A culture method for improving the yield of tacrolimus fermentation

Abstract

The application discloses a culture method for improving the fermentation yield of tacrolimus and belongs to the technical field of tacrolimus production. The culture method for improving the fermentation yield of tacrolimus comprises the following steps: inoculating Streptomyces tsukuba into a fermentation culture medium for fermentation culture to obtain a tacrolimus fermentation liquor; and performing separation and purification on the tacrolimus fermentation liquor to obtain tacrolimus; and adding macroporous adsorption resin into the fermentation culture medium according to the fermentation time during the fermentation process. The method for improving the fermentation yield of tacrolimus controls the carbon and nitrogen source concentration and pH in the fermentation process and supplements macroporous resin, improves the secondary metabolism efficiency, adsorbs tacrolimus in the fermentation liquor through the macroporous adsorption resin, reduces the negative feedback inhibition effect on the secondary metabolism of the production strain caused by high-concentration tacrolimus, and significantly improves the tacrolimus yield.

Description

Technical Field

[0001] This invention relates to the field of tacrolimus production technology, specifically to a cultivation method for increasing tacrolimus fermentation yield. Background Technology

[0002] Tacrolimus (FK506, brand name Prograf) is an immunomodulatory 23-membered macrolide antibiotic widely used in clinical treatment. It works by inhibiting the production of interleukin-2, thereby preventing T-cell activation. Its immunosuppressive activity is 10-100 times that of cyclosporine A. Clinically, it is often used as an immunosuppressant after organ transplantation and also plays an important role in the treatment of diseases caused by immune system abnormalities such as lupus erythematosus and psoriasis, showing broad application prospects. In China, tacrolimus sales have grown rapidly, from approximately 1.3 billion yuan in 2013 to approximately 5.8 billion yuan in 2022.

[0003] Previous studies have shown that FK506 is synthesized in *Streptomyces tsukuba* through a process where polyketide synthase (PKS) recognizes 4,5-dihydroxy-1-cyclohexenylcarboxylic acid (DHCHD) derived from branched acid, leading to a complete polyketide chain through a series of elongations. Subsequently, non-ribosomal peptide synthase (NRPS) introduces L-piperidinecarboxylic acid, synthesized from L-lysine, to form a cyclic polyketide backbone. Finally, substituent modification is completed under the action of O-methyltransferase and P450 enzymes. While there are methods for the complete in vitro synthesis of FK506 via chemical synthesis, the complexity, high cost, and low yield mean that microbial fermentation remains the primary method for production.

[0004] Patent CN118064517A discloses a method for increasing the yield of tacrolimus synthesized by *Streptomyces tsukuba* fermentation. Specifically, it discloses fermentation under conditions of timed and quantitative addition of exogenous factors. The conclusion was that when dimethyl phthalate was added for 0 hours at a concentration of 0.2 g / L, and 3,4-dihydroxy-1-phenylalanine was added for 72 hours at a concentration of 0.05 g / L, the yield of FK506 reached a maximum of 81.6 ± 1.8 mg / L, an increase of 45.9%. Xia et al., through metabolomics analysis, identified key metabolites closely related to tacrolimus synthesis and determined a reasonable feeding strategy, adding a final mass concentration at 96 hours. Sodium succinate at a concentration of 1.5 g / L increased the yield of FK506 by 28% to 321 mg / L. Adding 1.5 g / L shikimic acid, 0.1 g / L sodium phenylpropionate, tyrosine, and tryptophan over 72 hours further increased the yield by up to 41%. Patent CN114214240A discloses a mutant strain of *Streptomyces tsukuba* and its application in the co-production of tacrolimus and dienotacrolimus, specifically revealing that a strain with a maximum yield of 582 mg / L was obtained through mutagenesis of a tacrolimus-producing strain. Huang et al. increased the FK506 concentration by approximately 255% to 398 mg / L by combining overexpression of the fkbO, fkbP, fkbL, and fkbM genes in a strain. However, both of these patents suffer from complex processes, high costs, and low yields.

[0005] Due to the wide range of effective pharmacological activities and clinical applications of FK506, despite the fact that nearly forty years have passed since FK506 was first discovered and officially launched, the optimization of fermentation methods aimed at increasing FK506 yield has never stopped, but the scale-up production in fermenters is still unsatisfactory. Summary of the Invention

[0006] The purpose of this invention is to provide a cultivation method for increasing the fermentation yield of tacrolimus, thereby solving the technical problems of low fermentation efficiency and low yield in the scale-up production of tacrolimus in fermenters in the prior art.

[0007] To achieve the above objectives, one embodiment of the present invention provides a cultivation method for increasing tacrolimus fermentation yield, comprising the following steps:

[0008] Streptomyces tsukuba was inoculated into a fermentation medium and fermented to obtain tacrolimus fermentation broth;

[0009] Tacrolimus fermentation broth was separated and purified to obtain tacrolimus;

[0010] During fermentation, macroporous adsorption resin is added to the fermentation medium according to the fermentation time.

[0011] In one preferred embodiment of the present invention, the pH of the fermentation system is controlled at 6.7-6.9 during the fermentation process.

[0012] In one preferred embodiment of the present invention, maltodextrin solution is added during the fermentation process according to the glycogen concentration in the fermentation broth.

[0013] In one preferred embodiment of the present invention, *Streptomyces tsukuba* requires slant and seed culture expansion before fermentation culture. The slant and seed culture expansion of *Streptomyces tsukuba* includes:

[0014] Streptomyces tsukuba was inoculated onto slant culture medium and cultured to obtain slant spores;

[0015] Slant spores were inoculated into seed culture medium and cultured for 20-40 hours to obtain seed liquid, which was then transferred to fermentation medium.

[0016] In one preferred embodiment of the present invention, macroporous adsorption resin is added to the fermentation culture medium after 24 hours of fermentation.

[0017] In one preferred embodiment of the present invention, the amount of macroporous adsorption resin added is 10g / L-50g / L.

[0018] In one preferred embodiment of the present invention, when the concentration of residual glycogen in the fermentation broth is less than 20 g / L, maltodextrin solution is added to the fermentation broth.

[0019] One preferred embodiment of the present invention includes fermentation culture conditions of 25℃-35℃ and shaking speed of 180r / min-280r / min.

[0020] In one preferred embodiment of the present invention, the fermentation medium comprises 40 g / L-80 g / L maltodextrin, 10 g / L-30 g / L yeast powder, 0.5 g / L-1 g / L (NH4)2SO4, 0.5 g / L-1 g / L CaCO3, with a pH of 7.5-8.5, and sterile water as the solvent.

[0021] One preferred embodiment of the present invention is that the slant culture medium comprises: 5g / L-20g / L malt extract, 2g / L-8g / L yeast extract, 1g / L-6g / L glucose, 5g / L-30g / L agar, with a pH of 7.0-7.4, and sterile water as the solvent.

[0022] In one preferred embodiment of the present invention, the seed culture medium comprises: glucose 1g / L-3g / L, corn starch 5g / L-15g / L, soybean meal 10g / L-40g / L, CaCO3 1g / L-5g / L, pH 7.0-7.3, and sterile water as the solvent.

[0023] In one preferred embodiment of the present invention, the Streptomyces tsukuba is Streptomyces tsukuba NRRL18488.

[0024] In summary, the beneficial effects of the present invention are as follows:

[0025] 1. The cultivation method for increasing tacrolimus fermentation yield of the present invention improves secondary metabolism efficiency by controlling the concentration of carbon and nitrogen sources and pH during fermentation and supplementing macroporous resin. Then, the tacrolimus in the fermentation broth is adsorbed by macroporous adsorption resin, which reduces the negative feedback inhibition of secondary metabolism of the producing strain caused by high concentration of tacrolimus, thus significantly increasing tacrolimus yield. Furthermore, under the experimental conditions of the present invention, the tacrolimus fermentation unit is increased to 2560 μg / mL, thereby improving economic benefits.

[0026] 2. The cultivation method of the present invention for increasing tacrolimus fermentation yield can significantly increase the unit yield of tacrolimus synthesized by Streptomyces tsukuba by controlling the seed growth time, residual sugar concentration and pH during fermentation and supplementing macroporous resin. It has important application value and solves the problem of low fermentation efficiency.

[0027] Other features and advantages of the invention will be set forth in the description which follows, and will be apparent in part from the description, or may be learned by practicing the invention. The objects and other advantages of the invention will be apparent from the effects described in the description and the accompanying drawings. Attached Figure Description

[0028] Figure 1 This is a high-performance liquid chromatogram of tacrolimus obtained in Example 1 of the present invention;

[0029] Figure 2 This is a high-performance liquid chromatogram of tacrolimus obtained from culture in Comparative Example 1 of this invention. Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0031] The endpoints and any values ​​of the ranges disclosed in this invention are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this invention.

[0032] This invention provides a cultivation method for increasing tacrolimus fermentation yield, comprising the following steps:

[0033] Step (1): Inoculate Streptomyces tsukubaensis into a fermentation medium and ferment to obtain tacrolimus fermentation broth; specifically, inoculate Streptomyces tsukubaensis.NRRL18488 into a fermentation medium and ferment at 25℃-35℃ and 180r / min-280r / min to obtain tacrolimus fermentation broth. Further, the fermentation conditions include: fermentation temperature of 28℃, shaking speed of 250r / min, fermentation medium including maltodextrin 40g / L-80g / L, yeast powder 10g / L-30g / L, (NH4)2SO4 0.5g / L-1g / L, CaCO3 0.5g / L-1g / L, pH of 7.5-8.5, and sterile water as solvent;

[0034] Before fermentation culture, *Streptomyces tsukuba* requires slant and seed culture expansion, specifically including:

[0035] S1: Inoculate Streptomyces tsukubaensis onto a slant culture medium to obtain slant spores; specifically, inoculate Streptomyces tsukubaensis.NRRL18488 onto a slant culture medium and culture at 28℃ for 7-10 days to obtain slant spores; wherein, the slant culture medium includes: malt extract 5g / L-20g / L, yeast extract 2g / L-8g / L, glucose 1g / L-6g / L, agar 5g / L-30g / L, pH 7.0-7.4, and sterile water as the solvent;

[0036] S2: Slant spores are inoculated into seed culture medium to obtain seed liquid, and then the seed liquid is transferred to fermentation medium. Specifically, fresh slant spores are inoculated into primary seed culture medium and cultured on a shaker at 28℃ and 250r / min for 20h-40h. Then, the inoculation is transferred to fermentation medium at a volume concentration of 7%-12%. The seed culture medium includes: glucose 1g / L-3g / L, corn starch 5g / L-15g / L, soybean meal 10g / L-40g / L, CaCO3 1g / L-5g / L, pH 7.0-7.3, and sterile water as the solvent.

[0037] Step (2): The tacrolimus fermentation broth was separated and purified to obtain tacrolimus;

[0038] During fermentation, macroporous adsorption resin is added to the fermentation medium according to the fermentation time. Specifically, macroporous adsorption resin is added to the fermentation medium after 24 hours of fermentation, and the amount of macroporous adsorption resin added is 10g / L-50g / L. Furthermore, the macroporous adsorption resin is added by mixing macroporous adsorption resin with 200mL of sterile water, sterilizing it at 121℃ for 30min, and then replenishing it after 24 hours of fermentation.

[0039] Furthermore, the pH of the fermentation system needs to be controlled during the fermentation process; specifically, after fermentation begins, the pH of the fermentation system should be controlled between 6.7 and 6.9.

[0040] Furthermore, during the fermentation process, maltodextrin solution needs to be added according to the glycogen concentration in the fermentation broth. Specifically, when the remaining glycogen concentration in the fermentation broth is detected to be below 20 g / L, maltodextrin solution is added to the fermentation broth, and the maltodextrin concentration in the maltodextrin solution is 500 g / L. Furthermore, the maltodextrin solution is added as follows: maltodextrin is dissolved in sterile water and the volume is adjusted to 800 mL, and then sterilized together with the fermentation broth at a high temperature of 121°C for 30 min.

[0041] For experimental methods not specifically described in the following examples, follow conventional methods and conditions, or select according to the product instructions. Additionally, in each example, "mg" is the unit of weight "milligram"; "min" is the unit of time "minute"; "mL" is the unit of volume "milliliters"; "mg / mL" is the unit of concentration "milligrams per milliliter"; "r / min" is the rotation speed in revolutions per minute; and "L" is the unit of volume "microliter".

[0042] Example 1

[0043] (1) Preparation of strain spore suspension

[0044] Streptomyces tsukuba NRRL-18488 was inoculated onto agar slant culture medium and cultured at 28°C for 8 days to obtain slant cells. The slant was washed with sterile water, and the eluent was transferred to a grinder for thorough grinding. The ground spore suspension was filtered through a sterile funnel into sterile EP tubes for later use. The cell concentration was adjusted to 10⁻⁶ cells / mL with sterile water. 7 CFU / mL, which is the spore suspension;

[0045] The slant culture medium consisted of 10 g / L malt extract, 4 g / L yeast extract, 4 g / L glucose, and 20 g / L agar, with a pH of 7.2 and sterile water as the solvent.

[0046] (2) Preparation of seed liquid

[0047] Add 500 μL of the spore suspension prepared in step (1) to the seed culture medium and culture it in a shaker at 28℃ and 250 r / min for 24 h to obtain a mature seed culture.

[0048] The seed culture medium consisted of 1.5 g / L glucose, 10 g / L corn starch, 20 g / L soybean meal, and 2 g / L CaCO3, with a pH of 7.0 and sterile water as the solvent.

[0049] (3) Preparation and yield determination of tacrolimus

[0050] ① Fermentation of Tacomos

[0051] The mature seed liquid prepared in step (2) was transferred to a 5L fermenter containing 2.5L of fermentation medium at an inoculation rate of 10% by volume. The fermentation culture was carried out at 28℃ and 250r / min for 10 days, and the pH was controlled at 6.7-6.9. After 24 hours of inoculation, macroporous adsorption was added at 15g / L of culture medium. When the reducing sugar concentration in the fermentation system was lower than 20g / L, maltodextrin solution was added to maintain the reducing sugar concentration in the tank at 10g / L-19g / L. After the fermentation was completed, the fermentation liquid was taken for tacomoric acid yield detection.

[0052] The fermentation medium consisted of 60 g / L maltodextrin, 18 g / L yeast powder, 1 g / L (NH4)2SO4, and 1 g / L CaCO3, with a pH of 7.5 and sterile water as the solvent.

[0053] ②High-performance liquid chromatography detection conditions

[0054] Column: Hypersil ODS C18, 4.6 mm × 250 mm

[0055] Mobile phase: Acetonitrile: Water = 60:40

[0056] Detection wavelength: 210nm

[0057] Column temperature: 60℃

[0058] Flow rate: 1 mL / min

[0059] Injection volume: 20 μL

[0060] Time: 30 min

[0061] ③ Tacomoril production determination

[0062] Take the fermentation broth as a sample, add 4 times the volume of methanol, mix thoroughly, place in a shaker at 14℃ and 120 rpm for 2 hours, let stand, take 1 mL of the supernatant, centrifuge at 12000 rpm for 5 minutes, filter through a 0.22 μm filter membrane, inject into a liquid chromatograph, and record the chromatogram. The high-performance liquid chromatogram of tacrolimus obtained in Example 1 is shown below. Figure 1 As shown;

[0063] Place 25 mg of tacrolimus standard in a 100 mL volumetric flask, add acetonitrile, mix thoroughly, and dilute to the mark with acetonitrile. Calculate the tacrolimus yield based on the ratio of sample peak area / standard solution peak area × standard solution concentration × sample dilution factor.

[0064] Comparative Example 1: Using seeds grown for 72 hours, fermentation medium without pH adjustment, and without the addition of macroporous resin and maltodextrin solution as Comparative Example 1, mature seed culture was obtained according to the method in step (2) of Example 1. The seed culture was transferred to a 5L fermenter containing 2.5L of fermentation medium at an inoculum concentration of 8%. Fermentation was carried out at 28℃ and 250r / min for 10 days. The fermentation broth was then used to detect tacrolimus yield, and the fermentation unit was measured to be 861μg / mL. The high-performance liquid chromatogram of tacrolimus obtained from Comparative Example 1 is shown below. Figure 2 As shown;

[0065] 1. Controlling seed growth time

[0066] Using seeds at different growth stages as experimental groups, mature seed solutions were prepared according to steps (1) and (2) in Example 1. The prepared mature seed solutions were transferred to a 5L fermenter containing 2.5L of fermentation medium at an inoculation rate of 10% by volume for fermentation. The fermentation was carried out at 28℃ and 250r / min for 10 days. The fermentation broth was then used to detect the tacomuslate yield. The measured fermentation units are shown in Table 1.

[0067] Table 1. Effects of different seed growth times on tacrolimus yield

[0068]

[0069]

[0070] As shown in Table 1, controlling the seed growth time can promote the tacrolimus production capacity of Streptomyces tsukuba. When the seed growth time is 24 h, the tacrolimus production can reach 1755 μg / mL.

[0071] 2. Add macroporous adsorption resin to the fermentation medium.

[0072] The experimental groups were formed by adding 15 g / L macroporous adsorption resin to the fermentation medium at different times after the start of fermentation. Mature seed liquid was prepared according to steps (1) and (2) in Example 1. The prepared mature seed liquid was transferred to a 5L fermenter containing 2.5L fermentation medium at an inoculation rate of 10% by volume for fermentation. The fermentation was carried out at 28℃ and 250 r / min for 10 days. The fermentation broth was used to detect the tacosimylate yield. The fermentation units were measured as shown in Table 2.

[0073] Table 2. Effects of macroporous adsorption resin addition at different times after fermentation on tacrolimus yield.

[0074] Group Add macroporous resin time (h) Tacrolimus unit (pg / mL) 1 48 1398 2 24 1741 3 0 1167 Control No addition 861

[0075] Table 2 shows that adding a certain proportion of macroporous adsorption resin to the fermentation medium can promote the production of tacrolimus by Streptomyces tsukuba. When the macroporous adsorption resin is added for 24 hours, the tacrolimus production can reach 1741 μg / mL.

[0076] 3. Control pH during fermentation

[0077] During fermentation, different pH levels of the fermentation medium were maintained as experimental groups. Mature seed culture was prepared according to steps (1) and (2) in Example 1. The prepared mature seed culture was transferred to a 5L fermenter containing 2.5L of fermentation medium at an inoculation rate of 10% by volume. Shake-flask fermentation was carried out at 28℃ for 10 days. The fermentation broth was then used to detect the tacomagnesia yield. The fermentation units were measured as shown in Table 3.

[0078] Table 3. Effects of maintaining different pH levels after fermentation on tacrolimus yield.

[0079] Group pH Tacrolimus unit (pg / mL) 1 8.0 302 2 7.5 654 3 6.8 1549 4 6.3 1285 Control No control 861

[0080] Table 3 shows that controlling the pH of the fermentation medium within a certain range during the fermentation cycle can promote the tacrolimus production capacity of Streptomyces tsukuba. When the pH is controlled at 6.8, the tacrolimus production can reach 1549 μg / mL.

[0081] 4. Controlling residual sugar concentration during fermentation by adding feed during fermentation.

[0082] During fermentation, the residual sugar concentration in the fermentation medium was maintained as the experimental group. Mature seed culture was prepared according to steps (1) and (2) in Example 1. The prepared mature seed culture was transferred to a 5L fermenter containing 2.5L of fermentation medium at an inoculation rate of 10% by volume for fermentation. The fermentation was carried out at 28℃ for 10 days. The fermentation broth was taken for tacomuslate yield detection. The fermentation units were measured as shown in Table 4.

[0083] Table 4. Effects of maintaining different residual sugar concentrations on tacrolimus yield after fermentation

[0084] Group Remaining carbon source concentration (g / L) Tacrolimus unit (pg / mL) 1 30-40 407 2 20-29 974 3 10-19 1588 Control No control 861

[0085] Table 4 shows that controlling the residual sugar concentration in the fermentation medium within a certain range during the fermentation cycle can promote the tacrolimus production capacity of Streptomyces tsukuba. When the residual sugar concentration is controlled at 10 g / L-19 g / L, the tacrolimus production can reach 1588 μg / mL.

[0086] 5. During fermentation, simultaneously control seed growth time, pH, residual sugar concentration, and the timing of macroporous resin addition.

[0087] During fermentation, the seed growth time, pH, residual sugar concentration, and macroporous resin addition time were controlled simultaneously in the experimental group. The parameters in Example 1 were all selected from the optimal values ​​obtained by controlling the influencing factors of seed growth time, pH, residual sugar concentration, and macroporous resin addition. Mature seed culture was prepared according to steps (1) and (2) in Example 1. The prepared mature seed culture was transferred to a 5L fermenter containing 2.5L of fermentation medium at a volume concentration of 10% for fermentation. Fermentation was carried out at 28℃ for 10 days. The fermentation broth was then used to detect tacomuslate yield, and the fermentation units were measured as shown in Table 5.

[0088] Table 5 shows the effects of simultaneously controlling seed growth time, pH, residual sugar concentration, and the time of macroporous adsorption resin addition after fermentation on tacrolimus yield.

[0089]

[0090] As shown in Table 5, controlling the seed growth time, pH, residual sugar concentration, and the time of adding macroporous adsorption resin after fermentation can promote the production of tacrolimus by Streptomyces tsukuba, and the tacrolimus yield can reach 2586 μg / mL.

[0091] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A cultivation method for increasing tacrolimus fermentation yield, characterized in that, Includes the following steps: Streptomyces tsukuba was inoculated into a fermentation medium and fermented to obtain tacrolimus fermentation broth; Tacrolimus fermentation broth was separated and purified to obtain tacrolimus; During fermentation, macroporous adsorption resin is added to the fermentation medium according to the fermentation time.

2. The cultivation method for increasing tacrolimus fermentation yield as described in claim 1, characterized in that: The pH of the fermentation system is controlled at 6.7-6.9 during the fermentation process.

3. The cultivation method for increasing tacrolimus fermentation yield as described in claim 1, characterized in that: During the fermentation process, maltodextrin solution is added according to the glycogen concentration in the fermentation broth.

4. The cultivation method for increasing tacrolimus fermentation yield as described in claim 1, characterized in that: The *Streptomyces tsukuba* strain requires slant and seed culture expansion before fermentation. The slant and seed culture expansion of *Streptomyces tsukuba* includes: Streptomyces tsukuba was inoculated onto slant culture medium and cultured to obtain slant spores; Slant spores were inoculated into seed culture medium and cultured for 20-40 hours to obtain seed liquid, which was then transferred to fermentation medium.

5. The cultivation method for increasing tacrolimus fermentation yield as described in claim 1, characterized in that: After 24 hours of fermentation, macroporous adsorption resin was added to the fermentation medium.

6. A cultivation method for increasing tacrolimus fermentation yield as described in claim 1 or 5, characterized in that: The amount of macroporous adsorption resin added is 10g / L-50g / L.

7. The cultivation method for increasing tacrolimus fermentation yield as described in claim 3, characterized in that: When the residual glycogen concentration in the fermentation broth is below 20 g / L, maltodextrin solution is added to the fermentation broth.

8. The cultivation method for increasing tacrolimus fermentation yield as described in claim 1, characterized in that: The fermentation conditions include: a fermentation temperature of 25℃-35℃ and a shaking speed of 180r / min-280r / min.

9. The cultivation method for increasing tacrolimus fermentation yield as described in claim 1, characterized in that: The fermentation medium comprises 40g / L-80g / L maltodextrin, 10g / L-30g / L yeast powder, 0.5g / L-1g / L (NH4)2SO4, and 0.5g / L-1g / L CaCO3, with a pH of 7.5-8.5, and sterile water as the solvent.

10. The cultivation method for increasing tacrolimus fermentation yield as described in claim 4, characterized in that: The slant culture medium comprises: 5g / L-20g / L malt extract, 2g / L-8g / L yeast extract, 1g / L-6g / L glucose, 5g / L-30g / L agar, with a pH of 7.0-7.4, and sterile water as the solvent.

11. The cultivation method for increasing tacrolimus fermentation yield as described in claim 4, characterized in that: The seed culture medium comprises: glucose 1g / L-3g / L, corn starch 5g / L-15g / L, soybean meal 10g / L-40g / L, CaCO3 1g / L-5g / L, pH 7.0-7.3, and sterile water as the solvent.

12. The cultivation method for increasing tacrolimus fermentation yield as described in claim 1, characterized in that: The Streptomyces Tsukuba is Streptomyces Tsukuba NRRL18488.

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