Myxofactins compound derived from myxococcus as well as preparation method and application of myxofactins compound

Abstract

The invention discloses a myxofactins compound derived from myxococcus as well as a preparation method and application of the myxofactins compound, and belongs to the technical field of biological medicines. The structure of the myxofactins compound derived from myxofactins provided by the invention is shown as a formula (I), a formula (II) or a formula (III). Formula (I). The myxofactins compound provided by the invention has no obvious cytotoxic activity, can be used as an antibiotic adjuvant, remarkably improves the sensitivity of gram-negative bacteria such as acinetobacter baumannii to antibiotics, and provides a new thought for solving the problem of multidrug resistance of gram-negative bacteria and improving the curative effect of existing antibiotics. The preparation method provided by the invention realizes high yield through a heterologous expression technology, has the advantages of mild culture conditions, simple and feasible culture medium preparation, short fermentation time and the like, and is convenient for large-scale production.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to a myxococcus-derived compound, its preparation method, and its application. Background Technology

[0002] The information disclosed in the background section of this invention is intended only to enhance the understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Myxobacteria, as a group of microorganisms with complex social behaviors, have long attracted attention for their natural products. Early studies focused on active ingredients with direct therapeutic value; for example, some myxobacterial metabolites have been shown to have anticancer and enzyme-inhibiting functions. Others act as signaling molecules in community communication, participating in the regulation of physiological processes such as sporulation and cell aggregation, which are crucial for maintaining the ecological balance of the community. However, regarding fatty acid compounds in myxobacteria with "suicide activity," current research can only confirm their existence; their specific chemical structures have not been clearly elucidated. Furthermore, it is generally believed that these substances are mainly secreted and act within the bacterial membrane, limiting research on their functional mechanisms and practical applications. In addition, research on myxobacterial secondary metabolites secreted extracellularly as novel biological resources remains relatively weak, and their potential value in chemical ecology and medicine urgently needs to be explored.

[0004] Currently, multidrug resistance in Gram-negative bacteria has become a major threat to global public health. Existing antibiotics (such as polymyxins) have seen a significant decline in efficacy against resistant strains due to long-term overuse, while the development of novel antibiotics faces bottlenecks—the development of traditional terrestrial microbial resources is nearing saturation, and although marine extremophile microorganisms hold great promise, the development of their metabolites still requires breakthroughs in technology and resources. Against this backdrop, finding functional substances with unique mechanisms of action (such as adjuvants that can enhance the efficacy of existing antibiotics) has become a key direction for solving the problem of drug-resistant bacteria. Myxobacteria, as a microbial group rich in secondary metabolites, have not yet fully explored the application potential of their secretory metabolites in the treatment of drug-resistant bacteria, especially the development of compounds that combine safety and functional activity, which remains a gap. Summary of the Invention

[0005] In view of this, the present invention provides a myxococcal-derived myxococcal compound, its preparation method and application. The myxococcal-derived myxococcal compound provided by the present invention has no obvious cytotoxic activity and can be used as an antibiotic adjuvant to improve the efficacy of existing antibiotic-resistant Gram-negative bacteria.

[0006] In a first aspect, the present invention provides a myxofactins compound derived from Myxococcus, the structure of which is shown in formula (I), formula (II) or formula (III): Formula (I); Equation (II); Equation (III); In the compound of formula (I), R1 is selected from hydroxyl, amino, or any of the following substituents: , , or ; R2 is selected from hydrogen or methyl, R3 is selected from hydrogen or methyl, R4 is selected from hydrogen or methyl, and R5 is selected from hydrogen or methyl.

[0007] In formula (I), " "" indicates that the double bond is either Z-configuration or E-configuration.

[0008] Preferably, the compound of formula (I) is selected from any of the following structures: , , , , , , , or .

[0009] Secondly, the present invention provides a method for preparing the above-mentioned myxofactins compound derived from Myxococcus, comprising the following steps: Myxococci with accession number CCTCC NO: M 2021520 Myxococcus DNA fragments containing the myxofactin gene cluster were obtained by constructing a BAC library from sp.SDU36, with the GenBank accession number of the myxofactin gene cluster being CP077414.1; In vitro, scarless modification of DNA fragments containing the myxofactin gene cluster, including promoter substitution and / or gene knockout; The modified myxofactin gene cluster was introduced into a heterologous host bacterium. Myxococcus xanthus Fermentation broth was obtained by fermentation culture in DK1622; The fermentation broth was sequentially extracted and separated to obtain the myxofactins compound.

[0010] myxofactin gene cluster ( mftA ~ mftPThe sequence of ) is shown in SEQ ID NO. 1, and the specific sequence is as follows:

[0011] Preferably, the in vitro scarless modification step includes: releasing the myxofactin gene cluster using restriction endonucleases Xba I and Spe I; and linking the linearized vector pBAC-TnpA containing a Tn5 transposon cassette to the gene cluster using Red / ET recombination technology to form the recombinant vector pBAC-tnpA-mft. The recombinant vector pBAC-tnpA-mft was transformed into *E. coli* GBred-gyrA462 expressing Redαβ recombinase, and Redαβ recombinase activity was induced by L-arabinose. Anti-selection was performed using the AMP-CcdB fragment, and the AMP-CcdB fragment was removed using the restriction endonuclease PacI. Promoter replacement was performed using the Gibson assembly method; alternatively, promoter replacement and scarless knockout were performed using the Gibson assembly method. mftF Manipulation of genes.

[0012] Preferably, the fermentation medium used in the fermentation culture comprises per liter: 8-12g casein, 1.5-2.5g MgSO4·7H2O, 8-12mL 1M Tris-HCl, 8-12mL 100mM PBS, with a pH of 7.4-7.8; and the fermentation culture temperature is 28-32℃.

[0013] Preferably, the extraction operation is as follows: the fermentation broth is adsorbed using a macroporous adsorption resin, washed with water, and then the resin is eluted with an organic solvent to obtain a crude fermentation extract.

[0014] Furthermore, the separation operation is as follows: the crude fermentation extract is sequentially separated by reversed-phase medium-low pressure liquid chromatography, normal-phase chromatography, and semi-preparative high-performance liquid chromatography to obtain the myxofactins compound.

[0015] Thirdly, the present invention provides the use of the above-mentioned myxoactins compounds derived from Myxococcus in the preparation of drugs that improve the antibiotic sensitivity of bacteria.

[0016] Preferably, the bacteria include Acinetobacter baumannii, Staphylococcus aureus, Escherichia coli, or Burkholderia; the antibiotics include β-lactam antibiotics, quinolone antibiotics, and aminoglycoside antibiotics.

[0017] Fourthly, the present invention provides a drug for improving the antibiotic sensitivity of bacteria, comprising an effective amount of the above-mentioned myxococcal-derived compound and an antibiotic; The bacteria include Acinetobacter baumannii, Staphylococcus aureus, Escherichia coli, or Burkholderia; the antibiotics include β-lactam antibiotics, quinolone antibiotics, and aminoglycoside antibiotics.

[0018] Compared with the prior art, the present invention has achieved the following beneficial effects: (1) The myxofactins compound provided by this invention is a class of unsaturated fatty acid amide compounds that can be secreted into the extracellular space. For the first time, the specific structure of fatty acids with suicide activity in myxobacteria has been clarified. It is also a secondary metabolite with suicide activity that can be secreted into the extracellular space in myxobacteria, providing an important new microbial source for the research and development of chemical ecology mediated by small molecules in myxobacteria.

[0019] (2) The myxofactins compound provided by the present invention has no obvious cytotoxic activity and can be used as an antibiotic adjuvant to significantly improve the sensitivity of Gram-negative bacteria such as Acinetobacter baumannii to antibiotics, providing a new approach to solving the problem of multidrug resistance of Gram-negative bacteria and improving the efficacy of existing antibiotics.

[0020] (3) The method for preparing myxofactins compounds provided by the present invention achieves high yield through heterologous expression technology and has the advantages of mild culture conditions, simple and easy culture medium preparation and short fermentation time, which facilitates large-scale production and provides a feasible path for the large-scale preparation of myxofactins compounds and their development and application in the field of multidrug resistance of Gram-negative bacteria. Attached Figure Description

[0021] The accompanying drawings, which form part of this specification, are used to provide a further understanding of the invention. The illustrative embodiments and descriptions of the invention are used to explain the invention and do not constitute an undue limitation thereof. Obviously, those skilled in the art can obtain other drawings based on these drawings without any inventive effort.

[0022] Figure 1 This is the structural formula of the myxofactins compound identified in Example 3 of this invention; Figure 2 This is a diagram showing the experimental results of combining some myxofactins compounds with polymyxin in Example 5 of this invention. Detailed Implementation

[0023] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0024] The technical solution of the present invention will be further described below with reference to specific embodiments. The present invention does not impose any special restrictions on the source of reagents used in the following embodiments; commercially available products well known to those skilled in the art can be used.

[0025] The culture media used in the following examples are as follows: In each liter of CTT solid medium: 10 g casein, 1.97 g MgSO4·7H2O, 10 mL 1M Tris-HCl, 10 mL 100 mM PBS, 20 g agar powder, pH adjusted to 7.6.

[0026] In each liter of CTT liquid medium: casein 10 g, MgSO4·7H2O 1.97 g, 1M Tris-HCl 10 mL, 100 mM PBS 10 mL, pH adjusted to 7.6.

[0027] Example 1 This embodiment provides DK1622- mft and DK1622-Δ mft Obtaining strain F.

[0028] 1. DK1622- mft Acquisition: (1) Myxococcus with accession number CCTCC NO: M 2021520 Myxococcus sp. SDU36 cells (with patent CN113402509A) were cultured in CTT liquid medium for 48 hours, and then collected by centrifugation at 8000 rpm for 5 min. The genome was sent to Eight Star Biotechnology (Wuhan) Co., Ltd. to construct a BAC library. A genome with 20-fold coverage was generated, and each BAC clone had an average insertion of a ~120 kb DNA fragment.

[0029] (2) BAC clones containing the complete myxofactin gene cluster (GenBank accession number CP077414.1, sequence shown in SEQ ID NO. 1) were screened by PCR, and BGC was released using restriction enzymes Xba I and Spe I. The vector pBAC-TnpA containing the Tn5 transposon cassette was linearized by PCR and used with a pair of transposable transposons containing the myxofactin gene cluster (PGC). mft Gene cluster homology arm (40 bp) primer.

[0030] (3) Two linear DNA fragments were co-transformed into electrocompetent Escherichia coli GB2005-dir (purchased from Shanghai Murong Biotechnology Co., Ltd.) for Red / ET recombination, which enabled specific extraction of... mft A continuous DNA sequence from A to P (~43 kb) was obtained by removing redundant sequences, and pBAC-tnpA- was constructed. mft Plasmid; confirmed by restriction enzyme digestion and Sanger sequencing. mft Correct assembly on the carrier pBAC-tnpA.

[0031] (4) PBAC-tnpA- mft The plasmid was transformed into *E. coli* GBred-gyrA462 (purchased from Shanghai Yuchun Biotechnology Co., Ltd.), where the Redαβ recombinase was induced by 10% L-arabinose. Then, the AMP-CcdB fragment containing 40 homologous arms was electroporated into the plasmid containing pBAC-tnpA- mft In plasmid GBred-gyrA462, the selection condition was 100 μg / mL AMP (ampicillin). The restriction endonuclease PacI was used to extract pBAC-tnpA- mft The AMP-CcdB fragment was removed from the plasmid. After assembly using the Gibson assembly method (Nanjing Novizan ClonExpress Ultra one-step cloning kit), the linear plasmid was desalted and transformed into Escherichia coli GB2005 (purchased from Baosai Biotechnology), which was used for anti-selection of CcdB.

[0032] (5) The correct engineering design of the promoter substitution was verified by PCR and Sanger sequencing. Finally, different versions of pBAC-tnpA-mft were tested in the model strain Xanthomonas. Myxococcus xanthus DK1622 was introduced via electroporation under the selective condition of 40 μg / mL kanamycin, thus obtaining DK1622- mft Mutant strain.

[0033] 2. DK1622-Δ mft Obtaining strain F: (1) Myxococcus with accession number CCTCC NO: M 2021520 Myxococcus sp. SDU36 cells (with patent CN113402509A) were cultured in CTT liquid medium for 48 hours, and then collected by centrifugation at 8000 rpm for 5 min. The genome was sent to Eight Star Biotechnology (Wuhan) Co., Ltd. to construct a BAC library. A genome with 20-fold coverage was generated, and each BAC clone had an average insertion of a ~120 kb DNA fragment.

[0034] (2) BAC clones containing the complete myxofactin gene cluster were screened by PCR, and BGC was released using restriction enzymes Xba I and Spe I. The vector pBAC-TnpA containing the Tn5 transposon cassette was linearized by PCR and used with a pair of transposable cassettes containing the myxofactin gene cluster. mft Gene cluster homology arm (40 bp) primer.

[0035] (3) Two linear DNA fragments were co-transformed into electrocompetent Escherichia coli GB2005-dir (purchased from Shanghai Murong Biotechnology Co., Ltd.) for Red / ET recombination, which enabled specific extraction of... mft A continuous DNA sequence from A to P (~43 kb) was obtained by removing redundant sequences, and pBAC-tnpA- was constructed. mft Plasmid; confirmed by restriction enzyme digestion and Sanger sequencing. mft Correct assembly on the carrier pBAC-tnpA.

[0036] (4) PBAC-tnpA- mft The plasmid was transformed into *E. coli* GBred-gyrA462 (purchased from Shanghai Yuchun Biotechnology Co., Ltd.), where the Redαβ recombinase was induced by 10% L-arabinose. Then, the AMP-CcdB fragment containing 40 homologous arms was electroporated into the plasmid containing pBAC-tnpA- mft In plasmid GBred-gyrA462, the selection condition was 100 μg / mL AMP (ampicillin). The restriction endonuclease PacI was used to extract pBAC-tnpA- mft The AMP-CcdB fragment was removed from the plasmid. After assembly using the Gibson assembly method (Nanjing Novizan ClonExpress Ultra one-step cloning kit), the plasmid was desalted and transformed into *E. coli* GB2005 (purchased from Baosai Biotechnology). This strain was used for anti-selection of CcdB. mftF The construction of the knockout plasmid replaces AMP-CcdB. mftF Genes are knocked out.

[0037] (5) The correct engineering design of the promoter substitution was verified by PCR and Sanger sequencing. Finally, different versions of pBAC-tnpA-mft were tested in the model strain Xanthomonas. Myxococcus xanthus DK1622 was transduced using electroporation with a selectable concentration of 40 μg / mL kanamycin, thus obtaining... Myxococcus xanthus DK1622-Δ mft strain F.

[0038] Example 2 This embodiment provides a method for preparing the myxofactins compound.

[0039] 1. Fermentation by bacterial strain (1) Plate culture: The plate culture prepared in Example 1 was used to culture the plate culture. Myxococcus xanthus DK1622- mft or Myxococcus xanthus DK1622- mft - Δmft Strain F was inoculated into CTT solid medium and cultured at 30°C for 3 days to obtain activated strains.

[0040] (2) Seed culture: The activated bacterial cells were inoculated into CTT liquid medium and cultured on a shaker to obtain seed culture solution. The shaker culture conditions were: 30℃, 200 rpm for 3 days.

[0041] (3) Expanded fermentation: The prepared seed culture medium was transferred to fresh CTT liquid medium and placed in a shaker for expanded fermentation. The expanded fermentation culture conditions were: 3 L conical flask, 30℃, 200 rpm for 7 days; the composition of CTT liquid medium was the same as that of the medium in (2).

[0042] 2. Fermentation broth treatment and extraction After fermentation, the fermentation broth was adsorbed with XAD-16 macroporous resin. After adsorption, the surface culture medium components were washed off with water, and the resin was eluted with redistilled methanol. The broth was then evaporated to dryness under reduced pressure at 40°C to obtain the crude fermentation extract. The crude extract was dissolved in a small amount of methanol to obtain the methanol extract, and liquid chromatography-contrast analysis was performed.

[0043] 3. Separation and preparation The methanol extract from step 2 was subjected to gradient elution by reversed-phase medium-low pressure liquid chromatography with water and methanol as the mobile phase. The gradient elution was performed sequentially with 20% methanol, 40% methanol, 60% methanol, 70% methanol, 80% methanol, 90% methanol, and 100% methanol, yielding seven fractions from Fr.1 to Fr.7. Fraction Fr.3 was further fragmented into seven segments (Fr.3-1 to Fr.3-7) using Sephadex LH-20. Finally, Fr.4 to Fr.7 and Fr.3-5 to Fr.3-6 were purified by high performance liquid chromatography using methanol-water as the mobile phase. By comparing with the crude extract HPLC, the components containing the target compound were identified. The target myxofactins compound was obtained by semi-preparative HPLC purification using an Agilent 1260 HPLC system. The purification was performed using a Phenomenex Luna 5 μm C18(2) 100 Å, 250×10 mm column, with a flow rate of 1.8 mL / min, a gradient of 65%-80% methanol, a column temperature of 30℃, and UV spectra of 210nm, 254nm, 280nm, and 365nm. The enriched target myxofactins compound was detected by high-resolution mass spectrometry, nuclear magnetic resonance, partial infrared spectroscopy, and ECD.

[0044] Example 3 This example provides the structural identification of the myxofactins compounds prepared in Example 2. Compounds myxofactins 1-11 and 16 are derived from DK1622- mft The compound myxofactin12-15 was isolated from the fermentation broth and derived from DK1622-Δ mft F was obtained by separating the fermentation broth.

[0045] Myxofactin3 is a yellow crystalline substance with the molecular formula C3. 14 H 23 NO2 is characterized by its HRESIMS peak [M+H]. + of m / z The calculated value is 238.1822 (C). 14 H 24 NO2) was identified. This compound was found in λ. max The presence of ultraviolet absorption at 300 nm confirms the existence of a long conjugated system. 1 The 1H NMR spectrum (Table 1) shows the four conjugated allyl protons in d H 7.54 (d, J = 15.6 Hz), 7.11(dd, J = 15.6, 11.4 Hz), 6.09 (t, J = 11.4 Hz), and 5.36 (d, J = 10.2 Hz); a characteristic single peak in d H 5.19; One methoxy group in d H 3.70; and the three methyl doublets at d H 0.99 (d, J = 6.6 Hz), 0.90(d, J = 6.6 Hz), and 0.88 (d, J = 6.6 Hz). Consistent with these signals, 13 The C NMR spectrum showed seven sps 2 Hybridized carbon, including five alkene carbons, three methyl doublets, and two sp... 3 Methane carbon, a methylene group, and a methoxy group. The structure of myxofactin3 was clearly elucidated by combining COSY, HSQC, and HMBC signals. Specifically, a long-spin system from C-4 to C-13 containing two conjugated double bonds was identified by the COSY signal. D 4,5 and D 6,7 . β The -methoxyacrylate moiety was determined by the HMBC signal, from the oxymethyl singlet (H-13) and the allyl proton singlet (H-2) to... d C 125.4 (C-4) and 164.7 (C-3), etc. H-2 and d CAdditional HMBC correlations between the carbonyl groups at 164.9 (C-6) completed the identification of the overall structure of myxofactin3. (Double bond) D 4,5 and D 6,7 The geometric shapes are respectively judged as E and Z ,according to 1 H- 1 H coupling constant. The key NOESY correlation between H-13 and H-2 establishes the double bond. D 2,3 of E Configuration. The absolute configuration of C-8 was determined based on ECD calculations as follows: S This aligns with its biosynthetic origin. Therefore, myxofactin3 was identified as a linearly functionalized fatty acid amide with... a,b, c,d,e,g Unsaturated, explaining its position in λ max The characteristic ultraviolet absorption at 300 nm has the following structural formula: Figure 1 As shown in Figure 3, R1 = NH2, R2 = CH3. 6,7 =Z.

[0046] Myxofactin4 is a yellow crystalline substance with the molecular formula C0. 14 H 23 NO2, according to HRESIMS [M+H] + peak m / z 238.1809 (calculated value is C) 14 H 24 NO2) confirmed. 1 H and 13 The C10 NMR spectra are very similar to those of 3, and detailed 2D NMR interpretation confirms that they have the same C10-C10 connectivity. However, based on the coupling constant of 15.2 Hz between H-6 and H-7, the C10 NMR spectra of 4 are significantly different. D 6,7 The geometric shape is E Instead Z Its structural formula is as follows Figure 1 As shown in Figure 4, R1 = NH2, R2 = CH3. 6,7 =E.

[0047] myxofactin1 and myxofactin2 are yellow oily substances. 1 The H NMR spectrum contains myxofactin 3 and myxofactin 4. 1All signals were detected by ¹H NMR, but the chemical shift values ​​varied slightly. The elution times of the compounds differed according to HPLC-DAD detection. HRESITOFMS [M+H] was used to further analyze the results. + peak m / z 239.1649 was determined to be C. 14 H 23 O3. The structural formulas of myxofactin1 and myxofactin2 are as follows: Figure 1 As shown in 1 and 2, in myxofactin1, R1 = OH, R2 = CH3, 6,7 =Z; In myxofactin2, R1=OH, R2=CH3 6,7 =E.

[0048] The 1H NMR spectra of compounds myxofactin 1-6 and 16 are summarized in Table 1, and their structural formulas are as follows: Figure 1 As shown in 1~6 and 16.

[0049] Table 1. Proton NMR data of compounds myxofactin 1~6 & 16 ( d H , m, J )

[0050] Note: In Table 1, the NMR data of myxofactin 3, 5, and 6 are in deuterated methanol as solvent, and the NMR data of myxofactin 4 are in deuterated chloroform as solvent.

[0051] The carbon spectral data of myxofactin 3~11 are shown in Table 2.

[0052] Table 2. Carbon spectral data (δ) of myxofactin 3~11 C (type)

[0053] myxofactin5 is a yellow oily substance with a molecular formula determined by HRESITOFMS as C10. 13 H 21 NO2, [M+H] + peak m / z 224.1647, 1 H and 13 The C10 NMR spectrum is very similar to that of 1, as confirmed by COSY, HSQC, and HMBC. d H1.3) The integral is 2, which is one more H than 1, and there is only one methyl signal associated with it. Its structural formula is as follows: Figure 1 As shown in Figure 5, R1 = NH2, R2 = H. 6,7 =Z.

[0054] myxofactin6 is a yellow oily substance with a molecular formula determined by HRESITOFMS as C60. 14 H 24 O5, [M+H] + peak m / z 273.1713, this compound in λ max The presence of UV absorption at 260 nm confirms the existence of a conjugated system, and a blue shift occurs in the UV absorption. The calculated saturation is 3. 1 The H NMR spectrum (Table 1) shows that only two conjugate signals are present. d H 7.55 (d, J = 15.6 Hz, H-4), 6.46 (dd, J = 15.6, 7.2Hz, H-5), a characteristic singlet at d H 5.15 (s, H-2), one methoxy group in d H Signals 3.64 (s, H-13) are indistinguishable from those of signal 3, indicating that signal 6 has undergone significant changes in H-6 and H-7 compared to signal 3. The structure of signal 6 is clearly elucidated by combining COSY, HSQC, and HMBC data. Specifically, the conjugated double bonds... D 6,7 The signal disappeared. d H 4.16 (t, J = 6.6 Hz, H-6), d H 3.3 (m, H-7), d H 1.69 (m, H-8), COSY and HMBC signals show a correlation. Their structural formula is as follows: Figure 1 As shown in Figure 6.

[0055] Myxofactin 7 is a yellow oily substance with a molecular formula determined by HRESITOFMS as C10. 18 H 29 NO3. Although 7 1 The 1H NMR spectrum contained all three signals found, but three additional signals appeared, including two methyl groups in... d H2.17 (s, H-4') and 1.31 (d, J = 6.6 Hz, H-1'), and 4.38 (d, J = 7.2 Hz, H-2'). 7 of 13 The C NMR spectrum has four more carbon signals than 3, including a ketone group. d C 210.5, and three carbons in d C 26.2 (C-4'), 56.1 (C-2'), and 16.4 (C-1') correspond to three new signals resolved by HSQC. This strongly suggests that 7 is a derivative of 3 with an additional attachment at the carboxyl terminus. The identity of the new structural unit was determined to be 3-aminobutane-2-one, based on the key HMBC correlations from H-1' and H-4' to C-2' and C-3'. Although no H-2' / C-1 ( d C The HMBC correlation between 165.4) and [other related information] is considered, but given the molecular formula and the chemical shift of H-2', the connection between C-1 and C-2' should be through a nitrogen atom. Considering that 3-aminobutane-2-one originates from L-alanine, the absolute configuration of C-2' is determined to be [other information]. S The structure of number 7 represents the first natural topological structure combining aminobutane-2-one with a fatty acyl group. Its structural formula is as follows: Figure 1 As shown in Figure 7, R1=0, R2=H. 6,7 =Z.

[0056] The proton NMR spectra of compounds myxofactin 7-11 are shown in Table 3.

[0057] Table 3. 1H NMR data of compounds myxofactin 7-11 ( d H , m, J )

[0058] myxofactin8 is a yellow oily substance with a molecular formula determined by HRESITOFMS as C10. 18 H 31 NO3, [M+H] + peak m / z 310.2374. Although 8 1 H NMR contained seven of the detected signals, but one additional signal appeared. d H 3.74 (m, 1Based on the key HMBC correlations at H-1', H-2', H-4', and 70.8 (C-3'), the carbonyl group of the ketone is missing its C signal, and the carbonyl group at the 3' position is replaced by a hydroxyl group. Its structural formula is as follows: Figure 1 As shown in Figure 8, R1 = OH, R2 = H. 6,7 =Z.

[0059] Myxofactin9 is a yellow oily substance with a molecular formula determined by HRESITOFMS as C10. 18 H 31 NO3, [M+H] + peak m / z 310.2373. and 8. 1 Similar to H NMR, detailed 2D NMR interpretation confirmed they have the same C-C connectivity. However, based on the coupling constant of 15 Hz between H-6 and H-7, the NMR in 9... D 6,7 The geometric shape is E Instead Z Its structural formula is as follows: Figure 1 As shown in Figure 9, R1 = OH, R2 = H. 6,7 =E.

[0060] Myxofactin 10 is a yellow oily substance with a molecular formula determined by HRESITOFMS as C10. 19 H 31 NO3, [M+H] + peak m / z 322.2378.10 1 The 1H NMR spectrum is very similar to that of 7, but a change has been made in its new structural unit, 3-aminobutane-2-one. d H 2.58 (m, H-4') d H 1.04 (t, H-5') and d C Both 213 (C-3') have a key HMBC correlation and a COSY correlation, indicating that the methyl group at H-4' has been replaced by an ethyl group attached to H-3'. Its structural formula is as follows: Figure 1 As shown in Figure 10, R1=O, R2=CH3. 6,7 =Z.

[0061] Myxofactin 11 is a yellow oily substance with a molecular formula determined by HRESITOFMS as C10. 19 H 31 NO3, [M+H] + peakm / z 322.2378. and 10. 1 Similar to H NMR, detailed 2D NMR interpretation confirmed they have the same C-C connectivity. However, based on the coupling constant of 15 Hz between H-6 and H-7, the [missing information - likely a specific value or parameter] in [missing information - likely a specific value or parameter]... D 6,7 The geometric shape is E Instead Z Its structural formula is as follows: Figure 1 As shown in Figure 10, R1=O, R2=CH3. 6,7 =E.

[0062] Myxofactin12 is a yellow, oily substance, according to HRESIMS [M+H] + peak m / z 318.2061 (Calculated value is C) 19 H 28 NO3) has been determined to have the molecular formula C. 19 H 27 The NMR data for NO3.12 contain the entire fatty acyl group of 3, indicating that 12 is another amide derivative of 3. Five additional carbons (C-1'~C-5') present in 12 but not in 3 were identified using a combination of COSY and HMBC experiments. The COSY experiment detected a continuous spin system including a masked doublet methyl group (…). d H 1.43, H-1', d, J = 6.6 Hz), an aliphatic methyl group H-2', and two allylmethane protons H-3' and H-4'. H-2' ( d H 4.87, m) and C-2' ( d C The chemical shift of 59.5) diagnosed the connection with the heteroatom. H-4' ( d H 6.08, dd, J =6.0, 1.8 Hz) and H-3' ( d H 7.41, dd, J The chemical shift and coupling constants (6.0, 1.8 Hz) indicate that they form a Z-configuration double bond, thus forming a... a,b -Unsaturated amide function. This part is derived from the protons H-3' and H-4' to... d CThe HMBC correlation of the carbonyl carbon at 171.9 (C-1') was confirmed. Further HMBC correlation revealed the formation of a closed pyrrolidone ring between H-2' and C-5'. Although no HMBC correlation was observed between H-2' and C-1, given the molecular formula of 12, the pyrrolidone ring is connected through the carbonyl carbon ( d C 169.8) is linked to a 3-fatty acyl group. Similar to 3, based on ECD calculations and the biosynthetic origin of this carbon from L-alanine, the absolute configuration of C-2' in 12 is designated as S. This stereochemical allocation is further supported by the presence of the same structural unit in ypaoamide C, palmyrrolinone, eliamid, and jamaicinamide C. Its structural formula is as follows: Figure 1 As shown in Figure 12, R=H. 6,7 =Z.

[0063] The proton NMR data of myxofactin 12-15 are shown in Table 4.

[0064] Table 4. 1H NMR data of myxofactin 12-15 ( d H , m, J )

[0065] Table 5 shows the carbon spectral data of myxofactin 12-16.

[0066] Table 5. Carbon spectral data of myxofactin 12-16

[0067] Myxofactin 13 and 12 have the same molecular formula C. 19 H 27 NO3.13 1 H and 13 The C10 NMR spectrum is very similar to that of 12. They show similarities in the double bond. D 6,7 The geometry differs slightly, based on the split patterns of the H-6 and H-7. 1 H- 1 H is the coupling constant. The structural formula of myxofactin13 is as follows: Figure 1 As shown in Figure 13, R=H. 6,7 =E. It is worth noting that the variety of natural products with pyrrolidone characteristics is very limited, especially myxofactin12 and myxofactin13, which further increase the complexity of natural products.

[0068] Myxofactin 14 is a yellow oily substance with a molecular formula determined by HRESITOFMS as C10. 20 H 29 NO3, [M+H] + peak m / z 332.2229.14 1 The 12H NMR spectrum is very similar to that of 12H NMR, but changes have been made to its pyrrolidone ring. 1 1H NMR showed an additional fatty acid methyl signal in the high-field region. d H 0.81 (t, J = 7.2 Hz, H-6'), H-6' and C-1' (23.4), C-2' have HMBC correlation, H-6' and H-1' have COSY correlation, indicating that the methyl group of H-1' has been converted to an ethyl group and attached to H-2'. The structural formula of myxofactin14 is as follows Figure 1 As shown in 14, R=CH3, 6,7 =Z.

[0069] Myxofactin 15 is a yellow oily substance with a molecular formula determined by HRESITOFMS as C10. 19 H 29 NO4, [M+H] + peak m / z 336.2176. The fatty amide moiety is the same as 12. The double bond signal of the pyrrolidone ring disappears. The HMBC signal shows a H-2' / C-1 correlation, and the H-3' chemical shift value is... d H 3.41 (1H), H-4' chemical shift values d H Based on mass spectrometry predictions of the chemical formula at 2.38 and 2.46 (2H), it is inferred that a hydroxyl group is attached to the 3' end, and H-4' forms a lactam with the amide. Its structural formula is as follows: Figure 1 As shown in 15.

[0070] Myxofactin 16 is a yellow powder with a molecular formula determined by HRESITOFMS as C16. 11 H 18 O2, [M+H] + peak m / z 183.1394. 1 ¹H NMR showed the disappearance of the singlet of the methoxy group, indicating that the four conjugated allyl protons were in the middle. d H 7.62 (dd, J =15.6 Hz, 12Hz), 6.13 (t,J = 10.8Hz), 5.86 (d, J = 15Hz), and 5.64(t, J = 10.8Hz), the COSY signal determines their connection method, and 2D NMR ultimately determines their structure. Its structural formula is as follows: Figure 1 As shown in 16.

[0071] Example 4 This embodiment provides an experiment to detect the cytotoxic activity of the myxofactins compound identified in Example 3 above.

[0072] 1. Tumor cell lines The three tumor cell lines used in the experiment included human prostate cancer cells PC-7, human breast cancer cells MCF-7, and human cervical cancer cells HeLa.

[0073] 2. Principle of cytotoxicity (CCK-8 assay): The CCK-8 assay was used for detection. The detection principle is that the CCK-8 reagent contains WST-8, which, under the action of the electron carrier 1-methoxy-5-methylphenazine sulfate dimethyl ester (1-Methoxy PMS), is reduced by dehydrogenases in the cell mitochondria to a highly water-soluble yellow formazan product. The amount of formazan generated is directly proportional to the number of live cells.

[0074] 3. Experimental methods: (1) Cell inoculation: Three types of tumor cells were prepared into single-cell suspensions using culture medium containing 10% fetal bovine serum, and then inoculated into each well of a 96-well plate. Adherent cells were inoculated at a rate of 90 μL / well (5 × 10⁶ cells / well). 4 Inoculate at a rate of 90 μL / well (9 × 10⁶ cells / mL), and inoculate suspension cells at a rate of 90 μL / well (9 × 10⁶ cells / mL). 4 Inoculate at a rate of 1 per mL and pre-culture at 37°C for 24 h under 5% CO2 conditions.

[0075] (2) Add the sample solution of the myxofactins compound to be tested: Add 10 μL of sample solution to each well. Set one concentration for each sample for the initial activity screening, and set three replicates. Determine eight concentrations (including 0 concentration) by IC50, and set three replicates for each concentration. Incubate in an incubator for 48 h. The experiment includes a blank group, a control group, and a drug group.

[0076] (3) Color development: Aspirate the old culture medium and drug solution from the adherent cells (add 10 μL of original CCK-8 solution directly to the suspended cells), add 100 μL of CCK-8 solution diluted ten times to each well, and continue to culture at 37℃ and 5% CO2 for 1-4 h (operate in the dark and observe in real time).

[0077] (4) Detection: The absorbance at 450 nm was measured using an ELISA reader, and the raw data results were recorded. The raw data were standardized using Excel software. The cell proliferation inhibition rate was initially calculated based on the OD value of each well, using the formula: = (OD...) / (...) Control -OD Drug ) / (OD Control -OD Blank ) × 100%, statistical inhibition rate. IC 50 The experimental results were calculated using GraphPad Prism 8 (version 8.0.2, GraphPad Software Inc.) and are expressed as ±SD.

[0078] (6) Positive control: Docetaxel (Doc).

[0079] 4. Experimental Results The cytotoxic activity test results of some myxofactins compounds (myxofactin3, 7, 12) against three cancer cell lines are shown in Table 6. These three compounds represent three different types of unsaturated fatty acid amides of myxofactins, none of which have cytotoxic activity.

[0080] Table 6. Cytotoxic activity tests of selected myxofactins compounds against three cancer cell lines.

[0081] Example 5 This embodiment provides an experiment on the combined use of myxofactins compounds with antibiotics.

[0082] Although myxofactin8 itself has no antibiotic activity, its status as a potent surfactant and membrane permeabilizer suggests that it may enhance the efficacy of clinical antibiotics. To verify this, a checkerboard dilution assay was performed between myxofactin8 and several FDA-approved antibiotics, including chloramphenicol, cefotaxime, gentamicin, tetracycline, polymyxin, neomycin, azithromycin, and rifampin. Gram-negative Acinetobacter baumannii 07AC366 56, resistant to polymyxin, was used as an indicator strain. The results, shown in Table 7, indicate that 0.1 mg / mL of myxofactin8 can improve the sensitivity of this pathogen to most of the tested antibiotics. Among these combinations, myxofactin8 exhibited the best synergistic effect with polymyxin, reducing its minimum inhibitory concentration (MIC) by up to 128-fold, with a FIC (partial inhibitory concentration) index of 0.064.

[0083] Table 7 Results of combined use of myxofactin 8 and antibiotics

[0084] Note: a. MIC refers to the MIC value of antibiotics only, without myxofactin8; b. MIC for combined use refers to the MIC value of antibiotics containing both myxofactin8 and antibiotics.

[0085] Further research revealed that different colistin homologues (myxofactin 2, 3, 8, 10, 11, 12, 13) enhanced the potency of polymyxins, with compounds myxofactin 2 and 3 showing the highest activity (e.g., ...). Figure 2 (As shown). However, compounds myxofactin2 and 3 exhibited significant hemolytic toxicity at a concentration of 0.1 mg / mL, while myxofactin8 did not show such toxicity at the same concentration, suggesting its potential as a novel antibiotic adjuvant against the growing polymyxin resistance.

[0086] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A compound of myxococci derived from Myxococcus, characterized in that, Its structure is shown in equation (I), equation (II), or equation (III): Formula (I); Equation (II); Equation (III); In the compound of formula (I), R1 is selected from hydroxyl, amino, or any of the following substituents: , , or ; R2 is selected from hydrogen or methyl, R3 is selected from hydrogen or methyl, R4 is selected from hydrogen or methyl, and R5 is selected from hydrogen or methyl.

2. The myxococcal-derived myxoactins compound as described in claim 1, characterized in that, Compounds of formula (I) are selected from any of the following structures: , , , , , , , or .

3. The method for preparing myxobolus-derived myxococcal compounds as described in claim 1 or 2, characterized in that, Includes the following steps: DNA fragments containing the myxofactin gene cluster were obtained from Myxococcus sp. SDU36, which has the accession number CCTCC NO: M 2021520, through BAC library construction. The myxofactin gene cluster has the GenBank accession number CP077414.

1. In vitro, scarless modification of DNA fragments containing the myxofactin gene cluster, including promoter substitution and / or gene knockout; The modified myxofactin gene cluster was introduced into the heterologous host bacterium Myxococcus xanthus DK1622 for fermentation culture to obtain fermentation broth; The fermentation broth was sequentially extracted and separated to obtain the myxofactins compound.

4. The preparation method according to claim 3, characterized in that, The in vitro scarless modification step includes: releasing the myxofactin gene cluster using restriction endonucleases Xba I and Spe I; and connecting the linearized vector pBAC-TnpA containing the Tn5 transposon cassette to the gene cluster using Red / ET recombination technology to form the recombinant vector pBAC-tnpA-mft. The recombinant vector pBAC-tnpA-mft was transformed into *E. coli* GBred-gyrA462 expressing Redαβ recombinase, and Redαβ recombinase activity was induced by L-arabinose. Anti-selection was performed using the AMP-CcdB fragment, and the AMP-CcdB fragment was removed using the restriction endonuclease PacI. Promoter replacement was performed using the Gibson assembly method; alternatively, promoter replacement and scarless knockout were performed using the Gibson assembly method. mftF Manipulation of genes.

5. The preparation method according to claim 3, characterized in that, The fermentation medium used in the fermentation culture comprises per liter: 8-12g casein, 1.5-2.5g MgSO4·7H2O, 8-12mL 1M Tris-HCl, 8-12mL 100mM PBS, with a pH of 7.4-7.8; the fermentation culture temperature is 28-32℃.

6. The preparation method according to claim 3, characterized in that, The extraction process involves: adsorbing the fermentation broth with macroporous adsorption resin, washing with water, eluting the resin with an organic solvent, and concentrating to obtain the crude fermentation extract.

7. The preparation method according to claim 6, characterized in that, The separation operation is as follows: the fermentation crude extract is sequentially separated by reversed-phase medium-low pressure liquid chromatography, normal-phase chromatography, and semi-preparative high-performance liquid chromatography to obtain the myxofactins compound.

8. The use of the myxococcal-derived myxoactins compound as described in claim 1 or 2 in the preparation of drugs that enhance bacterial susceptibility to antibiotics.

9. The application as described in claim 8, characterized in that, The bacteria include Acinetobacter baumannii, Staphylococcus aureus, Escherichia coli, or Burkholderia; the antibiotics include β-lactam antibiotics, quinolone antibiotics, and aminoglycoside antibiotics.

10. A drug for improving the sensitivity of bacteria to antibiotics, characterized in that, Contains an effective amount of the myxococcal-derived myxobolus compound and antibiotic as described in claim 1 or 2; The bacteria include Acinetobacter baumannii, Staphylococcus aureus, Escherichia coli, or Burkholderia; the antibiotics include β-lactam antibiotics, quinolone antibiotics, and aminoglycoside antibiotics.

Images