阅读说明：本技术 一种高效低毒四霉素b衍生物及其制备和应用 (High-efficiency low-toxicity tetramycin B derivative and preparation and application thereof ) 是由 康前进 盛勇 白林泉 于 2019-08-16 设计创作，主要内容包括：本发明公开了一种高效低毒四霉素B衍生物及其制备和应用；所述四霉素B衍生物,分子式为C<Sub>35</Sub>H<Sub>55</Sub>NO<Sub>12</Sub>,化学结构式为：<Image he="517" wi="678" file="DDA0002168804510000011.GIF" imgContent="drawing" imgFormat="GIF" orientation="portrait" inline="no"></Image>该四霉素B衍生物是以基因工程改造菌株刺孢吸水链霉菌(Streptomyces hygrospinocus var.beijingensis)SY02为原料,经发酵液萃取、菌体浸泡、MCI胶柱层析及高效液相色谱制备分离获得。本发明化合物经体外抗真菌活性及溶血毒性测定,发现其抗真菌活性较好,溶血毒性较低,可作为抗真菌药物和食品防腐剂,具有良好的临床应用前景与经济价值。(The invention discloses a high-efficiency low-toxicity tetramycin B derivative, a preparation method and an application thereof, wherein the molecular formula of the tetramycin B derivative is C 35 H 55 NO 12 , the chemical structural formula of the tetramycin B derivative is , and the tetramycin B derivative is obtained by taking a genetically engineered strain Streptomyces hygroscopicus var. beijingensis SY02 as a raw material and performing fermentation liquor extraction, thallus soaking, MCI (cellulose-activated silica gel) column chromatography and high performance liquid chromatography preparation and separation.)

1. A high-efficiency low-toxicity tetramycin B derivative with molecular formula of C₃₅H₅₅N0₁₂The chemical structural formula is as follows:

2. A process for preparing the highly potent and low toxicity tetramycin B derivative according to claim 1, which comprises the steps of:

s1, performing in-frame deletion on polyketone synthase gene nysI in a nystatin biosynthetic gene cluster in a strain streptomyces spinosa Beijing mutant CGMCC4.1123 to obtain a mutant strain with the gene nysI deleted in the same frame;

S2, inactivating a P450 monooxygenase gene tetrG in a tetramycin biosynthesis gene cluster in the same-frame deletion mutant strain to obtain a gene tetrG inactivated mutant strain;

S3, fermenting and culturing the gene tetrG inactivated mutant strain, and preparing and separating by fermentation liquor extraction, thallus soaking, MCI gel column chromatography and high performance liquid chromatography to obtain the high performance low toxicity tetramycin B derivative.

3. The method for preparing the high-efficiency low-toxicity tetramycin B derivative according to claim 2, wherein the step S2 specifically comprises the following steps:

S21, constructing a P450 monooxygenase gene tetrG inactivation plasmid pJQK513 used in the tetramycin biosynthesis gene cluster;

S22, inactivating the P450 monooxygenase gene tetrG in the gene nysI in-frame deletion mutant strain by the intergeneric conjugative transfer of the streptomycete and escherichia coli on the plasmid pJQK 513;

S23, carrying out PCR verification on the genomic DNA extracted from the mutant strain obtained in the step S22 to obtain the mutant strain with the inactivated P450 monooxygenase gene tetrG in the tetramycin biosynthesis gene cluster.

4. The method of claim 3, wherein the inactivation of the P450 monooxygenase gene tetrG in the tetramycin biosynthesis gene cluster is performed by mutating the active site amino acid Cys340 of the gene tetrG to Ala in step S21.

5. The process for preparing high-potency low-toxicity tetramycin B derivatives according to claim 3, wherein the plasmid pJQK503 is constructed by inserting right and left sequenced homologous arm fragments C340A-tetrG-L and C340A-tetrG-R between EcoRI/XbaI double enzyme cutting sites of plasmid pJTU 1278; and the homologous arm fragment C340A-tetrG-L is inserted between EcoRI/HindIII enzyme cutting sites, and the homologous arm fragment C340A-tetrG-R is inserted between HindIII/XbaI enzyme cutting sites.

6. The method for preparing the high-efficiency low-toxicity tetramycin B derivative according to claim 2, wherein the step S3 includes the following steps: centrifuging the obtained fermentation culture solution to respectively collect thalli and fermentation supernatant; and (3) carrying out ultrasonic treatment on the fermentation thalli by using methanol, fully soaking, extracting fermentation supernatant by using ethyl acetate with the same volume, combining extracting solutions, and carrying out reduced pressure concentration on a vacuum rotary evaporator to obtain a crude sample A.

7. The method for preparing the high-efficiency low-toxicity tetramycin B derivative according to claim 6, wherein the MCI gel column chromatography separation specifically comprises the following steps: dissolving the crude sample A with a small amount of methanol 2-4 times the weight ratio, filtering, separating, loading the separated concentrated solution onto MCI gel column chromatography, performing gradient elution with methanol aqueous solution with volume ratio gradient of 0: 1, 2: 8, 4: 6 and 1: 0, collecting the eluate eluted from methanol aqueous solution with volume ratio of 1: 0, and concentrating under reduced pressure to obtain crude sample B.

8. The process for preparing high performance low toxicity tetramycin B derivatives according to claim 6, wherein the preparative high performance liquid chromatography separation is to dissolve the crude sample B in methanol solution and then to obtain the high performance low toxicity tetramycin B derivatives by preparative high performance liquid chromatography separation; the preparation and separation conditions of the high performance liquid chromatography are as follows: acetonitrile and 0.1% formic acid aqueous solution are used as mobile phases, the ratio of the acetonitrile to the 0.1% formic acid aqueous solution is 4: 6, a BDSHYPERSIL C18 reverse semi-preparative column with the flow rate of 5mL/min and the flow rate of 250mm X10mm is used as a stationary phase, the detection wavelength of an ultraviolet detector is 304nm, the sample feeding amount is 100 mu l each time, and specific fractions of a sample are evaporated to dryness after directional accumulation through determining the fraction time of a target peak.

9. the high-efficiency low-toxicity tetramycin B derivative producing strain according to claim 1, which is Streptomyces hygroscopicus var beijing (beijijingningsis) SY04 with the preservation number of CGMCC NO 18369.

10. The use of the highly potent and low toxicity tetramycin B derivative of claim 1 in the preparation of an antifungal drug or food preservative.

Technical Field

The research relates to a technology for separating and preparing natural secondary metabolites of microorganisms, in particular to a high-efficiency low-toxicity tetramycin B derivative and preparation and application thereof.

background

the fungal infections caused by broad-spectrum antibiotics, immunosuppressants, organ transplantation and the like clinically cause the advanced mycoses and the death rate thereof to rise year by year. Due to the rapid development of resistance to fungal pathogens, there are very limited antifungal drugs available clinically for the treatment of systemic fungal infections. Polyene macrolide antibiotics are currently recognized as the most effective antifungal drugs due to their specific chemical structures and their good antifungal activities. Although polyene macrolide antibiotics are rarely resistant for over half a century of clinical application, the drugs have low water solubility and are easy to react with cholesterol on the cell membrane of mammals, so that serious hemolytic toxicity is caused, and the clinical application of the polyene macrolide antibiotics is seriously influenced.

Tetramycin B is a common polyene macrolide antibiotic, and has good prevention and treatment effects on fungal infection of agricultural and forestry plants according to related literature reports. But because of its lower antifungal activity compared with other antifungal drugs, and higher hemolytic toxicity. The disadvantage of this pharmacological property severely limits the widespread use of tetramycin B. Therefore, the adoption of genetic engineering means and biological synthesis strategy to effectively generate new natural antifungal drugs with higher biological activity and lower hemolytic toxicity is imminent.

Disclosure of Invention

the invention aims to provide a high-efficiency low-toxicity tetramycin B derivative, and preparation and application thereof.

The purpose of the invention is realized by the following technical scheme:

In a first aspect, the invention relates to a high-efficiency low-toxicity tetramycin B derivative with a molecular formula of C₃₅H₅₅NO₁₂The chemical structure formula is as follows:

In a second aspect, the present invention relates to a method for preparing the high-efficiency low-toxicity tetramycin B derivative, which comprises the following steps:

S1, performing in-frame deletion on polyketone synthase gene nysI in a nystatin biosynthetic gene cluster in a strain streptomyces spinosa Beijing mutant CGMCC4.1123 to obtain a mutant strain with the gene nysI deleted in the same frame;

S2, inactivating a P450 monooxygenase gene tetrG in a tetramycin biosynthesis gene cluster in the same-frame deletion mutant strain to obtain a gene tetrG inactivated mutant strain;

S3, fermenting and culturing the gene tetrG inactivated mutant strain, and preparing and separating by fermentation liquor extraction, thallus soaking, MCI (cellulose-activated silica gel) column chromatography and high performance liquid chromatography to obtain the high-efficiency low-toxicity tetramycin B derivative.

As one embodiment of the invention, the mutant strain with the gene nysI deletion in frame is a genetically engineered strain Streptomyces hygroscopicus Beijing variant (Streptomyces hygrospinococcus var. beijingensis) SY 02.

as one embodiment of the invention, the gene tetrG inactivated mutant strain is genetically engineered strain Streptomyces hygroscopicus Beijing variant (Streptomyces hygrospinococcus var. beijingensis) SY 04.

In the present invention, step S1 specifically includes the following steps:

S11, constructing a double-exchange plasmid pJQK503 for the in-frame deletion of the polyketide synthase gene nysI in the nystatin biosynthetic gene cluster;

s12, transferring the plasmid pJQK503 into receptor bacterium streptomyces spinosus Beijing variant CGMCC4.1123 through conjugation between escherichia coli and streptomyces;

s13, carrying out PCR verification on the genomic DNA of the mutant strain obtained in the step S12 to obtain the mutant strain with the same frame deletion of the polyketide synthase gene nysI in the nystatin biosynthetic gene cluster.

The construction of the plasmid pJQK503 is that a left and a right homologous arm fragments delta Nys-L and delta Nys-R which are correctly sequenced are respectively inserted between XbaI/HindIII double enzyme cutting sites of the plasmid pJTU 1278; and the homologous arm fragment delta Nys-L is inserted between the XbaI/BspTI enzyme cutting sites, and the homologous arm fragment delta Nys-R is inserted between the BspTI/HindIII enzyme cutting sites.

The sequence of the homologous arm fragment delta Nys-L is shown in SEQ ID NO. 5; the sequence of the homologous arm fragment delta Nys-R is shown in SEQ ID NO. 6.

In the present invention, step S2 specifically includes the following steps:

S21, constructing a P450 monooxygenase gene tetrG inactivation plasmid pJQK513 used in the tetramycin biosynthesis gene cluster;

S22, inactivating the P450 monooxygenase gene tetrG in the gene nysI in-frame deletion mutant strain by the intergeneric conjugative transfer of the streptomycete and escherichia coli on the plasmid pJQK 513;

S23, carrying out PCR verification on the genomic DNA extracted from the mutant strain obtained in the step S22 to obtain the mutant strain with the inactivated P450 monooxygenase gene tetrG in the tetramycin biosynthesis gene cluster.

In step S21, the inactivation of the P450 monooxygenase gene tetrG in the tetramycin biosynthetic gene cluster is achieved by mutating the active site amino acid Cys340 of the gene tetrG to Ala.

the sequence of the P450 monooxygenase gene tetrG in the tetramycin biosynthesis gene cluster is shown as SEQ ID NO. 15.

the construction of the plasmid pJQK503 is that right and left homologous arm fragments C340A-tetrG-L and C340A-tetrG-R which are correctly sequenced are respectively inserted between EcoRI/XbaI double enzyme cutting sites of the plasmid pJTU 1278; and the homologous arm fragment C340A-tetrG-L is inserted between EcoRI/HindIII enzyme cutting sites, and the homologous arm fragment C340A-tetrG-R is inserted between HindIII/XbaI enzyme cutting sites.

The sequence of the homologous arm fragment C340A-tetrG-L is shown in SEQ ID NO. 10; the sequence of the homologous arm fragment C340A-tetrG-R is shown in SEQ ID NO. 11.

In step S3, the fermentation culture is to inoculate the spore suspension of the mutant strain with the inactivated gene tetrG into a TBSY seed culture medium, culture at 220rpm and 30 ℃ for 24h, transfer the spore suspension to the fermentation culture medium in a proportion of 5%, and continuously culture at 220rpm and 30 ℃ for 120h to obtain a fermentation culture solution.

according to the preparation method, the TSBY seed culture medium is prepared from 30g of Oxoid tryptone bean soup powder, 103g of sucrose and 5g of Difco yeast extract, and distilled water is added to the mixture until the volume is 1L and the pH value is 7.2.

The fermentation medium comprises corn flour 10g, soluble starch 20g, soybean cake powder 10g, KH₂PO₄0.2g、 NaCl 3g、NH₄Cl 3g、CaCO₃4g, adding distilled water to a constant volume of 1L, and adjusting the pH value to 7.0 by using 1M sodium hydroxide solution.

In step S3, the yeast liquid extraction and the thallus immersion specifically include: centrifuging the obtained fermentation culture solution to respectively collect thalli and fermentation supernatant; and (3) carrying out ultrasonic treatment on the fermentation thalli by using methanol, fully soaking, extracting fermentation supernatant by using ethyl acetate with the same volume, combining extracting solutions, and carrying out reduced pressure concentration on a vacuum rotary evaporator to obtain a crude sample A.

The MCI gel column chromatography separation specifically comprises the following steps: dissolving the crude sample A with methanol of 2-4 times of the weight ratio for many times, filtering, separating, loading the separated concentrated solution onto MCI gel column chromatography, performing gradient elution with methanol aqueous solution of which the volume ratio gradient is 0: 1, 2: 8, 4: 6 and 1: 0 in sequence, collecting the eluent obtained by eluting with methanol aqueous solution of 1: 0, and concentrating under reduced pressure to obtain crude sample B.

Dissolving the crude sample B in a small amount of methanol solution, and performing high performance liquid chromatography to obtain the high performance low toxicity tetramycin B derivative; the preparation and separation conditions of the high performance liquid chromatography are that acetonitrile and 0.1% formic acid aqueous solution are used as mobile phases, the proportion of the acetonitrile to the 0.1% formic acid aqueous solution is 4: 6, a BDS HYPERSIL C18 reverse semi-preparative column with the flow rate of 5mL/min and the size of 250mm X10mm is used as a stationary phase, the detection wavelength of an ultraviolet detector is 304nm, the sample volume of each time is 100 mu l, and specific fractions of a sample are subjected to directional accumulation and then are evaporated to dryness by determining the fraction time of a target peak.

In a third aspect, the invention relates to a production strain of the high-efficiency low-toxicity tetramycin B derivative, wherein the strain is Streptomyces hygroscopicus var beijing (beijijingninsis) SY04 with the preservation number of CGMCC NO 18369.

in a fourth aspect, the invention relates to an application of the high-efficiency low-toxicity tetramycin B derivative in preparation of antifungal drugs or food preservatives.

The application of the high-efficiency low-toxicity tetramycin B derivative is realized as follows: and (3) respectively carrying out in-vitro antifungal activity and hemolytic toxicity detection on the tetramycin B and the derivatives thereof. Wherein the antifungal activity is detected by using Saccharomyces cerevisiae and Rhodotorula rubra as indicator strains, and obtaining MIC of tetramycin B by using Saccharomyces cerevisiae as indicator strain₅₀/MIC₉₀Respectively as follows: 2.74. + -. 0.19. mu.g/mL/6.01. + -. 0.04. mu.g/mL, MIC of Tetramycin B derivative₅₀/MIC₉₀Respectively as follows: 1.89 plus or minus 0.11 mu g/mL/3.11 plus or minus 0.02 mu g/mL; MIC of Tetramycin B when Rhodotorula glutinis is used as indicator strain₅₀/MIC₉₀Respectively as follows: 3.95. + -. 0.21. mu.g/mL/8.38. + -. 0.03. mu.g/mL, MIC of tetramycin B derivative₅₀/MIC₉₀Respectively as follows: 2.26. + -. 0.14. mu.g/mL/5.51. + -. 0.02. mu.g/mL. Wherein in vitro hemolytic toxicity detection is characterized by the hemolytic toxicity of the antibiotic by the capability of the antibiotic to cause erythrocyte lysis in defibrinated horse whole blood, and the obtained HC of tetramycin B and derivatives thereof₅₀Values were 98.39 + -6.97 μ g/mL and 167.0 + -4.20 μ g/mL, respectively. Therefore, the hemolytic toxicity of the tetramycin B derivative is reduced by 70 percent compared with that of the tetramycin B, and the antifungal activity of the derivative on beer yeast and red yeast is improved by about 1.5 times and 1.8 times respectively.

the mutant strain SY02 is Streptomyces hygroscopicus Beijing variant (Beijing. Beijing) which has been preserved in the general microbiological center of China Committee for culture Collection of microorganisms (Beijing city, Chaoyang district, Beicheng West Lu No.1, institute of microbiology, China academy of sciences) in 2019 at 17 th month, and the preservation number is CGMCC NO. 18241.

The mutant strain SY04 is Streptomyces hygroscopicus Beijing variant (Beijing. Beijing) which has been preserved in the general microorganism center of China Committee for culture Collection of microorganisms (No. 3 Beijing institute of microbiology, China academy of sciences) 8.6 days in 2019 with the preservation number of CGMCC NO 18369.

Compared with the prior art, the invention has the following beneficial effects:

The high-efficiency low-toxicity tetramycin B derivative has a stable structure, the antifungal activity is obviously improved, and the hemolytic toxicity is obviously reduced, so that the prepared high-efficiency low-toxicity tetramycin B derivative has a great clinical application prospect and can be widely applied to food preservatives and plant insecticides.

Drawings

other features, objects and advantages of the invention will become more apparent upon reading of the detailed description of non-limiting embodiments with reference to the following drawings:

FIG. 1 is a schematic diagram of construction of plasmid pJQK503 for the in-frame deletion group of nySi gene;

FIG. 2 is a same frame deletion mutant strain SY02PCR verification agarose gel electrophoresis picture;

FIG. 3 is a schematic diagram of the construction of plasmid pJQK513 for inactivating P450 monooxygenase gene tetrG in the tetramycin biosynthesis gene cluster;

FIG. 4 is a PCR-verified agarose gel electrophoresis image of a tetrG inactivated mutant strain SY 04;

FIG. 5 is a high performance liquid chromatography HPLC ultraviolet absorption chart of a tetramycin B derivative;

FIG. 6 is a mass spectrometric detection of a tetramycin B derivative;

FIG. 7 is a high performance liquid chromatography analysis chart of fermentation products of strains SY02 and SY 04;

FIG. 8 is the determination of the in vitro hemolytic toxicity and antifungal activity of tetramycin B and its derivatives; wherein a is tetramycin B and the derivative thereof for measuring the in-vitro antifungal activity of the beer yeast; b is the determination of the in vitro antifungal activity of the tetramycin B and the derivative thereof on the rhodotorula glutinis; c is the determination of the in vitro hemolytic toxicity of tetramycin B and derivatives thereof.

Detailed Description

The present invention will be described in detail with reference to examples. The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. It should be noted that it would be apparent to those skilled in the art that several modifications and improvements can be made without departing from the inventive concept. All falling within the scope of protection of the present invention. The following examples are examples of experimental methods not specified under specific conditions, according to conventional conditions or manufacturer's recommended conditions.