

In a preferred embodiment of the present invention, the primary seed cultivation time is 60 hours, the secondary seed cultivation time is 48 hours, the secondary seed inoculation amount is 1%, and the fermentation broth inoculation amount is 10%; the percentage is volume percentage.

On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.

The reagents and starting materials used in the present invention are commercially available.

The positive progress effects of the invention are as follows: the invention provides a fermentation medium and a corresponding fermentation method which are beneficial to efficient fermentation production of tacrolimus by streptomyces. By comprehensively proportioning the carbon-nitrogen source of the culture medium formula, the formula is simplified, the cost is reduced, and Cu in the prior art is reduced²⁺、Zn²⁺、Mn²⁺And Co²⁺The use of metal ions and pollution caused by the metal ions are avoided, in addition, the culture medium disclosed by the invention can improve the fermentation yield of the streptomyces tsukubaensis on the tacrolimus, has an improvement effect on different wild streptomyces tsukubaensis bacteria and genetic engineering bacteria for producing the tacrolimus, and is strong in universality. By combining the fermentation method provided by the invention, the fermentation titer of the tacrolimus can reach more than 1000mg/L at most, which is about 3 times of the fermentation titer of the tacrolimus in the prior art, and the method is beneficial to the industrial production of the tacrolimus.

Drawings

FIG. 1 is a liquid phase detection profile of tacrolimus.

FIG. 2 is a bar graph of relative titers obtained after various seed culture times and inoculum sizes were used.

Detailed Description

The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.

The material sources in the following examples are:

fish meal peptone, anhydrous magnesium sulfate, soluble starch, (NH)₄)₂SO₄Fructose, yeast extract, lactose and CaCO₃Purchased from the national drug group; l-arginine was purchased from Shanghai Wei Shuzo; the natural product of DIDEN is purchased from CHUANKANG medicine.

The following percentages are by volume.

Example 1 fermentation culture method and specification of Standard titer

1. Orthogonal experiment for determining seed growth time and inoculum size

1) Preparation of seeds

Under the aseptic condition, a Streptomyces tsukubaensis (CGMCC) strain CGMCC No.0083 (purchased from CGMCC) is inoculated to a slant culture medium, namely SIPI-F001, the slant of the inoculated strain is placed in a constant temperature incubator at 28 ℃, the passage period is 10-14d (days), the inoculation period is 18-20d, the strain is cultured for 15 days generally according to the growth condition of the colony, and mature spores are obtained. The mature spores were then inoculated into 250ml shake flasks (or 750ml shake flasks with 100ml seed medium) containing 30ml seed medium and cultured on a shaker at 240rpm and 28 ℃. Then, under the same conditions as other conditions, the culture time of the primary seed, the inoculation amount of the secondary seed, the culture time of the secondary seed, and the inoculation amount of the fermentation liquid were examined specifically according to table 1 below.

TABLE 1 orthogonal experiments on seed growth time and inoculum size

2) Composition of the culture Medium used

Slant medium (ISP 4): 10g/L of soluble starch, 2g/L of ammonium sulfate, 1g/L of dipotassium phosphate, 1g/L of magnesium sulfate heptahydrate, 0.01g/L of ferrous sulfate, 0.01g/L of manganese chloride, 0.01g/L of zinc sulfate, CaCO₃2g/L, agar 20g/L, pH7.2, 121 ℃ for 25 minutes.

Seed culture medium: 10g/L of soluble starch, 15g/L of oral glucose, 20g/L of beef extract, 3g/L of ammonium sulfate, 2.5g/L of yeast powder, 5g/L of peanut meal, CaCO₃2g/L, seed bottle, pH 7.0, 121 ℃ for 25 minutes.

The fermentation medium comprises the following components: 50g/L of soluble starch, 20g/L of fish meal peptone, 20g/L of yeast extract, 10g/L of fructose and 10g/L of lactose, (NH)₄)₂SO₄10g/L anhydrous magnesium sulfate 5g/L, L-arginine 5g/L and 10g/L, CaCO g/L natural sodium chloride₃ 1g/L、pH 6.5。

3) Fermentation of

The mature seeds in the prepared fermentation liquor are inoculated in a fermentation culture medium according to the inoculation amount shown in the table 1, the loading amount is 30mL/250mL, the mature seeds are cultured for 168 hours at 25 ℃ and 250rpm in a rotary shaking table, 1mL of the fermentation liquor is taken out, the fermentation liquor is centrifuged, and the supernatant is taken out for measuring the content of the tacrolimus by using High Performance Liquid Chromatography (HPLC).

The method comprises the following specific steps:

the instrument comprises the following steps: agilent 1200 high performance liquid phase;

and (3) analyzing the column: moon Xue

XB-C18(2.1×150mm，3um)；

Mobile phase: a: 10% acetonitrile contains 0.1% phosphoric acid, B: 100% acetonitrile;

column temperature: 40 ℃;

wavelength: 200 nm;

sample introduction amount: 5 mu l of the solution;

table 2 flush procedure:

time/min	B(％)	Flow rate ml/min	Pressure (bar)
				0	50	0.3	250
1.0	50	0.3	250
				13.0	85	0.3	250
15.0	85	0.3	250
				16.0	50	0.3	250
22.0	50	0.3	250

The liquid phase detection pattern of tacrolimus is shown in figure 1.

The results are shown in FIG. 2, in which the experimental group numbered 1 is the control group. Through comparison, the relative titer of the streptomyces tsukubaensis cultured under the inoculation amount and the culture time of the experiment with the number 6 is 1082.81mg/L at most, and the Tacrolimus (TAC) yield is 6.45 mg/h.

Table 3 results of seed orthogonality experiments: relative titer and TAC yield for different experiments

Therefore, in the following examples, the culture time of the first-stage seeds is 60 hours, the culture time of the second-stage seeds is 48 hours, the inoculation amount of the second-stage seeds is 1%, and the inoculation amount of the fermentation liquid is 10%.

Example 2

1. Cultivation of seeds

Under the aseptic condition, a strain of streptomyces tsukubaensis, namely SIPI-F001 is inoculated to a slant culture medium, the slant of the inoculated strain is placed in a constant temperature incubator at 28 ℃, and the culture is carried out for 15 days, so as to obtain mature spores. Then inoculating the mature spores into a 250ml shake flask filled with 30ml of seed culture medium, culturing for 60h on a shaking table at the speed of 240rpm and the temperature of 28 ℃ to obtain first-stage seeds, transferring the first-stage seeds into the 250ml shake flask filled with 30ml of seed culture medium according to the proportion of 1%, and culturing for 48h on second-stage seeds under the same conditions to obtain mature seeds. The seed medium and the slant medium were the same as in example 1.

2. Fermentation of

The mature seeds in the prepared fermentation broth were inoculated in the following fermentation medium at an inoculum size of 10%. The fermentation conditions were the same as in example 1.

The fermentation medium comprises the following components: 50g/L of soluble starch, 20g/L of fish meal peptone, 20g/L of yeast extract, 10g/L of fructose, 10g/L of lactose and (NH)₄)₂SO₄10g/L and anhydrous magnesium sulfate 5g/L, pH 6.5. The TAC titer is measured by high performance liquid chromatography to be 823.5317mg/L, and the TAC yield is 4.90 mg/h.

Example 3

The fermentation medium comprises the following components: 50g/L of soluble starch, 20g/L of fish meal peptone, 20g/L of yeast extract, 10g/L of fructose, 10g/L of lactose and (NH)₄)₂SO₄10g/L, anhydrous magnesium sulfate 5g/L and L-arginine 5g/L, pH 6.5. The same as in example 2. The TAC titer is measured by the high performance liquid phase to be 1031.169mg/L, and the TAC yield is 6.14 mg/h.

Example 4

The fermentation medium comprises the following components: 30g/L of soluble starch, 5g/L of fructose, 5g/L of lactose, 1g/L of fish meal peptone, 10g/L of yeast extract, (NH)₄)₂SO₄1g/L anhydrous magnesium sulfate 1g/L, L-arginine 1g/L, natural sodium chloride 1g/L and CaCO₃0.5g/L, pH6.2, as in example 2. The TAC titer is measured by high performance liquid chromatography to be 850.2672mg/L, and the TAC yield is 5.06 mg/h.

Example 5

The fermentation medium comprises the following components: 50g/L soluble starch, 10g/L fructose, 10g/L lactose, 20g/L fish meal peptone and 20g/L yeast extract, pH 6.5, the same as example 2. The TAC titer is 675.2141mg/L by high performance liquid chromatography, and the TAC yield is 4.024 mg/h.

Example 6

70g/L of soluble starch, 20g/L of fructose, 20g/L of lactose, 40g/L of fish meal peptone, 30g/L of yeast extract, (NH)₄)₂SO₄20g/L anhydrous magnesium sulfate 10g/L, L-arginine 10g/L, 20g/L natural sodium chloride and CaCO₃1.5g/L, pH6.8, as in example 2. The TAC titer is measured by high performance liquid chromatography to be 809.2933mg/L, and the TAC yield is 4.82 mg/h.

Comparative example 1

The fermentation medium comprises the following components: 50g/L soluble starch, 20g/L fish meal peptone, pH 6.5, the same as example 2. The TAC titer is measured by high performance liquid chromatography to be 242.6045mg/L, and the TAC yield is 1.44 mg/h.

Comparative example 2

The fermentation medium comprises the following components: 50g/L of soluble starch, 20g/L of fish meal peptone and 20g/L of yeast extract, pH 6.5, the same as in example 2. The TAC titer is measured by high performance liquid chromatography to be 416.73mg/L, and the TAC yield is 2.50 mg/h.

Effect example 1

The fermentation medium formula of example 1 is compared with the fermentation level of the strain TAC reported in the literature as follows:

reference documents:

[1]Singh B P,Behera B K.Regulation of tacrolimus production by altering primary source of carbons and amino acids[J].Letters in Applied Microbiology,2009,49(2):254-9.

[2]

J,Gajzlerska W,Klimaszewska M.Enhancement of tacrolimus productivity in Streptomyces tsukubaensis by the use of novel precursors for biosynthesis[J].Enzyme&Microbial Technology,2012,51(6-7):388-95.

[3]Mo S J,Ban Y H,Park J W,et al.Enhanced FK506 production in Streptomyces clavuligerus,CKD1119 by engineering the supply of methylmalonyl-CoA precursor[J].Journal of Industrial Microbiology&Biotechnology,2009,36(12):1473-1482.

[4]Kim H S,Park Y I.Lipase activity and tacrolimus production in Streptomyces clavuligerusCKD 1119 mutant strains.[J].Journal of Microbiology&Biotechnology,2007,17(10):1638-1644.

[5] (carbon source) Wangya, Huanghe, Bijiaxin, etc. the construction and preliminary fermentation process optimization of tacrolimus engineering bacteria [ J ] in China journal of pharmaceutical industry 2016,47(2): 152-.

[6]Gajzlerska W,Kurkowiak J,

J.Use of three-carbon chain compounds as biosynthesis precursors to enhance tacrolimus production in Streptomyces tsukubaensis[J].New Biotechnology,2015,32(1):32-39.

[7]Jung S,Moon S,Lee K,et al.Strain development of Streptomyces sp.for tacrolimus production using sequential adaptation.[J].Journal of Industrial Microbiology,2009,36(12):1467-71.

Effect example 2

Formula of a control group fermentation medium: 50g/L dextrin, 10g/L yeast powder, 5g/L corn steep liquor dry powder, 3g/L leucine and 1g/L, K piperidine-2-formic acid₂HPO₄ 1g/L、CuSO₄·5H₂O 0.003g/L、FeSO₄·7H₂O 0.0025g/L、MnCl₂·4H₂O 5×10^-4g/L、ZnSO₄·7H₂O 0.01g/L、CaCl₂·2H₂O 0.02g/L、CoCl₂·6H₂O 0.003g/L、CaCO₃ 1g/L、Mg₃(PO4)₂5g/L and 1g/L of natural enemy; pH7.2 (see reference [5] above)]) (ii) a The fermentation process, such as seed preparation, inoculum size, incubation temperature and incubation time, was the same as in example 2 above.

The fermentation medium formulation of example 1 was selected for the present invention.

The fermentation results were as follows:

	SIPI-F001	SIPI-F002	SIPI-F003
				control TAC production level (mg/L)	489.0435	402.1252	670.3715
Example 1 level of TAC production (mg/L)	1314.749	699.6951	793.0005

Wherein the genetically engineered bacteria SIPI-F002 and SIPI-F003 of Streptomyces tsukubaensis are obtained from Shanghai pharmaceutical industry research institute, or prepared by the method disclosed in the above reference [5], wherein the Streptomyces coelicolor M145 and Actinoplanes N902-109 for constructing SIPI-F002 and SIPI-F003 are obtained from Shanghai pharmaceutical industry research institute, and the vectors pLYW2 and pIJ8660 are obtained from GIBCO-BRL. The Streptomyces tsukubaensis No.9993 is CGMCC No. 0083.

As can be seen from the above table, the fermentation medium of the invention used for other engineering strains also has different degrees of promotion effects on the production of tacrolimus, is not limited to the experimental strain SIPI-F001, and has a certain degree of promotion effects on other strains producing the same target product.

Claims

1. A medium for fermenting tacrolimus comprising a carbon source, a nitrogen source and inorganic salts; it is characterized in that the carbon source comprises 30-70g/L of soluble starch, 5-20g/L of fructose and 5-20g/L of lactose, the nitrogen source comprises 1-40g/L of fish meal peptone and 10-30g/L of yeast extract, and the inorganic salt is (NH)₄)₂SO₄And anhydrous magnesium sulfate.

2. The culture medium of claim 1, wherein the carbon source is 30-70g/L soluble starch, 5-20g/L fructose and 5-20g/L lactose, and/or the nitrogen source is 1-40g/L fish meal peptone and 10-30g/L yeast extract.

3. The culture medium of claim 1, further comprising an amino acid in an amount of 1-10 g/L.

4. The culture medium of claim 3, wherein the amino acid is L-arginine.

5. The culture medium of claim 4, wherein the (NH) is₄)₂SO₄And the content of anhydrous magnesium sulfate is 1-20g/L and 1-10g/L respectively.

6. The culture medium of claim 3, wherein the inorganic salt further comprises CaCO₃。

7. The culture medium of claim 6, wherein the CaCO₃The content of (B) is 0.5-1.5 g/L.

8. The culture medium of any one of claims 1-7, wherein the culture medium further comprises bubbled.

9. The culture medium of claim 8, wherein said natural gas is present in an amount of 1-20 g/L.

10. The culture medium of claim 1, wherein the pH of the culture medium is 6.2 to 6.8.

11. A fermentation method of tacrolimus is characterized by comprising the following steps:

(1) inoculating a seed liquid of tacrolimus-producing Streptomyces tsukubaensis (CGMCC No.0083) into a culture medium of any one of claims 1 to 10 for liquid fermentation culture to obtain a fermentation liquid;

(2) isolating tacrolimus from the fermentation broth.

12. The fermentation method of claim 11, wherein the seed solution is prepared by culturing the first seed for 48-72 hours, culturing the second seed for 24-48 hours, inoculating the second seed with 1-2%, and inoculating the fermentation solution with 5-15%; the percentages are volume percentages.

13. The fermentation process of claim 12, wherein the primary seed is incubated for 60 hours, the secondary seed is incubated for 48 hours, the secondary seed is inoculated by 1%, and the broth is inoculated by 10%.