CN107603934B - An engineered strain expressing histone deacetylase inhibitor heterologously and its application - Google Patents

Abstract

The invention discloses an engineering strain that heterologously expresses the histone deacetylase inhibitor Thailandepsin A. The strain is named as engineering strain 7029-tdp-A. Burkholderia sp. DSM7029 is used as the starting strain, and the transposition method is used The biosynthetic gene cluster (tdp) integrating Thailandepsins into its genome was obtained. The invention also discloses the application of the engineering strain in preparing the histone deacetylase inhibitor Thailandepsin A. Experiments confirmed that compared with the wild strain Burkholderia thailandensis E264, the engineering strain 7029-tdp-A of the present invention has a doubled yield of Thailandepsin A, which lays a solid foundation for the large-scale preparation and development of histone deacetylase inhibitor drugs. Base.

Description

An engineered strain expressing histone deacetylase inhibitor heterologously and its application

technical field

The invention relates to an engineering strain and its construction and application, in particular to an engineering strain that heterologously expresses the histone deacetylase inhibitor Thailandepsin A and its construction and application, belonging to the technical field of biosynthesis.

Background technique

Histone deacetylases (HDACs) can remove acetyl groups from lysine residues at the N-terminus of histones to maintain a more open transcriptionally active state of chromatin, thereby regulating the expression of silent tumor suppressor genes . Romidepsin, the first HDAC inhibitor with antitumor activity, was originally isolated from the rod-shaped Gram-negative bacterium Chromobacterium violaceum in Japanese soil samples. Romidepsin's anticancer drug preparation (injection) is traded under the trade name of Istodax, which was developed by Gloucester Pharmaceuticals in the United States and was approved by the US FDA on November 9, 2009. Romidepsin, also known as FK228 or Depsipeptide, is a potent and selective HDAC inhibitor (HDACI).

Burkholderia thailandensis E264 (Burkholderia thailandensis E264) was isolated from paddy fields in Thailand, and it can synthesize compounds Thailandepsins with bicyclic depsipeptide structure, including Burkholdac A, Thailandepsin A and Thailandepsin B, etc. Among them, Thailandepsin A had the highest yield in its wild strain.

Thailandepsin A is a class of histone deacetylase inhibitors (HDACI), similar in structure to the drug Romidepsin. National Cancer Institute NCI60 data show that Thailandepsin A has a wide range of cancer cell proliferation inhibition activities, especially for colon cancer, melanoma, ovarian cancer and kidney cancer. Thailandepsin A is not only an effective anticancer drug, but it can also be used as a precursor of various anticancer substances. Therefore, Thailandepsin A, as a natural product and a potent HDAC inhibitor, is very promising in the development of oncology drugs.

Although Thailandepsin A is expected to be promoted as a commercial drug in the near future as a promising anticancer drug, how to obtain a pure substance in large doses is one of the biggest limitations today. On the one hand, the amount of product obtained by fermentation of wild strain Burkholderia thailandensis E264 is relatively limited. On the other hand, it is very difficult to prepare Thailandepsin A by artificial total synthesis method and has no production research value. In view of this, how to efficiently produce and purify Thailandepsin A is an urgent problem to be solved. After searching, there is no report about using the biosynthetic gene cluster (tdp) of Thailandepsins to express in the heterologous host strain Burkholderia sp. DSM7029 to obtain an engineered strain capable of expressing the histone deacetylase inhibitor Thailandepsin A.

SUMMARY OF THE INVENTION

Aiming at the difficulty of genetic manipulation of the wild strain Burkholderia thailandensis E264 that currently produces Thailandepsin A and the relatively limited amount of Thailandepsin A product obtained by fermentation, the problem to be solved by the present invention is to provide a kind of histone deacetylase inhibitor Thailandepsin capable of heterologous expression The engineering strain of A and its construction and application.

The engineering strain that heterologously expresses the histone deacetylase inhibitor Thailandepsin A of the present invention is characterized in that: the strain is named as engineering strain 7029-tdp-A, and its genotype is: [Polyangium]brachysporumstrain DSM 7029, apramycin resistance, tdpA, tdpB, tdpC1, tdpDE1, tdpC2, tdpE2, tdpF, tdpG, tdpH, tdpI and tdpJ, using Burkholderia sp. DSM7029 as the starting strain, the biosynthesis of Thailandepsins was integrated into its genome by transposition Gene cluster (tdp) acquisition.

The construction method of the engineering strain heterologously expressing histone deacetylase inhibitor Thailandepsin A according to the present invention, the steps are:

(1) The biosynthetic gene cluster (tdp) of Thailandepsins was directly cloned into the p15A-cm-tetR-tetO-hyg-ccdB vector using Red/ET DNA recombination technology, and the plasmid p15A-cm-tetR-tetO-tdp was constructed;

(2) inserting a transposition element into the plasmid p15A-cm-tetR-tetO-tdp constructed in step (1) to construct an expression plasmid p15A-tnpA-apra-tetR-tetO-tdp;

(3) The expression plasmid p15A-tnpA-apra-tetR-tetO-tdp constructed in step (2) was electroporated into Burkholderia sp. DSM7029, and the expression plasmid was in Burkholderia sp. (tdp) was integrated into the genome of Burkholderia sp. DSM7029 to obtain an engineered strain capable of heterologously expressing the histone deacetylase inhibitor Thailandepsin A, named as engineering strain 7029-tdp-A.

The application of the engineering strain heterologously expressing the histone deacetylase inhibitor Thailandepsin A of the present invention in the preparation of the histone deacetylase inhibitor Thailandepsin A.

The engineering strain 7029-tdp-A involved in the present invention has not been reported in the literature, and it is the first time that the biosynthetic gene cluster (tdp) of Thailandepsins is expressed in the heterologous host strain Burkholderia sp. DSM7029. Experiments confirm that: compared with the wild strain Burkholderia thailandensis E264, the engineered strain provided by the present invention has a doubled yield of Thailandepsin A. It is expected to provide a test and theoretical basis for the large-scale production of Thailandepsin A, which is of great value in the development of clinical applications.

Description of drawings

Figure 1: Direct cloning process of the Thailandepsins biosynthetic gene cluster (tdp).

Figure 2: Construction of the Thailandepsins expression plasmid p15A-tnpA-apra-tetR-tetO-tdp.

Figure 3: Enzyme cleavage identification of the Thailandepsins biosynthetic gene cluster (tdp) directly cloned recombinants.

Among them: (A) PstI digestion analysis of the directly cloned recombinant p15A-cm-tetR-tetO-tdp of the Thailandepsins biosynthetic gene cluster (tdp). In the 20 recombinants, no.1 and no.2 digestions were correct. The marker on the left of the restriction map is NEB 1kb. (B) Further digestion analysis of no.1 and no.2 with PstI. The band patterns generated by PstI digestion were 8526, 6789, 4977, 4162, 3690, 2736, 2650, 2539, 2156, 2122, 954bp, respectively. The marker on the left of the restriction map is NEB 1kb.

Figure 4: Enzyme digestion identification of the expression plasmid p15A-tnpA-apra-tetR-tetO-tdp recombinant.

Among them, the band patterns generated by NcoI digestion were 10336, 9833, 6201, 5734, 5241, 4824 and 1040bp, respectively. Except for No.12, the 23 recombinants were all cut correctly. The marker on the left of the restriction map is NEB 1kb. c is the control plasmid p15A-cm-tetR-tetO-tdp.

Figure 5: Colony PCR detection of the constructed engineered strain 7029-tdp-A.

Among them: DL5000marker is on the left side of the enzyme digestion map. 6 clones (No. 1, 2, 3, 4, 5, 6) were tested for each pair of primers, "-" was the negative control, that is, the starting strain Burkholderia sp. DSM7029 was used as the template, and "+" was the positive control, that is, the Plasmid p15A-tnpA-apra-tetR-tetO-tdp served as template. Using a1-tdp/a2-tdp as primers, the size of the colony PCR fragment is 720bp, using b1-tdp/b2-tdp as the primer, the size of the colony PCR fragment is 200bp, using c1-tdp/c2-tdp as the primer, the colony PCR fragment The size is 1231bp.

Figure 6: High-performance liquid chromatography-mass spectrometry detection of Thailandepsin A expressed in engineered strain 7029-tdp-A.

HPLC-MS analysis of fermented extracts of wild strain Burkholderia thailandensis E264, starting strain Burkholderi sp.DSM7029 and engineered strain 7029-tdp-A, the mass spectra showed [M+H] ⁺ peak of ThailandepsinA extracted ion current (EIC) (EIC 548.1917± 0.01+All MS).

Detailed ways

The present invention will be described in detail below with reference to the accompanying drawings and specific examples for better understanding of the present invention, but the content does not limit the protection content of the present invention.

General description: Recombinase expression strain GB05-dir, recombinase expression strain GB08-Red, recombinase expression plasmid pSC101-BAD-ETgA-tet, plasmid p15A-cm-tetR-tetO-hyg-ccdB involved in the following examples and pR6K-oriT-tnpA-apra were purchased from GeneBridges, Germany; the starting strain Burkholderia sp. DSM7029 was purchased from DSMZ, Germany (accession number: DSM7029); the wild strain Burkholderia thailandensis E264 was purchased from DSMZ, Germany (accession number: DSM13276). For the Burkholderia thailandensis E264 genome sequence, see the complete genome sequence published in NCBI. The Thailandepsins biosynthetic gene cluster (tdp) gene sequence is shown in the sequence published in NCBI. Gene sequencing in plasmid construction was completed by BGI. Other plasmids not mentioned are commercially available conventional plasmids, and the method for electrotransformation into recipient bacteria is a conventional method.

All other reagents and consumables involved are domestically produced. Unless otherwise specified, the experimental methods and reagents in the examples are conventional methods in the art and commercially available reagents.

Example 1: Construction of Thailandepsins Biosynthesis Gene Cluster (tdp) Expression Plasmid

(1) Direct cloning of the Thailandepsins biosynthetic gene cluster (tdp)

The process of direct cloning of Thailandepsins biosynthetic gene cluster (tdp) is shown in Figure 1.

The specific steps are: restriction endonuclease AvaI digests the plasmid p15A-cm-tetR-tetO-hyg-ccdB to obtain the fragment p15A-cm-tetR-tetO (the large fragment is recovered by enzyme digestion, the gel runs to the bottom and then the gel is cut, and the gel is recovered For specific methods, please refer to the instructions of Tiangen kit). Then, using p15A-cm-tetR-tetO as a PCR template, the fragment p15A-cm-tetR-tetO was PCR amplified with primers p15A-cm-tet-5 and p15A-cm-tet-3, and the resulting PCR product p15A-cm- The two ends of the tetR-tetO vector fortdp carry the homology arms of the two sequences of the Thailandepsins biosynthesis gene cluster (tdp). Then the PCR amplification product p15A-cm-tetR-tetO vector for tdp and the genomic DNA of Burkholderia thailandensis E264 after restriction endonuclease DraI digestion and purification were co-electrotransformed into the thermosensitive recombinase expression plasmid pSC101- BAD-ETgA-tet in strain GB05-dir.

p15A-cm-tet-5: GACCGAATCGGTCAATTCGATGTAGCCGATCTTTACCCACGTCTTTagatccgaaaaccccaagttacggat

p15A-cm-tet-3:TCCGACTTAACCTTGCTTTTCAGATGCGCATCGCTTCGGGGCTCGACCACagatcctttctcctctttagatcttt) (capital letters in primers are homology arms, lower case letters are primers).

The electrotransformation steps are as follows: the strain GB05-dir containing the thermosensitive recombinase expression plasmid pSC101-BAD-ETgA-tet was added to LB medium (low salt, 1% Triptone, 0.5% yeast extract, 4 μg/ml tetracycline) 0.1% NaCl) at 30°C overnight (OD ₆₀₀ =3-4). 40 μl of the overnight culture (OD ₆₀₀ =3-4) was transferred to 1.3 ml of LB supplemented with 4 μg/ml tetracycline, placed on an Eppendorf thermomixer at 30° C., and incubated at 950 rpm for 2 h (OD ₆₀₀ =0.35-0.4). 20 μl of 10% L-arabinose was added to the culture, placed on an Eppendorf thermomixer at 37° C., and incubated at 950 rpm for 40 min. Cells were harvested by centrifugation at 9,400g for 30sec. The supernatant was discarded, and the pellet was suspended with 1 ml of _H2O . Repeat centrifugation, resuspension, and centrifugation again, discard the supernatant, and resuspend the cells with 20 μl of H ₂ O. Add 200ng PCR amplification product p15A-cm-tetR-tetO vectorfor tdp and 1μg Burkholderia thailandensis E264 digested and purified genomic DNA with DraI, and transfer the mixture of cells and DNA into a 1mm electric shock cup , using Eppendorf electroporator2510 for electric shock, the voltage is 1350V, the capacitance is 10Mf, and the resistance is 600Ω. Add 1 ml of LB to the electric shock cuvette, wash the cells and transfer them to a 1.5 ml tube with holes, and place them on an Eppendorf thermomixer at 37° C. and incubate at 950 rpm for 1 h. Finally, all bacterial liquids were spread on LB plates supplemented with 30 μg/ml chloramphenicol, and cultured at 37°C overnight.

Under the mediation of recombinase, the p15A-cm-tetR-tetO vector for tdp undergoes linear homologous recombination with the Thailandepsins biosynthetic gene cluster (tdp). A single colony was picked and digested with the restriction endonuclease PstI (see Figure 3 for enzyme digestion electrophoresis analysis), and the correct recombinant plasmid p15A-cm-tetR-tetO-tdp was screened. 20 recombinant clones were picked for PstI digestion, and 2 were identified as correct clones. The recombinant plasmids with correct restriction endonuclease analysis were sequenced with primers p15A-tdp-1, p15A-tdp-2, p15A-tdp-3 and p15A-tdp-4 to ensure that the sequences located in the homology arm region did not change.

The primer sequences used to sequence the sequence of the homology arm region are as follows:

p15A-tdp-1: gattccgacctcattaagcagctc

p15A-tdp-2:cttcaatatccggctgcgcaac

p15A-tdp-3: cgaggaacaatgggaggatg

p15A-tdp-4:gagcgtagcgagtcagtgagcg.

The specific procedure for PCR amplification of the fragment p15A-cm-tetR-tetO with primers p15A-cm-tet-5 and p15A-cm-tet-3 is as follows:

PCR amplification system:

PCR program: pre-denaturation at 94°C for 2min; denaturation at 98°C for 15s; annealing at 58°C (set according to the Tm value of the primer) for 30s; extension at 72°C for 4min (the extension time is determined according to the amplified length, 1 kb/1min); cycle 30 times ; The last 72 ℃, 10min. The primers used during the experiments were p15A-cm-tet-5 and p15A-cm-tet-3. The template is the product of p15A-cm-tetR-tetO-hyg-ccdB linearized with the restriction enzyme AvaI.

(2) Construction of Thailandepsins biosynthetic gene cluster (tdp) expression plasmid p15A-tnpA-apra-tetR-tetO-tdp

The construction process of the expression plasmid p15A-tnpA-apra-tetR-tetO-tdp is shown in Figure 2.

The specific steps are: the plasmid pR6K-oriT-tnpA-apra is digested with restriction endonuclease NheI to obtain the fragment oriT-tnpA-apra (the large fragment is recovered by enzyme digestion, the glue runs to the bottom and then the gel is cut, and the specific method of gel recovery refers to Tiangen Reagent box manual). The fragment oriT-tnpA-apra has homology arms at both ends of the cm gene in plasmid p15A-cm-tetR-tetO-tdp. Then, 200 ng of DNA fragment oriT-tnpA-apra and 200 ng of plasmid p15A-cm-tetR-tetO-tdp were co-electrotransformed into 20 μl of 10% L-arabinose-induced strain GB08-red expressing Redα/β/γ recombinase. Wire loop reorganization. Under the action of recombinase, the cm on the plasmid p15A-cm-tetR-tetO-tdp was replaced by oriT-tnpA-apra, thereby obtaining the recombinant plasmid p15A-tnpA-apra-tetR-tetO-tdp. The recovered cells were plated on LB plates supplemented with apramycin (20 μg/ml) and cultured at 37°C overnight. Then a single colony was picked and digested with restriction endonuclease NcoI (see Figure 4 for enzyme digestion electrophoresis analysis), and the correct recombinant plasmid p15A-tnpA-apra-tetR-tetO-tdp was screened.

The expression plasmid p15A-tnpA-apra-tetR-tetO-tdp is based on p15A-cm-tetR-tetO-tdp with a transposition element added, namely IR-tps cassette, IR is an inverted repeat sequence, tps is MycoMar Transposase expression gene. After the plasmid p15A-tnpA-apra-tetR-tetO-tdp is transferred into Burkholderia sp. DSM7029, the gene cluster can be randomly inserted into the host genome under the action of transposase, so that the gene cluster can be inserted into the host bacteria can exist stably.

Example 2: Construction of an engineered strain that heterologously expresses the histone deacetylase inhibitor Thailandepsin A

The plasmid p15A-tnpA-apra-tetR-tetO-tdp was electro-transformed into Burkholderia sp. DSM7029, and the electro-transformation steps were: Burkholderia sp. DSM7029 was prepared in CYMG (Casitone 8g/L, MgCl ₂ 4g/L, Yeastextract 4g/L , 50% glycerol 10ml/L) medium at 30°C for 12h. 40 μl of the culture was transferred to CYMG medium (OD ₆₀₀ ≈ 0.1), placed on an Eppendorf thermomixer at 30° C., 950 rpm for 14 h (OD ₆₀₀ ≈ 1.8). Cells were harvested by centrifugation at 9,400g for 30sec. The supernatant was discarded, and the pellet was suspended with 1 ml of _H2O . Repeat centrifugation, resuspension, and centrifugation again to suspend cells in 20 μl _H2O . 1 μg of plasmid p15A-tnpA-apra-tetR-tetO-tdp was added, and the mixture of cells and DNA was transferred into a 1 mm electroporation cup, and electroporated with Eppendorf electroporator 2510, with a voltage of 1250 V, a capacitance of 10 μf, and a resistance of 600 Ω. 1 ml of CYMG medium was added to the electric shock cup, and the cells were washed and transferred to a 1.5 ml tube with a puncture hole, and then placed on an Eppendorf thermomixer at 30° C. and incubated at 950 rpm for 3 h. It was plated on CYMG plates supplemented with apramycin (20 μg/ml), and cultured at 30 degrees for 2-3 days until a single clone was grown.

Then three pairs of primers (a1-tdp/a2-tdp, b1-tdp/b2-tdp and c1-tdp/c2-tdp) were used for colony PCR identification respectively. The identification results are shown in Figure 5.

The sequences of the above colony PCR primers are:

a1-tdp: cgcatctgaaaagcaaggtt

a2-tdp: ggatgcgcgttcgcacagac

b1-tdp:cttaccgggcggaaccgcatc

b2-tdp:tcacttcggcgcatcacatga

c1-tdp: tagtgaaaaaccttgttggca

c2-tdp:ggatgcgcgttcgcacagac.

The specific method of colony PCR is as follows:

PCR amplification system:

PCR procedure:

Pre-denaturation at 94°C for 1min; denaturation at 98°C for 10s; annealing at 56-60°C (set according to the Tm value of the primer) for 15s; extension at 68°C (the extension time is determined according to the amplified length, 1kb/1min); cycle 30 times; finally 72°C, 10min. The primers used in the experiment were a1-tdp/a2-tdp, b1-tdp/b2-tdp and c1-tdp/c2-tdp, respectively. The template is the shaken bacterial liquid, the bacterial liquid can be clearly seen turbid, and then take out 100 μl and wash it twice with 1000 ml sterile water to wash the medium. Then boil in boiling water for 15min. It can be used as a template after cooling.

The results confirmed that the constructed expression plasmid p15A-tnpA-apra-tetR-tetO-tdp was electroporated into Burkholderia sp.

In DSM7029, the expression plasmid expresses transposase in Burkholderia sp. DSM7029, and the biosynthetic gene cluster (tdp) of Thailandepsins is integrated into the genome of Burkholderia sp. DSM7029 to obtain the heterologous expression of histone deacetylase inhibitor Thailandepsin A The engineering strain was named as engineering strain 7029-tdp-A.

Example 3: Application of the engineering strain 7029-tdp-A of the present invention in the preparation of histone deacetylase inhibitor Thailandepsin A

The engineered strain 7029-tdp-A was inoculated into CYMG medium supplemented with apramycin (20 μg/ml), and cultured at 30°C for 24h. The overnight cultures were inoculated into 50 ml of fresh CYMG medium (apramycin 20 μg/ml) at 1% by volume inoculum. After culturing for 2 days at 30° C., 200 rpm, XAD-16 macroporous adsorption resin with a final concentration of 2% was added, and the culture was continued for 2 days. Cells and macroporous adsorption resin were collected by centrifugation at 8000 rpm for 10 min, and then extracted with methanol. The methanol was removed by rotary evaporation and the remaining extract was dissolved in 1 ml methanol. After filtration with a 0.22 μm filter, 3 μl was taken for HPLC-MS analysis. The high performance liquid chromatograph model is UltiMate ^™ 3000 RSLC. Chromatographic conditions were: AcclaimTM RSLC 120C18, 5 μm, 4.6 × 250 mm; solvent A was ultrapure water (0.1% trifluoroacetic acid) and B acetonitrile (0.1% trifluoroacetic acid); solvent gradient was, 0–5 min, 5% B, 5–45 min, 5%–95% B, 45–60 min, 95% B; column flow rate was 0.75 ml/min. The model of the high-resolution mass spectrometer is Bruker microOTOF-Q II, ESI-Q-TOF MS (electrospray quadrupole time-of-flight mass spectrometer). Mass spectrometry conditions: AutoMS ² , Mass range (50-1500), precision ion 2.

The results showed that the main product of the Thailandepsins biosynthesis gene cluster (tdp) expressed in Burkholderia sp. DSM7029 was Thailandepsin A, and the results are shown in Figure 6 .

Example 4: Comparison of the amount of Thailandepsin A produced by the constructed engineered strain 7029-tdp-A and the wild strain Burkholderia thailandensis E264

The comparison of the amount of Thailandepsin A produced by the engineered strain 7029-tdp-A constructed by the present invention and the wild strain Burkholderia thailandensis E264 mainly adopts the peak area comparison method. ] ⁺ peak (EIC 548.1917±0.01+All MS) was integrated to obtain the peak area; then the ratio of the peak areas was performed, and the ratio was close to 2:1. Experiments show that, compared with the wild strain Burkholderiathailandensis E264, the engineered strain 7029-tdp-A expressing the histone deacetylase inhibitor Thailandepsin A constructed by the present invention has a doubled yield of Thailandepsin A.

Claims (1)

1. An engineering strain for heterogeneously expressing histone deacetylase inhibitor Thailandepsin A is characterized in that: the strain is named as an engineering strain 7029-tdp-A, and the genotype of the strain is as follows: [ Polyangium ] brachysporum strain DSM7029, apramycin resistance, tdpA, tdpB, tdpC1, tdpDE1, tdpC2, tdpE2, tdpF, tdpG, tdpH, tdpI and tdpJ, are obtained by using Burkholderia sp.DSM7029 as a starting strain and integrating Thailandepsins biosynthesis gene cluster (tdp) on the genome thereof by a transposition method, and the steps are as follows:

(1) directly cloning a biosynthesis gene cluster (tdp) of Thailandepsins to a p15A-cm-tetR-tetO-hyg-ccdB carrier by using a Red/ET DNA recombination technology to construct a plasmid p 15A-cm-tetR-tetO-tdp;

(2) inserting a transposable element into the plasmid p15A-cm-tetR-tetO-tdp constructed in the step (1) to construct an expression plasmid p 15A-tnpA-apra-tetR-tetO-tdp;

(3) the expression plasmid p15A-tnpA-apra-tetR-tetO-tdp constructed in the step (2) is electrically transferred into Burkholderia sp.DSM7029, and the expression plasmid expresses transposase in Burkholderia sp.DSM7029 to integrate the biosynthetic gene cluster (tdp) of Thailandepsins into the genome of Burkholderia sp.DSM7029, so as to obtain the engineering strain named 7029-tdp-A capable of heterologously expressing histone deacetylase inhibitor Thailandepsin A.

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