CN110343650B - A recombinant Streptomyces tuberculosis producing amphotericin B and its application - Google Patents

Abstract

The invention discloses a recombinant Streptomyces tuberculosis amphotericin B producing amphotericin B and an application thereof. The recombinant Streptomyces tuberculosis is a competitive branch of amphotericin B in knockout Streptomyces nodosus ZJB2016050 (Streptomyces nodosus ZJB2016050) Then, introduce the tandem sequences of functional genes shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, and SEQ ID NO.5 into the obtained; The supply of the precursors for the synthesis of the skeleton of mycin B is comprehensively increased, the utilization rate of nutrients by the bacteria is improved, and the flow of nutrients of the target products is changed. The production of AmB was increased by about 20% by knocking out the competitive branch, the by-product amphotericin A was decreased by 40%, and the three types of precursors acetyl-CoA, malonyl-CoA and methylmalonyl were increased by overexpression of tandem functional gene validation. Coenzyme A greatly increases the production of amphotericin B, and finally in a 5L fermenter, the production of amphotericin B can reach 16g/L.

Description

A recombinant Streptomyces tuberculosis producing amphotericin B and its application

(1) Technical field

The present invention relates to a recombinant Streptomyces tuberculosis producing amphotericin B and its application.

(2) Background technology

Amphotericin B (AmB) is a polyene broad-spectrum antifungal antibiotic produced by Streptomyces nodosus, a strain of which was isolated from soil samples from the Orinoco delta in Venezuela in 1955. Collect and separate. Launched in 1966, it is the first drug for deep fungal infections and has been used for nearly half a century. The molecular formula of AmB is C ₄₇ H ₇₃ NO ₁₇ , AmB belongs to the polydene macrolide antibiotics, and has broad-spectrum resistance to fungi, especially for life-threatening systemic fungal infections such as Candida albicans, Aspergillus spp. At the same time, it also has effective antiviral and parasitic properties, such as Nguyen virus, Leishmania, etc. The amphotericin B drugs currently circulating in the market include injections, tablets, etc. AmB is yellow or orange-yellow powder, odorless, hygroscopic, and easily damaged in sunlight. Can dissolve DMSO, basically insoluble in water, absolute ethanol, chloroform or ether, (pH 6-7) less than 1mg/L. The antifungal mechanism of AmB is: AmB can combine with ergosterol on the fungal cell membrane to form micropores on the membrane, change the permeability of the membrane, and eventually lead to important cellular contents K ions, nucleotides, The uncontrolled loss of amino acids, etc. causes bacterial death. However, AmB also interacts to a lesser extent with cholesterol on mammalian cell membranes, causing certain side effects, notably nephrotoxicity. Although it has certain side effects, AmB is still the most important antibiotic for the treatment of deep systemic fungal infections in humans. People have strengthened the research on new administration forms of AmB, such as Abelcet and Ambisome liposome drugs that appeared in the 1990s, in order to reduce toxicity and improve the efficiency of drugs reaching the area of action. There are also research progress in fermentation direction and traditional mutagenesis. For example, in 2017, Zhang Bo et al. mutated wild-type Streptomyces tuberculosis, which increased the yield of amphotericin B to 5.02g/L. optimization, and finally realize the industrialization of high-yield amphotericin B.

There are many ways of microbial metabolic modification, among which the commonly used methods include overexpression of functional genes, knockout of competitive branches, and heterologous expression of synthetic gene clusters. The overexpression of functional genes is one of the main methods in the metabolic transformation of strains, and the effect is quick. The main genetic method of Streptomyces tuberculosis is conjugative transfer. Existing reference materials show that the conjugative transfer rate of Streptomyces tuberculosis is lower than that of model Streptomyces. Among them, integration or thermosensitive plasmids such as pSET152 and PKC1139 are successful. The rate of operation and success rate is even lower. As an expression plasmid, pJTU1278 is the only plasmid successfully conjugated and transferred in our laboratory.

The amphotericin B backbone is mainly composed of 18 synthetic precursors and a starting unit. The starting unit is composed of a polyketide synthase module amphA combined with an acetyl-CoA, followed by 15 malonyl-CoA and 3 methyl groups. Malonyl-CoA stepwise composition. Among them, acetyl-CoA is mainly derived from the glycolysis pathway of glucose, malonyl-CoA is mainly synthesized by acetyl-CoA under the action of acetyl-CoA carboxylase, and methylmalonyl-CoA is synthesized by two pathways , one is synthesized by succinyl-CoA under the action of methylmalonyl isomerase and mutase, and the other is synthesized by propionyl-CoA under the action of propionyl-CoA carboxylase. Acetyl-CoA is the core substance of biological metabolism, and it is one of the main precursors of secondary metabolites in organisms with complex metabolic branches.

Streptomyces is a kind of actinomycetes, known as a natural treasure trove of secondary metabolites. A Streptomyces species often has multiple secondary metabolite synthesis gene clusters, some of which may be silent gene clusters. Expression can only be achieved under the action of stress. Another part may be in a different expression state, and there is also a competitive role. There are five PKS type synthetic gene clusters in Streptomyces tuberculosis, among which amphotericin B belongs to PKS I type. According to the prediction results of antiSMASH and the inference of KEGG data, it was found that the secondary metabolites of PKS5 may have a competitive effect on the synthesis of amphotericin B.

(3) Contents of the invention

The object of the present invention is to provide amphotericin B (AmB)-producing recombinant Streptomyces tuberculosis and its application. By expressing functional genes in Streptomyces tuberculosis, the amount of precursor substances in Streptomyces tuberculosis can be increased or the amount of The utilization rate of the material can effectively improve the production of AmB.

The technical scheme adopted in the present invention is:

A recombinant Streptomyces tuberculosis producing amphotericin B, after knocking out the PKS5 gene cluster shown in SEQ ID NO. Acetyl-CoA carboxylase 1 gene shown in SEQ ID NO.1, acetyl-CoA carboxylase 2 gene shown in SEQ ID NO.2, polyketide synthase PKSamphA gene shown in SEQ ID NO.3, SEQ ID NO. The tandem gene of the methylmalonyl-CoA mutase gene shown in 4 and the methylmalonyl-CoA isomerase gene shown in SEQ ID NO. 5 was obtained.

Preferably, the recombinant Streptomyces tuberculosis is Streptomyces tuberculosis ZJB2016050-RAAAmm (Streptomycesnodosus ZJB2016050-RAAAmm), which is preserved in China Center for Type Culture Collection, address: Wuhan University, Wuhan, China, zip code 430072, deposit number: CCTCC NO: M 2019341, deposited on May 9, 2019.

The method for introducing the recombinant vector of the present invention into the host bacteria is a method for conjugation transfer between species, and the method can be as follows:

Knockout steps:

1) Amplify the homology arm fragments (3000bp each) on both sides of the knockout gene by PCR, insert into the pJTU1278 vector plasmid multi-cloning site, and obtain the recovery vector pJTU1278-RE;

2) The kana resistance gene was cloned and inserted between the two homology arms of pJTU1278-Re as a knockout selection marker to obtain a recovery vector pJTU1278-DE.

Overexpression steps:

1) Insert the acetyl-CoA carboxylase 1 gene (acc1) and promoter obtained by PCR cloning into the multi-cloning site of the pJTU1278 vector plasmid to obtain the recombinant vector pJTU1278-acc1;

2) Transform the recombinant vector obtained in step 1) into Escherichia coli JM109, sequence the obtained transformant, and introduce the confirmed vector into the donor bacteria Escherichia coli ET12567/pUZ8002;

3) Insert the acetyl-CoA carboxylase 2 gene (acc2), polyketide synthase PKSamphA, and methylmalonyl-CoA mutase (mcm) on the constructed plasmid successively as described in steps 1) and 2). , methylmalonyl-CoA isomerase (mme). The plasmid pJTU1278-acc1-acc2-amphA-mcm-mme was finally obtained.

4) The donor bacteria Escherichia coli ET12567/pUZ8002/pJTU1278-acc1-acc2-amphA-mcm-mme containing the recombinant vector obtained in step 3) was transformed into the recipient bacteria (i.e. CCTCC NO. : M 2017426), a genetically engineered bacterium with high amphotericin B production was obtained.

The invention also relates to the application of the recombinant Streptomyces tuberculosis in the preparation of amphotericin B by microbial fermentation.

Specifically, the application is as follows: inoculating the amphotericin B-producing recombinant Streptomyces tuberculosis into a fermentation medium, fermenting and culturing at 25-30° C. and 200-500 rpm to obtain a fermentation broth containing amphotericin B, The fermentation broth is separated and purified to obtain the amphotericin B; the final concentration of the fermentation medium is composed of: glucose 60-80 g/L, beef extract 5-10 g/L, soybean protein powder 5-10 g/L, cottonseed powder 8～12g/L, CaCO ₃ 5～10g/L, KH ₂ PO ₄ 0.1～0.4g/L, solvent is water, pH 7.0.

The fermentation culture is usually carried out in a fermentor with a pressure of 0.05MPa and an aeration ratio of 0.08-1.5vvm.

Preferably, the amphotericin B-producing recombinant Streptomyces tuberculosis is seed cultured before fermentation culture, and then the seed liquid is inoculated into the fermentation medium with an inoculum volume concentration of 2-10%, and the seed culture is : Inoculate amphotericin B-producing recombinant Streptomyces tuberculosis on a GYM plate, incubate at 28°C for 7 days, take the gray-black spores, use a cotton swab to elute the surface spores into sterile water, wash the washed spores The suspension was filtered with a syringe containing cotton, centrifuged at 12,000 rpm for 5 min, and then the supernatant was removed. After the precipitate was resuspended in sterile water, it was re-eluted by centrifugation at 12,000 rpm for 5 min, and resuspended with sterile water as a spore suspension. Inoculated into the seed medium, cultivated at 28°C and 220rpm for 46h to obtain seed liquid; the final concentration of the GYM plate is composed of: glucose 4g/L, yeast powder 4g/L, malt extract 10g/L, calcium carbonate 2g/L , agar 18g/L, the solvent is water, pH 7.2; the final concentration of the seed liquid medium is composed of: peptone 10～20g/L, NaCl 5～10g/L, glucose 10～15g/L, yeast powder 5～ 10g/L, CaCO ₃ 0.5～1g/L, solvent is water, pH 7.0.

Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

1. On the basis of knockout strains, overexpressing the functional proteins related to precursor supply and increasing the supply of precursors can increase the production of amphotericin B by 20%.

2. Improve the utilization rate of the nutrients in the culture medium during the fermentation process, shorten the fermentation cycle and reduce the risk of infection.

3. The price of kanamycin is excellent and cheap, the antibacterial spectrum is wide, and the bactericidal effect is strong. It is suitable for industrial use and has a strong effect on improving the overall profitability of the enterprise. Taking the laboratory 5L tank as the calculation, it can reduce the cost by about 2,000 yuan / Can.

(4) Description of drawings

Fig. 1 is that the embodiment of the present invention 1 constructs replacement restoration plasmid map;

Fig. 2 is the construction of kan resistance replacement plasmid map in Example 1 of the present invention;

3 is a schematic diagram of the tandem construction of functional genes in Example 4;

Fig. 4 is the fermentation result of recombinant Streptomyces tuberculosis 5L fermentation tank in Example 7;

FIG. 5 is the standard curve for the detection of AmB in Example 9 by high performance liquid chromatography (HPLC).

(5) Specific implementation manner

The present invention is further described below in conjunction with specific embodiment, but the protection scope of the present invention is not limited to this:

Example 1: Knockout Vector Construction

1. Homologous arm 1 insertion

Using the whole genome of Streptomyces tuberculosis ATCC14899 as a template, primers TYB1-F and TYB1-R were designed, TYB1-F is the forward primer for homology arm 1, TYB1-R is the reverse primer for homology arm 1 gene, The homology arm 1 gene was cloned and amplified from the template. The size of the fragment is about 3000bp, which is consistent with the target fragment. After sequencing analysis, the results show that the amplified sequence is the same as the target gene sequence. This fragment was treated with endonucleases XbaI and BamHI. After enzyme digestion, clean-up this fragment for use, and recover the vector pJTU1278 with the same XbaI and BamHI endonucleases, and connect the recovered gene fragment with the digested pJTU1278 vector to obtain the name of the recombinant plasmid vector. is pJTU1278-TYB1.

Clone PCR system: add 1 μL of Streptomyces tuberculosis ATCC14899 whole genome template, add 25 μL of 2× PhantaMax Buffer, 5 μL of dNTP (2.5 mM), 1 μL of TYB1 forward and reverse primers, 1 μL of Phanta Max DNA polymerase, and make up to 50 μL of deionized water .

The cloning PCR program was as follows: denaturation at 98°C for 10s, annealing at 55-60°C for 15s, and extension at 72°C for 3 min, a total of 30 cycles. A final extension at 72°C for 10 min.

The ligation process: add 1 μL of T4 DNA ligase buffer into a sterilized PCR tube, add 4 μl of recovered DNA fragments and 1 μl of vector DNA, add 1 μl of T4 DNA ligase, add 3 μl of ddH ₂ O, and react at 16°C for 20 hours. The ligated product was transformed into JM109 E. coli competent, and the transformants were selected for verification by ampicillin resistance screening.

The primers used are as follows:

2. Homologous arm 2 insertion

Using the whole genome of Streptomyces tuberculosis ATCC14899 as a template, primers TYB2-F and TYB2-R were designed, TYB2-F was the forward primer for homology arm 2, TYB2-R was the reverse primer for homology arm 2 gene, The homology arm 2 gene was cloned and amplified from the template. The size of the fragment was about 3000bp, which was consistent with the target fragment. The sequencing analysis showed that the amplified sequence was the same as the target gene sequence. This fragment was treated with endonucleases HindIII and KpnI. After enzyme digestion, clean-up this fragment for standby use, and recover the vector pJTU1278-TYB1 with the same HindIII and KpnI endonucleases, and connect the recovered gene fragment with the digested pJTU1278-TYB1 vector to obtain The recombinant plasmid vector was named pJTU1278-TYB1-TYB2, as shown in Figure 1.

Clone PCR system: add 1 μL of Streptomyces tuberculosis ATCC14899 whole genome template, add 25 μL of 2× PhantaMax Buffer, 5 μL of dNTP (2.5 mM), 1 μL of TYB2 forward and reverse primers, 1 μL of Phanta Max DNA polymerase, and make up to 50 μL of deionized water .

The cloning PCR program was as follows: denaturation at 98°C for 10s, annealing at 55-60°C for 15s, and extension at 72°C for 3 min, a total of 30 cycles. A final extension at 72°C for 10 min.

The ligation process: add 1 μL of T4 DNA ligase buffer into a sterilized PCR tube, add 4 μl of recovered DNA fragments and 1 μl of vector DNA, add 1 μl of T4 DNA ligase, add 3 μl of ddH ₂ O, and react at 16°C for 20 hours. The ligated product was transformed into JM109 E. coli competent, and the transformants were selected for verification by ampicillin resistance screening.

The primers used are as follows:

3. Resistance screening tag insertion

Using pET28b as a template, design primers kan-F and kan-R, kan-F is the forward primer for the canna resistance gene, kan-R is the reverse primer for the canna resistance gene, clone and expand from the template. The Kana resistance gene was added, and the fragment size was about 900bp, which was consistent with the target fragment. After sequencing analysis, the results showed that the amplified sequence was the same as the target gene sequence. After the fragment was digested with endonucleases HindIII and BamHI, clean -up this fragment for later use, the vector pJTU1278-TYB1-TYB2 is also recovered with the same HindIII and BamHI endonucleases, and the recovered gene fragment is connected with the digested pJTU1278-TYB1-TYB2 vector to obtain recombination The plasmid vector was named pJTU1278-TYB1-kan-TYB2, as shown in Figure 2.

Clone PCR system: add 1 μL of Streptomyces tuberculosis ATCC14899 whole genome template, add 25 μL of 2× PhantaMax Buffer, 5 μL of dNTP (2.5 mM), 1 μL of each of the forward and reverse primers of Kana resistance gene, and 1 μL of Phanta Max DNA polymerase, make up Ionized water to 50 μL.

The cloning PCR program: denaturation at 98°C for 10s, annealing at 55-60°C for 15s, and extension at 72°C for 1 min, for a total of 30 cycles. A final extension at 72°C for 10 min.

The ligation process: add 1 μL of T4 DNA ligase buffer into a sterilized PCR tube, add 4 μl of recovered DNA fragments and 1 μl of vector DNA, add 1 μl of T4 DNA ligase, add 3 μl of ddH ₂ O, and react at 16°C for 20 hours. The ligated product was transformed into JM109 E. coli competent, and the transformants were selected for verification by ampicillin resistance screening.

The primers used are as follows:

Example 2: Resistance Genome Replacement

Recombinant vector pJTU1278-TYB1-kan-TYB2 conjugation transfer transformation recipient strain Streptomyces tuberculosis A) Preparation of E.coil ET12567/puz8002 donor strain containing recombinant vector pJTU1278-TYB1-kan-TYB2:

The constructed recombinant vector pJTU1278-TYB1-kan-TYB2 was introduced into E.coil ET12567/puz8002 E. coli competent, using ampicillin (Amp ⁺ , 50μg/mL), chloramphenicol (Cm ⁺ , 50μg/mL) , kanamycin (Kan ⁺ , 50μg/mL) resistance screening, pick positive transformants, and verify by M13 upstream and downstream primer colony PCR, the results prove that the recombinant vector pJTU1278-TYB1-kan-TYB2 was successfully transformed into E.coil ET12567 /puz8002. The specific operations are as follows:

E.coil ET12567/puz8002 Escherichia coli competent preparation method is as follows:

Take the E.coil ET12567/puz8002 E.coli bacterial solution from the glycerol cryopreservation tube of the strain and streak it on the LB plate, and cultivate it at 37°C until a single colony grows. Pick a single colony on the plate and transfer it to 2-5 mL of LB medium for overnight culture at 37°C and 200 rpm. Take 200 μL of the overnight cultured bacterial solution and add it to 20 mL of LB medium at 37° C. and cultivate at 200 rpm until the OD600 is 0.4 to 0.7. The cultured bacterial solution was transferred to a pre-cooled 50 mL centrifuge tube and placed on ice for 10 min. Centrifuge at 4°C, 2500×g, 5min. Discard the supernatant, add 4 mL of 0.1 mol/L CaCl ₂ , resuspend on ice and let stand for 10 min. Centrifuge at 4°C, 2500×g, 5min. The supernatant was discarded, 2 mL of 0.1 mol/L CaCl ₂ was added (the solution contained glycerol with a final concentration of 15%), the pellet was resuspended, and allowed to stand on ice for 30 min to obtain E.coil ET12567/puz8002 E. coli competent cells. Aliquot 100μL/tube and store at -80°C.

Preparation of E.coil ET12567/puz8002 donor bacteria containing recombinant vector pJTU1278-TYB1-kan-TYB2:

Take 1 E.coil ET12567/puz8002 E. coli competent cell, ice-bath for 5min, add 5μL pJTU1278-TYB1-kan-TYB2 vector plasmid at a concentration of 200ng/μL, ice-bath for 30min, water bath at 42°C, heat shock After 90 s, put it back into the ice bath for 1 min, add 600 μL of LB liquid medium, and incubate for 1 h at 37° C., 200 rpm. Pipette 200 μL, spread evenly on Kan+ (final concentration 50 μg/mL), Cm+ (final concentration 50 μg/mL), and Amp+ (final concentration 50 μg/mL) resistant LB solid plate, and cultivate in a 37 °C incubator for 14 h. To grow a single colony of E.coil ET12567/puz8002 containing the recombinant vector pJTU1278-TYB1-kan-TYB2.

M13 verification PCR system: pick a single colony, add 20 μL of sterile water, take a boiling water bath for 5-10 min, and centrifuge at 12,000 rpm for 1 min. Take 1 μL of the supernatant as a template, add 1 μL of 10×pfu Buffer, 0.1 μL of dNTP (2.5 mM), 0.1 μL of M13 forward and reverse primers, 0.1 μL of pfu DNA polymerase, and make up to 10 μL of deionized water.

M13 verification PCR program: denaturation at 98°C for 10s, annealing at 55-60°C for 15s, and extension at 72°C for 8 min, a total of 30 cycles. A final extension at 72°C for 10 min.

M13 primers are as follows:

M13(-21)F TGTAAAACGACGGCCAGT M13 R CAGGAAACAGCTATGAC

The ET12567/puz8002 Escherichia coli that had been introduced into the pJTU1278-TYB1-kan-TYB2 plasmid was streaked to isolate a single colony, cultured at 37 °C, and the single colony was picked into a test tube containing 5 mL of LB medium, and Kan ⁺ (final concentration 50 μg/ mL), Cm ⁺ (final concentration 50 μg/mL), Amp ⁺ (final concentration 50 μg/mL) antibiotics, cultured at 37°C for 14h. Transfer 500 μL into a 50 mL LB shake flask, and add Kan ⁺ (final concentration 50 μg/mL), Cm ⁺ (final concentration 50 μg/mL), and Amp ⁺ (final concentration 50 μg/mL) resistance at the same time, and cultivate at 37 °C to OD ₆₀₀ is 0.35. The donor bacteria were centrifuged with a 50 mL centrifuge tube, 4000 rpm, 5 min, washed twice with 50 mL of LB medium, resuspended with 5 mL of LB medium, and stored at 4°C for later use.

The LB medium was prepared by the following method: 10 g of peptone, 5 g of yeast powder, 5 g of sodium chloride, constant volume to 1 L with tap water, natural pH, and sterilized at 121 degrees for 20 min.

B) Preparation of recipient strain Streptomyces nodosus

Inoculate Streptomyces nodosus ZJB2016050 (CCTCC M 2017426) of laboratory strains on GYM plates or slant cultures, grow at 28°C for 10 days, obtain gray-black spores, and use cotton swabs to elute the surface spores to 10 mL of 2×YT medium The washed spore suspension was filtered with a syringe containing cotton, the filtered spores were centrifuged at 12,000 rpm for 5 min, the supernatant was removed, 10 mL of 2×YT medium was added to resuspend, and 12,000 rpm was centrifuged for 5 min. Resuspend in 2x YT medium. The resuspended spores were heat-shocked at 50°C for 15-20 min, and then used at room temperature.

The 2×YT medium was prepared by the following method: 16 g of peptone, 10 g of yeast powder, 5 g of sodium chloride, constant volume to 1 L with tap water, natural pH, and sterilized at 121 degrees for 20 min.

GYM solid medium preparation method: glucose 4g, yeast powder 4g, malt extract 10g, calcium carbonate 2g, agar 18g, tap water to 1L, pH 7.2, sterilization at 121 degrees for 20min.

C) Conjugation process of donor and recipient bacteria:

After mixing and resuspending 500 μL of the heat-shocked spore suspension in step B) and 500 μL of the donor E. coli suspension in step A), centrifuge at 6000 rpm for 2 min, remove 800 μL of the supernatant, and resuspend the remaining supernatant in a solution containing 10 mM. MgCl on MS solid medium plates. After culturing at 28°C for 20 hours, apply 1 mL of an aqueous solution containing 0.5 mg of naphthoric acid and 0.5 mg of thiostrepton antibiotic, and continue to culture at 28°C for 10 days until transformants appear.

Transformants were serially purified 3 times on GYM solid plates containing a final concentration of 50 μg/mL naphthenic acid and kanamycin resistance at a final concentration of 50 μg/mL until a single colony was obtained, using 16S RNA upstream and downstream primers (16S-8 and 16S-1541) and M13 upstream and downstream primers (M13(-21)F, M13R) were verified by PCR on the transformant colonies, and the sequencing and comparison analysis confirmed that the plasmid (pJTU1278-TYB1-kan-TYB2) had been introduced into the recipient strain Streptomyces nodosus ZJB2016050 , and finally obtained the genetically engineered bacteria producing AmB, namely recombinant Streptomyces tuberculosis pJTU1278-TYB1-kan-TYB2. Through several rounds of resistant culture, Kan ⁺ (final concentration 50 μg/mL) was added to obtain the finally successfully replaced strain Streptomyces nodosus ZJB2016050-De5.

The MS solid medium was prepared by the following method: soybean powder 20g, mannitol 20g, agar 20g, tap water to 1L, adjusted to pH 7.2 with sodium hydroxide, and sterilized at 121°C for 20min. Sterile magnesium chloride was added at a final concentration of 10 mM before use.

Wherein the M13 verification PCR operation is as described in step A).

16sRNA verification PCR system: pick a single colony, add 20 μL sterile water, take a boiling water bath for 30 min, and centrifuge at 12000 rpm for 1 min. Take 3 μL of supernatant as template, add 5 μL of 2× Phanta Max Buffer, 0.1 μL of dNTP (2.5 mM), 0.1 μL of 16S forward and reverse primers, 0.1 μL of Phanta Max DNA polymerase, and make up to 10 μL of deionized water.

The 16S RNA verification PCR program was as follows: denaturation at 98°C for 10s, annealing at 55-60°C for 15s, and extension at 72°C for 1min 30s, a total of 30 cycles. A final extension at 72°C for 10 min.

The primers used are as follows:

16S-8 AGAGTTTGATCCTGGCTCAG 16S-1541 AAGGAGGTGATCCAGCCGCA M13(-21)F TGTAAAACGACGGCCAGT

Example 3: Knockout Resistance Tag Recovery

Recombinant vector pJTU1278-TYB1-TYB2 conjugation and transfer transformation recipient bacterium Streptomycesnodosus ZJB2016050-De5

A) Preparation of E.coil ET12567/puz8002 donor bacteria containing recombinant vector pJTU1278-TYB1-TYB2:

The constructed recombinant vector pJTU1278-TYB1-TYB2 was introduced into E.coil ET12567/puz8002 E. coli competent, using ampicillin (Amp ⁺ , 50μg/mL), chloramphenicol (Cm ⁺ , 50μg/mL), Namycin (Kan ⁺ , 50μg/mL) resistance was screened, positive transformants were picked and verified by M13 upstream and downstream primer colony PCR. The results proved that the recombinant vector pJTU1278-TYB1-TYB2 was successfully transformed into E.coil ET12567/puz8002. The specific operations are as follows:

E.coil ET12567/puz8002 Escherichia coli competent preparation method is as follows:

Take the E.coil ET12567/puz8002 E.coli bacterial solution from the glycerol cryopreservation tube of the strain and streak it on the LB plate, and cultivate it at 37°C until a single colony grows. Pick a single colony on the plate and transfer it to 2-5 mL of LB medium for overnight culture at 37°C and 200 rpm. Take 200 μL of the overnight cultured bacterial solution and add it to 20 mL of LB medium at 37° C. and cultivate at 200 rpm until the OD600 is 0.4 to 0.7. The cultured bacterial solution was transferred to a pre-cooled 50 mL centrifuge tube and placed on ice for 10 min. Centrifuge at 4°C, 2500×g, 5min. Discard the supernatant, add 4 mL of 0.1 mol/L CaCl ₂ , resuspend on ice and let stand for 10 min. Centrifuge at 4°C, 2500×g, 5min. The supernatant was discarded, 2 mL of 0.1 mol/L CaCl ₂ was added (the solution contained glycerol with a final concentration of 15%), the pellet was resuspended, and allowed to stand on ice for 30 min to obtain E.coil ET12567/puz8002 E. coli competent cells. Aliquot 100μL/tube and store at -80°C.

Preparation of E.coil ET12567/puz8002 donor bacteria containing recombinant vector pJTU1278-TYB1-TYB2:

Take 1 E.coil ET12567/puz8002 E. coli competent cell, ice-bath for 5min, add 5μL of pJTU1278-TYB1-TYB2 vector plasmid with a concentration of 200ng/μL, ice-bath for 30min, water bath at 42°C, heat shock for 90s, Put it back in the ice bath for 1 min, add 600 μL of LB liquid medium, and incubate for 1 h at 37° C., 200 rpm. Pipette 200 μL, spread evenly on Kan+ (final concentration 50 μg/mL), Cm+ (final concentration 50 μg/mL), and Amp+ (final concentration 50 μg/mL) resistant LB solid plate, and cultivate in a 37 °C incubator for 14 h. To grow a single colony of E.coil ET12567/puz8002 containing the recombinant vector pJTU1278-TYB1-TYB2.

M13 verification PCR system: pick a single colony, add 20 μL of sterile water, take a boiling water bath for 5-10 min, and centrifuge at 12,000 rpm for 1 min. Take 1 μL of the supernatant as a template, add 1 μL of 10×pfu Buffer, 0.1 μL of dNTP (2.5 mM), 0.1 μL of M13 forward and reverse primers, 0.1 μL of pfu DNA polymerase, and make up to 10 μL of deionized water.

M13 verification PCR program: denaturation at 98°C for 10s, annealing at 55-60°C for 15s, and extension at 72°C for 8 min, a total of 30 cycles. A final extension at 72°C for 10 min.

M13 primers are as follows:

M13(-21)F TGTAAAACGACGGCCAGT M13 R CAGGAAACAGCTATGAC

The ET12567/puz8002 E. coli that had been introduced into the pJTU1278-TYB1-TYB2 plasmid was streaked to isolate a single colony, cultured at 37°C, and the single colony was picked in a test tube containing 5 mL of LB medium, and Kan ⁺ (final concentration 50 μg/mL) was added at the same time. , Cm ⁺ (final concentration 50 μg/mL), Amp ⁺ (final concentration 50 μg/mL) antibiotics, cultured at 37°C for 14h. Transfer 500 μL into a 50 mL LB shake flask, and add Kan ⁺ (final concentration 50 μg/mL), Cm ⁺ (final concentration 50 μg/mL), and Amp ⁺ (final concentration 50 μg/mL) resistance at the same time, and cultivate at 37 °C to OD ₆₀₀ is 0.35. The donor bacteria were centrifuged with a 50 mL centrifuge tube, 4000 rpm, 5 min, washed twice with 50 mL of LB medium, resuspended with 5 mL of LB medium, and stored at 4°C for later use.

The LB medium was prepared by the following method: 10 g of peptone, 5 g of yeast powder, 5 g of sodium chloride, constant volume to 1 L with tap water, natural pH, and sterilized at 121 degrees for 20 min.

B) Preparation of recipient strain Streptomyces nodosus

The laboratory strain Streptomyces nodosus ZJB2016050-De5 was inoculated on a GYM plate or slant culture, and grown at 28°C for 10 days to obtain gray-black spores. The surface spores were eluted into 10 mL of 2×YT medium using a cotton swab. The washed spore suspension was filtered with a syringe containing cotton, and the filtered spores were centrifuged at 12,000 rpm for 5 min to remove the supernatant, resuspended in 10 mL of 2×YT medium, and re-eluted by centrifugation at 12,000 rpm for 5 min. The medium was resuspended. The resuspended spores were heat-shocked at 50°C for 15-20 min, and then used at room temperature.

The 2×YT medium was prepared by the following method: 16 g of peptone, 10 g of yeast powder, 5 g of sodium chloride, constant volume to 1 L with tap water, natural pH, and sterilized at 121 degrees for 20 min.

GYM solid medium preparation method: glucose 4g, yeast powder 4g, malt extract 10g, calcium carbonate 2g, agar 18g, tap water to 1L, pH 7.2, sterilization at 121 degrees for 20min.

C) Conjugation process of donor and recipient bacteria:

After mixing and resuspending 500 μL of the heat-shocked spore suspension in step B) and 500 μL of the donor E. coli suspension in step A), centrifuge at 6000 rpm for 2 min, remove 800 μL of the supernatant, and resuspend the remaining supernatant in a solution containing 10 mM. MgCl on MS solid medium plates. After culturing at 28°C for 20 hours, apply 1 mL of an aqueous solution containing 0.5 mg of naphthoric acid and 0.5 mg of thiostrepton antibiotic, and continue to culture at 28°C for 10 days until transformants appear.

Transformants were serially purified 3 times on GYM solid plates containing a final concentration of 50 μg/mL naphthenic acid and kanamycin resistance at a final concentration of 50 μg/mL until a single colony was obtained, using 16S RNA upstream and downstream primers (16S-8 and 16S-1541) and M13 upstream and downstream primers (M13(-21)F, M13R) were verified by PCR on the transformant colonies, and the sequencing and comparison analysis confirmed that the plasmid (pJTU1278-TYB1-TYB2) had been introduced into the recipient strain Streptomyces nodosus ZJB2016050-De5 Finally, a genetically engineered bacteria producing AmB, the recombinant Streptomyces tuberculosis pJTU1278-TYB1-TYB2, was finally obtained. After several rounds of resistant culture, Kan ⁺ (final concentration 50 μg/mL) was added to obtain the finally successfully replaced strain Streptomyces nodosus ZJB2016050-Re5.

The MS solid medium was prepared by the following method: soybean powder 20g, mannitol 20g, agar 20g, tap water to 1L, adjusted to pH 7.2 with sodium hydroxide, and sterilized at 121°C for 20min. Sterile magnesium chloride was added at a final concentration of 10 mM before use.

Wherein the M13 verification PCR operation is as described in step A).

16sRNA verification PCR system: pick a single colony, add 20 μL sterile water, take a boiling water bath for 30 min, and centrifuge at 12000 rpm for 1 min. Take 3 μL of supernatant as template, add 5 μL of 2× Phanta Max Buffer, 0.1 μL of dNTP (2.5 mM), 0.1 μL of 16S forward and reverse primers, 0.1 μL of Phanta Max DNA polymerase, and make up to 10 μL of deionized water.

The 16S RNA verification PCR program was as follows: denaturation at 98°C for 10s, annealing at 55-60°C for 15s, and extension at 72°C for 1min 30s, a total of 30 cycles. A final extension at 72°C for 10 min.

Example 4: Construction of functional gene tandem vector

Using the whole genome of Streptomyces tuberculosis ATCC14899 as a template, the primers mcm-F and mcm-R were designed, and the acetyl-CoA carboxylase 1 gene (acc1) was cloned and amplified from the template. The fragment size was about 1776bp. Enzyme 2 gene (acc2), fragment size is about 1941bp, polyketide synthase PKSamphA, fragment size is about 4239bp, methylmalonyl-CoA mutase (mcm), fragment size is about 1737bp, methylmalonyl-CoA isoform The size of the constitutive enzyme (mme) fragment is about 441bp, which is consistent with the target fragment. After sequencing analysis, the results show that the amplified sequence is the same as the target gene sequence. The nucleotide sequence of the acetyl-CoA carboxylase 1 (acc1) gene is shown in SEQ. ID NO.1, the nucleotide sequence of the acetyl-CoA carboxylase 2 gene (acc2) is shown in SEQ ID NO.2, and the nucleotide sequence of the polyketide synthase PKSamphA gene is shown in SEQ ID NO.3 The nucleotide sequence of methylmalonyl-CoA mutase (mcm) gene is shown in SEQ ID NO.4, and the nucleotide sequence of methylmalonyl-CoA isomerase gene (mme) is shown in shown in SEQ ID NO.5. After this fragment was digested with endonucleases BamHI and HindIII, clean-up this fragment for later use, and the vector pJTU1278 was also recovered with the same BamHI and HindIII endonuclease digested gel, and the recovered gene fragment was digested with the enzyme. The pJTU1278 vector was then connected to obtain a recombinant plasmid vector named pJTU1278-acc1-acc2-amphA-mcm-mme. The schematic diagram is shown in Figure 3.

Clone PCR system: add 1 μL of genomic template, 25 μL of 2× Phanta Max Buffer, 5 μL of dNTP (2.5 mM), 1 μL of corresponding forward and reverse primers, 1 μL of Phanta Max DNA polymerase, and make up to 50 μL of deionized water.

The cloning PCR program was as follows: denaturation at 98°C for 10s, annealing at 55-60°C for 15s, and extension at 72°C for 2 min, a total of 30 cycles. A final extension at 72°C for 10 min.

The ligation process: add 1 μL of T4 DNA ligase buffer into a sterilized PCR tube, add 4 μL of recovered DNA fragments and 1 μL of vector DNA, add 1 μL of T4 DNA ligase, add 3 μL of ddH ₂ O, and react at 16°C for 20 hours. The ligated product was transformed into JM109 E. coli competent, and the transformants were selected for verification by ampicillin resistance screening.

Example 5: Functional gene expression

The constructed recombinant vector pJTU1278-acc1-acc2-amphA-mcm-mme was introduced into E.coilET12567/puz8002 Escherichia coli competent, using ampicillin (Amp ⁺ , 50μg/mL), chloramphenicol (Cm ⁺ , 50μg /mL), kanamycin (Kan ⁺ , 50μg/mL) resistance screening, positive transformants were picked and verified by M13 upstream and downstream primer colony PCR, the results proved that the recombinant vector pJTU1278-acc1-acc2-amphA-mcm-mme Successfully transformed into E.coil ET12567/puz8002. The specific operations are as follows:

E.coil ET12567/puz8002 Escherichia coli competent preparation method is as follows:

Take the E.coil ET12567/puz8002 E.coli bacterial solution from the glycerol cryopreservation tube of the strain and streak it on the LB plate, and cultivate it at 37°C until a single colony grows. Pick a single colony on the plate and transfer it to 2-5 mL of LB medium for overnight culture at 37°C and 200 rpm. Take 200 μL of the overnight cultured bacterial solution and add it to 20 mL of LB medium at 37° C. and cultivate at 200 rpm until the OD600 is 0.4 to 0.7. The cultured bacterial solution was transferred to a pre-cooled 50 mL centrifuge tube and placed on ice for 10 min. Centrifuge at 4°C, 2500×g, 5min. Discard the supernatant, add 4 mL of 0.1 mol/L CaCl ₂ , resuspend on ice and let stand for 10 min. Centrifuge at 4°C, 2500×g, 5min. The supernatant was discarded, 2 mL of 0.1 mol/L CaCl ₂ was added (the solution contained glycerol with a final concentration of 15%), the pellet was resuspended, and allowed to stand on ice for 30 min to obtain E.coil ET12567/puz8002 E. coli competent cells. Aliquot 100μL/tube and store at -80°C.

Preparation of E.coil ET12567/puz8002 donor strain containing recombinant vector pJTU1278-acc1-acc2-amphA-mcm-mme:

Take 1 E.coil ET12567/puz8002 E. coli competent cell, ice-bath for 5min, add 5μL of pJTU1278-TYB1-TYB2 vector plasmid with a concentration of 200ng/μL, ice-bath for 30min, water bath at 42°C, heat shock for 90s, Put it back in the ice bath for 1 min, add 600 μL of LB liquid medium, and incubate for 1 h at 37° C., 200 rpm. Pipette 200 μL, spread evenly on Kan+ (final concentration 50 μg/mL), Cm+ (final concentration 50 μg/mL), and Amp+ (final concentration 50 μg/mL) resistant LB solid plate, and cultivate in a 37 °C incubator for 14 h. To grow a single colony of E.coilET12567/puz8002 containing the recombinant vector pJTU1278-acc1-acc2-amphA-mcm-mme.

M13 verification PCR system: pick a single colony, add 20 μL of sterile water, take a boiling water bath for 5-10 min, and centrifuge at 12,000 rpm for 1 min. Take 1 μL of the supernatant as a template, add 1 μL of 10×pfu Buffer, 0.1 μL of dNTP (2.5 mM), 0.1 μL of M13 forward and reverse primers, 0.1 μL of pfu DNA polymerase, and make up to 10 μL of deionized water.

M13 verification PCR program: denaturation at 98°C for 10s, annealing at 55-60°C for 15s, and extension at 72°C for 8 min, a total of 30 cycles. A final extension at 72°C for 10 min.

M13 primers are as follows:

M13(-21)F TGTAAAACGACGGCCAGT M13 R CAGGAAACAGCTATGAC

The ET12567/puz8002 E. coli that had been introduced into the pJTU1278-acc1-acc2-amphA-mcm-mme plasmid was streaked to isolate a single colony, cultured at 37°C, and the single colony was picked into a test tube containing 5 mL of LB medium, and Kan ⁺ ( Final concentration of 50 μg/mL), Cm ⁺ (final concentration of 50 μg/mL), Amp ⁺ (final concentration of 50 μg/mL) antibiotics, cultured at 37°C for 14h. Transfer 500 μL into a 50 mL LB shake flask, and add Kan ⁺ (final concentration 50 μg/mL), Cm ⁺ (final concentration 50 μg/mL), and Amp ⁺ (final concentration 50 μg/mL) resistance at the same time, and cultivate at 37 °C to OD ₆₀₀ is 0.35. The donor bacteria were centrifuged with a 50 mL centrifuge tube, 4000 rpm, 5 min, washed twice with 50 mL of LB medium, resuspended with 5 mL of LB medium, and stored at 4°C for later use.

The LB medium is prepared by the following method: 10 g of peptone, 5 g of yeast powder, 5 g of sodium chloride, constant volume of tap water to 1 L, natural pH, and sterilization at 121 degrees for 20 minutes.

B) Preparation of recipient strain Streptomyces nodosus

Inoculate Streptomyces nodosus ZJB2016050-Re5 of laboratory strain on GYM plate or slant culture, grow at 28°C for 10 d, obtain gray-black spores, and use cotton swab to elute the surface spores into 10 mL of 2×YT medium. The washed spore suspension was filtered with a syringe containing cotton, and the filtered spores were centrifuged at 12,000 rpm for 5 min to remove the supernatant, resuspended in 10 mL of 2×YT medium, and re-eluted by centrifugation at 12,000 rpm for 5 min. Finally, use 500 μL of 2× YT medium. Resuspended. The resuspended spores were heat-shocked at 50°C for 15-20 min, and then used at room temperature.

The 2×YT medium was prepared by the following method: peptone 16g, yeast powder 10g, sodium chloride 5g, tap water to 1L, natural pH, sterilization at 121 degrees for 20min.

GYM solid medium preparation method: glucose 4g, yeast powder 4g, malt extract 10g, calcium carbonate 2g, agar 18g, tap water to 1L, pH 7.2, sterilization at 121 degrees for 20min.

C) Conjugation process of donor and recipient bacteria:

After mixing and resuspending 500 μL of the heat-shocked spore suspension in step B) and 500 μL of the donor E. coli suspension in step A), centrifuge at 6000 rpm for 2 min, remove 800 μL of the supernatant, and resuspend the remaining supernatant in a solution containing 10 mM. MgCl on MS solid medium plates. After culturing at 28°C for 20 hours, apply 1 mL of an aqueous solution containing 0.5 mg of naphthoric acid and 0.5 mg of thiostrepton antibiotic, and continue to culture at 28°C for 10 days until transformants appear.

Transformants were serially purified 3 times on GYM solid plates containing a final concentration of 50 μg/mL naphthenic acid and kanamycin resistance at a final concentration of 50 μg/mL until a single colony was obtained, using 16S RNA upstream and downstream primers (16S-8 and 16S-1541) and M13 upstream and downstream primers (M13(-21)F, M13R) were verified by PCR on the transformant colonies, and the sequencing and comparison analysis confirmed that the plasmid (pJTU1278-acc1-acc2-amphA-mcm-mme) had been introduced into the receptor In the bacterium Streptomyces nodosus ZJB2016050-Re5, a genetically engineered bacterium producing AmB, namely recombinant Streptomyces nodosus pJTU1278-TYB1-TYB2, was finally obtained. Through several rounds of resistant culture, Kan ⁺ (final concentration 50 μg/mL) was added to obtain the finally successfully replaced strain Streptomyces nodosus ZJB2016050-De5-pJTU1278-acc1-acc2-amphA-mcm-mme (ie CCTCCNO: M2019341).

The MS solid medium was prepared by the following method: soybean powder 20g, mannitol 20g, agar 20g, tap water to 1L, adjusted to pH 7.2 with sodium hydroxide, and sterilized at 121°C for 20min. Sterile magnesium chloride was added at a final concentration of 10 mM before use.

Wherein the M13 verification PCR operation is as described in step A).

16sRNA verification PCR system: pick a single colony, add 20 μL sterile water, take a boiling water bath for 30 min, and centrifuge at 12000 rpm for 1 min. Take 3 μL of supernatant as template, add 5 μL of 2× Phanta Max Buffer, 0.1 μL of dNTP (2.5 mM), 0.1 μL of 16S forward and reverse primers, 0.1 μL of Phanta Max DNA polymerase, and make up to 10 μL of deionized water.

The 16S RNA verification PCR program was as follows: denaturation at 98°C for 10s, annealing at 55-60°C for 15s, and extension at 72°C for 1min 30s, a total of 30 cycles. A final extension at 72°C for 10 min.

Example 6: Shake flask fermentation to produce AmB

(1) Preparation of spore suspension: The AmB-producing recombinant Streptomyces tuberculosis ZJB2016050-De5-pJTU1278-acc1-acc2-amphA-mcm-mme prepared in Example 5 was inoculated onto a GYM plate and cultured at 28°C for 7 days. Take the gray-black spores, use a cotton swab to elute the surface spores into 10 mL of sterile water, filter the washed spore suspension with a syringe containing cotton, centrifuge the filtered spores at 12,000 rpm for 5 min, remove the supernatant, and add After resuspending in 10 mL of sterile water, centrifuge at 12,000 rpm for 5 min to re-elute once, and resuspend with 5 mL of sterile water as a spore suspension.

(2) Preparation of seed liquid:

The spore suspension of step (1) is inoculated into the seed medium, and cultured at 28° C. and 220 rpm for 46 hours to obtain seed liquid.

The seed medium was prepared as follows: peptone 20g, NaCl 8g, glucose 15g, yeast powder 10g, CaCO ₃ 1g, add water to make up to 1L, pH 7.0, sterilize at 121°C for 20min.

(3) Fermentation culture

A 500mL shake flask was filled with 50mL fermentation medium, and the seed liquid was inoculated at a concentration of 2% by volume during fermentation, and the fermentation was cultured at 28°C and 220rpm for 168h.

Composition of fermentation medium: glucose 70g/L, beef extract 8g/L, soybean protein powder 8g/L, cottonseed powder 10g/L, CaCO ₃ 10g/L, KH ₂ PO ₄ 0.2g/L, the solvent is tap water, pH 7.0, sterilize at 121 degrees for 20 minutes.

The above-mentioned genetically engineered bacteria were produced by shaking flask fermentation and detected according to the method in Example 8, wherein the AmB content in the fermentation broth obtained from ZJB2016050-De5-pJTU1278-acc1-acc2-amphA-mcm-mme was 7.2 g/L.

Example 7: Production of AmB by fermentation in a 5L fermenter

Inoculate the spore suspension or slant culture of the AmB-producing genetically engineered bacteria (recombinant Streptomyces tuberculosis ZJB2016050-De5-pJTU1278-acc1-acc2-amphA-mcm-mme) prepared in Example 5 into the seed medium, 28°C, 220rpm Seed liquid was obtained after culturing for 48h.

Fermentation conditions: 3L of fermentation broth was placed in a 5L fermenter, and the inoculum was inoculated with seed liquid at a volume concentration of 5%, 28°C, pressure 0.05MPa, aeration ratio 1.2vvm, rotation speed 400rpm, and fermentation culture for 100h to obtain fermentation broth.

The seed medium was prepared as follows: peptone 20g, NaCl 8g, glucose 15g, yeast powder 10g, CaCO ₃ 1g, add water to make up to 1L, pH 7.0, sterilize at 121°C for 20min.

5L fermentation tank fermentation medium composition: glucose 70g/L, beef extract 8g/L, soybean protein powder 8g/L, cottonseed powder 10g/L, CaCO ₃ 10g/L, KH ₂ PO ₄ 0.2g/L, the solvent is Tap water, pH 7.0, sterilized at 121 degrees for 20 minutes.

Under the same conditions, the recombinant Streptomyces tuberculosis ZJB2016050 (ie, CCTCC NO: M 2017426) was used as a control for fermentation culture.

The fermentation conditions of the AmB-producing genetically engineered bacteria (recombinant Streptomyces tuberculosis ZJB2016050-De5-pJTU1278-acc1-acc2-amphA-mcm-mme) prepared in Example 5 and the control bacteria (Streptomyces tuberculosis ZJB2016050) in a 5L tank were compared. The above-mentioned genetically engineered bacteria were produced by fermentation in a 5L tank, and detected according to the method in Example 8. The AmB content in the fermentation broth obtained from the genetically engineered bacteria was 16.0 g/L, as shown in Figure 4, while the control strain AmB yield was 13.6 g/L.

Example 8: Preparation of AmB Finished Products

Take 500L of the fermentation broth obtained by the method in Example 7. The fermentation broth was filtered through the plate frame to obtain wet mycelium, dried to obtain 8.1kg dry bacteria weight, the dried mycelium was put into the extraction tank, 70L of methanol was added, the temperature was lowered to 4°C, and the pH was adjusted to 3.0 with hydrochloric acid to stabilize. One hour, the filtrate was filtered. The filtrate was put into the crystallization tank, 10 L of purified water was added, the pH was adjusted to 6.0 with lye, the temperature was raised to 25°C, and the temperature was kept for one hour. Filter to obtain solid (i.e. AmB crystalline powder), continuously add methanol for washing, remove impurities, finally dry and pulverize to obtain AmB crude product, the crude product is detected by liquid chromatography described in Example 8, and the output is 14.6g/L , the product purity is more than 94%.

Example 9: HPLC detection method of AmB

Take the fermentation broth prepared by the method in Example 5, mix it with DMSO at a volume ratio of fermentation broth:DMSO of 1:9, extract at room temperature for 20-30 minutes, centrifuge at 12,000 rpm for 5 minutes, take the supernatant and filter it with a 0.45 μm organic filter. Detection by high performance liquid chromatography (HPLC).

Detection method: The chromatographic column is a C18 column (150×4.6mm), the column temperature is 25°C, the flow rate is 1mL/min, the injection volume is 20μL, the chromatographic retention time is 30min, and the detection wavelength is 405nm. The peak time of AmB was 26.9 min.

The mobile phase preparation method: 1.1g EDTA-Na ₂ and 4.1g sodium acetate are diluted to 1L with distilled water, take 900mL of this solution, mix with 700mL acetonitrile and 400mL methanol, and adjust the pH to 5.0 with acetic acid;

Calculation method of AmB yield: Purchase the standard product of AmB from sigma company, prepare AmB standard solutions of different concentrations (0mg/L, 200mg/L, 400mg/L, 800mg/L, 1000mg/L) with DMSO, and mix the above standard solutions respectively. The peak area was detected by HPLC, and the standard curve calculated according to the peak area and the concentration of AmB standard solution was Y=0.0123X+2539.9, R ² =0.999 (wherein Y is the concentration of AmB, and X is the peak area). A peak area can be obtained by detecting the AmB sample of unknown concentration by HPLC, and the concentration can be obtained by bringing it into the above standard curve formula. The result of the standard curve is shown in Figure 5.

sequence listing

<110> Zhejiang University of Technology

<120> A high-yielding amphotericin B recombinant Streptomyces tuberculosis and its application

<160> 6

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1776

<212> DNA

<213> Streptomyces nodosus

<400> 1

tcagtccttg atctcgcaga tcgcggcgcc ggaggagagg gaggcgccga cctccgcggt 60

caggcccttg atcgtgccgg ctcggtgcgc gttgagcggc tgctccatct tcatggcctc 120

caggacgacg accaggtcgc cctcctggac ctcctggccc tcctcgaccg cgaccttcac 180

gatcgtgccc tgcatcgggg aggccagggt gtcgccggag accgagggac cggacttctt 240

cgccgcgcgc cgcttcggct tggcgcccgc cgccagaccc gtacgggcca gcgacatacc 300

gagcgacgcg ggcagcgaca cctcgagacg cttgccgccg acctcgacca cgaccgtctc 360

acggcccgcc gggtcctcct cggcctccgc gtccgcggcc gccgcgaacg gcttgatctc 420

gttgacgaac tcggtctcga tccagcgggt gtggaccgtg aaggggccct cggagccggt 480

cagttcgggc gcgaacgcgg tgtccctgac caccgcgcgg tggaacggga tcgccgtggc 540

catcccctcg acctggaact cgtccagggc gcgggcggcc cgctccagag cctccttgcg 600

ggtgcggccc gtcacgatca gcttggccag cagggagtcc cacgccgggc cgatgaccga 660

acccgactcc acacccgcgt ccagccgcac accggggccg gacggcggag cgaacgcggt 720

caccgtgccg ggggcgggca ggaagccccg gcccgggtcc tcgccgttga tccggaactc 780

gaaggaatgg ccgcgcagtt cggggtcgtc atagcccagt tcctcgccgt cggcgatacg 840

gaacatctca cggaccaggt cgatgccggc gacctcctcg gtgaccgggt gctcgacctg 900

gagccgggtg ttgacctcca ggaaggagat cgtcccgtcg ttgccgacca ggaactccac 960

cgtgcccgcg cccacatagc cggcctcctt gaggatggcc ttggacgccg agtacagctc 1020

ggcgacctgc ccgtccgaca ggaacggcgc gggggcctcc tcgaccagct tctggtggcg 1080

ccgctgcagc gagcagtcac gggtggagac caccacgacg ttgccgtgcc ggtcggccag 1140

gcactgtgtc tccacgtgcc ggggccggtc cagatagcgc tccacgaagc actcgccacg 1200

gccgaaggcg gcgaccgcct cacggaccgc cgactcgtac agctcgggga tctcctccag 1260

ggtgcgggcc accttcagac cgcgcccgcc accgccgaag gcggccttga tcgcgatcgg 1320

caggccgtgc tcctcggcga aggtgacgac ctcgtcggcg ccggacaccg ggtcgggcgt 1380

accggcgacc aggggggcac ccgcgcgctg cgcgatgtgc cgggccgcga ccttgtcacc 1440

gagatcgcgg atggcctgcg gcggcgggcc gatccagatc agaccggcgt ccaggacggc 1500

ctgggcgaag tcggcgttct cggagaggaa accgtagccg gggtggatgg cgtccgcgcc 1560

ggaatccttg gccgcctgaa ggaccttgtc gatgtccagg taactggtgg cgggcgtgtc 1620

tcctcccagg gcgaacgcct cgtccgcggc gcggacgtgc agggcgtccc ggtccgggtc 1680

ggcgtagacg gccacgctcg cgatcccggc gtcacgacag gcccgggcga cacggacagc 1740

gatttcgcca cggttggcga tcaacacctt gcgcac 1776

<210> 2

<211> 1941

<212> DNA

<213> Streptomyces nodosus

<400> 2

atgtttgaca cggtgctcgt ggccaaccgg ggcgagatcg cggtccgggt cgtccgcacc 60

ctgcgcgcgc tcggggtgcg ttcggtggcc gtcttctccg acgcggacgc cgacgcccgg 120

cacgtccggg aggccgacac ggcggtacgg atcggaccgg cgcccgcagc cgagagctat 180

ctgtccgtcg agcggctcct cgcggcggcg gcccgcaccg gcgcccaggc ggtgcacccg 240

ggatacggct tcctcgcgga gaacgccgcc ttcgcgaagg cgtgcgcgga ggcggggctg 300

gtcttcatcg ggccgcccgc cgaggcgatc tccctcatgg gcgacaagat ccgcgccaag 360

gagacggtgc gggcggccgg ggtgccggtc gtcccgggct ccgacggcag cgggctgacg 420

gacgagcagc tggccgaggc ggcccacacc atcggcatgc cggtgctgct gaagccgagc 480

gccggcgggg gcggcaaggg catgcggctg gtgcgggagc cggagcggct ggccgaggag 540

atcgccgcgg cccgccgtga ggcccgcgcc tccttcggcg acgacacgct cctggtcgag 600

cgctggatcg accggccccg gcatatcgag atccaggtcc tggccgactc ccacgggaac 660

gtggtgcatc tgggcgagcg cgagtgctcc ctccagcgcc gccaccagaa gctcatcgag 720

gaggcgccca gtgtgttcct cgacgaggcc acccgtgcgg cgatgggcga ggcggcggtc 780

caggcggccc gctcctgcgg ctaccggggc gcgggcacgg tggagttcat cgtcccgggc 840

aacgacccct ccgcctatta cttcatggag atgaacaccc gcctccaggt ggaacacccg 900

gtcaccgagc tggtcaccgg cctggacctg gtggaatggc agctgcgggt ggcggcgggc 960

gagccgctgt ccttcgggca ggacgacatc acgctcaccg ggcacgccgt ggaggcgcgg 1020

atctgcgccg aggaccccgc ccgcggcttc ctcccctccg gcggcacggt gctcgcgctg 1080

cacgaaccgg ggggcgacgg cctccgcacc gactcgggcc tgtccgaggg caccgaggtc 1140

ggcagcctct acgacccgat gctgtccaag gtcatcgccc acggccccga ccgggcgacc 1200

gcgctgcgca gactgcgcgc ggccctcggg gagaccgtca ccctgggcgt ggggaccaac 1260

gccggttttc tgcgccggct gctggcccat cccgcggtcg tggcgggcga actggacacc 1320

gggctggtgg aacgcgaggc ggacggcctc atcccggagg gggtgccgga ggaggtgtac 1380

gaggccgccg ccgccgtgcg cctggacgca ctgcggcccc ggggcgaggg ctggaccgac 1440

ccgttctcgg tgccggacgg ctggcgcctc ggcggcgagc ccgcgcccct gtccttcccc 1500

ctgcgggtgt ccgaaccggt ggagtactcc ccccggggca cccacacggt caccgaggac 1560

cgggtgtccg tggtgctgga cggggtgcgg cacaccttcc accgcgccgc cgactggctc 1620

ggccgggacg gcgacgcctg gcaggtgcgc gaccatgacc cggtcgccgc ctcgctcacc 1680

ggcgccgccc gagccggcac cgactcgctg accgcgccca tgcccggcac ggtcaccgtg 1740

gtgaaggtcg ccgtcgggga cgaggtggcc gcagggcaga gcctgctggt ggtcgaggcg 1800

atgaagatgg agcacgtcat ctccgcgccg cacgccggga ccgtcgccga actcgacgtc 1860

accccgggca ccacggtggt catggaccag gtgctggccg tgatcacccc gcacgaggag 1920

cacacggagg cggagcgatg a 1941

<210> 3

<211> 4239

<212> DNA

<213> Streptomyces nodosus

<400> 3

atgacgatcg gagccaacga cgatccggta gtggtcgtcg gaatggcctg ccgcttcccg 60

ggaggcgtcg aggggcccga ggacctgtgg gagctggtcc gcgacggccg cgacgccacc 120

gggccgttcc ccggcgaccg cggctgggac ctggccgccc tgaccggcga cgggccggac 180

cacagcgtga cccaccgagg cggattcctc gccgcggccg ccgacttcga cgccggcttc 240

ttcgggatgt cgccccgcga ggccgtctcc accgacccgc agcagcggct cgtcctggag 300

acctcctggg aggccctgga acacgccggc atcgacccgc acaccctgcg gggcacccgc 360

accggcgtct tcgtcggcac caacggccag gactacgcga ccgtcaccaa cgcctcccgc 420

gaggacctca ccgggcacgc cctcaccggt ctgtcgccga gcatcgcctc cgggaggctc 480

gcctacttcc tcggcctcga agggcccgcc gtcaccctcg acacggcgtc ctcctcgtcc 540

ctggtcgccc tccactacgc gctgcgctcg ctcaggtcgg gggagtgcac caccgcgctg 600

gccggcggcg tcaccgtgat gtccacaccg gtcgggttca tcgcctacac ccggcagggc 660

ggactcgccg ccgacggccg ctgcaaggtc ttctccgacg acgccgacgg caccacctgg 720

gcggagggcg ccggcatgat cgtgctggag cgcctgtcca ccgcccgcgc cgccgggcac 780

cgggtgctcg ccgtgctgcg cggctccgcc gtcaaccagg acggcgcctc cgacggtctc 840

accgccccca gcggaccggc ccaggaacga ctcgtccgcg aggccctcgc cgacgccgga 900

ctcggacccg ccgacatcga cctcgtcgag gcccacggca ccggcacccg gctcggcgac 960

cccatcgagg cccgggccct gctcgccacc tacggccagg accgcgacgg cggacagccg 1020

ctgcgcctcg gctccctgaa gtccaacatc gggcacgccc aggcagccgc cggcatcggc 1080

ggactcatca aggccgtcca ggcgctgcgc cacggcctga tgccggagac cctgaacctc 1140

tccacgccca cccggcacgt cgactggtcg gccggcgccg tcgaactcct caccgaggcc 1200

ctgccctggc ccggcaccgg ccgcccgcgc cgggccgccg tctcctcctt cggcatcagt 1260

ggcaccaacg cgcatgtcat cgtggaggaa gccccgacga ccgaccccgc cgccgcggtc 1320

cccgccgggc ccgcccaccg ggacgtggcc tcggccgccg actccgccgc gcggcccgct 1380

gccctcgccg gggagcccgc cgacacctct gctcccgccg ctgtcgacgc cggcccggcc 1440

gaccgcccgg tcacccccgc cgctcggctc gccgccctcg tgcccgcggc cgacgccgtc 1500

gcgtggccgg tgtccggggc ctccccggag gctctcgacg cgcaggtcga gcggctcacc 1560

tccttcgtcc gggaccaccc cggcgccgat ccgctggaca tcggtcactc gctggccacc 1620

gggcgggcgg cgttgcggca ccgtgcggtg ctggtgccgt ccggtgacgg tgtcgtggag 1680

atcgcccgcg gtgaggccgc cccccgcacc accgccgtcc tcttctccgg acagggctcc 1740

cagcggctcg gcatgggccg tgaactcgcc gcccgcttcc cggtcttcgc gaaggccctg 1800

gacaccgtcc tggccgccct cgacccccaa ctcgagcgtc cggtgcggtc cgtgatgtgg 1860

ggcgaggacc ccgccgaact cgaccgcacc gggtggaccc agcccgcgct gttcgccttc 1920

gaggtcgccc tgtaccggct cgccgagtcc ttcgggctgc gccccgacgc cgtcggcggc 1980

cactccgtcg gcgagatcgc cgccgcgcac atcgccggag tgctctcgct ggaggacgcc 2040

gcgcgtctgg tcgccgcccg cgccaccctg atgcaggccc tgcccgaggg cggcgccatg 2100

tccgccgtcg aggcctccga ggacgaggtg ctcccgctgc tcgacggcga tgtctcgctc 2160

gccgccgtca acggccccac cgcggtcgtc gtctccggcg ccgaggacgc cgtggagcgc 2220

gtctccgccc acttcgctgc ccaaggccgc cgcaccagcc gcctcgcggt ctcgcacgcc 2280

ttccactcgc cgctgatgga gccgatgctc gacgccttcc gggacgtcgt cgccggactc 2340

accttccatg agccgacgct gccggtgatg tccaacctca ccggtgaact tgccggtgcc 2400

gagatcgcca cccccgagta ctgggtgcgg catgtgcgcg gtaccgtccg cttcgccgac 2460

ggcgtgacgg ccctgcggga acacggcacc gacctgctgg tcgaactggg ccccggcagc 2520

gtcctgaccg ccctcgcccg caccgtcctc ggcccggaca ccccgggcgc ccctgtcgac 2580

gtggtgccca ccctccgcaa ggaccagccc gaggagaggg ctctcaccgc cgcgctcggc 2640

cggctccatg tcctcggcgc gaccgtcgac tggtccgccc tctacaccgg caccggagcc 2700

cgccgcaccg acctgccgac gtacgccttc cagcacgcgc ggtactggcc cgccccgggc 2760

cggcccggca ccggtaccgc gggcggcggg catccgctgc tcggcccggc cgtggaactc 2820

gccgacggcg gcacggtgtc gggcgccaca ctgtccgtcg ccacccaccc ctggctcgcc 2880

gaccatgtcg tcgccgggcg cgtcctgctg cccgccgccg tgctcgtgga actcgccgta 2940

cgcgcgggtg acgacaccgg atgcgacgtc ctgcacgaac tcgccctcgt cgaggcgccg 3000

gtcctggagg ccggcgacac cctggacctc caggtccggg tcggctccgc cgacgaggcc 3060

ggccggcgca ccctcaccgt ccactcccgt cccggcaact cccccgccga gccctggacc 3120

cagcgggccg gcggcctgct cggcaccgcg ccccgcaccg cggcggcccc cgacacctcc 3180

ttcgccgtcg cctggccccc gccgggcgcc gaacccctcg acctcgggga ccactacgag 3240

cggctcgtcg acgacggctt cgacctcggc cccgccttcc gcggtctgcg caccgcctgg 3300

cgccacgacg gcgcgttcct cgccgaggtc gaactcccgg ccggcaccac cgacgacccc 3360

ggcgcctacg gagtgcaccc cgcgctcctc gacgccgccc ggcacgccgc cctcaccacc 3420

accggcacac tcccggtcgc ctggcacggg gtgcggctgc acgccgtcgg cgccaccgcc 3480

ctgcgggtgc ggatccactc cgccgacgac ggtgccctga ccctgaccgc cgccgatgtc 3540

accggcgccc cggtgttcac cgccgaggcc gtcgtcgtac ggcagctcac cgagcaggag 3600

cgcaccgccc cccggccgct cacacgcgcc tggcaccagg acaccgcgac cccgcgccgc 3660

acccggcccg tcgccgcggc ccccggcgcc gccgccgagc cgtccgcctc ctcggcgccg 3720

gacagcttcg ccgccgaggc cgcggccctg gccccccgccg agcgtgaacg ccggctcatc 3780

ggcctggtac ggacccaggc ggcggcggtc ctcggccatc agggcccgga cgcggtcgga 3840

ccccgcgcgg tcttcaagga gctgggcttc gactcgctgg ccggcgtgga actcagtgac 3900

cgcctcaccg cgctcaccgg actgcggctg ccggccaccc tcgtcttcaa cttccccacc 3960

cccgagctcg ccgcccggcg tatcggggaa ctcctcgtcg tatccggctc ctcaccgcag 4020

ggatcgtgcg acgacgaact caccaggttc gaggccgtcg tgcagaccct gtcggccgac 4080

gaccccggac gccaggccgt cgccgaccgc ctggacgcac tcgtcgcctc gctccggcgg 4140

aattccgccc cgcaggagaa cttctccgac gaggacatcg aatcggtgtc ggtcgacaga 4200

ctgctcgaca tcatcgatga agagttcgaa atctcctag 4239

<210> 4

<211> 1737

<212> DNA

<213> Streptomyces nodosus

<400> 4

atggacgctg acgccatcgc ggagggccgc cgacgctggc aggcccgtta tgacgccgca 60

cgcacgcgtg aggcggccgg gggctccgag gcgaaacccc cgaggagctg gacgcgcacc 120

acgctctccg gcgaccccgt ggagcccgtg tacgggcccc ggcccaccga cagctacgag 180

ggcttcgagc ggatcggctg gccgggcgag taccccttca cccgcggtct gtatccgacc 240

ggctaccggg gccggagctg gaccgtccgc cagttcgccg ggttcgggaa cgccgagcag 300

accaatgagc gcttcaaggc gatcctggag gccggcggcg gaggcctgag cgtcgccttc 360

gacatgccga cgctgatggg ccgcgactcc gacgatccgc gctccctggg cgaggtcggg 420

cactgcgggg tggcgatcga ctcggcggcc gacatggagg tcctgttcca ggacatccag 480

ctgggtgagg ttacgacctc gatgacgatc agcggtccgg ccgtccccgt cttctgcatg 540

tatctggtcg ccgccgagcg gcagggcgtc gacccggccg tgctcaacgg cacgctccag 600

accgacatct tcaaggagta catcgcccag aaggagtggc tcttccggcc cgagccgcat 660

ctgcgtctga tcggcgacct gatggagcac tgcacggccg gcatccccgc ctacaagccg 720

ctgtccgtct ccggctacca catccgtgag gcgggcgcga cggccgcaca ggagctggcg 780

tacaccctgg cggacggctt cggatatgtg gagctggggc tcagccgcgg gctcgacgtg 840

gatgtcttcg ccccggggct gtccttcttc ttcgacgcgc atgtcgactt cttcgaggag 900

atcgccaagt tccgggccgc gcgccgcatc tgggcgcgct ggatgcgcga ggtgtacggc 960

gcgcggagcg acaaggcgca gtggctgcgc ttccacaccc agaccgccgg ggtctcgctg 1020

accgcccagc agccgtacaa caacgtggtg cgtacggcgg tggaggcgct ggcggcggtg 1080

ctgggcggga ccaactcgct gcacaccaac gcactggacg agacgctggc gctgccgagc 1140

gaacaggcgg cggagatcgc gctgcgcacc cagcaggtgc tgatggagga gaccggggtc 1200

gcccatgtcg cggacccgct gggcggttcg tggtacgtcg agcagctgac ggaccggatc 1260

gaggcggacg cggagaagat cttcgagcag atcaaggagc ggggactgcg ggcgcatccg 1320

gacggtcggc acccgatcgg cccgatgacc tccggcattc tgcgggggat cgaggacggc 1380

tggttcacgg gcgagatcgc ggagtccgcc ttccgctatc agcaggccct ggagaagggc 1440

gacaagcacg tggtgggcgt caatgtccac accggatcgg tcaccgggga cctggagatc 1500

ctgcgggtcg gccacgaggt ggagcgggag caggtgcggg tgctcgcggc gcgccgggcg 1560

gcgcgggacg agaccgcggt gcgtacggcg ctcgacggca tgctggcggc ggcacgcgac 1620

ggcaccgaca tgatcggccc catgctggac gcggtgcgcg cggaggcgac gctcggcgag 1680

atctgcgggg cgctgcggga cgagtggggg atctacacgg agccgccggg cttctga 1737

<210> 5

<211> 441

<212> DNA

<213> Streptomyces nodosus

<400> 5

atgctgacgc gaatcgacca catcgggatc gcctgcttcg acctggagaa gaccgtcgag 60

ttctacacct cgacctacgg cttctccgtg ttccacaccg agatcaacga ggaacagggc 120

gtccgcgagg ccatgctgaa gatcaatgac acgggcgacg gcggggcctc ctacctccag 180

ctgctggaac ccgtccgcga ggactccgcc gtggcgaagt ggctcgccaa gaacggggag 240

ggcgtacatc acatcgcctt cggcacggcg gacgtcgacg gggacgcgga ggccgtccgg 300

gacaagggcg tgcgcgtgct gtacgacgag ccacgacggg gttccatggg gtcccggatc 360

acctttctgc accccaagga ttgtcacgga gttctcacag aactggtcac atccgcggcc 420

gttgagtcac ctgagcactg a 441

<210> 6

<211> 1227

<212> DNA

<213> Streptomyces nodosus

<400> 6

atgaacgggt ccggcgtccg ggtcgccgtc accgggctgg gcgtcgtcgc ccccaacggc 60

ctgggcgcgg aggcctactg gacggcgacc cgcaagggaa ccagcggcat cggccgcatc 120

tcgcggttcg tccccgacgg gtatccggcc caactggccg gggagatcga gggattcgcc 180

gcggcggagt atctgccggg ccggctgctg ccgcagaccg accggatgac acagctcgcc 240

ctcgtggcag cggactgggc gttcgaggac gccgcggtgc gtcccgggga actgcccgag 300

ttcgagatgg gcgtgatcac ggccagttcc tcgggcggct tcgagttcgg ccagcgggag 360

ttgcaggccc tgtggagcca gggaagccgg tacgtcagcg cgtatcagtc gttcgcctgg 420

ttctacgccg tcaacagcgg ccagatctcc atccgcaacg gcatgcgagg cccgagcggc 480

gtggtcgtca gcgaccaggc cggcggactc gacgcggtcg cccaggcgcg gcgccagatc 540

cgcaagggca cccggctggt gatgtcaggg gccgtggacg cctcgatctg cccctggggc 600

tgggtcgccc agatggcgag caaccggctg agcaccagga ccgacccgga gcgggcgtat 660

ctgcccttcg atgccgcggc gaacggccac gtggccggcg agggcggcgc cctgctcgtc 720

ctggaggaac tggagcaggc ccgagcccgg ggcgcgaagc agatctacgg cgagatcgcc 780

gggtacggct ccacgctcga cccccgcccg ggcagcgagc gtcccgcggg tctgcgcaag 840

gcgatcgaac tggcgctggc cgatgccggg gccacgccgg gcgagatcga cgtggtgttc 900

gccgacgcgg ccgccatccc cgagctggac cggatcgagg ccgcggcgat caacgaggtg 960

ttcggagccg gggcggtgcc ggtgacggcg cccaagacga tgaccgggcg gctctactcg 1020

ggagcggctc ccctggacct ggccgccgcg ttcctcgcca tgcgggacgg ggtgatcccg 1080

ccgtccatcg gcgtcacgcc ctcccccgag cacggcctcg acctggtcgt cgaccaggag 1140

cggaccgcca cagtgcgctc cgccctggtg atcgcccgcg gccacggcgg tttcaactcc 1200

gcaatcgtgg tgcgctccgc cgcatag 1227

Claims (5)

1. Recombinant streptomyces tuberculatus (A) for producing amphotericin BStreptomyces nodosus) And wherein the recombinant Streptomyces nodosus consists of the accession number: CCTCC NO: m2017426 Streptomyces tuberculatus ZJB2016050 knocks out PKS5 gene cluster shown in SEQ ID NO.6, and then introduces acetyl coenzyme A carboxylase 1 gene shown in SEQ ID NO.1, acetyl coenzyme A carboxylase 2 gene shown in SEQ ID NO.2, polyketide synthase PKSamphA gene shown in SEQ ID NO.3, methylmalonyl coenzyme A mutase gene shown in SEQ ID NO.4 and methylmalonyl coenzyme A isomerase gene shown in SEQ ID NO. 5.

2. Use of the recombinant Streptomyces tuberculosus according to claim 1 for the microbial fermentation production of amphotericin B.

3. The use according to claim 2, characterized in that the use is: inoculating the recombinant streptomyces tuberculatus for producing amphotericin B to a fermentation culture medium, carrying out fermentation culture at 25-30 ℃ and 200-500 rpm to obtain fermentation liquor containing amphotericin B, and separating and purifying the fermentation liquor to obtain amphotericin B; the final concentration of the fermentation medium is as follows: 60-80 g/L of glucose, 5-10 g/L of beef extract, 5-10 g/L of soybean protein powder, 8-12 g/L of cotton seed powder and CaCO₃ 5～10g/L，KH₂PO₄0.1-0.4 g/L, water as solvent, and pH 7.0.

4. The use according to claim 3, characterized in that the fermentation is carried out in a fermenter at a pressure of 0.05MPa and an aeration ratio of 0.08 to 1.5 vvm.

5. The application of claim 3, wherein the recombinant streptomyces tuberculatus producing amphotericin B is subjected to seed culture before fermentation culture, and then a seed solution is inoculated to a fermentation medium in an inoculum size of 2-10% by volume concentration, and the seed culture is as follows: inoculating recombinant streptomyces tuberculatus producing amphotericin B to a GYM plate, culturing at 28 ℃ for 7 days, taking gray and black spores, eluting surface spores into sterile water by using a cotton stick, filtering washed spore suspension by using an injector containing cotton, centrifuging at 12000rpm for 5min, removing supernatant, adding sterile water into a precipitate for re-suspension, centrifuging at 12000rpm for 5min, re-eluting once, re-suspending with sterile water to serve as spore suspension, inoculating the spore suspension into a seed culture medium, and culturing at 28 ℃ and 220rpm for 46h to obtain seed liquid; the final concentration of the GYM plate is as follows: 4g/L of glucose, 4g/L of yeast powder, 10g/L of malt extract, 2g/L of calcium carbonate, 18g/L of agar and water as a solvent, wherein the pH value is 7.2; the final concentration of the seed liquid culture medium is as follows: 10-20 g/L of peptone, 5-10 g/L of NaCl, 10-15 g/L of glucose, 5-10 g/L of yeast powder and CaCO₃0.5-1 g/L, water as solvent, and pH 7.0.

Images