CN111057669A - Strain for improving yield of tetramycin Z and method for preparing tetramycin Z by using same - Google Patents
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Abstract
The invention discloses a strain for improving the yield of tetramycin Z and a method for preparing the tetramycin Z by using the strain, wherein the strain is classified and named as Streptomyces diastatochromogenes tz3-1, the preservation date is 2019, 12 and 3 days, and the preservation registration number is CGMCC No. 19069. The content of tetramycin Z in the fermentation liquid obtained by fermenting the streptomyces diastatochromogenes tz3-1 is 20.6 times higher than that of the streptomyces diastatochromogenes D, so that the foundation is laid for the industrial production of the tetramycin Z in the future.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to a strain for improving the yield of tetramycin Z and a method for preparing the tetramycin Z by using the strain.
Background
At present, many novel agricultural antibiotics developed autonomously only stay in the laboratory stage, and the key reason is that the production level of the production bacteria does not meet the industrial requirements. Therefore, a major problem currently put forward by researchers is to improve the production level of the existing and developing agricultural antibiotic producing bacteria and accelerate the industrialization process of agricultural antibiotics. The conventional microorganism mutation breeding is time-consuming and labor-consuming and has high randomness, the directed evolution of single-gene coded enzymes can be realized by means of genetic engineering, and the overexpression is carried out, but the improvement of the yield of natural products such as antibiotics and the like which need to be coded by clustered genes is difficult. A great deal of research shows that the utilization of the ribosome engineering technology is to introduce specific mutation into microbial ribosome or RNA polymerase, which is beneficial to improving the yield of secondary metabolites and can induce or activate the biosynthesis pathway of metabolites which are not produced originally by wild strains. The method generally employed is to obtain highly resistant strains by treating microorganisms with antibiotics (such as streptomycin, rifampicin, gentamicin, etc.) capable of acting on ribosomes or RNA polymerases. The ribosome engineering technology is simple and easy to implement, is convenient to combine, and has good application prospects in aspects of industrial microorganism breeding, microorganism resource potential mining and the like.
Disclosure of Invention
The invention utilizes ribosome engineering technology and combines with screening of drug-resistant mutant strains to obtain mutant strains of streptomyces diastatochromogenes D which have drug resistance to low-concentration streptomycin, high-concentration streptomycin and rifampicin, and the capability of the obtained mutant strains of streptomyces diastatochromogenes tz3-1 for synthesizing tetramycin Z is obviously improved compared with the capability of the obtained mutant strains of streptomyces diastatogenes D. The invention provides a reliable method for improving the yield of the tetramycin Z.
The invention aims to provide a method for improving the production level of tetramycin Z against the defect of low production level of tetramycin Z by a wild strain streptomyces diastatochromogenes D; another purpose of the invention is to provide a streptomyces diastatochromogenes tz3-1 with high yield of tetramycin Z.
The purpose of the invention is realized by the following technical scheme:
the invention firstly discloses a strain for improving the yield of tetramycin Z, which is characterized in that the strain is classified and named as Streptomyces diastatochromogenes tz3-1, the strain is preserved in the China general microbiological culture Collection center, the preservation address is No. 3 of Xilu No.1 of the morning district of Beijing city, the preservation date is 2019, 12 months and 3 days, and the preservation registration number is CGMCC No. 19069.
The invention also discloses a method for preparing tetramycin Z by fermenting the strain, which comprises the following steps:
the fermentation culture solution contains glucose 5g, yeast extract 4g, malt extract 6g, CaCO per 1000mL35g, NaCl2g and the balance of water; sterilizing the fermentation culture solution at 121 ℃ for 20min, cooling to 50 ℃, inoculating streptomyces diastatochromogenes tz3-1 under the aseptic condition, performing fermentation culture at 28 +/-1 ℃, centrifuging the fermentation liquor, taking the supernatant, and separating the supernatant to obtain tetramycin Z.
As a preferable scheme of the invention, the tetramycin Z is obtained by extracting, carrying out chromatography, separating and purifying the supernatant.
As a preferred scheme of the invention, the supernatant is added with equal volume of ethyl acetate for extraction, the lower layer is taken and extracted with equal volume of n-butanol, the upper layer is taken and concentrated to extract under the vacuum condition,
dissolving the extract with methanol, centrifuging to obtain supernatant, loading onto ODS column, gradient eluting with 10-90% methanol water solution, separating and purifying to obtain tetramycin Z (tetramycin Z).
As a preferable scheme of the invention, the inoculation amount of the streptomyces diastatochromogenes tz3-1 is 3-5% in volume percentage of the fermentation culture solution (namely, the inoculation concentration is 10 in each 100mL of fermentation culture medium)63-5 mL of spore liquid per mL).
In a preferred embodiment of the present invention, the fermentation time is 5 to 7 days.
The molecular formula of the tetramycin Z is C35H51NO13The chemical structural formula is as follows:
the compound is a novel tetraene macrolide compound which is named as Tetramycin Z and has the English name of Tetramycin Z. Chinese patent application 201910987578.3 explored its application. At present, chemical pesticides are mainly used for preventing and treating plant diseases to kill pathogenic bacteria, but the problems of toxic and side effects and residues of the chemical pesticides on human and livestock are still difficult to effectively solve so far. The compound has strong inhibition effect on common plant pathogenic fungi; is expected to add a new way for the development of new agricultural fungicides in the future.
The compound has strong inhibition effect on common plant pathogenic fungi; the invention provides a new way for developing new agricultural fungicides in the future.
The invention has the beneficial effects that:
firstly, the ability of the obtained mutant strain to synthesize the tetramycin Z is obviously improved compared with the streptomyces diastatochromogenes D by screening the combined drug-resistant mutant strain, so that a reliable method is provided for improving the yield of the tetramycin Z;
the invention provides the streptomyces diastatochromogenes mutant strain with high yield of tetramycin Z, and lays a foundation for the industrial production of tetramycin Z in the future.
Drawings
FIG. 1 is an ultraviolet absorption spectrum of tetramycin Z;
FIG. 2 is an infrared spectrum of tetramycin Z;
FIG. 3 is a high resolution mass spectrum of tetramycin Z;
FIG. 4 Streptomyces diastochromogenes D colony morphology;
FIG. 5 colony morphology of Streptomyces diastochromogenes D mutant tz 3-1.
Detailed Description
The present invention will be further described with reference to the following examples.
Example 1: acquisition of Low concentration streptomycin resistant mutants (first round of selection of resistant mutants)
Preparing a GyM solid plate with the final concentration of 30 mug/mL of low-concentration streptomycin,that is, the concentration of streptomycin in the GyM solid plate was 30. mu.g/mL. 100. mu.L of Streptomyces diastochromogenes D spore suspension (1X 10) was aspirated by pipette6one/mL) on a GYM solid plate, uniformly coating the mixture by using a sterile coating rod, culturing the mixture in an incubator at 28 ℃ for 7 days, respectively picking out single colonies to a new GYM solid plate containing 20 mu g/mL streptomycin (one single colony is drawn into one plate), culturing the single colony in the incubator at 28 ℃ for 5 days, and obtaining a strain which can grow on the new GYM solid plate again, namely a streptomycin resistant mutant strain. Obtaining 15 low-concentration streptomycin resistant mutant strains in total, wherein the serial numbers of the mutant strains are fz 1-fz 15 respectively, carrying out liquid fermentation culture on the 15 mutant strains according to the step (4) in the technical scheme, and detecting by HPLC (high performance liquid chromatography) fermentation liquor, wherein the yield of the 6 mutant strains of tetramycin Z is reduced, namely, negative mutation is generated, the yield of 2 mutant strains has no obvious difference compared with the original strain, the yield of the 7 mutant strains of tetramycin Z is improved to different degrees, and the mutant strain fz9 has the strongest capacity of producing the tetramycin Z;
example 2: acquisition of high-concentration streptomycin-resistant mutants (second round of selection of drug-resistant mutants)
A solid GyM plate with a final streptomycin concentration of 300. mu.g/mL was prepared, i.e., the final streptomycin concentration in the solid GyM plate was 300. mu.g/mL. mu.L of a low-concentration streptomycin-resistant mutant fz9 spore suspension (1X 10) was pipetted using a pipette12one/mL) on a GYM solid plate, uniformly coating the GYM solid plate by using an aseptic coating rod, culturing the GYM solid plate in an incubator at 28 ℃ for 10-15 days, respectively picking a single colony to a new GYM solid plate containing 300 mug/mL of streptomycin (one single colony is drawn into one plate), culturing the GYM solid plate in the incubator at 28 ℃ for 7-10 days, and growing a strain on the new GYM solid plate again to obtain the high-concentration streptomycin resistant mutant strain. Obtaining high-concentration streptomycin resistant mutant strains 3 in total, wherein the serial numbers are sz2-1, sz2-2 and sz2-3 respectively, carrying out liquid fermentation culture on the 3 mutant strains according to the step (4) in the technical scheme, and detecting fermentation liquor by HPLC (high performance liquid chromatography), wherein the sz2-2 mutant strain has reduced tetramycin Z synthesis capability, and sz2-1 and sz2-3 have improved tetramycin Z production capability, and have stronger tetramycin Z synthesis capability compared with the sz2-3 mutant strain.
Example 3: acquisition of Rifampicin resistant mutants (third round of selection of drug resistant mutants)
A GYM solid plate having a final concentration of rifampicin of 20. mu.g/mL was prepared, i.e., the concentration of rifampicin in the GYM solid plate was 20. mu.g/mL. 100. mu.L of the spore suspension (1X 10) of mutant sz2-3 obtained in the second round of selection was pipetted using a pipette10one/mL) on a GYM solid plate containing 20 mu g/mL rifampicin, uniformly coating the mixture by using a sterile coating rod, culturing the mixture in an incubator at 28 ℃ for 7 days, respectively picking out single colonies to a new GYM solid plate containing 20 mu g/mL rifampicin (one single colony is drawn into one plate), culturing the new GYM solid plate in the incubator at 28 ℃ for 5 days, and growing a strain on the new GYM solid plate again to obtain the rifampicin resistant mutant strain. A total of 2 rifampicin resistant mutants were obtained in this screen, numbered tz3-1 and tz 3-2.
Example 4: liquid fermentation of mutant strains and product characterization
The fermentation culture solution contains glucose 5g, yeast extract 4g, malt extract 6g, CaCO per 1000mL35g, NaCl2g and the balance of water; bottling 60mL of fermentation culture solution in each 300mL triangular flask, sterilizing at 121 ℃ for 20min after preparation, cooling to 50 ℃, and inoculating streptomyces diastatochromogenes tz3-1 spores under aseptic condition, wherein the inoculation amount of the spores is 3-5% (namely, the inoculation concentration in each 100mL of fermentation culture medium is 10)63-5 mL of spore liquid per mL). Fermenting and culturing for 5 days at 28 +/-1 ℃;
collecting fermentation liquor after fermentation, adding equal volume of ethyl acetate for extraction, taking a lower layer, extracting with equal volume of n-butanol, taking an upper layer, concentrating under vacuum condition to obtain an extract, dissolving the extract with methanol, centrifuging to obtain a supernatant, then loading on an ODS column, eluting with methanol and water (water/methanol: V/V, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9, 4L), and further separating and purifying to obtain an active compound Tetramycin Z (Tetramycin Z).
Tetramycin Z is structurally verified by ultraviolet spectroscopy (UV), infrared spectroscopy (IR), Mass Spectroscopy (MS) and Nuclear Magnetic Resonance (NMR). The specific analysis conditions and analysis structures are as follows:
the ultraviolet spectrum is shown in figure 1, and the compound has ultraviolet absorption at (291 +/-2) nm, (302 +/-2) nm and (320 +/-3) nm. Infrared spectrum shown in FIG. 2, infrared spectrum (IR, cm-1): 3423(-OH stretching vibration), 2933(-CH2 antisymmetric stretching), 1712 (conjugated ester C ═ O stretching vibration), 1630 (lactone C ═ O stretching vibration), 1571 (multiconjugated C ═ C stretching vibration), 1401 (multiconjugated C — H variable angle vibration). The high-resolution mass spectrum is shown in FIG. 3, and the mass spectrum conditions are as follows: ionization mode ESI +, capillary voltage 0.6kV, vaporization temperature: 450 ℃, scanning range m/z: 50-1000 amu. As can be seen, the molecular weight of tetramycin Z was determined to be about 693.34. The nmr data for tetramycin Z are shown in table 1.
TABLE 1 NMR data for Tetramycin Z
The compound is a new compound named as tetramycin Z, and the specific structural formula is shown in the following formula
Example 5: comparison of mutant Tetramycin Z production
Inoculating streptomyces diastatochromogenes D or corresponding mutant strains according to the fermentation method of the embodiment 4 in the technical scheme, carrying out liquid fermentation culture, and detecting fermentation liquor by HPLC.
And (4) HPLC detection: the fermentation liquor is centrifuged for 10min at 5000r/min, and the supernatant is used for measuring the content of tetramycin Z. Performing gradient elution with SHIMADZU C18 chromatographic column (150mm × 4.6mm, 5 μm), methanol as mobile phase A, water as mobile phase B, and eluting for 0-15min and 5% -37% A; 15-30min, 37% -100% A; 30-40min, 100% -5% A; the flow rate was 1.0mL/min, the detection wavelength was 279nm, and the column temperature was 30 ℃.
Wherein the tz3-1 mutant has remarkably improved tetramycin Z synthesis capability and sz2-3 ratio, and the tz3-2 mutant has reduced capability (see table 2).
The results show that: the content of tetramycin Z in the fermentation liquor of the streptomyces diastatochromogenes mutant tz3-1 is 20.6 times higher than that of the streptomyces diastatochromogenes D.
TABLE 2 ability of Streptomyces diastatochromogenes D and mutant to synthesize tetramycin Z
Example 6: colony morphology study
Compared with the colony morphology of the streptomyces diastatochromogenes D and the mutant tz3-1, the colony morphologies of the streptomyces diastatochromogenes D and the mutant tz3-1 are obviously different, the colony shape of the streptomyces diastatochromogenes D is shown in figure 4, the colony shape of the mutant tz3-1 and the colony shape of the mutant tz3-1 are shown in figure 5, compared with the colony shape of the streptomyces diastatogenes D, the colony shape of the mutant tz3-1 on a GYM solid plate is smaller, and a plurality of fine burrs are arranged on the periphery of the colony, and the shape of.
Example 7: (Tetramycin Z inhibitory action against Rhizoctonia solani or Botrytis cinerea)
(1) Preparing tetramycin Z solutions with various concentrations;
(2) and (3) determining the antibacterial activity: 1mL of tetramycin Z solution with each concentration is taken to be put in a sterile culture dish and is rapidly and uniformly mixed with 9 mL of PDA culture medium cooled to 50 ℃, 1 fungus cake of cucumber rhizoctonia solani or tomato botrytis cinerea with the diameter of 4mm is respectively placed on each plane of the culture medium after cooling, the fungus cake is connected to the center of the culture dish (the diameter of the culture dish is 9cm), the culture dish is placed in an incubator for 36h at the temperature of 28 ℃, and the steps are repeated for 3 times by taking conventional chemical agents and sterile water treatment as references; the colony diameter was measured by the cross method, and the inhibition rate was calculated by the following formula:
the control group used tetramycin P and carbendazim, respectively, and the experimental results are shown in Table 3. As seen from Table 1, the EC of tetramycin Z against Rhizoctonia solani and Botrytis cinerea50The values are all lower than those of carbendazim, and the inhibition effect is stronger than that of the conventional chemical agent carbendazim.
TABLE 3 inhibitory Effect of Tetramycin Z on Rhizoctonia solani and Botrytis cinerea
Different letters in the same row represent significant differences at the 0.05 level.
Claims (5)
1. The strain for improving the yield of the tetramycin Z is characterized in that the strain is classified and named as Streptomyces diastatochromogenes tz3-1, the strain is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, the preservation address is No. 3 of Xilu No.1 of Beijing Korean district, the preservation date is 2019, 12 months and 3 days, and the preservation number is CGMCC No. 19069.
2. A process for the fermentative preparation of tetramycin Z using the strain of claim 1, characterized in that:
the fermentation culture solution contains glucose 5g, yeast extract 4g, malt extract 6g, CaCO per 1000mL35g, NaCl2g and the balance of water; sterilizing the fermentation culture solution at 121 ℃ for 20min, cooling to 50 ℃, inoculating streptomyces diastatochromogenes tz3-1 under the aseptic condition, performing fermentation culture at 28 +/-1 ℃, centrifuging the fermentation liquor, taking the supernatant, and separating the supernatant to obtain tetramycin Z.
3. The fermentation process of claim 2, wherein: extracting, carrying out chromatography, separating and purifying the supernatant to obtain the tetramycin Z.
4. The fermentation process of claim 2, wherein: extracting the supernatant with equal volume of ethyl acetate, collecting the lower layer, extracting with equal volume of n-butanol, collecting the upper layer, concentrating under vacuum to obtain extract,
dissolving the extract with methanol, centrifuging to obtain supernatant, loading onto ODS column, gradient eluting with 10-90% methanol water solution, separating and purifying to obtain tetramycin Z (tetramycin Z).
5. The fermentation process of claim 2, wherein the fermentation incubation time is 5-7 days.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112640918A (en) * | 2020-12-14 | 2021-04-13 | 中国计量大学 | Application of streptomyces diastatochromogenes metabolite in preventing and treating dendrobium officinale diseases |
| CN112640917A (en) * | 2020-12-14 | 2021-04-13 | 中国计量大学 | Application of streptomyces diastatochromogenes metabolite in preventing and treating thunberg dwarf lilyturf black spot and anthracnose |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112640918A (en) * | 2020-12-14 | 2021-04-13 | 中国计量大学 | Application of streptomyces diastatochromogenes metabolite in preventing and treating dendrobium officinale diseases |
| CN112640917A (en) * | 2020-12-14 | 2021-04-13 | 中国计量大学 | Application of streptomyces diastatochromogenes metabolite in preventing and treating thunberg dwarf lilyturf black spot and anthracnose |
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