A polyketide compound efrotomycin, and a preparation method and application thereof

Abstract

The application discloses a polyketide compound efrotomycin and a preparation method and application thereof. A structural formula of the polyketide compound efrotomycin is shown as formula (I). Four new polyketide compounds efrotomycins A1-A4 (1-4) are separated from a fermentation culture of Actinomyces cihanbeyliensis DSM 45679, and a BAC plasmid pCSG8112 containing a biosynthesis gene cluster of the efrotomycin is successfully expressed in a heterologous host S. lividans SBT18, and a new compound efrotomycin B1 (5) is generated. The polyketide compounds efrotomycins (1-5) have activities against Micrococcus luteus and Streptomyces, and can be used for preparing active drugs against the Micrococcus luteus and the Streptomyces.

Description

Technical fields:

[0001] This invention belongs to the field of industrial microbiology, specifically relating to polyketide compounds etrotomycins A1-A4 and etrotomycin B1, their preparation methods, and applications. Background technology:

[0002] Efrotomycin belongs to the elfamycin family of antibiotics. It acts on bacterial elongation factors to inhibit protein synthesis, thus exhibiting narrow-spectrum antibacterial activity. In this invention, novel glycosylated elfamycin antibiotics, efrotomycins A1-A4, were isolated from the actinomycete *A. cihanbeyliensis* DSM 45679, and non-glycosylated efrotomycin B1 was obtained through heterologous expression of the efrotomycin biosynthetic gene cluster. Summary of the Invention:

[0003] The purpose of this invention is to overcome the deficiencies in the prior art and to provide the polyketide compound etrotomycin, its preparation method, and its applications.

[0004] The first object of the present invention is to provide polyketide compounds etrotomycins A1-A4 (1-4) and etrotomycin B1 (5), the structures of which are shown in formula (I):

[0005]

[0006] The second objective of this invention is to provide a recombinant strain S. lividans SBT18 / pCSG8112, characterized in that it is obtained by transforming the biosynthetic gene cluster of the polyketide compound etrotomycin into Streptomyces lividansSBT18.

[0007] A third object of the present invention is to provide the use of the actinomycete A. cihanbeyliensis DSM 45679 in the preparation of the polyketide compounds etrotomycins A1-A4.

[0008] A fourth object of the present invention is the use of the recombinant strain S. lividans SBT18 / pCSG8112 in the preparation of the polyketide compound etrotomycin B1.

[0009] The fifth object of the present invention is to provide a method for preparing the polyketide compounds etrotomycins A1-A4, characterized in that the polyketide compounds etrotomycins A1-A4 are isolated from the fermentation culture of the actinomycete A. cihanbeyliensis DSM 45679.

[0010] Preferably, the specific steps are as follows:

[0011] a) The actinomycete A. cihanbeyliensis DSM 45679 was fermented in AM3 medium to obtain the fermentation culture. The fermentation broth and mycelium were collected by centrifugation. The fermentation broth was adsorbed with macroporous resin XAD-16 and then eluted with acetone. The mycelium was extracted with methanol and the cells were sonicated to disrupt the cells. After the organic solvents were recovered from both, the remaining aqueous phase was extracted with ethyl acetate and concentrated to dryness under reduced pressure to obtain the crude extract of actinomycete A. cihanbeyliensis DSM 45679 in AM3 medium.

[0012] b. The crude extract was separated by normal-phase silica gel column chromatography, using chloroform / methanol as the eluent, eluting sequentially at v / v ratios of 100:0, 10:1, 4:1, 2:1, and 0:100. The fraction Fr.2 eluted at a chloroform / methanol v / v ratio of 10:1 was collected. Fraction Fr.2 was then separated by normal-phase silica gel column chromatography, eluted sequentially at v / v ratios of cyclohexane / ethyl acetate 2:1, cyclohexane / ethyl acetate 1:1, cyclohexane / ethyl acetate 1:2, ethyl acetate, and acetone. The ethyl acetate eluted fraction Fr2-4 was separated by gel column chromatography with chloroform / methanol 1:1, v / v to obtain fraction Fr2-4-1-Fr2-4-4; fraction Fr2-4-2 was subjected to medium-pressure reversed-phase chromatography with gradient elution using water and acetonitrile to obtain fraction Fr2-4-2-1-Fr2-4-2-13; fraction Fr2-4-2-8 was purified by semi-preparative HPLC to obtain compounds etrotomycin A1 and etrotomycin A2; fraction Fr2-4-2-7 was purified by semi-preparative HPLC to obtain compounds etrotomycin A3 and etrotomycin A4.

[0013] Preferably, the fermentation culture method of the actinomycete A. cihanbeyliensis DSM 45679 is as follows: Activated actinomycete A. cihanbeyliensis DSM 45679 is inoculated into AM3 medium and cultured at 28℃ and 200rpm for 3 days to obtain a seed culture; the seed culture is inoculated into AM3 medium at an inoculation rate of 10%, v / v, and cultured at 28℃ and 200rpm for 5-7 days to obtain the fermentation culture; AM3 medium consists of 5g soybean flour, 15g bacteriological peptone, 15g soluble starch, 15g glycerol, 2g CaCO3, 30g sea salt, and purified water to 1L, pH 7.2-7.4.

[0014] The sixth object of the present invention is to provide a method for preparing the polyketide compound etrotomycin B1, characterized in that the polyketide compound etrotomycin B1 is isolated from the fermentation culture of the recombinant strain S. lividans SBT18 / pCSG8112.

[0015] Preferably, the specific steps are as follows:

[0016] a) The recombinant strain S. lividans SBT18 / pCSG8112 was fermented in AM3 medium to obtain the fermentation culture. The fermentation broth and mycelium were collected by centrifugation. The fermentation broth was adsorbed with macroporous resin XAD-16 and then eluted with acetone. The mycelium was extracted with methanol and the cells were sonicated to disrupt the cells. After the organic solvents were recovered from both, the remaining aqueous phase was extracted with ethyl acetate and concentrated to dryness under reduced pressure to obtain the crude extract of recombinant strain S. lividans SBT18 / pCSG8112 in AM3 medium.

[0017] b. The crude extract was separated by normal-phase silica gel column chromatography, using gradient elution with chloroform, chloroform / methanol 4:1, chloroform / methanol 2:1, and methanol, v / v, to obtain fractions Fr1-Fr4 sequentially; fraction Fr2 eluted with chloroform / methanol 4:1, v / v was separated by gel column chromatography with chloroform / methanol 1:1, v / v as the eluent to obtain fractions Fr2-1-Fr2-4; fraction Fr2-2 was purified by semi-preparative HPLC to obtain the compound etrotomycin B1.

[0018] Preferably, the fermentation culture method of recombinant strain S. lividans SBT18 / pCSG8112 is as follows: recombinant strain S. lividans SBT18 / pCSG8112 is inoculated into AM3 medium and cultured at 28℃ and 200rpm for 5-7 days to obtain fermentation culture.

[0019] A seventh object of the present invention is to provide the use of the aforementioned polyketide compounds etrotomycins A1-A4, etrotomycin B1, or pharmaceutically acceptable salts thereof in the preparation of antibacterial drugs. The antibacterial drugs are those effective against Micrococcus luteus and Streptomyces.

[0020] This invention isolated four new compounds, etrotomycins A1-A4, from the fermentation culture of actinomycete A. cihanbeyliensis DSM 45679 in AM3 medium. Furthermore, the BAC plasmid pCSG8112, containing the gene cluster for the biosynthesis of the polyketide compound etrotomycin, was successfully expressed in the heterologous host S. lividans SBT18, resulting in the new compound etrotomycin B1. The polyketide compounds etrotomycins A1-A4 and etrotomycin B1 exhibit anti-Micrococcus luteus and Streptomyces activity and can be used to prepare active drugs against these bacteria.

[0021] The actinomycete *A. cihanbeyliensis* DSM 45679 of this invention is disclosed in the literature: Tatar, D.; Sazak, A.; Guven, K.; Cetin, D.; Sahin, N. Int. J. Syst. Evol. Microbiol. 2013, 63, 3739-3743. The applicant also holds this strain and guarantees its availability to the public for the next 20 years.

[0022] The heterologous expression host Streptomyces lividans SBT18 of this invention is disclosed in the literature: Peng, QY; Gao, GX; Lu, J.; Long, QS; Chen, XF; Zhang, F.; Xu, M.; Liu, K.; Wang, YM; Deng, ZX; Li, ZY; Tao, MFFront. Microbiol. 2018, 9. The applicant also holds this strain and guarantees its availability to the public for the next 20 years. Attached image description:

[0023] Figure 1 These are the compound structures of the polyketide compounds etrotomycins A1-A4 and etrotomycin B1.

[0024] Figure 2 This is a schematic diagram of the efrotomycin biosynthesis gene cluster.

[0025] Figure 3 This indicates the location of the efrotomycin biosynthesis gene cluster in the BAC plasmid pCSG8112.

[0026] Figure 4 This is an HPLC chromatogram of the efrotomycin biosynthetic gene cluster heterologously expressed in S. lividans SBT18. (i) Heterologous expression

[0027] The host bacterium S. lividans SBT18 contains plasmid pSET152; (ii) the heterologous expression host bacterium S. lividans SBT18 contains plasmid pCSG8112; and (iii) standards of the compound etrotomycins A1-A4 (1-4).

[0028] Figure 5 This is a paper diffusion experiment to test the toxicity of compounds 1-5 to Streptomyces.

[0029] Figure 6 This is the HR-ESI-MS spectrum of compound efrotomycin A1(1).

[0030] Figure 7 It is the compound efrotomycin A1(1) 1 H NMR (δ) H Spectra (7.70-4.20).

[0031] Figure 8 It is the compound efrotomycin A1(1) 1 H NMR (δ) H 4.20-0.50) spectrum.

[0032] Figure 9 It is the compound efrotomycin A1(1) 13 C and DEPT135 NMR spectra.

[0033] Figure 10 This is the HSQC spectrum of compound efrotomycin A1(1).

[0034] Figure 11 This is the COSY spectrum of compound efrotomycin A1(1).

[0035] Figure 12 This is the HMBC spectrum of compound efrotomycin A1(1).

[0036] Figure 13 This is the ROESY spectrum of compound efrotomycin A1(1).

[0037] Figure 14This is the J-HMBC spectrum of compound efrotomycin A1(1).

[0038] Figure 15 This is the HR-ESI-MS spectrum of compound efrotomycin A2(2).

[0039] Figure 16 It is the compound efrotomycin A2(2) 1 H NMR (δ) H Spectra (7.70-4.10).

[0040] Figure 17 It is the compound efrotomycin A2(2) 1 H NMR (δ) H 4.20-0.40) spectrum.

[0041] Figure 18 It is the compound efrotomycin A2(2) 13 C and DEPT135 NMR spectra.

[0042] Figure 19 This is the HSQC spectrum of compound efrotomycin A2(2).

[0043] Figure 20 This is the COSY spectrum of compound efrotomycin A2(2).

[0044] Figure 21 This is the HMBC spectrum of compound efrotomycin A2(2).

[0045] Figure 22 This is the ROESY spectrum of compound efrotomycin A2(2).

[0046] Figure 23 This is the J-HMBC spectrum of compound efrotomycin A2(2).

[0047] Figure 24 This is the HR-ESI-MS spectrum of compound efrotomycin A3(3).

[0048] Figure 25 It is the compound efrotomycin A3(3) 1 H NMR (δ) H Spectrum (7.70-4.00).

[0049] Figure 26 It is the compound efrotomycin A3(3)1 H NMR (δ) H 4.00-0.50) spectrum.

[0050] Figure 27 It is the compound efrotomycin A3(3) 13 C and DEPT135 NMR spectra.

[0051] Figure 28 This is the HSQC spectrum of compound efrotomycin A3(3).

[0052] Figure 29 This is the COSY spectrum of compound efrotomycin A3(3).

[0053] Figure 30 This is the HMBC spectrum of compound efrotomycin A3(3).

[0054] Figure 31 This is the ROESY spectrum of compound efrotomycin A3(3).

[0055] Figure 32 This is the J-HMBC spectrum of compound efrotomycin A3(3).

[0056] Figure 33 This is the HR-ESI-MS spectrum of compound efrotomycin A4(4).

[0057] Figure 34 It is the compound efrotomycin A4(4) 1 H NMR (δ) H Spectrum (7.70-4.00).

[0058] Figure 35 It is the compound efrotomycin A4(4) 1 H NMR (δ) H 4.10-0.60) spectrum.

[0059] Figure 36 It is the compound efrotomycin A4(4) 13 C and DEPT135 NMR spectra.

[0060] Figure 37 This is the HSQC spectrum of compound efrotomycin A4(4).

[0061] Figure 38 This is the COSY spectrum of compound efrotomycin A4(4).

[0062] Figure 39 This is the HMBC spectrum of compound efrotomycin A4(4).

[0063] Figure 40 This is the ROESY spectrum of compound efrotomycin A4(4).

[0064] Figure 41 This is the J-HMBC spectrum of compound efrotomycin A4(4).

[0065] Figure 42 This is the HR-ESI-MS spectrum of compound efrotomycin B1(5).

[0066] Figure 43 It is the compound efrotomycin B1(5) 1 H NMR (δ) H Spectrum (7.80-4.00).

[0067] Figure 44 It is the compound efrotomycin B1(5) 1 H NMR (δ) H 4.20-0.50) spectrum.

[0068] Figure 45 It is the compound efrotomycin B1(5) 13 C and DEPT135 NMR spectra.

[0069] Figure 46 This is the HSQC spectrum of compound efrotomycin B1(5).

[0070] Figure 47 This is the COSY spectrum of compound efrotomycin B1(5).

[0071] Figure 48 This is the HMBC spectrum of compound efrotomycin B1(5).

[0072] Figure 49 This is the ROESY spectrum of compound efrotomycin B1(5).

[0073] Figure 50 This is the J-HMBC spectrum of compound efrotomycin B1(5). Detailed implementation method:

[0074] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0075] 1. Isolation of etrotomycins A1-A4 from Actinomycete A. cihanbeyliensis DSM 45679

[0076] Through culture medium screening, AM3 medium was selected for scale-up fermentation culture of actinomycete A. cihanbeyliensis DSM 45679. After extraction, crude fermentation extract was obtained. Four polyketide compounds, etrotomycins A1-A4, were then isolated from the crude fermentation extract using normal-phase, reverse-phase, gel permeation, and HPLC separation methods. Figure 1 ).

[0077] 2. Localization and heterologous expression of the A1-A4 biosynthetic gene cluster of polyketide compounds etrotomycins.

[0078] Polyketides, specifically ferotomycins A1-A4, belong to the elfamycin family of antibiotics. Guided by the reported kirromycin biosynthesis gene cluster within the elfamycin family, comparative genomics was used to locate the ferotomycin biosynthesis gene cluster efr(A. cihanbeyliensis DSM 45679) in the genome of this actinomycete. Figure 2 To verify whether the efr gene cluster is responsible for the synthesis of efrotomycins, the BAC plasmid pCSG8112 containing the efr gene cluster, obtained through screening, was heterologously expressed. The results showed that the efr gene cluster was successfully expressed in the heterologous host *S. lividans* SBT18. Figure 4 ), and the heterologous expression product etrotomycin B1 was isolated.

[0079] 3. Evaluation of the bioactivity of polyketide compounds etrotomycins A1-A4 and etrotomycin B1

[0080] The antibacterial activity of polyketide compounds etrotomycins A1-A4 and etrotomycin B1 was evaluated using the Mueller-Hinton broth microdilution method. All five compounds showed moderate inhibitory activity against *Micrococcus luteus* SCSIO ML01. The toxicity of compounds 1-5 against three *Streptomyces* species was tested using a disc diffusion assay. All five compounds were found to be toxic to *Streptomyces albus* Del14, while etrotomycins A1(1) and etrotomycin B1(5) were also toxic to *Streptomyces lividans* SBT18 and *Streptomyces coelicolor* YF11. Figure 5 ).

[0081] The following are further implementation examples, which are intended to help understand the invention and are used for illustration only and not to limit the invention.

[0082] Example 1: Isolation and preparation of polyketide compounds efrotomycins A1-A4

[0083] 1. Scale-up fermentation culture

[0084] After activating the actinomycete A. cihanbeyliensis DSM 45679 on ISP4 agar plates, an appropriate amount of mycelium was scraped and inoculated into 50 mL of AM3 medium. The culture was then incubated at 28°C and 200 rpm for 3 days to obtain the seed culture. The seed culture was then inoculated into Erlenmeyer flasks containing 200 mL of AM3 medium (total 15 L) at a 10% v / v inoculation rate. The culture was then incubated at 28°C and 200 rpm for 5-7 days to obtain the fermentation culture.

[0085] ISP4 medium: 10g soluble starch, 2g (NH4)2SO4, 1g K2HPO4, 2g CaCO3, 1g NaCl, 1g MgSO4·7H2O, 1mL 1× trace element, 30g sea salt, add purified water to 1L, pH 7.2-7.4.

[0086] AM3 medium: 5g soybean flour, 15g bacteriological peptone, 15g soluble starch, 15g glycerol, 2g CaCO3, 30g sea salt, add purified water to 1L, pH 7.2-7.4.

[0087] 2. Extraction of fermentation broth

[0088] After centrifuging the fermentation culture at 3900 rpm for 20 min, the fermentation broth and mycelium were collected separately. The fermentation broth was adsorbed with 2 L of macroporous resin XAD-16 and then eluted with 15 L of acetone. The mycelium was extracted four times with 1 L of methanol, with each extraction followed by ultrasonic cell disruption for 1 h. The organic solvents in both fractions were then recovered using a rotary evaporator. The remaining aqueous phases were combined and extracted ten times with 1 L of ethyl acetate. After recovering the ethyl acetate using a rotary evaporator, the crude extract of actinomycete A. cihanbeyliensis DSM 45679 on AM3 medium was obtained.

[0089] 3. Isolation of compounds

[0090] The crude extract (30.0 g) of actinomycete A. cihanbeyliensis DSM 45679 on AM3 medium was dissolved in chloroform and methanol (1:1 v / v), and 70 mL of 100-200 mesh silica gel was added and rotary evaporated. The sample was packed into a column using a dry method with a silica gel ratio of 1:3 (70 mL for mixing) / 200 mL for separation. Gradient elution was performed using chloroform and methanol as eluent (chloroform, chloroform / methanol 10:1, chloroform / methanol 4:1, chloroform / methanol 2:1, methanol, v / v) to obtain fractions Fr1 to Fr5 in sequence. The fraction Fr2 (chloroform / methanol 10:1 eluent, 10.0 g) was dissolved again in chloroform and methanol (v / v). 20 mL of 100-200 mesh silica gel was added and the mixture was stirred by rotary evaporation. A dry column was packed with a 1:5 ratio of sample silica gel (20 mL) to separation silica gel (100 mL). A gradient elution was performed using cyclohexane / ethyl acetate as the eluent (cyclohexane / ethyl acetate 2:1, cyclohexane / ethyl acetate 1:1, cyclohexane / ethyl acetate 1:2, ethyl acetate, acetone, v / v) to obtain fractions Fr2-1 to Fr2-5 sequentially. Fraction Fr2-4 (ethyl acetate eluent) was separated by Sephadex LH-20 gel column chromatography (120 cm × 3 cm, chloroform / methanol 1:1, v / v). One 10 mL vial was collected, and fractions Fr2-4-1 to Fr2-4-4 were combined based on TLC results. Fraction Fr2-4-2 (bottles 6-13) was subjected to medium-pressure reversed-phase chromatography (YMC*GELODS-A-HG, 12nm S-50μm) with phase A / phase B = water / acetonitrile (v / v) according to the following program: 10% B (0-20 min), 10% B-20% B (20-30 min), 20% B (30-50 min), 20% B-30% B (50-60 min), 30% B (60-90 min), 30% B-40% B (90-110 min), 40% B (110-140 min). 0 min), 40% B-50% B (140-150 min), 50% B (150-170 min), 50% B-70% B (170-180 min), 70% B (180-200 min), 70% B-90% B (200-210 min), 90% B (210-240 min), to obtain fractions Fr2-4-2-1 to Fr2-4-2-13 in sequence.

[0091] The fraction Fr2-4-2-8 [40% B-50% B (140-150 min) elution fraction] was subjected to semi-preparative high-performance liquid chromatography (Phenomenex Kinetex C18, 250 mm × 10.0 mm, 5 μm; A phase was water, B phase was acetonitrile, 48% B phase was eluted isocratically; flow rate was 2.5 mL·min -1The compounds etrotomycin A1(1) (Rt = 20.0 min) and etrotomycin A2(2) (Rt = 22.0 min) were purified by detection at a wavelength of 360 nm. The fraction Fr2-4-2-7 [40% B (110-140 min) elution fraction] was subjected to semi-preparative high performance liquid chromatography (Phenomenex Kinetex C18, 250 mm × 10.0 mm, 5 μm; A phase was water, B phase was acetonitrile, 45% B phase was isocratic elution; flow rate was 2.5 mL·min -1 The compounds etrotomycin A3(3) (Rt = 18.0 min) and etrotomycin A4(4) (Rt = 19.5 min) were obtained by purification at a detection wavelength of 360 nm.

[0092] 4. Structural identification of compounds

[0093] The structures of compounds 1-4 were determined by HR-ESI-MS. 1 H NMR, 13 The NMR spectra of compound efrotomycin A1(1) were used for identification, and their NMR data are listed in Tables 1 and 2. The spectrum of compound efrotomycin A1(1) is shown in Table 1. Figure 6-14 The spectrum of efrotomycin A2(2) is shown below. Figure 15-23 The spectrum of efrotomycin A3(3) is shown below. Figure 24-32 The spectrum of efrotomycin A4(4) is shown below. Figure 33-41 .

[0094] Therefore, the structural formulas of compounds 1-4 are determined as follows:

[0095]

[0096] Table 1. Compounds 1 and 2 1 H (700MHz) and 13 C(175MHz) NMR data (CD3OD)

[0097]

[0098] Continued table

[0099] Table 2. Compounds 3 and 4 1 H (700MHz) and 13 C(175MHz) NMR data (CD3OD)

[0100]

[0101] Continued table

[0102]

[0103] Example 2: Localization of the A1-A4 biosynthetic gene cluster of polyketide compounds efrotomycins

[0104] Polyketides, specifically ferotomycins A1-A4, belong to the elfamycin family of antibiotics. Among the elfamycin family, the biosynthetic gene clusters of kirromycin, factumycin, and aurodox have been reported. Guided by the kirromycin biosynthetic gene cluster, comparative genomics was used to locate the ferotomycin biosynthetic gene cluster efr (GenBank accession no. OP381654) in the genome of actinomycete *A. cihanbeyliensis* DSM 45679 (GenBank accession no. VFML01000000). Figure 2 The efrotomycin gene cluster *efr* and the *kirromycin gene cluster *kir* show high similarity, containing 20 open reading frames (Table 3). Among them, five trans-AT PKS genes (efrAI, *efrAII*, and *efrAIV-efrAVI*), two trans-AT genes (efrCI and *efrCII*), one PKS / NRPS gene (efrAIII), one NRPS gene (efrB), and one cyclase gene (efrH) are responsible for the synthesis of the polyketide backbone. Five other genes, including two glycosyltransferase genes (efrGI and *efrGII*), two methyltransferase genes (efrMIII and *efrMIV*), and one reductase gene (efrOIV), are responsible for the synthesis and assembly of disaccharide units. The remaining two P450 enzyme genes (efrOI and *efrOII*), two methyltransferase genes (efrMI and *efrMII*), and one dioxygenase gene (efrOIII) may encode post-modification enzymes. However, genes encoding precursor supply proteins, transport proteins, regulatory proteins, and some proteins with unknown functions in the kir gene cluster are not present in the efr gene cluster (Table 3).

[0105] Table 3. Gene function annotations of the Efrotomycin biosynthesis gene cluster

[0106]

[0107] Continued table

[0108]

[0109] Example 3: Screening of BAC plasmids containing the efrotomycin biosynthesis gene cluster

[0110] Primers Efr-testF1 / R1 and Efr-testF2 / R2 (Table 4) were designed based on the upstream and downstream boundary genes of the efrtomycin biosynthesis gene cluster to screen the BAC library of *A. cihanbeyliensis* DSM 45679. A positive BAC plasmid pCSG8112 was obtained, and end sequencing confirmed that pCSG8112 contained all genes of the efrtomycin biosynthesis gene cluster. Figure 3 ).

[0111] Table 4. Primers used in this invention

[0112]

[0113] Example 4: Construction of recombinant strain S. lividans SBT18 / pCSG8112

[0114] The BAC plasmid pCSG8112 was introduced into the heterologous host *S. lividans* SBT18 via a triparental conjugation transfer method. The specific conjugation transfer process is as follows: After culturing *S. lividans* SBT18 on MS solid agar plates for 5-7 days, an appropriate amount of spores was collected using a sterilized bamboo skewer and added to 500 μL of TSB medium. The mixture was shaken and incubated, then heat-shocked at 50°C for 10 min and recovered at 28°C with a shaker at 200 rpm for 2 h. This incubator served as the recipient bacterium for conjugation transfer. The donor bacterium *Escherichiacoli* DH10B / pCSG8112 was inoculated into 5 mL of LB liquid medium (containing 50 μg / mL). -1 Apopramine), and simultaneously inoculated E. coli ET12567 / pUB307 into 5 mL LB liquid medium (containing 50 μg / mL). -1 Kanamycin and 50 μg·mL -1 Chloramphenicol was added, and the mixture was incubated overnight at 37°C with a shaker at 200 rpm. Then, 100 μL of the overnight cultured E. coli DH10B / pCSG8112 and E. coli ET12567 / pUB307 were transferred to 10 mL of LB broth (containing 50 μg / mL chloramphenicol). -1 Apopramine - LB liquid medium for donor bacteria, or containing 50 μg / mL -1 Kanamycin and 50 μg·mL -1Chloramphenicol (LB medium for assisted bacteria), incubate at 37°C on a shaker at 200 rpm for 3-4 hours until OD. 600 The value was approximately 0.6. Two bacterial strains were collected by centrifugation at 3900 rpm for 10 min, washed twice with 10 mL of antibiotic-free LB liquid medium, and then resuspended separately in 250 μL of LB liquid medium. Finally, 250 μL of donor bacteria, 250 μL of auxiliary bacteria, and 500 μL of recipient bacteria were mixed and spread onto Mg... 2+ Place the plates on antibiotic-free solid plates containing 20 mM ISP4 medium, air dry, and incubate upside down at 28°C for 18-20 hours. Remove the plates and dilute with water containing 50 μg / mL antibiotic. -1 Apopramine and 100 μg·mL -1 Cover the plate with trimethoprim, dry it, and incubate it upside down in a 28°C incubator for 5-7 days. Once the zygotes have grown, streak them onto MS medium solid plates (containing 50 μg / mL). -1 Apopramine and 100 μg·mL -1 (Trimethoprim). After incubation at 28°C with the mixture inverted for 2 days, genomic DNA of the conjugate was extracted and verified using detection primers Efr-testF1 / R1 and Efr-testF2 / R2 (Table 4). The positive clone was the recombinant strain S. lividans SBT18 / pCSG8112.

[0115] Example 5: Fermentation detection of recombinant strain S. lividans SBT18 / pCSG8112

[0116] The recombinant strain S. lividans SBT18 / pCSG8112 was plated on MS medium (containing 50 μg·mL⁻¹). -1 Apopramine and 100 μg·mL -1 After culturing with trimethoprim for 5 days, a small amount of bacterial cells was scraped off with a sterilized bamboo stick and inoculated into 50 mL of AM3 medium. After culturing at 28°C and 200 rpm for 5 days, 5 mL of bacterial cells were taken as a sample, and 5 mL of butanone was added for extraction. The mixture was ultrasonically disrupted for 30 min and centrifuged at 3900 rpm for 10 min. The supernatant was collected into a 2 mL centrifuge tube, and the organic solvent was evaporated by rotary evaporation. The remaining crude extract was dissolved in 50 μL of DMSO and injected for HPLC analysis.

[0117] HPLC conditions: Column: Phenomenex Kinetex C18, 150 mm × 4.6 mm, 5 μm; Mobile phase A: 10% acetonitrile / water (v / v) + 0.1% formic acid (v / v); Mobile phase B: 90% acetonitrile / water (v / v); Injection program: 5% B - 80% B (0-20 min), 80% B - 100% B (20-21 min), 100% B (21-25 min), 100% B - 5% B (25-26 min), 5% B (26-30 min); Detection wavelength: 360 nm; Flow rate: 1 mL / min. -1 .

[0118] The results showed that the recombinant strain *S. lividans* SBT18 / pCSG8112 could produce etrotomycin homologues with different retention times than etrotomycins A1-A4(1-4), while the control strain *S. lividans* SBT18 / pSET152 did not produce any etrotomycin homologues. Figure 4 ).

[0119] Example 6: Isolation and preparation of the polyketide compound etrotomycin B

[0120] 1. Scale-up fermentation culture

[0121] The recombinant strain S. lividans SBT18 / pCSG8112 was plated on MS medium (containing 50 μg·mL⁻¹). -1 Apopramine and 100 μg·mL -1 After activation with trimethoprim, an appropriate amount of mycelium was scraped and inoculated into an Erlenmeyer flask containing 50 mL of AM3 medium (total 20 L). The culture was then incubated at 28°C and 200 rpm for 5-7 days to obtain the fermentation culture.

[0122] 2. Extraction of fermentation broth

[0123] After centrifuging the fermentation culture at 3900 rpm for 20 min, the fermentation broth and mycelium were collected separately. The fermentation broth was adsorbed with 1 L of macroporous resin XAD-16 and then eluted with 10 L of acetone. The mycelium was extracted four times with 1 L of methanol, with each extraction followed by ultrasonic disruption for 1 h. The organic solvents in both fractions were then recovered using a rotary evaporator. The remaining aqueous phases were combined and extracted ten times with 1 L of ethyl acetate. After recovering the ethyl acetate using a rotary evaporator, the crude extract of the recombinant strain S. lividans SBT18 / pCSG8112 in AM3 medium was obtained.

[0124] 3. Isolation of compounds

[0125] The crude extract (13.2 g) of recombinant strain S. lividans SBT18 / pCSG8112 in AM3 medium was dissolved in chloroform-methanol (v / v 1:1), and 50 mL of 100-200 mesh silica gel was added and rotary evaporated. The sample was then packed into a dry column using a 1:3 ratio of 50 mL of sample-mixing silica gel to 150 mL of separating silica gel. A gradient elution was performed using chloroform / methanol as the eluent (chloroform, chloroform / methanol 4:1, chloroform / methanol 2:1, methanol, v / v), yielding fractions Fr1 to Fr4 sequentially. Fraction Fr2 (chloroform / methanol 4:1 eluted fraction) was separated using Sephadex LH-20 gel column chromatography (120 cm × 3 cm, chloroform / methanol 1:1, v / v), with 10 mL collected per flask. Fractions Fr2-1 to Fr2-4 were combined based on TLC results. Fraction Fr2-2 (bottles 14-25) was subjected to semi-preparative high-performance liquid chromatography (Phenomenex Kinetex C18, 250 mm × 10.0 mm, 5 μm; phase A: water, phase B: acetonitrile, 50% isocratic elution of phase B; flow rate: 2.5 mL / min). -1 The compound etrotomycin B1(5) was purified at a detection wavelength of 360 nm (Rt = 17.0 min).

[0126] 4. Structural identification of compounds

[0127] The structure of compound 5 was determined by HR-ESI-MS. 1 H NMR, 13 The NMR spectra of compound efrotomycin B1(5) were used for identification, and their NMR data are listed in Table 5. The spectrum of compound efrotomycin B1(5) is shown in Table 5. Figure 42-50 .

[0128] Therefore, the structural formula of compound 5 is determined as follows:

[0129]

[0130] Table 5. Compound 5 1 H (700MHz) and 13 C(175MHz) NMR data (CD3OD)

[0131]

[0132] Example 7: Determination of antibacterial activity of compounds 1-5

[0133] The inhibitory activities of polyketides etrotomycins A1-A4 (1-4) and etrotomycin B1 (5) against five Gram-positive bacteria (Enterococcus faecalis ATCC 29212, Staphylococcus aureus ATCC 29213, methicillin-resistant Staphylococcus aureus shhs-A1 (clinical sample), Micrococcus luteus SCSIO ML01, Bacillus subtilis 1064) and three Gram-negative bacteria (Acinetobacter baumannii 19606, E. coli ATCC 25922, Klebsiella pneumoniae ATCC 13883) were determined using the Mueller-Hinton broth microdilution method. Eight indicator bacteria were cultured in MH medium at 37°C on a shaker at 200 rpm for 16 h, and then diluted to OD200 using sterile MH medium. 600 The value is approximately 0.04-0.06, then diluted 10-fold and added to a 96-well plate; after adding the sample, dilute equally to a final sample concentration of 64-0.125 μg·mL. -1 Three replicates were performed for each concentration; the cells were incubated at 37°C for 18 hours, and the absorbance of each well at 600 nm was measured using a microplate reader. The minimum inhibitory concentration (MIC) of each compound was calculated. The inhibition rate (%) was calculated as [1 - (sample A - sample A background) / (negative control A - blank control A)] × 100%. The concentration of the sample with an inhibition rate > 80% was the MIC value. The results showed that the polyketide compounds etrotomycins A1-A4 (1-4) and etrotomycin B1 (5) had moderate inhibitory effects on Micrococcus luteus SCSIO ML01 (Table 6).

[0134] Table 6. Antibacterial activity of compounds 1-5

[0135]

[0136] a. Van: Vancomycin, a positive control for Gram-positive bacteria; b. Cip: Ciprofloxacin, a positive control for Gram-negative bacteria.

[0137] Example 8: Streptomycin toxicity assay of compounds 1-5

[0138] The toxicity of polyketides etrotomycins A1-A4 (1-4) and etrotomycin B1 (5) to three Streptomyces species, *S. lividans* SBT18, *S. albus* Del14, and *S. coelicolor* YF11, was tested using the disc diffusion method. After culturing *S. lividans* SBT18, *S. albus* Del14, and *S. coelicolor* YF11 on MS solid plates for 5-7 days, appropriate amounts of spores were collected using sterilized bamboo sticks and added to 500 μL of TSB medium. The mixture was then spread onto MS solid plates, dried, and 6 mm diameter sterilized filter paper discs were placed on the plate surface. 5 μL of the sample (concentration 2 mg / mL) was added. -1 The samples were placed on filter paper and incubated upside down at 28°C for 24 hours. DMSO and apramycin were used as negative and positive controls, respectively. The results showed that compounds 1-5 were significantly toxic to *Streptomycins albus* Del14, and compounds etrotomycins A1(1) and etrotomycin B1(5) were also toxic to *Streptomycins lividans* SBT18 and *Streptomycins coelicolor* YF11. Figure 5 ).

Claims

1. The polyketide compound etrotomycin B1, the structure of which is shown in formula (I): Formula (I).

2. The use of the polyketide compound etrotomycin B1 or its pharmaceutical salt as described in claim 1 in the preparation of an antibacterial drug, wherein the antibacterial drug is an antimicrobial agent against Micrococcus luteus or Streptomyces.

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