A short rod bacterium tyropeptide derivative and application thereof

Abstract

The application discloses a short bacillus tyrocidine derivative and application thereof. The derivative is a secondary metabolite natural product produced by Brevibacillus brevis, and a structural formula of the derivative is as follows: cyclo- (O D FPL D FNQYVOL. The derivative has good bacteriostatic activity on various plant pathogenic fungi, and has practical application value.

Description

Technical Field

[0001] This invention belongs to the field of biology, and more specifically, relates to a derivative of short bacillus casein and its applications. Background Technology

[0002] In 1939, Dubos et al. discovered that crude extracts of peptides from the fermentation broth of *Bacillus shorthair* possessed antibiotic properties and named them tyrothricin. In subsequent studies, Dubos named the neutral fraction co-precipitated with ethanol gramicidin and the alkaline fraction tyrocidine. Even today, tyrothricin (a mixture of gramicidin and tyrocidine) is still used to treat skin and throat infections, and the accumulated resistance of target pathogens to tyrothricin is negligible. Against the backdrop of an increasingly severe public health crisis caused by multidrug-resistant (MDR) pathogens, this phenomenon has attracted close attention from researchers.

[0003] Tyrocidine is a collective term for a class of cyclic decapeptide compounds with similar skeletal structures, with tyrocidine A, tyrocidine B, and tyrocidine C being the main components. Tyrocidine can inhibit important pathogens in animals, plants, and humans, such as Listeria monocytogenes, Aspergillus fumigatus, Candida albicans, Fusarium solani, Botrytis cinerea, and Plasmodium falciparum, indicating that tyrocidine has good application potential and development prospects in the fields of agricultural antibiotics and biomedicine. Studies have shown that the antibacterial activity of tyrocidine is closely related to its primary structure: (1) polar amino acids and positively charged amino acids increase the amphiphilicity and electrostatic adsorption capacity of tyrocidine, thereby improving its antibacterial activity; (2) aromatic amino acids in the peptide chain increase the surface area of ​​the side chain, which helps it to bind to the glycosyl groups on the cell membrane of the target pathogen, thereby disrupting the integrity of the cell membrane.

[0004] In conclusion, discovering new tyrocidine derivatives can not only enrich our understanding of their structural and functional diversity, but also provide high-quality candidate compound resources for the development of novel agricultural fungicides. Summary of the Invention

[0005] This invention first utilizes Red-ET homologous recombination technology to knock out the biosynthesis gene clusters (BGCs) of antibacterial active substances in wild-type Bacillus brevis B011. By comparing the antibacterial activity of the gene-knockout strain against target bacteria, the biosynthetic gene clusters of the antibacterial active substances are identified. Second, the antibacterial active substances of wild-type B011 and the gene-knockout strain are extracted with ethanol, and the differences in fermentation spectra of each strain are compared using high-performance liquid chromatography (HPLC) to determine the differential chromatographic peaks and their retention times. Third, the primary structure of the new derivative tyrocidine F is resolved using HPLC-MS / MS. Tyrocidine F is synthesized in vitro in a one-pot method, and the compound is purified by HPLC. Plate contrast experiments show that this derivative has good antibacterial activity against a variety of plant pathogenic fungi. This completes the invention.

[0006] This invention first provides a derivative of short bacillus casein, with the chemical formula C. 63 H 89 N 13 O 13 The structural formula is: cyclo-( D FPL D FNQYVOL), where F = Phe, P = Pro, L = Leu, N = Asn, Q = Gln, Y = Tyr, V = Val, O = Orn. ​​The superscript D indicates a D-type amino acid, with an exact molecular weight of 1235.6703 and a mass-to-charge ratio of [M+H]. + The value is 1236.6776. Comprehensive analysis revealed that tyrocidine F is a novel derivative of tyrocidine A. Its structural formula can also be represented as:

[0007] .

[0008] The present invention therefore provides a method for preparing the derivative based on fermentation liquor, characterized in that it comprises the following steps:

[0009] S1 was obtained by culturing a mutant strain of *Brevibacillus brevis* with double knockout of the ede and gra genes to obtain fermentation broth;

[0010] S2. Add the fermentation broth to anhydrous ethanol, vortex to mix, and then let stand.

[0011] S3 was centrifuged at 8000-12000 r / min for 5-15 min and the supernatant was collected.

[0012] The powder is obtained after rotary vacuum drying with S4.

[0013] Optionally, it also includes S5: redissolving the powder in anhydrous ethanol and filtering it through a 0.22 μm filter for later use.

[0014] Specifically,

[0015] The culture medium used in S1 was NB liquid medium (10 g / L peptone, 3 g / L beef extract, 5 g / L NaCl, 10 g / L glucose); the culture conditions were 30℃, 180 r / min shaking culture for 72 h; the starting strain of Bacillus brevis was Bacillus brevis B011.

[0016] The S2 settling time is 20-28 h; the centrifugation conditions are 10000 r / min for 10 min to collect the supernatant; the vacuum drying is rotary vacuum drying.

[0017] The present invention also provides an in vitro preparation method for the aforementioned derivatives, characterized in that it comprises the following steps:

[0018] The derivative was obtained by using an enzymatic reaction component consisting of three enzymes, TycA, TycB and TycC, with amino acids L-Phe, L-Pro, L-Asn, L-Gln, L-Tyr, L-Val, L-Orn, L-Leu and L-Trp as substrates through a catalytic reaction.

[0019] Specifically, the reaction was carried out in Tris buffer containing 5 mM MgCl2, 100 mM NaCl, 1 mg / mL BSA, 4 mM TCEP and 50 mM ATP.

[0020] More specifically, the concentrations of each enzyme in the enzyme-catalyzed reaction component are 2–10 μM, and the substrate concentration is 1–5 mM.

[0021] The reaction was initiated by the addition of ATP and incubated overnight in a 37°C water bath;

[0022] Optionally, methanol is then added to terminate the reaction, followed by vortexing and centrifugation at 10,000-12,000 r / min for 5-15 min to remove precipitated proteins; the supernatant is then vacuum frozen and concentrated.

[0023] The present invention also provides the use of the derivatives in the preparation of antibacterial drugs or agricultural fungicides.

[0024] Specifically, the targets of antibacterial or bactericidal activity are Phytophthora parasitica var. nicotianae, Alternaria alternata, Phytophthora capsici, Colletotrichum scovillei, Fusarium graminearum, and Magnaphorthe oryzae.

[0025] The derivative obtained in this invention exhibits good antibacterial activity against various plant pathogenic fungi, demonstrating practical application value. This invention also completes the in vitro synthesis of this derivative, further facilitating its subsequent applications. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0027] Figure 1 This is a colony PCR double primer identification diagram of the Bacillus shortbread mutant strain obtained in this invention; M: DL5000 Marker; lanes 1-12 are transformants.

[0028] Figure 2 Wild-type B011 and various mutant strains were compared against pathogenic fungi on petri dishes. Among them, single knockout strains 011Δtyc, 011Δgra, and 011Δede, and double knockout strains 011Δede-tyc and 011Δede-gra; A was *Phytophthora parasitica* var. *nicotianae*, B was *Alternaria alternata*, C was *Phytophthora capsici*, D was *Colletotrichum scovillei*, E was *Fusarium graminearum*, and F was *Magnaporthe oryzae*.

[0029] Figure 3Chromatographic images of fermentation broth supernatant samples from wild-type B011 and various mutant strains. The mutant strain 011Δede-tyc, which had its tyrocidine gene cluster knocked out, lost the characteristic peak of tyrocidine; while the characteristic peak of tyrocidine was retained in the mutant strains 011Δede and 011Δede-gra, which did not have the tyrocidine gene cluster knocked out.

[0030] Figure 4 The diagram shows the in vitro total synthesis of tyrocidine F. A is an SDS-PAGE electrophoresis image of the purified TycA (128.8 kDa), TycB (408.9 kDa), and TycC (731.1 kDa) proteins; B is an HPLC chromatogram of the one-pot in vitro reaction; C is a structural diagram of tyrocidine; D is a diagram of the tyrocidine biosynthetic pathway; and E shows the tyrocidine biosynthetic gene cluster. Detailed Implementation

[0031] The technical solutions in the embodiments of the present invention will be clearly and completely described below.

[0032] Previous experiments revealed that *Bacillus brevis* B011 (NCBI accession number: NZ_CP041767.1) exhibits good antibacterial activity against various plant pathogenic fungi, but the specific antibacterial active substances it secretes were unknown. This invention employs reverse genetics to knock out the biosynthetic gene cluster of the antibacterial active substance, combined with antibacterial activity evaluation and liquid chromatography-mass spectrometry analysis, to clarify the chemical structure and antibacterial spectrum of its antibacterial active substance.

[0033] The biological materials used in the examples were sourced from the following sources: wild-type Bacillus shortbread B011 (see patent ZL201510066359): provided by the College of Plant Protection, Hunan Agricultural University; vector pE194 (see patent ZL202110329266.0), i.e. pBR322-ErmB-194ori, was preserved by the College of Plant Protection, Hunan Agricultural University / Hunan Provincial Institute of Microbiology; resistance gene Apra R Contains the apramycin resistance gene. R The plasmid pSET152 was preserved by the College of Plant Protection, Hunan Agricultural University / Hunan Institute of Microbiology.

[0034] Six plant pathogenic fungi—*Phytophthora parasitica var. nicotianae*, *Alternaria alternata*, *Phytophthora capsici*, *Cytospora ambiens*, *Colletotrichum scovillei*, *Colletotrichum gloeosporioides*, *Fusarium graminearum*, and *Magnaporthe oryzae*—were preserved by the Hunan Provincial Institute of Microbiology.

[0035] Restriction endonucleases BstZ17I and SmaI were purchased from NEB.

[0036] Example 1: Construction of the B011 mutant strain that synthesizes only tyrocidine F

[0037] Online analysis using antiSMASH 7.0 (https: / / antismash.secondarymetabolites.org / #! / start) identified 11 potential biosynthetic gene clusters from the B011 genome (NZ_CP041767). Clusters 3, 4, and 11 synthesize non-ribosomal peptides (NRPs); cluster 7 synthesizes polyketides (PKs); clusters 5, 6, and 9 synthesize PKS-NRP hybrid peptides; cluster 1 synthesizes siderophores; clusters 2 and 10 synthesize bacteriocins; and cluster 8 synthesizes terpenoids (Table 1). The knockout gene clusters 3 (responsible for tyrocidine synthesis), 4 (responsible for gramicidin synthesis), and 5 (responsible for edeine synthesis) are located in the genome at 2,897,423 - 2,970,327 nt, 3,019,366 - 3,086,604 nt, and 3,305,369 - 3,379,817 nt, respectively.

[0038] Table 1. Biosynthetic gene clusters of antibacterial active substances in the B011 genome

[0039] ;

[0040] Using wild-type Bacillus brevis B011 (NCBI accession number: NZ_CP041767.1) as the starting strain, knockout plasmids pE194-ede-ko-HAF-aprA-HAR (responsible for knocking out the gene cluster responsible for synthesizing edeine) and pE194-gra-ko-HAF-aprA-HAR (responsible for knocking out the gene cluster responsible for synthesizing gramicidin) were obtained using Red / ET homologous recombination technology. First, the pE194-ede-ko-HAF-aprA-HAR plasmid was transformed into wild-type B011 via electroporation, and the results were identified by PCR. Figure 1 The single knockout strain B011Δede was obtained. Then, the pE194-gra-ko-HAF-aprA-HAR plasmid was electroporated into the single knockout strain B011Δede, and the result was confirmed by PCR. Figure 1 The double knockout strain, namely B011Δede-gra, was obtained.

[0041] Subsequent experiments showed that the 011Δede-tyc knockout strain completely lost its antibacterial activity, indicating that Bacillus brevis B011 can synthesize tyrocidine and edeine. The tyrocidine and edeine products also correspond to the gene clusters. Therefore, a mutant strain B011Δede-gra, which synthesizes only tyrocidine, was successfully obtained.

[0042] Example 2: Comparison of antibacterial abilities between wild-type B011 and gene knockout strains

[0043] Mycelial blocks of *Phytophthora parasitica* var. *nicotianae*, *Alternaria alternata*, *Phytophthora capsici*, *Colletotrichum scovillei*, *Fusarium graminearum*, and *Magnaporthe oryzae* were inoculated into the center of NB plates (10 g / L peptone, 3 g / L beef extract, 5 g / L NaCl, 10 g / L glucose, 18 g agar). Single colonies of wild-type B011 and each mutant strain were inoculated at 2 cm depth onto the mycelial blocks, with three replicates. The plates were incubated at 30°C for 96 h, and the antibacterial activity of each strain was observed and compared.

[0044] The results of plate culture showed that the mutant strains 011Δtyc, 011Δgra, and 011Δede, which individually knocked out gene clusters 3 (tyc BGC), 4 (gra BGC), and 5 (ede BGC), exhibited good inhibitory activity against all three pathogens, as did WT (wild-type B011). The double-knockout mutant 011Δede-gra showed comparable antifungal activity against *C. ambiens*, the causal agent of pear rot, to WT, but significantly reduced antifungal activity against *G. piperatum* and *F. oxysprum*, indicating that gene clusters 4 and 5 may have synthesized gramicidin and edeine. The double-knockout mutant 011Δede-tyc completely lost its antifungal activity against all three pathogens, indicating that B011 synthesized tyrocidine and edeine, two antifungal active substances, on NB medium. Figure 2 Among them, the tyrocidine target peak was lost in the B011011Δtyc knockout strain; while the tyroc target peak was still present in the strains without tyc BGC knockout.

[0045] Six pathogenic fungi were activated and inoculated. Single colonies of the wild-type B011 and each mutant strain were inoculated into NB liquid medium for fermentation. After 72 h, the fermentation broth was filtered through a 0.22 μm filter for sterilization. Wells were punched 2 cm from the mycelial cake, and 100 μL of sterile fermentation broth was added to each well. The culture was then carried out as described above. Antimicrobial activity was observed and compared after 96 h. The results were completely consistent with the previous results. Figure 2 Among them, A is Phytophthora parasitica var. nicotianae, B is Alternaria alternata, C is Phytophthora capsici, D is Colletotrichum scovillei, E is Fusarium graminearum, and F is Magnaphorthe oryzae, indicating that B011 synthesized two antibacterial active substances, tyrocidine and edeine, on NB medium.

[0046] Example 3: Preparation of tyrocidine F based on fermentation broth

[0047] Wild-type B011 and the double knockout strain 011Δede-gra were inoculated into 200 mL of NB liquid medium (10 g / L peptone, 3 g / L beef extract, 5 g / L NaCl, 10 g / L glucose) and cultured at 30 °C with shaking at 180 r / min for 72 h. The fermentation broth was then added to 1000 mL of anhydrous ethanol and vortexed to mix, followed by standing for 24 h. The supernatant was collected by centrifugation at 10000 r / min for 10 min, dried under rotary vacuum, and the powder was redissolved in 8 mL of anhydrous ethanol and filtered through a 0.22 μm filter for later use.

[0048] The filtrates were analyzed by high-performance liquid chromatography (HPLC) using an Agilent 1260 Infinity II instrument and a Novapak HR C column. 18 (4.6 × 250 mm), mobile phase A is 0.1% trifluoroacetic acid, mobile phase B is 90% acetonitrile and 10% A, UV absorption wavelength is 280 nm, flow rate is 1 ml / min, linear gradient is 50% A to 80% B.

[0049] Analysis results as follows Figure 3 As shown, the retention times of the characteristic tyrocidine peaks in the ethanol extracts of 011Δede and 011Δede-gra were consistent, at 8.489 min, 16.640 min, 18.520 min, 20.236 min, and 22.604 min, respectively; while the mutant strain 011Δede-tyc, which had the tyrocidine gene cluster knocked out, lost these four characteristic peaks. Therefore, it is speculated that the double knockout strain 011Δede-gra can synthesize the antibacterial active substance tyrocidine F.

[0050] Example 4: In vitro synthesis of Tyrocidine F

[0051] Using purified TycA (128.8 kDa), TycB (408.9 kDa), and TycC (731.1 kDa) synthases ( Figure 4 A). Figure 4 Image A shows the SDS-PAGE electrophoresis image of purified TycA (128.8 kDa), TycB (408.9 kDa), and TycC (731.1 kDa) proteins. Tyrocidine F was synthesized in vitro via a one-pot enzymatic method under defined substrate conditions.

[0052] The reaction was carried out in Tris buffer (100 mM, pH 8.0) containing 5 mM MgCl2, 100 mM NaCl, 1 mg / mL BSA, 4 mM TCEP, and 50 mM ATP. The enzymatic reaction components included TycA (5 μM), TycB (5 μM), and TycC (5 μM). Each amino acid, L-Phe, L-Pro, L-Asn, L-Gln, L-Tyr, L-Val, L-Orn, L-Leu, and L-Trp, was added at a final concentration of 2 mM. The reaction was initiated by adding ATP and incubated overnight at 37°C. After the reaction was complete, 1.0 mL of methanol was added to every 200 μL of mixture to terminate the reaction, followed by vortexing and centrifugation at 12,000 r / min for 10 min to remove precipitated proteins. The supernatant was then vacuum frozen and concentrated to a final volume of 200 μL.

[0053] The tyrocidine F synthesized using this one-pot reaction system can be directly identified by subsequent secondary mass spectrometry. In the enzymatic one-pot reaction system, after adding essential amino acid substrates, cofactors, and enzymes for co-incubation, the reaction product was detected by HPLC-MS. A characteristic peak appeared at a retention time of 7.1 min, which is actually [M+H]. + The value was 1236.6782; when the reaction system lacked ATP, related proteins, or used heat-denatured proteins, no characteristic peak of tyrocidine F was detected (negative control). Figure 4 Figure B is the HPLC chromatogram after the one-pot in vitro reaction.

[0054] The structural formula of tyrocidine F is presumed to be [cyclo(DPhe 1 -Pro 2 -Leu 3 -DPhe 4 -Asn 5 -Gln 6 -Tyr 7 -Val 8 -Orn 9 -Leu 10 )]( Figure 4 For the structural identification of C, please refer to Example 5. Simultaneously, the enzymatic reaction product also exhibited antibacterial activity against the pathogens Cytospora ambiens (pear tree rot pathogen) and Gloeosporium piperatum (pepper red anthracnose). Figure 4 (D) In ​​summary, this indicates that the characteristic peak is the actual product synthesized by the tyrocidine biosynthesis gene cluster (E in Figure 4), and the product is named tyrocidine F.

[0055] Example 5: Identification of tyrocidine F

[0056] The ethanol extracts from Examples 3 and 4, and their in vitro synthetic product tyrocidine F, were analyzed by UPLC-MS / MS. The instrument used was a Waters Q-TOF Ultima mass spectrometer coupled to a Waters Acquity ultra-high performance liquid chromatograph, and the column was a Waters UPLC BEH C. 18 (2.1 × 50 mm, 1.7 μm spherical particles, Millipore-Waters, La Jolla, USA), using a gradient elution from 0.1% trifluoroacetic acid (A) to acetonitrile (B) (100% A for 30 s, 30 s to 60 s from 0% to 30% B, 1 to 10 min from 30% to 60% B, 10 to 15 min from 60% to 80% B, flow rate 300 μl / min), followed by column reequilibration to initial conditions.

[0057] Electrospray ionization mass spectrometry (ESMS) was performed using a Waters Q-Tof Ultima mass spectrometer. The sample loading volume was 10 μL, the solvent was 0.1% formic acid::50% acetonitrile, the flow rate was 300 μL / min, the capillary voltage was 3.5 kV, the source temperature was set to 100 °C, and the cone voltage was 35 V. Data acquisition was performed in positive mode, scanning the second analyzer (MS2), acquiring mass-to-charge ratios (m / z) ranging from 100 to 1999.

[0058] MS / MS analysis, based on [M+H] + The values ​​and fragment ion counts confirmed the primary structure of tyrocidine F, with mass deviations all within 5 ppm of the theoretical values. (Corresponding to Leu...) 3 Characteristic ions of the residues were observed at m / z 211.1430, 375.2016, 472.2554, 732.4039, 976.5231, and 1089.6053, and neutral loss fragments of daughter ions of NH3 (m / z 1219.6558) and H2O (m / z 1218.6626) were also found. The derived peptide sequence is: cyclo(DPhe 1 -Pro 2 -Leu 3 -DPhe 4 -Asn 5 -Gln 6 -Tyr 7 -Val 8 -Orn 9 -Leu 10The chemical formula of this compound is C. 63 H 89 N 13 O 13 The mass-to-charge ratio m / z is 1237.6776 [M+H] + The precise molecular weight is 1235.6703. Compared with the previously reported tyrocidine A, the third amino acid residue is replaced with Leu. Further comparison using the SciFinder database confirmed that it is a novel derivative of tyrocidine A not previously reported in the literature. The new derivative is named tyrocidineF, and its primary structure is as follows. Figure 4 As shown in C.

Claims

1. A derivative of short bacillus casein, having a cyclic decapeptide compound with the chemical formula C1. 63 H 89 N 13 O 13 The general structural formula is: cyclo-( D FPL D FNQYVOL), where F=Phe, P=Pro, L=Leu, N=Asn, Q=Gln, Y=Tyr, V=Val, O=Orn; the superscript D indicates D-type amino acid.

2. A method for preparing the derivative of claim 1 based on fermentation liquid, characterized in that, It includes the following steps: S1 was obtained by culturing a mutant strain of *Brevibacillus brevis* with double knockout of the ede and gra genes to obtain fermentation broth; S2. Add the fermentation broth to anhydrous ethanol, vortex to mix, and then let stand. S3 was centrifuged at 8000-12000 r / min for 5-15 min and the supernatant was collected. The powder is obtained after rotary vacuum drying with S4.

3. The method as described in claim 2, characterized in that, It also includes S5: redissolve the powder in anhydrous ethanol and filter it through a 0.22μm filter for later use.

4. The method as described in claim 2, characterized in that, The culture medium used in S1 was NB liquid medium (10 g / L peptone, 3 g / L beef extract, 5 g / L NaCl, 10 g / L glucose); the culture conditions were 30℃, 180 r / min shaking culture for 72 h; the starting strain of Bacillus brevis was Bacillus brevis B011. The S2 settling time is 20-28 h; the centrifugation conditions are 10000 r / min for 10 min to collect the supernatant; the vacuum drying is rotary vacuum drying.

5. A method for in vitro preparation of the derivative as described in claim 1, characterized in that, It includes the following steps: The derivative was obtained by using an enzymatic reaction component comprising three enzymes, TycA, TycB, and TycC, with L-Phe, L-Pro, L-Asn, L-Gln, L-Tyr, L-Val, L-Orn, L-Leu, and L-Trp as substrates through a catalytic reaction.

6. The in vitro preparation method according to claim 5, characterized in that, The reaction was carried out in Tris buffer containing 5 mM MgCl2, 100 mM NaCl, 1 mg / mL BSA, 4 mM TCEP and 50 mM ATP.

7. The in vitro preparation method according to claim 6, characterized in that, The concentrations of each enzyme in the enzymatic reaction component are 2-10 μM, and the concentration of each amino acid substrate is 1-5 mM, preferably 2 mM.

8. The in vitro preparation method according to claim 6, characterized in that, The reaction was initiated by the addition of ATP and incubated overnight in a 37°C water bath; Optionally, methanol is then added to terminate the reaction, followed by vortexing and centrifugation at 10,000-12,000 r / min for 5-15 min to remove precipitated proteins; the supernatant is then vacuum frozen and concentrated.

9. The use of a derivative as described in claim 1 in the preparation of antibacterial drugs or agricultural fungicides.

10. The application as described in claim 9, characterized in that, The targets of antibacterial or bactericidal agents are Phytophthora parasitica var. nicotianae, Alternaria alternata, Phytophthora capsici, Colletotrichum scovillei, Fusarium graminearum, and Magnaphorthe oryzae.

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