WO2023061391A1 - 一种安丝菌素p-3的发酵方法 - Google Patents

一种安丝菌素p-3的发酵方法 Download PDF

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WO2023061391A1
WO2023061391A1 PCT/CN2022/124781 CN2022124781W WO2023061391A1 WO 2023061391 A1 WO2023061391 A1 WO 2023061391A1 CN 2022124781 W CN2022124781 W CN 2022124781W WO 2023061391 A1 WO2023061391 A1 WO 2023061391A1
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fermentation
glucose
ansamitocin
powder
yeast extract
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PCT/CN2022/124781
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English (en)
French (fr)
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朱家骏
吴开凯
陈辅辰
张敏
冯富强
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杭州中美华东制药有限公司
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Priority to CN202280047420.8A priority Critical patent/CN117580957A/zh
Publication of WO2023061391A1 publication Critical patent/WO2023061391A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin

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  • the invention belongs to the technical field of medicine, and further belongs to the technical field of fermentation, and specifically relates to a fermentation method of ansamitocin, including seed cultivation, fermentation cultivation, feeding cultivation and the like.
  • Ansamitocin is a maytansinoid antibiotic, which is mainly fermented from microorganisms such as Actinomyces aurantiifolia, and has strong anti-tumor, anti-tuberculosis, anti-bacteria and other pharmacological activities.
  • Ansamitocin P-3 is the main fermentation product, which prevents cell mitosis and causes cell death by hindering the formation of microtubules, and has a significant anti-tumor effect in vitro and in tumor-bearing animals.
  • Ansamitocin is a polyketide compound, and the biosynthetic pathway of ansamitocin mainly consists of the following three main stages: I. Glucose undergoes a biological reaction to generate glucose-6-phosphate, with glucose-6-phosphate as the starting point of the reaction , produce erythrose-4-phosphate (E-4-P) and phosphoenolpyruvate (PEP) through the pentose phosphate pathway and glycolysis pathway respectively, and then erythrose-4-phosphate and phosphoric acid Enolpyruvate undergoes the aminoshikimic acid biosynthetic pathway to give 3-amino-5-hydroxybenzoic acid (AHBA). II.
  • AHBA 3-amino-5-hydroxybenzoic acid
  • CN 105907681B a mutant strain with high production of ansamitocin P-3 and the preparation method of ansamitocin P-3, the precious actinomycetes of orange fasciculata are screened out by ultraviolet lamp mutagenesis, and the aeration ratio is controlled to be 1.0 during the fermentation process ⁇ 2.0vvm, fermentation dissolved oxygen is greater than 30%, and pH is 6.0 ⁇ 8.0.
  • the fermentation period was 6-7 days, and the titer at the end of fermentation was 152.21mg/L.
  • CN 103805648 B high-yield ansamitocin fermentation process, precious actinomycetes orange, after 2 to 3 days of fermentation on the fermentation medium, add 0.10 to 0.30% (v/v) isobutanol to the fermentation liquid, and the fermentation proceeds After 4 to 6 days, add glucose to the fermentation broth at a rate of 3 to 10 g/L/d; after 8 to 12 days of fermentation, enter the stage of repeated fed-batch fermentation. The fermentation period is 20-21 days, and the titer at the end of fermentation is 410mg/L.
  • the method designs basic formula DOE experiments and precursor feed response surface experiments by analyzing biological metabolic pathways, adopts a clear fermentation formula and precursor supplement design, has simple operation, stable process control, and is suitable for industrial production.
  • Isobutanol is converted to isobutyric acid by isobutanol dehydrogenase (IDH) and further undergoes biological metabolism to produce isobutyryl-CoA, which is further converted to acetyl-CoA by isobutyryl-CoA mutase
  • IDH isobutanol dehydrogenase
  • acetyl-CoA isobutyryl-CoA mutase
  • malonyl-CoA by biological metabolism participates in the condensation reaction of the second stage of the biosynthetic pathway of ansamitocin to form the precursor ansamitocin compound.
  • Isobutanol also has the effect of activating the gene expression of glucose-6-phosphate dehydrogenase and citrate synthase, enhancing the glycolysis pathway, and affecting the first stage of the biosynthetic pathway of ansamitocin.
  • Methionine and isoleucine participate in the second stage of the biosynthetic pathway of ansamitocin through the biosynthesis of propionyl-CoA.
  • methionine is also synthesized by biological metabolism to form S-adenosyl-methionine, which participates in the modification step of polyketide synthase in the third stage of the biosynthetic pathway of ansamitocin. Controlling the feeding ratio and total amount of each precursor can effectively improve the efficiency of biological metabolism to synthesize ansamitocin (P-3).
  • the quick-acting carbon and nitrogen sources and the slow-release carbon and nitrogen sources in the fermentation medium, and the key precursors of each biological metabolic pathway are designed through DOE experiments, and suitable parameters are optimized. Complete the fermentation optimization control, and guide the carbon metabolic flow from the central metabolic pathway to the ansamitocin biosynthesis pathway.
  • the fermentation method provided by the invention has been verified by the fermentation system, and the fermentation cycle of 13 to 14 days can reach 711ug/ml in a tank.
  • a fermentation medium for ansamitocin P-3 comprising the following components:
  • Fermentation Medium Components Mass to volume ratio (W/V) available carbon source 0.2 ⁇ 5% slow release carbon source 0.5 ⁇ 5% Available Nitrogen Source 0.2 ⁇ 5% slow release nitrogen source 0.5 ⁇ 5% Inorganic salt 0.2 ⁇ 1.5%
  • the pH value of the fermentation medium is 6.5-7.5.
  • the fermentation medium is batched according to the above-mentioned process formula, water is added to the fermentation tank, and raw materials are put in while stirring;
  • the volume after the fermentation and feeding can be 0-50000L;
  • the temperature of the fermenter is 10-37°C, preferably 25-30°C;
  • the time for culturing in the fermenter is 12 to 14 days;
  • the fermented culture begins to feed immediately from the fermentation culture; the fed feed is calculated according to the amount of fed feed per day;
  • the feeding formula of described daily fluid replenishment is as follows:
  • the carbon source includes but is not limited to: glucose, sucrose, fructose, lactose, glycerin, maltodextrin, high maltose powder, corn flour, potato starch, tapioca starch, corn starch, corn steep liquor Dry powder, glutinous rice flour, soluble starch, yeast powder, etc.
  • the nitrogen source includes but not limited to: malt extract, yeast extract, yeast extract powder, yeast powder, yeast peptone, soybean peptone, corn gluten meal, soybean protein powder, cottonseed powder , soybean powder, soybean cake powder, etc.
  • the carbon source includes a quick-acting carbon source and a slow-release carbon source.
  • the nitrogen source includes a quick-acting nitrogen source and a slow-release nitrogen source.
  • the quick-acting carbon source includes, but is not limited to: glucose, fructose, sucrose, glycerin, lactose, and the like.
  • the slow-release carbon source includes, but is not limited to: corn flour, potato starch, tapioca starch, corn starch, glutinous rice flour, soluble starch and the like.
  • the quick-acting nitrogen source includes but not limited to: malt extract, yeast extract, yeast extract powder and the like.
  • the slow-release nitrogen source includes, but is not limited to: corn gluten powder, soybean protein powder, cottonseed powder, soybean powder, soybean cake powder, dry corn steep powder, etc.
  • the inorganic salts include but are not limited to: carbonates, sulfates, phosphates, chlorides and the like.
  • the inorganic salt is selected from calcium carbonate, potassium dihydrogen phosphate, potassium chloride, magnesium sulfate, zinc sulfate, and ferrous sulfate.
  • the present invention also provides a fermentation method of ansamitocin P-3, comprising the following steps:
  • Shake flask seed culture including the preparation, subpackaging, sterilization, inoculation and cultivation of shake flask seeds to obtain the shake flask seed bacterial liquid;
  • Seed tank cultivation including ingredients for seed cultivation, sterilization, inoculation and cultivation to obtain seed bacterial liquid;
  • Fermentation culture including the ingredients of the fermentation medium, sterilization, inoculation and cultivation, to obtain ansamicin fermentation liquid;
  • Feed-feeding culture start feeding from the beginning of fermentation culture, and the feeding is calculated according to the amount of feeding per day;
  • the formula of the fermentation medium of the present invention is as follows:
  • Fermentation Medium Components Mass to volume ratio (W/V) glucose 0.1 ⁇ 2%; glycerin 0.1 ⁇ 2%; fructose 0.1 ⁇ 2%; soluble starch 0.5 ⁇ 2.5%; Potato starch 0.5 ⁇ 2.5%; yeast extract 0.1 ⁇ 1%; Cottonseed Powder 0.1 ⁇ 1%; calcium carbonate 0.1 ⁇ 1%; Potassium dihydrogen phosphate 0.01 ⁇ 0.1%; magnesium sulfate 0.01 ⁇ 0.1%; ferrous sulfate 0.01 ⁇ 0.1%;
  • the way of fermentation and cultivation is to take each component according to the ratio for batching, add water to the fermenter, add raw materials while stirring, adjust the pH with sodium hydroxide, and then carry out real tank sterilization and pipeline After sterilization, the seeds are hydraulically pumped into fermenters with sterile air.
  • the feeding culture method of the present invention is as follows: 30-60% glucose is supplemented daily, 0.5-1% of residual sugar in the fermentation system is maintained, and the precursor is supplemented daily, and the precursor is Ansamitocin synthetic pathway precursor.
  • the precursor is selected from isobutanol, methionine and isoleucine.
  • the content of the fluid precursor is 0.02-0.2%.
  • the content of the fluid precursor methionine is 0.01-0.1%, preferably 0.03-0.06%, more preferably 0.05%.
  • the content of the fluid supplement precursor isoleucine is 0.01-0.1%, preferably 0.03-0.06%, more preferably 0.04-0.05%.
  • the content of the isobutanol precursor is 0.01-0.1%, preferably 0.01-0.04%, more preferably 0.015-0.02%.
  • Ansamitocin P-3 fermentation method comprises the following steps:
  • Fermentation culture Weigh each raw material and calculate by weight volume ratio, glucose 0.1-2%, fructose 0.1-2%, glycerin 0.1-2%, potato starch 0.5-2.5%, soluble starch 0.5-2.5%, yeast 0.1-1% extract, 0.1-1% cottonseed powder, 0.1-1% calcium carbonate, 0.01-0.1% potassium dihydrogen phosphate, 0.01-0.1% magnesium sulfate and 0.01-0.1% ferrous sulfate; Add defoamer and water, add raw materials while stirring, adjust the pH to 6.8-7.5 with sodium hydroxide, sterilize, keep warm and pressurize; use sterile air to press the seeds into the fermenter, and the fermenter is kept at a temperature of 25-30 Cultivation at °C;
  • Feed culture after the start of fermentation culture, daily feed is carried out, with 30-60% glucose, and the precursors isobutanol, methionine and isoleucine;
  • the present invention cooperates with the biosynthesis of API by adding an appropriate amount of part of the precursor in the process of biological metabolic synthesis.
  • some precursors such as isobutanol, will inhibit the growth of bacterial cells if excessive fluid supplementation.
  • This patent obtains an optimal solution in the selection of the precursor components and the total amount of the fermentation system through the response surface experiment.
  • the fermentation method provided by the present invention has been verified by the fermentation system, and the fermentation period of 12 to 14 days can reach more than 600ug/ml in the tank.
  • the classification of the Ansamitocin P-3 strain used in the present invention is named Actinosynnema pretiosum, and it has been preserved in the General Microbiology Center of China Microbiological Culture Collection Management Committee (address) on December 11, 2020 : No. 3, No. 1 Yard, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences), the preservation number is: CGMCC NO.21355.
  • strains described in the present invention are preserved in two forms of glycerol tube and inclined plane.
  • Embodiment 1 fermentation culture
  • ingredients are prepared according to the above-mentioned process formula, defoamer and water are added into the fermentation tank, raw materials are put in while stirring, and the pH is adjusted to 6.5-7.0. Sterilize the steam into the real tank, the tank temperature is 118-122°C, the tank pressure is 0.09-0.12MPa, keep the temperature and pressure for 30 minutes; carry out pipeline sterilization, and the sterilization time is 60 minutes.
  • the seeds are hydraulically pumped into the fermenter with sterile air, and the condition of the seeds is inspected and recorded prior to inoculation.
  • the fermentation broth is kept at a tank temperature of 30°C and a tank pressure of 0.04-0.05MPa.
  • the glucose preparation ratio in the supplementation feed is 30-60% of glucose
  • the supplementation includes 0.1% of ansamitocin biosynthetic precursor, wherein the precursor is isobutyric acid an equal mixture of alcohol, methionine and isoleucine.
  • the fermentation titer of Ansamectin P-3 obtained by the fermentation method described in Example 1 was 639ug/ml.
  • the fermentation broth is kept at a tank temperature of 30°C and a tank pressure of 0.04-0.05MPa.
  • the glucose preparation ratio in the supplementation feed is 30-60% of glucose
  • the supplementation includes the biosynthetic precursor of ansamitocin, of which 0.03% isobutanol, formazan Thionine 0.03% and Isoleucine 0.03%.
  • the fermentation titer of Ansamicin P-3 obtained by the fermentation method described in Example 2 was 642ug/ml.
  • the fermentation broth is kept at a tank temperature of 30°C and a tank pressure of 0.04-0.05MPa.
  • the glucose preparation ratio in the supplementation feed is 30-60% of glucose
  • the supplementation includes the biosynthetic precursor of ansamitocin, of which 0.01% isobutanol, formazan Thionine 0.05% and Isoleucine 0.03%.
  • the fermentation titer of Ansamectin P-3 obtained by the fermentation method described in Example 3 was 652ug/ml.
  • the glucose preparation ratio in the supplementation feed is 30-60% of glucose
  • the supplementation includes the biosynthetic precursor of ansamitocin, of which 0.03% isobutanol, formazan Thionine 0.05% and Isoleucine 0.05%.
  • the fermentation titer of Ansamectin P-3 obtained by the fermentation method described in Example 4 was 650ug/ml.
  • the glucose preparation ratio in the supplementation feed is 30-60% of glucose
  • the supplementation includes the biosynthetic precursor of ansamitocin, of which 0.018% isobutanol, formazan Thionine 0.05% and Isoleucine 0.043%.
  • the fermentation titer of Ansamectin P-3 obtained by the fermentation method described in Example 5 was 711ug/ml.
  • Embodiment 6 the investigation of fermentation culture material
  • This experiment is intended to screen the choice of carbon and nitrogen sources in the fermentation culture formula.
  • the components of the inorganic salt are calcium carbonate 0.5%; potassium dihydrogen phosphate 0.05%; magnesium sulfate 0.05%; ferrous sulfate heptahydrate 0.001%.
  • Fermentation medium formula (W/V) is lactose 0.5%; soluble starch 2.0%; malt extract 0.5%; yeast extract 0.5%; soybean powder 1%;
  • the fermentation medium formula (W/V) is 0.5% glucose; 2.0% soluble starch; 0.5% malt extract; 0.5% yeast extract; 1% soybean powder;
  • Fermentation medium formula (W/V) is 0.5% fructose; 2.0% soluble starch; 0.5% malt extract; 0.5% yeast extract; 1% soybean flour;
  • Fermentation medium formula (W/V) is glucose 0.5%; glycerol 2.0%; malt extract 0.5%; yeast extract 0.5%; soybean powder 1%;
  • Fermentation medium formula (W/V) is glucose 0.5%; high maltose powder 2.0%; malt extract 0.5%; yeast extract 0.5%; soybean powder 1%;
  • the fermentation medium formula (W/V) is 0.5% glucose; 2.0% maltodextrin; 0.5% malt extract; 0.5% yeast extract; 1% soybean powder;
  • Fermentation medium formula (W/V) is glucose 0.5%; corn flour 2.0%; malt extract 0.5%; yeast extract 0.5%; soybean flour 1%;
  • Fermentation medium formula (W/V) is glucose 0.5%; potato starch 2.0%; malt extract 0.5%; yeast extract 0.5%; soybean powder 1%;
  • Fermentation medium formula (W/V) is glucose 0.5%; glutinous rice flour 2.0%; malt extract 0.5%; yeast extract 0.5%; soybean flour 1%;
  • Fermentation medium formula (W/V) is glucose 0.5%; soluble starch 2.0%; malt extract 0.5%; soybean powder 1%;
  • Fermentation medium formula (W/V) is glucose 0.5%; soluble starch 2.0%; yeast extract 0.5%; soybean flour 1%;
  • Fermentation medium formula (W/V) is glucose 0.5%; soluble starch 2.0%; soybean flour 1%;
  • Fermentation medium formula (W/V) is glucose 0.5%; soluble starch 2.0%; malt extract 0.5%; yeast extract powder 0.5%; soybean powder 1%;
  • Fermentation medium formula (W/V) is glucose 0.5%; soluble starch 2.0%; malt extract 0.5%; yeast powder 0.5%; soybean powder 1%;
  • Fermentation medium formula (W/V) is 0.5% glucose; 2.0% soluble starch; 0.5% malt extract; 0.5% yeast peptone; 1% soybean powder;
  • Fermentation medium formula (W/V) is glucose 0.5%; soluble starch 2.0%; soybean peptone 0.5%; soybean powder 1%;
  • the fermentation medium formula (W/V) is 0.5% glucose; 2.0% soluble starch; 0.5% yeast extract; 0.5% yeast powder; 1% dry corn steep liquor;
  • Fermentation medium formula (W/V) is glucose 0.5%; soluble starch 2.0%; yeast extract 0.5%; yeast powder 0.5%; corn gluten powder 1%;
  • Fermentation medium formula (W/V) is 0.5% glucose; 2.0% soluble starch; 0.5% yeast extract; 0.5% yeast powder; 1% hydrolyzed vegetable protein;
  • Fermentation medium formula (W/V) is 0.5% glucose; 2.0% soluble starch; 0.5% yeast extract; 0.5% yeast powder; 1% soybean protein powder;
  • Fermentation medium formula (W/V) is 0.5% glucose; 2.0% soluble starch; 0.5% yeast extract; 0.5% yeast powder; 1% cottonseed powder;
  • Fermentation medium formula (W/V) is glucose 0.5%; soluble starch 2.0%; yeast extract 0.5%; yeast powder 0.5%; soybean cake powder 1%;
  • the quick-acting carbon source of the present invention can be glucose, fructose, glycerol
  • the slow-release carbon source can be maltodextrin, corn flour, potato starch, soluble starch;
  • the quick-acting nitrogen source can be yeast extract powder, yeast extract, malt extract, yeast powder, yeast peptone;
  • the slow-release nitrogen source can be Corn Steep Steep Powder, Corn Gluten Powder, Hydrolyzed Vegetable Protein, Soy Protein Powder, Soybean Flour, Cottonseed Powder, Soybean Cake Powder.
  • the carbon source, nitrogen source, and composite carbon and nitrogen source of the fermenter and feed of the present invention can be selected from glucose, glycerin, dextrin, wheat bran, bean cake powder, raw soybean powder, corn steep liquor, and yeast powder.
  • Embodiment 7 the investigation of the proportioning of fermentation culture material based on shake flask experiment
  • Example 6 Based on the selection of carbon and nitrogen sources in Example 6, further test the reasonable ratio of glucose, fructose, glycerin, potato starch, soluble starch, yeast extract, malt extract, and cottonseed powder in the fermentation medium .
  • the components of the inorganic salt are calcium carbonate 0.5%; potassium dihydrogen phosphate 0.05%; magnesium sulfate 0.05%; ferrous sulfate heptahydrate 0.001%.
  • the fermentation medium formula (W/V) is 0.5% of glucose; 2.0% of glycerin; 0.5% of fructose; 2.0% of soluble starch; 2.0% of potato starch; 0.5% of yeast extract; 1.0% of cottonseed powder;
  • the fermentation medium formula (W/V) is glucose 0.5%; glycerol 4.0%; fructose 1.0%; soluble starch 4.0%; potato starch 4.0%; yeast extract 1.0%; cottonseed powder 2.0%;
  • the fermentation medium formula (W/V) is glucose 0.5%; glycerol 6.0%; fructose 2.0%; soluble starch 6.0%; potato starch 6.0%; yeast extract 2.0%; cottonseed powder 3.0%;
  • the fermentation medium formula (W/V) is 1.0% of glucose; 2.0% of glycerol; 0.5% of fructose; 4.0% of soluble starch; 4.0% of potato starch; 2.0% of yeast extract; 3.0% of cottonseed powder;
  • Fermentation medium formula (W/V) is glucose 1.0%; glycerol 4.0%; fructose 1.0%; soluble starch 6.0%; potato starch 6.0%; yeast extract 0.5%; cottonseed powder 1.0%;
  • the fermentation medium formula (W/V) is glucose 1.0%; glycerin 6.0%; fructose 2.0%; soluble starch 2.0%; potato starch 2.0%; yeast extract 1.0%; cottonseed powder 2.0%;
  • the fermentation medium formula (W/V) is 2.0% of glucose; 2.0% of glycerin; 1.0% of fructose; 2.0% of soluble starch; 6.0% of potato starch; 1.0% of yeast extract; 3.0% of cottonseed powder;
  • the fermentation medium formula (W/V) is glucose 2.0%; glycerol 4.0%; fructose 2.0%; soluble starch 4.0%; potato starch 2.0%; yeast extract 2.0%; cottonseed powder 1.0%;
  • the fermentation medium formula (W/V) is 2.0% of glucose; 6.0% of glycerol; 0.5% of fructose; 6.0% of soluble starch; 4.0% of potato starch; 0.5% of yeast extract;
  • Embodiment 8 the investigation of the precursor of the fermented feeding culture based on shake flask
  • the components of the inorganic salt are calcium carbonate 0.5%; potassium dihydrogen phosphate 0.05%; magnesium sulfate 0.05%; ferrous sulfate heptahydrate 0.001%.
  • the fermentation medium formula (W/V) is glucose 0.5%; glycerin 2.0%; fructose 2.0%; soluble starch 6.0%; potato starch 4.0%; yeast extract 1.0%; cottonseed powder 1.0%; Precursor 0.1%;
  • the fermentation medium formula (W/V) is glucose 0.5%; glycerin 4.0%; fructose 0.5%; soluble starch 2.0%; potato starch 6.0%; yeast extract 2.0%; cottonseed powder 2.0%; Precursor 0.1%;
  • the fermentation medium formula (W/V) is 0.5% of glucose; 6.0% of glycerin; 1.0% of fructose; 4.0% of soluble starch; 2.0% of potato starch; 0.5% of yeast extract; 3.0% of cottonseed powder; Precursor 0.1%;
  • the fermentation medium formula (W/V) is glucose 1.0%; glycerol 2.0%; fructose 1.0%; soluble starch 6.0%; potato starch 2.0%; yeast extract 2.0%; cottonseed powder 2.0%; Precursor 0.1%;
  • the fermentation medium formula (W/V) is glucose 1.0%; glycerol 4.0%; fructose 2.0%; soluble starch 2.0%; potato starch 4.0%; yeast extract 0.5%; cottonseed powder 3.0%; Precursor 0.1%;
  • the fermentation medium formula (W/V) is 1.0% of glucose; 6.0% of glycerol; 0.5% of fructose; 4.0% of soluble starch; 6.0% of potato starch; 1.0% of yeast extract; 2.0% of cottonseed powder; Precursor 0.1%;
  • the fermentation medium formula (W/V) is glucose 2.0%; glycerol 2.0%; fructose 2.0%; soluble starch 4.0%; potato starch 6.0%; yeast extract 1.0%; cottonseed powder 1.0%; Precursor 0.1%;
  • the fermentation medium formula (W/V) is glucose 2.0%; glycerol 4.0%; fructose 0.5%; soluble starch 6.0%; potato starch 2.0%; yeast extract 1.0%; cottonseed powder 3.0%; Precursor 0.1%;
  • the fermentation medium formula (W/V) is 2.0% of glucose; 6.0% of glycerin; 1.0% of fructose; 2.0% of soluble starch; 4.0% of potato starch; 2.0% of yeast extract; Precursor 0.1%;
  • Example 9 Investigation, data analysis and optimization of the components and proportions of fermentation culture materials screened by Taguchi experiment
  • the ranking range obtained by signal-to-noise ratio response analysis was A precursor mixture, B glucose, E soluble starch, F potato starch, C glycerol, D fructose, and G yeast extract.
  • the rank range of the mean response is A precursor mixture, F potato starch, G yeast extract, H cottonseed flour, E soluble starch, D fructose, C glycerol, B glucose.
  • Mean effect analysis ranked as A precursor mixture, F potato starch, G yeast extract, H cottonseed powder, E soluble starch, D fructose, C glycerol, B glucose.
  • Embodiment 10 the investigation of adding precursor ratio in the culture medium
  • Example 9 Based on Example 9, adding the precursor mixture (0.3% of total methionine, 0.3% of isoleucine, 0.3% of isobutanol) in the basal medium shows that in the signal-to-noise ratio effect and the mean effect, it should be added precursor mixture. However, some precursors, such as isobutanol, will inhibit the growth of bacterial cells if excessive fluid supplementation. Further, the daily supplementation of methionine, isoleucine and isobutanol in the fermentation culture process was carried out by response surface three-factor Box-Behnken investigation.
  • the above-mentioned factors are respectively: 1. the balance between the feed rate of glucose and the feedback of glucose repression, it is necessary to screen the feed process rate; The phosphoenolpyruvate combined to form AHBA, it is necessary to combine AHBA precursor feeding experiment, valine, isobutanol, methionine precursor pathway for secondary response surface analysis; 3. Under adversity, microbial The metabolic pathway will be more biased towards the pentose phosphate pathway.
  • the experimental design is as follows:

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Abstract

一种安丝菌素的发酵方法，包括发酵培养和自发酵培养起即始的每日流补，所述每日流补所添加的补料按质量体积比包括30~60%葡萄糖及安丝菌素代谢合成前体0.02~0.2%，所述安丝菌素代谢合成前体包括甲硫氨酸、异亮氨酸和异丁醇。所述发酵培养的培养基按质量体积比包括：速效碳源0.2~5%，缓释碳源0.5~5%，速效氮源0.2~5%，缓释氮源0.5~5%，无机盐0.2~1.5%。发酵效价可稳定在600ug/ml以上。

Description

一种安丝菌素P-3的发酵方法 技术领域
本发明属于医药技术领域，更进一步的属于发酵技术领域，具体涉及一种安丝菌素的发酵方法，包括种子培养、发酵培养、补料培养等。
背景技术
安丝菌素是一种美登素类抗生素，其主要是从微生物比如橙色珍贵束丝放线菌发酵生产的，具有极强的抗肿瘤、抗结核杆菌、抗细菌等多种药理活性。
安丝菌素P-3为主要发酵产物，其通过阻碍微管形成从而阻止细胞的有丝分裂使细胞死亡，在体外及荷瘤动物中具有显著抗肿瘤作用。
安丝菌素的化学结构式为：
Figure PCTCN2022124781-appb-000001
安丝菌素为聚酮类化合物，安丝菌素的生物合成途径主要由以下三个主要阶段构成：Ⅰ.葡萄糖经生物反应生成葡萄糖-6-磷酸，以葡萄糖-6-磷酸为反应起始点，分别经过磷酸戊糖途径和糖酵解途径产出赤藓糖-4-磷酸(E-4-P)和磷酸烯醇式丙酮酸(PEP)，再由赤藓糖-4-磷酸和磷酸烯醇式丙酮酸经氨基莽草酸生物合成途径，从而得到3-氨基-5-羟基苯甲酸(AHBA)。Ⅱ.以3-氨基-5-羟基苯甲酸(AHBA)为生物代谢起点，Ⅰ型聚酮合酶的催化下继续添加丙酸单元、乙酸单元、聚酮碳链延伸单元等，缩合反应形成前体安丝菌素化合物。Ⅲ.前体安丝菌素化合物经过6个聚酮合酶的修饰步骤最终合成P-3。
目前大多数专利和文献，主要有以下现有技术述及，
CN 105907681B，一种高产安丝菌素P-3的突变株及安丝菌素P-3的制备方法，紫外线灯诱变筛选出珍贵橙色束丝放线菌，控制发酵过程中通气比为1.0～2.0vvm，发酵溶氧大于30％，pH为6.0～8.0。发酵周期6-7天，发酵终点效价152.21mg/L。
CN 103805648 B，高产安丝菌素发酵工艺，珍贵橙色束丝放线菌，发酵培养基发酵2～3天后，向发酵液中加入0.10～0.30％(v/v)的异丁醇，发酵进行4～6天后，向发酵液中流加葡萄糖，流加速率为3～10g/L/d；发酵进行8～12天后，进入重复补料分批发酵阶段。发酵周期20-21天，发酵终点效价410mg/L。
其余文献有零星描述通过正交等试验设计优化安丝菌素(P-3)发酵配方试验，进行配方优化，且配方优化主要集中于基础粮食物料配比，未针对代谢过程、生物代谢途径关键前体等进行优化。
发明内容
本方法通过分析生物代谢途径设计基础配方DOE实验和前体补料响应面实验，采用明确的发酵配方和前体补量设计，操作简单、过程控制稳定，适合工业化生产。
在对安丝菌素生物合成途径前体的影响研究中异丁醇、甲硫氨酸和异亮氨酸作用位点和代谢机理较为重要。异丁醇通过异丁醇脱氢酶(IDH)转化为异丁酸进一步经过生物代谢生产异丁酰辅酶A，异丁酰辅酶A通过异丁酰辅酶A变位酶转化为乙酰辅酶A进一步经过生物代谢生产丙二酰辅酶A参与至安丝菌素的生物合成途径Ⅱ阶段缩合反应形成前体安丝菌素化合物中。异丁醇同时还具备激活葡萄糖-6-磷酸脱氢酶和柠檬酸合成酶基因表达的作用，增强糖酵解途径，影响安丝菌素的生物合成途径Ⅰ阶段。甲硫氨酸和异亮氨酸通过生物合成丙酰辅酶A，参与至安丝菌素的生物合成途径Ⅱ阶段中。同时甲硫氨酸还经生物代谢合成形成S-腺苷-蛋氨酸参与至安丝菌素的生物合成途径Ⅲ阶段聚酮合酶的修饰步骤。控制各前体补入配比和总量，可以有效提高生物代谢合成安丝菌素(P-3)效率。
本发明通过将发酵培养基内速效碳氮源和缓释碳氮源、各生物代谢途径关键前体通过DOE实验设计，优选适宜参数。完成发酵优化控制，将碳代谢流从中心代谢途径向安丝菌素生物合成途径引导。本发明提供的发酵方法，经过发酵体系验证，在13～14天发酵周期，放罐可达到711ug/ml。
一种安丝菌素P-3的发酵培养基，包括以下组分：
发酵培养基组分 质量体积比(W/V)
速效碳源 0.2～5％
缓释碳源 0.5～5％
速效氮源 0.2～5％
缓释氮源 0.5～5％
无机盐 0.2～1.5％
所述发酵培养基的pH值为6.5～7.5。
发酵培养基按上述工艺配方进行配料，发酵罐内加水，边搅拌边投入原料；
所述的发酵投料后的体积可为0～50000L；
所述的发酵罐温度为10～37℃，优选为25～30℃；
所述的发酵罐培养的时间为12～14天；
所述的发酵培养自发酵培养起即开始流补；所述的流补按每日流补量计算；
所述每日流补的补料配方如下：
Figure PCTCN2022124781-appb-000002
作为一种具体的实施方案，所述的碳源包括但不限于：葡萄糖、蔗糖、果糖、乳糖、甘油、麦芽糊精、高麦芽糖粉、玉米粉、马铃薯淀粉、木薯淀粉、玉米淀粉、玉米浆干粉、糯米粉、可溶性淀粉、酵母粉等。
作为一种具体的实施方案，所述的氮源包括但不限于：麦芽提取物、酵母提取物、酵母浸粉、酵母粉、酵母蛋白胨、大豆蛋白胨、玉米蛋白粉、大豆蛋白粉、棉籽精粉、大豆粉、黄豆饼粉等。
作为一种具体的实施方案，所述的碳源包括速效碳源和缓释碳源。
作为一种具体的实施方案，所述的氮源包括速效氮源和缓释氮源。
作为一种具体的实施方案，所述的速效碳源包括但不限于：葡萄糖、果糖、蔗糖、甘油、乳糖等。
作为一种具体的实施方案，所述的缓释碳源包括但不限于：玉米粉、马铃薯淀粉、木薯淀粉、玉米淀粉、糯米粉、可溶性淀粉等。
作为一种具体的实施方案，所述的速效氮源包括但不限于：麦芽提取物、酵母提取物、酵母浸粉等。
作为一种具体的实施方案，所述的缓释氮源包括但不限于：玉米蛋白粉、大豆蛋白粉、棉籽精粉、大豆粉、黄豆饼粉、玉米浆干粉等。
作为一种具体的实施方案，所述的无机盐包括但不限于：碳酸盐、硫酸盐、磷酸盐、氯化物等。
作为一种具体的实施方案，所述的无机盐选自碳酸钙、磷酸二氢钾、氯化钾、硫酸镁、硫酸锌、硫酸亚铁。
本发明还提供一种安丝菌素P-3的发酵方法，包括以下步骤：
1.摇瓶种子培养：包括摇瓶种子的配制、分装、灭菌、接种和培养，得到摇瓶种子菌液；
2.种子罐培养：包括种子培养的配料、灭菌、接种和培养，得到种子菌液；
3.发酵培养：包括发酵培养基的配料、灭菌、接种和培养，得到安丝菌素发酵液；
4.补料培养：自发酵培养起始即开始流补，所述的流补以每日流补量计算；
5.放罐。
作为一种具体的实施方案，本发明的发酵培养基的配方如下：
发酵培养基组分 质量体积比(W/V)
葡萄糖 0.1～2％；
甘油 0.1～2％；
果糖 0.1～2％；
可溶性淀粉 0.5～2.5％；
马铃薯淀粉 0.5～2.5％；
酵母抽提物 0.1～1％；
棉籽精粉 0.1～1％；
碳酸钙 0.1～1％；
磷酸二氢钾 0.01～0.1％；
硫酸镁 0.01～0.1％；
硫酸亚铁 0.01～0.1％；
作为一种具体的实施方案，发酵培养的方式为，按配比取各组分进行配料，发酵罐内加入水，边搅拌边加入原料，用氢氧化钠调节pH，依次进行实罐灭菌和管道灭菌后，用无菌空气把种子液压入发酵罐。
作为一种具体的实施方案，本发明的补料培养方式如下：每日流补30～60％葡萄糖，维持发酵体系残糖0.5～1％，并每日流补前体，所述前体为安丝菌素合成途径前体。
作为一种具体的实施方案，所述的前体选自异丁醇、甲硫氨酸和异亮氨酸。
作为一种具体的实施方案，所述流补前体的含量为0.02～0.2％。
作为一种具体的实施方案，所述流补前体甲硫氨酸的含量为0.01～0.1％，优选为0.03～0.06％，更优选为0.05％。
作为一种具体的实施方案，所述流补前体异亮氨酸的含量为0.01～0.1％，优选为0.03～0.06％，更优选为0.04～0.05％。
作为一种具体的实施方案，所述流补前体异丁醇的含量为0.01～0.1％，优选为0.01～0.04％，更优选为0.015～0.02％。
本发明提供的安丝菌素P-3发酵方法，包括以下步骤：
1.发酵培养：称取各原料，以重量体积比计为，葡萄糖0.1～2％、果糖0.1～2％、甘油0.1～2％、马铃薯淀粉0.5～2.5％、可溶性淀粉0.5～2.5％、酵母抽提物0.1～1％、棉籽精粉0.1～1％、碳酸钙0.1～1％、磷酸二氢钾0.01～0.1％、硫酸镁0.01～0.1％和硫酸亚铁0.01～0.1％；发酵罐内加入消泡剂和水，边搅拌边投入原料，用氢氧化钠调节pH至6.8～7.5，灭菌，保温保压；用无菌空气把种子液压入发酵罐，发酵罐于罐温25～30℃培养；
2.补料培养：发酵培养开始后即进行每日流补，流补30～60％葡萄糖，以及前体异丁醇、甲硫氨酸和异亮氨酸；
3.放罐：发酵培养12～14天后，发酵结束。
本发明的有益效果为：
1.发酵培养基内速效/缓释碳源，速效/缓释氮源种类筛选和比例筛选。影响菌体初级代谢增长菌体，和利用碳氮源次级代谢产生API，以安丝菌素(P-3)为例，生物代谢途径中初始碳源为葡萄糖。但在基础培养基中加入过量葡萄糖，引起葡萄糖阻遏效应，分解代谢产物阻遏某些编码诱导酶体系的基因的转录，从而影响其对其他碳源的利用效率，细胞生长受到抑制。本专利对初始碳氮源进行种类筛选，并使用田口实验完成发酵配方各组分比例优选。
2.本发明相较于现有技术，通过在生物代谢合成过程中适量添加部分前体，配合API的生物合成。但部分前体如异丁醇，流补过量对菌体生长会产生抑制作用。本专利通过响应面实验，在发酵体系流补前体组分、总量选择中，得出优解。
3.本发明提供的发酵方法，经过发酵体系验证，在12～14天发酵周期，放罐可达到600ug/ml以上。
附图说明
图1-实施例5所得发酵产物的HPLC图谱
图2-实施例10所述的mintab多响应预测结果图
图3～5-实施例10响应面预测中P-3效价与前体％的等值线图
具体实施方式
下面结合具体实施例对本发明作进一步的详细说明。以下实施例用于理解本发明的 方法和核心思想，对于本领域的技术人员来说，在不脱离本发明构思的前提下，进行任何可能的变化或替换，均属于本发明的保护范围。本发明实施例中未注明具体条件的实验方法，通常为常规条件，或按照原料或商品制造厂商所建议的条件；未注明来源的原料和试剂，通常为通过商业途径可购得的常规试剂。
菌种及保存方法
本发明所使用的安丝菌素P-3菌株的分类命名为珍贵束丝放线菌(Actinosynnema pretiosum)，已于2020年12月11日保藏于中国微生物菌种保藏管理委员会普通微生物中心(地址：北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所)，保藏编号为：CGMCC NO.21355。
本发明所述的菌种以甘油管和斜面两种形式保藏。
实施例1:发酵培养
发酵培养基配方(W/V)：葡萄糖1.0％；甘油6.0％；果糖0.5％；可溶性淀粉4.0％；马铃薯淀粉6.0％；酵母抽提物1.0％；棉籽精粉2.0％；碳酸钙0.5％；磷酸二氢钾0.05％；硫酸镁0.05％；七水硫酸亚铁0.001％。
按上述工艺配方进行配料，发酵罐内加入消泡剂和水，边搅拌边投入原料，并调节pH至6.5～7.0。进汽实罐灭菌，罐温118-122℃，罐压0.09-0.12MPa，保温保压30min；进行管道灭菌，灭菌时间60min。用无菌空气把种子液压入发酵罐，接种前检查并记录种子情况。
发酵液于罐温30℃，罐压0.04～0.05MPa。
自发酵培养起始即开始每日流补，其中流补的补料中葡萄糖配制比例为葡萄糖30～60％，流补中包括安丝菌素生物合成前体0.1％，其中前体是异丁醇、甲硫氨酸和异亮氨酸的等量混合。
本实施例1所记载的发酵方法获得的安丝菌素P-3的发酵效价为639ug/ml。
实施例2
发酵培养基配方(W/V)：葡萄糖0.5％；甘油3.2％；果糖0.2％；可溶性淀粉2％；马铃薯淀粉3.2％；酵母抽提物0.5％；棉籽精粉0.4％；碳酸钙0.5％；磷酸二氢钾0.05％；硫酸镁0.05％；七水硫酸亚铁0.001％。
按上述工艺配方进行配料，发酵罐内加入消泡剂和水，边搅拌边投入原料，并调节pH至6.5～7.0。
发酵液于罐温30℃，罐压0.04～0.05MPa。
自发酵培养起始即开始每日流补，其中流补的补料中葡萄糖配制比例为葡萄糖30～60％，流补中包括安丝菌素生物合成前体，其中异丁醇0.03％、甲硫氨酸0.03％和异亮氨酸0.03％。
本实施例2所记载的发酵方法获得的安丝菌素P-3的发酵效价为642ug/ml。
实施例3
发酵培养基配方(W/V)：葡萄糖0.5％；甘油0.8％；果糖0.5％；可溶性淀粉2.0％；马铃薯淀粉3.2％；酵母抽提物0.8％；棉籽精粉0.4％；碳酸钙0.5％；磷酸二氢钾0.05％；硫酸镁0.05％；七水硫酸亚铁0.001％。
按上述工艺配方进行配料，发酵罐内加入消泡剂和水，边搅拌边投入原料，并调节pH至6.5～7.0。
发酵液于罐温30℃，罐压0.04～0.05MPa。
自发酵培养起始即开始每日流补，其中流补的补料中葡萄糖配制比例为葡萄糖30～60％，流补中包括安丝菌素生物合成前体，其中异丁醇0.01％、甲硫氨酸0.05％和异亮氨酸0.03％。
本实施例3所记载的发酵方法获得的安丝菌素P-3的发酵效价为652ug/ml。
实施例4
发酵培养基配方(W/V)：葡萄糖0.5％；甘油0.8％；果糖0.5％；可溶性淀粉2.0％；马铃薯淀粉3.2％；酵母抽提物0.8％；棉籽精粉0.4％；碳酸钙0.5％；磷酸二氢钾0.05％；硫酸镁0.05％；七水硫酸亚铁0.001％。
按上述工艺配方进行配料，发酵罐内加入消泡剂和水，边搅拌边投入原料，并调节pH至6.5～7.0。发酵液于罐温30℃，罐压0.04～0.05MPa。
自发酵培养起始即开始每日流补，其中流补的补料中葡萄糖配制比例为葡萄糖30～60％，流补中包括安丝菌素生物合成前体，其中异丁醇0.03％、甲硫氨酸0.05％和异亮氨酸0.05％。
本实施例4所记载的发酵方法获得的安丝菌素P-3的发酵效价为650ug/ml。
实施例5
发酵培养基配方(W/V)：葡萄糖0.5％；甘油0.8％；果糖0.5％；可溶性淀粉2.0％；马铃薯淀粉3.2％；酵母抽提物0.8％；棉籽精粉0.4％；碳酸钙0.5％；磷酸二氢钾0.05％；硫酸镁0.05％；七水硫酸亚铁0.001％。
按上述工艺配方进行配料，发酵罐内加入消泡剂和水，边搅拌边投入原料，并调节pH至6.5～7.0。发酵液于罐温30℃，罐压0.04～0.05MPa。
自发酵培养起始即开始每日流补，其中流补的补料中葡萄糖配制比例为葡萄糖30～60％，流补中包括安丝菌素生物合成前体，其中异丁醇0.018％、甲硫氨酸0.05％和异亮氨酸0.043％。
本实施例5所记载的发酵方法获得的安丝菌素P-3的发酵效价为711ug/ml。
附图1为本实施例4发酵终点产物HPLC图谱。
实施例6：发酵培养物料的考察
实验设计：
本次实验意在筛选发酵培养配方中碳氮源的选择。
除碳氮源外，无机盐的组分为碳酸钙0.5％；磷酸二氢钾0.05％；硫酸镁0.05％；七水硫酸亚铁0.001％。
下述每个筛选的实验方案，除发酵碳源氮源和前体的组分与比例外，其余方案均与实施例1相同。
实验方案：
方案1-1：发酵培养基配方(W/V)为乳糖0.5％；可溶性淀粉2.0％；麦芽提取物0.5％；酵母抽提物0.5％；大豆粉1％；
方案1-2：发酵培养基配方(W/V)为葡萄糖0.5％；可溶性淀粉2.0％；麦芽提取物0.5％；酵母抽提物0.5％；大豆粉1％；
方案1-3：发酵培养基配方(W/V)为果糖0.5％；可溶性淀粉2.0％；麦芽提取物0.5％；酵母抽提物0.5％；大豆粉1％；
方案1-4：发酵培养基配方(W/V)为葡萄糖0.5％；甘油2.0％；麦芽提取物0.5％；酵母抽提物0.5％；大豆粉1％；
方案1-5：发酵培养基配方(W/V)为葡萄糖0.5％；高麦芽糖粉2.0％；麦芽提取物0.5％；酵母抽提物0.5％；大豆粉1％；
方案1-6：发酵培养基配方(W/V)为葡萄糖0.5％；麦芽糊精2.0％；麦芽提取物0.5％；酵母抽提物0.5％；大豆粉1％；
方案1-7：发酵培养基配方(W/V)为葡萄糖0.5％；玉米粉2.0％；麦芽提取物0.5％；酵母抽提物0.5％；大豆粉1％；
方案1-8：发酵培养基配方(W/V)为葡萄糖0.5％；马铃薯淀粉2.0％；麦芽提取物0.5％；酵母抽提物0.5％；大豆粉1％；
方案1-9：发酵培养基配方(W/V)为葡萄糖0.5％；糯米粉2.0％；麦芽提取物0.5％；酵母抽提物0.5％；大豆粉1％；
方案1-10：发酵培养基配方(W/V)为葡萄糖0.5％；可溶性淀粉2.0％；麦芽提取物0.5％；大豆粉1％；
方案1-11：发酵培养基配方(W/V)为葡萄糖0.5％；可溶性淀粉2.0％；酵母抽提物0.5％；大豆粉1％；
方案1-12：发酵培养基配方(W/V)为葡萄糖0.5％；可溶性淀粉2.0％；大豆粉1％；
方案1-13：发酵培养基配方(W/V)为葡萄糖0.5％；可溶性淀粉2.0％；麦芽提取物0.5％；酵母浸粉0.5％；大豆粉1％；
方案1-14：发酵培养基配方(W/V)为葡萄糖0.5％；可溶性淀粉2.0％；麦芽提取物0.5％；酵母粉0.5％；大豆粉1％；
方案1-15：发酵培养基配方(W/V)为葡萄糖0.5％；可溶性淀粉2.0％；麦芽提取物0.5％；酵母蛋白胨0.5％；大豆粉1％；
方案1-16：发酵培养基配方(W/V)为葡萄糖0.5％；可溶性淀粉2.0％；大豆蛋白胨0.5％；大豆粉1％；
方案1-17：发酵培养基配方(W/V)为葡萄糖0.5％；可溶性淀粉2.0％；酵母抽提物0.5％；酵母粉0.5％；玉米浆干粉1％；
方案1-18：发酵培养基配方(W/V)为葡萄糖0.5％；可溶性淀粉2.0％；酵母抽提物0.5％；酵母粉0.5％；玉米蛋白粉1％；
方案1-19：发酵培养基配方(W/V)为葡萄糖0.5％；可溶性淀粉2.0％；酵母抽提物0.5％；酵母粉0.5％；水解植物蛋白1％；
方案1-20：发酵培养基配方(W/V)为葡萄糖0.5％；可溶性淀粉2.0％；酵母抽提物0.5％；酵母粉0.5％；大豆蛋白粉1％；
方案1-21：发酵培养基配方(W/V)为葡萄糖0.5％；可溶性淀粉2.0％；酵母抽提物0.5％；酵母粉0.5％；棉籽精粉1％；
方案1-22：发酵培养基配方(W/V)为葡萄糖0.5％；可溶性淀粉2.0％；酵母抽提物0.5％；酵母粉0.5％；黄豆饼粉1％；
实验结果：
Figure PCTCN2022124781-appb-000003
实验结论：配方中不足量的速效氮源会致使发酵效价不佳；配方中以乳糖为速效碳源会致使发酵效价不佳；基本确认本发明的速效碳源可以是葡萄糖、果糖、甘油；缓释碳源可以是麦芽糊精、玉米粉、马铃薯淀粉、可溶性淀粉；速效氮源可以是酵母浸粉、酵母抽提物、麦芽提取物、酵母粉、酵母蛋白胨；缓释氮源可以是玉米浆干粉、玉米蛋白粉、水解植物蛋白、大豆蛋白粉、大豆粉、棉籽精粉、黄豆饼粉。
基本确认本发明发酵罐与补料的碳源、氮源、复合碳氮源的选择可以为葡萄糖、甘油、糊精、麦麸、豆饼粉、生豆粉、玉米浆、酵母粉。
实施例7：基于摇瓶实验的发酵培养物料配比的考察
实验设计：基于实施例6中关于碳氮源的选择，进一步测试合理的葡萄糖、果糖、甘油、马铃薯淀粉、可溶性淀粉、酵母抽提物、麦芽提取物、棉籽精粉在发酵培养基中的比例。
除碳氮源外，无机盐的组分为碳酸钙0.5％；磷酸二氢钾0.05％；硫酸镁0.05％；七水硫酸亚铁0.001％。
下述每个筛选的实验方案，除发酵碳源氮源和前体的组分与比例外，其余方案均与实施例1相同。
方案2-1：发酵培养基配方(W/V)为葡萄糖0.5％；甘油2.0％；果糖0.5％；可溶性淀粉2.0％；马铃薯淀粉2.0％；酵母抽提物0.5％；棉籽精粉1.0％；
方案2-2：发酵培养基配方(W/V)为葡萄糖0.5％；甘油4.0％；果糖1.0％；可溶性淀粉4.0％；马铃薯淀粉4.0％；酵母抽提物1.0％；棉籽精粉2.0％；
方案2-3：发酵培养基配方(W/V)为葡萄糖0.5％；甘油6.0％；果糖2.0％；可溶性淀粉6.0％；马铃薯淀粉6.0％；酵母抽提物2.0％；棉籽精粉3.0％；
方案2-4：发酵培养基配方(W/V)为葡萄糖1.0％；甘油2.0％；果糖0.5％；可溶性淀粉4.0％；马铃薯淀粉4.0％；酵母抽提物2.0％；棉籽精粉3.0％；
方案2-5：发酵培养基配方(W/V)为葡萄糖1.0％；甘油4.0％；果糖1.0％；可溶性淀粉6.0％；马铃薯淀粉6.0％；酵母抽提物0.5％；棉籽精粉1.0％；
方案2-6：发酵培养基配方(W/V)为葡萄糖1.0％；甘油6.0％；果糖2.0％；可溶性淀粉2.0％；马铃薯淀粉2.0％；酵母抽提物1.0％；棉籽精粉2.0％；
方案2-7：发酵培养基配方(W/V)为葡萄糖2.0％；甘油2.0％；果糖1.0％；可溶性淀粉2.0％；马铃薯淀粉6.0％；酵母抽提物1.0％；棉籽精粉3.0％；
方案2-8：发酵培养基配方(W/V)为葡萄糖2.0％；甘油4.0％；果糖2.0％；可溶性淀粉4.0％；马铃薯淀粉2.0％；酵母抽提物2.0％；棉籽精粉1.0％；
方案2-9：发酵培养基配方(W/V)为葡萄糖2.0％；甘油6.0％；果糖0.5％；可溶性淀粉6.0％；马铃薯淀粉4.0％；酵母抽提物0.5％；棉籽精粉2.0％；
实验结果：
Figure PCTCN2022124781-appb-000004
实验结论：试验显示，葡萄糖1％，甘油4％，果糖0.5％，可溶性淀粉6％，马铃薯淀粉4％，酵母抽提物1％和棉籽精粉1％为相对较优的物料配比选择，为后续精选配比提供基础。
实施例8：基于摇瓶的发酵流补培养前体的考察
实验设计：基于实施例6和7中关于碳氮源的选择和配比，考察是否需加入安丝菌素P-3代谢途径关键前体混合物(总甲硫氨酸0.3％、异亮氨酸0.3％、异丁醇0.3％)为因子，并筛选前体的比例。
除碳氮源外，无机盐的组分为碳酸钙0.5％；磷酸二氢钾0.05％；硫酸镁0.05％；七水硫酸亚铁0.001％。
下述每个筛选的实验方案，除发酵碳源氮源的组分与比例外，其余方案均与实施例1相同。
方案3-1：发酵培养基配方(W/V)为葡萄糖0.5％；甘油2.0％；果糖2.0％；可溶性淀粉6.0％；马铃薯淀粉4.0％；酵母抽提物1.0％；棉籽精粉1.0％；前体0.1％；
方案3-2：发酵培养基配方(W/V)为葡萄糖0.5％；甘油4.0％；果糖0.5％；可溶性淀粉2.0％；马铃薯淀粉6.0％；酵母抽提物2.0％；棉籽精粉2.0％；前体0.1％；
方案3-3：发酵培养基配方(W/V)为葡萄糖0.5％；甘油6.0％；果糖1.0％；可溶性淀粉4.0％；马铃薯淀粉2.0％；酵母抽提物0.5％；棉籽精粉3.0％；前体0.1％；
方案3-4：发酵培养基配方(W/V)为葡萄糖1.0％；甘油2.0％；果糖1.0％；可溶性淀粉6.0％；马铃薯淀粉2.0％；酵母抽提物2.0％；棉籽精粉2.0％；前体0.1％；
方案3-5：发酵培养基配方(W/V)为葡萄糖1.0％；甘油4.0％；果糖2.0％；可溶性淀粉2.0％；马铃薯淀粉4.0％；酵母抽提物0.5％；棉籽精粉3.0％；前体0.1％；
方案3-6：发酵培养基配方(W/V)为葡萄糖1.0％；甘油6.0％；果糖0.5％；可溶性淀粉4.0％；马铃薯淀粉6.0％；酵母抽提物1.0％；棉籽精粉2.0％；前体0.1％；
方案3-7：发酵培养基配方(W/V)为葡萄糖2.0％；甘油2.0％；果糖2.0％；可溶性淀粉4.0％；马铃薯淀粉6.0％；酵母抽提物1.0％；棉籽精粉1.0％；前体0.1％；
方案3-8：发酵培养基配方(W/V)为葡萄糖2.0％；甘油4.0％；果糖0.5％；可溶性淀粉6.0％；马铃薯淀粉2.0％；酵母抽提物1.0％；棉籽精粉3.0％；前体0.1％；
方案3-9：发酵培养基配方(W/V)为葡萄糖2.0％；甘油6.0％；果糖1.0％；可溶性淀粉2.0％；马铃薯淀粉4.0％；酵母抽提物2.0％；棉籽精粉1.0％；前体0.1％；
实验结果：
Figure PCTCN2022124781-appb-000005
实验结论：试验显示，引入安丝菌素生物合成路径前体有助于其发酵培养。
实施例9：田口实验筛选发酵培养物料组分和配比的考察及数据分析和优化
实验设计：实施例6～8中筛选的物料，通过mintab进行静态田口设计，将葡萄糖、果糖、甘油、马铃薯淀粉、可溶性淀粉、酵母抽提物、棉籽精粉在发酵培养基中所占比例，以及每日流补中是否加入安丝菌素P-3代谢途径关键前体混合物(总甲硫氨酸0.3％、异亮氨酸0.3％、异丁醇0.3％)为因子。筛选物料比例，其正交因子如下。
因子符号 因子名称 水平1 水平2 水平3
A 前体混合 不添加 添加 N/A
B 葡萄糖 0.2％ 0.5％ 0.8％
C 甘油 0.8％ 2％ 3.2％
D 果糖 0.2％ 0.5％ 0.8％
E 可溶性淀粉 0.8％ 2％ 3.2％
F 马铃薯淀粉 0.8％ 2％ 3.2％
G 酵母抽提物 0.2％ 0.5％ 0.8％
H 棉籽精粉 0.4％ 1％ 1.6％
下述每个筛选的实验方案，除发酵罐与补料配方中的碳源氮源的组分与比例外，其余方案均与实施例1相同。
编号 A水平 B水平 C水平 D水平 E水平 F水平 G水平 H水平
4-1 1 1 1 1 1 1 1 1
4-2 1 1 2 2 2 2 2 2
4-3 1 1 3 3 3 3 3 3
4-4 1 2 1 1 2 2 3 3
4-5 1 2 2 2 3 3 1 1
4-6 1 2 3 3 1 1 2 2
4-7 1 3 1 2 1 3 2 3
4-8 1 3 2 3 2 1 3 1
4-9 1 3 3 1 3 2 1 2
4-10 2 1 1 3 3 2 2 1
4-11 2 1 2 1 1 3 3 2
4-12 2 1 3 2 2 1 1 3
4-13 2 2 1 2 3 1 3 2
4-14 2 2 2 3 1 2 1 3
4-15 2 2 3 1 2 3 2 1
4-16 2 3 1 3 2 3 1 2
4-17 2 3 2 1 3 1 2 3
4-18 2 3 3 2 1 2 3 1
实验结果：
实验编号 P-3效价1(ug/ml) P-3效价2(ug/ml)
4-1 266 303
4-2 321 332
4-3 255 302
4-4 465 462
4-5 416 419
4-6 124 129
4-7 237 282
4-8 320 326
4-9 170 151
4-10 497 501
4-11 577 598
4-12 380 390
4-13 514 510
4-14 409 415
4-15 636 642
4-16 545 543
4-17 475 483
4-18 571 573
使用mintab预测田口结果
设置(优化前)：
Figure PCTCN2022124781-appb-000006
设置(优化后)：
Figure PCTCN2022124781-appb-000007
使用mintab预测田口结果，S/N(信躁比)和标准差明显提升，发酵过程稳定性和抗干扰能力明显提升。均值提升，预期放罐效价值也显著提升。
根据信噪比响应表和均值响应表，对各因子的影响显著性进行分析。
通过信噪比响应分析得到排秩极差为A前体混合，B葡萄糖，E可溶性淀粉，F马铃薯淀粉，C甘油，D果糖，G酵母抽提物。
均值响应的排秩极差为A前体混合，F马铃薯淀粉，G酵母抽提物，H棉籽精粉，E可溶性淀粉，D果糖，C甘油，B葡萄糖。
均值效应分析排列为A前体混合，F马铃薯淀粉，G酵母抽提物，H棉籽精粉，E可溶性淀粉，D果糖，C甘油，B葡萄糖。
按S/N效应排列为A前体混合，B葡萄糖，E可溶性淀粉，F马铃薯淀粉，C甘油，D果糖，G酵母抽提物。
综合分析S/N效应(调节因子)和均值效应(分散度因子)，田口试验最终确定的各项因素最优水平为：A2、B2、C1、D2、E2、F3、G3、H1。
实施例10：培养基中加入前体配比的考察
基于实施例9，在基础培养基中加入前体混合物(总甲硫氨酸0.3％、异亮氨酸0.3％、异丁醇0.3％)在信噪比效应与均值效应中，均显示应加入前体混合物。但部分前体如异丁醇，流补过量对菌体生长会产生抑制作用。进一步对发酵培养过程甲硫氨酸、异亮氨酸、异丁醇每日流补量进行响应面三因素Box-Behnken考察。
上述的因素分别为：1.葡萄糖的补入速度与葡萄糖阻遏反馈的平衡，有必要筛选补料过程速率；2.磷酸戊糖途径产出的赤藓糖-4-磷酸与糖酵解途径产出的磷酸烯醇式丙酮酸结合形成AHBA，有必要结合AHBA前体喂养实验，缬氨酸、异丁醇、甲硫氨酸前体途径进行二次响应面分析；3.逆境状态下，微生物会代谢路径会更加偏向磷酸戊糖途径。
以此为基础筛选甲硫氨酸、异亮氨酸和异丁醇比例，其正交因子如下，
Figure PCTCN2022124781-appb-000008
实验设计如下：
实验编号 X水平(％) Y水平(％) Z水平(％) AP-3效价(ug/ml)
5-1 -1 -1 0 368
5-2 1 -1 0 472
5-3 -1 1 0 386
5-4 1 1 0 650
5-5 -1 0 -1 398
5-6 1 0 -1 652
5-7 -1 0 1 344
5-8 1 0 1 490
5-9 0 -1 -1 392
5-10 0 1 -1 502
5-11 0 -1 1 306
5-12 0 1 1 380
5-13 0 0 0 524
5-14 0 0 0 550
5-15 0 0 0 532
实验分析：根据所得数据进行多元回归分析，得到响应变量(X甲硫氨酸、Y异亮氨酸、Z异丁醇每日流补量)与响应值(AP-3效价(ug/ml))的多元二次回归方程；
AP-3效价(ug/ml)＝103.7+3100X甲硫氨酸(％)+10725Y异亮氨酸(％)+10425Z异丁醇(％)+12083X甲硫氨酸(％)*X甲硫氨酸(％)-177917Y异亮氨酸(％)*Y异亮氨酸(％)-172917Z异丁醇(％)*Z异丁醇(％)+100000X甲硫氨酸(％)*Y异亮氨酸(％)-67500X甲硫氨酸(％)*Z异丁醇(％)-22500Y异亮氨酸(％)*Z异丁醇(％)
各响应变量(X甲硫氨酸、Y异亮氨酸、Z异丁醇每日流补量)与响应值(AP-3效价(ug/ml))关系的显著性，由方差分析表中本设计模型的整体F值为173，P值为0(＜0.05％)，故本模型设计是显著的。
进而对X甲硫氨酸、Y异亮氨酸、Z异丁醇各响应变量，响应变量平方，响应变量双因子交互作用F值与P值进行分析。得出X甲硫氨酸、Y异亮氨酸、Z异丁醇、Y异亮氨酸*Y异亮氨酸、Z异丁醇*Z异丁醇、X甲硫氨酸*Y异亮氨酸、X甲硫氨酸*Z异丁醇之间对模型影响呈显著性。
各响应变量(X甲硫氨酸、Y异亮氨酸、Z异丁醇每日流补量)交互作用等值线图和曲面图分析以及使用mintab多响应预测，模型预测结果见附图2，其中P-3发酵效价(ug/ml)与前体量(％)的等值线图见附图3～5；
本方案具体预测结果为：
Figure PCTCN2022124781-appb-000009
实验结论：经过上述流补前体正交实验和数据分析可知，每日流补前体的配比为，甲硫氨酸0.05％，异亮氨酸0.043％，异丁醇0.018％。

Claims (10)

  1. 一种安丝菌素P-3的发酵方法，其包括发酵培养和自发酵培养起即始的每日流补，所述每日流补所添加的补料按质量体积比包括30～60％葡萄糖及安丝菌素代谢合成前体0.02～0.2％；所述的安丝菌素代谢合成前体包括甲硫氨酸、异亮氨酸和异丁醇。
  2. 一种如权利要求1所述的安丝菌素P-3的发酵方法，其中，所述流补前体甲硫氨酸的含量为0.01～0.1％，优选为0.03～0.06％，更优选为0.05％；
    和/或，所述流补前体异亮氨酸的含量为0.01～0.1％，优选为0.03～0.06％，更优选为0.04～0.05％；
    和/或，所述流补前体异丁醇的含量为0.01～0.1％，优选为0.01～0.04％，更优选为0.015～0.02％。
  3. 一种如权利要求1所述的安丝菌素P-3的发酵方法，其中，所述的发酵培养的培养基按质量体积比包括：速效碳源0.2～5％，缓释碳源0.5～5％，速效氮源0.2～5％，缓释氮源0.5～5％，无机盐0.2～1.5％；
    和/或，所述的速效碳源选自葡萄糖、果糖、蔗糖、甘油或乳糖中的一种或多种；
    和/或，所述的缓释碳源选自玉米粉、马铃薯淀粉、木薯淀粉、玉米淀粉、糯米粉或可溶性淀粉中的一种或多种；
    和/或，所述的速效氮源选自麦芽提取物、酵母提取物或酵母浸粉中的一种或多种；
    和/或，所述的缓释氮源选自玉米蛋白粉、大豆蛋白粉、棉籽精粉、大豆粉、黄豆饼粉或玉米浆干粉中的一种或多种；
    和/或，所述的无机盐选自碳酸盐、硫酸盐、磷酸盐或氯化物，优选为碳酸钙、磷酸二氢钾、氯化钾、硫酸镁、硫酸锌和硫酸亚铁。
  4. 一种如权利要求1所述的安丝菌素P-3的发酵方法，其中，所述发酵培养的培养基按质量体积比包括：葡萄糖0.1～2％，甘油0.1～2％，果糖0.1～2％，可溶性淀粉0.5～2.5％，马铃薯淀粉0.5～2.5％，酵母抽提物0.1～1％，棉籽精粉0.1～1％，碳酸钙0.1～1％，磷酸二氢钾0.01～0.1％，硫酸镁0.01～0.1％，硫酸亚铁0.01～0.1％。
  5. 一种如权利要求1所述的安丝菌素P-3的发酵方法，其中，所述发酵培养基的pH为6.5～7.5；
    和/或，所述发酵培养的温度为25～30℃；
    和/或，所述的发酵投料后的体积可为0～50000L；
    和/或，所述的发酵培养周期为12～14天。
  6. 一种如权利要求1所述的安丝菌素P-3的发酵方法，其包括以下步骤：
    a.发酵培养：称取各原料，以重量体积比计为，葡萄糖0.1～2％、果糖0.1～2％、甘油0.1～2％、马铃薯淀粉0.5～2.5％、可溶性淀粉0.5～2.5％、酵母抽提物0.1～1％、棉籽精粉0.1～1％、碳酸钙0.1～1％、磷酸二氢钾0.01～0.1％、硫酸镁0.01～0.1％和硫酸亚铁 0.01～0.1％；发酵罐内加入消泡剂和水，边搅拌边投入原料，用氢氧化钠调节pH至6.8～7.5，灭菌，保温保压；用无菌空气把种子液压入发酵罐，发酵罐于罐温25～30℃培养；
    b.每日流补：发酵培养开始后即进行每日流补，流补30～60％葡萄糖，维持发酵体系残糖检测0.5％～1％，并流补0.02～0.2％的安丝菌素生物合成前体异丁醇、甲硫氨酸和异亮氨酸；
    c.放罐：发酵培养12～14天后，发酵结束。
  7. 一种如权利要求1所述的安丝菌素P-3的发酵方法，其中，所述发酵培养基按质量提及比包括葡萄糖0.5％，甘油0.8％，果糖0.5％，可溶性淀粉2％，马铃薯淀粉3.2％，酵母抽提物0.8％，棉籽精粉0.4％，磷酸二氢钾0.05％，碳酸钙0.5％，硫酸镁0.05％，硫酸亚铁0.001％；
    和/或，葡萄糖0.5％；甘油3.2％；果糖0.2％；可溶性淀粉2％；马铃薯淀粉3.2％；酵母抽提物0.5％；棉籽精粉0.4％；碳酸钙0.5％；磷酸二氢钾0.05％；硫酸镁0.05％；硫酸亚铁0.001％；
    和/或，葡萄糖1.0％；甘油6.0％；果糖0.5％；可溶性淀粉4.0％；马铃薯淀粉6.0％；酵母抽提物1.0％；棉籽精粉2.0％；碳酸钙0.5％；磷酸二氢钾0.05％；硫酸镁0.05％；硫酸亚铁0.001％。
  8. 培养基在制备安丝菌素P-3中的应用，所述的培养基按质量体积比包括：葡萄糖0.1～2％，甘油0.1～2％，果糖0.1～2％，可溶性淀粉0.5～2.5％，马铃薯淀粉0.5～2.5％，酵母抽提物0.1～1％，棉籽精粉0.1～1％，碳酸钙0.1～1％，磷酸二氢钾0.01～0.1％，硫酸镁0.01～0.1％，硫酸亚铁0.01～0.1％。
  9. 如权利要求8所述的培养基在制备安丝菌素P-3中的应用，所述的培养基按质量体积比包括：葡萄糖0.5％，甘油0.8％，果糖0.5％，可溶性淀粉2％，马铃薯淀粉3.2％，酵母抽提物0.8％，棉籽精粉0.4％，磷酸二氢钾0.05％，碳酸钙0.5％，硫酸镁0.05％，硫酸亚铁0.001％；
    和/或，葡萄糖0.5％；甘油3.2％；果糖0.2％；可溶性淀粉2％；马铃薯淀粉3.2％；酵母抽提物0.5％；棉籽精粉0.4％；碳酸钙0.5％；磷酸二氢钾0.05％；硫酸镁0.05％；硫酸亚铁0.001％；
    和/或，葡萄糖1.0％；甘油6.0％；果糖0.5％；可溶性淀粉4.0％；马铃薯淀粉6.0％；酵母抽提物1.0％；棉籽精粉2.0％；碳酸钙0.5％；磷酸二氢钾0.05％；硫酸镁0.05％；硫酸亚铁0.001％。
  10. 一种如权利要求1所述的安丝菌素P-3的发酵方法，其中，所述流补前体的含量为甲硫氨酸0.05％，异亮氨酸0.043％，异丁醇0.018％。
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