Method for synergistically improving fermentation yield of daptomycin based on metabolic flow regulation

Abstract

The invention provides a method for synergistically improving fermentation yield of daptomycin based on metabolic flux regulation and control, which comprises the following steps: inoculating a daptomycin seed solution into a daptomycin fermentation culture medium for fermentation, supplementing a capric acid precursor in stages in the fermentation process, coupling a carbon source for starvation induction, adding acetyl coenzyme A to relieve a metabolic bottleneck at the same time, and carrying out fermentation to obtain the daptomycin fermentation yield. And feeding the corn steep liquor and the amino acid powder in a fermentation secondary metabolism stage, and controlling the synthesis direction in combination with feeding, so that the metabolic flux and the precursor utilization rate are improved, and the daptomycin is obtained. In the fermentation process, capric acid precursors are supplemented in stages, carbon source starvation induction is coupled, meanwhile, acetyl coenzyme A is added to relieve the metabolic bottleneck, corn steep liquor and amino acid powder are fed in the fermentation secondary metabolism stage, the synthesis direction is controlled in combination with material supplementation, the metabolic flux and the precursor utilization rate are improved, and therefore main components are increased, and impurities are reduced.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of microbial fermentation engineering, and particularly relates to a method for synergistically improving the fermentation yield of daptomycin based on metabolic flow regulation. BACKGROUND

[0002] Daptomycin is a novel lipopeptide antibiotic fermented by microorganism Streptomyces roseosporus. It is composed of a decanoyl side chain attached to the N-terminal tryptophan of a cyclic 13-amino acid peptide. The pure product of daptomycin is a light yellow crystalline powder, which is easily soluble in water and small-molecule organic solvents such as methanol, ethanol, butanol, etc. In alkaline solution and acidic solution with pH>3.5, daptomycin can be dissolved in large quantities; it is slightly soluble in organic solvents such as acetone and chloroform. Daptomycin interferes with the phospholipid structure of the cell membrane, hinders the synthesis of the main component of the bacterial cell wall, peptidoglycan, leading to cell rupture and bacterial death. Due to its novel mechanism of action, daptomycin has the characteristics of high bactericidal efficiency, low drug resistance, small dosage, and small side effects, etc., and stands out among numerous antibiotic drugs, becoming the best substitute for vancomycin. Daptomycin injection is used for the treatment of complex skin and soft tissue infections caused by gram-positive bacteria, including methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus, as well as bacteremia and right-sided endocarditis caused by Staphylococcus aureus.

[0003] The amino acid nucleus of daptomycin is synthesized by non-ribosomal peptide synthetase (NRPSs), and there are mostly fatty acyl coenzyme A ligases and acyl carrier proteins (ACPs) near the synthesis gene cluster, which participate in the activation of fatty acids. In the initial stage of the synthesis of cyclic lipopeptide antibiotics, fatty acyl coenzyme A ligase first catalyzes the reaction of fatty acid with ATP or acetyl coenzyme A to generate fatty acyl AMP or fatty acyl coenzyme A, and then continues to catalyze the combination of fatty acyl AMP or fatty acyl coenzyme A with acyl carrier protein (ACP). After the activation of the fatty acyl group, it is condensed with the amino group of the amino acid (tryptophan, aspartic acid or asparagine) under the action of NRPS, and the extension of the initial peptide chain.

[0004] The fermentation content of daptomycin is limited by the following factors: traditional carbon source (glucose) triggers catabolite repression, which inhibits the extension of the peptide chain; excessive addition of decanoic acid precursor leads to cell toxicity; competition between primary metabolism and secondary metabolism for resources causes unbalanced distribution of product metabolic flow; the problems of carbon source inhibition, precursor toxicity and metabolic flow competition cannot be solved by the medium formula and the feeding strategy, which limits the content improvement.

[0005] At present, the conventional method for producing daptomycin in the industry is mainly through fermentation of Streptomyces roseosporus strain. In the synthesis of daptomycin by fermentation method, first, the mother nucleus compound A21978C of daptomycin is generated by Streptomyces roseosporus, then the precursor capric acid is added to the fermentation broth to provide a side chain, and the final product is synthesized by biological acylation. The precursor compounds of capric acid include capric acid, capric acid salt, capric acid ester, capric aldehyde, etc. The addition mode of the precursor is the step that restricts the biosynthesis of daptomycin. The patent for invention with publication number CN111892647A discloses a feeding method for improving the fermentation yield of daptomycin, that is, at the time when the mycelial morphology begins to differentiate and enters the metabolic phase, a capric acid-oleic acid methyl ester solution with a volume ratio of 1:1 is added to the fermentation broth at a constant rate gradient. The patent for invention with publication number CN117210520A discloses a method for improving the fermentation level of daptomycin, using capric acid triglyceride as the precursor substance for daptomycin fermentation, adding phosphate to maintain the pH during the fermentation process, and at the same time, increasing the proportion of nitrogen source in the fermentation medium. The patent for invention with publication number CN109593808A discloses a daptomycin fermentation medium and a preparation method thereof. By improving the carbon and nitrogen sources in the fermentation medium, the production cost is reduced, and the yield of daptomycin is improved.

[0006] In the above-mentioned disclosed methods, the focus is mainly on the substance and addition mode of the precursor, and the types of precursors are basically capric acid. Since capric acid is toxic to the bacteria when in excess, there is no related report on the proportion of the main component. SUMMARY

[0007] The purpose of the present application is to solve the shortcomings in the prior art. The present application provides a method for improving the fermentation yield of daptomycin based on metabolic flow regulation.

[0008] The technical scheme adopted by the present application is as follows: a method for improving the fermentation yield of daptomycin based on metabolic flow regulation, comprising the following steps: inoculating daptomycin seed liquid into daptomycin fermentation medium for fermentation, adding capric acid precursor in stages during the fermentation process and coupling carbon source starvation induction, adding acetyl coenzyme A at the same time to remove metabolic bottleneck, adding corn syrup and amino acid powder in the fermentation secondary metabolic stage, combining feed control to synthesize direction, improving metabolic flux and precursor utilization rate, and obtaining daptomycin.

[0009] Preferably, the fermentation medium is prepared from the following materials in mass percentage: 1.0-1.4% glucose, 7.0-7.4% malt dextrin, 0.7-0.74% sugarcane molasses, 3.1-3.5% soybean hydrolysate, 1.1-1.5% amino acid powder, 0.04-0.06% ferrous ammonium sulfate hexahydrate, 0.04-0.06% defoaming agent, and the balance is water; the pH of the daptomycin fermentation medium is 6.5-6.7.

[0010] Preferably, in the fermentation process of daptomycin, the tank pressure is 0.04 MPa, the culture temperature is 30℃, the aeration ratio is 0.8 vvm, the dissolved oxygen is in the range of 30-50%, ammonia water is added after the pH drops to 6.5, the pH is controlled at 6.4-6.6, glucose is added when the glucose content drops to below 0.5%, and the glucose content is controlled at 0.3-0.5%. After 30h of product synthesis, a mixture of 1:1 n-decanoic acid and methyl oleate is added at a flow rate of 0.2-0.4 ml / L / h from 30h to 48h, and at a flow rate of 0.4-0.6 ml / L / h from 48h to the end of the fermentation.

[0011] Preferably, in the fermentation process of daptomycin,

[0012] A. The fermentation medium is supplemented with feed one starting at 30h; wherein, in terms of mass percentage, feed one consists of: 1-2% acetyl-CoA, and the balance is water; the flow rate of feed one is: 0.1-0.2 ml / L / h from 30h to 48h, 0.2-0.4 ml / L / h from 48h to 160h, and 0.05-0.1 ml / L / h from 160h to the end of the fermentation;

[0013] B. The fermentation medium is supplemented with feed two starting at 30h; wherein, in terms of mass percentage, feed two consists of: 8-10% corn syrup, 10-20% amino acid powder, and the balance is water; the flow rate of feed two is: 4-6 ml / L / h from 30h to 48h, and 6-8 ml / L / h from 48h to the end of the fermentation;

[0014] C. Feed three is added starting at 48h when product synthesis begins; wherein, in terms of mass percentage, feed three consists of: 3-5% calcium chloride, and the balance is water; the flow rate of feed three is: 3-5 ml / L / h from 48h to the end of the fermentation.

[0015] Preferably, the method for preparing daptomycin seed liquid comprises the following steps:

[0016] The prepared roseofinus solonifaciens seed bottle fermentation seed liquid is inoculated into the daptomycin seed culture medium at an inoculation amount of 0.2%, and cultured. When the bacterial concentration is above 20%, the mycelium is dense and reticular, the pH value is 6.7-6.9, and the culture period is 20-25h, the daptomycin seed liquid is obtained.

[0017] Preferably, the daptomycin seed medium is made of the following materials in mass percentage: 1.0-1.4% glucose, 7.0-7.4% maltodextrin, 0.7-0.74% cane molasses, 3.1-3.5% soybean hydrolysate, 0.4-0.6% amino acid powder, 0.08-0.1% ferrous ammonium sulfate hexahydrate, 0.04-0.06% antifoaming agent, and the rest is water; the pH of the daptomycin seed medium is 6.5-6.7.

[0018] Preferably, during the preparation of the daptomycin seed liquid, the tank pressure is 0.04 MPa, the culture temperature is 30°C, the aeration ratio is 0.8 vvm, and the dissolved oxygen is controlled at 30-50%.

[0019] The application has the following beneficial effects: the application optimizes the combination of carbon and nitrogen sources, designs a glucose-cane molasses-maltodextrin compound carbon source combined with soybean hydrolysate and amino acid powder compound nitrogen source, glucose provides rapid energy to promote cell growth, cane molasses and maltodextrin are slowly decomposed to continuously provide carbon skeletons (avoiding glucose repression), amino acid powder provides readily available amino acids, and soybean hydrolysate contains sulfur amino acids to promote coenzyme A synthesis. During the fermentation process, decanoic acid precursors are added in stages and coupled with carbon source starvation induction, acetyl coenzyme A is added at the same time to remove metabolic bottlenecks, corn syrup and amino acid powder are added in the fermentation secondary metabolism stage, the synthesis direction is combined with fed-batch control, the metabolic flux and precursor utilization rate are improved, thereby the main components are increased and impurities are reduced. The addition of acetyl coenzyme A improves the primary metabolic flux and removes metabolic bottlenecks; the use of secondary metabolism promoters (corn syrup and amino acid powder) removes key node restrictions and improves the utilization rate of precursors; calcium chloride is added at a flow rate of 3-5 ml / L / h starting from 48 h, which significantly improves the cyclic peptide polymerase activity and the utilization efficiency of precursors, and at the same time enhances the cell membrane permeability. Compared with the traditional process, the content of daptomycin is significantly improved without genetic modification, and the application is suitable for industrial production. DETAILED DESCRIPTION

[0020] The technical solutions in the embodiments of the application will be clearly and completely described below. Obviously, the described embodiments are only part of the embodiments of the application, rather than all the embodiments.

[0021] I. Embodiments

[0022] Embodiment 1

[0023] Seed culture: the culture medium volume is 20 L.

[0024] The seed medium is made of the following materials in mass percentage: 1.0% glucose, 7.0% maltodextrin, 0.7% cane molasses, 3.1% soybean hydrolysate, 0.4% amino acid powder, 0.08% ferrous ammonium sulfate hexahydrate, 0.04% antifoaming agent, and the rest is water; the pH of the daptomycin seed medium is 6.5-6.7.

[0025] The seed liquid of daptomycin monensin is prepared, the seed bottle of the cultured rose chain streptomyces is inoculated into the seed culture medium at the inoculation amount of 0.2% to culture, when the bacterial concentration (centrifugal speed 3000 rpm for 10 min) reaches more than 20%, the mycelium is dense and reticular, the pH value is 6.7-6.9, and the culture period is 20-25 h, the daptomycin seed liquid is obtained.

[0026] Fermentation culture: the volume of the culture medium is 60 L.

[0027] The fermentation medium, in terms of mass percentage, contains 1.0% glucose, 7.0% malt dextrin, 0.7% sugarcane molasses, 3.1% soybean hydrolysate, 1.1% amino acid powder, 0.04% ferrous ammonium sulfate hexahydrate, 0.04% defoaming agent, and the rest is water; the pH of the daptomycin fermentation medium is 6.5-6.7.

[0028] The fermentation culture of daptomycin is carried out under the conditions of tank pressure 0.04 MPa, culture temperature 30°C, aeration ratio 0.8 VVM, and the range of dissolved oxygen 30-50%, and after the pH decreases to 6.5, ammonia water is added, the pH is controlled at 6.4-6.6, and after the glucose content decreases to less than 0.5%, glucose is added, and the content is controlled at 0.3-0.5%. After the product starts to be synthesized at 30 h, a mixture of 1:1 n-decanoic acid and methyl oleate is added at a flow rate of 0.2-0.4 ml / L / h at 30-48 h, and at a flow rate of 0.4-0.6 ml / L / h at 48 h to tank.

[0029] (1) The first feeding is added at a flow rate of 0.1 ml / L / h at 30-48 h, 0.2 ml / L / h at 48-160 h, and 0.05 ml / L / h at 160 h to fermentation end, wherein the first feeding, in terms of mass percentage, contains 1% acetyl coenzyme A, and the rest is water.

[0030] (2) The second feeding is added at a flow rate of 4 ml / L / h at 30-48 h and 6 ml / L / h at 48 h to fermentation end, wherein the second feeding, in terms of mass percentage, contains 8% corn syrup and 10% amino acid powder, and the rest is water.

[0031] (3) The third feeding is added at a flow rate of 3 ml / L / h at 48 h to fermentation end, wherein the third feeding, in terms of mass percentage, contains 3% calcium chloride, and the rest is water.

[0032] After the fermentation culture ends, the fermentation period is 210 h, and the content of daptomycin is 5031 mg / mL, and the main component accounts for 50.8%.

[0033] Example 2

[0034] Seed culture: 20L of culture medium.

[0035] The seed culture medium, by mass percentage, consists of 1.2% glucose, 7.2% maltodextrin, 0.72% cane molasses, 3.3% soybean hydrolysate, 0.5% amino acid powder, 0.09% ferrous ammonium sulfate hexahydrate, 0.05% antifoaming agent, with the remainder being water; the pH of the daptomycin seed culture medium is 6.5-6.7.

[0036] To prepare daptomycin monensin seed culture, the cultured Streptomyces roseum seed culture was inoculated into seed culture medium at an inoculation rate of 0.2%. When the bacterial concentration (centrifugation speed 3000 rpm for 10 min) reached more than 20%, the mycelium formed a dense network, the pH value was 6.7-6.9, and the culture period was 20-25 h, daptomycin seed culture was obtained.

[0037] Fermentation culture: 60L of culture medium.

[0038] The fermentation medium, by mass percentage, consists of 1.2% glucose, 7.2% maltodextrin, 0.72% cane molasses, 3.3% soybean hydrolysate, 1.3% amino acid powder, 0.05% ferrous ammonium sulfate hexahydrate, 0.05% antifoaming agent, with the balance being water; the pH of the daptomycin fermentation medium is 6.5-6.7.

[0039] Fermentation culture of daptomycin was conducted at a tank pressure of 0.04 MPa, a culture temperature of 30°C, an aeration ratio of 0.8 vvm, and a dissolved oxygen range of 30-50%. Ammonia was added after the pH dropped to 6.5, and the pH was maintained at 6.4-6.6. Glucose was added again when the glucose content dropped below 0.5%, maintaining a content of 0.3-0.5%. After 30 hours, when product synthesis began, a 1:1 mixture of decanoic acid and methyl oleate was added. The flow rate was 0.2-0.4 ml / L / h from 30-48 hours, and 0.4-0.6 ml / L / h from 48 hours onwards.

[0040] (1) Feed 1 is added to the fermentation medium starting at 30h; wherein, by mass percentage, the composition of feed 1 is: 1.5% acetyl coenzyme A, with the remainder being water; the feed 1 has the following flow rate: 0.15ml / L / h from 30-48h, 0.3ml / L / h from 48-160h, and 0.075ml / L / h from 160h to the end of fermentation.

[0041] (2) Feed 2 is added to the fermentation medium starting at 30h; wherein, by mass percentage, the composition of feed 2 is: 9% corn steep liquor, 15% amino acid powder, and the remainder is water; the feed 2 is added at a rate of 5ml / L / h from 30-48h, and 7ml / L / h from 48h to the end of fermentation.

[0042] (3) After the product begins to synthesize at 48h, feed 3 is added: The composition of feed 3 by mass percentage is: 4% calcium chloride, with the remainder being water; The flow rate of feed 3 is: 4 ml / L / h from 48h to the end of fermentation.

[0043] Fermentation culture was completed after 210 hours. The daptomycin content was 5251 mg / mL, accounting for 51.2% of the main component.

[0044] Example 3

[0045] Seed culture: 20L of culture medium.

[0046] The seed culture medium, by mass percentage, consists of 1.4% glucose, 7.4% maltodextrin, 0.74% cane molasses, 3.5% soybean hydrolysate, 0.6% amino acid powder, 0.1% ferrous ammonium sulfate hexahydrate, 0.06% antifoaming agent, with the remainder being water; the pH of the daptomycin seed culture medium is 6.5-6.7.

[0047] To prepare daptomycin monensin seed culture, the cultured Streptomyces roseum seed culture was inoculated into seed culture medium at an inoculation rate of 0.2%. When the bacterial concentration (centrifugation speed 3000 rpm for 10 min) reached more than 20%, the mycelium formed a dense network, the pH value was 6.7-6.9, and the culture period was 20-25 h, daptomycin seed culture was obtained.

[0048] Fermentation culture: 60L of culture medium.

[0049] The fermentation medium, by mass percentage, consists of 1.4% glucose, 7.4% maltodextrin, 0.74% cane molasses, 3.5% soybean hydrolysate, 1.5% amino acid powder, 0.06% ferrous ammonium sulfate hexahydrate, 0.06% antifoaming agent, and the balance being water; the pH of the daptomycin fermentation medium is 6.5-6.7.

[0050] Fermentation culture of daptomycin was conducted at a tank pressure of 0.04 MPa, a culture temperature of 30°C, an aeration ratio of 0.8 vvm, and a dissolved oxygen range of 30-50%. Ammonia was added after the pH dropped to 6.5, and the pH was maintained at 6.4-6.6. Glucose was added again when the glucose content dropped below 0.5%, maintaining a content of 0.3-0.5%. After 30 hours, when product synthesis began, a 1:1 mixture of decanoic acid and methyl oleate was added. The flow rate was 0.2-0.4 ml / L / h from 30-48 hours, and 0.4-0.6 ml / L / h from 48 hours onwards.

[0051] (1) Feed 1 is added to the fermentation medium starting at 30h; wherein, by mass percentage, the composition of feed 1 is: 2% acetyl coenzyme A, with the remainder being water; the feed 1 has the following flow rate: 0.2ml / L / h from 30-48h, 0.4ml / L / h from 48-160h, and 0.1ml / L / h from 160h to the end of fermentation.

[0052] (2) Feed 2 is added to the fermentation medium starting at 30h; wherein, by mass percentage, the composition of feed 2 is: 10% corn steep liquor, 20% amino acid powder, and the remainder is water; the feed 2 is added at a rate of 6ml / L / h from 30-48h, and 8ml / L / h from 48h to the end of fermentation.

[0053] (3) After the product begins to synthesize at 48h, feed 3 is added: The composition of feed 3 by mass percentage is: 5% calcium chloride, with the remainder being water; the flow rate of feed 3 is: 5 ml / L / h from 48h to the end of fermentation.

[0054] Fermentation culture was completed after 210 hours. The daptomycin content was 5102 mg / mL, accounting for 51.8% of the main component.

[0055] Comparative Example 1

[0056] Seed culture: 20L of culture medium.

[0057] The seed culture medium, by mass percentage, consists of 1.2% glucose, 7.2% maltodextrin, 3.3% soybean meal, 0.09% ferrous ammonium sulfate hexahydrate, 0.05% antifoaming agent, and the remainder is water; the pH of the daptomycin seed culture medium is 6.5-6.7.

[0058] To prepare daptomycin monensin seed culture, the cultured Streptomyces roseum seed culture was inoculated into seed culture medium at an inoculation rate of 0.2%. When the bacterial concentration (centrifugation speed 3000 rpm for 10 min) reached more than 20%, the mycelium formed a dense network, the pH value was 6.7-6.9, and the culture period was 20-25 h, daptomycin seed culture was obtained.

[0059] Fermentation culture: 60L of culture medium.

[0060] The fermentation medium, by mass percentage, consists of 1.2% glucose, 7.2% maltodextrin, 3.3% soybean meal, 0.05% ferrous ammonium sulfate hexahydrate, 0.05% antifoaming agent, and the remainder is water; the pH of the daptomycin fermentation medium is 6.5-6.7.

[0061] Fermentation culture of daptomycin was conducted at a tank pressure of 0.04 MPa, a culture temperature of 30°C, an aeration ratio of 0.8 vvm, and a dissolved oxygen range of 30-50%. Ammonia was added after the pH dropped to 6.5, and the pH was maintained at 6.4-6.6. Glucose was added again when the glucose content dropped below 0.5%, maintaining a content of 0.3-0.5%. After 30 hours, when product synthesis began, a 1:1 mixture of decanoic acid and methyl oleate was added. The flow rate was 0.2-0.4 ml / L / h from 30-48 hours, and 0.4-0.6 ml / L / h from 48 hours onwards.

[0062] (1) Feed 1 is added to the fermentation medium starting at 30h; wherein, by mass percentage, the composition of feed 1 is: 1.5% acetyl coenzyme A, with the remainder being water; the feed 1 has the following flow rate: 0.15ml / L / h from 30-48h, 0.3ml / L / h from 48-160h, and 0.075ml / L / h from 160h to the end of fermentation.

[0063] (2) Feed 2 is added to the fermentation medium starting at 30h; wherein, by mass percentage, the composition of feed 2 is: 9% corn steep liquor, 15% amino acid powder, and the remainder is water; the feed 2 is added at a rate of 5ml / L / h from 30-48h, and 7ml / L / h from 48h to the end of fermentation.

[0064] (3) After the product begins to synthesize at 48h, feed 3 is added: The composition of feed 3 by mass percentage is: 4% calcium chloride, with the remainder being water; The flow rate of feed 3 is: 4 ml / L / h from 48h to the end of fermentation.

[0065] Fermentation culture was completed after 210 hours. The daptomycin content was 4073 mg / mL, accounting for 46.2% of the main component.

[0066] Comparative Example 2

[0067] Seed culture: 20L of culture medium.

[0068] The seed culture medium, by mass percentage, consists of 1.2% glucose, 7.2% maltodextrin, 0.72% cane molasses, 3.3% soybean hydrolysate, 0.09% ferrous ammonium sulfate hexahydrate, 0.05% antifoaming agent, and the balance being water; the pH of the daptomycin seed culture medium is 6.5-6.7.

[0069] To prepare daptomycin monensin seed culture, the cultured Streptomyces roseum seed culture was inoculated into seed culture medium at an inoculation rate of 0.2%. When the bacterial concentration (centrifugation speed 3000 rpm for 10 min) reached more than 20%, the mycelium formed a dense network, the pH value was 6.7-6.9, and the culture period was 20-25 h, daptomycin seed culture was obtained.

[0070] Fermentation culture: 60L of culture medium.

[0071] The fermentation medium, by mass percentage, consists of 1.2% glucose, 7.2% maltodextrin, 0.72% cane molasses, 3.3% soybean hydrolysate, 0.05% ferrous ammonium sulfate hexahydrate, 0.05% antifoaming agent, with the remainder being water; the pH of the daptomycin fermentation medium is 6.5-6.7.

[0072] Fermentation culture of daptomycin was conducted at a tank pressure of 0.04 MPa, a culture temperature of 30°C, an aeration ratio of 0.8 vvm, and a dissolved oxygen range of 30-50%. Ammonia was added after the pH dropped to 6.5, and the pH was maintained at 6.4-6.6. Glucose was added again when the glucose content dropped below 0.5%, maintaining a content of 0.3-0.5%. After 30 hours, when product synthesis began, a 1:1 mixture of decanoic acid and methyl oleate was added. The flow rate was 0.2-0.4 ml / L / h from 30-48 hours, and 0.4-0.6 ml / L / h from 48 hours onwards.

[0073] (1) Feed 1 is added to the fermentation medium starting at 30h; wherein, by mass percentage, the composition of feed 1 is: 1.5% acetyl coenzyme A, with the remainder being water; the feed 1 has the following flow rate: 0.15ml / L / h from 30-48h, 0.3ml / L / h from 48-160h, and 0.075ml / L / h from 160h to the end of fermentation.

[0074] (2) Feed 2 is added to the fermentation medium starting at 30h; wherein, by mass percentage, the composition of feed 2 is: 9% corn steep liquor, 15% amino acid powder, and the remainder is water; the feed 2 is added at a rate of 5ml / L / h from 30-48h, and 7ml / L / h from 48h to the end of fermentation.

[0075] (3) After the product begins to synthesize at 48h, feed 3 is added: The composition of feed 3 by mass percentage is: 4% calcium chloride, with the remainder being water; The flow rate of feed 3 is: 4 ml / L / h from 48h to the end of fermentation.

[0076] Fermentation culture was completed after 210 hours. The daptomycin content was 4321 mg / mL, accounting for 48.7% of the main component.

[0077] Comparative Example 3

[0078] Seed culture: 20L of culture medium.

[0079] The seed culture medium, by weight percentage, consists of 1.2% glucose, 7.2% maltodextrin, 0.72% cane molasses, 3.3% soybean hydrolysate, 0.5% amino acid powder, 0.09% ferrous ammonium sulfate hexahydrate, 0.05% antifoaming agent, and the balance being water; the pH of the daptomycin seed culture medium is 6.5-6.7.

[0080] To prepare daptomycin monensin seed culture, the cultured Streptomyces roseum seed culture was inoculated into seed culture medium at an inoculation rate of 0.2%. When the bacterial concentration (centrifugation speed 3000 rpm for 10 min) reached more than 20%, the mycelium formed a dense network, the pH value was 6.7-6.9, and the culture period was 20-25 h, daptomycin seed culture was obtained.

[0081] Fermentation culture: 60L of culture medium.

[0082] The fermentation medium, by mass percentage, consists of 1.2% glucose, 7.2% maltodextrin, 0.72% cane molasses, 3.3% soybean hydrolysate, 1.3% amino acid powder, 0.05% ferrous ammonium sulfate hexahydrate, 0.05% antifoaming agent, with the balance being water; the pH of the daptomycin fermentation medium is 6.5-6.7.

[0083] Fermentation culture of daptomycin was conducted at a tank pressure of 0.04 MPa, a culture temperature of 30°C, an aeration ratio of 0.8 vvm, and a dissolved oxygen range of 30-50%. Ammonia was added after the pH dropped to 6.5, and the pH was maintained at 6.4-6.6. Glucose was added again when the glucose content dropped below 0.5%, maintaining a content of 0.3-0.5%. After 30 hours, when product synthesis began, a 1:1 mixture of decanoic acid and methyl oleate was added. The flow rate was 0.2-0.4 ml / L / h from 30-48 hours, and 0.4-0.6 ml / L / h from 48 hours onwards.

[0084] (1) Feed 2 is added to the fermentation medium starting at 30h; wherein, by mass percentage, the composition of feed 2 is: 9% corn steep liquor, 15% amino acid powder, and the remainder is water; the feed 2 is added at a rate of 5ml / L / h from 30-48h, and 7ml / L / h from 48h to the end of fermentation.

[0085] (2) After the product begins to synthesize at 48h, feed 3 is added: The composition of feed 3 by mass percentage is: 4% calcium chloride, with the remainder being water; the flow rate of feed 3 is: 4 ml / L / h from 48h to the end of fermentation.

[0086] Fermentation culture was completed after 210 hours. The daptomycin content was 4517 mg / mL, accounting for 44.3% of the main component.

[0087] Seed culture: 20L of culture medium.

[0088] The seed culture medium, by weight percentage, consists of 1.2% glucose, 7.2% maltodextrin, 0.72% cane molasses, 3.3% soybean hydrolysate, 0.5% amino acid powder, 0.09% ferrous ammonium sulfate hexahydrate, 0.05% antifoaming agent, and the balance being water; the pH of the daptomycin seed culture medium is 6.5-6.7.

[0089] To prepare daptomycin monensin seed culture, the cultured Streptomyces roseum seed culture was inoculated into seed culture medium at an inoculation rate of 0.2%. When the bacterial concentration (centrifugation speed 3000 rpm for 10 min) reached more than 20%, the mycelium formed a dense network, the pH value was 6.7-6.9, and the culture period was 20-25 h, daptomycin seed culture was obtained.

[0090] Fermentation culture: 60L of culture medium.

[0091] The fermentation medium, by mass percentage, consists of 1.2% glucose, 7.2% maltodextrin, 0.72% cane molasses, 3.3% soybean hydrolysate, 1.3% amino acid powder, 0.05% ferrous ammonium sulfate hexahydrate, 0.05% antifoaming agent, with the balance being water; the pH of the daptomycin fermentation medium is 6.5-6.7.

[0092] Fermentation culture of daptomycin was conducted at a tank pressure of 0.04 MPa, a culture temperature of 30°C, an aeration ratio of 0.8 vvm, and a dissolved oxygen range of 30-50%. Ammonia was added after the pH dropped to 6.5, and the pH was maintained at 6.4-6.6. Glucose was added again when the glucose content dropped below 0.5%, maintaining a content of 0.3-0.5%. After 30 hours, when product synthesis began, a 1:1 mixture of decanoic acid and methyl oleate was added. The flow rate was 0.2-0.4 ml / L / h from 30-48 hours, and 0.4-0.6 ml / L / h from 48 hours onwards.

[0093] (1) Feed 1 is added to the fermentation medium starting at 30h; wherein, by mass percentage, the composition of feed 1 is: 1.5% acetyl coenzyme A, with the remainder being water; the feed 1 has the following flow rate: 0.15ml / L / h from 30-48h, 0.3ml / L / h from 48-160h, and 0.075ml / L / h from 160h to the end of fermentation.

[0094] (2) After the product begins to synthesize at 48h, feed 3 is added: The composition of feed 3 by mass percentage is: 4% calcium chloride, with the remainder being water; the flow rate of feed 3 is: 4 ml / L / h from 48h to the end of fermentation.

[0095] Fermentation culture was completed after 210 hours. The daptomycin content was 4618 mg / mL, accounting for 43.8% of the main component.

[0096] II. Experimental Results

[0097]

[0098] As can be seen from the above data, compared with the method for synergistically improving daptomycin fermentation yield based on metabolic flux regulation described in Examples 1-4, the daptomycin content is around 4300 mg / mL, which is far less than the data in Examples 1-3 of this invention (around 5100 mg / mL). The advantages of this invention are that by optimizing the carbon and nitrogen source combination, a combination of glucose-sugarcane molasses-maltodextrin complex carbon source and soybean hydrolysate and amino acid powder complex nitrogen source is designed. Glucose provides rapid energy to promote cell growth, while sugarcane molasses and maltodextrin slowly decompose to continuously provide carbon skeletons (avoiding glucose repression). Amino acid powder provides readily available amino acids, and sulfur-containing amino acids from soybean hydrolysate promote coenzyme A synthesis. During fermentation, decanoic acid precursor is added in stages and coupled with carbon source starvation induction. Acetyl coenzyme A is added simultaneously to relieve metabolic bottlenecks. Corn steep liquor and amino acid powder are added during the secondary metabolic stage of fermentation. Combined with feeding to control the direction of synthesis, metabolic flux and precursor utilization are improved, thereby increasing the main component and reducing impurities. Adding acetyl-CoA increases primary metabolic flux and removes metabolic bottlenecks; secondary metabolism promoters (corn steep liquor and amino acid powder) remove key node limitations and improve precursor utilization; starting at 48h, calcium chloride is added at 3-5 ml / L / h, which significantly improves cyclic peptide polymerase activity and precursor utilization efficiency, while enhancing cell membrane permeability.

[0099] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for synergistically increasing daptomycin fermentation yield based on metabolic flux regulation, characterized in that: The process includes the following steps: Daptomycin seed culture is inoculated into daptomycin fermentation medium for fermentation. During fermentation, decanoic acid precursor is added in stages and coupled with carbon source starvation induction. At the same time, acetyl-CoA is added to relieve metabolic bottleneck. Corn steep liquor and amino acid powder are added during the secondary metabolic stage of fermentation. Combined with feeding to control the direction of synthesis, improve metabolic flux and precursor utilization, and obtain daptomycin.

2. The method for synergistically increasing daptomycin fermentation yield based on metabolic flux regulation according to claim 1, characterized in that: The fermentation medium is made from the following materials by mass percentage: 1.0-1.4% glucose, 7.0-7.4% maltodextrin, 0.7-0.74% cane molasses, 3.1-3.5% soybean hydrolysate, 1.1-1.5% amino acid powder, 0.04-0.06% ferrous ammonium sulfate hexahydrate, 0.04-0.06% defoamer, balance water; the pH of the daptomycin fermentation medium is 6.5-6.

7.

3. The method for synergistically improving daptomycin fermentation yield based on metabolic flux regulation according to claim 1, characterized in that: During the fermentation of daptomycin, the tank pressure was 0.04 MPa, the culture temperature was 30℃, the aeration ratio was 0.8 vvm, the dissolved oxygen range was 30-50%, ammonia was added after the pH dropped to 6.5, and the pH was controlled at 6.4-6.

6. When the glucose content dropped below 0.5%, glucose was added, and the content was controlled at 0.3-0.5%. After the product synthesis began at 30 h, a 1:1 mixture of decanoic acid and methyl oleate was added. The flow rate was 0.2-0.4 ml / L / h from 30-48 h, and 0.4-0.6 ml / L / h from 48 h onwards.

4. A method for synergistically increasing daptomycin fermentation yield based on metabolic flux regulation according to any one of claims 1-3, characterized in that: During the fermentation culture of daptomycin, A. Feeding of the first feedstock begins after 30 hours of fermentation. The first feedstock, by mass percentage, consists of 1-2% acetyl-CoA, with the remainder being water. The feedstock flow rate is: 0.1-0.2 ml / L / h for 30-48 hours, 0.2-0.4 ml / L / h for 48-160 hours, and 0.05-0.1 ml / L / h from 160 hours until the end of fermentation. B. Feeding of the second feed begins after 30 hours of fermentation. The composition of the second feed, by mass percentage, is: 8-10% corn steep liquor, 10-20% amino acid powder, and the remainder is water. The feed rate of the second feed is: 4-6 ml / L / h from 30 to 48 hours, and 6-8 ml / L / h from 48 hours to the end of fermentation. C. After the product synthesis begins at 48h, feed 3 is added: feed 3 consists of 3-5% calcium chloride and the remainder is water by mass percentage; the flow rate of feed 3 is 3-5 ml / L / h from 48h to the end of fermentation.

5. The method for synergistically increasing daptomycin fermentation yield based on metabolic flux regulation according to claim 1, characterized in that: The method for preparing the daptomycin seed solution includes the following steps: The fermented seed culture of Streptomyces roseum in the culture bottle was inoculated into daptomycin seed medium at an inoculation rate of 0.2% and cultured. The culture was centrifuged at 3000 rpm for 10 min. When the bacterial concentration reached more than 20%, the mycelium formed a dense network, the pH value was 6.7-6.9, and the culture period was 20-25 h, the daptomycin seed culture was obtained.

6. The method for synergistically increasing daptomycin fermentation yield based on metabolic flux regulation according to claim 5, characterized in that: The daptomycin seed culture medium is prepared by the following materials in weight percentage: 1.0-1.4% glucose, 7.0-7.4% maltodextrin, 0.7-0.74% cane molasses, 3.1-3.5% soybean hydrolysate, 0.4-0.6% amino acid powder, 0.08-0.1% ferrous ammonium sulfate hexahydrate, 0.04-0.06% defoamer, with the balance being water; the pH of the daptomycin seed culture medium is 6.5-6.

7.

7. The method for synergistically increasing daptomycin fermentation yield based on metabolic flux regulation according to claim 6, characterized in that, During the preparation of the daptomycin seed culture, the tank pressure was 0.04 MPa, the culture temperature was 30°C, the aeration ratio was 0.8 vvm, and the dissolved oxygen was controlled at 30-50%.

Images