CN111304225B - Alkaloid cyanamide biosynthesis gene cluster and application thereof - Google Patents

Abstract

The invention discloses a biosynthesis gene cluster of alkaloid cyanamide and application thereof. The nucleotide sequence of the cyanogramid biosynthetic gene cluster derived from actinomycetes A. cyanogriseus WH1-2216-6 (the preservation number is CCTCC NO: M2009277) is shown as the 393-13440 base sequence of SEQ ID NO.1 and comprises 10 genes; the nucleotide sequence of the P450 oxidase gene cyaH is shown as the reverse complementary sequence of the base sequence of 10626-11843 th site of SEQ ID NO.1, and the P450 oxidase cyaH is coded and can catalyze cyanogram amide D to generate cyanogram amide through skeleton rearrangement by taking Fdx, Fdr and NADPH as cofactors. The invention provides all gene and protein information related to the biosynthesis of cyanamide, which can help people to understand the biosynthesis mechanism of natural products of cyanamide family and provide materials and knowledge for further genetic modification. The gene and the protein thereof provided by the invention can also be used for searching and discovering compounds or genes and proteins which can be used for medicine, industry or agriculture.

Description

Alkaloid cyanamide biosynthesis gene cluster and application thereof

The technical field is as follows:

the invention belongs to the field of microbial genetic engineering, and particularly relates to a biosynthetic gene cluster of an oxindole spiro-alkaloid compound cyanogamide and application thereof.

Background art:

cyanogramide (1 in figure 1) is isolated from the culture of Actinoalloteichous cyanogycereus WH1-2216-6, belongs to alkaloid compounds, and has a unique oxoindole spiro-structure. Cyanogramide (1) has activity to reverse resistance of tumor cells, and the corresponding mechanism is being studied intensively.

The combinatorial biosynthesis technology developed over the last decade has provided new ideas and methods for the engineering of complex natural products. On the basis of clarifying the biosynthesis pathway in nature, cloning and identifying the biosynthesis gene cluster of natural products of microorganisms, the combination biosynthesis technology is adopted to delete, recombine and heterologously express the discovered biosynthesis genes, so that the 'non-natural' structural analogs of the natural products can be produced, the yield of the natural products can be improved, or the required natural products can be directionally accumulated, and the molecular and activity diversity is provided for the discovery and drug development of the natural products.

So far, the clone identification of the biosynthesis gene cluster of the cyanamide (1), the construction of a recombinant strain to generate the cyanamide (1) and homologues, and the formation mechanism of the spiro ring in the structure of the cyanamide (1) and the like have not been reported at home and abroad.

Disclosure of Invention

The first object of the present invention is to provide a biosynthetic gene cluster of an alkaloid compound, cyanogramide (1), which is derived from a. cyangolieus WH1-2216-6.

The biosynthesis gene cluster of cyanamide (1) is characterized in that the nucleotide sequence of the biosynthesis gene cluster is shown as the 393-13440 base sequence of SEQ ID NO.1, and the biosynthesis gene cluster comprises 10 genes, and specifically comprises the following steps:

(1) the genes responsible for the synthesis of the beta-carboline skeleton, namely 3 genes in total, cyaA, cyaB and cyaC:

the cyaA is located at 393-1946 bases of the gene cluster nucleotide sequence, has the length of 1554 base pairs, encodes fatty acid CoA ligase, and has 517 amino acids;

cyaB is located at 3455-4399 bases of the gene cluster nucleotide sequence, is 945 base pairs in length, encodes Pictet-Spengler cyclase and has 314 amino acids;

cyaC is located at 4420-6111 th base of the gene cluster nucleotide sequence, has the length of 1692 base pairs, codes decarboxylase and 563 amino acids;

(2) responsible for methylation genes, i.e., 3 genes in total for cyaD, cyaE, and cyaF:

the cyaD is located at bases 6159-7190 of the nucleotide sequence of the gene cluster, is 1032 base pairs in length, encodes methyltransferase and is 343 amino acids;

cyaE is located at 7226-8008 bases of a gene cluster nucleotide sequence, is 783 base pairs in length, encodes methyltransferase and is 260 amino acids;

cyaF is located at 8583-9311 basic groups of a gene cluster nucleotide sequence, is 729 base pairs in length, encodes methyltransferase and has 242 amino acids;

(3) the genes responsible for oxidation, cyaG, cyaH and cyaI, total 3 genes:

the cyaG is positioned at the 9326-10579 base position of the gene cluster nucleotide sequence, has the length of 1254 base pairs, codes cytochrome p450 oxidase and has 417 amino acids;

the cyaH is positioned at 10626-11843 th base of the gene cluster nucleotide sequence, has the length of 1218 base pairs, codes cytochrome p450 oxidase and has 405 amino acids;

cyaI is located at 12226-13440 bases of the nucleotide sequence of the gene cluster, is 1215 base pairs in length, encodes cytochrome p450 oxidase, 404 amino acids;

(4) other genes

orf1 is located at 2019-3218 th base of the gene cluster nucleotide sequence, has the length of 1200 base pairs, codes cytochrome p450 oxidase and has 399 amino acids;

the complementary sequence of the base sequence from position 1 to position 15458 of SEQ ID NO.1 can be obtained at any time according to the principle of DNA base complementarity. The nucleotide sequence or partial nucleotide sequence from position 1 to position 15458 of SEQ ID NO.1 can be obtained by Polymerase Chain Reaction (PCR) or by digestion of the corresponding DNA with suitable restriction endonucleases or by in vitro synthetic techniques of DNA or by other suitable techniques. The invention provides a way to obtain a recombinant DNA vector comprising at least part of the DNA sequence in positions 1 to 15458 of SEQ ID NO. 1.

The invention provides a way for creating other gene modification such as heterologous expression of cyanamide (1) biosynthesis genes, wherein at least one gene comprises a DNA fragment in the 393-th 13440 position of a sequence shown in SEQ ID NO. 1.

The nucleotide sequence or partial nucleotide sequence provided by the invention can be used for re-screening and obtaining the cyanamide (1) biosynthesis gene cluster homologous gene from other organisms by using a PCR probe method, a Southern hybridization technology and other technologies.

The DNA fragment containing the nucleotide sequence or at least part of the nucleotide sequence provided by the invention can be modified by in vivo and in vitro mutation and the like, and comprises insertion, replacement, deletion, error-prone polymerase chain reaction, site-specific mutation, recombination of different sequences, directed evolution and the like.

The gene or gene cluster containing the nucleotide sequence or partial nucleotide sequence provided by the invention can construct a recombinant vector through a DNA recombination technology so as to obtain a novel biosynthesis pathway, and can also obtain other novel structural compounds based on the biosynthesis pathway through insertion, replacement, deletion or inactivation.

The invention provides application of a cyanamide (1) biosynthesis gene cluster in A cyanamide WH1-2216-6 in preparation of cyanamide (1) and analogues thereof.

The invention provides the use of the combination of genes cyaA, cyaB, cyaC, cyaD, cyaE, cyaF, cyaG, cyaH and cyaI for the preparation of cyanogramide (1) and analogues thereof.

A genetically engineered bacterium comprising genes cyaA, cyaB, cyaC, cyaD, cyaE, cyaF, cyaG, cyaH and cyaI.

The analogue of the cyanologramide is cyanologramide B (2), cyanologramide C (3), cyanologramide D (4), mrinaccarbolines E (5) and mrinaccarbolines F (6):

the invention provides a P450 oxidase gene cyaH, which is characterized in that the nucleotide sequence is shown as the base of 10626-11843 th site of SEQ ID NO. 1.

P450 oxidase CyaH coded by the P450 oxidase gene cyaH and application of the CyaH in preparing cyanogamide (1).

The invention also provides the application of the P450 oxidase in catalyzing the conversion of a substrate cyanogamide D into a product cyanogamide;

in conclusion, the invention provides all gene and protein information related to biosynthesis of cyanamide (1), which helps people to understand the key mechanism of the generation of spiro ring by oxidation rearrangement of alkaloid natural products, and provides material and theoretical basis for further genetic modification.

The strain actinomycetes a. cyanogrieus WH1-2216-6 produced by cyanogramide (1) of the present invention was deposited in chinese type culture collection (CCTCC) at 11/28 th 2009, address: the preservation number of the Wuhan university in Wuhan City of China is as follows: m2009277, which is disclosed in patent application No. 201710181976.7, the name of 5,5, 6-polycyclic macrocyclic lactam compound containing tetramic acid, and the preparation method and application thereof. Details of this actinomycete strain WH1-2216-6 have been reported in the (Fu Peng, Wang Shuxia, Hong Kui, Li Xia, Liu Peipei, Wang Yi, Zhu Weiming. cytotoxic bipyridines from the marine-derived actinomycetes WH1-2216-6.J Nat Prod 2011,74(8),1751-6) literature, and this application is not related to biological preservation.

The heterologous expression host Streptomyces coelicolor YF11 of the invention is disclosed in the literature: YIGUANG Zhu, Peng Fu, Qinheng Lin, Guangtao Zhuang, Haibo Zhuang, Sumei Li, Jianhua Ju, Weiming Zhu, and Changsheng Zhuang Identification of Calulomycin A Gene Cluster implants a Taiiling amino hydroside. org. Lett, 2012,14(11), pp 2666-. The strain the applicant also holds, warranting supply to the public since 20 years.

Description of the drawings:

FIG. 1 shows the structural formula of Cyanogramide (1) and its homologue.

FIG. 2 is a schematic diagram of the biosynthetic gene cluster of Cyanogramide (1).

FIG. 3 is a schematic representation of the vector construction required herein. pCSG2309 is derived from cosmid plasmid pCSG2030, i.e., a fragment related to conjugative transfer of pSET152 is incorporated into pCSG2030 to produce plasmid pCSG 2309; pCSG2310 is derived from plasmid pCSG2309, i.e. the transposase gene in pCSG2309 is deleted in frame; pSCSG2319, namely inserting cyaA-D into an EcoRI restriction site of pSET152, introducing an ermE p promoter before cyaA, inserting cyaE-I into an XbaI restriction site of pSET152, introducing an ermE p promoter before cyaE, and introducing a kasO p promoter before cyaF; pSCSG2320-pSCSG2325 are derived from pSCSG2319, i.e., the cyaD-I genes are deleted on the basis of pSCSG2319 respectively.

FIG. 4 is an HPLC assay for heterologous expression of the Cyanogramide (1) biosynthetic gene cluster. (i) The heterologous expression host bacterium YF11 comprises plasmid pSET 152; (ii) the heterologous expression host strain YF11 comprises a plasmid pCSG 2309; (iii) the heterologous expression host strain YF11 comprises a plasmid pCSG 2310; (iv) the heterologous expression host strain YF11 comprises a plasmid pCSG 2319; (ii) a standard substance of the compound cyanoramide (1).

FIG. 5 shows the results of the compound cyanoramide B (2) HR-ESI/MS.

FIG. 6 Compound cyanoramide B (2) H¹Nuclear magnetic resonance spectroscopy.

FIG. 7 Compound cyanoramide B (2) C¹³Nuclear magnetic resonance spectroscopy.

FIG. 8 shows the nuclear magnetic resonance spectrum of the compound cyanoramide B (2) DEPT 135.

FIG. 9 shows the Cyanogramide B (2) COSY NMR spectrum.

FIG. 10 shows the HSQC NMR spectrum of the compound cyanoramide B (2).

FIG. 11. Compound cyanoramide B (2) HMBC NMR spectrum.

FIG. 12 shows the results of the compound cyanoramide C (3) HR-ESI/MS.

FIG. 13 Compound cyanoramide C (3) H¹Nuclear magnetic resonance spectroscopy.

FIG. 14 Compound cyanogramide C (3) C¹³Nuclear magnetic resonance spectroscopy.

FIG. 15 shows the Cyanogramide C (3) COSY NMR spectrum.

FIG. 16 shows the nuclear magnetic resonance spectrum of the compound cyanoramide C (3) HSQC.

FIG. 17 shows the compound cyanoramide C (3) HMBC NMR spectrum.

FIG. 18 shows the results of the compound cyanoramide D (4) HR-ESI/MS.

FIG. 19. Compound cyanoramide D (4) H¹Nuclear magnetic resonance spectroscopy.

FIG. 20 Compound cyanoramide D (4) C¹³Nuclear magnetic resonance spectroscopy.

FIG. 21 shows the nuclear magnetic resonance spectrum of the compound cyanoramide D (4) DEPT 135.

FIG. 22 shows the Cyanogramide D (4) COSY NMR spectrum.

FIG. 23 shows the nuclear magnetic resonance spectrum of the compound cyanoramide D (4) HSQC.

FIG. 24. Compound cyanoramide D (4) HMBC NMR spectrum.

FIG. 25 shows the results of the compound cyanoramide E (5) HR-ESI/MS.

FIG. 26 Compound cyanoramide E (5) H¹Nuclear magnetic resonance spectroscopy.

FIG. 27 Compound cyanoramide E (5) C¹³Nuclear magnetic resonance spectroscopy.

FIG. 28 shows the nuclear magnetic resonance spectrum of the compound cyanoramide E (5) DEPT 135.

FIG. 29 shows the Cyanogramide E (5) COSY NMR spectrum of a compound.

FIG. 30 shows the HSQC NMR spectrum of the compound cyanoramide E (5).

FIG. 31. Compound cyanoramide E (5) HMBC NMR spectrum.

FIG. 32 shows the results of the compound cyanoramide F (6) HR-ESI/MS.

FIG. 33. Compound cyanoramide F (6) H¹Nuclear magnetic resonance spectroscopy.

FIG. 34 Compound cyanoramide F (6) C¹³Nuclear magnetic resonance spectroscopy.

FIG. 35 shows the nuclear magnetic resonance spectrum of the compound cyanoramide F (6) DEPT 135.

FIG. 36 shows the Cyanogramide F (6) COSY NMR spectrum.

FIG. 37 shows the HSQC NMR spectrum of the compound cyanoramide F (6).

FIG. 38. Compound cyanoramide F (6) HMBC NMR spectrum.

FIG. 39 HPLC detection of Compounds 1-4 by chiral column analysis. (i) The compound cyanoramide B (2); (ii) compound cyanogramide B (2) methylation-derived compound cyanogramide C (3); (iii) compound cyanogramide C (3); (iv) the compound cyanoramide D (4); (v) the compound cyanoramide (1);

FIG. 40 HPLC detection of mutant strains of the biosynthetic gene CyaD-I. (i) YF11/pCSG2320(Δ cyaD); (2) YF11/pCSG2322(Δ cyaF); (iii) YF11/pCSG2325(Δ cyaI); (iv) YF11/pCSG2321(Δ cyaE); (v) YF11/pCSG2323(Δ cyaG); (vi) YF11/pCSG2324(Δ cyaH).

FIG. 41 deduced cyanogramide (1) biosynthetic pathway

FIG. 42. PAGE detection map of P450 oxidase CyaH expression purification.

FIG. 43 HPLC chart of activity detection of P450 oxidase CyaH. (i) No addition of CyaH, (ii) no addition of coenzyme Fdx/Fdr; (iii) CyaH catalyzes the conversion of the substrate, cyanoogamide D (4), to the product, cyanoogamide (1); (iv) a standard for cyanogamide (1).

The specific implementation mode is as follows:

the following is a further description of the invention and is not intended to be limiting.

1. Cloning analysis of the biosynthetic Gene Cluster of cyanogramide (1)

Cyanogramide (1) (FIG. 1) belongs to the class of oxoindole spirocyclic alkaloid compounds, and a 15.4kb Cyanogramide (1) biosynthetic gene cluster (nucleotide sequence shown in SEQ ID NO. 1) containing 10 Open Reading Frames (ORFs) was identified by genome-wide scanning and annotation of Actinoallothecus cyanogenesis WH1-2216-6 (Table 1, FIG. 2). According to bioinformatics analysis, cyaA, cyaB and cyaC are responsible for generation of a beta-carboline base skeleton structure; cyaD, cyaE and cyaF are responsible for methylation modification during biosynthesis; cyaG, cyaH, and cyaI are responsible for oxidation and rearrangement modifications during biosynthesis.

TABLE 1 Cyanogramide (1) biosynthesis genes and functional analysis

Gene	Size(aa)	Predicted function
			cyaA	571	fatty acid CoA ligase
orf1	399	IS110 family transposase
			cyaB	314	Pictet-Spengler cyclization
cyaC	563	glutamate decarboxylase
			cyaD	343	O-methyltransferase
cyaE	260	methyltransferase
			cyaF	242	methyltransferase
cyaG	417	cytochrome P450
			cyaH	405	cytochrome P450
cyaI	404	cytochrome P450

Heterologous combinatorial expression of Cyanogramide (1) biosynthetic genes in the model strain Streptomyces YF11

In view of the difficulty in genetic manipulation of the wild strain WH1-2216-6 and the low yield of the cyanamide (1), the strategy of heterologous expression and promoter engineering is adopted to activate the expression of the cyanamide (1) and realize high yield. Specific primers CyaSF/SR (Table 2) were designed and the clone pCSG2030, which contains the complete cyanogramide (1) biosynthetic gene cluster, was selected from the genomic cosmid library of strain A. cyanogriseus WH1-2216-6. Using molecular biology techniques, pSET 152-based vectors were constructed: pCSG2309 (from pCSG2030, i.e., pCSG2030 inserted into the pSET152 vector), pCSG2310 (from pCSG2309, i.e., in-frame deletion of transposase gene orf1 in pCSG 2309) and pCSG2319 (from pCSG2310, i.e., cloning cyaA-I onto pSET152 with addition of the ermE p promoter in front of cyaA and cyaE and kasO in front of cyaF)₃P promoter) (fig. 3). Subsequently, streptomycete YF11 was transformed with the empty vectors of the constructed plasmids pCSG2309, pCSG2310, pCSG2319 and the control plasmid pSET152 respectively to obtain corresponding transformants of streptomycete YF 11.

TABLE 2 primers used in the present invention

No new compounds were produced in pCSG2039 and pCSG2310 compared to the Streptomyces transformants containing the empty vector pSET152 (due to the lack of a strong promoter)The promoter of the strain is not recognized in a heterologous host), the streptomyces YF11 transformant of pCSG2319 successfully expresses the cyanogramide (1) in a heterologous way, and simultaneously generates a known compound 7 and 5 new compounds (compounds 2-6 shown in figure 4) through the transformation¹H、¹³C. The results of HSQC, HMBC, and COSY NMR spectra confirmed the 5 novel compounds as cyanoramide B (2) (FIGS. 5-11), cyanoramide C (3) (FIGS. 12-17), cyanoramide D (4) (FIGS. 18-24), marinacarbolines E (5) (FIGS. 25-31), and marinacarbolines F (6) (FIGS. 32-38). Compounds 1-4 were analyzed by chiral column as enantiomers in different ratios (fig. 39).

The invention fully confirms the integrity of the cyanogram ramide (1) biosynthesis gene cluster, successfully realizes the production of the cyanogram (1) and the structural analogue thereof in the heterologous cell factory YF11 by the cyanogram (1) biosynthesis gene cluster, and provides a new strategy for creating the cyanogram (1) and the intermediate compound thereof (figure 4).

3. In vivo functional verification of post-modifier gene cyaD-I

6 post-modified genes (cyaD-I) are respectively constructed on the basis of the plasmid pCSG2319 by a molecular biological method: deletion mutant plasmids pCSG2320(Δ cyaD, deletion cyaD), pCSG2321(Δ cyaE, deletion cyaE), pCSG2322(Δ cyaF, deletion cyaF), pCSG2323(Δ cyaG, deletion cyaG), pCSG2324(Δ cyaH, deletion cyaH), and pCSG2325(Δ cyaI, deletion cyaI) (FIG. 3), and the plasmids constructed were respectively introduced into a heterologous host strain YF11 to generate different metabolic maps (FIG. 40). The results show that cyaD is independent of the synthesis of cyanogram (1), cyaE-I is an essential gene for post-modification, and the biosynthesis pathway of cyanogram (1) is deduced by combining metabolite identification and bioinformatics analysis, as shown in FIG. 41.

Biochemical function identification of Cyanogramide (1) biosynthesis gene CyaH and application thereof

The expression vector pCSG2294 of cyaH is constructed, soluble expression is obtained in an expression host (figure 42), the purified protein is brownish red, and a characteristic absorption peak appears at 450nm when CO (carbon monoxide) is added, which indicates that the purified CyaH is active P450 oxidase. CyaH catalyzes the conversion of the substrate cyanogamide D (6) to the final product cyanogamide (1) in the presence of the cofactors ferredoxin (Fdx), ferredoxin reductase (Fdr) and NADPH (FIG. 43).

The following further provides examples which are useful for understanding the present invention, and are intended to be illustrative only and not limiting as to the scope of the invention.

Example 1: extraction of genomic DNA of Cyanogramide (1) and related compound-producing bacterium WH1-2216-6

Fresh WH1-2216-6 mycelium was inoculated to 50mL of 1^#Culturing in culture medium (starch 10g, yeast powder 4g, bacteriological peptone 2g, sea salt 10g, adding water to constant volume of 1L, pH 7.2-7.4), shaking at 28-30 deg.C for about 5 days, centrifuging at 4000rpm for 10min, and collecting mycelium. The mycelia were washed twice with STE solution (NaCl 75mM, EDTA 25mM, Tris-Cl 20mM), 30mL of STE solution and lysozyme at a final concentration of 3mg/mL were added to the washed mycelia, vortexed uniformly, incubated at 37 ℃ for 3 hours, proteinase K at a final concentration of 0.1-0.2mg/mL was added, mixed well, incubated at 37 ℃ for 10 minutes, SDS at a final concentration of 1-2% was added, mixed well, placed in a 55 ℃ water bath for about 1 hour, and the phases were reversed several times. An equal volume of phenol-chloroform-isoamyl alcohol (V/V25: 24:1) was added, mixed well, and cooled on ice for 30 minutes. Centrifugation was carried out at 12000rpm at 4 ℃ for 10 minutes, and then the supernatant was carefully aspirated into a new centrifuge tube using a cut large-diameter pipette tip, and the treatment was repeated 3 times in the same manner, followed by washing twice with an equal volume of chloroform at 12000rpm and centrifugation at 4 ℃ for 10 minutes. The water phase is sucked out by a cut large-caliber gun head and transferred to a new centrifuge tube, 1/10 volume of 3mol/L NaAc (pH5.2) is added, after mixing, equal volume of isopropanol is added, after mixing, the mixture is placed on ice, and DNA is precipitated. The DNA fiber mass was carefully transferred to a new centrifuge tube with a glass rod, washed twice with 70% ethanol, the liquid was decanted, slightly dried at 37 ℃, dissolved with 5mL of TE, and 3-5U of RNase was added, thereby obtaining WH1-2216-6 genomic DNA.

Example 2: establishment of genomic library of Cyanogramide (1) producer WH1-2216-6

The amount of restriction endonuclease Sau3A I used was first determined by a series of dilution experiments in a 20. mu.L system containing 17. mu.L of Micromonospora SCSIO N160 genomic DNA, 2. mu.L of 10 × reaction buffer and 1. mu.L of different dilutions of Sau3A I, which stopped the reaction at 4. mu.L of 0.5mol/L EDTA and appropriate loading buffer. The enzyme activity unit of 0.025-0.05U is determined to be more appropriate by groping. On the basis, a large number of parts are digested to obtain 30-42 kb genome DNA fragments, and dephosphorylation treatment is carried out by using dephosphorylation enzyme.

The SuperCos l plasmid, the vector used for the construction of the library, was first cleaved with the restriction endonuclease Xba I from the middle of the two cos sequences, followed by dephosphorylation, and cleavage with the restriction endonuclease BamH I from the multiple cloning site to obtain two arms. The treated vector was ligated overnight with the previously prepared partially digested 30-42 kb genomic DNA fragment, with a ligation system of 10. mu.L containing 1.25. mu.g of the prepared genomic DNA fragment and 0.5. mu.g of the treated SuperCos 1 plasmid, 1. mu.L of 10 Xbuffer, 0.3U of ligase. The ligation product was treated at 65 ℃ for 15 minutes to inactivate the ligase. A tube of the packaging mixture (50. mu.L) was taken out of the freezer at-80 ℃ on ice, the packaging mixture was rapidly thawed between the fingers, half of the packaging mixture (25. mu.L) was carefully pipetted into a new centrifuge tube, 10. mu.L of the heat-treated ligation product was added, and the remaining packaging mixture was stored at-80 ℃. Carefully mix, incubate at 30 ℃ for 90 minutes, add the other half of the packaged mixture (25 μ L), incubate at 30 ℃ for an additional 90 minutes. Add 500. mu.L phage dilution buffer (100mmol/L NaCl, 10mmol/L MgCl)₂10mmol/L Tris-HCl pH 8.3), followed by addition of 25. mu.L chloroform, gentle mixing and storage at 4 ℃.

The strain E.coli LM392MP frozen at-80 ℃ was plated on LB medium for recovery. One day before the packaging reaction, a single clone was selected and inoculated into LB medium (supplemented with 0.2% maltose and 10mM MgSO₄) The cells were cultured overnight with shaking at 37 ℃ and, on the day of the packaging reaction, 5mL of the overnight-cultured broth was added to 50mL of fresh LB medium (supplemented with 0.2% maltose and 10mM MgSO. sub.MgSO.)₄) Shaking at 37 ℃ and 200rpm to OD of the culture₆₀₀When the temperature reaches 0.8-1 ℃, storing at 4 ℃ for later use. Mixing 100 μ L of the above treated host cell suspension and 100 μ L of appropriately diluted packaging solution, incubating at 37 deg.C for 15 min, and spreading on a container containing 100 μ g/mL ampicillin and 50 μ g/mL kanamycinThe cells were cultured overnight at 37 ℃ on LB plates containing biotin. The single grown clone was spotted with a sterile toothpick on 30 96-well plates containing LB medium described above, cultured overnight at 37 ℃, added with 20% final concentration glycerol, mixed well, and stored at-80 ℃. Thus, a genomic library of cyanamide-producing strain WH1-2216-6 was obtained.

Example 3: screening of clones containing the Cyanogramide (1) biosynthetic Gene Cluster

Specific primers CyaSF and CyaSR (primer sequences shown in Table 2) were designed based on genomic information and bioinformatic analysis, and clone pCSG2030 was selected from the genomic library of strain WH1-2216-6 and was subjected to end sequencing to confirm that pCSG2030 contains all the genes of the cyanamide (1) biosynthetic gene cluster (sequences shown in SEQ ID NO. 1).

Example 4: construction of heterologous expression vectors pCSG2309 and pCSG2310

Elements (oriT, aac (3) IV and Int φ C31) related to neutralization and conjugative transfer of pSET152 were used to replace the neo gene in pCSG2030 by PCR-targeting method to obtain expression vector pCSG2309 (i.e., pCSG2030 was inserted into pSET152 vector); the orf1 gene of pCSG2309 is used for replacing aadA and oriT in a PCR-targeting way, and the obtained plasmid is subjected to SpeI enzyme digestion self-ligation to generate a heterologous expression plasmid pSCG2310.

The deletion of orf1 illustrates the PCR-targeting method. A pair of deletion primers for orf1 gene was designed, the sequences of which are shown in Table 2 as orf1 deletion primer orf1-TarF/orf 1-TarR. Then constructing an in-vitro knockout plasmid by referring to a PCR-targeting method and transferring the knockout plasmid into a conjugately transferred donor bacterium. The method comprises the following specific steps: (1) transferring the plasmid pCSG2309 into E.coli BW25113/pIJ790 to obtain E.coli BW25113/pIJ790/pCSG2309, inducing the expression of a lambda/red recombination system by using 10 mmol/L-arabinose, and preparing the recombinant into electrotransformation competent cells for later use. (2) The plasmid pIJ778 was digested with the endonucleases EcoR I and Hind III, and a DNA fragment of about 1.4kb containing the transfer origin and the spectinomycin resistance gene was recovered as a PCR template, and a 1.4kb PCR product was amplified by PCR using the primers orf1-TarF/orf1-TarR, and 50. mu.L of a PCR reaction system: 3U of high-fidelity DNA polymerase, 5 mu L of 10 multiplied by Buffer, 0.5mmol/L of dNTPs, 2.5 mu L of DMSO, 0.5 mu mol/L of each primer and about 1ng of DNA template, and water is added to supplement the volume to 50 mu L. The PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 5 min; the amplification cycle is denaturation at 94 ℃ for 45s, annealing at 58 ℃ for 45s, and extension at 72 ℃ for 90s for 30 cycles; finally, extension is carried out for 10min at 72 ℃. The 1.4kb PCR product was recovered and purified for use. (3) The PCR product was electroporated into the competent cells prepared in step (1) to be recombined, plated on LB screening plates (containing 100. mu.g/mL ampicillin, 100. mu.g/mL spectinomycin, 50. mu.g/mL apramycin), and cultured overnight at 37 ℃. Positive single clones were picked from the plate, plasmids were extracted, and the recombinant plasmids were digested with SpeI to self-ligate to produce plasmid pCSG2310 in which the orf1 gene was deleted in frame. (4) The constructed recombinant mutant plasmid pCSG2310 is transformed into E.coli ET12567/pUZ8002 to construct E.coli ET12567/pUZ8002/pCSG2310 as a donor bacterium for conjugal transfer.

Example 5: bond transfer

The conjugative transfer method is described as plasmid pCSG 2310. Actinomycetes YF11 were streaked on an SFM medium plate for 3 to 5 days, and the grown spores were collected with a sterile cotton swab in a TSB medium and vortexed to disperse the spores. The mycelia and spores were separated by filtration, suspended in 5mL of TSB medium, heat-shocked at 50 ℃ for 10min, and then germinated at 28 ℃ for 2 hours as receiver bacteria for conjugative transfer. Coli ET12567/pUZ8002/pCSG2310 Donor bacteria were grown to OD at 37 ℃ in 50mL LB liquid medium containing 100. mu.g/mL 25. mu.g/mL chloramphenicol and 50. mu.g/mL apramycin₆₀₀When the value was about 0.8, the cells were collected by centrifugation (4000rpm, 10min), washed 3 times with LB, suspended in 300. mu.L of LB medium, and used as donor cells for conjugative transfer. And uniformly mixing 400 mu L of the recipient bacterium and 100 mu L of the donor bacterium, coating the mixture on ISP4 solid culture medium without any antibiotic, drying the mixture by blowing, and culturing the mixture for 18 to 20 hours at 28 ℃. Then the plates were taken out, covered with water containing antibiotics to a final concentration of 35. mu.g/mL apramycin and 50. mu.g/mL trimethoprim, blow-dried, placed in an incubator at 28 ℃ and observed after 5-7 days of incubation.

After the colonies grow on the conjugative transfer plate, the strains are transferred to an ISP4 plate containing 50 mug/mL of apramycin and 50 mug/mL of trimethoprim by using a sterile toothpick, after 3 days of culture at 28 ℃, the genome DNA of each mutant strain is extracted, and a positive clone is obtained by PCR detection by using a detection primer orf1-testF/orf1-testR (table 2), namely the pCSG2310 heterologous expression strain is obtained.

Example 6: construction of heterologous expression vector pCSG2319-pCSG2325

Heterologous expression vectors pCSG2319-pCSG2325 were obtained by a one-step cloning kit (Vazyme Biotech co., Ltd) to construct pCSG2319 as illustrated by the following steps. (1) Design primer pair CyaABCD-EF/CyaABCD-EF1 and CyaABCD-ER1/CyaABCD-ER (Table 2), amplify 300bp of promoter region of ermE P (which can be used as template in pUWL201PW, which is disclosed in Doumith M, Weingarten P, Wehmeier UF, Salah-Bey K, Benhamou B, Capdevila C, Michel JM, Pie persberg W, Raynal MC. analysis of genes encapsulated in 6-deoxyhexose biosyntheses and transfer in Saccharopolyspora erythraea. mol Gen Genet.2000; 264(4) EcoRI-477-85), and 5.3kb of cyaA-D fragment (which is used as template in pCSG 152) and generate plasmid pSA-D by one-step cloning into the site of the pCET 4; (2) designing primer pairs CyaE-ermEp-EF/CyaE-ermEp-EF1 and CyaE-ermEp-ER1/CyaE-ermEp-ER (Table 2), respectively amplifying 300bp ermE p promoter region (which can use pUWL201PW as a template) and 1kb cyaE fragment (which can use pCSG2030 as a template), and integrating the two fragments into the EcoRI site of pSET152 by a one-step cloning kit to generate a plasmid pCSG 2316; (3) designing primer pairs CyaFGHI-EF/CyaFGHI-EF1 and CyaFGHI-ER1/CyaFGHI-ER (Table 2), respectively amplifying a 100bp kasO p promoter region (taking pCSG2053 as a template) and a 5.5kb cyaF-I fragment (taking pCSG2030 as a template), and integrating the two fragments into an XbaI site of pSET152 by using a one-step cloning kit to generate a plasmid pCSG 2317; (4) primer pairs cyaEFGHI-EF/cyaEFGHI-EF1 and cyaEFGHI-ER1/cyaEFGHI-ER (Table 2) were designed, and 1.3kb ermE pcyaE fragment (using pCSG2316 as a template) and 5.8kb kasO pcyaFGHI fragment (using pCSG2017 as a template) were amplified and integrated into the XbaI site of pCSG2314 by a one-step cloning kit to generate plasmid pCSG 2319.

Example 7: biological fermentation and detection of Cyanogramide (1) and derivatives thereof

After activating the heterologous expression strain and the gene deletion mutant strainInoculating 5% of the inoculum size into 50mL of fermentation medium actinomycetes 2 respectively^#(glucose 20g, starch 10g, beef extract 3g, yeast extract 10g, peptone 10g, K)₂HPO₄ 0.5g，MgSO₄ 0.5g，CaCO₃2g, adding water to a constant volume of 1L, adjusting the pH value to 7.0, sterilizing), culturing at 28 ℃ for 4-6 days, adding butanone of the same volume, carrying out ultrasonic treatment for 30min to break cells, stirring for 30min, and then standing for layering. Separating butanone extract from water phase, evaporating butanone to dryness by using a rotary evaporator, dissolving residue in dimethyl sulfoxide (DMSO) to form a sample, and performing High Performance Liquid Chromatography (HPLC) detection under the following detection conditions: phenomex C184.6X 150mm reversed-phase column, wherein the mobile phase A is 10% acetonitrile and contains 0.8% TFA, and the mobile phase B is 90% acetonitrile; the flow rate was 1mL/min and the detection wavelength was 300 nm. HPLC procedure: 0-20min, 5% -100% of phase B; 20-24 min, 100% phase B; 24-25min, 100% -5% of phase B; 5% B in 25-30 min.

Example 8: use of the Cyanogramide (1) biosynthetic Gene Cluster-genetic deletions to biosynthetic genes result in structural analogs:

by the methods described in example 4, example 5 and example 6, the biosynthetic gene cluster of cyanamide (1) was heterologously expressed, 6 genes of cyaD-I in the gene cluster were knocked out and heterologously expressed, and the heterologously expressed strain and the gene deletion mutant strain were biologically fermented and tested according to the method of example 7, and the following results were obtained:

(1) the complete gene cluster heterologous expression strain (YF11/pCSG2319) produced the compounds cyanamide (1), cyanamide B (2), cyanamide C (3), cyanamide D (4), marinacarbolines E (5) and marinacarbolines F (6) (FIG. 1, FIG. 4, FIG. 41);

(2) the methyltransferase cyaE deletion mutant produces the compounds cyanamide B (2), marinacarbolines E (5) and marinacarbolines F (6) and 7 (FIG. 1, FIG. 40, FIG. 41);

(3) the methyltransferase cyaF deletion mutant produced compounds 7, 8 and 9 (FIG. 1, FIG. 40, FIG. 41);

(4) the P450 oxidase cyaG deletion mutant produces the compounds cyanograms B (2), cyanograms C (3), marinacarbolines E (5) and marinacarbolines F (6) and 7 (FIG. 1, FIG. 40, FIG. 41);

(5) the P450 oxidase cyaH deletion mutant produces the compounds cyanograms B (2), cyanograms C (3), cyanograms D (4), marinacarbolines E (5) and marinacarbolines F (6) and 7 (FIG. 1, FIG. 40, FIG. 41);

(6) the P450 oxidase cyaI deletion mutant produces the compounds cyanograms (1), cyanograms D (4), marinacarbolines E (5) and marinacarbolines F (6) and 7 (FIG. 1, FIG. 40, FIG. 41).

Example 9: cyanogramide B (2) chemical derivatization to form cyanogramide C (3)

To achieve methylation of the specific 12-hydroxyl group of the compound cyanoramide B (2), 5mg of the compound cyanoramide B (2) and 5mg of Amberlyst-15 ion exchange resin were dissolved in 2mL of methanol solution, left to stand at room temperature for 1.5h with stirring, followed by centrifugation at 10000 rpm for 10min, the supernatant was concentrated in vacuo and redissolved with isopropanol, and the methylation modification was checked with a chiral column.

Example 10: chiral column analysis of Compounds 1-4

The chiral column HPLC detection conditions are as follows: the column model is Lux Amylose-2,5 mu, 250x 4.6mm, Phenomenex, the mobile phase A is 10% acetonitrile, and the mobile phase B is 100% acetonitrile; the flow rate was 1mL/min and the detection wavelength was 300 nm. HPLC procedure: 0-65 min, 50% of phase B.

Example 11: construction of P450 oxidase CyaH high-efficiency expression engineering bacteria

The cyaH gene (the nucleotide sequence of which is shown as 10626-11843 base of SEQ ID NO. 1) is obtained by amplifying the nucleotide sequence of cosmid pCSG2309 by using a PCR method, and PCR fragments (taking pCSG2030 as a template) are amplified by using high fidelity enzyme and primers CyaH-EF and CyaH-ER (Table 2). The vector pET28a was digested with NdeI/EcoRI, and the amplified PCR product and the digested vector were used to construct an expression plasmid pCSG2294 using a one-step cloning kit. The constructed expression plasmid pCSG2294 is transferred into escherichia coli DH5a for storage, and simultaneously transferred into escherichia coli BL21(DE3) to be used as an initial strain for inducing the expression of CyaH protein.

Example 12: in vitro Activity study of P450 oxidase CyaH

Coli BL21(DE3) containing pCSG2294 plasmid was cultured at 37 ℃ to OD in LB medium containing 50. mu.g/mL kanamycin₆₀₀About 0.7, isopropyl-. beta. -D-thiogalactopyranoside (IPTG) was added to the medium at a final concentration of 0.1mM, and the culture was continued at 16 ℃ for 18 to 20 hours to induce protein expression. The cells were harvested by centrifugation, resuspended in buffer (50mM Tris-HCl, pH 8.0), and the contents released by sonication. The separation and purification of recombinant FlsO2 were performed by fast protein liquid chromatography, and the electrophoretogram of the purified protein is shown in FIG. 42. Specifically, the supernatant after cell disruption was applied to a HisTrap column of 1mL standard, and then eluted with a 250mM imidazole solution. The purified protein was desalted by PD-10 column and stored in an enzyme storage buffer (50mM Tris-HCl, pH 8.0; 100mM NaCl; 10% glycerol) at-80 ℃ to thereby obtain P450 oxidase CyaH.

A reaction solution containing 100. mu.L of oxidase CyaH was prepared by: mu.M cyanogamide D (4), 10. mu.M CyaH, 50. mu.M Fdx, 50. mu.M Fdr and 2mM NADPH were reacted at 30 ℃ for 5 min. No CyaH or Fdx/Fdr was added to the reaction system of the control group. After the reaction solution was terminated with an equal volume of methanol, it was centrifuged at 13500rpm for 10min at 4 ℃ and the supernatant was analyzed by HPLC under the following conditions: phenomex C184.6X 150mm reversed-phase column, wherein the mobile phase A is 10% acetonitrile and contains 0.8% TFA, and the mobile phase B is 90% acetonitrile; the flow rate was 1mL/min and the detection wavelength was 300 nm. HPLC procedure: 0-20min, 5% -100% of phase B; 20-24 min, 100% phase B; 24-25min, 100% -5% of phase B; 5% B in 25-30 min. As shown in FIG. 43, CyaH can convert compound cyanoramide D (4) into the final product cyanoramide (1) in the presence of coenzyme Fdx and Fdr, with NAD (P) H as a cofactor.

Sequence listing

<110> Nanhai oceanic research institute of Chinese academy of sciences, China oceanic university

<120> alkaloid cyanamide biosynthesis gene cluster and application thereof

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 15458

<212> DNA

<213> Heteromura paucicostata WH1-2216-6(Actinoalloteichus cyanoriseus)

<400> 1

ccgggctcct cctggttggt gtcggctact gcggcaggtg ggccggcagc gagtggccca 60

ccgtgactga atgtcgaatc ctacccggtg ggggccctca acccggtagc gcccgcgtgg 120

tgagaggcct cactgtcggg cgacgcgggt cccccggacg ggttgacggt caaccgttca 180

ggaaaagcgg cgacccccac acgggaatac cccgagcaaa cagccggttc cgtgacccac 240

ggtcgtgtcc gatgctcggc gagagggaac ggaagatggc gttgcgtatt ccgatttcgc 300

ccctggcgag tattccttgg ttccatcggg tctccgtcac cgaaatatcg agtatcgctc 360

attgcgcctc cgttcgcctt tcggaatcgt cgttgtggcg caacccgatc cccccggaac 420

caacgtcgga ggaactggtg gactacgtgc gacgggtctt agccggaatc gccccccagg 480

acgacgtcct cgtttccgag aaccggtcgc tgaatggcgc cgaaacccat cgtctggtcg 540

ccggtatggc tgcggtcctc gcgcggttcg gagtgggccc cggagtccgg gtcgcctgcc 600

tgcacgggaa cacgccggag gccgtcctcc tccggctcgc ggtccaatgg gcggggggct 660

gctacgtcgg cctgcgcccg atgttctccc ccaccgtgca cgcggcgtgc ctcgcccacg 720

cggcgccgga ggtgctcgtc cacgaccagc agcgcgagga gcgcgccacc gaactgctcc 780

gccgcgcgtc ggtgccccgc gtcctctcgc tgggtccctc cgctctcggc gaggacctgg 840

ccgcactcgc cgccgccgcc gtggccgacg ctcccacgct ccccgccgcg ctcgacaggc 900

cggcgtcgct ggcctacacc agcgggacga ccggcagccc caagggcgtc gtccacggca 960

ccaccgccat ggcggcgtgc ctcgacgtgg cccgcgccat gtacggaccg ccaccctggc 1020

gcttcctggt ggccatcccg ctctccgacc tgggcggaga gctcgcccag tggatcctcg 1080

cctgcggcgg ggtggtggtg ctgcgggagg acctggaccc cctggcgacg ctcgcgctgc 1140

tggagcggga acgcatcacc cacctgttca ccgcgcccag ctcgctctac cagctcgcgg 1200

agcaccccga cctcgaccac ttcgacctca cggccctgcg actcgccgtc tacggagggg 1260

cccccgccgt gccagcccgc acgtccgccg ccctgcgccg gctcggcccc cgcctcatgc 1320

agaactacgg cacccaggag accgggttcg tgtgcgcgct cctgcccgag gaccacgggc 1380

accccgagct gctggaccac gccggtcggc tgctcccggg agtcgaggtg gagatccggg 1440

acacctcggg cgcggtcgtc ccgccggggg tggtcggcga gatctgggtc cgctccccga 1500

tgacgatgag cgggtaccac gccgatccgg cccggacggg cgaggtgctg gtggacgggt 1560

gggtccgcac cggtgacctg ggccacctct ccgaggcgga cctgctccgc atcctcgacc 1620

gcgccaagga cctcatcatc gtcgaggcgt acaacgtcta ctcccgtcgg gtggaggacg 1680

tcctctgcca ccacccgaag gtcctccagg ccggggtcgt aggcctttcc gacgtcgacc 1740

gaggtgagcg ggtctgcgcg gcggtcgtcc tcgcgagcgg aaccgcggac gccgacgagc 1800

tccgcgaaca cgtcgaggcg caactcggtg aaccacacgt accccgcctc atccgattcg 1860

tcgacaccat tccactcacc gacgggggaa aaccggacaa gtcggcatta cgcgccctgt 1920

tcgcccccga caccccctcc cactgatccc acgaccaacc ggacgaaagg attcccaatg 1980

aagttgtcaa gcggtgaagg tgtcgggccg tgagctcgtc aggccgctgc ggcttcgggt 2040

tgcggcgtgc cggtctggaa ggcgcggttg tcgcggatga gtgcccacaa cacgttcagg 2100

cgtcggcggg ccagcgcgag gacggcctgt ttgtgcgcct tgccttcgcg ccttttcctg 2160

tcgtagaagg ctttcgacgt cgggcaacag cgggcggcga cctgcgcgga caggtagaac 2220

atgcgcagca ggcgccggtt gtagcggcgt ggccggcgca ggttgccccg gactcgtccg 2280

gagtccttgg gcaccggcgc caggcccgcg acaccggcca gccggtcggc gctgccgaac 2340

gcggccatgt cgccgccggt cagcgcgatg aactcggctc cgagcagggg cccgacgccg 2400

ggcatgctca ggaccgcgac ggcgtgacgg tgtcggcgaa accggccctc gatgagcgcg 2460

tcggtctccg cgatctcgtc atcgagggcc atcaccccct tggcgagccg ggccaccatg 2520

gccgcggccg tcatctctcc ggggacggtg gtgaactggg cttcggcggc ggcgactgcg 2580

gcggcggcga ccgtcgcgct gttgcggacc ttccgggcgc gcagccagtc ggcgagctgt 2640

tcccggccgc gctcacgcag tgcggcgggg gtctggtggc cggtgagcag gaccagggcg 2700

gccttggagg tgctgtagtc gaaggcccgt tccaacgcgg ggaagtactc cagcagttgg 2760

gcgcgcagcc ggttgatcgc ccgggtgcgg tcggcggcca ggtcgtagcg gcgggcggtg 2820

aggatccgca actccaccgc gatctcgtcg ccggcggtca ccgggtgcag gtcacggcgc 2880

atccgggcct ggtcggcgat gacgaaggcg tccttggagt cgctcttgcc gtccccacga 2940

taggtgccgg cggcgcggtg gacggtgcga ccggggatgt agagcagctt ctgtccgtgg 3000

ttgaccagca atgcgatcag cagcgcggcc ccgccggagt tcaggtcgat cgcccacagc 3060

acgtccgggg ccaaagcccg tacgtcaccg atcagctcca acaggtcggc ctcgttgttg 3120

gcgacccggc gggaaagcac cttcgtgccg tcggtatcaa tcaccgtgca gtggtgcgcg 3180

gccttgcccg cgtccacgcc ggcccagagt tcgggcacgg ccacctccgt cgtcgttcgt 3240

gcggaacacc cggcagacga cctcgccggc acgtccttat gcagcgatcg gttcgctcat 3300

cccaatcagc ggtcgagtcg tcgcgggacc tcgggcggcc aatcctttca agccacacca 3360

acggcgacca catgacagcc acacccgaag tccccgggcc ctcccgatct tacgactgac 3420

cagaacagac ccgacttaga agataaggaa cagcatgcgg cagatcgaga tcgaatgggt 3480

gcagcccgga atcaccgtca ccgccgacct gaacgaggaa cgcaatccgc ggctggcgga 3540

tctgctgtgg aacgaactgc tcccgtacaa cagcctccag aatcacgcac tggtatccgg 3600

gaaccacctc taccacctcg ttccccacca ccagctcgtc tacacccgcg ccgagtacaa 3660

ggaggaccgg acgaagtccc cggacggaac ggtgttcctc tcccagctgc aacacctcgc 3720

cgtcaagtac gggccgctca gcgagtacct ccccgccgcc ccggtcgggc acgtggtacc 3780

ggaggacgtc gaggccttgc gcgccgccgg ccgcgcctgc tgggaggcgg cgtggagcag 3840

caagcaggtg atcgaggtcc gggtccggcg caagggggag gacgtgcgga acttcgtgct 3900

cccgcgcacc gccccggtgg actcccccgc cgtgcagaaa ctggtggagg agatccacga 3960

cgaggtcgcg cgggtgtgga tcgatccacc ccaggagatc gtcgccatgc acgaggggcg 4020

gatcgccagc cgcgccggta gttacgacca gtacttctcc acgctcgtct tcctcaacgg 4080

cgaggtgcgg ccgctgggct actccgccct caacggcctg gtgacgatgt cccgcaccac 4140

cgacatctcc ctcaccgacc tcctccgcat cacaccgatg ctgatcaaga cgcccgccga 4200

gttcctcggc tacaccgggc tcaacacctt gtgggacttc acccaacgcg tcctggagac 4260

gctgcccggc gtggagacgc gtgacgagta cttcgccctg atcaacgcgc tcgcgctgta 4320

cgccaacgtc ctgaacacct ggaacctgca cttctttccc tggcgagccg gcgccgacca 4380

cgcctacgcg accgtctgat ccggtcgaag gagtcagcca tgcccagcag ggaattcgtc 4440

gagacgctga ccgaactgcg tcaccgcacg ctgcatccca cggagcacaa ccgaggtcag 4500

cttcccgaac ggctcagcca cgcgctccgc acgctggccg tgccctccca tcccgacggt 4560

gcctggctag gcacggctcc tcccgacctc ggctggctgc gggacttcga ggcggtgccc 4620

gagcacatgt ccgaccccga cctggtgttg cgcggggtga ccgccgggct gggcgcgcaa 4680

ctgcgcgccc acgcccccgg caacctgttc aacatcttcc cggtcccgct gttcgacgcg 4740

gtgaccgcgg ccacgctggc ccagctctac acccccaacg cgctgtggga cctcctggcc 4800

ggtggcacgc tcgagatcga acgccagctg gtccgacagc tcgccgacct ggctggctgg 4860

ccacgcgacg acgcgggcgg caccttctcc ttcggcggca aggccggtct gatgtacgcc 4920

gtccggatcg ggctgaaccg gtgcctgccc gagtccggcc ggcgcgggct ggccggtggg 4980

gccgcgccgg tggtggtcac gaccgagcac aaccacgaca gcgtggagcc ggtgtgcgct 5040

ctcctcgggc tgggttcgga ggcgtgcgtg cgcgtgccgg caccgggacg ggaccgggcg 5100

accaccgccg agcgggtcct caccacggtg gccgacctgg tcgcacgggg aactccggtg 5160

gcctgcgtgg tgctctccgg cgggagcatc atggaccaca ccatcgatcc ggtcggcctg 5220

gtcgccgccg gcctgagcag tctgagcggt tcgctgccct accggcctta cctgcacttc 5280

gacaccgcgc acggctggcc gtggttgttc ttccgcgact acgacttccc cggcaacccg 5340

ttggagatcg acccggtgac gctcgccgcc ctccgcgagg tcagcgacct cgtcgcggag 5400

gcggcctggg cggactcgat gacggccgac ttccacaagg ccggcttctg cccctacccc 5460

tccagtgcct tcctggccag gaacgccgcc gagctgcaca ccctgcacga ggacccgcac 5520

accgccgagt ggcggccgtg gggcggcaac ctggtactgc accacaccat cgagcactcc 5580

cggggcgcgg cgggaatcct cgcggccttc gccgcgctcc acagcgtggg gaggaccggg 5640

ttccgggtcc acctcgccca cgtcaccgcg atgaacaacg tgttccgcgc gcacctcccg 5700

gagttcggct acgaactggt gaaccccacg agccccggga tggcgtcggt gttctggccg 5760

gtggcgccgg ggggcccggc caccttcgcg gaactgggcc gggcggaacc cctgctcgtc 5820

gacgacgcca accgttacac gcaggccctc taccagcgcc tggctgggct ggacccccag 5880

cacgaggtcg ccgcgccgat cgtgctggga ttcctgcccg cgttcacgca cggcgcgcac 5940

ggccatccgc tgtcggcgct gcgcctgctc cccaactcgc cccaccacga cgaggtgacc 6000

gtcgcggcga tcgcggaccg gatggccacc gccaaggcgg agttcgaccg ctccgtcacc 6060

tcccgcaccg gtcccctcgc cggcctccgc ctccgccacg tcccggagtg agccggcacc 6120

cgccggccct ccaggaccga caccccgagg agttcgaggt gaccgcatcc ccccgcccgt 6180

gcccagccca ggttcccgac tcgtccgcgg cgttgcggct cgcccggttc ttcgccgcgg 6240

gatgcctggg acaggcactc gccgctctgg tcagggcggg cgtcgtccgg gagatggcca 6300

gggaccggcc ctccgtcgac gaactcgccc accggaccgg gaccgatccg gcgacgctgt 6360

cgcggttcct gcgcgccgga gaggcggctg gcgtgttcgt ggaggaccca ccacaccact 6420

tcgccctgtc ggacacggga gaactgctgg cggaggggcc gggatcgctg ggtggtctcc 6480

tcgaactggc gaacacggcc ccggtgttgg ccgcgtggtc cgccgccgag cacaccctgc 6540

gcaccgggga gtccgccttc gcggcggtgc acggctcgtc cgtgttcgac cacctcgcgc 6600

aggcgccgac cctggccggc gcggtggggc gggcgatggg ggccagcgtc gccgcggaac 6660

tcgtcccggc ctcggtggat ctcccctcag cgcgccacgt ggtcgacgtg ggcgcggggg 6720

gcgcgcgcct cctcacggcg ctgctcgccg cccaaccgca cctcgtgggg acgctggtcg 6780

gcgtgcccac ggcggctcgg gagcaggtcg ccgaggagct gcggcggtcg ggggtgcgtt 6840

cccgctgccg cctgcccgac ggcgaggtcc tcccccagga cggggacgtc tacctgttgt 6900

gccacgtgct gccgtgcctc gacgacgagg acgcggtctc cctgctccga tcgctcgtcc 6960

ccgtgctgcg ggagggcgcc cggctgctgg tgatcggctt cgttccctcg gctcgggacc 7020

gcaccccggt gatccccgcg ttggacgtgt ggatggcggc gatgtgcggc ggacgtcaac 7080

gcaccgaaca cgagtactcg gctctcctcg accgagccgg attgctgcta cacgcccggc 7140

acggggtccg ccccgacacc gagtgcgtcc tcgaggtggt tcgccggtag tcgacgccac 7200

cggccgggag ggcgttcgcg gacgctcacc cggcccggcg gggcaaccgc ccgatgatca 7260

tgccggcctc gccgtggtcg gtgaagtgcc cgcccgcgag gacggctcgc agcctgccct 7320

ccgcggcggt gaggaagtcc tcccagacag cgtcggccgg gagcaggtgt cgccacgcgg 7380

gacgcaggga gtccaggtac ccgagcaggg gttcgagctc gtcgcagacg accgtgtacg 7440

cgcgtcgccg taccgccacc tccccgaagg cggccgccag gaggcgcgcg tggtgcaggc 7500

cgaaccgcgc gtccggccga ggcgggacgg gcccgccgcc gacgtcggcg aacgcgtggt 7560

cggccagcgt gtacagctcg gtcagcgagc tcgagtcgtt ggtggtggcc accgcgatgc 7620

cgtcgggccg cagggctcgg gccatctccc ggaccgccgt cacgatgtcg ggcacgtggt 7680

agagcatgtg catggccacc acggcgccgg ccgcgccgtc ggcgaccggg aacgcctgga 7740

cgtccccgac cacgtcgggc gccatgccgg tggagacatc gacaccgaag aggtccaggt 7800

cgggtctgcg ggcggcgagc cgggcgcgca gcagaccgtt cccgcacccc acgtcgatga 7860

cgggtcccgg cgcggtggcg accacggagg cgacgtcgtc gtggaggtcg aaccgaggac 7920

gctggaagtc gaaaaggcgt tggcgtgccc gcaggatcct ggtggtcccg tagtactcgt 7980

ccgccaccag ctgccggtcg gtgtgcatac cggaatgcta ttccccgacg ggggctccgt 8040

ggtctcctcg agctccgatt ccggaattcg ggcgacggaa gcgtcccgcc cggtggacgg 8100

ctctcaccga ccgtgccgac acgacctccc gcactgctcc aaccgacgga gcggaaagcc 8160

ccccgttcag gattatcccg aaccccacga ccgcgcggaa aacgacgcgg aacacgtcat 8220

cccaggagtc aggaacggcc gttcacccga tcagcgaggc cgggttcacg aagcgcaccg 8280

ggcgagcgac cacccgccca tcagccaatt caccctactc ggatggggga aacagccacc 8340

ctgccaccca ctcgttcttg ccgacgggtc accgacgttg gtatatctct attcagcggt 8400

tcacgtcatg gattccacca ccggcccacg actccgcatt tccgatgtcg ccattcctga 8460

ccacccggtt cgtggacgca ccagcaccca ccacagaaaa ccgggcccgc gccacctgct 8520

tccccgtggc ccgttccggc ggccgcgatc agcgcgcacc gttccgcacg aggagaccgc 8580

acgtgattct ggattccggc cgaaacaacg gggtcaactg ggaacgttac tggcgggacg 8640

tcaccagcga cgccggcact cccagcccgc cctggcactt cggttcgggt gccgagatgg 8700

ctccctacct ccccgtcatg ctcgaccacc tcgctcccga gctccccctg gtcgacctcg 8760

ggtgcggcga cggtctcctg accgaccacc tcgccaggca ctacccggtg gtcgtgggcg 8820

tggacgtctc cccggcggcg atcgccgccg cgcgcggccg ggcccgtccc ggtctgtcgt 8880

tcgacgtcct cgacgccacc gacgtggagg ccgcggcgag gttgcgcgcc acggtcggtg 8940

aggccaacgt ccacctgcgc ggggtgctgc acgcgatgga cccggccgac tggcccgccg 9000

ccctgacgac actggcgacg ctcaccggcc gacgggggcg ggtcttcgac atcgagatca 9060

ccccggcctt ctccgacgcc gtggaggaga tgctcgggaa gttcgccgcc ccgccaccgg 9120

ggatggccgc ggtcgcccgc agcggcctgc ggccgacgga gctggactcg ccgagcctgc 9180

gagccctgta cgaggacacc ggctggacgg tggtggccgc cgaggaactg acgggacgtt 9240

ccaccatgcg gctgcccgac ggctcctact tcgagtaccc gttcacctac ctcgtggccg 9300

ggcgacggtg agctcggagg gctccgtggc gggcggagcc gaggtggagg gtgtcccgcg 9360

ctcaccggac ccgtccggtc cggtcttcac cctcaacgag gtcccggccc tcgacagcga 9420

tccgctgttc gcgctgttcc ggcgggagca cccgctgagc cggatccggc tcccccacgg 9480

cggggagggc tggctggtca gccgttacca ggacgtgcgc accgtgatga cccacccggc 9540

gttcagcgcg gccgccgccg ccagcgacca ggtaccccgg atgacggtgg tggcgggccg 9600

gcccccggag accatcgccg gcctcgaccc accggaacac acccggctcc gccggctcgc 9660

cgcgcccacg ttccgggcga gccgcatcgc gaccttccgg ccgctggcca cccgtatcgc 9720

cgtggacacc ctgaccgagt tggtggacgc cgggcgaccc gccgacttcg tacggcactt 9780

cgccctcccg ttccccgtcc ggttgatctg cacggtcctc ggcgttccct acgaggacca 9840

ctcccgcttc gtgccctggt cggaggccgt gctctccacg acggggctca gcggcgccga 9900

gtcagcccag gcgttggagg agatgaagga gtacttccgc cacctcatcg ccgaacgccg 9960

acgcgccccg cacgacgacc tgctcgccga cctcgtcgcc gcccgcgacg ccacacccgg 10020

ccccggggag agcgatcagc tcagcgagga ggagctggtg atgttcgcgg tcctgctgct 10080

catcgccggt tacgagacct cgatcaacca gatcggcgct tcctgcttcc tgctgttgga 10140

acggcgttcc cggtgggagc agctggtggc gcacccgcga ctcgtcaccc gggccgtgga 10200

ggagttgctg cggttcgtgc cgctgatcaa cggggtgacg ctgcccaggg tggcggtcag 10260

cgacgtcgag gtcgcgggcg gcacgatccg ggccggtgag gcggtgttcg tctccatccc 10320

ctcggcgaac cgcgacgagc ggatcttcga cgaccccgac accttcgacc cgacccgggt 10380

ccacaaccag cacgtggcct tcgggcacgg gccccactac tgcctcgggg cgcacctcgc 10440

ccgcctcgaa ctccaggtgg cactgaccga gctgatccgg gtgctcccct cgctgcggct 10500

ggccacgccc agggacgacg tgccctggcg caccggtctg gtggcgcgcg ggccgaccgc 10560

gctcgccgtc tcgtggtgag caccgcgacg ttcctccgca tccgttccga ccaggagacg 10620

agacgatgag tcaggaagac gcgtcggggg tgtgcttcct cgaggagatg gacgccctcg 10680

agatcgaccc ccacttcgcg atggcccgac gtgaacggcc gctgagccgg atcaaactgc 10740

tctacgggga cgagggctgg ttggtcaccc ggtacgagga cgtgaagctg gtgctgtcgg 10800

acccccgctt cagcgcggtg gcggcggcca acgacaacgt tccccgcatg acggcggtgg 10860

ggaaaccggt cgacaccctc gctgggctcg atccgcccga gcacacgcgg ttgcgccggc 10920

tcgccacccc ggccttcacc gtgtcccgcc tcgagtcctt ccgcccccag gcagtgcgga 10980

tcgccaccga gctgctcgac gcgctgcgcg aggccgggcc acccggagac gtcgtgcagg 11040

ggttggcgct cccgttcccg gtgctggtca tctgcgagat gctcggcgtc ccctacgagg 11100

accgcgcgcg gttcctgccc tggtccgaca cgatcctggc cacgacggcg cactcccccg 11160

aggaggccgt cgtggcgttg gaggagatga aggagtactt ccgccacctc atcgccgaac 11220

gccgacgcgc cccgcacgac gacctgctcg ccgacctcgt cgccgcccgc gacgccacac 11280

ccggccccgg ggagagcgat cagctcagcg aggacgaact ggtgatgttc gcctcggtgc 11340

tgctgatcgc cggtcacgag acgtccgtga accagatcgg ggactcctgc ttcctcctgc 11400

tgcgggaccg ccaccggtgg gagctcctcc agcggcgtcc cgaactgctc cccaaggtcg 11460

tggaggagct gctccgctac gtgccgctcg tcaacggggt gatcctcccc cgcgtcgcga 11520

cggaggacgt cgaggtggcg gggggtgtca tccgggcggg cgaggcggtg ttcgcctcga 11580

cggcggcggc caaccgggac gagcggttct tcgacagggc cgatgagctc gacctgctcc 11640

gccagcgcaa tccgcacctg gccttcggct acggaccgca ctactgcctt ggcgcgcacc 11700

tcgccaggat ggaactgcgg atcgccctgg gcgaactgct gcgcacgttc cccgcgctgc 11760

ggttggcgga cccgcccgag gaggtcaggt ggcggagtgg gttggtcatg cgggggccgg 11820

tcgagctgcg cgtcacgtgg tgataccgcg ggcggccggg acggtgggcc aggaccctgc 11880

cgcgatgctc gcgcccccgc cccggatcca ccccgggcgc tggtcgggcc ggtggtcccg 11940

ggtgtcccgc cgcccgaggc gccccgcccc gcccctggcg gggaggtgcg ccaggcccgg 12000

gcacgacctg cggtccgcgc ctcgtggcag gcgggcgagg cggcgggccg cgcggcccac 12060

gggcgaccgc gaggcgtgct ggaccggatc ggtcccgccc ctggtccacc gggggtgccc 12120

cagcccggac gtcccgggga gccgcgttgg agcggacctc cgatccgacg gcgcacccgc 12180

gagtggcggg cgcacggacc ctgttgatcg acgaacggac gagagatgac ggacacgacc 12240

acggacatgg tcttcggcct gtccgagatc gacgggatcg agatcgaccc gctcttcggg 12300

cggctccgcc gcgaacgacc gctcgccagg atccgcatgc ccttcggcgg tgagggctgg 12360

ttggtcaccc gctacgagga cgtgcgcacc gtcctggccg acccccggtt cagcgcggcg 12420

gccgccgccg gcgactcggt tccccggatg accgccgtgg gcaggccgga cgacacgatc 12480

gccggcctgg acccaccgga ccacagtcgg ctccgccggt tggcggcacc ggcgttcagc 12540

cccgcccgcc tggagtcgtt ccgcccccgg gccaccagga tcgcctcgga cctcctggac 12600

gcggtggagc gcacggggcc gcccgccgac ctcgtcacgg cgctgggcct cccgttcccg 12660

gtgcaggtga tctgcgagat cctcggggtt ccctacgagg accgggcgaa cttcctcccg 12720

tggtcggacg ccgtcctctc gaccaccgcg tacaccgccg agcaggcggc ggcgtcgctc 12780

gcggagatga aggcgtactt cgcccacctg gtggaacggc atcgcgtcca ccgccacgac 12840

gatctcctgg cggatctggt ggcggccagg gacaccaccg tgccgggcga ggcggaccgg 12900

ctctccgagg acgaactggt catgttcgcg ctcgtcctgc tgatcgcggg ccacgagacg 12960

acggccaacc agataggcaa ctcctgcttc ctgttgctct cggaccgctc gcgctgggaa 13020

cagctggtcg accagcccga actcatcccc cgggcggtgg acgaactgtt gcggttcgtg 13080

ccactggtca acggcgtcac gctgcccagg gtggcgaagg tggacgtgga gatcgcgggc 13140

gggctggtga gggcaggaga ggcggtgttc acctcgacgt cggcggcgaa ccgggacgag 13200

cgggtgttcg aggacccgga gcgcctggac ctgactcggc ggcacaaccc gcacgtcgcg 13260

ttcggccacg ggccccacca ctgcatcggc gcgcagctgg cgaaggtgga gctgcagatc 13320

gccctcggcg aactcgtcag acggttcccg accctgcggc tggccggccc ggccgaggag 13380

gtgccgtggc ggcgggggct ggtgatgcgg gggccgaccg agctgcgggt gtcgtggtga 13440

ggccgggcca cgcgcccggt cgccacgtcc ccgcgacgcg agccggtccg cctcccggga 13500

tgcgggccgc caccgccgcg ccgcgtacct cctcccgacg tcctgacctt ggccagcgcc 13560

gccgggtcac gatccgagcg gccggagcgt ggctcaccgc gtcccggcgc tccgccgtcg 13620

agctggagca ccgtgaacag cggttccgcg tcggccgccg gggccgggcg gtggctggtc 13680

ccggcaacgg ggacccgttg cgtcccggtg agcgcggttg gctaaagtaa gggcagcaac 13740

gcccccgtag cccaatcggc agaggcagcg gactcaaaat ccgtccagtg tgcgttcgag 13800

tcgcaccggg ggcactcgcc tttcgccggg cgatacggcc cctgaccagc atgttctggt 13860

caggggccgc ttggcgtcgg cggtaggcgg tgtccggtgg cggcacctgt tcggcagtcg 13920

accgcgtgct cgagcagccg ctccgccggc cgtcgtcggg cggttcggca gcccatgctg 13980

gtacctccgc gctcctcccg ggaagccctg ccgcgcgcac ggactcgccc gtcgatctcg 14040

gccaagcgag gccggtctcc tcggcccgct cgacgcggcg gatccgagtg cgagccagac 14100

gacgccaggg tgtcctcgtg acgttcggga acggtgctcg cctggggcgg cggggtcagg 14160

tgtcgcaggc ggtctcccgt tcggccgcca gggcgcggag ggccgcgcgg tcggtcttgc 14220

cggtgccggc caccgggaac gccgggaggg tgcggcacgt ggcgggtacc ttggcggctt 14280

cgaggaaccg ccgcagttcc cgcaggacct ggtggctccc cagctccgtc acggcgaaca 14340

ggaccgcgtc ccggccgccc gccgggggca ccaccacggc gtcggtgacc tcggggacct 14400

gacgcgccgc cgcctcgatc tcggcggcgc tggtgcgcac gccgcgttgc ttgaagatgt 14460

cgtcccggcg gccgtggaag tacaggtggc cgtcgctgtc gagacggccg tggtcgccgg 14520

tgcgcaggag gcgttctccc gtgcgcgggt cccggccgaa cgtcctcgtg gtcagcgatt 14580

cgtcacgcca gtagccggcc atgacgtgcg gcccggagac gacgatctca ccctcggtcc 14640

cggccggcac cggccggcca gccgggtcca ggatcgacac cgacgtcccg ggtagtgggg 14700

ttccgagcga accgggccgg accaggtcgc tgtcggcctc ggcgatggtg acccgtttgc 14760

attcggtgat gccgtacatc agtcgcacgg cggcggtcgg gaaggtccgc cggaggtcct 14820

cgatgcccgt cggggtcagc tcctggcccg tgttggtcag caggcgaacg ctgggcgcgg 14880

gggcgcgccg gccgagccgg gtgagcacgg cggccagcgg gggcacgacc gggacgacgg 14940

tgacctcgtg gcggcggatg gtcgcgagca ggccggcgtc gtcgcctggc ccgcgcagca 15000

ccaccgccgc gcccacccgg gcggccagga gtgcctggta gaggccgtag tcgaaggaca 15060

acggcagccg gcacaggatg acgtcgtccg ctcggtaccc gagggtgttg ccgatcgcgc 15120

gcacggcgaa catgaccgcg cggtgggggc agacgacggc cttcggttcg gaggtgctcc 15180

cggaggtgta gagcaggatg gcggcggtgt cggggtccgg ggcgcggggc ggcgtgggtg 15240

gtcggcgcgg gccaccggtg gtggggggag gttcggtcag gaccagtgtc ggctccgcgt 15300

cggaggtgat ccgctcgagt cgctggggtg gggcctccgg gttgaggagc acggcggcga 15360

cgccggcccg ggagcacgcg tagagggcgg cgagtaccca cgcccgcgcg ctggccctga 15420

tcgcgacccg gtccccgggg cgtgctcccg agcggacc 15458

Claims (6)

1. A biosynthetic gene cluster of cyanogamide, characterized in that the nucleotide sequence is represented by the nucleotide sequence 393 to 13440 th bits of SEQ ID NO. 1.
2. 2. Use of the cyanamide biosynthetic gene cluster of claim 1 for the preparation of the compound cyanamide and analogs thereof;

   the analogue of the cyanologramide is cyanologramide B, cyanologramide C, cyanologramide D, mrinaccarbolines E and mrinaccarbolines F:

   
3. 3. the use of the combination of genes cyaA, cyaB, cyaC, cyaD, cyaE, cyaF, cyaG, cyaH and cyaI for the preparation of cyanogamide and analogues thereof;

   cyaA is shown as 393-1946 bases in SEQ ID NO. 1;

   cyaB is shown as 3455-4399 bases of SEQ ID NO. 1;

   cyaC is shown as 4420-6111 base of SEQ ID NO. 1;

   cyaD is shown as 6159-7190 bases of SEQ ID NO. 1;

   cyaE is shown as 7226-8008 bases of SEQ ID NO. 1;

   cyaF is shown as 8583-9311 bases in SEQ ID NO. 1;

   cyaG is shown as the 9326-10579 bases of SEQ ID NO.1

   cyaH is shown as 10626-11843 bases in SEQ ID NO. 1;

   cyaI is shown as 12226-13440 bases in SEQ ID NO. 1;

   the analogue of the cyanologramide is cyanologramide B, cyanologramide C, cyanologramide D, mrinaccarbolines E and mrinaccarbolines F:

   
4. 4. a genetically engineered bacterium comprising the genes cyaA, cyaB, cyaC, cyaD, cyaE, cyaF, cyaG, cyaH and cyaI according to claim 3.
5. 5. The use of the genetically engineered bacterium of claim 4 for the preparation of cyanogamide and analogues thereof;

   the analogue of the cyanologramide is cyanologramide B, cyanologramide C, cyanologramide D, mrinaccarbolines E and mrinaccarbolines F:

   
6. use of the P450 oxidase CyaH for catalyzing the conversion of a substrate, cyanoogamide D, to a product, cyanoogamide;

   

   the nucleotide sequence of the coding gene CyaH of the P450 oxidase CyaH is shown as the 10626-11843 base of SEQ ID NO. 1.

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