CN110564718A - Method and strain for screening high-yield amphotericin B-tubercle streptomycete by high-throughput mutagenesis - Google Patents

Abstract

The present invention relates to a method for screening high-yielding amphotericin B Streptomyces tuberculosis by high-throughput mutagenesis, a screened mutant strain, a recombinant engineering bacterium obtained from the mutant strain and its application. A high-yielding amphotericin B strain of Streptomyces nodosus obtained by screening in the present invention-Streptomyces nodosus ZJB2016050. The beneficial effects of the present invention are mainly reflected in: 1. Through the high-throughput screening method for high-yielding amphotericin B strains, the yield of amphotericin B can be conveniently, quickly and efficiently detected, and the selection of mutant breeding and genetic engineering strains is improved or verify efficiency. 2. Compared with the original strain N3, the Streptomyces tuberculosis N5 obtained by mutation breeding has a 20% increase in the yield of amphotericin B, and can be used as the original strain of genetically engineered bacteria. 3. The production of amphotericin B was increased by overexpressing the functional gene, among which acetyl-CoA carboxylase 1 (acc1) increased the production of amphotericin B by 20%. 4. 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 enterprises. Taking the laboratory 5L tank as the calculation, it can reduce the cost by about 2,000 yuan / Can.

Description

High-throughput mutagenesis screening method and strain of high-yielding amphotericin B Streptomyces tuberculosis

(1) Technical field

The invention relates to a method for screening high-yielding amphotericin B Streptomyces tuberculosis by high-throughput mutagenesis, a screened mutant strain, a recombinant engineering bacterium obtained from the mutant strain and its application.

(2) Background technology

Polyene macrolide antibiotics are a class of antibiotics mainly composed of secondary metabolites of Streptomyces. These antibiotics have broad-spectrum antifungal activity and little tolerance, making them the most effective antifungal drugs so far, and are widely used in the treatment of various infectious diseases caused by fungi. Amphotericin B (AmB) was first discovered in 1955. Amphotericin B was first isolated from Streptomyces nodosus in the water and soil samples of Orinoco River in Venezuela. Amphotericin B was extracted from the fermentation broth of Streptomyces tuberculosis in 1959, and it was launched on the market in 1966. Amphotericin B was the first clinically used drug for deep fungal infections, and it is still an irreplaceable antifungal drug. AmB has broad-spectrum resistance to fungi, especially for life-threatening systemic fungal infections such as Candida albicans, Aspergillus, etc., and also has effective antiviral and parasitic properties, such as Ruan virus, Shemania et al. AmB is mostly used in patients with damaged or weak immune systems, such as organ transplant recipients, HIV patients, and tumor patients using immune-suppressing drugs.

Studies have shown that amphotericin B initially aggregates on the fungal cell membrane and inserts into the cell membrane to form a V-shaped pore. The amphotericin B molecule is tilted so that the terminal OH group on the polyenolide ring faces the center of the lipid bilayer, but this process does not No sterol is involved, and the V-shaped pore does not penetrate the cell membrane, as shown in Figure 1. In this conformation, V-shaped channels can be loaded with ions or small molecule non-electrolytes (such as urea), but ions do not occur until a brief "open" state (amphotericin B molecules are perpendicular to the membrane plane, allowing diffusion of solutes) through the membrane. When the concentration of amphotericin B is above the threshold, the thickness of the lipid bilayer is further reduced, and sterol molecules and amphotericin B molecules together form transmembrane pores. It is worth noting that the sterol molecules are directly involved in the formation of the transmembrane pore, rather than assisting the V-shaped pore to penetrate the membrane. The massive loss of intracellular ions and small molecules through these transmembrane pores causes cell death.

With the changes in the environment and living standards and the enhancement of drug resistance, the problem of fungal infection is becoming more and more serious. In recent years, the proposal of topical amphotericin B and the launch of various amphotericin B derivatives have reduced the rate of amphotericin B. Toxic and side effects on the human body, expanded the scope of application of amphotericin B. The broad market prospect makes the study of amphotericin B production still of great significance and economic value. The chemical structure of amphotericin B is complex, and it is mostly produced by microbial fermentation. Therefore, screening of excellent amphotericin B high-yielding strains is an important precursor. At present, the methods for obtaining high-yielding strains generally fall into two categories: traditional mutagenesis breeding and genetically engineered strains. Traditional breeding is divided into physical mutagenesis, chemical mutagenesis, mixed mutagenesis and other methods; genetically engineered strains generally improve yield by changing the metabolic pathways of precursors, energy metabolism, and cutting off competitive branches in the strains. At the same time, both types of methods require an efficient, convenient and rapid high-throughput screening method for preliminary screening to improve the efficiency of breeding or construction of strains.

The antibacterial zone method, also known as the diffusion method, uses the drug to be tested diffuse in the agar plate to inhibit the growth of the surrounding bacteria to form a transparent circle, that is, the antibacterial zone. a method. The zone of inhibition method is convenient, simple and easy to operate, with low cost and accurate and reliable results. Tool bacteria generally use antibiotic-sensitive bacteria. If the tested bacteria can secrete some substances that inhibit the growth of tool bacteria, such as antibiotics, a zone of inhibition will be formed around the colony where tool bacteria cannot grow, which can be easily identified.

(3) Contents of the invention

Object of the Invention The object of the present invention is a high-throughput mutagenesis method for screening high-yielding amphotericin B Streptomyces tuberculosis, the screened high-yielding amphotericin B mutant strain, the recombinant engineering bacteria obtained from the mutant strain and the application.

The technical scheme adopted in the present invention is:

A method for high-throughput mutagenesis screening of high-yielding amphotericin B Streptomyces tuberculosis, the method comprising:

(1) Inoculate Streptomyces tuberculosis on a GYM plate at 25-26°C for 5-7 days, take gray-black spores, use a cotton swab to elute the surface spores into sterile water, and suspend the washed spores Filter with a syringe containing cotton, centrifuge the filtered spores and remove the supernatant, add sterile water to resuspend, centrifuge, re-eluate once, and resuspend with sterile water as a spore suspension;

(2) Take the diluted spore suspension and place it under a UV lamp at 20-30 cm for 1-2 minutes, then inoculate it in GYM medium, and cultivate it in the dark at 25-26°C for 24-32 hours, and collect the mutagenized cells , treated with NTG, treatment method: each 1 mL of bacterial suspension was treated with 50 mM PBS buffer containing 5 mg/mL NTG for 0.5 h, centrifuged, collected and washed 3 times with sterile water, resuspended and coated on GYM solid In the medium, cultivate at 26°C in the dark until the mutant strain is obtained;

(3) The mutant strains were spread on GMY solid medium, cultured at 26°C for 4-7 days until a single colony could be observed, punctured the single colony with a sterile toothpick, and placed in a new GYM solid medium, in order Re-single colonies for culture and preservation;

(4) The yeast X33 was used as a sensitive strain, inoculated in GYM liquid medium, cultivated at 28° C., 200 rpm for 24 hours, and coated on GYM solid medium; single colony of Streptomyces tuberculosis was used aseptic punch or sterile A 200 μL pipette tip was punched to separate the entire medium agar block, and the colonies were placed upside down on the solid medium coated with yeast X33. After culturing at 26°C for 20 hours, the size of the inhibition zone was observed, and high-yielding amphotericin B colonies were obtained by screening. Streptomyces sp.

The high-yielding amphotericin B Streptomyces tuberculosis obtained in step (4) can be used again as an initial strain to repeat steps (1) to (4) for the next round of screening.

The invention utilizes the inhibition zone method, uses yeasts as sensitive strains, performs preliminary screening on agar plates, and ferments the strains whose transparent zone is larger than the control, and can also use high performance liquid chromatography (HPLC) for accurate detection.

The invention also relates to a high-yielding amphotericin B strain of Streptomyces tuberculosis - Streptomyces nodosus ZJB2016050 (Streptomyces nodosus ZJB2016050) obtained by screening, which is preserved in the China Type Culture Collection on July 17, 2017, The deposit number is CCTCC NO: M 2017426, and the deposit address is Wuhan University, Wuhan, China, zip code 430072.

The present invention also relates to a recombinant Streptomyces tuberculosis producing amphotericin B, obtained by introducing one of the following exogenous genes into the aforementioned Streptomyces nodosus ZJB2016050 (Streptomyces nodosus ZJB2016050):

(1) acetyl-CoA carboxylase 1 gene shown in SEQ ID NO.1;

(2) acetyl-CoA carboxylase 2 gene shown in SEQ ID NO.2;

(3) polyketide synthase PKS amphA gene shown in SEQ ID NO.3;

(4) methylmalonyl-CoA mutase gene shown in SEQ ID NO.4;

(5) The methylmalonyl-CoA isomerase gene represented by SEQ ID NO.5.

Preferably, the exogenous gene is the acetyl-CoA carboxylase 1 gene shown in SEQ ID NO.1.

More preferably, the recombinant Streptomyces tuberculosis is Streptomyces tuberculosis ZJB2016050-ACC1 (Streptomycesnodosus ZJB2016050-ACC1), which is preserved in China Center for Type Culture Collection, address: Wuhan University, Wuhan, China, zip code 430072, deposit number: CCTCC NO: M 2019343, deposited on May 9, 2019.

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

Specifically, the application is as follows: inoculating the amphotericin B-producing 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, and fermenting the amphotericin B liquid separation and purification 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.

Preferably, the fermentation medium is composed as follows: glucose 70g/L, soybean protein powder 8g/L, cottonseed powder 10g/L, CaCO ₃ 10g/L, KH ₂ PO ₄ 0.2g/L, the solvent is distilled water or tap 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.

The beneficial effects of the present invention are mainly reflected in:

1. The method of high-throughput screening of high-yielding amphotericin B strains can conveniently, quickly and efficiently detect the production of amphotericin B, and improve the screening or verification efficiency of mutagenesis breeding and genetically engineered strains.

2. Compared with the original strain N3, the Streptomyces tuberculosis N5 (namely CCTCC NO: M 2017426) obtained by mutation breeding has a 20% increase in the yield of amphotericin B, and can be used as the original strain of genetically engineered bacteria.

3. The production of amphotericin B was increased by overexpressing the functional gene, among which acetyl-CoA carboxylase 1 (acc1) increased the production of amphotericin B by 20%.

4. 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 enterprises. Taking the laboratory 5L tank as the calculation, it can reduce the cost by about 2,000 yuan / Can.

(4) Description of drawings

Figure 1 shows the principle of amphotericin B inhibiting fungi.

Fig. 2 is the inhibition zone of amphotericin B in Example 1 of the present invention.

Fig. 3 is AmB structural formula;

Figure 4 is a map of the recombinant vector pJTU1278-acc1 constructed in Example 4 of the present invention;

Figure 5 is a map of the recombinant vector pJTU1278-acc2 constructed in Example 5 of the present invention;

Figure 6 is a map of the recombinant vector pJTU1278-amphA constructed in Example 6 of the present invention;

Fig. 7 is the standard curve of Amphotericin B in Example 12 of the present invention;

Fig. 8 is the influence of different pH on shake flask fermentation in the shake flask fermentation process among the embodiment 13;

Fig. 9 is the influence of different temperatures in the shake flask fermentation process among the embodiment 14;

FIG. 10 shows the effect of different rotational speeds in the fermentation process of shake flasks in Example 15. FIG.

(5) Specific implementation methods

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:

The experimental methods in the following examples are conventional methods unless otherwise specified.

The test materials used in the following examples are conventional biochemical reagents unless otherwise specified.

Example 1: Construction of a high-throughput screening method

(1) The mutagenized spores and the control strain Streptomyces tuberculosis N3 (ATCC14899) were spread or streaked on GYM solid medium, and cultured at 26°C for 7 days until a single colony was observed. Using a sterile toothpick or pipette tip, poke a single colony and streak a new plate of GYM solid medium. After numbering, culture at 26°C for 4-7 days for storage. Plates after retention treatment were used for zone of inhibition experiments.

(2) Using yeast X33 (preserved in the laboratory) as a sensitive strain, pick a single colony on GYM solid medium, cultivate it in GYM liquid medium for 24 hours, and take 100 μL of it and spread it on GYM solid medium. On the plate that has been preserved in (1), use a sterile hole punch or pipette tip to pick out the agar block containing a single colony, and place it upside down on the yeast-coated GYM solid medium, numbered. The treated plates were incubated at 28°C for 24 hours. According to the control strain, the diameter of the inhibition zone was measured by observation, as shown in Figure 3.

Example 2: Mutagenic strains with high AmB production

(1) Inoculate Streptomyces nodosus N3 (ATCC14899) on a GYM plate for 7 days at 26°C, take the gray-black spores, use a cotton swab to elute the surface spores into 10 mL of sterile water, wash the The spore suspension was filtered with a syringe containing cotton, and the filtered spores were centrifuged at 12,000 rpm for 5 min, and then the supernatant was removed. After adding 10 mL of sterile water to resuspend, centrifuged at 12,000 rpm for 5 min to re-eluate once, and resuspended with 5 mL of sterile water. Spore suspension.

(2) NTG-UV mutagenesis method: Take 10 mL of the diluted spore suspension and place it under a UV lamp (15W, 254 nm) at 20 cm for 1 min, then inoculate it in GYM medium, and cultivate at 26 °C for 24-32 h in the dark , collect the mutated cells and treat them with NTG. The treatment method is as follows: each 1 mL of the cell suspension is treated with 50 mM PBS buffer containing 5 mg/mL NTG for 0.5 h, centrifuged at 8000 rpm for 5 min, and the collected cells are washed with sterile water for 3 times. , resuspended and spread on GYM solid medium, and cultivated at 26°C in the dark until mutant strains were obtained. Three parallel groups were used in each experiment. For the high-yielding strains obtained by each round of mutagenesis, the next round of screening was carried out according to the same steps as the initial strains. The number of mutations, mutation rate and lethality are shown in Table 1.

Table 1: Nitrosoguanidine-UV Compound Mutagenesis Process

The high-yielding strains obtained in each round of mutagenesis were re-used as initial strains and subjected to compound mutagenesis according to the above method. Finally, the mutant strain N5 with a shake flask fermentation AmB yield of 8-12 g/L was obtained by screening and named as Streptomyces nodosus ZJB2016050, which was deposited in the China Center for Type Culture Collection on July 17, 2017 The deposit number is CCTCC NO: M 2017426, and the deposit address is Wuhan University, Wuhan, China, zip code 430072.

The present invention is not limited to the above-mentioned mutagenesis method.

The preparation of GYM solid medium: glucose 4g/L, yeast powder 4g/L, malt extract 10g/L, calcium carbonate 2g/L, agar 18g/L, distilled water, pH 7.2, sterilization at 121 degrees for 20 minutes .

Preparation of GYM liquid medium: glucose 4g/L, yeast powder 4g/L, malt extract 10g/L, distilled water as solvent, pH 7.2, sterilization at 121 degrees for 20min.

Example 3: Shake flask fermentation to produce AmB

(1) Preparation of seed solution: streak the high-yielding amphotericin B-producing Streptomyces tuberculosis mutagenic bacteria (i.e. CCTCC NO: M 2017426) prepared in Example 2 to a GYM plate, culture at 26°C for 4 days, and select a single colony , inoculated into the seed medium, cultivated at 26°C, 220rpm for 48h 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.

(2) Fermentation culture

A 500mL shake flask was filled with 100mL fermentation medium. During fermentation, the seed liquid was inoculated at a volume concentration of 4%, and the fermentation was carried out at 26°C and 220rpm for 144 hours. The yield of AmB in the fermentation broth could reach 12g/L.

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, distilled water as solvent, pH 7.0, sterilize at 121 degrees for 20 minutes.

Example 4: Construction of genetically engineered bacteria carrying acc1

1. Construction of recombinant vector

1. Construction of recombinant vector pJTU1278-acc1

Using the whole genome of Streptomyces tuberculosis ATCC14899 as the template, primers acc1-F and acc1-R were designed, acc1-F was the forward primer for the acc1 gene, and acc1-R was the reverse primer for the acc1 gene. The acc1 gene was added, and the fragment size was about 1776bp, 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. The nucleotide sequence of the acc1 gene was shown in SEQ ID NO.1. 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. The schematic diagram is shown in Figure 4.

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 acc1 forward and reverse primers, 1 μL of Phanta Max DNA polymerase, and make up to 50 μL of deionized water .

The cloning PCR program: 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.

The primers used are as follows:

acc1-F TCCAGCTTCACGCTGCCGCGTAGA acc1-R CTTCGAACGCTCCCAGAGTCCCGC

2. Recombinant vector pJTU1278-acc1 conjugation and transfer transformation recipient strain Streptomyces tuberculosis

A) Preparation of E.coil ET12567/puz8002 donor bacteria containing recombinant vector pJTU1278-acc1:

The constructed recombinant vector pJTU1278-acc1 was introduced into E.coil ET12567/puz8002 Escherichia coli competent, using ampicillin (Amp ⁺ , 50μg/mL), chloramphenicol (Cm ⁺ , 50μg/mL), kanamycin The positive transformants were selected and verified by M13 upstream and downstream primer colony PCR. The results proved that the recombinant vector pJTU1278 ^- acc1 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-acc1:

Take 1 E.coil ET12567/puz8002 E. coli competent cell above, ice-bath for 5min, add 5μL of pJTU1278-Kan vector plasmid with a concentration of 200ng/μL, ice-bath for 30min, water bath at 42°C, heat shock for 90s, put back 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-acc1.

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 2 min, a total of 30 cycles. A final extension at 72°C for 10 min.

M13 primers are as follows:

M13(-21)F TGTAAAACGACGGCCAGT M13R CAGGAAACAGCTATGAC

The ET12567/puz8002 E. coli that had been introduced into the pJTU1278-acc1 plasmid were 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), Cm ⁺ (final concentration 50μg/mL), Amp ⁺ (final concentration 50μg/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 the laboratory strain Streptomyces nodosus ZJB2016050 (CCTCC NO: M 2017426) on GYM plates or slant cultures, grow at 28°C for 10 days, and obtain gray-black spores, and use a cotton swab to elute the surface spores to 10 mL of 2×YT In the culture medium, the washed spore suspension was filtered with a syringe containing cotton, the filtered spores were centrifuged at 12,000 rpm for 5 min, and the supernatant was removed, and 10 mL of 2×YT medium was added to resuspend, and centrifuged at 12,000 rpm for 5 min to re-else once. Resuspend with 500 μL of 2×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-acc1) had been introduced into the recipient bacteria Streptomyces nodosus ZJB2016050, and finally obtained The genetically engineered bacteria that produces AmB, namely recombinant Streptomyces tuberculosis ZJB2016050-acc1.

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 M13R CAGGAAACAGCTATGAC

Example 5: Construction of Streptomyces tuberculosis genetically engineered bacteria carrying the acc2 gene

Using the whole genome of Streptomyces tuberculosis ATCC14899 as a template, primers acc2-F and acc2-R were designed, acc2-F was the forward primer for the acc2 gene, and acc2-R was the reverse primer for the acc2 gene. The acc2 gene was added, and the fragment size was about 1941bp, which was consistent with the target fragment. The sequencing analysis showed that the amplified sequence was the same as the target gene sequence. The nucleotide sequence of the acc2 gene was shown in SEQ ID NO.2. 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-Acc2. The schematic diagram is shown in Figure 5.

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 acc2 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 30s, 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 E. coli JM109 competent, and the ampicillin resistance was used to screen, and the transformants were selected for verification.

The primers used are as follows:

acc2-F ATGAAGAGTTCGAAATCTCCTAGGAC acc2-R GACACTCGCATGGACGTCTCAGTCC

According to the conjugation transfer method in Example 4, the constructed pJTU1278-acc2 plasmid was introduced into Streptomycesnodosus ZJB2016050 (CCTCC NO: M 2017426) to obtain recombinant Streptomyces tuberculosis ZJB2016050-acc2.

Example 6: Construction of Streptomyces tuberculosis genetically engineered bacteria carrying amphA gene

Using the whole genome of Streptomyces tuberculosis ATCC14899 as a template, the primers amphA-F and amphA-R were designed, amphA-F was a forward primer for the amphA gene, and amphA-R was a reverse primer for the amphA gene, cloned and amplified from the template. The amphA gene was added, and the fragment size was about 4239bp, which was consistent with the target fragment. The sequencing analysis showed that the amplified sequence was the same as the target gene sequence. The nucleotide sequence of the ampha gene was shown in SEQ ID NO.3. After this fragment was digested with endonucleases BamHI and HindIII, clean-up this fragment for subsequent 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 resulting pJTU1278 vector was connected to obtain a recombinant plasmid vector named pJTU1278-amphA. The schematic diagram is shown in Figure 6 .

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 amphA 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 5 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:

amphA-F GGACGTCTCAGTCCTTGATCTCGCAGA amphA-R GACGTCTCAGTCCTTGATCTCGCAGA

According to the conjugation transfer method in Example 4, the constructed pJTU1278-amphA plasmid was introduced into Streptomycesnodosus ZJB16050 (CCTCC NO: M 2017426) to obtain recombinant Streptomyces tuberculosis ZJB2016050-amphA.

Example 7: Construction of Streptomyces tuberculosis genetically engineered bacteria carrying mcm gene

Using the whole genome of Streptomyces tuberculosis ATCC14899 as the template, the primers mcm-F and mcm-R were designed, mcm-F was the forward primer for the mcm gene, and mcm-R was the reverse primer for the mcm gene. The mcm gene was added, and the fragment size was about 1737bp, which was consistent with the target fragment. The sequencing analysis showed that the amplified sequence was the same as the target gene sequence. The nucleotide sequence of the mcm gene was shown in SEQ ID NO.4. 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 ligated to obtain a recombinant plasmid vector named pJTU1278-mcm.

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 mcm 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.

The primers used are as follows:

mcm-F CTCTAGACGGTGGGGGAAATGATT mcm-R AGAACCGGCGGCGCGGTCCGGGGG

According to the conjugation transfer method in Example 4, the constructed pJTU1278-mcm plasmid was introduced into Streptomycesnodosus ZJB16050 (CCTCC NO: M 2017426) to obtain recombinant Streptomyces tuberculosis ZJB2016050-mcm.

Example 8: Construction of Streptomyces tuberculosis genetically engineered bacteria carrying mme gene

Using the whole genome of Streptomyces tuberculosis ATCC14899 as a template, design primers mme-F and mme-R, mme-F is the forward primer for mme gene, mme-R is the reverse primer for mme gene, clone and expand from the template. The mme gene was added, and the fragment size was about 441bp, which was consistent with the target fragment. The sequencing analysis showed that the amplified sequence was the same as the target gene sequence. The nucleotide sequence of the mcm gene was 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 ligated to obtain a recombinant plasmid vector named pJTU1278-mme.

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 mme 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.

The primers used are as follows:

mcm-F TAGACGGTGGGGGAAATGATTCACGGATT mcm-R GGCCGTGCGGTCCAGTGGTAGAAGCGCC

According to the conjugation transfer method in Example 4, the constructed pJTU1278-mme plasmid was introduced into Streptomycesnodosus ZJB16050 (CCTCC NO: M 2017426) to obtain recombinant Streptomyces tuberculosis ZJB2016050-mme.

Example 9: Shake flask fermentation to produce AmB

(1) Preparation of spore suspension: The AmB-producing recombinant Streptomyces tuberculosis ZJB16050-acc1 (ie CCTCC NO: M 2019343), ZJB16050-acc2, ZJB16050-amphA, ZJB16050-mcm, ZJB16050 prepared in Examples 4-8 -mme, inoculate on GYM plate, incubate 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, The filtered spores were centrifuged at 12,000 rpm for 5 min, and the supernatant was removed. After adding 10 mL of sterile water to resuspend, the spores were re-eluted by centrifugation at 12,000 rpm for 5 min, and resuspended 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 of Example 11, wherein the content of AmB in the fermentation broth obtained from ZJB2016050-acc1 was the highest, at 6.1 g/L.

Example 10: Production of AmB by fermentation in a 5L fermenter

The spore suspension or slant culture of the AmB-producing genetically engineered bacteria (recombinant Streptomyces tuberculosis ZJB2016050-acc1) prepared in Example 4 was inoculated into the seed medium, and cultivated at 28° C. and 220 rpm for 48 hours to obtain seed liquid.

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 screened in Example 1 was used as a control for fermentation culture.

The fermentation conditions of the AmB-producing genetically engineered bacteria (recombinant Streptomyces tuberculosis ZJB2016050-acc1) prepared in Example 4 and the control bacteria (recombinant 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 12, the AmB content in the fermentation broth obtained from the genetically engineered bacteria was 7.02 g/L, while the AmB content in the control bacteria fermentation broth was 5.04 g/L.

Example 11: 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 12, and the output is 15.3g/L , the product purity is more than 94%.

Example 12: HPLC detection method of AmB

Take the fermentation broth prepared by the method in Example 9, mix it with DMSO according to the volume ratio of fermentation broth:DMSO to 1:9, extract at room temperature for 20-30 minutes, centrifuge at 12000 rpm for 5 minutes, take the supernatant and filter it with a 0.45 μm organic filter membrane. 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 HPLC detection of an AmB sample of unknown concentration, which is brought into the above standard curve formula to obtain the concentration. The standard curve results are shown in Figure 7.

Example 13: pH optimization of shake flask fermentation system

The pH of the fermentation medium in Example 4 was adjusted to 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, and 9.0, respectively. Other operations were the same as those in Example 4. The fermentation broth AmB was detected according to the detection method in Example 12. The results are shown in Figure 8.

The pH of the fermentation process is preferably between 6.5 and 7.0. Under the condition of pH 6.5, the dry weight of mycelium and the yield of AmB are the highest. % and 15%. AmB production decreased by more than 30% during fermentation with pH higher than 8.5.

Example 14: Optimization of fermentation temperature of shake flask fermentation system

The fermentation temperature in Example 4 was changed to 25°C, 28°C, 30°C, 32°C, and 37°C, respectively. Other operations were the same as those in Example 4. The fermentation broth AmB was detected according to the detection method of Example 9. The results are shown in Figure 9.

The optimum fermentation temperature was 28℃, and the highest yield was about 6.4g/L in 144 hours. When the temperature exceeded 30 °C, the AmB production decreased early at 120 h.

Example 15: Optimization of rotating speed of shake flask fermentation system

The rotation speed of the shaker in Example 1 was changed to 50rpm, 100rpm, 150rpm, and 220rpm, respectively. Other operations were the same as those in Example 4. The fermentation broth AmB was detected according to the detection method of Example 12. The results are shown in Figure 10.

When the rotation speed was 200 rpm, the production of amphotericin B was larger, and when the rotation speed was higher than 200 rpm, the increase of the production was not obvious. When the rotation speed was lower than 200 rpm, the production of amphotericin B had an obvious downward trend. In the case of low rotation speed, insufficient dissolved oxygen would reduce the synthesis of amphotericin B.

sequence listing

<110> Zhejiang University of Technology

<120> High-throughput mutagenesis screening method and strain of high-yielding amphotericin B Streptomyces tuberculosis

<160> 5

<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

Claims (8)

1. A method for screening high-yielding amphotericin streptomyces B nodosus by high-throughput mutagenesis, the method comprising:

(1) Inoculating streptomyces tuberculatus to a GYM flat plate, culturing at 25-26 ℃ for 5-7 days, taking gray and black spores, eluting the surface spores into sterile water by using a cotton stick, filtering the washed spore suspension by using a syringe containing cotton, centrifuging the filtered spores, removing supernatant, adding sterile water for re-suspension, centrifuging and re-eluting once, and re-suspending by using sterile water to obtain spore suspension;

(2) Placing the diluted spore suspension under an ultraviolet lamp tube for irradiating for 1-2 min at a position of 20-30 cm, inoculating the spore suspension into a GYM culture medium, culturing at 25-26 ℃ in a dark place for 24-32h, collecting the mutagenized thallus, and treating by using NTG (nitrilotriacetic acid) in a treatment mode: treating 1mL of thallus suspension with 50mM PBS buffer solution containing 5mg/mL NTG for 0.5h, centrifuging, collecting thallus, washing with sterile water for 3 times, resuspending, spreading in GYM solid culture medium, and culturing at 26 deg.C in dark condition until mutant strain is obtained;

(3) Coating the mutant strain on a GMY solid culture medium, culturing at 26 ℃ for 4-7 days until a single colony can be observed, stabbing the single colony by using a sterile toothpick rod, and marking the single colony again for culture and preservation in a new GYM solid culture medium according to the sequence;

(4) inoculating yeast X33 as sensitive strain in GYM liquid culture medium, culturing at 28 deg.C and 200rpm for 24 hr, and coating on GYM solid culture medium; and (3) perforating a streptomyces tubercle single colony by using an aseptic perforator or an aseptic 200-mu-L gun head to separate an agar block of the whole culture medium, inversely placing the colony in a solid culture medium coated with yeast X33, culturing for 20 hours at 26 ℃, observing the size of a bacteriostatic zone, and screening to obtain the high-yield amphotericin B streptomyces tubercle.

2. the method according to claim 2, wherein the Streptomyces amphotericin B nodosus with high yield obtained in the step (4) is used as an initial strain to repeat the steps (1) to (4) for the next screening.

3. The high-yield amphotericin B Streptomyces tuberculatus strain screened by the method of claim 1, Streptomyces tuberculatus ZJB2016050(Streptomyces nodosus ZJB2016050), which is deposited in China center for type culture Collection with a deposition date of 2017, month 07, day 17 and a deposition number of CCTCC NO: m2017426, the preservation address is Wuhan, Wuhan university, China, zip code 430072.

4. A recombinant Streptomyces tuberculosus producing amphotericin B is obtained by introducing exogenous gene selected from Streptomyces nodosus ZJB2016050(Streptomyces nodosus ZJB 2016050):

(1) Acetyl coenzyme A carboxylase 1 gene shown in SEQ ID NO. 1;

(2) Acetyl coenzyme A carboxylase 2 gene shown in SEQ ID NO. 2;

(3) A polyketide synthase PKS amphA gene shown in SEQ ID No. 3;

(4) a methylmalonyl-CoA mutase gene shown in SEQ ID No. 4;

(5) The methylmalonyl-CoA isomerase gene shown in SEQ ID NO. 5.

5. the recombinant Streptomyces tuberculosus of claim 4, wherein said foreign gene is the acetyl-CoA carboxylase 1 gene of SEQ ID No. 1.

6. The recombinant Streptomyces tuberculosus according to claim 4, wherein said recombinant Streptomyces tuberculosus is Streptomyces tuberculosus ZJB2016050-ACC1(Streptomyces nodosus ZJB2016050-ACC1) deposited at the China center for type culture Collection under the address: wuhan university, Wuhan, China, zip code 430072, preservation number: CCTCC NO: m2019343, date of deposit 2019, 09/05 month.

7. Use of Streptomyces tuberculosus according to claim 3 or 4 for the fermentative preparation of amphotericin B.

8. The use according to claim 7, characterized in that the use is: inoculating the streptomyces tuberculatus producing amphotericin B into 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.