Application of alpha-pyrone compound in promoting conjugational transfer of streptomyces

Abstract

The invention discloses an application of an alpha-pyrone compound in promoting conjugation transfer of streptomycete, the alpha-pyrone compound can improve the conjugation transfer efficiency of streptomycete, the alpha-pyrone compound has a structure as shown in a formula I, in the formula I, R1 is CH3 or H, R2 is CH3 or H, and R3 is CH3. The compound can effectively solve the bottleneck problem that streptomycete is difficult to carry out genetic modification through conjugational transfer, and can improve the conjugational transfer efficiency of streptomycete, promote the development of novel antibiotics, improve the activity and yield of existing antibiotics and reduce the pharmaceutical cost, thereby improving the drug accessibility. # imgabs0 #

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to application of alpha-pyrone compounds in promoting conjugation transfer of Streptomyces. Background Art

[0002] α-pyrone, also known as chalcone, is a class of unsaturated six-membered ring lactone compounds produced by bacteria, fungi and plants. This family of compounds has diverse structures and has a variety of biological activities such as inhibiting spore germination, antibacterial, antifungal, antitumor, anticoagulant, HIV protease inhibitor, etc., and has great application potential. Germicidin A~D are the four most typical α-pyrone compounds produced by Streptomyces.

[0003] Streptomyces is a type of Gram-positive bacteria with a high G+C content and is the main source of antibiotics. Wild-type Streptomyces isolated from nature have problems such as low antibiotic production, insufficient compound structure diversity, low activity, and silencing of metabolite gene clusters, which are difficult to meet human needs for antibiotics. Therefore, it is necessary to genetically modify Streptomyces through genetic manipulation (mainly conjugation transfer).

[0004] Conjugative transfer is the process of horizontal transfer of plasmid DNA or other integrative and conjugative elements (ICE) from donor bacteria to recipient bacteria through direct contact. There are different ways of conjugative transfer between different bacteria, but almost all Gram-positive and Gram-negative bacteria use the type IV secretion system to form conjugative pores to mediate the transfer of plasmid DNA from donor bacteria to recipient bacteria. However, actinomycetes represented by Streptomyces do not rely on the type IV secretion system. It mainly forms conjugative pores through a plasmid-encoded protein TraB to mediate the transfer of plasmid DNA.

[0005] However, due to the widespread restriction modification system in Streptomyces, many Streptomyces are difficult to use as recipient bacteria for genetic modification through conjugation transfer, which greatly limits the development and utilization of Streptomyces resources and leads to great restrictions on the development of new antibiotics.

[0006] Meanwhile, the existing methods for promoting the efficiency of conjugative transfer of Streptomyces are mainly to optimize the conditions of conjugative transfer, such as the culture state of the donor bacteria and the recipient bacteria, heat shock, pre-germination time, the ratio of the donor bacteria to the recipient bacteria, the concentration of metal ions and the culture medium used for conjugative transfer (Choi SU, Lee CK, Hwang YI, et al. Intergeneric conjugal transfer of plasmid DNA from Escherichia coli to Kitasatospora setae, abafilomycin B1 producer [J]. Arch Microbiol, 2004, 181 (4): 294-298.). These optimized conditions are suitable for the conjugative transfer of a certain Streptomyces, but Streptomyces is a highly differentiated species and has a restriction modification system, so these conditions may not be applicable to other Streptomyces. For some Streptomyces that are difficult to conjugate, despite attempts to optimize the conditions of conjugative transfer, conjugates still cannot grow. There is no universal conjugative transfer condition that can solve the problem of conjugative transfer of most Streptomyces, which leads to the fact that genetic manipulation of Streptomyces is still very difficult. Therefore, how to improve the efficiency and universality of Streptomyces conjugation transfer has become a technical problem that needs to be solved urgently. Summary of the invention

[0007] The technical problem to be solved by the present invention is to overcome the shortcomings of the prior art and provide a class of compounds, germicidin A to D, which can promote the conjugation transfer of Streptomyces. The compounds greatly improve the conjugation transfer rate of Streptomyces, can effectively break through the bottleneck that Streptomyces is difficult to genetically transform through conjugation transfer, and provide a new and effective way for obtaining strains with better activity and higher yield through genetic transformation of Streptomyces.

[0008] The technical solution adopted by the present invention to solve the technical problem is: an application of an α-pyrone compound in promoting conjugation transfer of Streptomyces, wherein the α-pyrone compound can improve the conjugation transfer efficiency of Streptomyces, and the α-pyrone compound has a structure as shown in Formula I:

[0009]

[0010] In formula I, R1 is CH3 or H, R2 is CH3 or H, and R3 is CH3.

[0011] Further, the α-pyrone compounds are germicidin A, germicidin B, germicidin C, and germicidin D, wherein the germicidin A corresponds to formula I in which R1 and R2 are both CH3, and C7 is in S configuration, and its structural formula is shown in formula II; the germicidin B corresponds to formula I in which R1 is CH3 and R2 is H, and its structural formula is shown in formula III; the germicidin C corresponds to formula I in which R1 is H, R2 is CH3, and C7 is in S configuration, and its structural formula is shown in formula IV; the germicidin D corresponds to formula I in which R1 and R2 are both H, and its structural formula is shown in formula V;

[0012]

[0013]

[0014] Furthermore, in the conjugation transfer step of Streptomyces, Streptomyces is used as the recipient bacterium, and a conjugation transfer plate containing α-pyrone compounds is used to carry out conjugation transfer between Streptomyces and the donor bacterium.

[0015] Further, the joint transfer specifically includes the following steps:

[0016] 1) Activation of recipient bacteria and preparation of spore suspension;

[0017] 2) Activation of donor bacteria and preparation of bacterial suspension;

[0018] 3) Preparation of conjugation transfer plates: Add MgCl2 and α-pyrone compounds to ISP4 medium to make Mg 2+ The final concentrations of α-pyrone compounds were 30 mM and 2-10 nM, respectively. Pour the mixture into a plate;

[0019] 4) Conjugation transfer: first, mix the spore suspension of the recipient bacteria and the bacterial suspension of the donor bacteria evenly to obtain a mixed bacterial suspension; then spread the mixed bacterial suspension on the conjugation transfer plate, blow dry and invert it in an incubator for culture; then take out the cultured plate, evenly cover it with Apr antibiotics and TMP antibiotics, blow dry and invert it in an incubator for culture.

[0020] Preferably, the final concentration of the α-pyrone compound is 2 to 5 nM.

[0021] Most preferably, the final concentration of the α-pyrone compound is 2 nM.

[0022] Furthermore, in the step 4), the volume ratio of the spore suspension of the recipient bacteria to the bacterial suspension of the donor bacteria is 100:1; when the mixed bacterial suspension is coated on the conjugation transfer plate, the volume ratio of the mixed bacterial suspension to the culture medium in the conjugation transfer plate is 1:40, and the temperature for inverted culture after coating and drying is 28°C, and the time is 16-20h.

[0023] Furthermore, in step 4), the final concentrations of Apr antibiotic and TMP antibiotic are 50 μg / mL and 100 μg / mL respectively, and the temperature for inverted culture after Apr antibiotic and TMP antibiotic are evenly covered and blown dry is 28° C., and the culture time is 3-5 days.

[0024] Furthermore, the α-pyrone compound is isolated and prepared from the fermentation culture of Streptomyces coelicolor A3(2).

[0025] Furthermore, the method for separating and preparing α-pyrone compounds from the fermentation culture of Streptomyces coelicolor A3(2) comprises the following steps:

[0026] a) preparing seed solution;

[0027] b) fermentation culture to obtain a fermentation culture;

[0028] c) preparing a crude extract: separating the fermentation broth and mycelium of the fermentation culture, extracting the fermentation broth with ethyl acetate, distilling and concentrating it to obtain extract A, extracting the mycelium with acetone, and treating it with ultrasound, collecting the extract, distilling and concentrating it to obtain extract B, combining extracts A and B to obtain extract C, and obtaining a crude extract;

[0029] d) Separation: After the extract C is dissolved in methanol, it is subjected to silica gel column chromatography, and gradient elution is performed using dichloromethane-methanol in a volume ratio of 80:1 to 0:100 as the mobile phase, and the fraction Fr.2 containing compound germicidin AD eluted from the gradient elution with a volume ratio of dichloromethane / methanol of 50:1 is collected; fraction Fr.2 is then subjected to silica gel column chromatography, and dichloromethane / ethyl acetate is used as the eluent, and gradient elution is performed from a volume ratio of 80:1 to 0:100, and fractions Fr.2-2 and Fr.2-3 containing compound germicidin AD eluted from the gradient elution with a volume ratio of dichloromethane / ethyl acetate of 60:1 are collected; fractions Fr.2-2 and Fr.2-3 are then separated by semi-preparative HPLC, and freeze-dried by a centrifugal concentrator to obtain compounds germicidin A, B, C and D.

[0030] Further, the HPLC conditions in step d) are as follows: the chromatographic column is an Agilent Ultimate XB-C18 column; the mobile phase includes phase A and phase B, mobile phase A: 10% (v / v) acetonitrile + 0.08% (v / v) formic acid, the solvent is water; mobile phase B: 90% (v / v) acetonitrile, the solvent is water; the gradient elution program is as follows:

[0031] Time (min) Mobile phase A (%) Mobile phase B (%) 0～20 95→0 5→100 20～23 0→0 100→100 23～25 0→95 100→5 25～30 95→95 5→5

[0032] The detection wavelength was 298 nm and the flow rate was 1 mL / min.

[0033] Furthermore, any one of the germicidin A, germicidin B, germicidin C and germicidin D is used as an active ingredient for promoting conjugative transfer of Streptomyces to prepare a reagent for promoting conjugative transfer of Streptomyces.

[0034] The invention discloses a beneficial effect of using an alpha-pyrone compound in promoting conjugation transfer of Streptomyces: the invention applies an alpha-pyrone compound (germicidin A, germicidin B, germicidin C, germicidin D) to the conjugation transfer of Streptomyces. During the application process, the Streptomyces and a donor bacterium are inoculated into a conjugation transfer plate containing the alpha-pyrone compound. A set of experimental procedures can not only significantly improve the conjugation transfer efficiency of the Streptomyces, but also significantly broaden the applicable host range of the Escherichia coli-Streptomyces conjugation transfer system. The compound has great application value in promoting the conjugation transfer of Streptomyces, provides a new idea and method for developing biological research reagents for improving the conjugation transfer efficiency, can effectively solve the bottleneck problem that Streptomyces and actinomycetes are difficult to genetically modify through conjugation transfer, promotes the development of new antibiotics and improves the activity and yield of existing antibiotics, reduces pharmaceutical costs, thereby improving drug accessibility, and has broad application prospects and universality. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 — is the chromatogram of extract C;

[0036] Figure 2 — is a high-resolution mass spectrometry analysis diagram of compound 1 (germicidin A) and its ultraviolet absorption characteristic diagram;

[0037] Figure 3 — is the high-resolution mass spectrometry analysis diagram of compound 2 / 3 (germicidin C, germicidin B) and its ultraviolet absorption characteristic diagram;

[0038] Figure 4— is the high-resolution mass spectrometry analysis diagram of compound 4 (germicidin D) and its ultraviolet absorption characteristic diagram. DETAILED DESCRIPTION

[0039] The present invention is further described below in conjunction with the accompanying drawings and examples, but these specific implementation schemes do not limit the protection scope of the present invention in any way.

[0040] The strains, culture media and reagents used in the present invention are described as follows:

[0041] Bacterial species-receptor bacteria:

[0042] Streptomyce coelicolor A3(2), which is a strain known in the prior art, such as reference (Aoki Y, Matsumoto D, Kawaide H, Natsume M. Physiological role of germicidins in spore germination and hyphal elongation in Streptomyces coelicolor A3(2). J Antibiot (Tokyo). 2011 Sep; 64(9): 607-11. doi: 10.1038 / ja.2011.59. Epub 2011 Jul 27. PMID: 21792209.);

[0043] Streptomyce coelicolor A3(2)△sco7221 (this strain is a mutant strain, in which the sco7221 gene is knocked out based on the A3(2) strain, wherein sco7221 is a type III PKS gene, which is a germicidin A to D synthesis gene), as shown in reference (Aoki, Y., Matsumoto, D., Kawaide, H. et al. Physiological role of germicidins in spore germination and hyphal elongation in Streptomyces coelicolor A3(2). J Antibiot 64, 607-611 (2011));

[0044] S.avermitilis ATCC31271 is a Streptomyces avermitilis that produces the agricultural antibiotic avermectin. It is a strain in the prior art and was donated by Wuhan University;

[0045] S. hygroscopicus ATCC21705, a Streptomyces hygroscopicus that produces the immunosuppressant rapamycin, is a strain in the prior art and was donated by Wuhan University;

[0046] S. roseosporus NRRL11379 is a strain of Streptomyces roseosporus that produces daptomycin, an important antibiotic for clinical antibacterial infection. It is a strain in the prior art and was donated by Wuhan University.

[0047] S. spectabilis NRRL2494, a species of Streptomyces spectabilis that produces varicose streptococcin, is a gift from Wuhan University.

[0048] Bacteria-Donor bacteria:

[0049] PKC1139 / ET12356 (PUZ8002) strain; this strain is a methyl-deficient Escherichia coli and is also a common tool bacterium, as shown in the reference (Mo Hongbo, Bai Linquan, Wang Shenglan, et al. Construction and application of efficient Escherichia coli-Streptomyces conjugative vector [J]. Journal of Biotechnology, 2004, (05): 662-666.); most Streptomyces can distinguish between their own DNA and foreign DNA through the methyl modification system, so the plasmid that needs to be transferred into Streptomyces must use ET12567 (pUZ8002) to enter the Streptomyces body through conjugation transfer. PKC1139 is a temperature-sensitive plasmid with an Apr resistance gene, and Apr is used for preliminary screening of positive conjugates.

[0050] Culture medium:

[0051] Seed culture medium and fermentation medium: each liter contains 20g starch, 10g glucose, 5g bacteriological peptone, 5g yeast extract, 5g CaCO3, and the balance is water, pH 7.2, 115℃, sterilization for 30min;

[0052] SFM medium: Each liter of medium contains 20g soybean powder, 20g mannitol, 18g agar powder, and the balance is water, pH 7.2, and high-pressure steam sterilization at 115℃ for 30min;

[0053] ISP4 culture medium: each liter of culture medium contains 5 g soluble starch, 0.5 g bacteriological peptone, 1 g (NH4)2SO4, 0.5 g K2HPO4, 0.5 g NaCl, 1 g CaCO3, 0.5 mL trace elements, 18 g agar powder, and the balance is water, pH 7.2-7.4, and sterilized by high-pressure steam at 115°C for 30 min; trace elements: weigh FeSO4·7H2O 2.5 g, ZnSO4·7H2O 0.85 g, CoCl2·6H2O11.85 g, and MnSO4·H2O 0.8 g, make up to 500 mL with ddH2O, and sterilize by high-pressure steam at 115°C for 30 min.

[0054] Screening plate: SFM medium + abra antibiotics, the final concentration of abra antibiotics is 50 μg / ml;

[0055] TSB medium: Each liter of medium contains 30g Tryptic Soy Broth, the balance is water, and is sterilized by high-pressure steam at 121℃ for 15min;

[0056] LB medium: Each liter of culture medium contains 10g Tryptone, 5g Yeast extract, 10g NaCl, and the balance is water, pH 7.2-7.4, and is sterilized by high-pressure steam at 121℃ for 20min.

[0057] Reagents:

[0058] 3M MgCl2: weigh 57 g of anhydrous magnesium chloride, dilute to 200 mL with ddH2O, and sterilize with high pressure steam at 115°C for 30 min;

[0059] Abramycin sulfate antibiotic (Apr): working concentration 50 μg / ml;

[0060] Trimethoprim antibiotic (TMP): working concentration 100 μg / ml;

[0061] Kannamycin Sulfate antibiotic (Kan): working concentration 50 μg / ml;

[0062] Chloramphenicol antibiotic (Chl): working concentration 50 μg / ml

[0063] Germicidin A, germicidin B, germicidin C, and germicidin D used in the following examples and experimental examples were prepared by the following method:

[0064] a) Preparation of seed solution: A single colony of Streptomyce coelicolor A3(2) activated on a culture dish was inoculated into a 300 mL conical culture flask containing 50 mL of seed culture medium and incubated at 28°C and 200 rpm. -1 , culture for 72 hours to obtain seed solution;

[0065] b) Fermentation culture: The seed solution was inoculated with 5% of the inoculum (volume percentage) into a 2L conical culture flask containing 400ml of fermentation medium at 28°C and 200r / min. -1 , shaking culture for 120 h to obtain a fermentation culture;

[0066] c) Preparation of crude extract: The fermentation culture was first carried out at 4000 r·min -1 The mixture was centrifuged for 10 min to obtain a supernatant (fermentation liquid) and mycelium; the supernatant was extracted with ethyl acetate in a ratio of 1:1, and the extraction was performed 3-4 times. The ethyl acetate extract layer was concentrated by distillation to obtain extract A. The mycelium was extracted with acetone and subjected to ultrasonic treatment to allow the solid phase and the liquid phase to fully contact. The extract was collected and concentrated by distillation to obtain extract B. Extracts A and B were combined to obtain extract C (5.92 g). Extract C was a crude extract, and its chromatogram was as shown in FIG. Figure 1 As shown;

[0067] d) Separation: After the extract C was dissolved in methanol, it was subjected to silica gel column chromatography, using dichloromethane-methanol in a volume ratio of 80:1 to 0:100 as the mobile phase for gradient elution, and collecting the fraction Fr.2 (61.8 g) containing the compound germicidin AD eluted by the gradient elution of dichloromethane / methanol in a volume ratio of 50:1; fraction Fr.2 was then subjected to silica gel column chromatography, using dichloromethane / ethyl acetate as the eluent, and gradient eluted from a volume ratio of 80:1 to 0:100, and collecting the fractions Fr.2-2 and Fr.2-3 containing the compound germicidin AD eluted by the gradient elution of dichloromethane / ethyl acetate in a volume ratio of 60:1. Fractions Fr.2-2 and Fr.2-3 were then separated by semi-preparative HPLC, and freeze-dried by a centrifugal concentrator to obtain compounds 1, 2, 3 and 4.

[0068] The HPLC conditions are as follows: the chromatographic column is an Agilent Ultimate XB-C18 column; the mobile phase includes phase A and phase B, mobile phase A: 10% (v / v) acetonitrile + 0.08% (v / v) formic acid, the solvent is water; mobile phase B: 90% (v / v) acetonitrile, the solvent is water; the gradient elution program is as follows:

[0069] Time (min) Mobile phase A (%) Mobile phase B (%) 0～20 95→0 5→100 20～23 0→0 100→100 23～25 0→95 100→5 25～30 95→95 5→5

[0070] The detection wavelength was 298 nm and the flow rate was 1 mL / min.

[0071] The inventors conducted structural identification and analysis on four compounds (Compound 1, Compound 2, Compound 3, and Compound 4) prepared from the fermentation culture of Streptomyce coelicolor A3 (2), and the results are as follows:

[0072] Compound 1: Colorless oil, analyzed by quadrupole-electrostatic field orbital trap high-resolution mass spectrometry liquid-mass spectrometry, the high-resolution mass spectrum analysis of compound 1 and its UV absorption characteristic diagram are shown in Figure 2 As shown, m / z C 11 H 16 O3(measured value [M-Na] + 219.09842, calculated value is 219.09917), the above data are consistent with the m / z and ultraviolet absorption characteristics of the known compound germicidin A in the literature [Aoki Y, Matsumoto D, Kawaide H, Natsume M. Physiological role of germicidins in spore germination and hyphalelongation in Streptomyces coelicolor A3(2). J Antibiot (Tokyo), 2011, ;64:607-11.], and the results suggest that compound 1 and the known compound germicidin A are the same substance, and the structural formula is shown in Formula II.

[0073] Compounds 2 and 3: Colorless oil, analyzed by quadrupole-electrostatic field orbital trap high-resolution mass spectrometry liquid-mass spectrometry, the high-resolution mass spectrometry analysis diagrams of compounds 2 and 3 and their UV absorption characteristic diagrams are shown in Figure 3 As shown, m / z C 10 H 14 O3(measured value [M-Na] + 205.08287, calculated value is 205.08352), the above data are consistent with the m / z and ultraviolet absorption characteristics of the known compound germicidin B in the literature published by Aoki Y, and the results suggest that compounds 2, 3 and the known compound germicidin B / C are the same substance. According to the high-performance liquid chromatogram of the crude extract in the literature, it is determined that the one with greater polarity is germicidin B, and the one with less polarity is germicidin C. It can be determined that compound 2 is germicidin C, and compound 3 is germicidin B, and the structural formulas are shown in formulas III and IV, respectively.

[0074] Compound 4: colorless crystals, analyzed by quadrupole-electrostatic field orbital trap high-resolution mass spectrometry liquid-mass spectrometry, the high-resolution mass spectrum analysis of compound 4 and its ultraviolet absorption characteristic diagram are shown in Figure 4 As shown, m / z C9H 12 O3(measured value [M-Na] + The above data are consistent with the m / z and ultraviolet absorption characteristics of the known compound germicidin D in the literature published by Aoki Y. The results suggest that compound 4 and the known compound germicidin D are the same substance, and the structural formula is shown in Formula V.

[0075] Therefore, it was determined that compound 1, compound 2, compound 3, and compound 4 prepared from the fermentation culture of Streptomyce coelicolor A3 (2) were germicidin A, germicidin C, germicidin B, and germicidin D, respectively. Germicidin A corresponds to formula I in which R1 and R2 are both CH3, and C7 is in S configuration, and its structural formula is shown in formula II; germicidin B corresponds to formula I in which R1 is CH3 and R2 is H, and its structural formula is shown in formula III; germicidin C corresponds to formula I in which R1 is H, R2 is CH3, and C7 is in S configuration, and its structural formula is shown in formula IV; germicidin D corresponds to formula I in which R1 and R2 are both H, and its structural formula is shown in formula V.

[0076] Example 1

[0077] The invention discloses an application of an α-pyrone compound in promoting conjugation transfer of Streptomyces. In the conjugation transfer step of Streptomyces coelicolor A3(2)△sco7221, Streptomyces coelicolor A3(2)△sco7221 is used as a recipient bacterium, and a conjugation transfer plate containing germicidin A is used to carry out conjugation transfer between Streptomyces coelicolor A3(2)△sco7221 and a donor bacterium. The invention specifically comprises the following steps:

[0078] 1) Activation of recipient bacteria and preparation of spore suspension: Streptomyces coelicolor A3(2)△sco7221 was streaked on an SFM medium plate and cultured at 30°C for 3-5 days. After spores grew, the spores were collected with a sterile cotton swab or bamboo stick and suspended in 10-15 mL TSB medium. The spores were vortexed to fully disperse the spores. The spores were filtered with a sterile spore filter, heat-shocked at 55°C for 10 min, and recovered at 30°C for about 1-2 h. The spores were collected by centrifugation and suspended in 3 mL TSB medium. The suspension was diluted to 108 CFU / mL, and obtain the spore suspension of the recipient bacteria for later use;

[0079] 2) Activation of donor bacteria and preparation of bacterial suspension: The donor bacteria PKC1139 / ET12356 (PUZ8002) strain was inoculated in 20 mL of LB medium containing Kan, Chl, and Apr and cultured at 37°C until OD600 was about 0.6-0.8, then washed 2-3 times with LB medium, and finally suspended in 0.5 mL of LB medium to obtain a bacterial suspension of the donor bacteria for later use;

[0080] 3) Preparation of conjugation transfer plates: Add MgCl2 and α-pyrone compounds to ISP4 medium to make Mg 2+ The final concentrations of α-pyrone compounds were 30 mM and 5 nM, respectively, and the mixture was poured onto a plate;

[0081] 4) Conjugation transfer: first, the spore suspension of the recipient bacteria and the bacterial suspension of the donor bacteria were mixed evenly at a volume ratio of 100:1 to obtain a mixed bacterial suspension; then, 0.5 mL of the mixed bacterial suspension was spread on a conjugation transfer plate containing 20 mL of culture medium, blown dry in a clean bench, and inverted in a 28°C incubator for 16-20 h; then, the incubated plate was taken out, and evenly covered with Apr antibiotics at a final concentration of 50 μg / mL and TMP antibiotics at a final concentration of 100 μg / mL, blown dry in a clean bench, and inverted in a 28°C incubator for 3-5 days, the number of colonies was counted, and transformants were randomly selected, and the whole genome was extracted for PCR verification; then the ability of conjugation transfer was evaluated.

[0082] Example 2

[0083] The difference between this embodiment and embodiment 1 is that in the conjugation transfer process between Streptomyces coelicolor A3(2)Δsco7221 and the donor bacteria, a conjugation transfer plate containing germicidin B is used.

[0084] Example 3

[0085] The difference between this embodiment and embodiment 1 is that in the conjugation transfer process between Streptomyces coelicolor A3(2)Δsco7221 and the donor bacteria, a conjugation transfer plate containing germicidin C is used.

[0086] Example 4

[0087] The difference between this embodiment and embodiment 1 is that in the conjugation transfer process between Streptomyces coelicolor A3(2)Δsco7221 and the donor bacteria, a conjugation transfer plate containing germicidin D is used.

[0088] Example 5

[0089] The difference between this embodiment and embodiment 1 is that Streptomyces avermitilis ATCC31271 is used as the recipient bacteria.

[0090] Example 6

[0091] The difference between this embodiment and embodiment 5 is that: in the conjugation transfer process between Streptomyces S. avermitilis ATCC31271 and the donor bacteria, a conjugation transfer plate containing germicidin B is used.

[0092] Example 7

[0093] The difference between this embodiment and embodiment 5 is that: in the conjugation transfer process between Streptomyces avermitilis ATCC31271 and the donor bacteria, a conjugation transfer plate containing germicidin C is used.

[0094] Example 8

[0095] The difference between this embodiment and embodiment 5 is that in the conjugation transfer process between Streptomyces avermitilis ATCC31271 and the donor bacteria, a conjugation transfer plate containing germicidin D is used.

[0096] Example 9

[0097] The difference between this embodiment and embodiment 1 is that Streptomyces hygroscopicus ATCC21705 is used as the recipient bacteria.

[0098] Example 10

[0099] The difference between this embodiment and embodiment 9 is that in the conjugation transfer process between Streptomyces S. hygroscopicus ATCC21705 and the donor bacteria, a conjugation transfer plate containing germicidin B is used.

[0100] Embodiment 11

[0101] The difference between this embodiment and embodiment 9 is that in the conjugation transfer process between Streptomyces S. hygroscopicus ATCC21705 and the donor bacteria, a conjugation transfer plate containing germicidin C is used.

[0102] Example 12

[0103] The difference between this embodiment and embodiment 9 is that in the conjugation transfer process between Streptomyces S. hygroscopicus ATCC21705 and the donor bacteria, a conjugation transfer plate containing germicidin D is used.

[0104] Example 13

[0105] The difference between this embodiment and embodiment 1 is that Streptomyces roseosporus NRRL11379 is used as the receptor bacteria.

[0106] Embodiment 14

[0107] The difference between this embodiment and embodiment 13 is that in the conjugation transfer process between Streptomyces S. roseosporus NRRL11379 and the donor bacteria, a conjugation transfer plate containing germicidin B is used.

[0108] Embodiment 15

[0109] The difference between this embodiment and embodiment 13 is that: in the conjugation transfer process between Streptomyces S. roseosporus NRRL11379 and the donor bacteria, a conjugation transfer plate containing germicidin C is used.

[0110] Example 16

[0111] The difference between this embodiment and embodiment 13 is that in the conjugation transfer process between Streptomyces S. roseosporus NRRL11379 and the donor bacteria, a conjugation transfer plate containing germicidin D is used.

[0112] Embodiment 17

[0113] The difference between this embodiment and embodiment 1 is that Streptomyces spectabilis NRRL2494 is used as the recipient bacteria.

[0114] Embodiment 18

[0115] The difference between this embodiment and embodiment 17 is that in the conjugation transfer process between Streptomyces spectabilis NRRL2494 and the donor bacteria, a conjugation transfer plate containing germicidin B is used.

[0116] Embodiment 19

[0117] The difference between this embodiment and embodiment 17 is that in the conjugation transfer process between Streptomyces spectabilis NRRL2494 and the donor bacteria, a conjugation transfer plate containing germicidin C is used.

[0118] Embodiment 20

[0119] The difference between this example and Example 17 is that during the conjugation transfer process between Streptomyces spectabilis NRRL2494 and the donor bacteria, a conjugation transfer plate containing germicidin D was used.

[0120] The differences in the recipient bacteria and α-pyrone compounds used in the conjugation transfer plates corresponding to the above Examples 1-20 are shown in Table 1.

[0121] The present invention measures the conjugation transfer efficiency of the Streptomyces corresponding to Examples 1-20, and the specific detection method is: transfer the conjugate to a screening plate, culture for 3-4 days, extract the genome after spores grow, and randomly perform PCR verification. The conjugation transfer efficiency is equal to the number of conjugative transfers (cfu) divided by the number of recipient bacteria (cfu). Control groups 1-5 are conjugation transfer plates without adding α-pyrone compounds and use Streptomyces coelicolor A3 (2) △ ​​sco7221, S.avermitilisATCC31271, S.hygroscopicus ATCC21705, S.roseosporus NRRL11379, S.spectabilisNRRL2494 as recipient bacteria for conjugation transfer. The results of the Streptomyces conjugation transfer efficiency are shown in Table 1:

[0122] Table 1 Acceptor bacteria and α-pyrone compounds used in Examples 1-20 and Control Groups 1-6 and the corresponding Streptomyces conjugation transfer efficiency

[0123]

[0124]

[0125] From the results of control group 1 and examples 1-4 in Table 1, it can be seen that in the conjugation transfer process of Streptomyces coelicolor A3(2)△sco7221 and pKCSG332 / ET12356(PUZ8002), the conjugation transfer efficiency of control group 1 was significantly lower than that of examples 1-4, and its corresponding conjugation transfer efficiency was lower than that of wild-type Streptomyces coelicolor A3(2) (the conjugation transfer efficiency without adding α-pyrone compounds was 86×10 -6 ), indicating that, in the conjugation transfer process of Streptomyces coelicolor A3(2)△sco7221, the conjugation transfer experiment can be greatly promoted by using a conjugation transfer plate containing germicidins A to D, and that germicidin A has the most significant effect in promoting the conjugation transfer of Streptomyces.

[0126] To illustrate the universality of germicidin AD promoting conjugative transfer of Streptomyces, Examples 5-20 studied a variety of Streptomyces that produce important antibiotics but are difficult to genetically modify by conjugative transfer, including Streptomyces avermitilis ATCC31271 that produces the agricultural antibiotic avermectin (Examples 5-8), Streptomyces hygroscopicus ATCC21705 that produces the immunosuppressant rapamycin (Examples 9-12), Streptomyces roseosporus NRRL11379 that produces daptomycin, an important antibiotic for clinical anti-bacterial infections (Examples 13-16), and Streptomyces spectabilis NRRL2494 that produces varicose streptococcus (Examples 17-20). The results of control groups 2-5 and examples 5-20 indicate that germicidin AD can improve the conjugation transfer efficiency of the corresponding Streptomyces, and can promote the conjugation transfer of a variety of Streptomyces that produce important antibiotics and are difficult to transform through conjugation transfer, indicating that germicidin AD has a universal technical effect in the conjugation transfer process of Streptomyces, and can be applied to the conjugation transfer of a variety of Streptomyces.

[0127] Experimental example

[0128] This experimental example refers to the application of Example 1 to study the effect of different concentrations (0, 2nM, 5nM, 10nM) of germicidin AD on the conjugative transfer efficiency of Streptomyces A3(2)△sco7221. The results are shown in Table 2:

[0129] Table 2 Effects of different α-pyrone compound concentrations on the conjugation transfer efficiency of Streptomyces A3(2)△sco7221

[0130]

[0131]

[0132] As can be seen from the above table, with the increase in the concentration of α-pyrone compounds (germicidin AD), the conjugation transfer efficiency of Streptomyces showed a trend of first increasing and then decreasing, and the most preferred concentration was 2 nM.

[0133] It should be noted that germicidin AD are α-pyrone compounds existing in the prior art, and in addition to being prepared by the method of the present invention, they can also be obtained by direct purchase.

[0134] The above description is only a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Although the present invention has been disclosed as a preferred embodiment as above, it is not intended to limit the present invention. Any technician familiar with the profession can make some changes or modifications to equivalent embodiments of equivalent changes using the technical contents disclosed above without departing from the scope of the technical solution of the present invention. However, any simple modification, equivalent change and modification made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention still falls within the scope of the technical solution of the present invention.

Claims

1. An application of an α-pyrone compound in promoting conjugation transfer of Streptomyces, characterized in that: The α-pyrone compound can improve the conjugation transfer efficiency of Streptomyces, and the α-pyrone compound has a structure as shown in Formula I: In formula I, R1 is CH3 or H, R2 is CH3 or H, and R3 is CH3.

2. The use of an α-pyrone compound in promoting conjugation transfer of Streptomyces as claimed in claim 1, characterized in that: The α-pyrone compounds are germicidin A, germicidin B, germicidin C, and germicidin D. In the germicidin A corresponding to Formula I, R1 and R2 are both CH3, and C7 is in S configuration; in the germicidin B corresponding to Formula I, R1 is CH3 and R2 is H; in the germicidin C corresponding to Formula I, R1 is H, R2 is CH3, and C7 is in S configuration; in the germicidin D corresponding to Formula I, R1 and R2 are both H.

3. The use of an α-pyrone compound in promoting conjugation transfer of Streptomyces as claimed in claim 1, characterized in that: In the conjugation transfer step of Streptomyces, Streptomyces is used as a recipient bacterium, and a conjugation transfer plate containing α-pyrone compounds is used to carry out conjugation transfer between Streptomyces and donor bacteria.

4. The use of an α-pyrone compound in promoting conjugation transfer of Streptomyces as claimed in claim 3, characterized in that: The joint transfer specifically comprises the following steps: 1) Activation of recipient bacteria and preparation of spore suspension; 2) Activation of donor bacteria and preparation of bacterial suspension; 3) Preparation of conjugation transfer plates: Add MgCl2 and α-pyrone compounds to ISP4 medium to make Mg 2+ The final concentrations of α-pyrone compounds were 30 mM and 2-10 nM, respectively. Mix well and pour into a plate; 4) Conjugation transfer: first, mix the spore suspension of the recipient bacteria and the bacterial suspension of the donor bacteria evenly to obtain a mixed bacterial suspension; then spread the mixed bacterial suspension on the conjugation transfer plate, blow dry and invert it in an incubator for culture; then take out the cultured plate, evenly cover it with Apr antibiotics and TMP antibiotics, blow dry and invert it in an incubator for culture.

5. The use of an α-pyrone compound in promoting conjugation transfer of Streptomyces as claimed in claim 4, characterized in that: In the step 4), the volume ratio of the spore suspension of the recipient bacteria to the bacterial suspension of the donor bacteria is 100:1; when the mixed bacterial suspension is coated on the conjugation transfer plate, the volume ratio of the mixed bacterial suspension to the culture medium in the conjugation transfer plate is 1:40, and the temperature for inverted culture after coating and drying is 28°C, and the time is 16-20h.

6. The use of an α-pyrone compound in promoting conjugation transfer of Streptomyces as claimed in claim 4, characterized in that: In the step 4), the final concentrations of Apr antibiotic and TMP antibiotic are 50 μg / mL and 100 μg / mL respectively, and after Apr antibiotic and TMP antibiotic are evenly covered and blown dry, the temperature for inverted culture is 28° C. and the culture time is 3-5 days.

7. The use of an α-pyrone compound in promoting conjugation transfer of Streptomyces as claimed in claim 2, characterized in that: The α-pyrone compound is separated and prepared from the fermentation culture of Streptomyces coelicolor A3 (2).

8. The use of an α-pyrone compound in promoting conjugation transfer of Streptomyces as claimed in claim 7, characterized in that: The method for separating and preparing α-pyrone compounds from the fermentation culture of Streptomyces coelicolor A3(2) comprises the following steps: a) preparing seed solution; b) fermentation culture to obtain a fermentation culture; c) preparing a crude extract: separating the fermentation broth and mycelium of the fermentation culture, extracting the fermentation broth with ethyl acetate, distilling and concentrating it to obtain extract A, extracting the mycelium with acetone, and treating it with ultrasound, collecting the extract, distilling and concentrating it to obtain extract B, combining extracts A and B to obtain extract C, and obtaining a crude extract; d) Separation: After the extract C is dissolved in methanol, it is subjected to silica gel column chromatography, and gradient elution is performed using dichloromethane-methanol in a volume ratio of 80:1 to 0:100 as the mobile phase, and the fraction Fr.2 containing compound germicidin AD eluted from the gradient elution with a volume ratio of dichloromethane / methanol of 50:1 is collected; fraction Fr.2 is then subjected to silica gel column chromatography, and dichloromethane / ethyl acetate is used as the eluent, and gradient elution is performed from a volume ratio of 80:1 to 0:100, and fractions Fr.2-2 and Fr.2-3 containing compound germicidin AD eluted from the gradient elution with a volume ratio of dichloromethane / ethyl acetate of 60:1 are collected; fractions Fr.2-2 and Fr.2-3 are then separated by semi-preparative HPLC, and freeze-dried by a centrifugal concentrator to obtain compounds germicidin A, B, C and D.

9. The use of an α-pyrone compound in promoting conjugation transfer of Streptomyces as claimed in claim 8, characterized in that: The HPLC conditions in step d) are as follows: the chromatographic column is an Agilent Ultimate XB-C18 column; the mobile phase includes phase A and phase B, mobile phase A: 10% (v / v) acetonitrile + 0.08% (v / v) formic acid, the solvent is water; mobile phase B: 90% (v / v) acetonitrile, the solvent is water; the gradient elution program is as follows: The detection wavelength was 298 nm and the flow rate was 1 mL / min.

10. The use of an α-pyrone compound in promoting conjugation transfer of Streptomyces as claimed in claim 2, characterized in that: Any one of the germicidin A, germicidin B, germicidin C and germicidin D is used as an active ingredient for promoting conjugation transfer of Streptomyces to prepare a reagent for promoting conjugation transfer of Streptomyces.

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