Biosynthetic gene cluster of isocyanide indole alkaloids and use thereof

Abstract

The present application relates to the field of biotechnology, and particularly relates to a biosynthesis gene cluster of isonitrile indole alkaloids and application thereof. The present application successfully reconstitutes the biosynthesis gene cluster of Brasilidine isonitrile indole alkaloids, and obtains a compound having a structure as shown in formula I and an isonitrile group hydrolysis product thereof from a fermentation culture of a recombinant bacterium. The compound has selective inhibitory activity on multiple drug-resistant Staphylococcus aureus, pathogenic yeast and Cryptococcus neoformans, and part of the compounds have a clear antibacterial spectrum and an activity superior to existing agricultural antibiotics. Meanwhile, the activity difference between different members of the compound provides a key clue for structure-activity relationship research, and also provides an important reference for the discovery of new antibacterial drugs and pesticide lead compounds.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a biosynthetic gene cluster of isonitrile indole alkaloids and its applications. Background Technology

[0002] Isocyanide indole alkaloids are a class of natural secondary metabolites with unique chemical structures, characterized by the direct connection between the indole skeleton and the isonitrile functional group. This special structural unit endows the molecule with excellent lipophilicity and metal chelating ability, which is an important structural basis for its various biological activities, such as antibacterial, antimalarial, and cytotoxic effects. They hold significant research value in the field of discovering novel drug lead compounds.

[0003] With advancements in microbial genomics and synthetic biology, strategies based on gene mining and heterologous expression have been widely applied to the discovery and preparation of microbial secondary metabolites. By performing bioinformatics analysis and functional identification of biosynthetic gene clusters in the genome, gene clusters that are silenced or expressed at low levels under natural conditions can be activated in a targeted manner, thereby achieving the heterologous synthesis of the corresponding products.

[0004] However, the number of biosynthetic gene clusters of isonitrile indole alkaloids that have been functionally identified is currently limited, and the systematic understanding of the molecular basis and functional elements of the biosynthetic pathways of these compounds remains insufficient. Standardized elements available for synthetic biology modification are also very limited. Therefore, discovering and analyzing novel biosynthetic gene clusters of isonitrile indole alkaloids, and clarifying their gene composition and element arrangement, is of great significance for promoting the efficient preparation and drug development of these compounds. Summary of the Invention

[0005] In view of this, the present invention proposes a biosynthetic gene cluster of isonitrile indole alkaloids and its application.

[0006] The technical solution of this invention is implemented as follows:

[0007] In a first aspect, the present invention provides an isonitrile indole alkaloid or a pharmaceutically acceptable salt thereof, said isonitrile indole alkaloid having the structure shown in Formula I:

[0008] ;

[0009] The isonitrile indole alkaloid is either Brasilidine B or Brasilidine C:

[0010] .

[0011] Secondly, the present invention provides an isonitrile hydrolysate of an isonitrile indole alkaloid, or a pharmaceutically acceptable salt thereof, wherein the isonitrile indole alkaloid is Brasilidine C; the structural formula of the isonitrile hydrolysate (i.e., the isonitrile hydrolysate of Brasilidine C) is:

[0012] .

[0013] Thirdly, the present invention provides a biosynthetic gene cluster for the isonitrile indole alkaloid.

[0014] The biosynthetic gene cluster from the 5' end to the 3' end includes the following (a1) or (a2):

[0015] (a1) Promoter kasOp*, braA gene, braB gene, braC gene, promoter SF14p, first RBS sequence, braD gene, second RBS sequence and braE gene;

[0016] (a2) Promoter kasOp*, braA gene, braB gene, braC gene, promoter SF14p, first RBS sequence and braD gene;

[0017] The nucleotide sequence of the first RBS sequence is shown in SEQ ID NO: 10;

[0018] The nucleotide sequence of the second RBS sequence is as shown in SEQ ID NO: 9;

[0019] The braA, braB, braC, braD, and braE enzymes encode isopentenyl pyrophosphate isomerase, isonitrile synthase, α-ketoglutarate-dependent oxidase, isopentenyl transferase, and cytochrome P450 enzyme, respectively, and their amino acid sequences are shown in SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15, respectively.

[0020] Fourthly, the present invention provides an expression cassette for a biosynthetic gene cluster of isonitrile indole alkaloids, the expression cassette containing the aforementioned biosynthetic gene cluster.

[0021] Fifthly, the present invention provides an expression vector for a biosynthetic gene cluster of isonitrile indole alkaloids, the expression vector containing the expression cassette described above.

[0022] Sixthly, the present invention provides a recombinant bacterium containing a biosynthetic gene cluster of isonitrile indole alkaloids, wherein the recombinant bacterium contains the aforementioned expression vector. Further, the host cell of the recombinant bacterium is Streptomyces.

[0023] In a seventh aspect, the present invention provides a method for the biosynthesis of isonitrile indole alkaloids, comprising the following steps:

[0024] A recombinant bacterium containing the aforementioned biosynthetic gene cluster was constructed, and the recombinant bacterium was fermented to obtain a fermentation culture. The fermentation culture was extracted, separated, and the product was purified to obtain the isonitrile indole alkaloid.

[0025] In some embodiments of the present invention, the following two recombinant strains were fermented to obtain Brasilidine B, Brasilidine C, and Brasilidine D, respectively:

[0026] The strain S. albus J1074::pSET152-braA-D was used to prepare Brasilidine C and Brasilidine D;

[0027] The strain S. albus J1074::pSET152-braA-E was used to prepare Brasilidine B.

[0028] Furthermore, the recombinant expression vector pSET152-braA-E contains two independent transcription units, driven by the promoters kasOp* and SF14p, respectively. The elements are arranged in the following order from the 5' end to the 3' end: kasOp*-BraA-BraB-BraC-promoterSF14p-(RBS-cpmF)-BraD-(RBS-cpmH)-BraE.

[0029] In some embodiments of the present invention, the nucleotide sequence of the recombinant expression vector pSET152-braA-E is shown in SEQ ID NO: 1, wherein the RBS-cpmF and RBS-cpmH sequences are shown in SEQ ID NO: 10 and SEQ ID NO: 9, respectively. pSET152-braA-E is introduced into S. albus J1074 to obtain the recombinant strain S. albus J1074::pSET152-braA-E.

[0030] In the recombinant expression vector pSET152-braA-D, the arrangement of elements along the transcriptional direction is as follows:

[0031] The promoter kasOp-BraA-BraB-BraC-promoter SF14p-RBS-cpmF-BraD was introduced into S. albus J1074 to obtain the recombinant strain S. albus J1074::pSET152-braA-D.

[0032] Eighthly, the present invention provides the use of the aforementioned isonitrile indole alkaloid or a pharmaceutically acceptable salt thereof in the preparation of antibacterial drugs.

[0033] Furthermore, the antimicrobial agent has antimicrobial activity against at least one of the following microorganisms: Candida albicans BNCC 337321, Candida albicans BNCC 186382, Cryptococcus neoformans BNCC 225501, Cryptococcus neoformans ATCC 32045, Staphylococcus aureus ATCC 29213, Acinetobacter baumannii ATCC 19606, Escherichia coli DH5α, and Klebsiella pneumoniae CMCC 46117.

[0034] The beneficial effects of the present invention include at least the following:

[0035] This invention employs a heterologous expression strategy to completely reconstruct the biosynthetic gene cluster of isonitrile indole alkaloids belonging to the Brasilidine class, and successfully achieves the biosynthesis and preparation of Brasilidine B, Brasilidine C, and Brasilidine D (the isonitrile hydrolysis product of Brasilidine C).

[0036] The antibacterial activity evaluation results of this invention show that Brasilidine C has a potent inhibitory activity (MIC) against Cryptococcus neoformans. 80 = 0.125 μg / mL), possessing the potential for drug development against cryptococcal infections; maintaining moderate inhibitory activity against multidrug-resistant Klebsiella pneumoniae (MIC 0.125 μg / mL). 80 = 8 μg / mL), and resistant to all commonly used clinical antibiotics, suggesting that Brasilidine C has a unique antibacterial mechanism and is worthy of further research.

[0037] Compound Brasilidine B exhibited significant inhibitory activity against Candida albicans (MIC80 = 16 μg / mL) and Cryptococcus neoformans (MIC80 = 8 μg / mL). In contrast, Brasilidine D showed no significant inhibitory activity against any of the tested bacterial and fungal strains (MIC80 > 32 μg / mL). This difference in activity among the three compounds provides an important basis for elucidating the structure-activity relationship of this class of compounds. Attached Figure Description

[0038] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0039] Figure 1 Schematic diagram of the structures of pSET152-braA-E and pSET152-braA-D plasmids.

[0040] Figure 2 The plasmid maps are for pSET152-braA-E and pSET152-braA-D.

[0041] Figure 3 Mass spectrometry (A) of fermentation extracts from recombinant strains S. albus J1074::pSET152-braA-D and S. albus J1074::pSET152-braA-E, and UV spectra of compounds Brasilidine B (B) and Brasilidine C (C).

[0042] Figure 4-9 These are the compounds Brasilidine B. 1 1H-NMR (H-NMR) 13 C-NMR (carbon nuclear magnetic resonance), DEPT135 (distortion-free polarization transfer enhancement), COSY (homnuclear correlation), HSQC (heteronuclear single quantum coherence), and HMBC (heteronuclear multi-bond correlation) spectra.

[0043] Figure 10-15 These are the compounds Brasilidine C. 1 H-NMR, 13 C-NMR, DEPT135, COSY, HSQC, and HMBC spectra.

[0044] Figure 16-21 These are the compounds Brasilidine D. 1 H-NMR, 13 C-NMR, DEPT135, COSY, HSQC, and HMBC spectra.

[0045] Figure 22-24 These are schematic diagrams of key 2DNMR (two-dimensional nuclear magnetic resonance) data for compounds Brasilidine B, Brasilidine C, and Brasilidine D, respectively.

[0046] Figure 25 The diagram shows the X-ray crystal structure of compounds Brasilidine B and Brasilidine C, where A represents Brasilidine B and B represents Brasilidine C. Detailed Implementation

[0047] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall be followed. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0048] Table 1 Sequences of SEQ ID NO: 1-15

[0049]

[0050]

[0051]

[0052]

[0053] Example 1: Screening of the biosynthetic gene cluster of isonitrile indole alkaloid (Brasilidine)

[0054] Using the isocyanide synthase IsnA, first identified by Sean Brady in 2005, as a query probe, the species were limited to the genus *Nocardia* for BLAST homology search. This analysis yielded a series of gene sequences of IsnA homologous proteins and their corresponding phylogenetic information. The probe sequence was obtained from the literature: Brady SF, Clardy J. Cloning and heterologous expression of isocyanide biosynthetic genes from environmental DNA[J]. Angewandte Chemie International Edition, 2005, 44(43): 7063-7065.

[0055]

[0056] For each returned sequence, bioinformatics tools were used to analyze approximately 10 kb of the genomic regions upstream and downstream to preliminarily determine the boundaries and gene composition of candidate gene clusters.

[0057] Candidate gene clusters were screened and eliminated step by step according to the following criteria: (1) If the gene boundaries of a candidate cluster are incomplete, resulting in the truncation of key biosynthetic genes, it will be eliminated; (2) If it lacks an essential enzyme gene that matches the functions of isonitrile formation, isopentenyl transfer, or cyclization, it will also be eliminated; (3) If the structure of a gene cluster is highly homologous to known clusters reported in the literature, it will also be eliminated. Those that meet any of the above conditions will not be included in the subsequent analysis.

[0058] After the above screening, the only remaining target gene cluster originated from *Nocardia brasiliensis*, which has a complete gene structure, reasonable functional composition, and no complete functional reports have been found. This target gene cluster was then chemically synthesized to obtain a complete DNA fragment for subsequent heterologous expression and functional studies.

[0059] This gene cluster (named the bra gene cluster) contains the following:

[0060] The genes for isopentenyl pyrophosphate isomerase (braA), isonitrile synthase (braB), α-ketoglutarate-dependent oxidase (braC), isopentenyl transferase (braD), and cytochrome P450 enzyme (braE) are listed.

[0061] The amino acid sequences encoded by the above genes are shown in SEQ ID NO: 11-15 (see Table 1).

[0062] Example 2 Construction of recombinant expression vector and recombinant strain

[0063] In this embodiment, S. albus J1074, helper Escherichia coli pUB307, and plasmid pSET152 are all commercially available strains and plasmids commonly used in the field.

[0064] The synthesis of relevant sequences, construction of expression vectors, and sequencing during the construction process were outsourced to Qingke Biotechnology Co., Ltd. In this invention, when expressing a gene or the protein encoded by a gene, the "vector" can be a plasmid; in some cases, the terms "vector" and "plasmid" can be used interchangeably.

[0065] 2.1 Construction of the recombinant expression vector pSET152-braA-E

[0066] This embodiment is constructed as follows: Figure 1The multi-gene expression unit shown (i.e., the bra gene cluster) contains two independent transcription units, driven by the promoters kasOp* and SF14p, respectively. The elements are arranged in the following order from the 5' to the 3' end: kasOp*-BraA-BraB-BraC-promoter SF14p-(RBS-cpmF)-BraD-(RBS-cpmH)-BraE. The specific construction steps are as follows:

[0067] 1) Fragment Assembly: After obtaining the recombinant plasmid containing partial gene cluster fragments delivered by the synthesis company, double enzyme digestion was performed first. Following standard usage guidelines for restriction endonucleases, a 20 μL double digestion reaction system was prepared (containing 1 μg plasmid DNA, 2 μL 10× digestion buffer, 1 μL XbaI restriction enzyme, 1 μL LEcoRI restriction enzyme, and purified water to a final volume of 20 μL), and incubated at 37°C to obtain the BraA-C gene fragment. A 20 μL double digestion reaction system was also prepared (containing 1 μg plasmid DNA, 2 μL 10× digestion buffer, 1 μL BamHI restriction enzyme, 1 μL LEcoRI restriction enzyme, and purified water to a final volume of 20 μL), and incubated at 37°C to obtain the BraD-E gene fragment. After separation and purification by agarose gel electrophoresis, T4 DNA ligase was added, and the two double-digested products were ligated overnight at 16°C to obtain a complete bra gene cluster fragment assembled from the two fragments. The ligation product was then separated and purified by agarose gel electrophoresis.

[0068] 2) Vector Assembly: The pSET152 plasmid was double-digested using XbaⅠ and BamHI restriction endonucleases. The 50 μL double digestion reaction system is shown in Table 2. After gel purification of the digestion products, the pSET152 plasmid double digestion products and the bra gene cluster fragment were ligated overnight at 16°C using T4 DNA ligase. The ligation reaction system is shown in Table 3, yielding the pSET152-braA-E plasmid.

[0069] 3) Transform the ligation product into E. coli DH5α competent cells and spread it evenly on LB agar plates containing apramycin (final concentration 50 μg / mL). Incubate overnight at 37°C. Pick single colonies of transformants for initial colony PCR screening. Clones with positive PCR results are cultured in liquid culture, and recombinant plasmids with correct sequencing results are selected for subsequent experiments.

[0070]

[0071] 4) Conversion, screening and validation

[0072] The recombinant product was transformed into *E. coli* DH5α competent cells using a heat shock method, plated on LB agar plates containing apramycin (50 μg / mL), and incubated overnight at 37°C. Single colonies were picked and inoculated into LB liquid medium containing apramycin for expansion. Plasmids were extracted and verified by PCR and sequencing. The recombinant plasmid with correct sequencing results was named pSET152-braA-E.

[0073] 2.2. Construction of recombinant strain S. albus J1074::pSET152-braA-E

[0074] The recombinant expression vector pSET152-braA-E was introduced into the Streptomyces host S. albus J1074 via triparental conjugation. The specific steps are as follows:

[0075] 1) Culture of donor and helper Escherichia coli

[0076] Pick fresh transformants containing pSET152-braA-E or inoculate 5 μL of DH5α-pSET152-braA-E from the preservation tube into 5 mL of LB medium containing apramycin (50 μg / mL) and incubate overnight at 37°C. Take 5 μL of helper plasmid pUB307 from the preservation tube and inoculate it into 5 mL of LB liquid medium containing kanamycin (25 μg / mL) and chloramphenicol (25 μg / mL) and incubate overnight at 37°C.

[0077] 2) Preparation of donor and auxiliary bacteria

[0078] Take 500 μL of overnight culture of DH5α (containing pSET152-braA-E) and overnight culture of pUB307 and inoculate them into 50 mL of fresh LB containing the corresponding antibiotics. Incubate at 37°C until the OD is 0.4-0.5. Centrifuge at 5000 rpm / min for 4 min to collect the cells, wash once with LB, and finally suspend in 1 mL of LB.

[0079] 3) Preparation of recipient bacteria

[0080] Streptomyces albus J1074 was streaked on MS agar plates for 5-7 days. Streptomyces spores were collected using sterile swabs in culture tubes containing 20% ​​glycerol. For conjugation transfer, 20-200 μL of the Streptomyces albus J1074 spore culture solution was centrifuged at 10,000 rpm for 2 min, the glycerol was discarded, and the spores were washed once with 1 mL of TSB medium. The supernatant was removed by centrifugation (10,000 rpm, 5 min). The spores were dispersed and resuspended in 500 μL of TSB medium using a pipette tip. The mixture was heat-shocked in a 50°C water bath for 15 min, followed by shaking recovery at 30°C for 4 h. The mixture was then washed once with TSB and resuspended in 100 μL of TSB as the recipient bacteria for conjugation transfer.

[0081] 4) Triparental conjugation transfer

[0082] Take 100 μL of donor Escherichia coli DH5α-pSET152-braA-E, 200 μL of helper Escherichia coli pUB307 and 100 μL of Streptomyces S. albus J1074 and mix them evenly. Spread the mixture on Modified ISP4 plates (add 30 mM MnCl2 before pouring) and incubate at 28 °C.

[0083] 5) Resistance coverage and conjugate screening

[0084] After culturing for 16-20 hours, cover the plate with apramycin (50 mg / mL stock solution) and naphthylpyridinic acid (25 mg / mL stock solution). Add 25 μL of apramycin and 50 μL of naphthylpyridinic acid to 1 mL of sterile water, mix well, and cover the entire plate (25 mL). The plate should contain a final concentration of 50 μg / mL apramycin and 50 μg / mL naphthylpyridinic acid. Blow the plate in a clean bench for about 1 hour until completely dry, and then incubate at 28°C.

[0085] 6) Purification and verification of conjugates

[0086] Five to seven days after covering, the zygotes that grow are picked and placed on MS plates containing apramycin. Single colonies are isolated and selected for PCR verification. The PCR-positive single colonies are transferred to MS plates containing apramycin for amplification and culture, and stored for later use. This strain is named S. albus J1074::pSET152-bra.

[0087] The formulations for each culture medium are as follows:

[0088] The TSB medium is prepared by adding 20g of TSB to 1L of water and adjusting the pH to 7.2-7.4.

[0089] The MS medium consists of 20g soybean flour, 20g mannitol, and 20g technical agar powder added to 1L of water, with a pH of 7.2-7.4, and sterilized at 121℃ for 30min.

[0090] The Modified ISP4 medium consists of the following components: 10g soluble starch, 2g (NH4)2SO4, 1g K2HPO4, 1g MgSO4, 2g CaCO3, 0.5g yeast extract, 1g peptone, 20g agar, and 1mL inorganic salt solution (1g / L FeSO4, 1g / L MnCl2, 1g / L ZnSO4) added to 1L of water, and the pH adjusted to 7.2-7.4.

[0091] 2.3 Construction of recombinant strain S. albus J1074::pSET152-braA-D

[0092] The element arrangement order of the pSET152-braA-D gene cluster is: promoter kasOp*-BraA-BraB-BraC-promoter SF14p-(RBS-cpmF)-BraD.

[0093] Construction of the BraE gene knockout vector pSET152-braA-D

[0094] Based on the expression vector pSET152-braA-E, the braE gene was knocked out using reverse PCR and homologous recombination techniques. The specific procedure is as follows:

[0095] (1) Knockout strategy and primer design

[0096] Using reverse PCR amplification technology and the pSET152-bra plasmid as a template, a pair of reverse amplification primers with 15-20 bp complementary homologous arms were designed: upstream primer P152-F and downstream primer P152-R. This primer pair extends outwards to amplify the plasmid backbone and remaining gene cluster sequences, excluding the braE gene sequence, and to form homologous complementary sequences at both ends of the product for subsequent self-circularization.

[0097]

[0098] (2) Reverse PCR amplification of the target fragment

[0099] PCR amplification was performed using purified pSET152-braA-E plasmid as a template in a 50 μL volume. The reaction mixture consisted of: 1 μL template plasmid, 2 μL each of forward and reverse primers (10 μM), 25 μL 2 × KeyPo Master Mix, 2.5 μL DMSO, and ddH2O to a final volume of 50 μL. The amplification program was as follows: 95℃ pre-denaturation for 5 min; followed by 30 cycles of amplification (98℃ denaturation for 10 s, 60℃ annealing for 5 s, 72℃ extension at 5 s / kb); and a final extension at 74℃ for 5 min. PCR products were detected by agarose gel electrophoresis and purified using a kit.

[0100] (3) Seamless circularization and transformation of linear segments

[0101] The linearized fragment was circularized using a one-step cloning kit. A 10 μL recombinant reaction mixture (on ice) was prepared by adding 5 μL of 2 × CE Mix V3, 1 μL of the purified linearized fragment, and then bringing the remaining volume to ddH2O. The mixture was incubated at 50°C for 30 min, then transferred to ice to terminate the reaction. The ligation product was then transformed into E. coli DH5α competent cells and plated on LB agar plates (ampromycin (50 μg / mL)) for culture.

[0102] (4) Screening and verification of positive transformants

[0103] Transformants were picked from antibiotic-resistant plates and subjected to preliminary screening using colony PCR. The clones selected after preliminary screening were then sent to a sequencing company for sequencing to confirm whether the braE gene sequence had been precisely knocked out.

[0104] Subsequently, following the triparental conjugation transfer method described in Section 2.2, pSET152-braA-D was introduced into S. albus J1074 to obtain the recombinant strain S. albus J1074::pSET152-braA-D.

[0105] 2.5 HPLC (High Performance Liquid Chromatography) Detection Method for Fermentation Products of Recombinant Strains

[0106] (1) Sample preparation

[0107] Spores of the recombinant strain S. albus J1074::pSET152-braA-D were inoculated onto MS solid medium plates (9 cm in diameter) containing apramycin (final concentration 50 μg / mL) and incubated at 28°C for 5 days. Spores were collected with sterile swabs, suspended in a preservation solution containing 20% ​​(v / v) glycerol, and stored at -80°C for later use.

[0108] Take 20 μL of the frozen spore solution and inoculate it onto an ISP2 solid medium plate, incubating at 28°C for 5 days. In a clean bench (sterilized with UV for 30 min before operation), use a 1 mL pipette tip (approximately 0.6 cm in diameter) to cut two agar blocks (approximately 0.5 mL in volume) and transfer them to 2 mL centrifuge tubes. Add 1 mL of ethyl acetate to the centrifuge tubes and extract by sonication for 1 h. Remove the blocks and evaporate the extract to dryness under vacuum at 50°C (approximately 50 min). Add 100 μL of methanol to the residue, sonicate for 10 min, centrifuge at 12000 rpm for 2 min, and take 80 μL of the supernatant for HPLC analysis.

[0109] (2) HPLC detection conditions

[0110] The chromatographic column was a Luna® C18 (2) reversed-phase column (5 μm, 100 Å, 250 × 4.6 mm, Phenomenex).

[0111] The mobile phase consists of phase A and phase B. Mobile phase A is composed of 90% water, 10% acetonitrile, and 0.08% (volume fraction) formic acid. Mobile phase B is composed of 90% acetonitrile, 10% water, and 0.08% (volume fraction) formic acid.

[0112] Injection program: 0-20 min, mobile phase ratio A / B (volume ratio): 100:0-0:100; 20-25 min, mobile phase ratio A / B (volume ratio): 0:100; 25.01-30 min, mobile phase ratio A / B (volume ratio): 100:0; flow rate: 1 mL / min; HPLC was performed using an Agilent 1260 binary pump system.

[0113] Example 3 Preparation of isonitrile indole alkaloids Brasilidine B, C and D

[0114] 3.1 Scale-up fermentation culture of recombinant strains

[0115] The following two recombinant strains were fermented separately:

[0116] Strain 1: S. albus J1074::pSET152-braA-D (used for the preparation of Brasilidine C and Brasilidine D)

[0117] Strain 2: S. albus J1074::pSET152-braA-E (used for the preparation of Brasilidine B)

[0118] Each recombinant strain was stored in a preservation tube at -80℃ and inoculated into a culture dish containing MS agar medium with a final concentration of 50 mg / mL apramycin. After culturing at 28℃ for 5 days, the spores on the MS medium were collected into a 2 mL centrifuge tube containing 1.8 mL of 20% glycerol and stored at -80℃ as seed culture.

[0119] Take 25 μL of the above seed culture and inoculate it onto a medium containing 25 mL of ISP2 agar. Incubate at 28 °C for 5 days to obtain fermentation cultures of the two recombinant strains.

[0120] The formulations for each culture medium are as follows:

[0121] The MS seed culture medium consists of 20g soybean powder, 20g mannitol, and 20g technical agar powder added to 1L of water, adjusted to pH 7.2-7.4, and sterilized at 121℃ for 30min.

[0122] The ISP2 fermentation medium consists of: 10g malt extract, 4g glucose, 4g yeast extract, 5g CaCO3, and 20g agar added to 1000mL of water, adjusted to pH 7.2-7.4, and sterilized.

[0123] 3.2. Extraction of solid fermentation products

[0124] The solid fermentation culture was first chopped and then extracted three times with equal volume of dichloromethane at room temperature for 3 hours each time. The extracts were combined and distilled under reduced pressure with dichloromethane to obtain crude extracts of strain 1, strain 2 and strain 3, respectively.

[0125] 3.3. Isolation and purification of target compounds

[0126] (1) Isolation of Brasilidine C (from strain 1)

[0127] Crude extracts of strain 1 were dissolved in methanol and separated by 200-300 mesh silica gel. The fractions were eluted sequentially with three gradients: 100% cyclohexane, cyclohexane:ethyl acetate = 9:1, and cyclohexane:ethyl acetate = 8:2. One flask was collected for every 15 mL of fractions, and the fractions were collected sequentially.

[0128] The fractions were analyzed by TLC (thin-layer chromatography), and fluorescent spots were observed under a UV lamp at 254 nm. The fractions containing the target compounds were then identified using HPLC. The results showed that Brasilidine C was mainly distributed in the cyclohexane-ethyl acetate (9:1) elution fraction of the crude extract of the strain.

[0129] Fractions containing Brasilidine C from strain 1 were combined (each fraction was individually combined and concentrated under reduced pressure). The residues were dissolved in methanol and separated by C18 reversed-phase column chromatography with gradient elution using acetonitrile aqueous solutions of different concentrations. Fractions were collected in 15 mL increments and analyzed by HPLC. Fractions containing a single target compound were combined, concentrated to remove organic solvents, frozen at -80°C for 3 h, and then freeze-dried to remove moisture. The resulting solid was dissolved in methanol and further purified by semi-preparative HPLC to obtain pure Brasilidine C in a yield of 0.5 mg / L.

[0130] Semi-preparative HPLC conditions: The high-performance liquid chromatograph was a Ruihe brand, and the column was a YMC-Pack ODS-A reversed-phase column (5 μm, 12 nm, 250 × 10.0 mm). The mobile phase consisted of phase A and phase B. Mobile phase A was pure water, and mobile phase B was 100% acetonitrile. The injection program was as follows: 0-10 min, mobile phase ratio A / B (volume ratio): 50:50-10:90; 10-20 min, mobile phase ratio A / B (volume ratio): 10:90; 21-26 min, mobile phase ratio A / B (volume ratio): 50:50; detection wavelength: 280 nm; flow rate: 3 mL / min. The target peak fraction was collected, and acetonitrile was evaporated to obtain pure Brasilidine C with a yield of 0.5 mg / L.

[0131] (2) Isolation of Brasilidine D (from strain 1)

[0132] The crude extract of strain 1 was dissolved in methanol and separated by silica gel column chromatography with a mesh size of 200-300. The extract was eluted sequentially with three gradients: 100% cyclohexane, cyclohexane-ethyl acetate (9:1), and cyclohexane-ethyl acetate (8:2). One flask was collected for every 15 mL of the extract.

[0133] TLC and HPLC analysis confirmed that Brasilidine D was mainly distributed in the elution fraction of cyclohexane-ethyl acetate (8:2). This fraction was combined, concentrated under reduced pressure to remove the solvent, and the residue was dissolved in methanol. Separation was performed by C18 reversed-phase column chromatography with gradient elution using acetonitrile aqueous solutions of different concentrations. Fractions containing Brasilidine D were combined, concentrated, freeze-dried, dissolved in methanol, and further purified under the same semi-preparative HPLC conditions as in (1) above. The target peak was collected, and acetonitrile was evaporated to obtain pure Brasilidine D with a yield of 1 mg / L.

[0134] (3) Isolation of Brasilidine B (from strain two)

[0135] The crude extract of strain 2 was dissolved in methanol and separated using 200-300 mesh silica gel. Elution was performed sequentially with three gradients: 100% cyclohexane, cyclohexane:ethyl acetate = 9:1, and cyclohexane:ethyl acetate = 8:2. One 15 mL flask was collected, and the fractions were collected sequentially. TLC analysis was performed on the fractions. The dried TLC plate was placed in a sealed container containing iodine crystals and developed in iodine vapor for several minutes until the spots were clear. It was determined that Brasilidine B was mainly distributed in the cyclohexane:ethyl acetate = 9:1 fraction. This fraction was combined.

[0136] The concentrate was subjected to gel electrophoresis again using a 200-300 mesh silica gel column to remove unsaturated fatty acids from Brasilidine B. It was eluted sequentially with five gradients: 100% cyclohexane, cyclohexane:chloroform = 9.5:0.5, cyclohexane:chloroform = 9:1, cyclohexane:chloroform = 8:2, cyclohexane:chloroform = 7:3, and cyclohexane:chloroform = 6:4. TLC and HPLC analysis confirmed that the compound Brasilidine B was distributed in the elution fractions of cyclohexane:chloroform = 7:3 and cyclohexane:chloroform = 6:4. All fractions containing Brasilidine B were combined, concentrated under reduced pressure to remove the solvent, and the residue was dissolved in methanol. It was then further purified under the same semi-preparative HPLC conditions as in (1) above. The target peak was collected, and acetonitrile was evaporated to remove it, thus obtaining pure Brasilidine B.

[0137] Example 4: Structural identification of isonitrile indole alkaloids Brasilidine B, C, and D

[0138] The structure of compound Brasilidine D was determined by 1 H-NMR, 13 C-NMR, DEPT135, COSY, HSQC, and HMBC spectra (corresponding to...) Figures 16-21 ) Identification.

[0139] The structures of compounds Brasilidine B and Brasilidine C were determined by... 1 H-NMR, 13 C-NMR, COSY, HSQC, and HMBC spectra (corresponding to Brasilidine B) Figures 4-9 Brasilidine C corresponds to Figures 10-15 ), key two-dimensional nuclear magnetic resonance (2D NMR) related spectra ( Figure 22 , Figure 23 , Figure 24 These are Brasilidine B, Brasilidine C, and Brasilidine D, respectively, combined with X-ray single-crystal diffraction analysis ( Figure 25 In the figure, A is Brasilidine B, and B is Brasilidine C) together confirm this.

[0140]

[0141]

[0142]

[0143] Based on the above results, the structural formulas of Brasilidine B, Brasilidine C, and Brasilidine D are shown below:

[0144]

[0145] Example 5: Determination of the antibacterial activity of compounds Brasilidine B, Brasilidine C, and Brasilidine D

[0146] 5.1 Test strains

[0147] A total of 11 indicator bacteria were used in this embodiment, which were purchased from commercial channels of bacterial culture collection centers such as ATCC (American Center for Type Culture Collection), CMCC (China Center for Medical Bacteriological Culture Collection), and BNCC (Beina Biotechnology). Specific information is as follows:

[0148] Bacterial strains (LB medium, 37°C): E. faecalis ATCC 29212, E. faecalis ATCC51299, E. coli DH5α, S. aureus ATCC 29213, A. baumannii ATCC 19606, P. aeruginosa ATCC 27853, Klebsiella pneumoniae CMCC 46117.

[0149] Yeast strains (YPD medium, cultured at 28℃): C. albicans BNCC 186382, C. albicans BNCC 337321, C. neoformans BNCC 225501, C. neoformans ATCC 32045.

[0150] 5.2 Test Compounds and Positive Controls

[0151] The test compounds were Brasilidine B, Brasilidine C, and Brasilidine D prepared in this invention. Positive control drugs were: terbinafine hydrochloride, fluconazole, linezolid, tetracycline, amphotericin B, ciprofloxacin, chloramphenicol, phenazine-1-carboxylic acid (PCA), erythromycin, gentamicin, streptomycin, vancomycin, and kasugamycin.

[0152] The test compounds and positive control drugs were dissolved in appropriate solvents to prepare stock solutions with a concentration of 1.28 mg / mL. The solvents used for each compound are as follows:

[0153]

[0154] 5.3 Preparation of bacterial culture

[0155] Single colonies of the test strains were picked from fresh plates and inoculated into the corresponding culture media. The cultures were then incubated with shaking at 220 rpm for 16–48 h (see the test strain information for incubation temperature and culture medium). Overnight cultures or spore suspensions were then transferred to fresh culture media at an inoculation rate of 1‰ (v / v) to prepare bacterial stock solutions for later use.

[0156] 5.4 MIC 80 Determination methods

[0157] The minimum inhibitory concentration (MIC) was determined using the 96-well plate microdilution method. 80 This refers to the minimum compound concentration required to inhibit the growth of 80% of microorganisms. The specific steps are as follows:

[0158] In a 96-well plate, 100 μL of bacterial stock solution was added to each well in columns 2-12; 195 μL of bacterial stock solution was added to each well in column 1; and column 12 was a bacterial control (negative control) without the compound.

[0159] Add 5 μL of each compound stock solution to the corresponding well in column 1. Mix thoroughly using a multichannel pipette, then transfer 100 μL to column 2. Perform serial dilutions of 2-fold up to column 11, discarding the 100 μL of mixture transferred from column 11. The final concentration range for each compound is 0.03125–32 μg / mL.

[0160] Incubate the 96-well plate at a suitable temperature for 12-48 hours. Before measuring absorbance, thoroughly mix the culture using a microplate shaker to reduce measurement error. Measure the absorbance of each well at an appropriate wavelength using a microplate reader.

[0161] Wherein, the inhibition rate is calculated as inhibition rate (%) = [1 - (A)] 样品 -A 样品背景 ) / (A 阴性对照 -A 空白对照 Calculate using 100% × 100%. 样品背景 The absorbance of the blank well containing the compound but not the bacterial culture is given. The minimum concentration with an inhibition rate ≥80% is defined as the MIC. 80 .

[0162] 5.5 Results of antibacterial activity assay

[0163] MICs of each compound and positive control drug against 11 indicator strains 80 As shown in the table below:

[0164]

[0165] 5.6 Results Analysis

[0166] (1) Inhibitory activity of compound Brasilidine C against pathogenic yeast

[0167] The compound Brasilidine C exhibits significant and selective inhibitory activity against pathogenic yeast.

[0168] Regarding its effect against Cryptococcus neoformans, Brasilidine C showed a MIC of [missing information - likely a value] against strain BNCC 225501. 80 The MIC for strain ATCC 32045 was 0.125 μg / mL. 80 The concentration was 1 μg / mL. The MIC of the positive control drug fluconazole against the two strains mentioned above was... 80 The concentrations were 16 μg / mL and 8 μg / mL, respectively, indicating that the activity of Brasilidine C was 8–128 times higher than that of fluconazole. The MICs of amphotericin B and terbinafine hydrochloride against the two strains were also determined. 80 At a concentration of 0.5 μg / mL, Brasilidine C showed superior activity against BNCC 225501 strain compared to the other two, while its activity against ATCC 32045 strain was approximately half that of the other two.

[0169] Regarding its effectiveness against Candida albicans, Brasilidine C showed a MIC of [missing value] against strains BNCC 186382 and BNCC 337321.80 The concentrations of 2 μg / mL and 4 μg / mL were respectively superior to those of fluconazole (corresponding to MIC). 80 Both are 16 μg / mL), but weaker than amphotericin B (MIC). 80 (0.125–0.25 μg / mL) and terbinafine hydrochloride (MIC) 80 (2–4 μg / mL).

[0170] In addition, the MICs of agricultural antibiotics kasugamycin and phenazinamide against all tested yeast strains were... 80 Both were ≥32 μg / mL, indicating that they were essentially ineffective against pathogenic yeasts, further highlighting the advantages of Brasilidine C as an antifungal compound.

[0171] (2) Inhibitory activity of compound Brasilidine C against bacteria

[0172] Brasilidine C exhibits significant selectivity in its inhibitory activity against bacteria, displaying distinct antibacterial spectrum characteristics between Gram-positive and Gram-negative bacteria.

[0173] For Gram-positive bacteria: Brasilidine C MIC against Staphylococcus aureus ATCC 29213 80 The concentration was 4 μg / mL, consistent with linezolid (4 μg / mL) and slightly lower than vancomycin (2 μg / mL). Regarding Enterococcus faecalis, the MIC of Brasilidine C was 4 μg / mL, consistent with both linezolid and vancomycin-sensitive ATCC 29212 strain and ATCC 51299 strain, which was linezolid-sensitive but vancomycin-resistant. 80 Both were >32 μg / mL, showing no detectable inhibitory activity. The MIC of the control drug linezolid against the above two Enterococcus faecalis strains was... 80 All were 2 μg / mL; the MIC of vancomycin against ATCC 29212 strain 80 The concentration was 4 μg / mL, while for ATCC 51299 resistant strains it was >32 μg / mL.

[0174] For Gram-negative bacteria: Brasilidine C has a MIC against Acinetobacter baumannii (ATCC 19606), Escherichia coli (DH5α), and Klebsiella pneumoniae (CMCC 46117). 80 All concentrations were 8 μg / mL, indicating a moderate degree of inhibition.

[0175] Of particular note is the comparison of the MICs of all tested clinical control antibiotics (ciprofloxacin, gentamicin, chloramphenicol, erythromycin, streptomycin, tetracycline, vancomycin, linezolid) and agricultural antibiotics kasugamycin and phenazinamide against Klebsiella pneumoniae strain CMCC 46117. 80 The concentrations of all samples were >32 μg / mL, while Brasilidine C maintained an antibacterial activity of 8 μg / mL, suggesting that this compound has a unique inhibitory advantage against multidrug-resistant strains. On the other hand, Brasilidine C showed no inhibitory activity (MIC) against Pseudomonas aeruginosa ATCC 27853. 80 >32 μg / mL), while the control drugs ciprofloxacin (0.5 μg / mL) and gentamicin (2 μg / mL) remained effective against this strain.

[0176] (3) Deactivation of compounds Brasilidine B and Brasilidine D

[0177] Compared to Brasilidine C, Brasilidine B showed only weak activity against Candida albicans (16 μg / mL) and Cryptococcus neoformans (8 μg / mL, against BNCC 225501) at higher concentrations, and was ineffective against other tested strains (MIC). 80 >32 μg / mL). Brasilidine D showed no inhibitory activity against any of the tested strains (MIC). 80 All >32 μg / mL).

[0178] In summary, Brasilidine C is an isonitrile indole alkaloid with selective antibacterial activity, exhibiting potent inhibitory activity against Cryptococcus neoformans (MIC). 80 = 0.125 μg / mL), possessing the potential for drug development against cryptococcal infections; maintaining moderate inhibitory activity against multidrug-resistant Klebsiella pneumoniae (MIC 0.125 μg / mL). 80 = 8 μg / mL), and resistant to commonly used clinical antibiotics, suggesting that it has a unique antibacterial mechanism that is worth exploring in depth.

[0179] Brasilidine B exhibited inhibitory activity against Candida albicans (MIC80 = 16 μg / mL) and Cryptococcus neoformans (MIC80 = 8 μg / mL) at higher concentrations. Brasilidine D showed no significant inhibitory activity against any of the tested bacterial and fungal strains (MIC80 > 32 μg / mL). These differences in activity provide crucial clues for studying the structure-activity relationship of this class of compounds.

[0180] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An isonitrile indole alkaloid or a pharmaceutically acceptable salt thereof, characterized in that, The isonitrile indole alkaloid has the structure shown in Formula I: ； The isonitrile indole alkaloid is either Brasilidine B or Brasilidine C: 。 2. An isonitrile indole alkaloid's isonitrile hydrolysis product, or a pharmaceutically acceptable salt thereof, characterized in that, The isonitrile indole alkaloid is Brasilidine C as described in claim 1; the structural formula of the isonitrile hydrolysis product is: 。 3. A biosynthetic gene cluster for isonitrile indole alkaloids as described in claim 1, characterized in that, The biosynthetic gene cluster from the 5' end to the 3' end includes the following (a1) or (a2): (a1) Promoter kasOp*, braA gene, braB gene, braC gene, promoter SF14p, first RBS sequence, braD gene, second RBS sequence and braE gene; (a2) Promoter kasOp*, braA gene, braB gene, braC gene, promoter SF14p, first RBS sequence and braD gene; The nucleotide sequence of the first RBS sequence is shown in SEQ ID NO: 10; The nucleotide sequence of the second RBS sequence is as shown in SEQ ID NO: 9; The braA, braB, braC, braD, and braE enzymes encode isopentenyl pyrophosphate isomerase, isonitrile synthase, α-ketoglutarate-dependent oxidase, isopentenyl transferase, and cytochrome P450 enzyme, respectively, and their amino acid sequences are shown in SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15, respectively.

4. An expression cassette for a biosynthetic gene cluster of isonitrile indole alkaloids, characterized in that, The expression cassette contains the biosynthetic gene cluster as described in claim 3.

5. An expression vector for a biosynthetic gene cluster of isonitrile indole alkaloids, characterized in that, The expression vector contains the expression cassette as described in claim 4.

6. A recombinant bacterium containing a biosynthetic gene cluster of isonitrile indole alkaloids, characterized in that, The recombinant bacteria contain the expression vector as described in claim 5.

7. The recombinant bacteria as described in claim 6, characterized in that, The host cell is Streptomyces.

8. A method for the biosynthesis of isonitrile indole alkaloids, characterized in that, Includes the following steps: A recombinant bacterium containing the biosynthetic gene cluster as described in claim 3 is constructed, the recombinant bacterium is fermented to obtain a fermentation culture, and the fermentation culture is extracted, separated and purified to obtain the isonitrile indole alkaloid.

9. The use of the isonitrile indole alkaloid as described in claim 1 or a pharmaceutically acceptable salt thereof in the preparation of antibacterial drugs.

10. The application as described in claim 9, characterized in that, The antimicrobial agent has antimicrobial activity against at least one of the following microorganisms: Candida albicans BNCC 337321, Candida albicans BNCC 186382, Cryptococcus neoformans BNCC 225501, Cryptococcus neoformans ATCC 32045, Staphylococcus aureus ATCC 29213, Acinetobacter baumannii ATCC 19606, Escherichia coli DH5α, and Klebsiella pneumoniae CMCC 46117.

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