A method for efficiently producing geldanamycin by fermentation

Abstract

The present application belongs to the technical field of microbial engineering, and particularly relates to a method for efficiently fermenting and producing geldanamycin based on soil-derived Streptomyces geldanamycinus. The present application is based on the geldanamycin-producing Streptomyces geldanamycinus FIM18-0592, and provides a fermentation medium and culture condition for promoting the production of extracellular geldanamycin by the Streptomyces geldanamycinus FIM18-0592, which can significantly improve the yield of geldanamycin in the fermentation broth of the Streptomyces. The method has the advantages of simple operation, low production cost, and is suitable for the industrial production and application of geldanamycin.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of microbial engineering, and particularly relates to a method for efficiently producing geldanamycin based on soil-derived Streptomyces hygroscopicus. BACKGROUND

[0002] Geldanamycin (GA) was first isolated from the fermentation broth of Streptomyces hygroscopicus by Deboer et al. in 1970, and is a benzoquinone ansamycin antibiotic with a molecular formula of C 29 H 40 N2O9. Geldanamycin is a benzoquinone ansamycin antibiotic derived from Streptomyces hygroscopicus, and has good biological activity, such as anti-tumor, anti-viral, antibacterial and anti-inflammatory activity. Among them, geldanamycin, as a specific inhibitor of heat shock protein Hsp90, can specifically inhibit the ATP / PADP domain of Hsp90, down-regulate various target proteins of Hsp90, and has a particularly significant effect on anti-tumor, and has a wide application prospect and development value in the medical and health field.

[0003]

[0004] GA is the first Hsp90 N-terminal inhibitor discovered, and currently, a plurality of derivatives have entered the clinical research stage, such as 17-AAG, 17-DMAG, Ganetespib (STA-9090), etc. Among them, 17-AAG is the earliest Hsp90 inhibitor entering the clinical stage, and is currently in the clinical phase III, but its further application is limited due to poor solubility and serious toxic side effects. The water-soluble analog 17-MDAG is obtained by modification of 17-AAG in the art, 17-DMAG improves the oral bioavailability in clinical animal models, and has more significant anti-tumor activity, and is currently in the clinical phase II. At the same time, Ganetespib has entered the clinical phase III, and shows clinical activity in breast cancer, gastric cancer, melanoma, colon cancer and non-small cell lung cancer, etc.

[0005] The current reported geldanamycin producing strains are various, and the production of geldanamycin by fermentation has made obvious progress, but there is still a large space for improvement. For example, Wang Jimin screened the fermentation formula of Streptomyces hygroscopicus N5300, and the highest fermentation unit reached 615 U / mL after optimization. For example, Bai Quanhong carried out single factor experiment on the culture process of Streptomyces hygroscopicus SIPI.A.2039, which can significantly improve the yield of geldanamycin. Lin Huimin et al. further carried out orthogonal experiment on the fermentation conditions of Streptomyces hygroscopicus SIPI.A.2039, and the fermentation titer of GA reached 2500 μg / mL in a shake flask, and the fermentation titer reached 3700 μg / mL in a 50L fermenter. For example, Zhu Wan-yi et al. screened Streptomyces melanosporofaciens 101 through resistance screening experiment, and further optimized the formula, and the fermentation titer was 10 mg / mL after batch feeding fermentation. He Weiqing et al. used gene blocking technology to destroy the naphthoquinone AHBA biosynthesis gene cluster (shnSOP) in Streptomyces hygroscopicus 17997, and the peak area ratio of the genetically engineered strain was 185% higher than that of the original strain.

[0006] In summary, the fermentation level of geldanamycin produced by microorganism fermentation in the art is still low, and one of the main reasons for this problem is that the fermentation activity and stability of the existing fermentation strains are poor. Therefore, it is of great significance to develop a high-efficiency fermentation process for producing geldanamycin for the fermentation production of geldanamycin. SUMMARY

[0007] Therefore, the technical problem to be solved by the present application is to provide a method for high-efficiency fermentation production of geldanamycin based on soil-derived geldanamycin Streptomyces.

[0008] To solve the above technical problems, the method for high-efficiency fermentation production of geldanamycin comprises the step of inoculating the fermentation strain into a suitable fermentation medium for fermentation culture.

[0009] The fermentation strain is geldanamycin Streptomyces FIM18-0592, which is classified and named as

[0010] Streptomyces geldanamycininus, which has been preserved in the China General Microbiological Culture Collection Center, located at No. 1, Beichen West Road, Haidian District, Beijing, with a preservation number of CGMCC NO. 27634.

[0011] The present application obtains a geldanamycin Streptomyces FIM18-0592 with high yield of geldanamycin through strain screening.

[0012] FIM18-0592, the main biological characteristics are: colony morphology round, wavy edge, colony diameter size is about 3-5mm, colony surface rough, with furrows, slightly concave in the middle, substrate mycelium developed, closely combined with the culture medium, color is green white, light gray yellow, yellow white and green white, aerial mycelium is light green gray, green gray, green white, light gray yellow, gray yellow green and green gray, spore production is rich, early green white, later turn dark green, later turn green gray, with soluble pigment.

[0013] Specifically, the geldanamycin Streptomyces FIM18-0592, the strain is a geldanamycin high-yield strain.

[0014] Specifically, the method for high-efficiency fermentation production of geldanamycin, the fermentation medium comprises the following components in mass content: soybean meal 1-2wt%, ammonium sulfate 0.1-0.3wt%, lactic acid 0.1-0.3wt%, glycerol 2-6wt%, magnesium sulfate 0.1-0.3wt%, calcium carbonate 0.1-0.4wt%, glucose 6-15wt%, pH 6.8-7.2.

[0015] Specifically, the method for high-efficiency fermentation production of geldanamycin, the conditions of the fermentation culture step include: fermentation temperature 26-28℃, fermentation rotation speed 140-240r / min, culture time 48-168h.

[0016] Specifically, the method for high-efficiency fermentation production of geldanamycin, further comprising the step of inoculating the geldanamycin Streptomyces FIM18-0592 in the seed culture medium for seed liquid culture;

[0017] The seed culture medium comprises the following components in mass content: glucose 1-2wt%, malt dextrin 1-2wt%, soybean meal 2-3wt%, MgSO4·7H2O 0.1-0.5wt%, K2HPO4·3H2O 0.1-0.5wt%, yeast powder 0.5-1.0wt%, pH 6.8-7.2.

[0018] Specifically, the method for high-efficiency fermentation production of geldanamycin, the conditions of the seed liquid culture step include: culture temperature 26-28℃, control rotation speed 140-240r / min, culture time 24-48h.

[0019] Specifically, the method for high-efficiency fermentation production of geldanamycin, further comprising the step of inoculating the geldanamycin Streptomyces FIM18-0592 in the slant culture medium for bacterial activation;

[0020] The slant medium comprises ISP2, ISP3, ISP4, ISP5, G1, PDA or CM0011.

[0021] Specifically, the method for efficiently producing geldanamycin through fermentation further comprises a step of purifying the geldanamycin in the fermentation liquor.

[0022] Specifically, the method for efficiently producing geldanamycin through fermentation, the purification step comprises: taking the fermentation liquor to perform solid-liquid separation, collecting the supernatant and mixing with ethanol, and performing adsorption treatment with an HZ816 macroporous adsorption resin column.

[0023] Gradient elution is performed with 20%, 40% and 60% ethanol respectively, and then elution is performed with 80% and 100% ethanol, each gradient elution is performed for two column volumes, and the elution filtrate is collected in sections and subjected to HPLC detection.

[0024] The samples with a purity higher than 90% in the HPLC are collected and concentrated and dried to obtain the crude geldanamycin.

[0025] The obtained crude product is dissolved in acetone and left to stand, and then is subjected to suction filtration and vacuum drying.

[0026] Specifically, the method for efficiently producing geldanamycin through fermentation, the purification step comprises: taking the fermentation liquor to perform solid-liquid separation, collecting the supernatant and mixing with ethanol, and performing adsorption treatment with an HZ816 macroporous adsorption resin column.

[0027] Specifically, the method for efficiently producing geldanamycin through fermentation further comprises a step of detecting the fermentation titer of the geldanamycin through HPLC.

[0028] Specifically, the method for efficiently producing geldanamycin through fermentation, the HPLC detection conditions comprise:

[0029] The chromatographic column is a Syncronis C18 chromatographic column.

[0030] The mobile phase is methanol-water (75:25).

[0031] Injection volume: 10 muL;

[0032] Flow rate: 1 mL / min;

[0033] Detection wavelength: 304 nm;

[0034] Elution time: 30 min.

[0035] The gilvoseptin streptomyces FIM18-0592 with high gilvoseptin yield is obtained through strain screening, and the strain characteristics are excellent, and the gilvoseptin production capacity is significantly improved compared with other strains in the prior art, the strain can effectively improve the gilvoseptin titer in the fermentation liquor, improve the fermentation process level, and is beneficial to realize large-scale production.

[0036] The gilvoseptin streptomyces FIM18-0592 with high gilvoseptin yield is obtained through strain screening, and the strain characteristics are excellent, and the gilvoseptin production capacity is significantly improved compared with other strains in the prior art, the strain can effectively improve the gilvoseptin titer in the fermentation liquor, improve the fermentation process level, and is beneficial to realize large-scale production. BRIEF DESCRIPTION OF DRAWINGS

[0037] In order to make the content of the application more easily understood, the application will be further described in detail below according to specific embodiments of the application and in combination with the drawings, wherein,

[0038] Figure 1 FIM18-0592 strain in ISP4 medium mycelium morphology (100x);

[0039] Figure 2 FIM18-0592 strain in ISP1, ISP2, ISP3, ISP4, ISP5, ISP6, ISP7, G1, PDA, CM008, CM0011 and Czapek-Sucrose 12 kinds of slant medium culture characteristics diagram; wherein, (A) is the surface culture characteristics, (B) is the back culture characteristics;

[0040] Figure 3 FIM18-0592 strain phylogenetic tree;

[0041] Figure 4 The influence result of the single factor of the culture condition on the relative titer of gilvoseptin in example 5;

[0042] Figure 5 The influence result of the single factor of the culture condition on the relative titer of gilvoseptin in example 5;

[0043] Figure 6 Response surface and contour plot for Example 6;

[0044] Figure 7 Difference of fermentation titer before and after optimization for Example 6. DETAILED DESCRIPTION

[0045] In the following examples of the present application, the materials and reagents used below are commercially available unless otherwise specified.

[0046] In the following examples of the present application, the DNA extraction kit is purchased from Nanjing Nuo Weizan Biological Technology Co., Ltd.

[0047] Strain source and culture of Example 1

[0048] The Streptomyces strain described in the present application is screened and preserved by the laboratory from the soil of Longhai Mangrove Nature Reserve in Zhangzhou, Fujian, and named Streptomyces FIM18-0592.

[0049] Prepare ISP2 culture medium, sterilize at 121℃ for 30min, and place the test tube slant after sterilization is completed. The original strain is cultured on the ISP2 slant medium in the dark at a temperature of 28℃ and a humidity of 40-50% for 8-10d, and the slant is placed at 4℃ for use.

[0050] Prepare seed culture medium, the formula includes: glucose 1.8wt%, malt dextrin 1.0wt%, soybean meal 2.25wt%, MgSO4·7H2O 0.1wt%, K2HPO4·3H2O 0.1wt%, yeast powder 0.5wt%, the pH of the culture medium is 7.2, the liquid volume of the triangular flask is 30mL / 250mL, and the preparation is placed at 121℃ for sterilization for 30min.

[0051] Seed culture: under sterile conditions, spores on the ISP2 slant medium are inoculated into the seed liquid culture medium, cultured in the dark at a temperature of 28℃ and a humidity of 40-50% and a shaking speed of 240r / min for 48h to obtain seed fermentation liquor.

[0052] Prepare fermentation culture medium, the formula includes: glucose 10.0wt%, glycerol 4.0wt%, soybean meal 3.0wt%, yeast powder 0.5wt%, MgSO4·7H2O 0.1wt%, K2HPO4·3H2O 0.1wt%, calcium carbonate 0.4wt%, the pH of the culture medium is 7.2, and the liquid volume of the triangular flask is 60mL / 500mL. Among them, glucose is sterilized at 115℃ for 30min, and the rest of the culture medium is sterilized at 121℃ for 30min.

[0053] Fermentation culture: under sterile conditions, the seed culture after 48h culture was inoculated into the fermentation culture medium at 5% inoculation amount, and the culture was carried out at 28℃, humidity 40-50%, rotation speed 240r / min, and light shielding for 120-168h, and then observed.

[0054] Example 2

[0055] This example is directed to verifying the morphological characteristics, culture characteristics and physiological and biochemical characteristics of the screened strain FIM18-0592.

[0056] Morphological characteristics of FIM18-0592

[0057] ISP4 medium was prepared, sterilized at 121℃ for 30min, and after sterilization, cooled to 50-65℃ and poured into plates. Streptomyces FIM18-0592 was inoculated into the ISP4 medium for plug culture, and cultured at 28℃ for 10d, and then observed under an optical microscope (100x), and the results are shown in Figure 1 It was found that the intracellular hyphae grew well, the aerial hyphae grew vigorously, the branches were more, and the spore filaments were stretched.

[0058] Cultivation characteristics

[0059] Slope culture media were prepared, and ISP1, ISP2, ISP3, ISP4, ISP5, ISP6, ISP7, G1, PDA, CM008, CM0011 and Czapek-Sucrose were respectively prepared, sterilized at 121℃ for 30min, and immediately placed on test tube slopes after sterilization.

[0060] The stored strain FIM18-0592 was respectively cultured in ISP1, ISP2, ISP3, ISP4, ISP5, ISP6, ISP7, G1, PDA, CM008, CM0011 and Czapek-Sucrose slope culture media at a temperature of 28℃ and humidity of 40-50% under light shielding for 21d. During the period, the culture characteristics of the strain were observed and recorded every 7d, and the surface and back morphological characteristics of the strain FIM18-0592 are shown in Figure 2 Figures (A) and (B) respectively. It can be seen that the strain FIM18-0592 screened in the present application grows well in ISP2, ISP3, ISP4, ISP5, G1, PDA and CM0011 media, and the spores are thicker.

[0061] Physiological and biochemical experiments were carried out on the strain according to "Actinomycetes Rapid Identification and Systematic Classification", and the hyphae and substrate colors were compared according to the American ISCC-NBS standard color name and color value (267 kinds of non-luminous body color).

[0062] The specific growth of the strain FIM18-0592 is shown in Table 1 below.

[0063] Table 1 Culture characteristics of Streptomyces FIM18-0592 (21 days)

[0064]

[0065] Note: ++ means that the mycelium can grow with a small amount of spores; +++ means that the mycelium grows well with a large amount of spores; ++++ means that the mycelium grows best with abundant spores

[0066] It can be seen that the Streptomyces FIM18-0592 colony in this embodiment is round, with wavy edges, and the colony diameter is about 3-5 mm. The colony surface is rough with grooves, slightly concave in the middle, with developed substrate mycelium closely combined with the culture medium. The color is greenish white, light gray yellow, yellowish white and greenish white. The aerial mycelium is light greenish gray, greenish gray, greenish white, light gray yellow, grayish green and greenish gray. The spore production is rich, with greenish white in the early stage, turning dark blue in the later stage, and mostly turning greenish gray in the later stage. The strain produces pigments on ISP2, ISP4, ISP7, G1 and PDA culture media.

[0067] Physiological and biochemical characteristics

[0068] (1) Utilization of carbon sources

[0069] In this embodiment, the utilization of 23 carbon sources by Streptomyces FIM18-0592 was detected without adding carbon sources as a control, which were: α-D-glucose, D-mannitol, D-raffinose, D-sorbitol, glycerol, D-maltose, D-fructose, sucrose, α-D-lactose, inositol, glycine, ribose, starch, D-trehalose, pine triose, dulcitol, erythritol, xylose, L-rhamnose, D-arabinose, D-melibiose, D-cellobiose, D-galactose, and the results are shown in Table 2 below.

[0070] Table 2 Utilization of carbon sources by Streptomyces FIM18-0592

[0071] Carbon source Growth Carbon source Growth Alpha-D-glucose + Starch + D-mannitol + D-trehalose + D-raffinose + Cellobose - D-sorbitol + Duanol + Glycerol + Erythritol + D-maltose + Xylose + D-fructose + L-rhamnose + Sucrose + D-arabinose + Alpha-D-lactose + D-melibiose + Myo-inositol + D-cellobiose + Glycine - D-galactose + Ribose +

[0072] Note: + means promoting mycelial growth; - means inhibiting mycelial growth.

[0073] It can be seen that the strain FIM18-0592 is positive for the utilization of α-D-glucose, starch, D-mannitol, D-trehalose, D-raffinose, D-sorbitol, dulcitol, glycerol, erythritol, D-maltose, xylose, D-fructose, L-rhamnose, sucrose, D-arabinose, α-D-lactose, D-melibiose, myo-inositol, D-cellobiose, D-galactose, ribose, and is negative for the utilization of pine triose and glycine.

[0074] (2) Physiological and biochemical characteristics

[0075] In this embodiment, the physiological and biochemical characteristics of the strain FIM18-0592 were detected according to the Rapid Identification and Systematic Classification of Actinomycetes, and the results are shown in Table 3 below.

[0076] Table 3 Physiological and biochemical characteristics of the strain FIM18-0592

[0077] Physiological and biochemical characteristics Results Gelatin liquefaction - Starch hydrolysis - Cellulose hydrolysis - Melanin production + Hydrogen sulfide production -

[0078] Note: + represents positive; - represents negative

[0079] It can be seen that the strain FIM18-0592 is negative for gelatin liquefaction, starch hydrolysis, cellulose hydrolysis, and hydrogen sulfide production, and can produce melanin.

[0080] (3) Effect of pH

[0081] In this embodiment, the effect of different pH values on the growth of the strain was investigated using ISP4 as the basic medium, and the results are shown in Table 4 below.

[0082] Table 4 Effect of pH on the strain FIM18-0592

[0083] pH 4 5 6 7 8 9 10 11 Growth ++ +++ +++ +++ ++ + + -

[0084] Note: + represents weak mycelial growth; ++ represents mycelial growth; +++ represents good mycelial growth; - represents no mycelial growth

[0085] It can be seen that the strain FIM18-0592 grows well on ISP4 medium, with thick spores. When the pH of the ISP4 medium is adjusted to 4-11 and cultured at 28°C for 7-10 days, the strain can grow at a pH of 4-10, and the mycelial growth is vigorous at a pH of 5, 6, and 7.

[0086] (4) Temperature tolerance

[0087] In this embodiment, the growth of the strain FIM18-0592 at 25-35°C was investigated using ISP4 as the basic medium, and the results are shown in Table 5 below.

[0088] Table 5 Effect of temperature on Streptomyces FIM18-0592

[0089] Temperature (°C) 20 25 30 35 40 Growth - ++ +++ +++ -

[0090] Note: + represents weak mycelial growth; ++ represents mycelial growth; +++ represents good mycelial growth; - represents no mycelial growth

[0091] It can be seen that the Streptomyces FIM18-0592 does not grow at 20℃ and below and 40℃ and above, and the mycelial growth is vigorous at 30℃ and 35℃.

[0092] (5) Salt tolerance test

[0093] This example uses Gauze agar as the base medium, and the growth at different salt concentrations is investigated by culturing at 28℃ for 7-10d. The results are shown in Table 6 below.

[0094] Table 6 Effect of NaCl concentration on Streptomyces FIM18-0592

[0095] NaCl concentration (%) 1 2 3 4 5 6 Growth ++ +++ +++ ++ + -

[0096] Note: + represents weak mycelial growth; ++ represents mycelial growth; +++ represents good mycelial growth; - represents no mycelial growth

[0097] It can be seen that the Streptomyces FIM18-0592 grows at 1%-5% NaCl, and grows well at 2%-3% NaCl, and does not grow at more than 6%.

[0098] Example 3 Strain identification

[0099] (1) 16S rDNA sequence analysis of strain FIM18-0592

[0100] The mycelium in the fermentation broth is collected, and the genomic DNA of the above strain is extracted using Nuai Zhan Fast Pure Bacteria DNA Isolation MiniKit. The DNA is subjected to PCR amplification using the following 16S rRNA specific primers:

[0101] 27F (5'-AGAGTTTGATCCTGGCTCAG-3');

[0102] 1492R (5'-GGTTACCTTGTTACGACTT-3').

[0103] The PCR amplification system and procedure are shown in Table 7 and Table 8.

[0104] Table 7 PCR reaction system

[0105] PCR reaction components PCR reaction volume (μL) ddH2O 9 Forward primer 1 Reverse primer 1 Dimethyl sulfoxide 1 Premix 12.5 Template 0.5

[0106] Table 8 PCR reaction procedure

[0107] Temperature (°C) Time (s) Cycles 95 300 1 95 15 1 55 20 1 72 90 1 95 15 29 72 360 1

[0108] The amplification products were detected by 0.7% agarose gel electrophoresis, and after purification, they were sent to Fuzhou Shangya Biotechnology Co., Ltd. for sequence determination.

[0109] In this embodiment, the 16S rDNA sequence of FIM18-0592 was compared with the typical strain in the EzBioCloud database (https: / / www.ezbiocloud.net / identify) online, and the typical strain sequence with high homology was obtained, from which the target strain with good activity was screened, and the results are shown in Table 9.

[0110] Table 9 Homology of strains and typical strains

[0111]

[0112]

[0113] It can be seen that by sequencing the 16S rDNA sequence of Streptomyces FIM18-0592 and comparing it with the homologous sequence in the EzBioCloud database, it was found that it had a high homology of 99.57% with Streptomyces geldanamycininus. Combined with the apparent morphological characteristics, the strain FIM18-0592 was identified as Streptomyces geldanamycininus. The DNA sequence of the Streptomyces geldanamycininus FIM18-0592 is shown as SEQ ID No. 1, and the phylogenetic tree of the Streptomyces geldanamycininus FIM18-0592 is shown in FIG. 1. Figure 3

[0114] The above-mentioned Streptomyces geldanamycininus FIM18-0592 is preserved in the China General Microbiological Culture Collection Center (CGMCC) located at No. 1, Beichen West Road, Beijing City, Chaoyang District, Block 3, and its preservation number is CGMCC NO. 27634, and the preservation date is June 14, 2023. The strain is a high-yield strain of geldanamycin.

[0115] Example 4

[0116] The preserved Streptomyces FIM18-0592 was inoculated in ISP2 slant medium and cultured in darkness at 26-28°C for 10 days.

[0117] ​Seed culture medium (g / mL) for flask preparation: glucose 1.8wt%, malt dextrin 1.0wt%, soybean cake powder 2.25wt%, MgSO4·7H2O 0.1wt%, K2HPO4·3H2O 0.1wt%, yeast powder 0.5wt%, pH adjusted to 7.2, sterilized at 121℃ for 30min.

[0118] The fresh slant culture was inoculated into the seed culture medium and cultured in the dark for 24-48h at a temperature of 26-28℃ and a rotation speed of 140-240r / min with a liquid volume of 30-60mL.

[0119] Fermentation medium (g / mL) for flask preparation: glucose 10.0wt%, glycerol 4.0wt%, soybean cake powder 3.0wt%, yeast powder 0.5wt%, MgSO4·7H2O 0.1wt%, K2HPO4·3H2O 0.1wt%, calcium carbonate 0.4wt%, pH adjusted to 7.2. The glucose was sterilized at 115℃ for 30min, and the rest of the medium was sterilized at 121℃ for 30min.

[0120] The seed liquid was inoculated into a 60-120mL flask for fermentation at a temperature of 26-28℃ and a rotation speed of 140-240r / min in the dark for 48-168h with a liquid volume of 60-120mL.

[0121] In this embodiment, the fermentation titer of the strain was determined by HPLC method, and the characteristic peak was determined by comparing the ultraviolet absorption spectrum and retention time with the geldanamycin standard. The specific conditions include:

[0122] Chromatographic column: Syncronis C18 chromatographic column (Agilent, 4.6mm×250mm, filler particle size 5μm);

[0123] Mobile phase: methanol-water (75:25);

[0124] Injection volume: 10μL;

[0125] Flow rate: 1mL / min;

[0126] Detection wavelength: 304nm;

[0127] Elution time: 30min.

[0128] The fermentation broth was centrifuged at 4000 r / min for 30 min, and the precipitate was discarded. Ethanol was added to the supernatant to a final concentration of 20%, and the sample was loaded onto an HZ816 macroporous adsorption resin column. Adsorption was performed at a flow rate of 6 mL / min, and the sample was eluted with 20%, 40% and 60% ethanol gradient elution, respectively. The sample was washed with 80% and 100% ethanol, and two column volumes of each gradient were used for elution. The eluate was collected and subjected to HPLC detection. The samples with a purity higher than 90% in HPLC were combined and concentrated in a rotary evaporator to obtain a crude geldanamycin product. The crude product was dissolved in acetone and left overnight. The product was obtained by vacuum filtration, and the dry product was obtained by drying in a vacuum drying oven at 40°C to obtain the pure product.

[0129] Example 5

[0130] In this example, the slant culture and seed culture were the same as in Example 4, and the fermentation culture was optimized.

[0131] In this example, the culture conditions such as the rotation speed (140, 160, 180, 200, 220, 240 r / min), the liquid volume (60, 80, 100, 120 mL), the inoculation amount (2%, 3%, 5%, 7%, 9%) and the fermentation time (72, 96, 120, 144, 168 h) during the fermentation process were optimized. The potency was detected according to the method in Example 4, and the relative potency of geldanamycin was used as an index. The results are shown in Table 1. Figure 4

[0132] It can be seen that the Streptomyces geldanamycin FIM18-0592 was more suitable for culture under the conditions of a rotation speed of 140 r / min, a liquid volume of 60 mL, an inoculation amount of 7% and a fermentation time of 144 h.

[0133] In this example, the nutritional formula such as the carbon source (glucose, starch, malt dextrin, sucrose, fructose, mannitol), the nitrogen source (soybean cake powder, peanut cake powder, soybean meal, yeast powder, soybean powder, cottonseed hulls) and the inorganic salt ((NH4)2SO4, K2HPO4, MgCl2, KH2PO4, NH4Cl, NH4HPO4) was optimized. The relative potency of geldanamycin was used as an index, and the results are shown in Table 2. Figure 5

[0134] It can be seen that the Streptomyces geldanamycin FIM18-0592 was more suitable for culture under the conditions of a carbon source of glucose, a nitrogen source of soybean cake powder and an inorganic salt of (NH4)2SO4.

[0135] Example 6

[0136] ​​The present example further optimizes the single-factor experiment results by the steepest ascent test and the response surface test to explore the optimal culture conditions for improving geldanamycin production.

[0137] In the present example, the relative potency of geldanamycin is used as an index, and based on the single-factor experiment results, glucose, soybean meal powder and ammonium sulfate are determined as the factors for the steepest ascent test, and the experimental design and results are shown in Table 10.

[0138] Table 10 Steepest ascent test design and results

[0139] Group Glucose (g / L) Soybean meal (g / L) Ammonium sulfate (g / L) Relative titer 1 60 10 2.0 104％ 2 80 15 2.5 114％ 3 100 20 3.0 136％ 4 120 25 3.5 31％ 5 140 30 4.0 15％

[0140] As shown in Table 10, with the increase of the contents of glucose, soybean meal powder and ammonium sulfate, the content of geldanamycin first increases and then decreases, reaching the maximum in the third group, indicating that the third group is the optimal medium component content, which can be used as the center point of the response surface test, and the second and fourth groups are used as the other two levels for the response surface test.

[0141] In the present example, based on the single-factor experiment and the steepest ascent test results, three-factor three-level response surface test is designed with glucose (A), soybean meal powder (B) and ammonium sulfate (C) as the independent variables and the relative potency of geldanamycin (Y) as the response value, and the experimental results are shown in Table 11.

[0142] Table 11 Response surface experiment design and results

[0143] Group A: Grape (g / L) B: Soybean meal (g / L) C: Sulfuric acid (g / L) Relative titer (Y) 1 1 1 0 145.79±37.27 2 0 0 0 163.10±10.45 3 -1 -1 0 146.09±11.86 4 1 0 -1 134.75±13.15 5 0 0 0 165.58±1.63 6 0 0 0 153.91±16.84 7 0 1 1 25.25±0.59 8 0 0 0 158.94±5.90 9 -1 1 0 37.32±1.45 10 1 -1 0 138.31±1.15 11 1 0 1 135.49±7.29 12 -1 0 -1 153.49±1.61 13 0 1 -1 146.57±13.94 14 0 -1 -1 140.41±2.33 15 -1 0 1 49.91±8.92 16 0 -1 1 148.60±12.41 17 0 0 0 154.04±1.46

[0144] As shown in Table 11, the relative potency is higher in the group with 100 g / L of glucose, 20 g / L of soybean meal powder and 3.0 g / L of ammonium sulfate. The response surface fitting equation obtained by analyzing the results is Y (relative potency) = 159.12 + 20.94A - 27.31B - 27.00C + 29.60AB + 26.08AC - 32.38BC - 19.52A 2 -22.72B 2 -21.19C 2 The variance analysis of the above regression equation results is shown in Table 12.

[0145] Table 12 Variance analysis and significance error of regression equation

[0146] Source Sum of squares Degrees of freedom Mean square F P Model 31930.65 9 3547.85 83.14 <0.0001 A - glucose 3508.21 1 3508.21 82.21 <0.0001 B - soybean meal 5967.02 1 5967.02 139.83 <0.0001 C - ammonium sulfate 5830.98 1 5830.98 136.64 <0.0001 AB 3378.32 1 3378.32 79.17 <0.0001 AC 2721.11 1 2721.11 63.76 <0.0001 BC 4193.53 1 4193.53 98.27 <0.0001 A 2 ]]> 1604.43 1 1604.43 37.60 0.0005 B 2 ]]> 2173.81 1 2173.81 50.94 0.0002 [C 2 ]]> 1890.01 1 1890.01 44.29 0.0003 Lack of fit 188.18 3 62.73 2.27 0.2225 Residual error 298.72 7 42.67 [R 2 = 0.9907 Pure error 110.54 4 27.64 Adj R 2 = 0.9788 Total 32229.37 16

[0147] Note: P < 0.05 indicates significant difference, and P < 0.01 indicates extremely significant difference.

[0148] As shown in Table 12, the model P<0.0001, indicating that the model has extremely significant. The regression equation correlation coefficient R 2 =0.9907, the correction coefficient Adj R 2 =0.9788, and the P value of the misfit is 0.2225>0.05, the misfit is not significant, indicating that the response surface model fitting degree is good, the actual value and the predicted value have strong correlation, and the relationship between the independent variables glucose (A), soybean meal powder (B), ammonium sulfate (C) and the relative potency of geldanamycin (Y) can be better reflected. In the first order term, the effects of glucose (A), soybean meal powder (B) and ammonium sulfate (C) on the relative potency of geldanamycin are extremely significant (P<0.0001), and the effects of A 2 , B 2 , C 2 and AB, AC, BC in the second order term are also extremely significant (P<0.0001). By comparing the F value, the order of the effects of the factors on the relative potency of geldanamycin is: soybean meal powder (B)>ammonium sulfate (C)>glucose (A).

[0149] In this embodiment, the response surface graph and the contour graph are shown in Figure 6 , the response surface curve is obviously curved, and the contour is elliptical, indicating that the interaction of the three factors is strong. The slope on one side of soybean meal powder and ammonium sulfate is steep, while the slope on the glucose side is gentle, indicating that both have a large effect on the production of geldanamycin, which is consistent with the above results in Table 12.

[0150] In this embodiment, the highest relative potency predicted by the response surface model is 166.72%, and the best fermentation formula is glucose 10.42%, soybean meal powder 1.68%, ammonium sulfate 0.3%, lactic acid 0.3%, glycerol 4%, magnesium sulfate 0.1%, calcium carbonate 0.4%. The culture conditions are 140 r / min of rotation speed, 60 mL of liquid volume, and 7% (v / v) of inoculum amount for 144 h of culture. Under this culture condition, the actual highest fermentation potency of geldanamycin in a flask is 3032.35 μg / mL (as shown in Figure 7 Scheme II). Under this scheme, the yield of geldanamycin is 1.80 times of the original fermentation process (as shown in Figure 7 Scheme I).

[0151] Obviously, the above embodiments are only examples for clear illustration, and are not limitations on the embodiments. Based on the above description, other different forms of changes or variations can be made by those skilled in the art. Here, all the embodiments cannot be exhausted, and the obvious changes or variations derived therefrom are still within the protection scope of the present application.

Claims

1. A method for efficiently producing geldanamycin by fermentation, characterized in that: The method comprises the steps of inoculating the fermentation strain into a suitable fermentation medium for fermentation culture; The fermentation strain is Streptomyces geldanamycininus FIM18-0592, which has been deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms with a deposit number of CGMCC NO.27634.

2. The method for producing geldanamycin by efficient fermentation according to claim 1, characterized in that: The fermentation medium comprises the following components by weight: 1-2 wt% soybean meal, 0.1-0.3 wt% ammonium sulfate, 0.1-0.3 wt% lactic acid, 2-6 wt% glycerol, 0.1-0.3 wt% magnesium sulfate, 0.1-0.4 wt% calcium carbonate and 6-15 wt% glucose, with a pH of 6.8-7.

2.

3. The method for producing geldanamycin by efficient fermentation according to claim 2, characterized in that: The conditions of the fermentation culture step include: fermentation temperature of 26-28° C., fermentation speed of 140-240 r / min, and culture time of 48-168 h.

4. The method for producing geldanamycin by efficient fermentation according to any one of claims 1 to 3, characterized in that: Before the fermentation culture step, the method further includes inoculating the geldanamycin Streptomyces FIM18-0592 into a seed culture medium for seed liquid culture; The seed culture medium comprises the following components by weight: 1-2 wt% glucose, 1-2 wt% maltodextrin, 2-3 wt% soybean cake powder, 0.1-0.5 wt% MgSO4·7H2O, 0.1-0.5 wt% K2HPO4·3H2O and 0.5-1.0 wt% yeast powder, with a pH value of 6.8-7.

2.

5. The method for producing geldanamycin by efficient fermentation according to claim 4, characterized in that: The conditions of the seed liquid culture step include: a culture temperature of 26-28° C., a controlled rotation speed of 140-240 r / min, and a culture time of 24-48 h.

6. The method for producing geldanamycin by efficient fermentation according to claim 4, characterized in that: Before the seed liquid culture step, the method further includes inoculating the geldanamycin Streptomyces FIM18-0592 into a slant culture medium to activate the bacteria; The slant culture medium includes ISP2, ISP3, ISP4, ISP5, PDA or CM0011.

7. The method for producing geldanamycin by efficient fermentation according to any one of claims 1 to 3, characterized in that: After the fermentation and culturing step, the method further includes a step of purifying geldanamycin in the fermentation broth.

8. The method for producing geldanamycin by efficient fermentation according to claim 7, characterized in that: The purification step comprises: The fermentation broth was separated into solid and liquid, and the supernatant was collected and mixed with ethanol, and then adsorbed on a HZ816 macroporous adsorption resin column; Gradient elution was performed using 20%, 40% and 60% ethanol for 3 column volumes, followed by elution using 80% and 100% ethanol, with each gradient washing two column volumes. The elution filtrate was collected in sections and tested by HPLC. The samples with a purity higher than 90% in HPLC were collected and combined, concentrated and evaporated to dryness to obtain crude geldanamycin; The obtained crude geldanamycin product is dissolved in acetone and allowed to stand, and then filtered and vacuum-dried to obtain pure geldanamycin.

9. The method for producing geldanamycin by efficient fermentation according to any one of claims 1 to 3, characterized in that: After the fermentation and culturing step, the method further includes a step of detecting the fermentation titer of geldanamycin in the fermentation broth by using HPLC.

Images