CN113121545A - Polycyclic macrocyclic lactam compound and preparation method and application thereof - Google Patents

Abstract

The invention relates to a polycyclic macrocyclic lactam compound and a preparation method and application thereof. The invention constructs engineering bacteria S001-cbm-OX4-sgr810 and S001-cbm-OX4-sshg _05717 for producing polycyclic macrocyclic lactam compounds. Realizes the preparation of the polycyclic macrocyclic lactam compound. The polycyclic macrocyclic lactam compound provided by the invention has stronger inhibitory activity on the secretion of virulence protein of bacteria III type secretion system, and has concentration gradient dependence, and the III type secretion system widely exists in gram-negative pathogenic bacteria and has highly conservative structure, so the polycyclic macrocyclic lactam compound can be used for preparing medicaments for resisting the secretion of virulence protein of gram-negative pathogenic bacteria.

Description

Polycyclic macrocyclic lactam compound and preparation method and application thereof

Technical Field

The invention relates to a polycyclic macrocyclic lactam compound and a preparation method and application thereof, belonging to the technical field of natural pharmaceutical chemistry and microorganisms.

Background

The emergence of new drug-resistant bacteria and pathogenic bacteria continuously threatens the health and economic development of human beings, and the research and development of new active and new action mechanism antibiotics are imminent. Natural products are important resources for the development of new antibiotics, and polycyclic tetramic acid macrolactam (PoTeM) is a macrolactam compound with a tetramic acid structural unit and a polycyclic system. The structural diversity of the PoTeMs is mainly reflected in the difference of polycyclic systems, such as 5/6/5 tricyclic ring, 5/5/6 tricyclic ring, 5/5 bicyclic ring and the like, and in addition, the structural diversity is further enriched by post-modification of hydroxylation, carbonylation, epoxidation and the like catalyzed by cytochrome P450 enzymes. The PoTeMs have various biological activities of resisting bacteria, fungi, protozoans, ulcers and tumors, inhibiting the secretion of virulence proteins of a III-type secretion system and the like.

The action mechanism of the traditional antibiotics is mainly bacteriostasis or sterilization, while the action mechanism of inhibiting the secretion of virulence protein of a III-type secretion system of gram-negative pathogenic bacteria is mainly to make the pathogenic bacteria lose virulence, pathogenic capability and survival competitive capability by targeting the virulence protein, so that the antibiotic is a novel action mechanism against pathogenic bacteria. The PoTeMs gradually form a new antibiotic family with complex structures, rich biological activity and unique action mechanisms, and have potential medicinal value.

Chinese patent document CN106008531A discloses the pancreatic cancer resistant application of polycyclic condensed macrocyclic lactam compound. Chinese patent document CN108623607A discloses a 5,5, 6-polycyclic tetramic acid-containing macrocyclic lactam compound and a preparation method and application thereof. However, the compounds provided in these two patents are functional as antitumor and antifungal and are not related to antibacterial aspects.

A published article, "polycyclic tetramic acid macrocyclic lactam produced by Streptomyces S001 metabolism", has been reported to be the unexpected activation of the host' S own PoTeMs gene cluster mtm (GenBank No. MN817126) upon expression of recombinant plasmids in heterologous hosts, resulting in the reported compound montamide A, which has weak cytotoxic activity but no reported activity of inhibiting the secretion of bacterial virulence proteins.

At present, only one literature report about PoTeMs compounds with antibacterial toxicity and the preparation of the compounds is that Targeted discovery and combinatorial biosynthesis of multicyclic tetramate maize combamides A-E, although the combamides biosynthesis gene cluster cbm (GenBank No. MH167394) is modified and expressed heterologously by using a combination biosynthesis means, the cytochrome P450 enzyme gene contained in the engineering bacteria constructed in the paper is cbmD.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a polycyclic macrocyclic lactam compound and a preparation method and application thereof.

The technical scheme of the invention is as follows:

the structural formula of the polycyclic macrocyclic lactam compound is shown as a formula (I) or (II):

the engineering bacteria for producing the polycyclic macrocyclic lactam compound are characterized in that the metabolite of the engineering bacteria contains the polycyclic macrocyclic lactam compound shown as the formula (I) or (II).

According to the invention, the engineering bacteria preferably contain a polyketide-non-ribosomal peptide synthase gene cbmA responsible for synthesis of PoTeMs polyene skeletons, and the nucleotide sequence is shown as SEQ ID NO. 1; the flavin-dependent oxidoreductase gene cbmB responsible for the synthesis of the middle five-membered ring of the polycyclic system has a nucleotide sequence shown in SEQ ID No. 2; the flavin-dependent oxidoreductase gene cbmC responsible for five-membered ring synthesis outside the polycyclic system has a nucleotide sequence shown in SEQ ID No. 3; an NADPH-dependent oxidoreductase gene OX4 responsible for synthesis of six-membered rings inside the polycyclic system, the nucleotide sequence of which is shown in SEQ ID No. 4; the cytochrome P450 enzyme gene sgr810 or cytochrome P450 enzyme gene sshg _05717 responsible for post-oxidation modification has the nucleotide sequences shown as SEQ ID NO.5 and SEQ ID NO.6, respectively.

The construction method of the engineering bacteria for producing the polycyclic macrocyclic lactam compound comprises the following steps:

(1) carrying out PCR amplification by taking the genomic DNA of Streptomyces sp.S10 as a template to obtain flavin-dependent oxidoreductase gene cbmB and flavin-dependent oxidoreductase gene cbmC gene segments with NdeI and SpeI enzyme cutting sites respectively introduced at two ends of the sequence; carrying out PCR amplification by taking the genome DNA of Lysobacter enzymogenes C3 as a template to obtain an NADPH dependent oxidoreductase OX4 gene segment with NdeI and SpeI enzyme cutting sites respectively introduced into the two ends of the sequence; performing PCR amplification by taking Streptomyces albus J1074 genome DNA as a template to obtain a cytochrome P450 enzyme gene sshg _05717 gene fragment with NdeI and SpeI enzyme cutting sites respectively introduced at two ends of the sequence; the four PCR products are subjected to double enzyme digestion by NdeI and SpeI to obtain cbmB-NdeI/SpeI, cbmC-NdeI/SpeI, OX4-NdeI/SpeI and sshg-05717-NdeI/SpeI fragments; artificially synthesizing a cytochrome P450 enzyme gene sgr810 and cloning the gene into a vector fragment pMV-AMP to obtain a plasmid pMV-sgr810, wherein the plasmid is subjected to double enzyme digestion by NdeI and SpeI to obtain a sgr810-NdeI/SpeI fragment; cbmB-NdeI/SpeI, cbmC-NdeI/SpeI, OX4-NdeI/SpeI, sgr810-NdeI/SpeI and sshg _05717-NdeI/SpeI are respectively connected with vector fragments pUC-kasOp/ermEp-NdeI/SpeI through ligase, the ligation products are respectively transformed into escherichia coli DH5 alpha competent cells, and plasmids of pUC-cbmB, pUC-cbmC, pUC-OX4, pUC-sgr810 and pUC-sshg _05717 are obtained after verification and extraction;

(2) carrying out double enzyme digestion on plasmids pUC-cbmB and pUC-OX4 respectively to obtain cbmB-MfeI/SpeI and OX4-MfeI/SpeI fragments; plasmids pUC-cbmC, pUC-sgr810 and pUC-sshg _05717 are subjected to double digestion by XbaI and PstI to obtain cbmC-XbaI/PstI, sgr810-XbaI/PstI and sshg _05717-XbaI/PstI fragments; the cbmB-MfeI/SpeI, the cbmC-XbaI/PstI and the vector fragment pSET5035-MfeI/PstI are connected by ligase to obtain a plasmid pSET 5035-cbmB-C; OX4-MfeI/SpeI, sgr810-XbaI/PstI and the vector fragment pSET5035-MfeI/PstI are connected by ligase to obtain a plasmid pSET5035-OX4-sgr 810; OX4-MfeI/SpeI, sshg _05717-XbaI/PstI and the vector fragment pSET5035-MfeI/PstI are connected by ligase to obtain a plasmid pSET5035-OX4-sshg _ 05717;

(3) carrying out double enzyme digestion on plasmid pUC-cbmA by MfeI and SpeI to obtain cbmA-MfeI/SpeI; the plasmid pSET5035-cbmB-C is subjected to double digestion by XbaI and PstI to obtain a cbmB-C-XbaI/PstI fragment; the cbmA-MfeI/SpeI, the cbmB-C-XbaI/PstI and the vector fragment pSET5035-MfeI/PstI are connected by ligase to obtain a plasmid pSET 5035-cbmA-B-C;

(4) plasmid pSET5035-cbmA-B-C is subjected to MfeI and SpeI double enzyme digestion to obtain a cbmA-B-C-MfeI/SpeI fragment; plasmids pSET5035-OX4-sgr810 and pSET5035-OX4-sshg _05717 are subjected to double digestion by XbaI and PstI respectively to obtain OX4-sgr810-XbaI/PstI and OX4-sshg _05717-XbaI/PstI fragments; the cbmA-B-C-MfeI/SpeI, the OX4-sgr810-XbaI/PstI and the vector fragment pSET5035-MfeI/PstI are connected by ligase to obtain an expression vector pSET5035-cbm-OX4-sgr 810; the cbmA-B-C-MfeI/SpeI, OX4-sshg _05717-XbaI/PstI and the vector fragment pSET5035-MfeI/PstI are connected by ligase to obtain an expression vector pSET5035-cbm-OX4-sshg _ 05717;

(5) respectively electrically transforming the expression vectors pSET5035-cbm-OX4-sgr810 and pSET5035-cbm-OX4-sshg _05717 obtained in the step (4) into escherichia coli S17-1 competent cells, transferring the expression vectors into Streptomyces sp.S001 through escherichia coli S17-1 donor bacteria, and obtaining the engineering bacteria S001-cbm-OX4-sgr810 and S001-cbm-OX4-sshg _05717 for producing the polycyclic macrocyclic lactam compound after zygote purification and PCR verification.

Preferably, in step (1), the PCR amplification primer sequences are as follows:

NdeI-cbmB-F:5’-cggacatatgcggcgtagtaacagggtg-3’

SpeI-cbmB-R:5’-attactagttcacctcatgctcttgtcg-3’

NdeI-cbmC-F:5’-cggacatatgcctcgcaagccccg-3’

SpeI-cbmC-R:5’-attactagttcagcggacgtgaacgg-3’

NdeI-OX4-F:5’-aggcggacatatgaagaccgaacgttgggtgg-3’

SpeI-OX4-R:5’-attactagttcacacccgcaccagcacct-3’

NdeI-sshg_05717-F:5’-cggacatatgtccgccgcaacacccgcc-3’

SpeI-sshg_05717-R:5’-attactagtcaccaggtgaccgggagcc-3’

PCR amplification conditions: pre-denaturation at 95 deg.C for 5 min; denaturation at 95 ℃ for 30sec, annealing at 65 ℃ for 30sec, elongation at 72 ℃ for 2min, and 30 cycles; final extension, 5min at 72 ℃.

In the invention, the vector fragment pUC-kasOp/ermEp-NdeI/SpeI is obtained by double digestion of plasmid pUC-kasOp/ermEp with NdeI and SpeI; the vector fragment pSET5035-MfeI/PstI is obtained by carrying out MfeI/PstI double digestion on the plasmid pSET 5035-KT.

The artificial synthesis of cytochrome P450 enzyme gene sgr810 in the invention is carried out according to the prior art, and the vector fragment pMV-AMP is the prior plasmid vector.

Preferably, in step (3), the plasmid pUC-cbmA is prepared as follows:

carrying out PCR amplification by using fosmid XIV-12H containing polyketide-non-ribosomal peptide synthase gene cbmA as a template to obtain partial cbmA fragments with NdeI and SpeI enzyme cutting sites respectively introduced into two ends of the sequence; carrying out double enzyme digestion on the obtained PCR product by NdeI and SpeI to obtain a cbmA-part1-NdeI/SpeI fragment, connecting the cbmA-part1-NdeI/SpeI fragment with a vector fragment pUC-ermEp-NdeI/SpeI by using a ligase, converting a ligation product into an Escherichia coli DH5 alpha competent cell, verifying and extracting to obtain a plasmid pUC-cbmA-part1, and carrying out double enzyme digestion on the plasmid by using PstI and SphI to obtain a cbmA-part1-PstI/SphI fragment; carrying out double enzyme digestion on Fosmid XIV-12H by PstI and SphI to obtain a cbmA-part2-PstI/SphI fragment, connecting the cbmA-part1-PstI/SphI fragment and the cbmA-part2-PstI/SphI fragment by using ligase, converting a connecting product into escherichia coli DH5 alpha competent cells respectively, and obtaining a plasmid pUC-cbmA after verification and extraction;

wherein, the PCR amplification primer sequence is as follows:

NdeI-cbmA-LF:5’-cggacatatgtcggacctatgcaaggtc-3’

SpeI-cbmA-LR:5’-attactagtctacgccgcatgcgagattgatgtggggtggga-3’

PCR amplification conditions: pre-denaturation at 95 deg.C for 5 min; denaturation at 95 ℃ for 30sec, annealing at 65 ℃ for 30sec, elongation at 72 ℃ for 2min, and 30 cycles; final extension, 5min at 72 ℃.

The preparation method of the polycyclic macrocyclic lactam compound comprises the following steps:

(1) respectively inoculating engineering bacteria S001-cbm-OX4-sgr810 and S001-cbm-OX4-sshg _05717 on a YMG solid agar culture medium, and carrying out amplification culture at 25-35 ℃ for 8-15 days;

(2) cutting a YMG solid agar culture medium and thalli of engineering bacteria S001-cbm-OX4-sgr810 into blocks, soaking and extracting for 2-4 times by using ethyl acetate-methanol-glacial acetic acid (80:15:5, v/v/v) of an extracting solution, then combining leaching liquor, concentrating the leaching liquor under reduced pressure to 200-400 mL, extracting for 2-4 times by using ethyl acetate with the same volume, combining extracted ethyl acetate, and then concentrating under reduced pressure to be dry to obtain an ethyl acetate extract A;

(3) cutting a YMG solid agar culture medium and engineering bacteria S001-cbm-OX4-sshg _05717 into blocks, soaking and extracting an extracting solution ethyl acetate-methanol-glacial acetic acid (80:15:5, v/v/v) overnight for 2-4 times, then combining leaching solutions, respectively combining a water phase and an organic phase of the leaching solutions, concentrating under reduced pressure to 200-400 mL, then respectively extracting with ethyl acetate of the same volume for 2-4 times, combining extracted ethyl acetate phases, concentrating under reduced pressure to dryness to obtain an ethyl acetate extract B;

(4) extracting the ethyl acetate extract A with petroleum ether and methanol of the same volume to obtain a methanol phase and a petroleum ether phase, and washing a light yellow precipitate separated out from the methanol phase with methanol for 2-4 times to obtain the polycyclic macrocyclic lactam compound shown in the formula (I); and extracting the ethyl acetate extract B with petroleum ether and methanol in equal volume to obtain a methanol phase and a petroleum ether phase, washing a light yellow precipitate separated from the methanol phase for 2-4 times by using methanol-dichloromethane (2:1, v/v), and then separating by using sephadex column chromatography to obtain a component B1, and preparing the component B1 by using HPLC to obtain the polycyclic macrocyclic lactam compound shown in the formula (II).

According to the invention, in the step (1), the amplification culture is 8-10L, and the culture condition is 30 ℃ and 12 d.

Preferably, in step (1), the formula of the YMG solid agar medium is: yeast extract 4g, malt extract 10g, glucose 4g, 10M NaOH to adjust pH to 7.2-7.4, pure water to 1L, 2% agar, and autoclaving at 115 deg.C for 30 min.

The polycyclic macrocyclic lactam compound is applied to the preparation of medicaments for resisting gram-negative bacteria virulence protein secretion.

An anti-gram-negative bacterial virulence protein secretion medicament comprising a polycyclic macrocyclic lactam compound of the invention and one or more pharmaceutically acceptable carriers or excipients.

Advantageous effects

1. The polycyclic macrocyclic lactam compound provided by the invention has stronger inhibitory activity on the secretion of virulence protein of bacteria III type secretion system, and has concentration gradient dependence, and the III type secretion system widely exists in gram-negative pathogenic bacteria and has highly conservative structure, so the polycyclic macrocyclic lactam compound can be used for preparing medicaments for resisting the secretion of virulence protein of gram-negative pathogenic bacteria.

2. The invention constructs a polypeptide containing a polyketide-non-ribosomal peptide synthase gene cbmA responsible for synthesis of a PoTeMs polyene skeleton, a flavin-dependent oxidoreductase gene cbmB responsible for synthesis of a five-membered ring in a multi-ring system, a flavin-dependent oxidoreductase gene cbmC responsible for synthesis of a five-membered ring outside the multi-ring system, an NADPH-dependent oxidoreductase gene OX4 responsible for synthesis of a six-membered ring inside the multi-ring system, expression vectors pSET5035-cbm-OX4-sgr810 and pSET5035-cbm-OX 4-sshg-05717 responsible for post-oxidation modification of a cytochrome P450 gene sgr810 or sshg-05717, realizes heterologous expression of core skeleton genes, OX4 genes and different cytochrome P450 enzyme genes in Streptomyces sp.S001, and then separating and purifying the metabolites of the engineering bacteria S001-cbm-OX4-sgr810 and S001-cbm-OX4-sshg _05717 to obtain the polycyclic macrocyclic lactam compound. The invention utilizes a combined biosynthesis means to obtain the 'non-natural' PoTeM compound by modifying and heterogeneously expressing the combamides biosynthesis gene cluster cbm.

Drawings

FIG. 1 is a schematic diagram of the construction process of the engineering bacteria for producing the polycyclic macrocyclic lactam compound.

FIG. 2 is a gene PCR amplification agarose gel electrophoresis diagram of the engineering bacteria for producing polycyclic macrocyclic lactam compounds according to the invention;

in the figure: band 1 is a cbmB gene fragment; band 2 is cbmC gene fragment; lane 3 is a fragment of OX4 gene; lane 4 is sshg _05717 gene fragment.

FIG. 3 shows PCR-verified agarose gel electrophoresis of engineering bacteria for producing polycyclic macrocyclic lactams.

FIG. 4 is a HPLC check chart of metabolites of engineered bacteria of the present invention for the production of polycyclic macrocyclic lactams.

FIG. 5 is a graph of the effect of polycyclic macrocyclic lactams on Salmonella growth in accordance with the present invention.

FIG. 6 shows the inhibitory activity of polycyclic macrocyclic lactam compounds of the present invention against the secretion of virulence proteins of the Salmonella type III secretion system in vitro.

Detailed Description

The invention is further illustrated with reference to the following figures and examples.

Example 1 construction of plasmid pUC-cbmA

Carrying out PCR amplification on a cbmA-part1 fragment by using fosmid XIV-12H containing a polyketide-non-ribosomal peptide synthase gene cbmA as a template, designing primers by using bioinformatics software, and respectively introducing NdeI and SpeI single enzyme cutting sites at two ends of the sequence, wherein the sequences of the primers are as follows:

NdeI-cbmA-LF:5’-cggacatatgtcggacctatgcaaggtc-3’

SpeI-cbmA-LR:5’-attactagtctacgccgcatgcgagattgatgtggggtggga-3’

preparing a PCR amplification system according to the kit instruction;

PCR amplification conditions: pre-denaturation at 95 deg.C for 5 min; denaturation at 95 ℃ for 30sec, annealing at 65 ℃ for 30sec, elongation at 72 ℃ for 2min, and 30 cycles; final extension, 5min at 72 ℃.

The obtained PCR product is recovered by a gel recovery kit, the PCR product and the vector plasmid pUC-kasOp/ermEp are respectively subjected to double enzyme digestion by NdeI and SpeI, the cbmA-part1-NdeI/SpeI fragment obtained by enzyme digestion and the vector fragment pUC-ermEp-NdeI/SpeI are connected by ligase, the connection product is introduced into escherichia coli DH5 alpha competent cells by a chemical transformation method, the competent cells are coated on an LB solid agar plate containing 50 mu g/mL kanamycin and cultured overnight at 37 ℃, positive recombinants are picked up, and the plasmid pUC-cbmA-part1 is extracted from the positive recombinants by a plasmid extraction kit. The plasmid pUC-cbmA-part1 was digested simultaneously with PstI and SphI to obtain a cbmA-part1-PstI/SphI fragment. The plasmid pUC-cbmA-part 2-PstI/SphI fragment is obtained by carrying out double enzyme digestion on the Fosmid XIV-12H through PstI and SphI, the cbmA-part1-PstI/SphI fragment and the cbmA-part2-PstI/SphI fragment are connected through ligase, a connection product is introduced into an escherichia coli DH5 alpha competent cell through a chemical transformation method, the competent cell is spread on an LB solid agar plate containing 50 mu g/mL kanamycin and cultured overnight at 37 ℃, a positive recon is picked up, and the plasmid pUC-cbmA is extracted from the positive recon through a plasmid extraction kit.

Example 2 construction of plasmids pUC-cbmB, pUC-cbmC, pUC-OX4, pUC-sgr810 and pUC-sshg _05717

S10 genome DNA of Streptomyces sp.S10 is taken as a template to carry out PCR amplification on a cbmB fragment, bioinformatics software is utilized to design primers, NdeI and SpeI single enzyme cutting sites are respectively introduced into two ends of a sequence, and the sequences of the primers are as follows:

NdeI-cbmB-F:5’-cggacatatgcggcgtagtaacagggtg-3’

SpeI-cbmB-R:5’-attactagttcacctcatgctcttgtcg-3’

s10 genome DNA of Streptomyces sp.S10 is taken as a template to carry out PCR amplification on a cbmC fragment, bioinformatics software is utilized to design primers, NdeI and SpeI single enzyme cutting sites are respectively introduced into two ends of a sequence, and the sequences of the primers are as follows:

NdeI-cbmC-F:5’-cggacatatgcctcgcaagccccg-3’

SpeI-cbmC-R:5’-attactagttcagcggacgtgaacgg-3’

carrying out PCR amplification on OX4 fragment by using Lysobacter enzymogenes C3 genome DNA as a template, designing primers by using bioinformatics software, and respectively introducing NdeI and SpeI single enzyme cutting sites at two ends of the sequence, wherein the sequences of the primers are as follows:

NdeI-OX4-F:5’-aggcggacatatgaagaccgaacgttgggtgg-3’

SpeI-OX4-R:5’-attactagttcacacccgcaccagcacct-3’

PCR amplification is carried out on sshg _05717 fragments by taking Streptomyces albus J1074 genome DNA as a template, primers are designed by bioinformatics software, NdeI and SpeI single enzyme cutting sites are respectively introduced at two ends of the sequence, and the sequences of the primers are as follows:

NdeI-sshg_05717-F:5’-cggacatatgtccgccgcaacacccgcc-3’

SpeI-sshg_05717-R:5’-attactagtcaccaggtgaccgggagcc-3’

preparing a PCR amplification system according to the kit instruction;

PCR amplification conditions: pre-denaturation at 95 deg.C for 5 min; denaturation at 95 ℃ for 30sec, annealing at 65 ℃ for 30sec, elongation at 72 ℃ for 2min, and 30 cycles; final extension, 5min at 72 ℃.

The obtained PCR product was analyzed and detected by 0.8% agarose gel electrophoresis as shown in FIG. 2, wherein the electrophoresis band 1 of 1681bp is cbmB gene fragment, the electrophoresis band 2 of 1750bp is cbmC gene fragment, the electrophoresis band 3 of 1075bp is OX4 gene fragment, the electrophoresis band 4 of 1215bp is sshg _05717 gene fragment, and the above target fragments were recovered by a gel recovery kit.

Artificially synthesizing cytochrome P450 enzyme gene sgr810 according to the prior art, and then cloning the cytochrome P450 enzyme gene into a vector fragment pMV-AMP to obtain a plasmid pMV-sgr 810; PCR fragments of cbmB, cbmC, OX4 and sshg-05717, plasmid pMV-sgr810 containing artificially synthesized cytochrome P450 enzyme gene sgr810 and vector plasmid pUC-kasOp/ermEp were double-digested with NdeI and SpeI, respectively, and the resulting digested cbmB-NdeI/SpeI, cbmC-NdeI/SpeI, OX4-NdeI/SpeI, sshg-05717-NdeI/SpeI and sgr810-NdeI/SpeI were ligated with vector fragments pUC-kasOp/ermEp-NdeI/SpeI, respectively, by ligase, ligation products were introduced into E.coli DH5 alpha competent cells by chemical transformation, spread on solid agar plates containing 50. mu.g/mL kanamycin, pUC-pUC, cultured at 37 ℃ to obtain positive plasmids, and recombinant plasmids were extracted from the recombinant plasmids by using an extraction kit to obtain cbMC-4, cbO-853-CmC-mOX, pUC-sgr810 and pUC-sshg _ 05717.

Example 3: construction of expression vectors pSET5035-cbm-OX4-sgr810 and pSET5035-cbm-OX4-sshg _05717

Plasmids pUC-cbmA, pUC-cbmB, pUC-OX4 and vector plasmid pSET5035-KT are subjected to double enzyme digestion by MfeI and SpeI to obtain cbmA-MfeI/SpeI, cbmB-MfeI/SpeI, OX4-MfeI/SpeI fragments and vector fragment pSET 5035-MfeI/PstI.

Plasmids pUC-cbmC, pUC-sgr810 and pUC-sshg _05717 were double digested with XbaI and PstI to obtain cbmC-XbaI/PstI, sgr810-XbaI/PstI and sshg _05717-XbaI/PstI fragments, respectively.

The cbmB-MfeI/SpeI, cbmC-XbaI/PstI and the vector fragment pSET5035-MfeI/PstI are connected by ligase to obtain a plasmid pSET 5035-cbmB-C.

The plasmid pSET5035-cbmB-C is subjected to double digestion by XbaI and PstI to obtain a cbmB-C-XbaI/PstI fragment; the cbmA-MfeI/SpeI, cbmB-C-XbaI/PstI and the vector fragment pSET5035-MfeI/PstI are connected by ligase to obtain a plasmid pSET 5035-cbmA-B-C.

OX4-MfeI/SpeI, sgr810-XbaI/PstI and the vector plasmid pSET5035-MfeI/PstI are connected by ligase to obtain a plasmid pSET5035-OX4-sgr 810.

OX4-MfeI/SpeI, sshg _05717-XbaI/PstI and the vector plasmid pSET5035-MfeI/PstI are connected by ligase to obtain a plasmid pSET5035-OX4-sshg _ 05717.

After plasmid pSET5035-cbmA-B-C is subjected to double enzyme digestion by MfeI and SpeI, a cbmA-B-C-MfeI/SpeI fragment is obtained.

Plasmids pSET5035-OX4-sgr810 and pSET5035-OX4-sshg _05717 are digested by XbaI and PstI respectively to obtain OX4-sgr810-XbaI/PstI and OX4-sshg _05717-XbaI/PstI fragments.

The cbmA-B-C-MfeI/SpeI, OX4-sgr810-XbaI/PstI and the vector plasmid pSET5035-MfeI/PstI are connected by ligase to obtain an expression vector pSET5035-cbm-OX4-sgr 810.

The cbmA-B-C-MfeI/SpeI, OX4-sshg _05717-XbaI/PstI and the vector plasmid pSET5035-MfeI/PstI are connected by ligase to obtain an expression vector pSET5035-cbm-OX4-sshg _ 05717.

Example 4: construction of engineering bacteria S001-cbm-OX4-sgr810 and S001-cbm-OX4-sshg _05717

Respectively electrotransforming expression vectors pSET5035-cbm-OX4-sgr810 and pSET5035-cbm-OX4-sshg _05717 into competent cells of Escherichia coli S17-1, transferring the heterologous expression vectors pSET5035-cbm-OX4-sgr810 and pSET5035-cbm-OX4-sshg _05717 into Streptomyces sp.S001 via the competent cells of Escherichia coli S17-1, plating on SFM solid agar medium, performing inversion culture at 30 ℃ for 15 hours, covering each zygote with 1mg of nalidixic acid and 1mg of arabino antibiotic, picking up the zygotes and inoculating on YMG solid agar medium containing 25. mu.g/mL of nalidixic acid and 30. mu.g/mL of arabino antibiotic at 30 ℃ for about 5 days, purifying on YMG LB solid agar medium containing 25. mu.g/mL of nalidixic acid and 30. mu.g/mL of arabino antibiotic at 30 ℃ for about 5 days, pure zygote genomic DNA was extracted by culturing at 37 ℃ and 220rpm for about 3 days. Design and adopt primer InS10PKSNRPS-verF/InS10-verR to carry out PCR verification to the genomic DNA of zygote, PCR verification agarose gel electrophoresis is shown in figure 3, the expected size of the PCR product of the engineering bacteria S001-cbm-OX4-sgr810 is 6629bp, and the expected size of the PCR product of the engineering bacteria S001-cbm-OX4-sshg _05717 is 6621 bp. The spliceosomes with the expected sizes of the PCR products are engineering bacteria S001-cbm-OX4-sgr810 and S001-cbm-OX 4-sshg-05717 for producing the polycyclic macrocyclic lactam compound.

The SFM solid agar culture medium comprises the following components: 20g of soybean cake powder (boiled with soft fire for half an hour, filtered by 4 layers of gauze), 20g of mannitol, adding pure water to a constant volume of 1L, adjusting the pH value to 7.2-7.4, adding 2% agar, and sterilizing at 121 ℃ for 30min under high pressure.

The formula of the YMG solid agar culture medium is as follows: yeast extract 4g, malt extract 10g, glucose 4g, 10M NaOH to adjust pH to 7.2-7.4, pure water to 1L, 2% agar, and autoclaving at 115 deg.C for 30 min. The extraction of pure zygotic DNA was performed according to the prior art, and the PCR verified primer sequences were as follows:

InS10PKSNRPS-verF:5’-ccagggactacatcgccttctgc-3’

InS10-verR:5’-gctcactcaaaggcggtaatacgg-3’

PCR conditions were as follows: pre-denaturation at 95 deg.C for 5 min; denaturation at 95 ℃ for 30sec, annealing at 61 ℃ for 30sec, elongation at 72 ℃ for 1min, and 30 cycles; final extension, 5min at 72 ℃.

Example 5: fermentation culture of engineering bacteria for producing polycyclic macrocyclic lactam compound

The engineered bacteria S001-cbm-OX4-sgr810 and S prepared in example 4 were used001-cbm-OX4-sshg _05717 respectively inoculating to YMG solid agar culture medium, culturing at 30 deg.C for 10 days, cutting the culture medium and mycelium, soaking in ethyl acetate-methanol-glacial acetic acid (80:15:5, v/v/v) overnight, extracting once, concentrating the extractive solution at 25 deg.C under reduced pressure to obtain crude extract, and adding CH₃OH is dissolved, and the supernatant is taken for HPLC detection.

The formula of the YMG solid agar culture medium is as follows: yeast extract 4g, malt extract 10g, glucose 4g, 10M NaOH to adjust pH to 7.2-7.4, pure water to 1L, 2% agar, and autoclaving at 115 deg.C for 30 min.

As shown in FIG. 4, in the HPLC test of this example, it can be seen from FIG. 4 that both of the engineered bacteria S001-cbm-OX4-sgr810 and S001-cbm-OX4-sshg _05717 can produce two compounds, referred to as compound 1 and compound 2, respectively, but the engineered bacteria S001-cbm-OX4-sgr810 produce compound 1 at a higher yield, and the engineered bacteria S001-cbm-OX4-sshg _05717 produce compound 2 at a higher yield.

Example 6: preparation of Compounds 1 and 2

The preparation method of the polycyclic macrocyclic lactam compound comprises the following steps:

(1) inoculating engineering bacteria S001-cbm-OX4-sgr810 to YMG solid agar medium, and performing inverted culture at 30 ℃ for 12 days; inoculating the engineering bacteria S001-cbm-OX4-sshg _05717 to a YMG solid agar culture medium, and performing inverted culture at 30 ℃ for 12 days;

(2) cutting YMG solid agar culture medium and engineering bacteria S001-cbm-OX4-sgr810 into small blocks, soaking and extracting for 3 times in an extracting solution of ethyl acetate-methanol-glacial acetic acid (80:15:5, v/v/v) overnight, then combining leaching liquor, concentrating the leaching liquor at 25 ℃ under reduced pressure to 300mL, extracting for 3 times with equal volume of ethyl acetate, combining extracted ethyl acetate phases, and concentrating under reduced pressure to dryness to obtain ethyl acetate extract A;

(3) cutting YMG solid agar culture medium and engineering bacteria S001-cbm-OX4-sshg _05717 into small blocks, soaking and extracting in ethyl acetate-methanol-glacial acetic acid (80:15:5, v/v/v) overnight for 3 times, mixing the leaching solutions, mixing the water phase and the organic phase of the leaching solution, concentrating under reduced pressure at 25 deg.C to 300mL, extracting with ethyl acetate of the same volume for 3 times, mixing the extracted ethyl acetate phases, and concentrating under reduced pressure at 25 deg.C to dryness to obtain ethyl acetate extract B;

(4) extracting the ethyl acetate extract A with petroleum ether and methanol of the same volume to obtain a methanol phase and a petroleum ether phase, and washing a light yellow precipitate separated from the methanol phase with methanol for 3 times to obtain a compound 1; extracting ethyl acetate extract B with petroleum ether and methanol to obtain methanol phase and petroleum ether phase, washing light yellow precipitate separated from methanol phase with methanol-dichloromethane (2:1, v/v) for 3 times, separating by Sephadex LH-20 (60 g, methanol-dichloromethane, 10s/d, 3 mL/tube) to obtain component B1, and separating by semi-preparative HPLC (90% CH-H/C) for B1₃CN, 4mL/min, UV 320nm) to give Compound 2.

Example 7: structural characterization of Compound 1 and Compound 2

The compound 1 and the compound 2 prepared in example 6 were subjected to spectroscopic data analysis by 1D and 2D NMR, UV, IR and HRMS, and the analysis results are shown in table 1.

TABLE 1 preparation of Compound 1 and Compound 2¹H and¹³C NMR(Pyridine-d₅) Data of

]25+126.17(c 1.00,CH₃OH-CH₂Cl₂ 1:1,v/v)；UV-vis(CH₃CN)λ_max:232,321nm；IR:3319, ]25+167.56(c 1.00,CH₃OH-CH₂Cl₂ 1:1,v/v)；UV-vis(CH₃CN)λ_max:215,323nm；IR:3332, 1691,1656,1360cm^-1。

As shown in Table 1, the molecular formula of Compound 1 is C as determined by high resolution Mass Spectrometry (HR-ESIMS)₂₉H₄₀O₅N₂(m/z 497.3011 [M+H]⁺,calcd.for C₂₉H₄₁O₅N₂ ⁺,497.3015). The molecular formula of the compound 2 is C measured by high resolution mass spectrum (HR-ESIMS)₂₉H₄₀O₄N₂(m/z 481.3067[M+H]⁺,calcd.for C₂₉H₄₁O₄N₂ ⁺,481.3066). Determining that the compound 1 and the compound 2 are both polycyclic macrocyclic lactam compounds, and the specific structural formula is shown as the following formula:

example 8: testing the Effect of Compound 1 and Compound 2 on the growth of Salmonella typhimurium

Taking a salmonella typhimurium overnight culture bacterial solution at 37 ℃, and mixing the salmonella typhimurium and the culture medium according to a volume ratio of 1: 30 were transferred to LB liquid medium, then Compound 1, Compound 2 and positive control DMSO prepared in example 6 were added, respectively, and incubated at 37 ℃ with shaking at a shaking temperature of 37 ℃ and a shaking rotation speed of 220 rpm. Each sample was subjected to three replicates and OD was measured every 2h₆₀₀Values, 12h salmonella growth curves were plotted, and the graph is shown in fig. 5.

As can be seen from FIG. 5, the polycyclic macrocyclic lactams 1 and 2 had little effect on the growth of Salmonella typhimurium at concentrations of 100. mu.M and 50. mu.M, respectively.

Example 9: testing the inhibitory Effect of Compound 1 and Compound 2 on the secretion of virulence proteins of the Salmonella type III secretion System in vitro

Testing materials:

salmonella typhimurium UK-1(χ 8956); compound 1 and Compound 2 prepared in example 6 were dissolved in DMSO respectively to prepare stock solutions at concentrations of 20mM and 4mM, respectively; the positive control Csn-B was synthesized according to the prior art.

The specific test method is as follows:

(1) inoculating 10 mu L of salmonella stored in glycerinum tubing into 1mL of LB liquid medium for activation, and mixing the activated bacterium liquid according to the volume ratio of 1: transferring 10 of the transfer amount to 1mL of fresh LB liquid culture medium, and performing shake culture at the culture temperature of 37 ℃, the rotation speed of a shaking table of 220rpm and the culture time of 7 h;

(2) taking the culture liquid obtained in the step (1) to mix according to the volume ratio of 1: transferring the transfer amount of 10 into an LB liquid culture medium, wherein the transfer amount is 15 tubes, and the sample culture system of each tube is 1 mL; respectively adding stock solutions of the compound 1 into tubes 1-6 to ensure that the concentration of the compound 1 is 100, 50, 25, 12.5, 6.25 and 3.125 mu M respectively; adding the stock solutions of the compound 2 into tubes 7-12 respectively to make the concentrations of the compound 2 respectively 100, 20, 4, 0.8, 0.16 and 0.032 mu M; taking a 13-14 tube as a blank control, and adding DMSO with the same volume as that of stock solution required by 100 mu M of the compound to be detected; 15 tubes are used as positive control, and Csn-B with the final concentration of 100 mu M is added; performing shake culture at the culture temperature of 37 ℃ and the shaking table rotating speed of 220rpm for 7 h;

(3) centrifuging the bacterial liquid cultured in the step (2) for 10min at 4 ℃ and 12000rpm, taking 700 mu L of supernatant, adding 150 mu L of trichloroacetic acid solution, mixing the supernatant and the trichloroacetic acid solution gently, and standing the mixture on ice for 30 min; centrifuging at 12000rpm at 4 deg.C for 10min, discarding supernatant, inverting, draining, adding 400 μ L precooled acetone, gently shaking, and standing on ice for 10 min; continuously centrifuging at 12000rpm for 10min at 4 deg.C, discarding supernatant, inverting, draining, adding 25 μ L1 × loading buffer, dissolving protein by vortex, and heating at 95 deg.C for 5 min;

(4) and (3) taking 7.5 mu L of the protein sample prepared in the step (3), carrying out protein electrophoresis detection by 10% SDS-PAGE, dyeing by using 0.1% (w/v) Coomassie brilliant blue R250, decoloring, and carrying out imaging observation by using a gel imaging system, wherein the protein sample is shown in FIG. 6.

As seen in FIG. 6, compound 1 and compound 2 have inhibitory activity on the secretion of virulence proteins of the Salmonella type III secretion system and have concentration gradient dependence, compared to the positive control Csn-B (100. mu.M). The Minimum Inhibitory Concentration (MIC) of Compound 1 was 50. mu.M, and the MIC value of Compound 2 was 20. mu.M.

In conclusion, the polycyclic macrocyclic lactam compound has no inhibition on the growth of the salmonella, but has stronger inhibitory activity on the secretion of virulence proteins of a salmonella III type secretion system and has concentration gradient dependence. Therefore, the compound has the potential of being used as a novel antibacterial virulence drug.

SEQUENCE LISTING

<110> Shandong university

<120> polycyclic macrocyclic lactam compound and preparation method and application thereof

<160> 6

<170> PatentIn version 3.5

<210> 1

<211> 10005

<212> DNA

<213> Streptomyces sp. S10

<400> 1

atgtcggacc tatgcaaggt cgccacccgc gacaagatcg cgatcatcgg aatcggctgc 60

cgcctgcccg gcggcgcctc ggaccaccgg acgttctggc agaacctgat cgacggcagg 120

gactgcatca ccgcgacgcc cggaagccgg tatgacatcg cgaccctcgc cagccgggac 180

aagaccaagc ccggccgact ggtcggcggg cgcgggggtt acatcgacgg ttttgacgaa 240

ttcgacccgg cgttcttcgg catcagcccc cgcgaggccg cgcacatgga cccgcagcag 300

cgcaagttgc tggaggtggc ctgggaggcg ctggaggacg gcgggcagaa gccggccgag 360

ctggccgggc aggacgtcgg cgtcttcgtc ggcgcgttca cgctggacta caagatcctg 420

caattcgccg acctggggtt cgagacgctg gccgcccaca ccgcgaccgg cacgatgatg 480

acgatggtgt cgaaccgcat ctcgtactgc ttcgacttcc gcggccccag cgtctccctc 540

gacacggcgt gcagttcctc gctggtggcg gtccacctgg cctgccagag cctgagccgt 600

ggcgagagca gcctcgcgct cgccggcggc acgctgctgc acatggcgcc gcagtacacc 660

atcgccgaga ccaagggcgg cttcctctcc cccgagggcc gctcccgcac cttcgacgcg 720

tcggccgacg gttacgtgcg tgccgaaggc gtcggcgtgg tcgccctcaa gcgcctggac 780

gacgccctgc gcgacggcga tccgatccac gcggtggtca tcggcagcgg cgtcaaccag 840

gacggccgta ccaccggcat caccgtgccg aacgccgacg cccaggtcgc cctgatcgaa 900

cgggtctgca ccgaggcggg catcgcgccc ggcagcctgc agtacgtcga ggcccacggc 960

acgtcgaccc cggtcggcga cccgatcgag gcgggcgcgt taggccgggc gctgtcgatc 1020

gggagagaac ccggcgccgc ctgctacgtg ggctcggtca agaccaacat cgggcacacc 1080

gaatcggccg ccggcatcgc cgggttgatc aagacagcgc tctgcctgaa gcaccggaag 1140

atcccacccc acatcaatct cgaccagctc aatcccgcga tcgacctgga cgcgctgccc 1200

taccggatcc cgaccagcac ggtcgactgg ccggagcacg acggacccgc ccgcgccggg 1260

gtcaactcct tcggtttcgg cggcaccaac gcccacgtgc tgctcgaagc ggcaccggcc 1320

cgtgcggagg ccgcgaggaa cacggcagcg gccacgacga gcgcggcagc gggtacggca 1380

gcggccacgg agggcgctga aggcgcggca gcggcttcaa agggcagttc ggcagagggc 1440

gcgacgagca cggcagggac ttcgggcagt gcggcggcgg ccgttgtggg cgcgggggcc 1500

gaggtgggcg tgggggccgg gggctcggcg gcagcggttc cgccttcgta ctccatcctt 1560

ccgctgacca cgcgtgaccc ggcgctgctc cccgaggcgg cggccggcat ccgcaaggag 1620

ctggccgggt ccggccacgc tccggccgcc tcgctgaccg acatcggcta caccctggcc 1680

cgccgtcggc agcacctcga atcgcggctg tcggtcgtct acgactcgcg tacgtcgctc 1740

gaagcggccc tcgacgccta tctgcgcggc gaccagcacc cccatgtgct ggtcgaccag 1800

cggcgcgagg gcgagcagcg gcggctggtc tgggtcttca ccgggatggg gccgcagtgg 1860

tgggccatgg gccggcagtt gttcgagacc gagccggtct accgcgctgc ggtcgaacgc 1920

tgcgaccgcg agatccgtga cctcaccggc tggtcgctgg tggaggagtt gtccgccgac 1980

gaggcggact cgaagatgag cgagacctgg ctggcccagc ccgccaactt cgccgtccag 2040

gtcgggctcg cggccctgtg gcgcagccgc ggcgtccgtc cggacgccgt cgtcggccac 2100

agcaccggcg aggtcgcggc gttctacgaa gcgggcgtct acaccctgcg ggacgcggtc 2160

acggtcgtcg tgcaccgcag ccgcctccag cagaagctcg tggacaccgg cgcgatgctg 2220

gccgtgagtc tgtccgagga ggaggccgag cggcgggtgg cgccgtacgc ggaccgcgtg 2280

tcggtggccg ccgtcaacag cccgacggcg atcaccctcg ccggggacaa ggacgcgctc 2340

gcggagctgg ccctgcaact ccaggcggag gacgtcttcg ccaagttcct caccgtacga 2400

gtgccttacc acagcccccg tatggatccg atcaaggacg agttgctgtc ctcgctcgcc 2460

gcgctggagc cgcgcgccgc gcgggtgccg ctgtatctga cgggagggcc gcaggccgcg 2520

gcccgcggcc gggcggaagg gcccgagctg gacgcggact actggtggcg caacgtccgc 2580

gacagcgtgc gcttccggga cgccgtcgac cgcctcgtcg aggacggcca tggcctcttc 2640

ctggagatcg gcccgcatcc cgtgctgggt cactcgatcg ccgaatgcct gaccgccaag 2700

ggcgccgagg ggcgcagcct gccctcgatc cggcgccagg aggacgagcg cgcgcggttc 2760

accctctccc tcgcggctct ccacaacgcg ggcgtcgaca tcgcctggga ggtactgcac 2820

ccgcggggca ggccggtgcc gctgccccgc tacccctgga agcgggaccg gcactggacg 2880

gagccgaagg cggtcgagca gatccggctc ggcaggcgcg aacacccgct gctgggccgt 2940

cggctggcca cggcggaacc ggcctgggag gcccgactcg acgtcgaggc cctgccctat 3000

ctgctcgacc accgcatcca gggcagtgcg ctgttcccgg cggcgggcta cgtggagatg 3060

gcgacgcagg cggtgcgcgc gatgaccggc ggcggggagg ccgtgatcgc cgacatcgag 3120

ctgcgcaagg cgctgttcct gtcggccggt tcggacggcg ggaacgcccc ggaggagtcc 3180

ggcggtgacc cggccccggc cgccttgggc ggcgacaccg ccaccgtgca gctctccttc 3240

tccgcggaga acgccgcgtt caccgtcgcc acggtgcacg ccgacgactc ccgggagcgg 3300

accgtgcacg cacgcggagt cgtccgtacc gggcaacgcg ccgccggtac ggcccggctc 3360

gacgccggcc cggttaaggc gcgcagcctg cgccgcctcg acggcgccga ctgctatgcc 3420

gccctcagga cgctgggcta ccactacggc ccggcgttcc agggcatcga ggaggtgtgg 3480

atcggccggg acgaggcgct ggcccggatc cgtccgacgg agccgatcgg tgacgacgcc 3540

acccgccacc acatgcaccc ggtcctcctc gacgcctgct tccagaccct gctgaccccg 3600

cagatcgtcg agaccggtga cgacgccggg gacaccgcgg gcggcatccg gctgccgttg 3660

tcgatcgacg aggtgcgcat ggccgccgtg ggcgaccgtg cgctgtgggc gcacgccgtc 3720

gtcacccgcc gggcggacgg tgaactcgtc ggtgacatcg ccgtccacga cgacgacggt 3780

gcgccgctcg gccggatcag cggcttccgc gccgccgaca tcgagaaagc ctccgccgcc 3840

gtcagcctga acaccgtgga ctcctggctg gccgacgtca cctgggtcca cacgccgtac 3900

gcggcggacg tcgacacccc gtacgcggca gacgtcgacg ccccggacac accgggcggc 3960

gacaccgcgt ccgcggcacg caccgacgcc ctccacgcag cggacgccgg cacatcgcgt 4020

acggcggacc cggtcgctcc gtacggggcg gaagccgtcg cgccgtaccc gggagagacc 4080

gtcaccccgt tcccggcaga ggccgtagcc gcgggcggga cggccgcgcc ccgctggctc 4140

gtcctcgccg acggccgcgg cgtcggcgac gcgctcgcgg cgctgatcgc cgggcgcggc 4200

gggcactgcc acctggtacg cccaggagcc gcctaccgcg tcgccgacga cctgcgggag 4260

tcgacggtcc ggccggactc cgccgccgac ctggaccgtc tcctcgccga cctgcgcgcc 4320

gcgggcggcc ggccgtacga ccacgtcgtg cacctgtgga acctcgacct gcccacgttc 4380

gacgcgacgg ccgccgccga cgagctgcgt ggactctccg gtgtcggcgg ctactcgctg 4440

atcgcgctcg cccagacgct gccgtccgcg ctgccgggcg gcaggctgca catcgtcacc 4500

cgcggcgccc aggccgtcgt accgggtgat ccggtcgagc cgctgtccgc tccggcctgg 4560

ggcatcgggc gcgtgctgtg gcagcaggaa ctcaccgccc accgcgggaa gctgatcgat 4620

ctcgatcccg cgtccgcctc gtgcgaggcc gacgccgagg cactgctgcg ggaggtgccg 4680

gccgccggtg ggaccgccga cgacgagatc gcgctgcgcg ccggccaccg ctacaccagc 4740

cgcctcaccc cggtcgccgg ccggctgacc cgtccgctgc cgctgcggct gcgcgccgac 4800

ggcgcctacc tggtgaccgg tgccttcggc gcgctgggcc gggtgctctg ccgcgccctg 4860

gtcgcctggg gcgcacggcg gctgatcctg gtgggccgga cccggctgcc cggccgggag 4920

cactggcgcg agaccgaccc cggctccgcg gccggccgcg ccgtccggtt cgtgcgggag 4980

ctggaggcgc tgggcgccca gcccgtcctc gcgcccctcg acatcagcga cgaggcgagc 5040

ctgacggcct ggctggcgga gtaccggcgg ctcgcgtccg cgccgatccg cggtgtcttc 5100

cacctcgcgg gccaggtccg cgacaccctg ctgcccgaca tggaccggga gaccttcgac 5160

gcggtccacg atcccaaggt cctcggcgcg tacctcctgc accggcatct cggcgcggaa 5220

ccgctcgacc acttcgtgct cttctcctcg atcgcctccg tgctgacgac cgccgggcag 5280

accaactacg cggcgggcaa cgcgttcctc gacgcgctgg cgcaccaccg ccgggcccgc 5340

ggcctgcccg gtctcagcct ggactggggt ccctgggcga ccggcatgat cgaggagctg 5400

gggctcatcg agcactaccg caggaaccgc ggcatgagtt ccctgtcgcc cgaggcgggg 5460

acggccgtgc tggaacgcgt catcgggcag gaccgggccc agctgctcgt cgccaccgtc 5520

gtcgactggc cggtcttcct cgcctggtac gcctccccgc cgccgctggt gagcgagctg 5580

gccgcggccc tgggcggtcc gtccgccgca cagggggaac agagcagctt cctcgacgcg 5640

ttccggggcg ccgatgacgc ggcccgtcgg accctggtgg ccgagcgctt cacggcggtg 5700

gtcgcgggtg tgctgcgggt ggcgaccgac caggtcgagc tcgaccacgc actgggcgga 5760

ctgggcctgg actcgctgct cgcgatggag ctgcgctccc gcgtgcacgc ggaactcggc 5820

ctcgcgctcc cggtcgtcac cctgctcagc agcgccccgg tgggcgaact gatcgaccgg 5880

ctccacgagg gcctggccga gctggtggcg gccggtgcgg acgaggccgc cggcggtgcc 5940

gcggtcgagc tgttccgcga cgagggacgg cacccgctga cccagaacca gaaggcgctg 6000

tggttcctca agcagctcaa ccccgacggt ttcgcctaca acatcggcgg cgcggtcgag 6060

gtgcgcgtgg aactcgaacc ggagctgatg ttcgacgcgt tccgggtgct gatcgagcgc 6120

catcccagcc tgcgcgccaa cttcacgcac gagcaggggc agccggtgca gaaggtcgcc 6180

cccgccgccg aggccgcgct gcgccgggac gtcgccctct tcgacgtgga gggcgtggcg 6240

tgggacgacg tccacgcgat gatcgtgcgc gagtaccgca agccgtacga cctcgaacgc 6300

gacccgctga tccggttgcg cctgttccgg cgcggtcccg accgctgggt gctgatgaag 6360

gccgtccacc acatcatctc cgacgcgatc tctaccttca ccttcatcga ggagctgctc 6420

gcggtctacg aggcgctgcg ccggggccgc gcccccgagc tgccgccggt ctcggcccgc 6480

tacctcgact tcctgaactg gcagaaccag ttcctcgccg gacccgaggc gcgccggatg 6540

ctcgaccact ggaagggccg gctgccggcc gacgtcccgg tgctcggcct gcccaccgac 6600

aagccccgtc ccgcggtgca gacccacaac ggcgcgtccg agttcttcgt gctcgacgcc 6660

gacctcagcg cccgcgtcca cgacgtggcc cgcgagcaca acgtcaccgt gttcatggtg 6720

ctgctcagcg cctactacct gctgctccac cactactccg ggcaggacga catcatcgtc 6780

ggcagcccgg tgacgggccg tacccaggag gagttctcct cggtctacgg ctacttcgtc 6840

aatccgctgc cgctgcacgt cgatctgggg gacggcccgt cggtggagca gttgctcgcc 6900

cgggtccggg atgtcgtgct cggcggcctg gacaaccagg agtacccgtt cgtgctgctg 6960

gtggaggagc tgggcctcca gcacgacccg agccggtccg cggtcttcca agccatgttc 7020

atcctgctca cccacaaggt ggccaccgag aagtacggct accgcctgga gtacatcgaa 7080

ctgcccgagg aggagggcca gttcgacctg acgctgtcgg tgtacgagga cgaggcggac 7140

cggcgtttcc actgcgtctt caagtacaac accgacctgt tcctggccga gacgatgcgg 7200

cggctgtccg ggcactacgt caacgtcctc gacgccctga cccgcgcccc cgccgcgcag 7260

ccggccggcc ggctggaact gctgggcgcc cgtgagcgcc ggcagatcct cggcgactgg 7320

agcggggccg gacgccgggc cgccgagccc ggtgtgccgg tgcacgagct gatcgcccgg 7380

gccgccgccg agcaccccgg ggcgaccgcc gtctgcgtgc ccgccgagga cggcacgacc 7440

gagcgcatga cctacgccgc actcgaccgc cgcagccgcg accgcgcgcg acggctgcgc 7500

gaactgggcg tccgcgacgg cgcggtggtc gccgtctgca tggagaagtc ggccgagctg 7560

gtggtcaccc tgctggcggt cctccgggcg ggcggcgcct atctgccgct cgaccccggc 7620

aacccggccg accggctggc ctacatggcc gagcacgccg gggccgcgcg cgtcgtcgtg 7680

gacggggcgg gcggtgcgcg cttcgccggg cattccggcg tgctcaccct ggcggagctg 7740

tccgacgcgg cgggcgcgcc cggcacggcg gccgccccgc caaaaccggc ggggcaggcg 7800

gaaccggccg ggccgacgga ccagatcgag cccaccgggc aggccaggcc ggtcgcggcc 7860

ggtactgcgg ctgccgcggc cgacgcagcc gatccgaccg acccgaccga cccactcgtc 7920

ggcatggacg gcccggccta cgtcatctac acctccggct ccacgggccg tcccaaggcg 7980

gtgcaggtca gccatcgcaa cctggcgtcc gcctacgccg cctggcgagc ggagtaccgc 8040

ctcgacacgg acgcccgggt ccatctccag atggccggcc cggcgttcga cgtgttcacc 8100

ggtgacctgg tccgcgccct gtgctccggc gggaccctgg tgctggtcgg ccgtgagctg 8160

ctgttcaaca cggcccggct gtacgcgacc atgcgcgccg agcgggtcga ctgcgccgag 8220

ttcgtgccct ccgtcgtgcg cggcctgatg gcccactgcg aacgcgacgg cgcccggctg 8280

gacttcatgc gcctgctgat cgtcggctcg gacacctgga aggtggagga gtacgaccgg 8340

ctgcgcggcc tgaccggacc ggacacccgc gtggtcaact cctacggcct caccgaggcc 8400

accgtggaca gcacctactt cgagggcccg accgacggcc tggaaccgag ccagatggtg 8460

ccgatcggcc ggccgttccc cggtgccgag gtgtacatcc tggacgggca cggcgcaccg 8520

gtgccgccgg gcgtcgcggg cgagctgtgg atcggcggcg ccggggtcgc cctcggttat 8580

ctcgccgatc ccgggcacac cgccgagcgt ttcgtcagcc gcaccctgga ccacggcagc 8640

ggcgcggcac cggtacggct gtaccggacc ggcgacctgg cccgctggga caccggcggc 8700

ggagtccacc tgctcggccg ggccgactca caggtcaagg tcggcggcca ccgggtggag 8760

atcggcgaga tcgagttcca gctcgcggaa tggcccgcgc tggcccaggc ggtcgtcgtc 8820

gtccggccgg accgcaacgg cgagaacacg ctgtgcgcgt acggcgtcgc cgcacccggc 8880

gcgaagctcg actggcggga gctgcgccgg cacctcggcg agcacctgcc gacgtatctg 8940

gtcccggcgg ctttcaccga gctgcccgcg ctgccgctca ccccgaacgg caaggtcgac 9000

gtcaacgcgc tgcccgagcc ccggttcgag accggcgagg acgcccacga gccgcccgtg 9060

acgctctacg agacgcggat ggccgaccac tgggcggcgc tgctcggtct cggggaggtc 9120

ggtctccagc acgacttctt cgaggtcggc ggcagctcca tcaagctgat cgagctgatc 9180

cacgggttgc aggacgagtt cgagatcagc atcccggtca gccagctgtt caaggtcacg 9240

acgctgcacg ggatggcccg gaccgtcgag cacatcatca ccggccggct gccgggcgcc 9300

cagccgttcc tccgcttcaa ccggggggcg ggtcccgccg tgttctgctt cccgcccgcg 9360

ggcgggcacg gcctggtcta ccggcggttc gccgagcagc tgccggagta cgaactcgtg 9420

gcgttcaact acgtcgccga ggacgacaag gtcgcccggt acgccgatct gatcgccgcg 9480

caccagcccg aggggccctg cgcgctgctc ggctactccc tcggcgggaa cctcgccttc 9540

gaggtggcca aggaactcga acggcgcggc cgtgaggtcg ccgacgtcgt catcgtcgac 9600

tcgtaccgca tcgatgccgc cttcaccccg ggcgccgagc acatcgaggc gttcgagcgg 9660

gagctgcgcg agcacctgca caagcacacc ggctccgaga ccctcgcgca ggagaccctc 9720

gaccaggcca gggactacat cgccttctgc ggccgtactc ccaacctcgg cacggtcggc 9780

gcggccgtca ccgtcgtctc cgacgagcag aaggtggcct tctacgccgc gggcgagcgc 9840

gggacctggc acggcacctc gaccgtgcgc accgaggtgc tgcgcggcgc cggcgtccac 9900

gcggacatgc tcgacccggg gtgcatcgag cacaacgccc ggctggtgcg cggcgtcctc 9960

acgcacaccg ccgtgatcga aggggcggcc ccgcatgcgg cgtag 10005

<210> 2

<211> 1665

<212> DNA

<213> Streptomyces sp. S10

<400> 2

atgcggcgta gtaacagggt gcagtggagc gaacgcgagc gtgcccccgg ggccaagccg 60

cgcgtcatca tcgtcggagc gggcctgggc gggctctccg ccggctgcta cgggcagatg 120

agcggcatgg agacccgtgt cttcgagaag cacgtgctgc cgggcggctg ctgcacggcc 180

tggtcccggg acggctacgt cttcgactac tgcatcgaat ggctgaccgg caccgccccg 240

ggcaacgagg cccatcaggt ctggcgcgag ctgggggccc tggacgggaa gaccatccgc 300

aacttcgagg tgttcaacac ggtcgtcgac gacgacggcc ggtcggtggt cttctacaac 360

gaccccgacc ggctggagcg gcacctgctg cggctctcgc cggcggacgc gccgctgatc 420

cggtcgttct gccgtgacct gcggcggttc accgacatcg acatcttccc gttcctgaag 480

ccggccccgc tgctgacgct gcgggagaag gccagggcgc tgcggaagat cctgccgctg 540

ctgaacctgt tccggcgcac cgccgggacc tccatggagt ccttcgccgc gcgcttcgag 600

gatccgctgc tgcgccgggc gctgccgttc gtcttcttcc aggaccacga ggtcttccca 660

ctgctgccgt acctgttcaa catggcgggg gcccaccagt ccaacgcggg cttcccgcag 720

ggcggttcgc tggggctggc ccggtcgatc gaggagcgct acacctcgct cggcggccgg 780

atcgactacc gcgcccgggt ggagcagatc ctggtcgagg acggccgggc gatcggcgtg 840

gaactgcgcg gcggcgagcg gcactacgcg gaccacgtgg tggcggcctg cgacggcgcc 900

accaccctgg accggttgct gaagggacgc tactccagcc cgcggaccga ccggctgttc 960

cagtcggtcc tcggcacccc gaagctggtc tacccggggg tggtgtcggt gttcgtcggg 1020

ttcgccgggg acgtggcgcc ggacgccccg cacagcgcga cctatctgct ctccgccgcc 1080

gacagcgccc ggctgcccgg cgtgctccag gacagtctgg tggtgcagtt gcgctcccgg 1140

ttctcggacg ggctggcacc gcccggcaag tcggtgatcc actgctccta cttcagcgac 1200

tacagctcct ggaaggaact gcgccgcacc gaccgccgcg cctaccgggc gcgcaagcag 1260

gaggccggcc ggttcgtgcg ggagttcctg gagcgccacc atccggggat cgccgcgcgc 1320

atcgaggtgg tcgacgtcgc cacgcccgcg accaccgagc gctacaccgg caatctgcac 1380

ggctccatcc tcgcctggaa gtcctacacc gaagccgacg gcctgatcca gcacctgatc 1440

gagaaggacc ggctgcggct gccggggctc gacgggctct cgctggcggg tcagtggttc 1500

accggcggcg ggctgatccg ggtggcggcc ggcggccggt tcgtggccca gtacctctgc 1560

gaggagctgg gcctcgagtt ccgggcgtgg gagagcacgg ccggtgagac gtggcatccc 1620

gggaagctgg gccacctgcc ccagctcgac aagagcatga ggtga 1665

<210> 3

<211> 1734

<212> DNA

<213> Streptomyces sp. S10

<400> 3

atgcctcgca agccccgcga gcgccagaca atgatcatca tcggggccgg tctcggcggg 60

ctgtccaccg gctgctacgc gcagatgaac ggctaccgga cccggatctt cgagatgcac 120

gagatcccgg gcggctgctg cacggcctgg gaccgtggcg acttcacctt cgactgctgc 180

gtcagctggc tgctcggcaa cgggcccggc aacgagatgc accagatctg gctggagctg 240

ggcgcgctcc agggcaagca gatgcggcac ttcgacgtct tcaacgtggt gcgcgggcgg 300

gacggccggt cggtctactt ctattccgac ccggaccggc tcgaagccca tctgctggag 360

atctccccgt ccgacgccag gctgatccga agtttctgct cggggctgcg gaagttccgc 420

aagtgccttg ccgcgtatcc cttcctcaaa ccggtgggac tgatgggccg cgccgaacgc 480

tggcgcatgc tcgcgtcgtt cgtcccctat ttcaatgtca tccgcaagtc gatcagcgtc 540

ctgatgacgg actattccgc gaagttcagg gatccgctgc tgcgcgaggc gttcaatttc 600

atcctctacg agaagcattc cgcttttccg gtgctgccgt tctatttcca gctgtccgcg 660

cacgccaatc tctcggccgg ggtgccggag ggcggctcac tggggctcgc gacctcgatc 720

gagcagcgct atctgcggct cggcggcgag gtcagctaca acacgaaggt cgaggagatc 780

ctcgtcgagg acgaccgggc ggtgggggta cggctcagcg acgggcggga gttccgcgcg 840

gacatcgtgg tctcggcctg cgacgggcac gccaccacga tgaagctcct gaagggccgt 900

tacctccccg aggagtaccg gcggctgtac acctcggtca tcgacgagcc gggcatggtc 960

ttccccggat acgtcaccct cttcctcggg ctgcggcgcc ccttccccga gggcgccccc 1020

tgcaccacgt acctgctgac cgaggaggag gccacccggc tggtcggcat ccgccatccg 1080

agcatcaacg tccagttccg cagcctgcac tacccggagc tgtcgcccga gcggtcgacc 1140

gtcgtctacg ccacctactt cagcgacgtc ggtccctggc gcgcgctgag cgacggcccg 1200

gagcagcgca cccgcagccg gggcggaacg gagctgcaca ccctgccggt gcgccgcggg 1260

cgcccgtact acgaggctaa gcaccaggtg cgcgacatgc tgctgggctt cctcgaccgg 1320

cggttccccg gcctgacgga cagcgtcgcg gtgcgggacg tgtcgacccc gctcacccag 1380

gtccgttaca ccgggaacta cgacgggacc gtcctcggct ggcagccgtt cgtggaaagc 1440

ggcgagacga tggagaaggt cgtcaagaag tacgggccgg ccctgcccgg cctcgcgaac 1500

ttctacctct ccggggtctg ggccaccacc ggcggcctca tccgcgccgc ggccgccggc 1560

cggcacgtca tgcagttcgt ctgccgcgac gacggcaggg cgttcaccgc acacatcgac 1620

gcgtcggggc cgctcccgac ccatgtcgtc atccccgtag gacccgatgg gaaggatgga 1680

gaccccgatg agcgaacgac cggcgccgct gccgtatccg ttcacgtccg ctga 1734

<210> 4

<211> 1056

<212> DNA

<213> Lysobacter enzymogenes C3

<400> 4

atgaagaccg aacgttgggt ggtacgcgaa cacgtcgagg gcgtgcccga tgccgcgcgc 60

atctacgaaa aagtcgagac cgaactgaac acgcgcctgg gcgaggagca gatgctgctc 120

aagaccctgt acgtgtcggt cgatccctac ctgcaaggca tctgcctgga cacgccgatc 180

ggcgaccaca tgggcgccga ctcgatcatg caggtgctcg atgccggccc gaacgcgccg 240

ttccggccgg gcgacctggt gcagggcttc ggcggctggc gcacgcacct ggtcagcgac 300

ggcaagccca agctgtggca gaccggcacc ttcccgatgg tgttcccggc ctatcgcaag 360

ctcgacctgc gccactacga cgacgccctg ccgctgtcga ccgcgctcgg cgtgatgggc 420

ggccccggca tgaccgcctg gggcacgatg accaaattca tgcaggtgcg tcccggcgac 480

accgtcgtgg tcagcggcgc ctcgggcatg atcggcaccc tggttgggca gatggccaag 540

cgcgccggcg cgcgcgtggt cggcaccgcc ggctcggccg gaaaggcccg ctacctgagc 600

cagctcggct tcgacgcggt gatcgactac aagctcgccg acgacgccga caagatgcgc 660

gaagcgctgc gcgaggccgc gcccgacggc gtggacaagt acttcgacag catcggcggc 720

agcgtcaccg acgcggtgtt ctcgatgctc aacgtcggca gccaggtcgc ggtgtgctgg 780

caatgggcga cccaggtcca gcgcgactac cacggtccgc gcctgctgcc ctacatcatg 840

ttcccgcgcg cgaccatccg cggcatcttc tcgctggagt ggttcaccga gcagaactgg 900

agcgcgctgc acgaggaact cggcgggctg gtgcggcggc aagagctggt cgcgcacgaa 960

accgtgcagg acgggttcga gcatattccc gccgcatacc agacgctgtt ctcggccagt 1020

gaaagcaacc gcggcaaggt gctggtgcgg gtgtga 1056

<210> 5

<211> 1191

<212> DNA

<213> Streptomyces griseus IFO 13350

<400> 5

atgaccacca ccgacccgac ccgcccggcc ccggtcccga tgcaccgcct gttcttcgag 60

gagccgggcc cgccgcgccc ggccgagctg ccgggcggcg acccggcctg gttggtgtcc 120

cgctacgccg acgtccgcca ggtcctgtcc gacccgcgct tcggccgcgc ccgcctgtac 180

gccccggagg ccccggccct gtccggcgtc ccggacctgg tcaacaaccc ggacctgatg 240

ttcaaccagg acggctccga ccacctgcgc ctgcgccgca ccctgcgccg cgccttcacc 300

ccgcgcgccg tcgcccgctg gcgcccgtgg atcgccgcca ccgtcgaggg catcctggac 360

cgcctggagt cccgcccgca gccggccgac gtcgtcgccg agttcgccct gccgctgccg 420

gtcgccgtca tctcccgcct gatgggcctg gacgagtccg tctgggaccg catgcgctac 480

tggtccgagc acgccttctc cgacggcacc cacgagcgcg agcaggtcgc cgccgccctg 540

aaggagttct ccgccttcgg cgcccacctg ctggccgagc gccgctccac cccgggcgag 600

gacctcgtgt ccggcctggt caccgccgcc gacgaggagg gcggcgtccc ggaggcccag 660

ctggtgtccc tggtctgcgg cctggtcgtc ggcggccacg actccaccat gaccatgctg 720

ggcaacgccc tgctgtacct gctgggcgag cgccgcgaga catggccgcg cctgggcgcc 780

gacgaggagg ccgccggcct gctggtcgag cgcctggtcc acctggtccc gctgggcgac 840

gaccgcggct ccacccgcca cgccgccgag gacgtcgaag tctccggcgt ccgcatcccg 900

gccggcgcca tcgtcatcgc cgactgcggc atggccaacc gcgacccgga ggtcttcccg 960

ccggccaccc tgtacgacct gttcgccccg ctggaggccc cgaccctgtc cttcggcgcc 1020

ggcccgcact actgcctggg cgcctggctg gcccgcaccg agctgcaact ggccctgcac 1080

cgcctggccg cccgcttccc ggagctgcgc ctggccgacc cggtcgacgc cgtcgtctgg 1140

cgcaccggca ccacctcccg ctccccgcgc cgcctgggcg tccgctggtg a 1191

<210> 6

<211> 1200

<212> DNA

<213> Streptomyces albus J1074

<400> 6

atgtccgccg caacacccgc ccccgccgga gccgttccgc cgctcgcccc gctccaccgc 60

cgggcgcccg ccgagccggg accgccccgg ccgtgcaccc tgcccgacgg ttcccccggc 120

tggctggtcg accgctacgc cgacgtacgg caggtgctca gcgacagccg gttcggccgc 180

gccgggctct acctccagga cggcccctcc cgctcccagg cggccggact ggtggacgac 240

ccggagctga tgttcaacca ggacggcgtc gagcacctgc ggctgcgccg caccctgcgc 300

cgggccttca ccccgagggc cgtcgcccgg tggcgcccct ggatcgcctc gatcgtcgac 360

cagctcctgg acgacctgtc ggcgcggagc ggaccggtgg acgccgtcgc cgagttcacc 420

ctgccgctgc cggtcgccgt gatcagccgc ctgatgggcc tcgacgcctc ggtgcgtggc 480

cggatgcgcc actggagcga acacgccttc tccgacggca cccggcccaa ggacgaggtg 540

gacgcggcgc tcgcggagtt caccgccttc ggcgcccgac tgctcgccca gcggcgccgg 600

gcccccggcg acgacctggt cagcagcctg gtgcgggccg ccgacgccga gggcggcatc 660

cccgaggacc gtctcgtcag cctggtctgc ggcctggtgg ccggcgggca cgacagcacc 720

atgacgatgc tcggcaactc cctgctctac ctgctcgcgg aacggcccga ggagtggccc 780

cggctggccg acgagccctc ggcggagctg gccgccgccc gcctgatcca cctgatcccg 840

ctcggcgacg acccgggcag cacccgctgc gccaccgagg acgccgaggt cgggggcgtc 900

ctcatcccgg ccggcgcggt ggtcctcgcc gactccacca ccgccaaccg cgacccgtcg 960

gtcttcccgg ccgcgcagac cgaggccctc ttcaccccgc tgccggcgcc caccctcgcc 1020

ttcggcgcag ggccccacta ctgcctgggc acctggctcg cccggctcga actccacctg 1080

gccctgcacc ggctggcggt ccgcttcccc gggctgcggc tcgcggaacc ggagaagccg 1140

gtccgctggc gcccggccgg cacctcacgc agccccgaac ggctcccggt cacctggtga 1200

Claims (10)

1. The polycyclic macrocyclic lactam compound is characterized in that the structural formula is shown as a formula (I) or (II):

2. an engineered bacterium for producing the polycyclic macrocyclic lactam of claim 1, wherein the metabolite of the engineered bacterium comprises the polycyclic macrocyclic lactam of claim 1.

3. The engineering bacterium of claim 2, wherein the engineering bacterium comprises a polyketide-nonribosomal peptide synthase gene cbmA responsible for synthesis of PoTeMs polyene backbone, and the nucleotide sequence is shown as SEQ ID NO. 1; the flavin-dependent oxidoreductase gene cbmB responsible for the synthesis of the middle five-membered ring of the polycyclic system has a nucleotide sequence shown in SEQ ID No. 2; the flavin-dependent oxidoreductase gene cbmC responsible for five-membered ring synthesis outside the polycyclic system has a nucleotide sequence shown in SEQ ID No. 3; an NADPH-dependent oxidoreductase gene OX4 responsible for synthesis of six-membered rings inside the polycyclic system, the nucleotide sequence of which is shown in SEQ ID No. 4; the cytochrome P450 enzyme gene sgr810 or cytochrome P450 enzyme gene sshg _05717 responsible for post-oxidation modification has the nucleotide sequences shown as SEQ ID NO.5 and SEQ ID NO.6, respectively.

4. The construction method of the engineering bacteria of claim 2, characterized by comprising the following steps:

(1) carrying out PCR amplification by taking the genomic DNA of Streptomyces sp.S10 as a template to obtain flavin-dependent oxidoreductase gene cbmB and flavin-dependent oxidoreductase gene cbmC gene segments with NdeI and SpeI enzyme cutting sites respectively introduced at two ends of the sequence; carrying out PCR amplification by using the genome DNA of Lysobacter enzymogenes C3 as a template to obtain an NADPH dependent oxidoreductase OX4 gene segment with NdeI and SpeI enzyme cutting sites respectively introduced into two ends of the sequence; performing PCR amplification by taking Streptomyces albus J1074 genome DNA as a template to obtain a cytochrome P450 enzyme gene sshg _05717 gene fragment with NdeI and SpeI enzyme cutting sites respectively introduced at two ends of the sequence; the four PCR products are subjected to double enzyme digestion by NdeI and SpeI to obtain cbmB-NdeI/SpeI, cbmC-NdeI/SpeI, OX4-NdeI/SpeI and sshg-05717-NdeI/SpeI fragments; artificially synthesizing a cytochrome P450 enzyme gene sgr810 and cloning the gene into a vector fragment pMV-AMP to obtain a plasmid pMV-sgr810, wherein the plasmid is subjected to double enzyme digestion by NdeI and SpeI to obtain a sgr810-NdeI/SpeI fragment; cbmB-NdeI/SpeI, cbmC-NdeI/SpeI, OX4-NdeI/SpeI, sgr810-NdeI/SpeI and sshg _05717-NdeI/SpeI are respectively connected with vector fragments pUC-kasOp/ermEp-NdeI/SpeI through ligase, the ligation products are respectively transformed into escherichia coli DH5 alpha competent cells, and plasmids of pUC-cbmB, pUC-cbmC, pUC-OX4, pUC-sgr810 and pUC-sshg _05717 are obtained after verification and extraction;

(2) carrying out double enzyme digestion on plasmids pUC-cbmB and pUC-OX4 respectively to obtain cbmB-MfeI/SpeI and OX4-MfeI/SpeI fragments; plasmids pUC-cbmC, pUC-sgr810 and pUC-sshg _05717 are subjected to double digestion by XbaI and PstI to obtain cbmC-XbaI/PstI, sgr810-XbaI/PstI and sshg _05717-XbaI/PstI fragments; the cbmB-MfeI/SpeI, the cbmC-XbaI/PstI and the vector fragment pSET5035-MfeI/PstI are connected by ligase to obtain a plasmid pSET 5035-cbmB-C; OX4-MfeI/SpeI, sgr810-XbaI/PstI and the vector fragment pSET5035-MfeI/PstI are connected by ligase to obtain a plasmid pSET5035-OX4-sgr 810; OX4-MfeI/SpeI, sshg _05717-XbaI/PstI and the vector fragment pSET5035-MfeI/PstI are connected by ligase to obtain a plasmid pSET5035-OX4-sshg _ 05717;

(3) carrying out double enzyme digestion on plasmid pUC-cbmA by MfeI and SpeI to obtain cbmA-MfeI/SpeI; the plasmid pSET5035-cbmB-C is subjected to double digestion by XbaI and PstI to obtain a cbmB-C-XbaI/PstI fragment; the cbmA-MfeI/SpeI, the cbmB-C-XbaI/PstI and the vector fragment pSET5035-MfeI/PstI are connected by ligase to obtain a plasmid pSET 5035-cbmA-B-C;

(4) plasmid pSET5035-cbmA-B-C is subjected to MfeI and SpeI double enzyme digestion to obtain a cbmA-B-C-MfeI/SpeI fragment; plasmids pSET5035-OX4-sgr810 and pSET5035-OX4-sshg _05717 are subjected to double digestion by XbaI and PstI respectively to obtain OX4-sgr810-XbaI/PstI and OX4-sshg _05717-XbaI/PstI fragments; the cbmA-B-C-MfeI/SpeI, the OX4-sgr810-XbaI/PstI and the vector fragment pSET5035-MfeI/PstI are connected by ligase to obtain an expression vector pSET5035-cbm-OX4-sgr 810; the cbmA-B-C-MfeI/SpeI, OX4-sshg _05717-XbaI/PstI and the vector fragment pSET5035-MfeI/PstI are connected by ligase to obtain an expression vector pSET5035-cbm-OX4-sshg _ 05717;

(5) respectively electrically transforming the expression vectors pSET5035-cbm-OX4-sgr810 and pSET5035-cbm-OX4-sshg _05717 obtained in the step (4) into escherichia coli S17-1 competent cells, transferring the expression vectors into Streptomyces sp.S001 through escherichia coli S17-1 donor bacteria, and obtaining the engineering bacteria S001-cbm-OX4-sgr810 and S001-cbm-OX4-sshg _05717 for producing the polycyclic macrocyclic lactam compound after zygote purification and PCR verification.

5. The method of claim 4, wherein in step (1), the PCR amplification primer sequences are as follows:

NdeI-cbmB-F:5’-cggacatatgcggcgtagtaacagggtg-3’

SpeI-cbmB-R:5’-attactagttcacctcatgctcttgtcg-3’

NdeI-cbmC-F:5’-cggacatatgcctcgcaagccccg-3’

SpeI-cbmC-R:5’-attactagttcagcggacgtgaacgg-3’

NdeI-OX4-F:5’-aggcggacatatgaagaccgaacgttgggtgg-3’

SpeI-OX4-R:5’-attactagttcacacccgcaccagcacct-3’

NdeI-sshg_05717-F:5’-cggacatatgtccgccgcaacacccgcc-3’

SpeI-sshg_05717-R:5’-attactagtcaccaggtgaccgggagcc-3’

PCR amplification conditions: pre-denaturation at 95 deg.C for 5 min; denaturation at 95 ℃ for 30sec, annealing at 65 ℃ for 30sec, elongation at 72 ℃ for 2min, and 30 cycles; final extension, 5min at 72 ℃.

6. The construction method according to claim 4, wherein in the step (3), the plasmid pUC-cbmA is prepared as follows:

carrying out PCR amplification by using fosmid XIV-12H containing polyketide-non-ribosomal peptide synthase gene cbmA as a template to obtain partial cbmA fragments with NdeI and SpeI enzyme cutting sites respectively introduced into two ends of the sequence; carrying out double enzyme digestion on the obtained PCR product by NdeI and SpeI to obtain a cbmA-part1-NdeI/SpeI fragment, connecting the cbmA-part1-NdeI/SpeI fragment with a vector fragment pUC-ermEp-NdeI/SpeI by using a ligase, converting a ligation product into an Escherichia coli DH5 alpha competent cell, verifying and extracting to obtain a plasmid pUC-cbmA-part1, and carrying out double enzyme digestion on the plasmid by using PstI and SphI to obtain a cbmA-part1-PstI/SphI fragment; carrying out double enzyme digestion on Fosmid XIV-12H by PstI and SphI to obtain a cbmA-part2-PstI/SphI fragment, connecting the cbmA-part1-PstI/SphI fragment and the cbmA-part2-PstI/SphI fragment by using ligase, converting a connecting product into escherichia coli DH5 alpha competent cells respectively, and obtaining a plasmid pUC-cbmA after verification and extraction;

wherein, the PCR amplification primer sequence is as follows:

NdeI-cbmA-LF:5’-cggacatatgtcggacctatgcaaggtc-3’

SpeI-cbmA-LR:5’-attactagtctacgccgcatgcgagattgatgtggggtggga-3’

PCR amplification conditions: pre-denaturation at 95 deg.C for 5 min; denaturation at 95 ℃ for 30sec, annealing at 65 ℃ for 30sec, elongation at 72 ℃ for 2min, and 30 cycles; final extension, 5min at 72 ℃.

7. The process for preparing polycyclic macrocyclic lactams according to claim 1, comprising the steps of:

(1) respectively inoculating engineering bacteria S001-cbm-OX4-sgr810 and S001-cbm-OX4-sshg _05717 on a YMG solid agar culture medium, and carrying out amplification culture at 25-35 ℃ for 8-15 days;

(2) cutting a YMG solid agar culture medium and thalli of engineering bacteria S001-cbm-OX4-sgr810 into blocks, soaking and extracting for 2-4 times by using ethyl acetate-methanol-glacial acetic acid (80:15:5, v/v/v) of an extracting solution, then combining leaching liquor, concentrating the leaching liquor under reduced pressure to 200-400 mL, extracting for 2-4 times by using ethyl acetate with the same volume, combining extracted ethyl acetate, and then concentrating under reduced pressure to be dry to obtain an ethyl acetate extract A;

(3) cutting a YMG solid agar culture medium and engineering bacteria S001-cbm-OX4-sshg _05717 into blocks, soaking and extracting an extracting solution ethyl acetate-methanol-glacial acetic acid (80:15:5, v/v/v) overnight for 2-4 times, then combining leaching solutions, respectively combining a water phase and an organic phase of the leaching solutions, concentrating under reduced pressure to 200-400 mL, then respectively extracting with ethyl acetate of the same volume for 2-4 times, combining extracted ethyl acetate phases, concentrating under reduced pressure to dryness to obtain an ethyl acetate extract B;

(4) extracting the ethyl acetate extract A with petroleum ether and methanol of the same volume to obtain a methanol phase and a petroleum ether phase, and washing a light yellow precipitate separated out from the methanol phase with methanol for 2-4 times to obtain the polycyclic macrocyclic lactam compound shown in the formula (I); and extracting the ethyl acetate extract B with petroleum ether and methanol in equal volume to obtain a methanol phase and a petroleum ether phase, washing a light yellow precipitate separated from the methanol phase for 2-4 times by using methanol-dichloromethane (2:1, v/v), and then separating by using sephadex column chromatography to obtain a component B1, and preparing the component B1 by using HPLC to obtain the polycyclic macrocyclic lactam compound shown in the formula (II).

8. The method according to claim 1, wherein in the step (1), the scale-up culture is 8-10L, and the culture conditions are 30 ℃ for 12 days;

the formula of the YMG solid agar culture medium is as follows: yeast extract 4g, malt extract 10g, glucose 4g, 10M NaOH to adjust pH to 7.2-7.4, pure water to 1L, 2% agar, and autoclaving at 115 deg.C for 30 min.

9. Use of a polycyclic macrocyclic lactam compound of claim 1 in the preparation of a medicament for the secretion of a virulence protein of a gram-negative bacterium.

10. An anti-gram-negative bacterial virulence protein secretion drug comprising a polycyclic macrocyclic lactam compound of claim 1 and one or more pharmaceutically acceptable carriers or excipients.

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