阅读说明：本技术 高通量诱变筛选高产两性霉素b结节链霉菌的方法及菌株 (Method and strain for screening high-yield amphotericin B-tubercle streptomycete by high-throughput mutagenesis ) 是由 柳志强 郑裕国 黄恺 张博 姜圣贤 张雨函 陈燏 于 2019-05-28 设计创作，主要内容包括：本发明涉及本发明涉及一种高通量诱变筛选高产两性霉素B结节链霉菌的方法,以及筛选到的诱变株,由诱变株获得的重组工程菌及应用。本发明筛选获得的一株高产两性霉素B结节链霉菌菌株——结节链霉菌(Streptomyces nodosus)ZJB2016050。本发明的有益效果主要体现在：1、通过高通量筛选高产两性霉素B菌种的方法,能够方便快速高效地检测两性霉素B的产量,提高了诱变育种和基因工程菌株的筛选或验证效率。2、诱变育种获得的结节链霉菌N5,相对原始菌株N3,两性霉素B产量提高了20％,能够作为基因工程菌的原始菌株。3、通过过表达功能基因分别提高两性霉素B的产量,其中乙酰辅酶A羧化酶1(acc1)提高两性霉素B产量20％。4、卡那霉素价格优廉、抗菌谱广、杀菌作用强,适用于工业中使用,对于企业总体收益率有较强提高作用,以实验室5L罐为计算,能降低成本约2000元/罐。(The invention relates to a method for high-throughput mutagenesis screening of high-yield amphotericin B streptomyces tubercle, a screened mutant strain, recombinant engineering bacteria obtained from the mutant strain and application. The screened high-yield amphotericin B Streptomyces tubercle strain-Streptomyces tubercle (Streptomyces nodosus) ZJB 2016050. The invention has the following beneficial effects: 1. the method for screening the high-yield amphotericin B strain at high throughput can conveniently, quickly and efficiently detect the yield of amphotericin B, and improve the screening or verification efficiency of mutation breeding and genetic engineering strains. 2. Compared with the original strain N3, the yield of the streptomyces tuberculatus N5 obtained by mutation breeding is improved by 20 percent, and the streptomyces tuberculatus N5 can be used as the original strain of a genetic engineering strain. 3. The yield of amphotericin B is respectively improved by over-expressing functional genes, wherein the yield of amphotericin B is improved by 20 percent by acetyl coenzyme A carboxylase 1(acc 1). 4. Kanamycin has the advantages of low price, wide antimicrobial spectrum and strong bactericidal action, is suitable for industrial use, has strong improvement effect on the total income rate of enterprises, and can reduce the cost by about 2000 yuan per tank by taking a laboratory 5L tank as calculation.)

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.

(I) technical field

the invention relates to a method for screening high-yield amphotericin B-tubercle streptomycete by high-throughput mutagenesis, a screened mutant strain, recombinant engineering bacteria obtained from the mutant strain and application.

(II) background of the invention

Polyene macrolide antibiotics are a class of antibiotics that consists primarily of the secondary metabolites of streptomyces. The antibiotics have broad-spectrum antifungal activity and very low tolerance, become the most effective antifungal medicines so far, and are widely applied to the treatment of various infectious diseases caused by fungi. Amphotericin B (ambotericin B, AmB) was first discovered in 1955 and was first isolated from Streptomyces nodosus (Streptomyces nodosus) in river soil samples of orinociaceae, venezuela. Amphotericin B was extracted from streptomyces tubercle fermentation broth in 1959 and was marketed in 1966. Amphotericin B is the first drug clinically applied to deep fungal infection and is still an irreplaceable antifungal drug up to now. AmB has a broad spectrum of fungal resistance, particularly against life-threatening systemic fungal infections such as candida albicans, aspergillus, etc., while also having potent antiviral, parasitic properties such as raney virus, leishmania, etc. AmB is mostly used for patients with damaged immune system or poor immunity, such as organ transplant recipients, HIV patients, tumor patients using immunity-inhibiting drugs, and the like.

Studies have shown that amphotericin B initially accumulates on fungal cell membranes and inserts into the cell membrane to form a V-shaped pore channel, the amphotericin B molecule tilts such that the terminal OH group on the polyene lactone ring is oriented toward the center of the lipid bilayer, but this process is not sterol involved and the V-shaped pore channel does not penetrate the cell membrane, as shown in FIG. 1. In this conformation, the V-shaped channels can be loaded with ionic or small molecule non-electrolytes (such as urea), but ion transport through the membrane does not occur until a transient "open" state (the amphotericin B molecule is perpendicular to the plane of the membrane, allowing diffusion of solutes). At amphotericin B concentrations above the threshold, the lipid bilayer thickness is further reduced and the sterol molecule and amphotericin B molecule together form a transmembrane pore. It is noteworthy that the sterol molecule participates directly in the formation of transmembrane channels, rather than assisting the V-channel in transmembrane. Massive loss of intracellular ions and small molecule substances through these transmembrane pores leads to cell death.

With the change of environment and living standard and the enhancement of drug resistance, the problem of fungal infection is increasingly severe, and in recent years, the proposal of the local application of amphotericin B and the marketing of various amphotericin B derivative drugs reduce the toxic and side effects of amphotericin B on human bodies and expand the application range of amphotericin B. The wide market prospect makes the research on the yield increase of amphotericin B still have great significance and economic value. The chemical structure of amphotericin B is complex, and at present, the amphotericin B is mostly produced by adopting a microbial fermentation method, so that the screening of excellent amphotericin B high-yield strains is an important precursor. The current methods for obtaining high producing strains are generally of two general types: traditional mutation breeding and genetic engineering to modify strains. Traditional breeding is divided into physical mutagenesis, chemical mutagenesis, mixed mutagenesis and other methods; genetically engineered strains generally improve yield by altering the pathways of precursor metabolism within the strain, energy metabolism, cutting off competing branches, and the like. Meanwhile, two major methods need an efficient, convenient and rapid high-throughput screening method for primary screening so as to improve the efficiency of breeding or strain construction.

the bacteriostatic ring method, also called as diffusion method, is a method of determining the bacteriostatic potency of a drug to be tested according to the size of the bacteriostatic ring, which is a transparent ring formed by inhibiting the growth of bacteria around the drug by diffusing the drug to be tested in an agar plate. The bacteriostatic loop method has the advantages of convenient operation, simplicity, easy implementation, low cost and accurate and reliable result, and is widely used for primary screening of strains of metabolites such as antibiotics. The tool bacteria are sensitive bacteria of antibiotics. If the tested bacterium secretes some substances for inhibiting the growth of the tool bacterium, such as antibiotics, a bacteriostasis zone where the tool bacterium cannot grow is formed around the colony, and the bacteria can be easily identified.

Disclosure of the invention

The invention aims to provide a method for screening high-yield amphotericin B tubercle streptomyces through high-throughput mutagenesis, a screened high-yield amphotericin B mutant strain, recombinant engineering bacteria obtained from the mutant strain and application.

The technical scheme adopted by the invention is as follows:

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.

and (4) repeating the steps (1) to (4) to perform the next round of screening by taking the high-yield amphotericin B streptomyces tubercle obtained in the step (4) as an initial strain again.

The invention uses the method of the zone of inhibition, uses yeast as a sensitive strain, carries out preliminary screening on an agar plate, carries out fermentation on the strain of which the transparent zone is larger than the control, and can also carry out accurate detection by using High Performance Liquid Chromatography (HPLC).

The invention also relates to a Streptomyces tubercle strain, namely Streptomyces nodosus ZJB2016050(Streptomyces nodosus ZJB2016050), which is obtained by screening and is preserved in China center for type culture collection with the preservation date of 2017, 07 and 17 days and the preservation number of CCTCC NO: m2017426, the preservation address is Wuhan, Wuhan university, China, zip code 430072.

The invention also relates to recombinant Streptomyces tuberculatus for producing amphotericin B, which is obtained by introducing one of the following exogenous genes into the Streptomyces tuberculatus 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.

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

More preferably, the recombinant Streptomyces sarcomere is Streptomyces sarcomere ZJB2016050-ACC1(Streptomyces nodosus ZJB2016050-ACC1) deposited in China center for type culture Collection, address: wuhan university, Wuhan, China, zip code 430072, preservation number: CCTCC NO: m2019343, date of deposit 2019, 09/05 month.

The invention also relates to application of the streptomyces tuberculatus in preparation of amphotericin B through microbial fermentation.

Specifically, the application is as follows: 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.

Preferably, the fermentation medium consists of: 70g/L of glucose, 8g/L of soybean protein powder, 10g/L of cotton seed powder and CaCO₃ 10g/L，KH₂PO₄0.2g/L, solvent is distilled water or tap water, pH 7.0.

The fermentation culture is usually carried out in a fermentation tank, the pressure of the fermentation tank is 0.05MPa, and the aeration ratio is 0.08-1.5 vvm.

preferably, before fermentation culture, the streptomyces tuberculatus producing amphotericin B is subjected to seed culture, and then a seed solution is inoculated to a fermentation medium in an inoculum size of 2-10% by volume concentration, wherein 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 pH7.0。

The invention has the following beneficial effects:

1. The method for screening the high-yield amphotericin B strain at high throughput can conveniently, quickly and efficiently detect the yield of amphotericin B, and improve the screening or verification efficiency of mutation breeding and genetic engineering strains.

2. Compared with the original strain N3, the yield of the streptomyces tubercle N5 (CCTCC NO: M2017426) obtained by mutation breeding is improved by 20 percent, and the streptomyces tubercle B can be used as the original strain of a genetic engineering bacterium.

3. The yield of amphotericin B is respectively improved by over-expressing functional genes, wherein the yield of amphotericin B is improved by 20 percent by acetyl coenzyme A carboxylase 1(acc 1).

4. Kanamycin has the advantages of low price, wide antimicrobial spectrum and strong bactericidal action, is suitable for industrial use, has strong improvement effect on the total income rate of enterprises, and can reduce the cost by about 2000 yuan per tank by taking a laboratory 5L tank as calculation.

(IV) description of the drawings

FIG. 1 shows the fungistatic principle of amphotericin B.

FIG. 2 shows the zones of inhibition of amphotericin B in example 1 of the present invention.

FIG. 3 is the AmB structural formula;

FIG. 4 is a map of recombinant vector pJTU1278-acc1 constructed in example 4 of the present invention;

FIG. 5 is a map of the recombinant vector pJTU1278-acc2 constructed in example 5 of the present invention;

FIG. 6 is a map of recombinant vector pJTU1278-amphA constructed in example 6 of the present invention;

FIG. 7 is a standard amphotericin B curve of example 12 of the present invention;

FIG. 8 is a graph showing the effect of different pH values on shake flask fermentation in example 13;

FIG. 9 is a graph showing the effect of different temperatures on shake flask fermentation in example 14;

FIG. 10 is a graph showing the effect of different rotation speeds on the shake flask fermentation process in example 15.

(V) detailed description of the preferred embodiments

The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:

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

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