A cyclo-lactone peptide compound aglomycin a biosynthetic gene cluster and application thereof

Abstract

The present application relates to a kind of cyclic ester peptide compound aglomycin A biosynthesis gene cluster and its application.The nucleotide sequence of the gene cluster is as shown in SEQ ID NO.1, and the use is to produce peptide compound aglomycin A or peptide compound aglomycin D.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a cyclic ester peptide compound aglomycin A biosynthetic gene cluster and its application. Background Technology

[0002] Natural products (NPs), a diverse class of bioactive metabolites synthesized by plants, animals, and microorganisms, constitute a rich resource and have inspired the development of over 65% of approved drugs to date. Halogenated natural products are a particularly noteworthy class of compounds, attracting significant attention due to the fact that the introduction of halogen atoms typically significantly enhances their bioactivity. From a pharmacological perspective, the introduction of halogens can also improve the metabolic stability and membrane permeability of drugs; currently, over 25% of approved drugs contain halogen groups. [14-16] In nature, halogenation is a widespread biosynthetic modification process involving the formation of over 10,000 natural products. Notably, over 2,400 of these originate from microorganisms, representing approximately 6.6% of known microbial natural products, and 98.5% of these compounds are chlorinated or brominated derivatives.

[0003] Streptomyces agglomeratus is a Streptomyces strain isolated from lichens in Tibet, my country, by the Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences. Through systematic chemical isolation and identification, a novel halogenated compound with antibacterial activity, aglomycin A, was discovered in the fermentation products of this strain.

[17] Aglomycin A exhibited significant antibacterial activity against multiple clinically resistant isolates of E. faecium and E. faecalis. Furthermore, aglomycin A showed a synergistic effect with linezolid, and their combined use significantly enhanced the antibacterial efficacy against VRE (vancomycin-resistant Enterococcus faecium). In a Galleria mellonella infection model, the combined application of aglomycin A and linezolid significantly improved survival rates.

[0004] Aglomycin A belongs to the monochlorocyclic peptide class of compounds, formed by the sequential condensation of 3-chloro-2-aminobenzoic acid, cysteine, threonine, proline, hexahydropyridazine-3-carboxylic acid, epoxyvaline, and N-methylvaline. It is a typical non-ribosomal peptide compound. Its structure contains an extremely rare epoxyvaline residue and a dipeptide side chain formed by the condensation of the uncommon 3-chloro-2-aminobenzoic acid and cysteine. Exploring the biosynthetic process based on the novelty of chemical structure has the following characteristics: Significance .

[0005] Nonribosomal peptides (NRPs) are natural products synthesized by nonribosomal peptide synthetases (NRPS) and are widely found in bacteria and fungi.

[18] A typical NRPS consists of multiple modules, each typically comprising three key domains: an adenylation domain (A domain), which recognizes and activates amino acids to form aminoacyl-adenosine; a condensation domain (C domain), which catalyzes the formation of an amide bond between two amino acids; and a thiolation domain (T domain), which carries the amino acid and the nascent peptide chain.

[19] In biosynthesis, NRPS perform the catalytic function of each module, assembling specific amino acid sequences into nonribosomal peptides with specific structures.

[0006] Based on the modularity and sequential assembly characteristics of NRPS, we used bioinformatics analysis of aglomycin A-related biosynthetic gene clusters and key enzymes to infer its possible biosynthetic pathway. Furthermore, the regulatory mechanism of aglomycin A biosynthesis has not yet been reported.

[0007] Based on this, the present invention is proposed. Summary of the Invention

[0008] This invention first relates to a gene cluster for the biosynthesis of compound aglomycin A, the nucleotide sequence of which is shown in SEQ ID No. 1, and which sequentially contains twelve functional genes: aglB, aglC, aglD, aglE, aglF, aglG, aglI, aglJ, aglK, aglL, aglM, and aglN, the nucleotide sequences of which are shown in SEQ ID No. 2 to 13, respectively.

[0009] In the aforementioned gene cluster,

[0010] AglB is a non-ribosomal peptide synthase responsible for the adenylation of the fourth amino acid, cyclooxyvaline or its precursor valine.

[0011] AglC is a tryptophan dioxygenase whose function is to catalyze the ring-opening of tryptophan to generate 3-chloroformylkynurenine.

[0012] AglD is a formamidinase whose function is to convert 3-chloroformylkynurenine to 3-chlorokynurenine.

[0013] AglE is a tryptophan halogenase that catalyzes the conversion of tryptophan to 7-chlorotryptophan.

[0014] AglF is kynurenase, whose function is to catalyze the production of 3-chlorokynurenine from 3-chloro-o-aminobenzoic acid;

[0015] AglG is a cytochrome P450 oxidase whose function is to catalyze the epoxidation of valine.

[0016] AglI is a type II thioesterase whose function is to present valine or its oxidation product cyclooxyvaline to the T domain of the fourth module of AglJ.

[0017] AglJ is a non-ribosomal peptide synthase, and its five modules are responsible for peptide chain assembly.

[0018] AglK (L-ornithine monooxygenase) and AglL (NN bond-forming enzyme) are responsible for the biosynthesis of pyridazine acid (Piz). Then, the third module of AglJ is responsible for activation and assembly onto the peptide chain.

[0019] AglM is an AMP-binding ligase that is responsible for activating 3-chloro-o-aminobenzoic acid;

[0020] AglN is a non-ribosomal peptide synthase that recognizes cysteine ​​residues and undergoes condensation and cyclization oxidation catalyzed by the Cy and Ox domains to generate thioesteryl 4-(2-amino-3-chloro-phenyl)thiazole.

[0021] The present invention also relates to proteins encoded by each gene aglB, aglC, aglD, aglE, aglF, aglG, aglI, aglJ, aglK, aglL, aglM and aglN in the aforementioned gene cluster, the amino acid sequences of which are shown in Seq ID No. 14 to 25.

[0022] The present invention also relates to the use of the aforementioned aglomycin A biosynthetic gene cluster or the aglomycin A biosynthetic gene cluster and / or its encoded protein in the catalytic synthesis of aglomycin A or its analogues.

[0023] This invention also relates to the use of the aglB gene or its encoded AglB protein in the synthesis of aglomycin A.

[0024] The structure of aglomycin A is shown in equation (2) below.

[0025]

[0026] This invention also relates to the peptide compound aglomycin D, the molecular formula of which is: C 10H7ClN2O2S has a molecular weight of 254; its chemical structure is shown in formula (1).

[0027]

[0028] The present invention also relates to a medicine or pharmaceutical composition comprising the compound aglomycin D, wherein the medicine or pharmaceutical composition comprises a therapeutically effective amount of the compound aglomycin D, and necessary pharmaceutical excipients.

[0029] The drug is an antibacterial drug, and preferably, the bacteria are vancomycin-resistant Enterococcus faecalis, Enterococcus faecium, or Staphylococcus aureus.

[0030] This invention also relates to the application of the compound aglomycin D in the preparation of a drug, wherein the drug is an antibacterial drug, preferably wherein the bacteria are vancomycin-resistant Enterococcus faecalis, Enterococcus faecalis, or Staphylococcus aureus.

[0031] The present invention also relates to a method for producing the compound aglomycin D, comprising the following steps:

[0032] (1) Knock out the AglB gene in the Streptomyces agglomeratus strain to obtain the knockout strain AglBKO;

[0033] (2) Fermentation of the knockout strain AglBKO, the fermentation conditions are ISP2 medium, 28℃ for seed culture, M3 medium, 28℃ for scale-up culture and harvesting of fermentation broth;

[0034] (3) Purification and concentration of fermentation broth: The supernatant of fermentation broth was collected by centrifugation and adsorbed using HP-20 macroporous adsorption resin. Then, it was eluted with 30% ethanol-water, 50% ethanol-water, 70% ethanol-water (5L each) and 100% ethanol. The elution solutions were concentrated under vacuum to obtain four components: Fr.A (30% ethanol fraction), Fr.B (50% ethanol fraction), Fr.C (70% ethanol fraction) and Fr.D (100% ethanol fraction);

[0035] (4) Purification of aglomycin D: Fr.A and Fr.B were further separated by reversed-phase column chromatography. The components containing aglomycin D were combined according to the liquid chromatography-mass spectrometry results, and then purified by high performance liquid chromatography. The eluent was collected according to the retention time and concentrated and dried under reduced pressure to obtain the compound aglomycin D shown in Formula 1.

[0036] Preferred,

[0037] The reversed-phase column chromatography method is a gradient elution method: the eluent phase A is 0.1% formic acid in water; the eluent phase B is 0.1% formic acid in acetonitrile.

[0038] The gradient elution parameters are:

[0039] 0min-30min, 10%-20% B;

[0040] 30.1min-60min,20%-50% B;

[0041] 60.1min-80min,50%-100% B;

[0042] The flow rate is 5 mL / min;

[0043] In the high-performance liquid chromatography preparation and purification steps described above,

[0044] The chromatographic column used was: YMC-C8, 10μm, 10×250mm;

[0045] The eluent was an aqueous solution containing 42% acetonitrile with 0.1% formic acid added.

[0046] The system fluid flow rate is 2.5 mL / min.

[0047] The beneficial effects of this invention are as follows:

[0048] (1) The complete gene cluster involved in the biosynthesis of aglomycin A was identified;

[0049] (2) By knocking out the specific protein AglB in the gene cluster, a stable aglomycin A precursor compound, Aglomycin D, was obtained. The compound was identified as having excellent antibacterial activity, especially against vancomycin-resistant Enterococcus faecalis, Enterococcus faecium, and Staphylococcus aureus. At the same time, the compound is non-toxic to eukaryotic cells and has good safety. Attached Figure Description

[0050] Figure 1 The chemical structure of Aglomycin D and its 1H-1H COSY (Bold) and HMBC (Arrow) related signals.

[0051] Figure 2 HRMS spectrum of Aglomycin D.

[0052] Figure 3 Following whole-genome sequencing of Streptomyces agglomeratus, bioinformatics analysis was used to predict the biosynthetic gene cluster of aglomycin A.

[0053] Figure 4 The amino acid sequence homology analysis of the CAL and A domains was performed. Differential amino acid residues in the A10 motif were highlighted in green. Conserved sequence alignment was performed using ClustalW 2.1 and visualization was performed using ESPript 3.014.

[0054] Figure 5 The possible biosynthetic pathway of compound aglomycin A.

[0055] Figure 6 A schematic diagram of constructing the aglB knockout plasmid.

[0056] Figure 7 Enzyme digestion identification of recombinant plasmid pKC-aglB;

[0057] Lane M, 1kb plus DNA ladder (10000,8000,6000,5000,4000,3000,2000,1500,1000,800,500,300bp);

[0058] Lane 1,pKC-aglB / HindIII+EcoRI;

[0059] Lane 2, pKC-aglB / HindIII+NdeI;

[0060] Lane 3,pKC-aglB / KpnI+EcoRI.

[0061] Figure 8 PCR verification of the aglB gene double exchange blocking strain, (A) AglBKO double exchange strain (B) wild strain.

[0062] Figure 9 Analysis of metabolites from the aglB-blocking strain AglBKO, the complement strain AglBKO / pL-aglB, and the wild-type strain Streptomyces agglomeratus.

[0063] Figure 10 Recombinant plasmid enzyme digestion identification: (A) pKC-aglA and (B) pKC-aglG, as shown in the figure:

[0064] Lane M, 1kb plus DNA ladder (10000,8000,6000,5000,4000,3000,2000,1500,1000,800,500,300bp);

[0065] Lane 1,pKC-aglA / HindIII+EcoRI;

[0066] Lane 2, pKC-aglA / HindIII+NdeI;

[0067] Lane 3, pKC-aglA / KpnI+EcoRI;

[0068] Lane 4, pKC-aglG / HindIII+EcoRI;

[0069] Lane 5,pKC-aglG / HindIII+NdeI;

[0070] Lane 6,pKC-aglG / KpnI+EcoRI.

[0071] Figure 11 PCR verification of the aglA gene double crossover blocking strain (left), (A) AglAKO double crossover strain (B) wild strain;

[0072] (Right) PCR verification of the aglG gene double crossover blocking strain, (A) AglGKO double crossover strain; (B) wild strain.

[0073] Figure 12 EIC mass spectrometry extraction images of metabolites from knockout strains AglAKO, AglGKO and wild-type strains (m / z 773.2848).

[0074] Figure 13 Analysis of metabolites from knockout strain AglGKO, replenishment strain AglGKO / pLP450, and wild-type strain Streptomyces agglomeratus.

[0075] Figure 14 Figure showing the cytotoxicity results of Aglomycin D on Huh-7, Vero, and 293T cell lines. Detailed Implementation

[0076] Materials and Methods

[0077] 1. Strains, plasmids, and culture methods

[0078] Wild-type strain Streptomyces agglomeratus was cultured and fermented on ISP2 medium, and conjugation was performed on MS medium. Both culture and fermentation were carried out at 28°C for 7 days. All strains were cultured on liquid TSB medium when genomic DNA was extracted.

[0079] E. coli ET12567 / pUZ8002 is a host for conjugation transfer between Escherichia coli and Streptomyces. [2] The cultures were incubated at 37°C using LB medium. When antibiotics were required, their working concentrations were as follows: apramycin (Am, 50 μg / ml), sulfanilamide (Thi, 50 μg / ml), kanamycin (Km, 50 μg / ml), chloramphenicol (Cm, 50 μg / ml), and aztreonam (Azt, 25 μg / ml).

[0080] All strains and plasmids used in this study are listed in Table 1, and primers are listed in Table 2.

[0081] Table 1. Strains and Plasmids

[0082]

[0083]

[0084] Table 2. Primers

[0085]

[0086]

[0087] 2. Extracting high-quality Streptomyces agglomeratus genome.

[0088] DNA extraction was performed using a bacterial DNA extraction kit. Streptomyces were picked with a sterile bamboo stick and inoculated into TSB medium containing glass beads. The mixture was incubated on a shaker at 200 rpm and 28°C for 2 days. 1 mL of the bacterial culture was transferred to a 1.5 mL centrifuge tube and centrifuged at 10000 rpm for 1 min to collect the bacterial cells. 220 μL of Buffer STE Plus, 30 μL of lysozyme, and 10 μL of RNase A were added. The mixture was incubated at room temperature for 10-20 minutes. Then, 250 μL of Buffer DL and 10 μL of Proteinase K were added, vortexed, and digested in a 70°C water bath for 10 min. After centrifugation for 3 min, the supernatant was transferred to a new centrifuge tube. 250 μL of anhydrous ethanol was added to the lysis buffer, and the mixture was vortexed for 15 seconds. The mixture was transferred to a 2 mL HiPure DNA Mini Column and centrifuged at 10000 rpm for 1 min. Discard the eluent, add 500 μL of Buffer GW1, centrifuge at 10000 rpm for 1 min, discard the eluent, add 650 μL of Buffer GW2, centrifuge at 10000 rpm for 1 min, discard the eluent, and centrifuge at 10000 rpm for 2 min. Transfer the column to a new 1.5 mL centrifuge tube, add 30–100 μL of preheated water (70°C) to the center of the column membrane, incubate for 3 min, centrifuge at 10000 rpm for 1 min to obtain the extracted DNA, and store at -20°C.

[0089] 3. Bonding transfer between Streptomyces agglomeratus and E. coli ET12567 / pUZ8002

[0090] The conjugation transfer method of Actinoplanes friuliensis reported in the literature is used as a reference. [5]A genetic operating system for *Streptomyces agglomeratus* was established. A small amount of *Streptomyces agglomeratus* spore suspension was spread on an agar slant and incubated at 28°C for 7 days. The spores were then scraped off with a spatula and placed in 3 mL of 20% glycerol. The mixture was poured into a homogenizer, shaken for 1 min as needed, and filtered. The resulting filtrate was the spore suspension. *E. coli* ET12567 / PUZ8002 containing the plasmid to be transferred was activated. A single colony was inoculated into 5 mL of LB liquid medium (with 25 μg / mL Cm and Km added, and the corresponding antibiotic, 50 μg / mL Am, added according to the resistance gene carried by the plasmid), and incubated overnight at 37°C with shaking. The next day, a 5% inoculum was transferred to 50 mL of LB liquid medium and incubated at 37°C with shaking for 6 h. 5 mL of the bacterial suspension was centrifuged at 5000 rpm for 10 min, and the bacterial cells were collected and washed twice with LB liquid medium. The bacterial cells were resuspended in 0.5 mL of LB liquid medium. Take 1 mL of Streptomyces spore suspension, centrifuge, discard the supernatant, add 1 mL of LB liquid medium to the cells, and wash twice. Resuspend the spore suspension in 0.5 mL of LB liquid medium. Heat shock in a 50°C water bath for 10 min, then cool in a water bath at room temperature. Incubate at 37°C with shaking for 2.5 h. Vigorously shake the spore suspension. Mix different proportions of E. coli ET12567 / pUZ8002 cells with the spore suspension, centrifuge, discard most of the supernatant, then resuspend the precipitated cells in the remaining supernatant and spread on MS plates supplemented with 10 mM MgCl2. Incubate the plates at 28°C for 16-20 h, then cover the surface of the plates with 1 mL of sterile water containing the corresponding antibiotic, evaporate to dryness, and incubate at 28°C for approximately 5-7 days before picking conjugates.

[0091] 4. Screening and identification of blocking strains

[0092] Pick conjugates into ISP2 culture dishes (50 μg / mL Am) r Incubate at 37°C. Pick potential single-exchange colonies and inoculate them into 2 mL of TSB medium (50 μg / mL Am). r The culture was carried out at 28°C with shaking for 2 days, and the whole genome was extracted. PCR verification was performed using identification primers. Successfully verified single-exchange strains were inoculated onto antibiotic-free ISP2 plates and subcultured at 37°C for relaxation. Strains that could grow on ISP2 plates without alpramycin but not on plates containing antibiotics were screened using a photocopying method; these were considered successfully relaxed strains. They were then inoculated into 2 mL of TSB medium (50 μg / mL Amino Acid). r The genome was cultured at 28°C with shaking for 2 days, and the whole genome was extracted and identified by PCR using identification primers.

[0093] Example 1: Bioinformatics Analysis of the Aglomycin A Synthetic Gene Cluster

[0094] We performed whole-genome DNA sequencing on the strain *Streptomyces agglomeratus* (conducted by BioMed Biotechnology Co., Ltd.). The whole-genome sequencing platform was used in PacBio HIFI mode. Assembly was performed using Hifiasm v0.12 software, followed by circularization and adjustment of the start site using Circlator v1.5.5 software. Further error correction was performed using Pilon v1.22 software with second-generation data to obtain the complete genome map. The *Streptomyces agglomeratus* genome is a linear chromosome of 8,182,762 bp in length, with a (G+C)mol percentage of 70.51%.

[0095] From a biosynthetic perspective, the 3-chloro-o-aminobenzoic acid structural unit likely originates from tryptophan, subsequently undergoing halogenation catalyzed by tryptophan halogenases. Therefore, we utilize the tryptophan halogenase PrnA [6] Using this as a probe, and combined with comprehensive bioinformatics analysis of genomic data using the online antiSMASH tool, a potential agl gene cluster was identified. AntiSMASH and BLAST annotations (Table 3) revealed that this agl gene cluster encodes two independent non-ribosomal peptide synthase macroenzymes (NRPSs), AglN and AglJ, containing two and five modules respectively, and are expected to be responsible for synthesizing dipeptide side chains and cyclic pentapeptide backbones. The CAL domain in the first module of AglN is predicted to activate the 3-chloro-o-aminobenzoic acid precursor and translocate it to the PCP domain. [7] However, detailed bioinformatics analysis revealed that the CAL domain lacked a lysine residue from the A10 conserved motif. Figure 4 This residue is crucial for adenylate esterification activity. [8] Meanwhile, a gene encoding an independent A domain was found near aglN, which may be used to compensate for the adenylation function of the CAL domain, thereby activating 3-chloro-o-aminobenzoic acid. Interestingly, an initiation condensation domain (C...) was found in the first module of AglJ. starter The Cs domain, typically found in lipopeptides, is used to condense a fatty acid with the first amino acid residue to initiate peptide chain elongation. [9] In this study, this domain is hypothesized to be used to condense a dipeptidyl group with a threonine acyl group to initiate the biosynthesis of cyclic pentapeptides.

[0096] In addition to NRPS, which is responsible for skeletal synthesis, enzymes used to synthesize these unusual building blocks have been identified in the agl gene cluster. It is speculated that the 3-chloro-o-aminobenzoic acid precursor may be synthesized by AglC, AglD, AglE, and AglF, which are respectively tryptophan dioxygenase, formamidinase, tryptophan halogenase, and kynurenase. Furthermore, the aglK and aglL genes are associated with their homologs KtzI and KtzT. [1] The similarities were 62% and 51%, respectively, encoding an L-ornithine N(5)-monooxygenase and an FMN-binding enzyme, presumably related to the biosynthesis of piperazine acid (Piz). Notably, the fourth module of AglJ lacks the A domain and contains only a CT double domain; meanwhile, an independent AT double domain (AglB) and a type II thioesterase gene aglI were found in the agl gene cluster. Similar to WS9326A

[10] legonmycin

[11] and rotihibin

[12] The synthetic pathway introduces non-natural amino acids, predicting their joint involvement in the introduction of the uncommon amino acid epoxyvaline. The fifth module then loads valine onto the peptide chain, followed by methylation catalysis by the methylation domain to N-methylvaline. Finally, the TE domain catalyzes the release and cyclization of the peptide chain, forming the esterified peptide compound aglomycin A. Figure 5 .

[0097] Example 2. Functional analysis of gene aglB

[0098] To further confirm the role of aglB in the biosynthesis of aglomycin A, we constructed the aglB knockout strain AglBKO, the complement strain AglBKO / pL-aglB, the control strain AglBKO / pSET152, and the wild-type strain, and performed UPLC-MS / MS analysis on their metabolites. Figure 6-8 ).

[0099] The results show ( Figure 9 ):

[0100] (1) Compared with the wild-type strain, the AglBKO knockout strain showed a complete disappearance of the signal of aglomycin A and its cyclic peptide backbone-related derivatives in its metabolite profile. At the same time, a new monochloro compound (aglomycin D) was observed at a mass-to-charge ratio m / z 254.9995.

[0101] (2) Further analysis of the metabolic profile of the AglBKO / pL-aglB supplemented strain revealed the reappearance of aglomycin A, while the signal of aglomycin D disappeared.

[0102] The structure of aglomycin D was subsequently isolated and identified as 2-(2-amino-3-chlorophenyl)-4-thiazocarboxylic acid, consistent with the hydrolysis products of the dipeptide side chain analyzed in previous bioinformatics studies. This indicates that the knockout of aglB leads to impaired elongation of the cyclic peptide chain, hydrolysis of the dipeptide side chain from the assembly line, and accumulation of the dipeptide product. Further, it is speculated that the NRPS encoded by the aglB gene is involved in the synthesis of the aglomycin A core pentapeptide backbone. Combined with the analysis of the aglomycin A biosynthetic gene cluster, the fourth module encoded by the aglJ gene is a CT double-domain module lacking the A domain.

[0103] In summary: The A domain encoded by aglB is responsible for the fourth amino acid, epoxyvaline or its precursor valine adenosine. change .

[0104] aglB, SEQ ID NO.2:

[0105]

[0106] AglB, SEQ ID NO.14:

[0107] MSGTAASVVEVFEQQAAKRPEAIAVRGAGGELTYAELDRRANQLAHELRARGVDVEVPVALLMRRSTDLIVTLVAVLKAGGVYLALDHRLPAERRATVLLDARPALVITDEREPSATDGGPPWCPLPELSACASLRPTTRPVTRTSP ESTAYIAYTSGSAGKPKGVMVPHRAIERLVVGAGYLPIGPDDVFLQLAPVAFDASTLEIWGPLLNGGKMVVAPEGQLPLGALADLVRGEGVTILWLTAGLFHHLVASGLVARLRGLRFLLAGGDVLSVESVNAAMAELTGTTLINGY GPTENTTFTCCAPLTEPVTAPRVPIGPAIDGTSVYVLDDAVGTAPDGQAGRIHAAGAGLAHGYLNDPALTAQRFVANPFSGVPGDRMYRTGDLGSRGEDGALDFHGREDGQVKIRGFRIEPGDVEHALRAHPDVADAAIVATSSGDE RSLVGFYVADEPLVSEHLRKHMETLVPPYMIPAVLVWLDALPLTANGKVDRDALTAYAPPGRGDLSSDYRGPGSPLEQWLAEMWGDLTQTSPVGVDDDFFELGGHSLMAVRIIVEISDRTGVEIDPQDFYACPTISELAALIAAGAPK

[0108] Example 3. Functional analysis of genes aglA and aglG

[0109] Furthermore, considering that valine epoxidation is extremely rare in nature, occurring only in the lipopeptide Streptocinnamide B (Scm B),...

[13] Upon observing this group, we conducted a systematic analysis of the related enzyme-encoding genes. The results showed that two genes, aglA and aglG, encode a FAD-dependent oxidase and a cytochrome P450 monooxygenase, respectively, which may form epoxide groups through a dehydrogenation-epoxidation cascade reaction, a common epoxidation mechanism in natural products.

[0110] To verify this hypothesis, we knocked out aglA and aglG genes, respectively, to construct mutant strains AgLAKO and AgLGKO. Figure 10 and Figure 11 The results show () Figure 12 ),

[0111] (1) The production of aglomycin A in the AglAKO mutant was almost unaffected, while the AglGKO mutant was completely unable to synthesize aglomycin A.

[0112] (2) After aglG was reintroduced into the AglGKO mutant (C-ΔaglG), the production of aglomycin A was restored to the wild-type level, further supporting the important role of aglG in the biosynthesis of aglomycin A.

[0113] Meanwhile, analysis of metabolites using UPLC-MS / MS showed ( Figure 13 The AglGKO mutant produced aglomycin E(m / z 781.2849 [M+Na)). + The substance was confirmed by MS / MS fragment analysis to be a valine analog of aglomycin A. The key role of aglG in the synthesis of epoxy groups was confirmed. .

[0114] aglG, SEQ ID NO.7:

[0115]

[0116] AglG, SEQ ID NO.19:

[0117] MSERTAPAASTDLDSLDLMDPRLYGSGDPHPIWTALRERAPLHLQSLPDGRRFWSVTKYHDVREVLRDHTRFTSSRGTLLSVLGSRDPAGGKMMAASDPPVHTMLHEPIAKLLSRRALEPFVPEVRRVAHRLLERLADGPRDLAELAAGFPMAFTGTLMGIPEQDWARLTVLTTAAIAPEDPDFRLGSGLNTLATAHHELFSYF SRRTRRTSREDGLIGALLGLEAGGRRLRHDELVYNCYNCYNCTTPHAVTQTVLAFIENPAEYRKLVDDPRLVPGAVEEGLRWSSPANHFLRHATRDTTLRGRRVREGDAVVTWLGSANRDEEVFSEPFRFHVTRSPNPHVAFGFGPHHCVGAALARMALNALFAEIAGSVERFDLAGPVEHLASDFAAGIKHMPVVTKLRTT

[0118] Example 4: Scale-up fermentation of knockout strain AglBKO and extraction and purification of fermentation broth

[0119] 1. Fermentation of the knockout strain AglBKO

[0120] The knockout strain AglBKO was cultured in ISP2 solid medium at 28°C for 7 days, then transferred to ISP2 solid medium for another 7 days. Afterwards, approximately 1 cm... 2 Solid cultures of various sizes were inoculated into 50 mL of ISP2 liquid medium and cultured at 220 rpm and 28 °C for 48 hours to obtain seed culture.

[0121] The aglG knockout strain was scaled up for fermentation using M3 medium. A total of 100 500mL Erlenmeyer flasks were inoculated, with 5mL of seed culture added to each flask. The flasks were cultured with shaking at 28°C for 8 days.

[0122] 2. Crude purification and concentration of fermentation broth

[0123] The fermentation broth (10 L) of the above strain was centrifuged at 8,000 rpm for 10 min, and the supernatant was collected. The supernatant was treated with HP-20 macroporous adsorption resin (column volume 1.0 L). It was washed with 5 L of deionized water until nearly colorless, and then eluted successively with 5 L of 30% ethanol-water, 5 L of 50% ethanol-water, 5 L of 70% ethanol-water, and 5 L of 100% ethanol. The eluents were concentrated under vacuum to obtain the following four fractions: Fr. A (30% ethanol fraction), Fr. B (50% ethanol fraction), Fr. C (70% ethanol fraction), and Fr. D (100% ethanol fraction).

[0124] 3. Preparation of the novel cyclic ester peptide compound aglomycin D

[0125] The above-mentioned components Fr.A and Fr.B were further separated by reversed-phase medium-pressure column chromatography (A phase: 0.1% formic acid in water; B phase: 0.1% formic acid in acetonitrile; 0 min-30 min, 10%-20% B; 30.1 min-60 min, 20%-50% B; 60.1 min-80 min, 50%-100% B, flow rate 5 mL / min). Based on the LC-MS results, the components containing aglomycin D were combined and then semi-preparatively purified by high-performance liquid chromatography (HPLC) using a Waters ACQUITY Arc 2D (YMC-C8, 10 μm, 10 × 250 mm, 42% acetonitrile / water with 0.1% formic acid, 2.5 mL / min). The eluent was collected based on the retention time and concentrated and dried under reduced pressure to obtain the final product. Formula 1 The compound shown is aglomycin D .

[0126] Example 5: Structural identification of the cyclic ester peptide compound aglomycin D

[0127] Aglomycin D(2) is a white powder that is readily soluble in methanol;

[0128] High-resolution mass spectrometry (HS-MS) Figure 3 The quasi-molecular ion peak is shown to be at m / z 254.9985 [M+H]. + (C 10 The theoretical calculated value of H8ClN2O2S is 254.9995 (with an error of 3.9 ppm), suggesting that the molecular formula of compound 2 is C. 10 H7ClN2O2S has an unsaturation degree of 9.

[0129] Analyzing the NMR spectrum of aglomycin D, its 1 H and 13 C10 NMR (DMSO-d6, Table 3) data show the presence of one ortho-trisubstituted phenyl group [δ]. C 115.1–142.4,δH 7.40, d(7.8); 6.67, dt(1.8, 7.8); 7.64, d(8.4)], 1 2,4-substituted thiazole (δ C 167.7, 148.6, 126.2; δ H 8.39,s).

[0130] Based on the mass spectrometry analysis results, we speculate that aglomycin D is the 2-(2-amino-3-chlorophenyl)-4-thiazocarboxylic acid moiety of aglomycin A.

[0131] 1 H- 1 1H COSY spectroscopy shows that compound 2 has one spin system. Figure 1 The structure is CH / CH / CH. Based on the HMBC spectrum, H-5 is associated with C-2, C-4, and C-1", H-6' is associated with C-2, C-1', C-2', C-3', and C-4', H-4' is associated with C-1', C-2', C-3', and C-6', and 2'-NH2 is associated with C-1' and C-3'. Therefore, its structure is inferred to be 2-(2-amino-3-chlorophenyl)-4-thiazolic acid.

[0132] Table 3. 1H and 13C NMR data of Aglomycin D

[0133]

[0134]

[0135] Example 6. Antibacterial activity of the cyclic ester peptide compound aglomycin D

[0136] 1. In vitro antibacterial activity of aglomycin D, a novel cyclic ester peptide compound

[0137] The antibacterial activity of aglomycin D against seven microorganisms—Enterococcus faecalis (ATCC 35667), Staphylococcus aureus (ATCC 29213), Escherichia coli (ATCC 25922), Klebsiella pneumoniae (ATCC BAA2470), Acinetobacter baumannii (ATCC 19606), Pseudomonas aeruginosa (ATCC 27853), and Candida albicans (ATCC 10231)—was evaluated using the microplate method (Table 4). The results showed that aglomycin D exhibited good antibacterial activity against Enterococcus faecalis (ATCC 35667) and Staphylococcus aureus (ATCC 29213) (MIC = 2-8 μg / mL). Aglomycin D showed no activity against Gram-negative bacteria or fungi, indicating that this compound is a narrow-spectrum antibiotic targeting Gram-positive bacteria.

[0138] Table 4. Results of in vitro antibacterial activity of compound aglomycin D

[0139]

[0140] 2. Antibacterial activity of the novel cyclic ester peptide compound aglomycin D against clinically isolated microorganisms.

[0141] Based on the good performance of compound aglomycin D against susceptible strains, its MIC against nine different clinically isolated drug-resistant bacteria was further determined. Aglomycin D significantly inhibited the growth of Enterococci. Therefore, nine clinically isolated Enterococci faecalis and Enterococcus faecium strains were selected, along with four clinically used first- or second-line antibiotics as control groups to further identify the antibacterial activity of compound aglomycin D (Table 5).

[0142] The results showed that aglomycin D had inhibitory effects on clinical isolates from different tissues, and maintained stable antibacterial activity against highly resistant strains of ampicillin, gentamicin, vancomycin, and daptomycin. It showed particularly strong activity against vancomycin-resistant Enterococcus faecalis, exhibiting activity against both vanA and vanB genotypes. Therefore, The antibacterial activity of compound aglomycin D is not limited to susceptible strains; its inhibitory effect on clinical isolates is comparable. Most commonly used clinical antibiotics .

[0143] Table 5. Antibacterial activity of Aglomycin D against clinically isolated Enterococci.

[0144]

[0145]

[0146] 3. Cytotoxicity assay of the novel cyclic ester peptide compound aglomycin D

[0147] The MTT assay is a commonly used cytotoxicity assay for evaluating the biological effects of compounds on eukaryotic cells. We used the MTT assay to detect the cytotoxicity of the compound aglomycin D. We determined the cytotoxicity of aglomycin D against Huh7 cells (human liver cancer cells), Vero cells (African green monkey kidney cells), and 293T cells (human embryonic kidney cells). Figure 14 The results showed that compound aglomycin D had an effect on the CC of the above-mentioned cells. 50 The concentrations were 106.9 μg / mL, 50.2 μg / mL, and 90.3 μg / mL, respectively. All of these values ​​far exceeded the MIC, initially indicating a certain degree of safety.

[0148] Finally, it should be noted that the above embodiments are only used to help those skilled in the art understand the essence of the present invention, and are not intended to limit the scope of protection of the present invention.

[0149] [References]

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[0151] 2. Paget, MS; Chamberlin, L.; Atrih, A.; Foster, SJ; Buttner, MJ, Evidence that the extracytoplasmic function sigma factorσE is required for normal cellwall structure in Streptomyces coelicolor A3(2). Journal of bacteriology 1999, 181(1), 204-11.

[0152] 3.Paget,M.S.;Chamberlin,L.;Atrih,A.;Foster,S.J.;Buttner,M.J.,Evidencethat the extracytoplasmic function sigma factor sigmaE is required for normalcell wall structure in Streptomyces coelicolor A3(2).J Bacteriol1999,181(1),204-11.

[0153] 4.Bierman,M.;Logan,R.;O'Brien,K.;Seno,E.T.;Rao,R.N.;Schoner,B.E.,Plasmid cloning vectors for the conjugal transfer of DNA from Escherichiacoli to Streptomyces spp.Gene 1992,116(1),43-9.

[0154] 5.Heinzelmann,E.;Berger,S.;Puk,O.;Reichenstein,B.;Wohlleben,W.;Schwartz,D.,A glutamate mutase is involved in the biosynthesis of thelipopeptide antibiotic friulimicin in Actinoplanes friuliensis.Antimicrobialagents and chemotherapy 2003,47(2),447-57.

[0155] 6.Selin,C.;Habibian,R.;Poritsanos,N.;Athukorala,S.N.;Fernando,D.;deKievit,T.R.,Phenazines are not essential for Pseudomonas chlororaphis PA23biocontrol of Sclerotinia sclerotiorum,but do play a role in biofilmformation.FEMS Microbiol Ecol 2010,71(1),73-83.

[0156] 7.Gulick,A.M.,Conformational dynamics in the Acyl-CoA synthetases,adenylation domains of non-ribosomal peptide synthetases.ACS Chem Biol 2009,4(10),811-27.

[0157] 8.Horswill AR,E.-S.J.,Characterization of the Propionyl-CoASynthetase(PrpE)Enzyme of Salmonella enterica Residue Lys592 Is Required forPropionyl-AMP Synthesis.Biochemistry 2002,41(7),2379-87.

[0158] 9.Rausch,C.;Hoof,I.;Weber,T.;Wohlleben,W.;Huson,D.H.,Phylogeneticanalysis of condensation domains in NRPS sheds light on their functionalevolution.BMC Evol Biol 2007,7,78.

[0159] 10.Kim,M.S.;Bae,M.;Jung,Y.E.;Kim,J.M.;Hwang,S.;Song,M.C.;Ban,Y.H.;Bae,E.S.;Hong,S.;Lee,S.K.;Cha,S.S.;Oh,D.C.;Yoon,Y.J.,UnprecedentedNoncanonical Features of the Nonlinear Nonribosomal Peptide SynthetaseAssembly Line for WS9326A Biosynthesis.Angew Chem Int Ed Engl 2021,60(36),19766-19773.

[0160] 11.Wang,S.;Brittain,W.D.G.;Zhang,Q.;Lu,Z.;Tong,M.H.;Wu,K.;Kyeremeh,K.;Jenner,M.;Yu,Y.;Cobb,S.L.;Deng,H.,Aminoacyl chain translocation catalysedby a type II thioesterase domain in an unusual non-ribosomal peptidesynthetase.Nat Commun 2022,13(1),62.

[0161] 12.Planckaert,S.;Deflandre,B.;Vries,A.M.;Ameye,M.;Martins,J.C.;Audenaert,K.;Rigali,S.;Devreese,B.,Identification of Novel RotihibinAnalogues in Streptomyces scabies,Including Discovery of Its BiosyntheticGene Cluster.Microbiol Spectr 2021,9(1),e00571-21.

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Claims

1. A gene cluster for the biosynthesis of compound aglomycin A, wherein the nucleotide sequence of the gene cluster is shown in SEQ ID No. 1, and it sequentially contains twelve functional genes: aglB, aglC, aglD, aglE, aglF, aglG, aglI, aglJ, aglK, aglL, aglM, and aglN, the nucleotide sequences of which are shown in SEQ ID No. 2 to 13, respectively. In the aforementioned gene cluster, AglB is a non-ribosomal peptide synthase responsible for the adenylation of the fourth amino acid, cyclooxyvaline or its precursor valine. AglG is a cytochrome P450 oxidase whose function is to catalyze the epoxidation of valine.

2. The proteins AglB and AglG encoded by the genes aglB and aglG in the gene cluster of claim 1 have amino acid sequences as shown in Seq ID No. 14 and 19, respectively.

3. The use of the aglomycin A biosynthesis gene cluster of claim 1 or the proteins AglB and AglG of claim 2 in the biosynthesis of aglomycin A or its analogues.

4. The use of the aglB gene or its encoded AglB protein as described in claim 2 in the synthesis of aglomycin A.

5. The peptide compound aglomycin D has the molecular formula: C 10 H7ClN2O2S has a molecular weight of 254; its chemical structure is shown in formula (1).

6. A drug or pharmaceutical composition comprising the peptide compound aglomycin D of claim 5, wherein the drug or pharmaceutical composition comprises a therapeutically effective amount of the peptide compound aglomycin D, and necessary pharmaceutical excipients.

7. The drug or drug composition according to claim 6, characterized in that, The drug is an antibacterial drug, and preferably, the bacteria are vancomycin-resistant Enterococcus faecalis, Enterococcus faecium, or Staphylococcus aureus.

8. The use of the peptide compound aglomycin D according to claim 5 in the preparation of a drug, wherein the drug is an antibacterial drug, preferably, the bacteria are vancomycin-resistant Enterococcus faecalis, Enterococcus faecium, or Staphylococcus aureus.

9. A method for producing the peptide compound aglomycin D of claim 5, comprising the following steps: (1) Knock out the AglB gene in the Streptomyces agglomeratus strain to obtain the knockout strain AglBKO; (2) Fermentation of the knockout strain AglBKO, fermentation conditions were: seed culture on ISP2 medium at 28℃, M3 medium at 28℃ for scale-up culture and harvesting of fermentation broth; (3) Purification and concentration of fermentation broth: The supernatant of fermentation broth was collected by centrifugation and adsorbed using HP-20 macroporous adsorption resin. Then, it was eluted with 30% ethanol-water, 50% ethanol-water, 70% ethanol-water (5L each) and 100% ethanol. The elution solutions were concentrated under vacuum to obtain four components: Fr.A (30% ethanol fraction), Fr.B (50% ethanol fraction), Fr.C (70% ethanol fraction) and Fr.D (100% ethanol fraction); (4) Purification of aglomycin D: Fr.A and Fr.B were further separated by reversed-phase column chromatography. The components containing aglomycin D were combined according to the liquid chromatography-mass spectrometry results, and then purified by high performance liquid chromatography. The eluent was collected according to the retention time and concentrated and dried under reduced pressure to obtain the compound aglomycin D shown in Formula 1. Preferred, The reversed-phase column chromatography method is a gradient elution method: the eluent phase A is 0.1% formic acid in water; the eluent phase B is 0.1% formic acid in acetonitrile. The gradient elution parameters are: 0min-30min, 10%-20% B; 30.1 min - 60 min, 20% - 50% B; 60.1min-80min,50%-100%B; The flow rate is 5 mL / min; In the high-performance liquid chromatography preparation and purification steps described above, The chromatographic column used was: YMC-C8, 10μm, 10×250mm; The eluent was an aqueous solution containing 42% acetonitrile with 0.1% formic acid added. The system fluid flow rate is 2.5 mL / min.

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