CN110551660B

CN110551660B - Synthetic culture medium and preparation method and application thereof

Info

Publication number: CN110551660B
Application number: CN201910875331.2A
Authority: CN
Inventors: 黄明志; 陈腾飞; 李敏超
Original assignee: East China University of Science and Technology
Current assignee: East China University of Science and Technology
Priority date: 2019-09-17
Filing date: 2019-09-17
Publication date: 2021-05-04
Anticipated expiration: 2039-09-17
Also published as: CN110551660A

Abstract

The invention discloses a synthetic culture medium and preparation and application thereof, and is characterized in that the synthetic culture medium comprises the following components in percentage by mass: glucose. H₂10-30 g/L of O, 0.2-1.0 g/L of xylose, 3-10 g/L of ammonium sulfate, 2-5 g/L of sodium nitrate, 0.2-0.8 g/L of potassium chloride, 1.0-3.0 g/L of monopotassium phosphate, 0.02-0.08 g/L of ferrous sulfate, 0.03-0.06 g/L of magnesium sulfate, 0.005-0.02 g/L of cobalt chloride, 2-10 g/L of calcium carbonate, 0.01-0.05 g/L of phenylalanine, 120.001-0.01 g/L of vitamin VB, 30.001-0.02 g/L of vitamin VB, 0.001-0.03 g/L of biotin, 0.01-0.1 g/L of betaine and the balance of water. The novel culture medium provided by the invention is used for culturing the small single cell bacteria, and has important significance for mechanism research of synthesizing secondary metabolites through small single cell bacteria fermentation and industrial production of gentamicin C1 a.

Description

Synthetic culture medium and preparation method and application thereof

Technical Field

The invention belongs to the technical field of fermentation, and particularly relates to a synthetic culture medium, and a preparation method and application thereof.

Background

Gentamicin C1a is a parent nucleus precursor of a new semi-synthetic drug, namely amicin, which is originally created in China and has independent intellectual property rights I. The traditional method for preparing C1a is to extract C1a single component from multi-component gentamicin by adopting a resin separation technology, and because the components have similar structures and similar physical and chemical properties, the extraction cost is high, and the high-purity C1a in the market has a high selling price. As gentamicin C1a is one of gentamicin multi-components, a large amount of organic nitrogen sources, carbon sources and other compounds are adopted in the industrial fermentation process, so that the relation between the product synthesis mechanism and the fermentation condition cannot be accurately judged.

Although some reports are made in domestic and foreign literatures on the synthetic culture of gentamicin, the synthetic content of the product is very low when the product is directly used for fermentation of gentamicin C1 a.

Therefore, a special synthetic medium suitable for gentamicin C1a needs to be developed to improve the synthetic content of the product of gentamicin C1a fermentation.

The medium generally includes complex media, semi-synthetic media, and synthetic media. The composite culture medium has the advantages of rich nutrient components and good culture effect, but has the defects of complex components, poor quality stability of raw materials and poor quality stability of produced products. At present, the gentamicin C1a is produced by using a composite culture medium in the field, and although the yield basically meets the requirement, no report of producing gentamicin C1a by using a synthetic culture medium exists in the field at present.

Therefore, in order to research the relationship between the fermentation conditions and the anabolism mechanism of the product, the development of a special synthetic medium suitable for gentamicin C1a has important practical significance.

Disclosure of Invention

In order to solve the problem that the synthetic content of the product of the gentamicin C1a fermentation in the prior art is very low, the invention provides a synthetic medium formula which is suitable for culturing the Micromonospora purpurea or Micromonospora echinospora so as to efficiently produce the gentamicin C1 a. The culture medium is suitable for growth of the small monad and can greatly improve the yield of gentamicin C1 a.

The synthetic culture medium provided by the invention comprises the following components in percentage by mass: glucose. H₂10-30 g/L of O, 0.2-1.0 g/L of xylose, 3-10 g/L of ammonium sulfate, 2-5 g/L of sodium nitrate, 0.2-0.8 g/L of potassium chloride, 1.0-3.0 g/L of potassium dihydrogen phosphate, 0.02-0.08 g/L of ferrous sulfate, 0.03-0.06 g/L of magnesium sulfate, 0.005-0.02 g/L of cobalt chloride, 2-10 g/L of calcium carbonate, 0.01-0.05 g/L of phenylalanine, 120.001-0.01 g/L of vitamin VB, and 0.001-0. 3g/L of vitamin VB

0.001-0.02 g/L, biotin 0.001-0.03 g/L, betaine 0.01-0.1 g/L, and the balance of water.

Further, the mass concentration of the components of the synthetic medium is preferably: glucose. H₂O20-28 g/L, xylose 0.4-0.8 g/L, ammonium sulfate 3-5 g/L, sodium nitrate 2-3 g/L, potassium chloride 0.4-0.6 g/L, potassium dihydrogen phosphate 1.5-2.5 g/L, ferrous sulfate 0.04-0.06 g/L, magnesium sulfate 0.04-0.05 g/L, cobalt chloride 0.01-0.015 g/L, calcium carbonate 4-8 g/L, phenylalanine 0.03-0.05 g/L, vitamin VB 120.005-0.008 g/L, vitamin VB 30.005-0.008 g/L, biotin 0.01-0.02 g/L, betaine 0.03-0.08 g/L, and the balance of water.

Still further, the optimal mass concentration values of the components of the synthetic medium are as follows: glucose. H₂28g/L of O, 0.5g/L of xylose, 3g/L of ammonium sulfate, 2.3g/L of sodium nitrate, 0.5g/L of potassium chloride, 2.2g/L of monopotassium phosphate, 0.05g/L of ferrous sulfate, 0.045g/L of magnesium sulfate, 0.012g/L of cobalt chloride, 4g/L of calcium carbonate, 0.05g/L of phenylalanine, 120.006g/L of vitamin VB30.0055g/L of vitamin, 0.015g/L of biotin, 0.05g/L of betaine and the balance of water.

The components of the culture medium are common chemical products, the price is low, the preparation is simple, and compared with the culture medium in the prior art, the novel culture medium can obviously promote the fermentation production of gentamicin C1a by using the Micromonospora, the yield of gentamicin C1a is obviously improved, and the research on metabolic mechanism and the production of gentamicin C1a is facilitated. Betaine is used as a methyl donor and added into the culture medium in a proper proportion, and has a positive effect on the product synthesis of fermentation. VB3 and biotin are very important influencing factors in the fermentation process.

The invention also provides a preparation method of the synthetic medium, which comprises the following steps:

adding xylose, ammonium sulfate, sodium nitrate, potassium chloride, potassium dihydrogen phosphate, ferrous sulfate, magnesium sulfate, cobalt chloride, phenylalanine, vitamin VB12, vitamin VB3, biotin and betaine into water, mixing, and adjusting the pH value of the solution to 7.2-7.5;

adding calcium carbonate into the above solution, sterilizing, and adding sterilized glucose H₂O to obtain a synthetic medium.

Preferably, the method comprises:

the calcium carbonate is added into the solution, the solution is uniformly mixed, and then the sterilization temperature is 121 ℃, and the sterilization time is 20-30 min.

Further, preferably, the sterilized glucose · H₂And the sterilization condition of O is 115 ℃, and the sterilization time is 20-30 min.

The invention also provides an application of the synthetic medium in producing gentamicin C1a by using the fermentation of the small monad.

Further, preferably, the micromonospora is micromonospora purpurea or micromonospora echinospora.

Advantageous effects

Due to the implementation of the technical scheme, compared with the prior art, the invention has the following advantages:

1. the novel medium component of the invention comprises glucose H₂O, ammonium sulfate, sodium nitrate, potassium chloride, monopotassium phosphate, ferrous sulfate heptahydrate, calcium carbonate, magnesium sulfate, cobalt chloride, VB3, phenylalanine, biotin, betaine and xylose are common reagents, and the preparation method is easy to obtain raw materials, low in dosage, low in cost, simple and beneficial to large-scale industrial production.

2. The novel culture medium is adopted to culture the stenotrophomonas (more preferably the stenotrophomonas or stenotrophomonas), the yield of the gentamicin C1a obtained by the strain in the novel culture medium is obviously improved, the amount of synthesized gentamicin C1a is 284 mug/mL, and the yield is improved by 6 times compared with 47 mug/mL before optimization. Has important significance for the mechanism research of synthesizing secondary metabolite by fermenting the small monad and the industrial production of the gentamicin C1 a.

Drawings

FIG. 1 is a graph showing the yield of gentamicin C1a in fermentation broth as the fermentation time changes during the fermentation process of producing gentamicin C1a by using the original synthetic medium before optimization and the optimized synthetic medium of the present invention in example 1.

FIG. 2 is a graph showing the dry weight of the cells in the fermentation broth as a function of the fermentation time during the fermentation process for producing gentamicin C1a by using the original synthetic medium and the optimized synthetic medium before optimization of the present invention in example 1.

FIG. 3 is a graph showing the yield of gentamicin C1a in fermentation broth, which is obtained by fermenting gentamicin C1a with the synthetic medium of examples 2-5, and the fermentation process is changed with the fermentation time.

Detailed Description

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the technical solutions of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The experimental procedures, for which specific conditions are not noted in the following examples, are generally performed according to conventional conditions such as those described in J. SammBruk et al, molecular cloning protocols, third edition, scientific Press, 2002 or according to the manufacturer's recommendations.

Example 1

The specific embodiment provides a synthetic culture medium, which comprises the following components in percentage by mass:

glucose. H₂28g/L of O, 0.5g/L of xylose, 3g/L of ammonium sulfate, 2.3g/L of sodium nitrate, 0.5g/L of potassium chloride, 2.2g/L of monopotassium phosphate, 0.05g/L of ferrous sulfate, 0.045g/L of magnesium sulfate, 0.012g/L of cobalt chloride, 4g/L of calcium carbonate, 0.05g/L of phenylalanine, 120.006g/L of vitamin VB30.0055g/L of vitamin, 0.015g/L of biotin, 0.05g/L of betaine and the balance of water.

The preparation method of the synthetic culture medium comprises the following steps:

adding calcium carbonate into the above solution, mixing, sterilizing at 121 deg.C for 25min, and adding sterilized glucose H into sterile operation table₂And O, obtaining a synthetic culture medium.

Glucose. H₂And the sterilization condition of O is 115 ℃, and the sterilization time is 20-30 min.

The fermentation culture method comprises the following steps:

6mL of cultured stenotrophomonas purpurea seed liquid is inoculated into a 500mL shake flask filled with 60mL of synthetic culture medium, the culture is carried out for 96h at the culture temperature of 35.5 +/-0.5 ℃, the rotating speed of 260rpm and the humidity of 40% -60%, the dry weight of the thalli and the product concentration are measured after the fermentation is finished, and the results are shown in fig. 1 and fig. 2.

Control group

The control group provides an original synthetic medium formula, which is as follows:

glucose. H₂20g/L of O, 3g/L of ammonium sulfate, 2g/L of sodium nitrate, 0.5g/L of potassium chloride, 1.5g/L of monopotassium phosphate, 0.05g/L of ferrous sulfate heptahydrate, 0.05g/L of magnesium sulfate and 0.01g/L of cobalt chloride are added into water to be mixed, then the pH is adjusted to be within the range of 7.8 +/-0.2 by using sodium hydroxide (1mol/L), then 4g/L of calcium carbonate is added into the mixture to be mixed, then the mixture is sterilized at 118 ℃ for 25min, and the mixture is cultured by a constant temperature shaking table at 35 ℃ for 96h to be used for fermenting and culturing gentamycin C1 a.

On the basis of the original synthetic medium formula, the influence of different carbon sources, nitrogen sources, amino acids, vitamins, metal ions and precursors on product synthesis and growth is investigated by adopting a single-factor addition experiment.

The specific implementation is that different substances with the concentration of 0.01-0.02% are added into a 24-pore plate culture medium, the plate shaking table is cultured for 96 hours at the rotating speed of 220rpm and the culture temperature of 35.5 +/-0.5 ℃, the dry weight (W) of a unit cell and the content of gentamicin C1a are measured, the product content of the unit cell is calculated on the basis, and the product content is improved by more than 10% compared with a control group to be used as a next experimental investigation object.

TABLE 1 Single factor addition test results

As can be seen from Table 1, Phe, glu, gln, Leu, Tyr, Asn, betaine, guanine, cytidine, VB3, biotin, 2-dox, xylose, and the like were substances that were screened to be improved by 10% or more according to the single-factor implementation results, indicating that these substances have a promoting effect on the gentamicin C1a content.

The substances which are screened according to the single-factor implementation result and improved by more than 10 percent are used as candidates to carry out a PB test, and by utilizing Minitab software, 24 groups of experiments are designed, wherein the high level is 1.5 times of the low level, and the PB test table and the results are shown in the following table. The unit thallus titer is used as a response parameter, the unit thallus titer is used as a response value by software for calculation, the model is obvious from the P value, and the model gives the relationship between gentamicin C1a and each component:

y (mg/g) ═ 3.711-0.239phe +0.363gly-0.356ile-0.657gln-0.548glu-0.153asp +0.345tyr-0.029leu-0.695vb3+0.668vb12-1.352 biotin-0.261 adenine +0.987 uracil-0.060 cytidine-0.490 guanine +2.289 betaine-0.049 uridine +0.153mo +0.0072 dox-1.507 xylose.

TABLE 2Plackett-Burman design factor levels and codings

As can be seen from Table 2, xylose, betaine and biotin have the most significant influence on the unit titer of the culture medium, and the components and concentrations of the synthetic culture are finally determined by integrating other experimental data: glucose. H₂O28g/L, xylose 0.5g/L, ammonium sulfate 3g/L, sodium nitrate 2.3g/L, potassium chloride 0.5g/L, potassium dihydrogen phosphate 2.2g/L, ferrous sulfate 0.05g/L, magnesium sulfate 0.045g/L, cobalt chloride 0.012g/L, calcium carbonate 4g/L, phenylalanine 0.05g/L, vitamin VB120.006g/L, vitamin VB30.0055g/L, biotin 0.015g/L, betaine 0.05 g/L.

Comparison of the original synthetic medium, i.e. the control, and the optimized synthetic medium, i.e. example 1, in the 5L fermenter for product synthesis and bacterial growth of gentamicin C1 a:

1. fermentation process

(1) Slant culture

The strain of Micromonospora echinospora (knock-out GenK, obtained from Wuhan university) placed in a refrigerator at 80 ℃ in glycerol storage was taken out, and 200. mu.l of the strain was uniformly applied to the slant of an eggplant bottle having a liquid loading of 50 mL. The slant culture medium comprises: starch 10g/L, KNO₃1g/L，K₂HPO₄·3H₂O 0.3g/L，MgSO₄·7H₂O0.5 g/L, NaCl 0.5g/L, L-asparagine 0.02g/L, CaCO₃1g/L, 17g/L of bran, 14g/L of agar powder, pH7.8, and high-pressure moist heat sterilization at 121 ℃ for 30 min. The sand soil spores are spread in eggplant bottles, cultured at 35 +/-0.5 ℃ for 8 days, and stored in a refrigerator at 4 ℃ for later use.

Seed culture medium: 15g/L of corn flour, 10g/L of starch, 1g/L of glucose, 10g/L of low-temperature soybean flour, 2g/L of peptone and KNO₃0.5g/L，CaCO₃5g/L, and performing high-pressure moist heat sterilization at 121 ℃ for 30 min. Digging agar with width of 1cm multiplied by 2cm and spores to insert into a shake flask with liquid loading capacity of 60mL/500mL, culturing at 35 +/-1 ℃ with shaking table rotation speed of 220rpm, and culturing period of 46 +/-3 h.

Fermentation culture: 60mL of cultured seeds are inoculated into a 5L fermentation tank (with stirring, pH, dissolved oxygen, weighing and the like) with the volume ratio of 3000mL/5000mL, the rotation speed is 300rpm, the culture temperature is 35.5 +/-0.5 ℃, the seeds are cultured for 96h, and the dry weight of the thalli and the product concentration are measured by sampling every 12 h.

Determination of cell dry weight: the cell concentration is determined by dry cell weight method, 5mL fermentation liquid is weighed in a peeled centrifuge tube, mycelium is obtained by filtering with a vacuum pump and a 0.8 μm microporous filter membrane with aperture of 47mm, and the filter cake with the mycelium is dried in an oven at 80 ℃ to constant weight.

And (3) product liquid phase determination: 0.3636g/L of sodium heptanesulfonate 250mL are prepared as the aqueous phase. According to the following methanol: water phase: preparing 1L of mobile phase with the volume ratio of 730, 225 and 45, ultrafiltering the prepared mobile phase through an organic phase filter membrane with the pore diameter of 0.22 mu m, and ultrasonically degassing for 10min for later use.

Preparation of the derivatizing agent OPA: and (4) preparing an OPA solution. Weighing 0.5g of phthalaldehyde, dissolving the phthalaldehyde in 2.5mL of methanol, adding 47.5mL of 0.4mol/L boric acid with the pH value of 10.4, uniformly mixing, adding 1mL of 3-mercaptopropionic acid, adjusting the uniformly mixed solution to 10.4 by using 45% NaOH, putting the solution into a brown wide-mouth bottle, and storing the bottle in a dark place.

Pretreating a standard product: 0.25mL of 1000U/mL gentamicin C1a standard sample is sucked by using a pipette, 3.75mL of ultrapure water, 1mL of a derivative of LOPA and 5mL of isopropanol are added in sequence, and the mixture is mixed uniformly. Placing into 60 deg.C water bath kettle, water bathing for 15min, and cooling at room temperature. 1mL of the derivatized sample was taken out by a 2mL syringe, filtered through a 0.22 μm filter, diluted to 200U/mL,400U/mL,600U/mL,800U/mL,1000U/mL and added to the sample separately for further use.

And (3) high performance liquid chromatography detection: the liquid inlet amount is set to be 20 mul, the detection wavelength is 330nm, the detection period is 10min, the temperature is 40 ℃, and the flow rate is 1.2 mL/min. The column pressure was stabilized by passing the mobile phase using 90% methanol water through a high performance liquid chromatography C18 strong particle exchange column ZORBAX SB-C18 (model 4.6X 150mm, 5 μm).

As can be seen from FIG. 1, the weight of the optimized synthetic medium, i.e., the dry cell weight of example 1, was greater than that of the original synthetic medium, i.e., the control, in any cycle, so the synthetic medium prepared in example 1 was advantageous for the culture and fermentation of Micromonospora parvum.

As can be seen from FIG. 2, the amount of the synthesized gentamicin C1a cultured on the optimized synthetic medium, i.e., the synthetic medium of EXAMPLE 1, was 284. mu.g/mL, which is 6-fold higher than the unit of 47. mu.g/mL in the control group before optimization.

Example 2

The present embodiment provides another embodiment of a synthetic culture medium, which comprises the following components by mass:

glucose. H₂10g/L of O, 0.2g/L of xylose, 3g/L of ammonium sulfate, 2g/L of sodium nitrate, 0.2g/L of potassium chloride, 1g/L of monopotassium phosphate, 0.02g/L of ferrous sulfate, 0.03g/L of magnesium sulfate, 0.005g/L of cobalt chloride, 2g/L of calcium carbonate, 0.01g/L of phenylalanine, 120.001g/L of vitamin VBE, 30.001g/L of vitamin VBE, 0.001g/L of biotin, 0.01g/L of betaine and the balance of water.

The fermentation culture method comprises the following steps:

6mL of cultured stenotrophomonas purpurea seed liquid is inoculated into a 500mL shake flask filled with 60mL of synthetic culture medium, the culture is carried out for 96h at the culture temperature of 35.5 +/-0.5 ℃, the rotating speed of 260rpm and the humidity of 40% -60%, and the product concentration is determined after the fermentation is finished, and the result is shown in figure 3.

Example 3

This embodiment provides a further embodiment of a synthetic medium, comprising the following components in mass concentration:

glucose. H₂30g/L of O, 1g/L of xylose, 10g/L of ammonium sulfate, 5g/L of sodium nitrate, 0.8g/L of potassium chloride, 3g/L of monopotassium phosphate, 0.08g/L of ferrous sulfate, 0.06g/L of magnesium sulfate, 0.02g/L of cobalt chloride, 10g/L of calcium carbonate, 0.05g/L of phenylalanine, 120.01g/L of vitamin VB30.02g/L, 0.03g/L of biotin, 0.1g/L of betaine and the balance of water.

The fermentation culture method comprises the following steps:

Example 4

This embodiment provides another embodiment of a synthetic medium, which comprises the following components in mass concentration:

glucose. H₂20g/L of O, 0.4g/L of xylose, 4g/L of ammonium sulfate, 2g/L of sodium nitrate, 0.4g/L of potassium chloride, 1.5g/L of monopotassium phosphate, 0.04g/L of ferrous sulfate, 0.04g/L of magnesium sulfate, 0.01g/L of cobalt chloride, 4g/L of calcium carbonate, 0.03g/L of phenylalanine, 120.05g/L of vitamin VB120, 30.05g/L of vitamin VB30, 0.01g/L of biotin, 0.03g/L of betaine and the balance of water.

The fermentation culture method comprises the following steps:

Example 5

glucose. H₂O28g/L, xylose 0.8g/L, ammonium sulfate 6g/L, sodium nitrate3g/L, 0.6g/L of potassium chloride, 2.5g/L of monopotassium phosphate, 0.06g/L of ferrous sulfate, 0.05g/L of magnesium sulfate, 0.015g/L of cobalt chloride, 8g/L of calcium carbonate, 0.05g/L of phenylalanine, 120.08g/L of vitamin VBE, 30.08g/L of vitamin VBE, 0.02g/L of biotin, 0.08g/L of betaine and the balance of water.

The fermentation culture method comprises the following steps:

As shown in FIG. 3, the weight of the optimized synthetic medium, i.e., the dry cell weight in examples 2 to 5, in any cycle is in a higher range, so that the synthetic medium prepared in examples 2 to 5 is beneficial to the culture and fermentation of Micromonospora parvum.

In summary, the invention provides a synthetic medium for producing gentamicin C1 a. The synthetic culture medium is more beneficial to analyzing the synthesis and metabolism regulation and control mechanism of the gentamicin C1a, and lays a foundation for further improving the industrial yield of the gentamicin C1 a.

It should be noted that the above embodiments can be freely combined as necessary. The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

1. A synthetic medium comprising the following components:

glucose. H₂10-30 g/L of O, 0.2-1.0 g/L of xylose, 3-10 g/L of ammonium sulfate, 2-5 g/L of sodium nitrate, 0.2-0.8 g/L of potassium chloride, 1.0-3.0 g/L of monopotassium phosphate, 0.02-0.08 g/L of ferrous sulfate, 0.03-0.06 g/L of magnesium sulfate, 0.005-0.02 g/L of cobalt chloride, 2-10 g/L of calcium carbonate, 0.01-0.05 g/L of phenylalanine, 120.001-0.01 g/L of vitamin VB, 30.001-0.02 g/L of vitamin VB, 0.001-0.03 g/L of biotin, 0.01-0.1 g/L of betaine and the balance of water.

2. The synthetic medium of claim 1 wherein said synthetic medium comprises the following components:

glucose. H₂20-28 g/L of O, 0.4-0.8 g/L of xylose, 3-5 g/L of ammonium sulfate, 2-3 g/L of sodium nitrate, 0.4-0.6 g/L of potassium chloride, 1.5-2.5 g/L of monopotassium phosphate, 0.04-0.06 g/L of ferrous sulfate, 0.04-0.05 g/L of magnesium sulfate, 0.01-0.015 g/L of cobalt chloride, 4-8 g/L of calcium carbonate, 0.03-0.05 g/L of phenylalanine, 0.008g/L of vitamin VB 120.005-0.008 g/L, 0.01-0.02 g/L of vitamin VB 30.005-0.008 g/L, 0.03-0.08 g/L of betaine and the balance of water.

3. The synthetic medium of claim 2 wherein said synthetic medium comprises the following components:

glucose. H₂28g/L of O, 0.5g/L of xylose, 3g/L of ammonium sulfate, 2.3g/L of sodium nitrate, 0.5g/L of potassium chloride, 2.2g/L of monopotassium phosphate, 0.05g/L of ferrous sulfate, 0.045g/L of magnesium sulfate, 0.012g/L of cobalt chloride, 4g/L of calcium carbonate, 0.05g/L of phenylalanine, 0.3 g/L of sodium nitrate, potassium chloride, potassium dihydrogen phosphate, potassium sulfate,vitamin VB120.006g/L, vitamin VB30.0055g/L, biotin 0.015g/L, betaine 0.05g/L, and the balance of water.

4. A method of preparing a synthetic medium according to any one of claims 1 to 3, comprising:

5. The method of claim 4, wherein the method comprises:

6. The method of claim 4, wherein the method comprises:

the sterilized glucose. H₂And the sterilization condition of O is 115 ℃, and the sterilization time is 20-30 min.

7. Use of a synthetic medium prepared by a method of preparing a synthetic medium according to any one of claims 4 to 6;

the small unicellular bacteria are utilized to ferment and produce the gentamicin C1 a.

8. Use of a synthetic medium according to claim 7, wherein:

the Micromonospora purpurea is Micromonospora purpurea (A)Micromonospora purpurea) Or Micromonospora echinospora (Micromonospora echinospora）。