CN114317361B - New streptomycete strain, and separation method and application thereof - Google Patents
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Abstract
The invention discloses a new strain of streptomycete, named streptomycete II-2-2-2, which is preserved in China general microbiological culture Collection center (CGMCC) No.23479 and has a preservation date of 2021, 9 months and 24 days. Streptomyces II-2-2-2 is yellow brown on a solid culture medium of Gao's No. I, is small and round, has no luster of surface folds, has a central bulge, has filamentous cells at the edge, has cream-colored aerial hyphae and dark brown matrix hyphae, has compact texture and dry and firm surface. Under microscopic observation, mycelium diameter was 0.7-0.9 μm, spore diameter was 0.4-0.6 μm, and gram staining was positive. Experiments prove that Streptomyces (Streptomyces sp.) II-2-2-2 has rich natural product production capacity, can be used for antibiotic discovery, and is worthy of further research and analysis on secondary metabolites and biosynthesis gene clusters.
Description
Technical Field
The invention belongs to the field of microbial pharmacy, and particularly relates to a novel streptomycete strain, and a separation method and application thereof.
Background
Actinomycetes are the largest, most numerous, and most widely distributed bacteria among known organisms. To date, lifestyle, morphology, physiological metabolic activity and genetic analysis of actinomycetes have become research hotspots. Research into new molecular biology techniques and methods based on actinomycete genome is the leading edge of biomedical science technology in the 21 st century. Since the 50 s of the 20 th century, many important antibiotics, antitumor and immunomodulatory drugs have been discovered and applied clinically, providing a material basis for humans to overcome intractable and serious diseases, thanks to the extensive study and screening of secondary metabolites of microorganisms in the terrestrial common habitat. However, due to the wide use of antibiotics over a period of time, even in local abuse, the resistance of pathogens increases dramatically, and even develops into super-resistant bacteria such as enterococcus faecium, staphylococcus aureus, klebsiella pneumoniae, acinetobacter baumannii, pseudomonas aeruginosa, enterobacter and other pathogenic bacteria. These pathogens are resistant to almost all antibiotics currently in clinical use, causing serious health hazards. Therefore, while administering drugs reasonably, there is an urgent need to find new structural chemical entities to develop new antibiotics to cope with the advent of the "post-antibiotic age". Over 70 years, a large number of species have been identified by isolation, active metabolites have been screened, and the probability of finding novel active compounds from common habitat microbial sources has been decreasing. Thus, researchers place the center of gravity on microbial resources of a particular habitat, such as deep sea, salt lakes, polar glaciers, alpine meadows, and the like.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a novel streptomycete strain, and a separation method and application thereof.
In order to achieve the aim of the invention, the invention adopts the following technical scheme:
a new strain of Streptomyces is provided, which is named as Streptomyces II-2-2-2 and is preserved in China general microbiological culture Collection center (CGMCC) No.23479, and the preservation date is 2021, 9 months and 24 days.
Further: streptomyces II-2-2-2 is yellow brown on a solid culture medium of Gao's No. I, is small and round, has no luster of surface folds, has a central bulge, has filamentous cells at the edge, has cream-colored aerial hyphae and dark brown matrix hyphae, has compact texture and dry and firm surface. Under microscopic observation, mycelium diameter was 0.7-0.9 μm, spore diameter was 0.4-0.6 μm, and gram staining was positive.
Further: a method for isolating a novel strain of streptomyces comprising the steps of:
(1) Sampling: collecting a soil sample from Ningxia saline-alkali soil;
(2) Dilution: adding 1g of soil sample and a proper amount of glass beads into 9mL of sterile water, oscillating for 10min, standing for 30s to prepare a soil stock solution, and recording the dilution as 10 -1 Adding 1mL of supernatant of the soil stock solution into a test tube containing 9mL of sterile water, and mixing thoroughly, wherein the dilution is 10 -2 Sucking 1mL from the test tube, adding into another test tube containing 9mL sterile water, mixing, and making into diluent with dilution degree of 10 -4 ；
(3) Coating: 100 mu L of diluents with different dilutions are respectively sucked by a pipette and uniformly coated on a Gao's No. 1 solid culture medium added with 100mg/L of sterile potassium dichromate, and the culture is carried out for 5-7d at 28 ℃;
(4) Screening: observing the growth state of the bacterial colony, and timely picking a new bacterial colony growing in the culture medium in an ultra-clean workbench for separation culture; obtaining pure streptomycete II-2-2-2 after multiple flat scribing.
Further: streptomyces II-2-2-2 consists of a 4409970bp linear DNA chromosome with a G+C content of 66.43%, and comprises 3974 predictive coding genes, 72 tRNA genes and 10 rRNA genes.
Further: streptomyces II-2-2-2 has five gene clusters involved in biosynthesis of known secondary metabolites, including LAP, riPP-like, NRPS, lanthinipeptides and Arylpolyene.
Further: streptomyces II-2-2-2 produces polyketides, non-ribosomal peptides and fatty acids; five radical ions in NPRS, PKS and terpenes can be further predicted.
Further: a fermentation process of a new strain of streptomyces comprising the steps of:
(1) Activating: taking solid culture medium containing Streptomyces II-2-2-2 at 1cm 2 SterileInoculating the strain into 50ml of bennite liquid culture medium under the environment, and performing shake culture at 28 ℃ and 220rpm for 48 hours until bacterial liquid suspension and the neck bacterial circle of the triangular flask grow well;
(2) Fermentation: 1ml of bennite liquid culture solution is inoculated into 50ml of non-iron Nahniki liquid culture medium under the aseptic environment, and shake culture is carried out for 7d at 28 ℃ and 180 rpm; filtering the bacterial liquid and then using C 18 Adsorbing by an adsorption column, and sequentially carrying out gradient elution on water, 50% methanol water and 95% methanol to obtain a streptomycete II-2-2-2 fermentation product.
Further: performing liquid analysis on a fermentation product of the new streptomycete strain by adopting high performance liquid chromatography and quadrupole time-of-flight tandem mass spectrometry;
(1) Mass spectrometry conditions: high-purity nitrogen is used as atomizing and drying gas, and high-purity helium is used as collision gas; the collection mode selects positive ions and negative ions to be detected separately; the capillary spray voltage is 3.5 kilovolts; the temperature of the atomizing chamber is set to 320 ℃, and the flow rate of the atomizing gas is 5.0L/min; the scanning range of the primary mass spectrum is between 300 and 2000, and the scanning range of the secondary mass spectrum is between 20 and 2000; adopting an automatic secondary mode to select the abundance to be more than 10 4 Carrying out secondary mass spectrum acquisition on ions;
(2) Liquid phase conditions: the liquid chromatography is an Agilent 1200 automatic sample injection system, and chromatographic column zorbax XDB-C18 is adopted for chromatographic separation; mobile phase is 0.1% formic acid water solution and methanol solution, gradient elution: increasing the methanol solution from 5% to 95% within 0-20 min, and then isocratically eluting with 95% methanol water solution for 5min; balancing the system with 5% methanol water solution for 5min; the flow rate was 1.0mL/min, the column temperature was set at 25℃and the sample volume was 10. Mu.L.
Further: the use of a new strain of Streptomyces in antibiotics.
The beneficial effects of the invention are as follows:
the invention discloses a new streptomycete strain II-2-2-2 and a complete genome sequence thereof, and researches on chemical diversity of the strain. The preservation number of the microbial strain is CGMCC No.23479 in the China general microbiological culture Collection center. Experiments prove that Streptomyces (Streptomyces sp.) II-2-2-2 has rich natural product production capacity, can be used as antibiotics, and is worthy of further research and analysis in secondary metabolic products and biosynthesis gene clusters.
Drawings
FIG. 1 shows the growth state of Streptomyces of the invention II-2-2 on solid culture medium No. 1 Gaoshi;
FIG. 2 shows the microscopic morphology of Streptomyces II-2-2-2 of the present invention;
FIG. 3 is a sequence diagram of 16S rRNA gene of Streptomyces II-2-2-2 of the present invention;
FIG. 4 is a phylogenetic tree of Streptomyces II-2-2-2 of the invention;
FIG. 5 shows the whole genome of Streptomyces II-2-2-2 of the invention;
FIG. 6 is a cluster of the level metabolite synthesis genes of Streptomyces II-2-2-2 of the invention;
FIG. 7 is a graph showing the networking cluster of the GNPS molecules of the secondary metabolite of Streptomyces II-2-2-2.
The specific embodiment is as follows:
the following description of the embodiments of the present invention is provided to facilitate understanding of the present invention by those skilled in the art, but it should be understood that the present invention is not limited to the scope of the embodiments, and all the inventions which make use of the inventive concept are protected by the spirit and scope of the present invention as defined and defined in the appended claims to those skilled in the art.
Iron-free liquid medium: naNO 3 3g、K 2 HPO 4 1g、MgSO 4 ·7H 2 0.5g of O, 0.5g of KCl, 30g of sucrose and distilled water to 1L, and sterilizing at 121 ℃ for 20min.
Bennett (Bennett) liquid medium: 1g of yeast extract, 1g of beef extract, 2g of casein amino acid, 10g of glucose and distilled water to a volume of 1L, adjusting the pH value to 7.3, and sterilizing for 30min at 115 ℃.
Solid medium No. 1 gao: dissolving soluble starch 20g and KNO 3 1g、K 2 HPO 4 0.5g、MgSO 4 ·7H 2 O0.5 g, naCl 0.5g and FeSO 4 ·7H 2 Dissolving O0.01 g in distilled water, adding agar 20g, and fixing with distilled waterHolding to 1L, adjusting pH to 7.4-7.6, and sterilizing at 121deg.C for 20min.
PBS buffer: 8.0g of NaCl, 0.2g of KCl and Na 2 HPO 4 1.44g、KH 2 PO 4 0.24g was dissolved in 800mL distilled water, the pH was adjusted to 7.4 with HCl, and distilled water was added to a volume of 1L.
The invention is described in further detail below with reference to the attached drawing figures:
example 1: isolation, identification and preservation of the strains of the invention
(1) Separation
Adding 1g of soil sample collected from Ningxia saline-alkali soil (106 deg.08 ', 38 deg.38') and appropriate amount of glass beads into 9mL of sterile water, oscillating for 10min, standing for 30s to obtain soil stock solution (the dilution is 10 at this time) -1 ). The supernatant of 1mL of the stock solution was added to a test tube containing 9mL of sterile water and thoroughly mixed (at a dilution of 10) -2 ) 1mL of the solution was taken from this tube, added to another tube containing 9mL of sterile water, and mixed well, and so forth to prepare a diluent (dilution of 10 -4 )。
Respectively sucking 100 mu L of diluents with different dilutions by a pipetting gun, uniformly coating the diluents on a Gao's No. 1 solid culture medium added with 100mg/L of sterile potassium dichromate, culturing for 5-7d at 28 ℃, observing the growth state of a colony, timely picking new colonies growing in the culture medium in an ultra-clean workbench, and performing isolated culture; obtaining pure streptomycete II-2-2-2 after multiple flat scribing.
The growth state of Streptomyces II-2-2 on solid culture medium of Gao's No. 1 is shown in FIG. 1.
(2) Morphological identification
It was observed that the colonies of Streptomyces II-2-2-2 were yellowish brown, small and round, with no luster of surface wrinkles, raised middle, with filamentous cells at the edges, with aerial hyphae of cream color and hyphae of dark brown matrix, with a dense texture, dry surface and firmness. The colony is white in the initial stage of culture, gradually turns yellow-brown after culture, and the culture medium turns brown.
Further, a sterilized cover slip was taken with tweezers for aseptic manipulation and inserted into a plate agar inoculation line at an angle of about 45 °, the plate was inverted, incubated at 28℃for 3-5d, the cover slip was carefully removed with tweezers, the back culture was wiped off, the bacterial face was placed face up on the slide, the morphology of Streptomyces II-2-2-2 was observed by microscopy, and the observation was performed using gram stain.
Further, the aseptic conductive film is inserted into a flat agar culture medium for 3d growth, cultured for 3-5d at 28 ℃, an electron microscope smear is manufactured, and the form of streptomycete II-2-2-2 is further observed through a scanning electron microscope.
The result shows that under microscopic observation, the streptomycete II-2-2-2 cells are in a filamentous branch, the hypha is slender and has no diaphragm, and the diameter is about 0.8 mu m; the spore filaments are arranged in a chain shape, the spores are elliptical, the surfaces are smooth, the middle part is concave, and the diameter is about 0.5 mu m. The microscopic morphology of Streptomyces II-2-2-2 is shown in FIG. 2.
(3) Molecular characterization
The 16S rDNA gene sequence of Streptomyces II-2-2-2 is shown in FIG. 3.
The 16S rDNA gene sequence of Streptomyces II-2-2-2 was subjected to homology alignment, specifically, a strain having high homology and Streptomyces II-2-2 were selected for phylogenetic analysis, and a phylogenetic tree was constructed by the adjacent method (Neighbor-Joining) in MEGA software, and the results are shown in FIG. 4. Finally, the genetic distance between the Streptomyces II-2-2 and the Streptomyces (Streptomyces sp.) is identified to be the nearest.
(4) Preserving
Streptomyces II-2-2 is preserved in China general microbiological culture collection center (CGMCC) with the preservation number of CGMCC No.23479 in 9 months and 24 days of 2021, wherein the address is CGMCC No. 1 and 3 in North Chen West Lu of the Korean area of Beijing city.
Example 2: determination of Streptomyces II-2-2-2 Whole genome
(1) Streptomyces II-2-2-2 was inoculated into sterilized Bennit liquid medium and shake-cultured at 28℃and 220rpm for 48 hours.
(2) And (3) centrifuging bacterial liquid, flushing the precipitate for 2-3 times by using PBS buffer solution, transferring the precipitate to a 1.5mL sterile freezing tube until the bacterial precipitate reaches the height of 0.5mL, enriching to obtain bacterial cells of streptomycete II-2-2-2, detecting the whole genome of the streptomycete II-2-2-2, and annotating part of genes.
Whole genome detection results: the final assembled genome of Streptomyces II-2-2-2 consisted of a 4409970bp linear DNA chromosome with a G+C content of 66.43% and included 3974 predictive coding genes, 72 tRNA and 10 rRNA genes. The cgview of the whole genome of Streptomyces II-2-2 is shown in FIG. 5.
Example 3: annotation of Streptomyces II-2-2-2 Secondary metabolite Synthesis Gene Cluster
Streptomyces II-2-2-2 whole genome sequence was annotated with an anti-SMASH 6.0.2 gene cluster involved in the biosynthesis of secondary metabolites. Functional analysis of AntiSMASH revealed that Streptomyces II-2-2-2 had five gene clusters involved in the synthesis of secondary metabolites, including LAP, riPP-like, NRPS, lanthinipeptides and Arylpolyene. The annotation of the Streptomyces II-2-2-2 secondary metabolite biosynthetic gene cluster is shown in FIG. 6.
Example 4: chemical diversity analysis of Streptomyces II-2-2-2 based on GNPS clusters
(1) Activating single colony of Streptomyces II-2-2-2, inoculating to 50mL of iron-free culture medium, shake culturing at 28deg.C at 180r/min for 7d, filtering the Streptomyces liquid, and adding C 18 Adsorbing by an adsorption column, and sequentially carrying out gradient elution on water, 50% methanol water and 95% methanol to obtain a streptomycete II-2-2-2 fermentation product.
(2) After pretreatment of the fermentation product, HPLC-Q-TOF-MS/MS analysis was performed.
Mass spectrometry conditions: high-purity nitrogen (liquid nitrogen) is used as atomizing and drying gas, and high-purity helium is used as collision gas; the collection mode selects positive ions and negative ions to be detected separately; the capillary spray voltage is 3.5 kilovolts; the temperature of the atomizing chamber is set to 320 ℃, and the flow rate of the atomizing gas is 5.0L/min; the scanning range of the primary mass spectrum is between 300 and 2000, and the scanning range of the secondary mass spectrum is between 20 and 2000; adopting an automatic secondary mode to select the abundance to be more than 10 4 And carrying out secondary mass spectrum acquisition on the ions.
Liquid phase conditions: liquid chromatography is an Agilent 1200 (united states) autosampling system. Chromatographic separation employs chromatographic column zorbax XDB-C 18 (4.6X105 mm,5.0 μm, agilent Technologies, santa Clara, calif., USA); the mobile phase is0.1% aqueous formic acid (a) +methanol (B), gradient elution: phase B was increased from 5% to 95% over 0-20 min, and then eluted isocratically with 95% methanol water for 5min. The system was equilibrated with 5% methanol water for 5min. The flow rate was 1.0mL/min, the column temperature was set at 25℃and the sample volume was 10. Mu.L.
(3) And after the original data is converted, the original data is imported into a molecular networking website to perform similar component search and molecular clustering. The networking parameters are set as follows: MS/MS ion error 0.5Da; parent ion error 1.0Da; the lowest ion pair scored 0.6; the minimum number of matching ions is 4; the minimum number of matching ions is 10 for the networked Top K. The molecular networking results were visualized in Cytoscape 3.7.0 software.
Results: six groups containing 79 nodes are classified into Polyketides (PKSs), non-ribosomal peptides (NPRS) and fatty acids based on the corresponding chemical structure under positive ionic conditions. In negative ion mode, five radical ions in NPRS, PKS and terpenes can be predicted by comparing their MS/MS data with those in GNPS online databases. All these preliminary and shallow chemical studies indicate that Streptomyces II-2-2-2 has a rich natural product production capacity and deserves further research and analysis.
The molecular network clustering analysis of the secondary metabolite GNPS of Streptomyces II-2-2 is shown in FIG. 7.
The above is only for illustrating the technical idea of the present invention, and the protection scope of the present invention is not limited by this, and any modification made on the basis of the technical scheme according to the technical idea of the present invention falls within the protection scope of the claims of the present invention.
Sequence listing
<110> university of national north
Yinchuan known microorganism technology Co.Ltd
Northwest Institute of ecological environment and resources, Chinese Academy of Sciences
<120> a novel strain of Streptomyces, and isolation method and application thereof
<141> 2021-12-31
<160> 1
<170> SIPOSequenceListing 1.0
<210> 2
<211> 1010
<212> DNA
<213> Streptomyces II-2-2 (Streptomyces sp. II-2-2)
<400> 2
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actttcgtga cgtgacgggc ggtgtgtaca aggcccggga acgtattcac cgcagcaatg 120
ctgatctgcg attactagca actccgactt catggggtcg agttgcagac cccaatccga 180
actgagaccg gctttttgag attcgctccg cctcgcggca tcgcagctca ttgtaccggc 240
cattgtagca cgtgtgcagc ccaagacata aggggcatga tgacttgacg tcgtccccac 300
cttcctccga gttgaccccg gcagtctcct gtgagtcccc atcaccccga agggcatgct 360
ggcaacacag aacaagggtt gcgctcgttg cgggacttaa cccaacatct cacgacacga 420
gctgacgaca gccatgcacc acctgtatac cgaccacaag gggggcacca tctctgatgc 480
tttccggtat atgtcaagcc ttggtaaggt tcttcgcgtt gcgtcgaatt aagccacatg 540
ctccgctgct tgtgcgggcc cccgtcaatt cctttgagtt ttagccttgc ggccgtactc 600
cccaggcggg gaacttaatg cgttagctgc ggcaccgacg acgtggaatg tcgccaacac 660
ctagttccca acgtttacgg cgtggactac cagggtatct aatcctgttc gctccccacg 720
ctttcgctcc tcagcgtcag taatggccca gagatccgcc ttcgccaccg gtgttcctcc 780
tgatatctgc gcatttcacc gctacaccag gaattccgat ctcccctacc acactctagc 840
tagcccgtat cgaatgcaga cccggggtta agccccgggc tttcacatcc gacgtgacaa 900
gccgcctacg agctctttac gcccaataat tccggacacg cttgcgccct acgtattacc 960
gcggctgctg gcacgtagtt agccggcgct tcttctgcag gtaccgtcac 1010
Claims (4)
1. New strain of streptomyceteStreptomyces sp.) The method is characterized in that: the new strain of streptomycete is named as streptomycete II-2-2-2, and is preserved in China general microbiological culture Collection center (CGMCC) No.23479, and the preservation date is 2021, 9 and 24 days.
2. A fermentation process of a novel strain of streptomyces according to claim 1, characterized in that: the method comprises the following steps:
(1) Activating: taking solid culture medium containing Streptomyces II-2-2-2 at 1cm 2 Inoculating to 50ml of Bennit liquid culture medium in a sterile environment, and performing shake culture at 28deg.C and 220rpm for 48 hr until the bacterial liquid suspension grows well on the neck of the triangular flask;
(2) Fermentation: 1ml of bennite liquid culture solution is inoculated into 50ml of non-iron Nahniki liquid culture medium under the aseptic environment, and shake culture is carried out for 7d at 28 ℃ and 180 rpm; filtering the bacterial liquid and then using C 18 Adsorbing by an adsorption column, and sequentially carrying out gradient elution on water, 50% methanol water and 95% methanol to obtain a streptomycete II-2-2-2 fermentation product.
3. The fermentation process of a novel strain of Streptomyces according to claim 2, wherein: performing liquid analysis by adopting high performance liquid chromatography and quadrupole time-of-flight tandem mass spectrometry;
(1) Mass spectrometry conditions: high-purity nitrogen is used as atomizing and drying gas, and high-purity helium is used as collision gas; the collection mode selects positive ions and negative ions to be detected separately; the capillary spray voltage is 3.5 kilovolts; the temperature of the atomization gas is set to 320 ℃, and the flow rate of the atomization gas is 5.0L/min; first-order mass spectrum scanning rangem/z300-2000, secondary mass spectrum scan rangem/z20-2000; adopting an automatic secondary mode to select the abundance to be more than 10 4 Carrying out secondary mass spectrum acquisition on ions;
(2) Liquid phase conditions: the liquid chromatography is an Agilent 1200 automatic sample injection system, and chromatographic column zorbax XDB-C is adopted for chromatographic separation 18 The method comprises the steps of carrying out a first treatment on the surface of the Mobile phase is 0.1% formic acid water solution and methanol solution, gradient elution: the methanol solution was increased from 5% to 95% over 0-20 minutes, followed by 95%Isocratic elution with aqueous methanol for 5min; balancing the system with 5% methanol water solution for 5min; the flow rate was 1.0. 1.0mL/min, the column temperature was set at 25℃and the sample volume was 10. Mu.L.
4. Use of a novel strain of streptomyces according to claim 1 for the preparation of an antibiotic.
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