CN111876344A - 24-membered ring macrolide high-yield strain and application thereof - Google Patents
24-membered ring macrolide high-yield strain and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of bioengineering, and particularly relates to a 24-membered ring macrolide high-yield strain and application thereof, in order to breed a strain which can generate 24-membered ring macrolide antibacterial substances with high yield and stability, the invention uses Siamese bacillus YB304 as an original strain to obtain a 24-membered ring macrolide high-yield strain UV-A2 after ultraviolet mutagenesis, the mutant strain is preserved in Guangdong province microorganism strain preservation center in 2020, 06, 17.A, and the preservation number is GDMCC NO: 61064. the 24-membered ring macrolide yield of the mutant strain is 2.77 times of that of an original strain, and the mutant strain has good antibacterial activity on escherichia coli, aspergillus flavus, bacillus subtilis and saccharomyces cerevisiae, can be used for fermenting 24-membered ring macrolide compounds, can be used for preparing antibacterial agents, and can be used for inhibiting escherichia coli, bacillus subtilis, saccharomyces cerevisiae and aspergillus flavus.
Description
Technical Field
The invention belongs to the technical field of bioengineering, and particularly relates to a 24-membered ring macrolide high-yield strain and application thereof.
Background
Bacillus is attracting more and more attention and favor as an important industrial microbial strain. With the continuous development of modern biotechnology and the intensive research on mutation breeding technology, the application of molecular biology technology to the research on bacillus will also become a hotspot. The probability of the microbial strains to mutate under natural conditions is low, the mutation amplitude is not large, and the gene structure is difficult to change. Therefore, the simple reliance on natural mutation of microbial populations to breed specific strains is far from meeting the needs of production and research. With the development of life science and the deepening of interdisciplinary science, the mutation breeding technology is continuously developed and innovated, thereby providing technical possibility and convenience for screening more efficient strains. The ultraviolet rays are the most common physical mutagen, adjacent pyrimidines can form dimers to cause the genetic material of the strain to be changed, and the method is simple to operate and low in cost.
Macrolide antibiotic drugs in a broad sense include: 14-to 16-membered macrolide antibiotics, 24-or 31-membered macrolide lactam antibiotics, polyene macrolide antibiotics, and 18-membered novel macrolide antibiotics, and the like. Wherein, the 24-membered ring macrolide compound has a plurality of conjugated double bonds, hydroxyl side chain groups, macrolide skeleton and other structures, and the antibiotics have the inhibiting effect on fungi and can form compound holes with sterol on a fungal cell membrane to enable micromolecules and ions of cell contents to escape from transmembrane holes to finally cause the fungal cellDeath of (1). Researches show that the 24-membered ring macrolide has stronger inhibitory activity on the germination of winter spores of ustilago sugarcane, and EC50Is 7.549 mug/mL, and the bacteriostasis effect is better than that of positive control difenoconazole missible oil. At present, 36 24-membered ring macrolides have been found in the world, most of which are obtained from marine microorganisms. The 24-membered ring macrolide compounds are important novel macrolide compounds, have chemical structures different from 14-to 16-membered macrolide antibiotics, and have various biological activities. However, the yield of the 24-membered ring macrolide compound which is the main metabolite of the natural strain is low, and the requirements of scientific research and production application can not be met, so that the method has very important significance in breeding the strain which can produce the 24-membered ring macrolide antibacterial substance and has high and stable yield.
Disclosure of Invention
In order to overcome the above-mentioned disadvantages of the prior art, the primary object of the present invention is to provide a 24-membered ring macrolide high-producing strain.
The second object of the present invention is to provide the use of a 24-membered macrolide high-producing strain for the production of a 24-membered macrolide compound.
It is a third object of the present invention to provide an antibacterial agent.
A fourth object of the present invention is to provide the use of a 24-membered ring macrolide highly producing strain for the preparation of an antibacterial agent.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
the invention provides a 24-membered ring macrolide high-yield strain, wherein the 24-membered ring macrolide high-yield strain is Siamese Bacillus mutant strain UV-A2 which is classified and named as Bacillus siamensis, the UV-A2 strain is collected in Guangdong province microorganism strain collection center in 2020, 06, 17, and the collection number is GDMCC NO: 61064.
the Siamese bacillus YB304 is separated from northern gulf white soil, the main metabolite of the Siamese bacillus YB304 is a 24-membered ring macrolide compound, and Siamese bacillus mutant bacteria UV-A2 are obtained after ultraviolet mutagenesis, the yield of the 24-membered ring macrolide of the mutant bacteria is 2.77 times of that of the original strain, the heritability stability is good, and the Siamese bacillus YB is expected to be applied to fermenting the 24-membered ring macrolide compound and preparing an antibacterial agent with an antibacterial effect.
The invention also provides application of the 24-membered ring macrolide high-producing strain in preparation of 24-membered ring macrolide compounds.
Preferably, the preparation method of the 24-membered ring macrolide compound comprises the following steps:
s1, inoculating the mutant strain UV-A2 into an MSM culture medium, and culturing to obtain a seed solution;
s2, inoculating the seed solution into a shake flask filled with an MSM culture medium, and culturing to obtain a fermentation liquid;
s3, extracting the fermentation liquor by using ethyl acetate, and collecting supernatant liquor;
s4, evaporating the supernatant to dryness, dissolving in methanol, filtering, collecting filtrate, and purifying to obtain the 24-membered ring macrolide compound.
Preferably, the culture conditions of the seed solution in step S1 are: culturing at 28-32 ℃ for 20-25 h at a rotating speed of 150-250 r/min.
Preferably, the culture conditions of the fermentation broth in step S2 are: culturing at 28-32 ℃ for 5-9 days at a rotating speed of 150-250 r/min.
Preferably, the inoculation amount of the seed solution in the step S2 is 5-10% by volume ratio.
Preferably, the extraction method in step S3 is: mixing the fermentation liquid with equal volume of ethyl acetate, extracting for 3 times, and combining the three supernatants.
Preferably, the filtration in step S2 is performed by using a 0.22 μm organic microporous membrane.
The invention also provides application of the 24-membered ring macrolide high-producing strain in preparation of an antibacterial agent. Specifically, the antibacterial spectrum of the antibacterial agent comprises Escherichia coli, bacillus subtilis, saccharomyces cerevisiae and aspergillus niger.
The present invention also provides an antibacterial agent whose active ingredient is produced by the 24-membered ring macrolide high-producing strain.
Preferably, the antibacterial spectrum of the antibacterial agent comprises escherichia coli, bacillus subtilis, saccharomyces cerevisiae and aspergillus niger.
The mutant bacterium UV-A2 has strong bacteriostatic activity on escherichia coli and aspergillus flavus, has medium bacteriostatic activity on bacillus subtilis and saccharomyces cerevisiae, and is expected to be applied to preparation of antibacterial agents. Of course, in order to increase the applicable scope of the antibacterial agent, the antibacterial agent can also comprise other acceptable auxiliary materials, and can be prepared into liquid or solid dosage forms.
Compared with the prior art, the invention has the beneficial effects that:
the invention provides a 24-membered ring macrolide high-yield strain which is Siamese bacillus mutant strain UV-A2 and is preserved in Guangdong province microorganism strain preservation center in 2020 and 17 th, wherein the preservation number is GDMCC NO: 61064. the strain is obtained by using Siamese bacillus YB304 as an original strain through ultraviolet mutagenesis, the yield of the 24-membered ring macrolide of the mutant strain is 2.77 times of that of the original strain, and the mutant strain has good bacteriostatic activity on escherichia coli, aspergillus flavus, bacillus subtilis and saccharomyces cerevisiae, not only can be used for fermenting 24-membered ring macrolide compounds, but also can be used for preparing antibacterial agents and inhibiting escherichia coli, bacillus subtilis, saccharomyces cerevisiae and aspergillus niger.
Drawings
Fig. 1 is an ultraviolet lethal curve of siamese bacillus YB 304;
FIGS. 2 to 4 are graphs of preliminary screening results of mutant strains;
FIG. 5 is a UV spectrum of a 24-membered ring macrolide;
FIG. 6 is a graph of 24-membered ring macrolide yield for 13 rescreened mutant strains;
FIG. 7 is a colony morphology diagram of Siamese bacillus mutant bacteria UV-A2.
Detailed Description
The following further describes the embodiments of the present invention. It should be noted that the description of the embodiments is provided to help understanding of the present invention, but the present invention is not limited thereto. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
The test methods used in the following experimental examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.
Example 1 acquisition of original Strain (Siamese Bacillus YB304)
The fruit of the Aricennia marina, a northern Orthosiphon, northern gulf, was used as a test material, and its endogenous bacteria were isolated by a conventional dilution coating method. Fermenting endophytic bacteria to obtain a secondary metabolite, analyzing by high performance liquid chromatography, generating 24-element macrolide by a strain YB304, and identifying by a 16S rRNA sequence, wherein the YB304 is Siamensis.
Example 2 ultraviolet mutagenesis of Siamese Bacillus YB304 (Siamese Bacillus mutant bacteria UV-A2 acquisition)
(1) Preparation of bacterial suspensions for mutagenesis
Inoculating 2-ring activated Siamese bacillus YB304 into a triangular flask (500mL) filled with 200mL ISP2 liquid culture medium, culturing for 16h at 30 ℃ to form a culture solution in a logarithmic growth phase, collecting a bacterial solution, and freezing and centrifuging for 10min at 4 ℃ at a rotating speed of 10000 r/min; then collecting the thallus and washing with sterile normal saline for 3 times, and finally suspending the thallus in the sterile normal saline and adjusting the cell concentration to 1 × 106And (3) taking the CFU/mL as a bacterial suspension to be treated, and preserving the bacterial suspension at 4 ℃ for later use to obtain the bacterial suspension for mutagenesis.
(2) Determination of the UV lethal Curve
In order to stabilize ultraviolet light waves, an ultraviolet lamp (25w) is started in advance to preheat for 20min, the irradiation distance is 25cm, 9mL of the prepared bacterial suspension is added into a sterile culture dish (90mm), a magnetic stirrer is started to enable bacterial liquid to rotate at a constant speed, the cover is opened for timing, and the irradiation Time (Time) is 0, 15, 30, 45, 60, 75, 90, 105, 120 and 135s respectively. Diluting the treated bacterial liquid to 10 degree with sterile normal saline-3、10-4、10-5Three dilutions were then applied to ISP2 plates at 200. mu.L of each bacterial dilutionCoating 3 plates with dilution, coating the plates with sterile normal saline as a blank control, wrapping the plates with tinfoil paper, culturing at 37 ℃ in a constant temperature and in a dark place for 2d, counting the number of colonies, and calculating the lethality (Mortality) according to the following formula by taking bacterial liquid without ultraviolet irradiation as a control:
as shown in figure 1, the Siamese bacillus YB304 is sensitive to ultraviolet irradiation, the death rate of the strain is increased along with the prolonging of the ultraviolet irradiation time, the survival rate is generally lower than 10% when the ultraviolet treatment time is longer than 105s, the bacteria lethality rate reaches 91.58% at 120s, and the positive mutation rate is the highest and is 12.5%. Therefore, the optimum ultraviolet irradiation time of the original strain YB304 is 120 s.
(3) Ultraviolet mutagenesis
Selecting 100s as the optimal time for ultraviolet mutagenesis according to a lethal curve, performing ultraviolet mutagenesis, namely turning on an ultraviolet lamp (25w) in advance for preheating for 20min, irradiating for 25cm, adding 9mL of prefabricated bacterial suspension into a sterile culture dish (90mm), starting a magnetic stirrer to enable bacterial liquid to rotate at a constant speed, opening a cover for timing, and taking the irradiation time as 100s, and then picking out bacterial colonies with larger bacterial colony diameter and irregular shapes for screening of 24-membered ring macrolide high-yield strains.
(4) Preliminary screening of 24-membered ring macrolide high-producing strains
Preparation of primary screening fermentation crude extract: the mutant strains with larger colony diameter and irregular shape are picked to ISP2 plate for streak enrichment, and are inoculated to 20mL MSM culture medium by one ring, and cultured for 7d at 30 ℃ and the rotating speed of 200 r/min. And (3) taking 10mL of fermentation liquor and ethyl acetate for isovolumetric mixed extraction, sucking supernatant liquor into a centrifuge tube for natural volatilization, and adding 200 mu LDMSO for dissolution to obtain a fermented crude extract for later use.
Primary screening: and primary screening is carried out by adopting an agar diffusion method. The growth speed of the escherichia coli is high, so that the bacterium screening period can be greatly shortened; and earlier experiments show that the main component playing a bacteriostatic action on the escherichia coli in the crude fermentation extract is 24-membered-ring macrolide, so that the escherichia coli is selected as an indicatorAnd (5) bacteria. Adjusting the concentration of the E.coli suspension to 105～106CFU/mL, 100. mu.L were applied evenly to ISP2 plates. Punching 3 holes on a flat plate by using a puncher, adding 50 mu L of the fermentation crude extract into each hole, taking DMSO solution as a blank control and taking an original strain as a control, setting 3 strains in parallel, culturing at 28 ℃, observing and recording inhibition zones, and judging the antibacterial activity. Selecting mutant strains with the diameter of the inhibition zone increased by 20 percent compared with that of the original strains, and then re-screening the mutant strains.
As shown in the preliminary screening result of FIG. 2, 13 mutant strains were selected and rescreened based on the increase of 20% in the zone of inhibition, namely UV-A18, A6, A24, A2, A23, C3, C6, C10, B38, B25, B12, B24 and B39.
(5) Double screening of 24-membered ring macrolide high-producing strains
Preparing a re-screened fermentation crude extract: inoculating mutant strain with 20% increased inhibition zone diameter compared with original strain into MSM culture medium, and culturing at 30 deg.C at 200r/min for 22h as seed liquid. The seed liquid is inoculated into a shake flask according to the standard of 7.5 percent inoculum size (volume ratio) and cultured for 7d at the rotating speed of 200r/min at the temperature of 30 ℃. Then mixing the fermentation liquor with equal volume of ethyl acetate, extracting for 3 times, combining the supernatant obtained by 3 times of extraction, and performing vacuum rotary evaporation at 43 ℃ to dryness.
Liquid chromatography analysis of the crude fermentation extract: dissolving the crude fermented extract with 2mL of methanol, filtering with 0.22 μm filter membrane, detecting with high performance liquid chromatography, determining whether the product is 24-membered macrolide according to ultraviolet spectrogram, determining 24-membered macrolide if the product has maximum ultraviolet absorption peak at 228nm and 262nm (as shown in FIG. 5), and calculating the yield of 24-membered macrolide according to the following standard curve formula:
y ═ x-279687)/177.4(x denotes macrolide yield, y denotes peak area).
The ultraviolet spectrogram shows that all 13 mutant strains entering the secondary screening are strains for producing 24-membered ring macrolide, and the mutant strains UV-B39, A23 and A2 are 24-membered ring macrolide high-producing strains according to the yield of the 24-membered ring macrolide (figure 6), wherein the yield of the mutant strain UV-B39 is 1.34 times that of the original strain, namely 46.69 mg/L; the yield of the mutant strain UV-A23 is 2.31 times of that of the original strain, namely 80.12 mg/L; the yield of the mutant strain UV-A2 was 2.77 times that of the original strain, i.e., 96.14 mg/L. The ultraviolet mutagenesis of this experiment has a significant effect on yield improvement.
(6) Genetic stability analysis of high producing strains
Carrying out continuous passage on mutant strains (mutant strains UV-B39, A23 and A2) of the 24-membered ring macrolide compound with high yield obtained by mutagenesis for 1, 5, 10, 15, 20, 25 and 30 times respectively on a plate, then carrying out shake flask fermentation, detecting the yield of the 24-membered ring macrolide compound, and checking genetic stability.
The experimental results in Table 1 show that the mutant strain UV-A2 is genetically stable and is a 24-membered ring macrolide high-producing strain with good genetic stability.
TABLE 1 genetic stability assay results for mutant strains UV-B39, A23, and A2
Example 3 bacteriostatic activity of Bacillus siamensis mutant UV-A2 (24-membered ring macrolide high-yield mutant UV-A2)
Escherichia coli, bacillus subtilis, saccharomyces cerevisiae and aspergillus niger are used as detection bacteria, and the inhibition activity of the 24-membered ring macrolide high-yield mutant bacteria UV-A2 on the Escherichia coli, the bacillus subtilis, the saccharomyces cerevisiae and the aspergillus niger is determined by an Oxford cup method. Adjusting the concentration of the bacterial suspension of the detection bacteria to 105～106CFU/mL, 100. mu.L of the suspension was uniformly spread on ISP2 plates as detection plates. Punching 3 holes on a flat plate by using a puncher, adding 80 mu L of mutant bacterium UV-A2 fermentation crude extract into each hole, setting 3 samples in parallel by using DMSO solution as blank control, culturing at 28 ℃, observing and recording inhibition zones, and judging the antibacterial activity. The judgment standard of the antibacterial activity is as follows:
(1) the diameter of the bacteriostatic zone is more than or equal to 20mm, and the bacteriostatic activity is strong;
(2) the diameter of the inhibition zone is less than 20mm when the diameter is less than 10mm, and the antibacterial activity is medium;
(3) the diameter of the bacteriostatic circle is less than or equal to 10mm when the diameter is less than 6mm, and the bacteriostatic activity is weaker;
(4) the diameter of the bacteriostatic zone is less than or equal to 6mm, and the bacteriostatic activity is weak.
The test results in Table 2 show that the mutant bacterium UV-A2 with high yield of 24-membered ring macrolide has strong bacteriostatic activity on escherichia coli and aspergillus flavus and moderate bacteriostatic activity on bacillus subtilis and saccharomyces cerevisiae, and thus the mutant bacterium UV-A2 is expected to be applied to preparation of antibacterial agents.
TABLE 2 bacteriostatic activity of mutant UV-A2
| Escherichia coli | Bacillus subtilis | Saccharomyces cerevisiae | Aspergillus flavus | |
| Diameter of bacteriostatic circle (mm) | 28.5±0.30 | 16.6±0.18 | 12.0±0.20 | 20.0±0.25 |
| Bacteriostatic activity | Is stronger | Medium and high grade | Medium and high grade | Is stronger |
Example 4 characterization of Bacillus siamensis mutant UV-A2 (24-membered ring macrolide high-yielding mutant UV-A2)
1. Plate shape
Round, milky white, irregular edges, wrinkled surfaces, dull on ISP2 medium, produced a large amount of viscous watery material by the fifth day of culture.
2. 16S rRNA sequence: as shown in SEQ ID NO: 1 is shown.
2. Physiological and biochemical properties
The results of physiological and biochemical tests show that the strain UV-A2 is an aerobic bacterium, can move and grow, can utilize sucrose, glucose, mannitol and galactose, and has positive contact enzyme, gelatin liquefaction, casein hydrolysis and V-P test, negative methyl red test and no production of hydrogen sulfide. According to the morphological characteristics and physiological and biochemical characteristics of the strain UV-A2, the strain can be preliminarily identified as Bacillus (Bacillus sp.).
Example preservation of 524-membered ring macrolide high-producing mutant UV-A2
The deposit information of the 24-membered ring macrolide high-producing mutant UV-A2 is as follows:
preservation time: 17 months 06 in 2020;
the name of the depository: guangdong province microbial strain preservation center;
the preservation number is: GDMCC No. 61064;
the address of the depository: building No. 59, building No. 5 of the first-furious Zhonglu 100 yard in Guangzhou city;
and (3) classification and naming: bacillus siamensis UV-A2.
The embodiments of the present invention have been described in detail, but the present invention is not limited to the described embodiments. It will be apparent to those skilled in the art that various changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, and the scope of protection is still within the scope of the invention.
Sequence listing
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ggtcgcaaga ctgaaactca aaggaattga cgggggcccg cacaagcggtggagcatgtg 900
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gtcggtgagg taac 1394
Claims (10)
1. A24-membered ring macrolide high-producing strain is characterized in that the 24-membered ring macrolide high-producing strain is Siamese bacillus mutant strain UV-A2, the UV-A2 strain is preserved in Guangdong province microorganism strain preservation center in 2020, 06 and 17 days, and the preservation number is GDMCC NO: 61064.
2. use of the 24-membered ring macrolide high-producing strain according to claim 1 for producing a 24-membered ring macrolide compound.
3. Use according to claim 2, wherein the 24-membered ring macrolide compound is prepared by a process comprising the steps of:
s1, inoculating the mutant strain UV-A2 into an MSM culture medium, and culturing to obtain a seed solution;
s2, inoculating the seed solution into a shake flask filled with an MSM culture medium, and culturing to obtain a fermentation liquid;
s3, extracting the fermentation liquor by using ethyl acetate, and collecting supernatant liquor;
s4, evaporating the supernatant to dryness, dissolving in methanol, filtering, collecting filtrate, and purifying to obtain the 24-membered ring macrolide compound.
4. The use of claim 3, wherein the seed solution of step S1 is cultured under the following conditions: culturing at 28-32 ℃ for 20-25 h at a rotating speed of 150-250 r/min.
5. The use according to claim 3, wherein the culture conditions of the fermentation broth of step S2 are: culturing at 28-32 ℃ for 5-9 days at a rotating speed of 150-250 r/min.
6. The use according to claim 3, wherein the seed solution is inoculated in an amount of 5-10% by volume in step S2.
7. The use of claim 3, wherein the extraction method of step S3 is: mixing the fermentation liquid with equal volume of ethyl acetate, extracting for 3 times, and combining the three supernatants.
8. The use of claim 3, wherein the filtration of step S2 is performed by using a 0.22 μm organic microporous membrane.
9. An antibacterial agent characterized in that an active ingredient of the antibacterial agent is produced from the 24-membered ring macrolide high-producing strain of claim 1.
10. The antimicrobial agent of claim 9, wherein the antimicrobial spectrum of the antimicrobial agent comprises escherichia coli, bacillus subtilis, saccharomyces cerevisiae, and aspergillus niger.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113151086A (en) * | 2021-04-24 | 2021-07-23 | 青海省农林科学院 | Siamese bacillus, microbial inoculum, extract and application thereof |
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| CN108060100A (en) * | 2017-12-29 | 2018-05-22 | 中国科学院南海海洋研究所 | A kind of multi-functional Siam bacillus and its preparation and application of bioactive substance |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108060100A (en) * | 2017-12-29 | 2018-05-22 | 中国科学院南海海洋研究所 | A kind of multi-functional Siam bacillus and its preparation and application of bioactive substance |
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| CHENGHAI GAO 等: "New 24-Membered Macrolactins Isolated from Marine Bacteria Bacillus siamensis as Potent Fungal Inhibitors against Sugarcane Smut", 《J. AGRIC. FOOD CHEM.》 * |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113151086A (en) * | 2021-04-24 | 2021-07-23 | 青海省农林科学院 | Siamese bacillus, microbial inoculum, extract and application thereof |
| CN113151086B (en) * | 2021-04-24 | 2023-04-07 | 青海省农林科学院 | Siamese bacillus, microbial inoculum, extract and application thereof |
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