Tumor-targeted drug-producing engineering strain as well as construction method and application thereof

Abstract

The invention belongs to the technical field of microbial genetic engineering, and particularly relates to a tumor-targeted drug-producing engineering strain as well as a construction method and application thereof. The invention discloses a series of tumor-targeted drug-producing engineering strains, the series of strains can generate an anti-cancer drug romidepsin in situ in a tumor, the series of strains can heterologously express romidepsin and can generate romidepsin in situ in a tumor of a tumor-bearing small animal, and the tumor-targeted drug-producing engineering strains have the activity of inhibiting tumor growth. That is, a series of Escherichia coli Nissle 1917 recombinant strains provided by the invention are expected to become anti-tumor viable bacteria drugs and are clinically applied in the future, so that the Escherichia coli Nissle 1917 recombinant strains have good practical application value.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial genetic engineering, and specifically relates to a tumor-targeted drug-producing engineering strain and a construction method and application thereof. Background Art

[0002] The information disclosed in the background of the invention is only intended to enhance the understanding of the overall background of the invention and should not be necessarily regarded as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Research on using synthetic biology methods to transform microorganisms and apply them to disease treatment and diagnosis is in the ascendant. Developing live bacterial drugs based on tumor-targeting bacteria can provide new solutions for tumor treatment. Endowing bacteria with biosynthetic pathways for anticancer drugs is an emerging strategy to improve the anti-tumor activity of bacteria.

[0004] Escherichia coli Nissle 1917 is a probiotic and also a tumor-targeting bacterium that has a preference for solid tumors and can proliferate in large numbers in solid tumors. Escherichia coli Nissle 1917, which has good tumor-targeting colonization and safety, has great potential to become a carrier for tumor-targeted therapy. However, the anti-tumor activity of Escherichia coli Nissle 1917 itself is weak.

[0005] At present, most studies at home and abroad use E. coli Nissle 1917 to heterologously express biomacromolecules such as active peptides to achieve the purpose of anti-tumor or adjuvant treatment of tumors. However, there are few reports on the use of E. coli Nissle 1917 or other tumor-targeting bacteria to biosynthesize small molecule drugs. Given that E. coli Nissle 1917 itself has a clear genetic background and a complete primary metabolic pathway, it has great potential to serve as a chassis to accommodate the biosynthetic pathway of small molecule drugs and achieve targeted production of small molecule drugs.

[0006] Romidepsin (FK228) is a small molecule anti-tumor drug isolated from Chromobacterium violaceum. Romidepsin inhibits the activity of histone deacetylases (HDACs) through a prodrug mechanism, thereby enhancing the transcription of tumor suppressor genes, and ultimately inhibiting the growth of tumor cells. Romidepsin has been approved by the U.S. Food and Drug Administration (FDA) for the treatment of cutaneous T cell lymphoma and peripheral T cell lymphoma. Romidepsin has a certain therapeutic effect on solid tumors such as breast cancer, pancreatic cancer, and lung cancer.

[0007] The biosynthetic gene cluster of romidepsin is about 36kb, including 12 genes, which are composed of non-ribosomal polypeptide synthetase (NPRS) and polyketide synthetase (PKS) coding genes, including 3 NPRS modules and 2 PKS modules, other synthesis-related genes and regulatory genes, etc. The inventors found that there are no patents or documents reporting the use of Escherichia coli Nissle 1917 to biosynthesize romidepsin and use it as a live bacterial drug to inhibit tumor growth. Summary of the invention

[0008] In view of the problems existing in the prior art, the purpose of the present invention is to provide a series of Escherichia coli Nissle1917 engineered strains containing a biosynthetic pathway of romidepsin, and the application of this series of strains. This series of Escherichia coli Nissle 1917 engineered strains containing a biosynthetic pathway of romidepsin can heterologously express romidepsin, can produce romidepsin in situ in tumors of tumor-bearing small animals, and has the activity of inhibiting tumor growth. Compared with wild-type Escherichia coli Nissle1917, the anti-tumor activity is improved. Based on the above research results, the present invention is completed.

[0009] Specifically, the technical solution of the present invention is as follows:

[0010] The first aspect of the present invention provides a tumor-targeted drug-producing engineered strain, which is obtained by using Escherichia coli Nissle1917 as a starting strain and introducing an exogenous romidepsin biosynthetic gene cluster; wherein the specific genes of the romidepsin biosynthetic gene cluster include depA, depB, depC, depD, depE, depF, depG, depH, depI, depJ, depK, depL, depM, depN, fabD1, and sfp.

[0011] Furthermore, the exogenous romidepsin biosynthesis gene cluster is regulated by a promoter, and the promoter includes one or more of a BAD promoter, a Tet promoter and a Pfnrs promoter.

[0012] Furthermore, the Escherichia coli Nissle 1917 may be a wild-type Escherichia coli Nissle 1917 after knocking out its own cryptic plasmids pMUT1 and pMUT2, or an Escherichia coli Nissle 1917 obtained after knocking out its own cryptic plasmids pMUT1 and pMUT2 and the araC gene in the genome.

[0013] The second aspect of the present invention provides a method for constructing the above-mentioned tumor-targeted drug-producing engineered strain, the construction method comprising:

[0014] The romidepsin biosynthetic gene cluster plasmid was constructed by Red / ET homologous recombination technology and introduced into Escherichia coli Nissle 1917;

[0015] Among them, the specific genes of the romidepsin biosynthetic gene cluster include depA, depB, depC, depD, depE, depF, depG, depH, depI, depJ, depK, depL, depM, depN, fabD1, and sfp.

[0016] The Escherichia coli Nissle 1917 may be a wild-type Escherichia coli Nissle 1917 after the cryptic plasmids pMUT1 and pMUT2 are knocked out, or an Escherichia coli Nissle 1917 obtained after the cryptic plasmids pMUT1 and pMUT2 and the araC gene in the genome are knocked out.

[0017] Furthermore, the construction method includes: introducing plasmids 23-p15A-km-pBAD-FK228, 24-p15A-km-pTet-FK228, 25-p15A-km-pFnrS-FK228, 40-p15A-km-△depK-pBAD-FK228, and 44-p15A-km-△depK-pTet-FK228 into Escherichia coli Nissle 1917 to construct an engineered strain.

[0018] The construction method of the plasmid 23-p15A-km-pBAD-FK228 includes: using the plasmid R6K-oriT-TnpA-km as a template, amplifying the kam and oriT fragments respectively, and then performing tandem PCR to obtain the target fragment kam-oriT; the plasmid p15A-cm-FK228CODON is digested with ScaI and AatII, precipitated with sodium acetate and ethanol, and then co-transformed with the PCR recovery product into E.coli GB05-dir for line-to-line recombination to obtain the obtained fragment;

[0019] The construction method of the plasmid p15A-cm-FK228CODON comprises:

[0020] S1, gene synthesis fragments OF2, OF4, OF5, OF6, OF7, OF8, OF9, OF10, OF11, OF12, OF13, the nucleotide sequences of OF2-OF13 are shown in SEQ ID NO.1-11;

[0021] S2. Construction of plasmid 01-p15A-cm-FK228CODON-F12-3-neoccdB: Using p15A-cm-tetR-tetO-hyg-ccdB as template, amplify the fragment p15A-cm; using pBBR1-BAD-luxABECD-kan plasmid as template, amplify the fragment BAD; using pBR322-amp-ccdB-rpsLneo as template, amplify the fragment neoccdB; the above fragments and OF2, OF12, and OF13 were co-transformed into the competent cell of E. coli GB05dir-gyrA462 containing the recombinase plasmid pSC101-BAD-ETgA-tet for multi-fragment assembly;

[0022] S3. Construction of plasmid 02-BR322-cmccdB-FK228codon-F47: Using R6K-cm-ccdB as template, amplify the fragment cm-ccdB, using pBR322-amp-ccdB-rpsLneo as template, amplify the fragment BR322-02, and transform the above fragments and OF4, OF5, OF6, and OF7 into the competent medium of E. coli GB05dir-gyrA462 containing the recombinase plasmid pSC101-BAD-ETgA-tet for multi-fragment assembly;

[0023] S4. Construction of plasmid 03-BR322-cmccdB-FK228codon-F811: Using R6K-cm-ccdB as template, amplify the fragment cm-ccdB, using pBR322-amp-ccdB-rpsLneo as template, amplify the fragment BR322-03, and transform the above fragments and OF8, OF9, OF10, and OF11 into the competent medium of E. coli GB05dir-gyrA462 containing the recombinase plasmid pSC101-BAD-ETgA-tet for multi-fragment assembly;

[0024] S5. Use SnaI to cut plasmid p15A-cm-FK228CODON-F12-3-neoccdB, plasmid BR322-cmccdB-FK228codon-F47 and plasmid BR322-cmccdB-FK228codon-F811 to obtain linearized plasmid fragments, and then co-transform them into E. coli GB05dir competent medium containing recombinase plasmid pSC101-BAD-ETgA-tet for multi-fragment assembly.

[0025] The construction method of the plasmid 24-p15A-km-Ptet-FK228 is as follows: using p15A-cm-tetR-tetO-hyg-ccdB as a template to amplify the fragment tet, using p15A-amyEF-amp-ccdB-specR-amyER as a template to amplify ccdB-amp, and then performing tandem PCR to obtain the target fragment ccdB-amp-tet, which is then transferred to E. coli GBred-gyrA462 containing p15A-cm-FK228CODON and induced by 10% L-arabinose, and the obtained recombinant is digested with XhoI, precipitated with sodium acetate and ethanol, and then T4 ligated and transferred to E. coli The second recombinant was obtained from GB2005; the plasmid R6K-oriT-TnpA-km was used as a template to amplify the kam and oriT fragments respectively, and then tandem PCR was performed to obtain the target fragment kam-oriT; the second recombinant was digested with ScaI and AatII, precipitated with sodium acetate and ethanol, and then co-transformed with kam-oriT into E. coli GB05-dir for linear recombination.

[0026] The construction method of the plasmid 25-p15A-km-PfnrS-FK228 comprises: using p15A-amyEF-amp-ccdB-specR-amyER as a template to amplify ccdB and amp-PfnrS fragments, then tandem PCR to obtain the target fragment ccdB-amp-PfnrS, and then transferring it to E. coli containing p15A-cm-FK228CODON and induced by 10% L-arabinose. GBred-gyrA462 was used to obtain a recombinant, which was digested with XhoI, precipitated with sodium acetate and ethanol, and then ligated with T4. The recombinant was then transferred to E.coli GB2005 to obtain a second recombinant. Plasmid R6K-oriT-TnpA-km was used as a template to amplify kam and oriT fragments, respectively, and then tandem PCR was performed to obtain the target fragment kam-oriT. The second recombinant was digested with ScaI and AatII, precipitated with sodium acetate and ethanol, and then co-transformed with kam-oriT into E.coli GB05-dir for linear recombination.

[0027] The construction method of the plasmid 40-p15A-km-△depK-pBAD-FK228 comprises: using pR6K-cm-ccdB as a template to amplify the cm fragment, and transferring it to E.coliGBred containing 23-p15A-km-BAD-FK228 and induced by 10% L-arabinose to obtain a recombinant;

[0028] The construction method of the plasmid 44-p15A-km-△depK-pTet-FK228 comprises: using pR6K-cm-ccdB as a template to amplify the cm fragment, and transferring it to E.coliGBred containing 24-p15A-km-ptet-FK228 and induced by 10% L-arabinose to obtain a recombinant.

[0029] The third aspect of the present invention provides the use of the above tumor-targeted drug-producing engineered strain in the preparation of anti-tumor drugs. The present invention has proved through experiments that the above engineered strain can produce the anticancer drug romidepsin in situ in the tumor and has anti-tumor activity in a small animal transplant tumor model, so it can be used as an anti-tumor drug.

[0030] The tumors include blood tumors and solid tumors, such as cutaneous T-cell lymphoma, peripheral T-cell lymphoma, breast cancer, pancreatic cancer and lung cancer, etc., which are not specifically limited here.

[0031] A fourth aspect of the present invention provides an anti-tumor drug, which comprises at least the above-mentioned tumor-targeted drug-producing engineered strain.

[0032] A fifth aspect of the present invention provides a method for producing romidepsin, the method comprising: culturing the tumor-targeted drug-producing engineered strain.

[0033] Beneficial technical effects of one or more of the above technical solutions:

[0034] The above technical scheme discloses a series of tumor-targeted drug-producing engineered strains, which can produce the anticancer drug romidepsin in situ in the tumor. The strains can heterologously express romidepsin and can produce romidepsin in situ in the tumor of tumor-bearing animals, and have the activity of inhibiting tumor growth.

[0035] In summary, the series of Escherichia coli Nissle 1917 recombinant strains provided by the above technical scheme are expected to become anti-tumor live bacterial drugs and be used in clinical practice in the future, so they have good practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0037] Figure 1 This is the HRMS spectrum of FK228 produced by the BAD series fermentation of the tumor-targeted drug-producing strain of the present invention.

[0038] Figure 2 The figure is a comparison of the yield of FK228 produced by the tumor-targeted drug-producing strain of the present invention.

[0039] Figure 3 The figure is a comparison of the FK228 yield in the fermentation supernatant and the bacterial cells of the tumor-targeted drug-producing strain of the present invention.

[0040] Figure 4 FK228 molecules were detected in the tumor sites of tumor-bearing mice to which the BAD series strains were administered according to the present invention.

[0041] Figure 5 This is the anti-tumor activity of the BAD series strains in the tumor-bearing mouse model of the present invention.

[0042] Figure 6 This is the HRMS spectrum of FK228 produced by fermentation of tumor-targeted drug-producing strains 24 and 44 of the present invention.

[0043] Figure 7 FK228 molecules were detected in the tumor sites of tumor-bearing mice to which strain EcN-24 of the present invention was administered.

[0044] Figure 8 The antitumor activity of strain EcN-24 in the tumor-bearing mouse model of the present invention.

[0045] Fig. 9 FK228 molecules were detected in the tumor sites of tumor-bearing mice to which strain EcN-44 of the present invention was administered.

[0046] Fig.10 The anti-tumor activity of strain EcN-44 in the tumor-bearing mouse model of the present invention.

[0047] Fig.11 This is the HRMS spectrum of FK228 produced by anaerobic fermentation of the tumor-targeted drug-producing strain EcN-25 of the present invention.

[0048] Fig.12 FK228 molecules were detected in the tumor sites of tumor-bearing mice to which strain 25 of the present invention was administered.

[0049] Fig.13 The anti-tumor activity of strain EcN-25 in the tumor-bearing mouse model of the present invention. DETAILED DESCRIPTION

[0050] It should be noted that the following detailed descriptions are all illustrative and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0051] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0052] The present invention is further explained by the following examples, but they are not intended to limit the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0053] The recombinase expression strains E. coli GB2005, E. coli GB05-dir, E. coli GB08-red, and E. coli GBdir-gyrA462 involved in the following examples were all purchased from GeneBridges, Germany; E. coli WM3064 and E. coli Nissle 1917 (endogenous cryptic plasmids pMUT1 and pMUT2 had been eliminated) were from Shandong University-Helmholtz Institute of Biotechnology.

[0054] The related plasmids involved, pSC101-BAD-ETgA-tet, p15A-cm-tetR-tetO-hyg-ccdB, p15A-amyEF-amp-ccdB-specR-amyER, pBBR1-BAD-luxABECD-kan, pBR322-amp-ccdB-rpsLneo, R6K-cm-ccdB, R6K-oriT-TnpA-km, pSC101-BAD-ccdA-Rha-gbaA-tet, RK2-BAD-Cre-SacB, etc., are all from Shandong University-Helmholtz Institute of Biotechnology.

[0055] The configuration of LB medium is: peptone 10g / L, NaCl 1g / L, yeast extract 5g / L; adding 1.2% agar powder can obtain a solid medium; in addition, increasing the NaCl to 10g / L can obtain LB medium for fermentation.

[0056] The sequence of the dep gene cluster of romidepsin biosynthesis gene is disclosed in NCBI.

[0057] The synthesis of gene fragments during plasmid construction was completed by Sangon Biotech (Shanghai) Co., Ltd. or Suzhou Jinweizhi Biotechnology Co., Ltd. Gene sequencing was completed by Sangon Biotech (Shanghai) Co., Ltd. or Beijing Ruiboxingke Biotechnology Co., Ltd. Other plasmids not mentioned are commercially available conventional plasmids, and the plasmid transformation method is generally the electroporation method.

[0058] Example 1: Construction of romidepsin biosynthetic pathway plasmid

[0059] (1) The specific steps of the construction process of plasmid p15A-cm-FK228CODON are as follows:

[0060] In the first step, codon optimization and synthesis of the dep biosynthetic gene cluster and other related genes required for FK228 synthesis were performed.

[0061] Gene synthesis fragments OF2 (SEQ ID NO.1), OF4 (SEQ ID NO.2), OF5 (SEQ ID NO.3), OF6 (SEQ ID NO.4), OF7 (SEQ ID NO.5), OF8 (SEQ ID NO.6), OF9 (SEQ ID NO.7), OF10 (SEQ ID NO.8), OF11 (SEQ ID NO.9), OF12 (SEQ ID NO.10), OF13 (SEQ ID NO.11). Among them, OF2 contains fabD1, sfp, depL, depN, depM, and depK, OF4 contains part of depA, OF5 contains part of depA and depB, OF6 contains part of depB, OF7 contains part of depB and depC, OF8 contains part of depC and depD, OF9 contains part of depD, OF10 contains part of depD and depE, OF11 contains depE, OF12 contains part of depE, depF, and depG, and OF13 contains depH, depI, and depJ.

[0062] Step 2: Construction of plasmid 01-p15A-cm-FK228CODON-F12-3-neoccdB

[0063] Using p15A-cm-tetR-tetO-hyg-ccdB as a template, primers F1-1 and P15A-FK228-CODON-2 were used to amplify the fragment p15A-cm. Using pBBR1-BAD-luxABECD-kan plasmid as a template, primers F4-1 and F4-2 were used to amplify the fragment BAD. Using pBR322-amp-ccdB-rpsLneo as a template, primers neoccdB-1 and neoccdB-2 were used to amplify the fragment neoccdB. The above fragments and OF2, OF12, and OF13 were co-transformed into the competent cell of E. coli GB05dir-gyrA462 containing the recombinase plasmid pSC101-BAD-ETgA-tet for multi-fragment assembly. After the cells were recovered, cm plates were coated and the recombinants were verified by restriction digestion and sequenced.

[0064] Step 3: Construction of plasmid 02-BR322-cmccdB-FK228codon-F47

[0065] Using R6K-cm-ccdB as a template, primers cmccdB-02-1 and cmccdB-2 were used to amplify the fragment cm-ccdB. Using pBR322-amp-ccdB-rpsLneo as a template, primers BR322-1 and BR322-02-2 were used to amplify the fragment BR322-02. The above fragments and OF4, OF5, OF6, and OF7 were co-transformed into the competent medium of E. coli GB05dir-gyrA462 containing the recombinase plasmid pSC101-BAD-ETgA-tet for multi-fragment assembly. After the cells were revived, cm plates were coated and the recombinants were verified by enzyme digestion and sequencing.

[0066] Step 4: Construction of plasmid 03-BR322-cmccdB-FK228codon-F811

[0067] Using R6K-cm-ccdB as a template, primers cmccdB-03-1 and cmccdB-2 were used to amplify the fragment cm-ccdB. Using pBR322-amp-ccdB-rpsLneo as a template, primers BR322-1 and BR322-03-2 were used to amplify the fragment BR322-03. The above fragments and OF8, OF9, OF10, and OF11 were co-transformed into the competent medium of E. coli GB05dir-gyrA462 containing the recombinase plasmid pSC101-BAD-ETgA-tet for multi-fragment assembly. After the cells were revived, they were coated on cm plates, and the recombinants were verified by enzyme digestion and sequenced.

[0068] Step 5: Use SnaI to digest plasmid p15A-cm-FK228CODON-F12-3-neoccdB, plasmid BR322-cmccdB-FK228codon-F47 and plasmid BR322-cmccdB-FK228codon-F811, respectively, to obtain linearized plasmid fragments, and then co-transform into E. coli GB05dir competent medium containing recombinase plasmid pSC101-BAD-ETgA-tet, perform multi-fragment assembly, apply cm plates after cell recovery, and perform enzyme digestion verification and sequencing of recombinants. The correct plasmid is p15A-cm-FK228CODON.

[0069] (2) The construction process of plasmid 23-p15A-km-pBAD-FK228 is as follows:

[0070] First, plasmid R6K-oriT-TnpA-km was used as a template to amplify kam and oriT fragments respectively, and then tandem PCR was performed to obtain the target fragment kam-oriT; the amplified fragment was purified by gel recovery. Plasmid p15A-cm-FK228CODON was digested with ScaI and AatII, precipitated with sodium acetate and ethanol, and then co-transformed with the PCR recovery product into E.coli GB05-dir for linear recombination. After cell recovery, kam resistance plates were coated, and the obtained recombinants were verified by enzyme digestion and sequenced.

[0071] (3) The construction process of plasmid 24-p15A-km-Ptet-FK228 is as follows:

[0072] In the first step, the fragment tet was amplified using p15A-cm-tetR-tetO-hyg-ccdB as a template, and ccdB-amp was amplified using p15A-amyEF-amp-ccdB-specR-amyER as a template, and then PCR was performed in series to obtain the target fragment ccdB-amp-tet. The amplified fragment was purified by gel recovery and electrotransferred to E.coli GBred-gyrA462 containing p15A-cm-FK228CODON and induced by 10% L-arabinose. After the cells were revived for 1 hour, they were coated on amp-resistant plates to obtain recombinants for enzyme digestion verification. The correct clone was selected to extract a large amount of plasmid, and then digested with XhoI, and then precipitated with sodium acetate ethanol at 37°C for 2.5 hours, and then T4 ligated in a PCR reactor, and then the reaction solution was desalted and electrotransferred to E.coli GB2005. After recovery, an appropriate amount was coated on cm-resistant plates, and the recombinants were digested and sequenced.

[0073] In the second step, plasmid R6K-oriT-TnpA-km was used as a template to amplify kam and oriT fragments respectively, and then tandem PCR was performed to obtain the target fragment kam-oriT; the amplified fragment was purified by gel recovery. The plasmid obtained in the first step was digested with ScaI and AatII, precipitated with sodium acetate and ethanol, and then co-transformed with the PCR recovery product into E. coli GB05-dir for linear recombination. After cell recovery, kam resistance plates were coated, and the obtained recombinants were verified by enzyme digestion and sequenced.

[0074] (4) The construction process of plasmid 25-p15A-km-PfnrS-FK228 is as follows:

[0075] In the first step, p15A-amyEF-amp-ccdB-specR-amyER was used as a template to amplify ccdB and amp-PfnrS fragments, and then tandem PCR was performed to obtain the target fragment ccdB-amp-PfnrS. The amplified fragment was purified by gel recovery and electrotransferred to E.coli GBred-gyrA462 containing p15A-cm-FK228CODON and induced by 10% L-arabinose. After the cells were revived for 1 hour, they were coated on amp-resistant plates to obtain recombinants for restriction digestion verification. The correct clone was selected to extract a large amount of plasmids, and then digested with XhoI, 37°C, 2.5 hours later, sodium acetate ethanol precipitation was performed, and then T4 ligation was performed in a PCR reactor, and then the reaction solution was desalted and electrotransferred to E.coli GB2005. After recovery, an appropriate amount was coated on cm-resistant plates, and the recombinants were subjected to restriction digestion verification and sequencing.

[0076] In the second step, plasmid R6K-oriT-TnpA-km was used as a template to amplify kam and oriT fragments respectively, and then tandem PCR was performed to obtain the target fragment kam-oriT; the amplified fragment was purified by gel recovery. The plasmid obtained in the first step was digested with ScaI and AatII, precipitated with sodium acetate and ethanol, and then co-transformed with the PCR recovery product into E. coli GB05-dir for linear recombination. After cell recovery, kam resistance plates were coated, and the obtained recombinants were verified by enzyme digestion and sequenced.

[0077] (5) The construction process of plasmid 40-p15A-km-△depK-pBAD-FK228 is as follows:

[0078] The cm fragment was amplified using pR6K-cm-ccdB as a template. The amplified fragment was purified by gel recovery and electroporated into E. coli GBred containing 23-p15A-km-BAD-FK228 and induced by 10% L-arabinose. After the cells were revived for 1 hour, they were coated on cm-resistant plates and the recombinants were obtained for enzyme digestion verification and sequencing.

[0079] (6) The construction process of plasmid 44-p15A-km-△depK-pTet-FK228 is as follows:

[0080] The cm fragment was amplified using pR6K-cm-ccdB as a template. The amplified fragment was purified by gel recovery and electroporated into E. coli GBred containing 24-p15A-km-ptet-FK228 and induced by 10% L-arabinose. After the cells recovered for 1 hour, they were coated on cm-resistant plates to obtain recombinants for enzyme digestion verification and sequencing.

[0081] Primer sequences used in the above process

[0082]

[0083]

[0084]

[0085] PCR amplification system:

[0086]

[0087] T4 ligation reaction system:

[0088] 10×T4 DNA ligase buffer 2μL T4 DNA ligase 1μL DNA precipitation fragment 17μL

[0089] 16℃ overnight / 25℃, 4h

[0090] Enzyme digestion system:

[0091] Plasmid 4μl 10X NEB buffer 1μl Enzyme 0.2μl <![CDATA[ddH2O]]> 4.8μl Total 10μl

[0092] Example 2: Construction of tumor-targeted drug-producing strains

[0093] (1) The construction process of strain EcN(△araC) is as follows:

[0094] The tool plasmid pSC101-BAD-ccdA-Rha-gbaA-tet was transformed into Escherichia coli Nissle 1917 by electroporation. The correct clone was selected by extracting the plasmid and verifying the restriction enzyme digestion. At the same time, the positive screening gene cmR was amplified by PCR, and the fragment carried the homologous arms (HA, 50bp) on both sides of the target gene araC and lox sites (lox66, lox71). After that, the correct clone was cultured overnight, and the inducer Rha was used to prepare the competent state. Then, the recovered 800ng PCR product was electroporated into the competent bacteria to mediate homologous recombination, and then 1mL LB was added and revived at 37℃ for 1h. After that, the three zones were marked on the LB plate containing cm for positive screening. Before that, it was cultured at 30℃ to prevent plasmid loss. After that, it was cultured at 37℃ to eliminate the plasmid pSC101-BAD-ccdA-Rha-gbaA-tet. The correct single clone was screened by colony PCR.

[0095] The plasmid RK2-BAD-Cre-SacB was transformed into the recombinant bacteria in the same way, and 10% Ara was added to the culture medium to induce the expression of Cre enzyme. The Cre enzyme recognized the two lox sites and recombined, thereby deleting the resistance screening marker between the two lox sites, and then the target knockout strain EcN (△araC) was obtained by colony PCR verification. The target knockout strain was subcultured 3 times in a culture medium containing sucrose, and three zones were streaked on an LB plate, and then double-plate streaking was used to finally obtain EcN (△araC) with the plasmid RK2-BAD-Cre-SacB eliminated.

[0096] The primers used in the above process are as follows:

[0097]

[0098] The colony PCR reaction system is

[0099]

[0100]

[0101] (2) A series of romidepsin biosynthetic pathway plasmids constructed in Example 1 (23-p15A-km-pBAD-FK228, 24-p15A-km-pTet-FK228, 25-p15A-km-pFnrS-FK228, 40-p15A-km-△depK-pBAD-FK228, 44-p15A-km-△depK-pTet-FK228) were introduced into Escherichia coli Nissle 1917 to construct an engineered strain. The construction process is as follows: first, the target plasmid is electrotransformed into E.coli WM3064. After the correct clone is obtained, the E.coli WM3064 and E.coli Nissle 1917 containing the target plasmid are cultured to the logarithmic growth phase, centrifuged and resuspended, and the two are mixed in about 100 μL of LB containing DAP liquid, and left to stand for 5 minutes. Drop the bacterial liquid on the DAP plate, wait for the liquid to evaporate, and culture it at 37°C overnight. Use an inoculation loop to scrape the colony from the plate and dissolve it in 1mL of antibiotic-free LB liquid, blow it evenly, take 100 μL of the bacterial liquid and draw three lines on the plate added with the corresponding antibiotics, and culture it overnight. Pick a single clone, perform colony PCR or extract plasmid enzyme digestion verification, and identify the correct E. coli Nissle 1917 engineering strain containing the romidepsin biosynthesis pathway. These engineered strains are the tumor-targeted drug-producing strains BAD series EcN-23 and EcN-40 containing the optimized romidepsin biosynthetic pathway plasmid regulated by the BAD promoter; the tumor-targeted drug-producing strains EcN-24 and EcN-44 containing the optimized romidepsin biosynthetic pathway plasmid regulated by the Tet promoter; and the tumor-targeted drug-producing strain EcN-25 containing the optimized romidepsin biosynthetic pathway plasmid regulated by the fnrs promoter.

[0102] The process of transforming the romidepsin biosynthetic pathway plasmid 23-p15A-km-pBAD-FK228 constructed in the example into the strain EcN (△araC) is the same as described above, and the resulting strain is characterized by containing an optimized romidepsin biosynthetic pathway plasmid regulated by the BAD promoter, and the tumor-targeted drug-producing strain EcN (△araC)-23 with the araC gene knocked out of the genome.

[0103] Example 3: Application of tumor-targeted drug-producing strain BAD series

[0104] The tumor-targeted drug-producing strain BAD series of this embodiment refers to E. coli Nissle 1917 or E. coli Nissle 1917 with the araC gene knocked out, containing the optimized, BAD promoter-regulated romidepsin biosynthesis pathway plasmid 23 or 40. Specifically, it refers to the EcN-23 strain, the EcN (△araC) -23 strain and the EcN-40 strain.

[0105] (1) The tumor-targeted drug-producing strain BAD series can be fermented to produce FK228.

[0106] As attached Figure 1 As shown, LC-MS / MS-HDMS showed that, compared with the retention time and secondary mass spectrometry of the FK228 standard, the tumor-targeted drug-producing strain BAD series can produce FK228 molecules after fermentation.

[0107] As attached Figure 2 As shown, through fermentation quantitative analysis, among the tumor-targeted drug-producing strains BAD series, the one with the highest yield was 40-BAD-EcN, with an average yield of 1.46 mg / L, followed by EcN(△araC)-23, with an average yield of 0.53 mg / L, and then EcN-23, with an average yield of 0.33 mg / L.

[0108] As attached Figure 3 As shown, most of the FK228 molecules produced by the fermentation of the tumor-targeted drug-producing strain BAD series can be secreted outside the cells and exist in the fermentation supernatant.

[0109] The fermentation and separation analysis process is as follows:

[0110] a. After the correct clone is activated on the plate, it is inoculated into 1.8 mL of liquid culture medium containing LB containing the corresponding antibiotics and cultured overnight at 37°C and 950 rpm.

[0111] b. Transfer the inoculum volume (500 μL) to a 50 mL LB conical flask (containing the corresponding resistance) at 1% and culture at 30°C and 200 rpm. Culture for 2 h, OD 600 At about 0.4, add arabinose inducer, whose working concentration can be between 10-150 mg. Add inducer again at the same time point the next day.

[0112] c. Continue culturing for two days, add 1 mL of treated sterile XAD-16 macroporous resin, calculate the time based on the seed solution, and stop fermentation after a total fermentation time of 96 hours.

[0113] d. Pour the obtained fermentation culture into a 50mL centrifuge tube, centrifuge at 8000rpm for 5min, discard the supernatant; add 40mL methanol, ultrasonicate for 30min, 37℃, 200rpm, soak for 2.5-3h; filter the liquid, evaporate and concentrate to obtain the extract. Use chromatographically pure methanol to fully dissolve the metabolites, filter and detect them by HPLC-MS after filtration.

[0114] e. If the content of compounds in the fermentation supernatant and the bacterial cells needs to be determined separately, the macroporous resin and bacterial precipitate are separated, methanol is added, ultrasonic is performed for 30 minutes, 37°C, 200rpm, and soaked for 2.5-3 hours; the liquid is filtered, and the extract is concentrated and dried by rotary evaporation. The metabolites are fully dissolved in chromatographically pure methanol, filtered, and then detected by high performance liquid chromatography-mass spectrometry.

[0115] (2) Antitumor activity of the BAD series of tumor-targeted drug-producing strains in mouse transplanted tumor models

[0116] The BAD series of tumor-targeted drug-producing strains, including EcN-23, EcN(△araC)-23 and EcN-40, can produce FK228 in situ in mouse model tumors, and the anti-tumor activity of the BAD series of engineered strains is significantly improved compared with wild-type Escherichia coli Nissle 1917.

[0117] Mouse breast cancer cells were used to construct a mouse breast cancer xenograft model, and the anti-tumor activity of the tumor-targeted drug-producing strain BAD series was evaluated in the mouse xenograft model. After the mice were given the strain and the corresponding inducer, romidepsin (FK228) could be detected in the tumor tissue, as shown in the attached Figure 4 As shown. The engineered strain BAD series was injected into the tail vein of mice, and the inducer was injected intraperitoneally 48 hours later. The mice were killed 6 hours or 24 hours later, and the target compound could be detected after sampling. The positive drug FK228 was used as a control. After 1 hour or 2 hours of intratumoral or intraperitoneal injection of the positive drug, the mice were killed, and the target compound could be detected after sampling. Therefore, the BAD series engineered strains described in the present invention can produce FK228 molecules in situ in the tumor.

[0118] As attached Figure 5 As shown in the figure, the results of the animal experiment on antitumor activity showed that the engineered strains had a significant inhibitory effect on tumor growth compared with the blank group without drug administration. EcN-23 had the best inhibitory activity, followed by EcN-40 and EcN(△araC)-23, but the inhibitory activities of 23 and 40 were close. Compared with the original E. coli Nissle 1917, the antitumor activity of the three engineered strains was significantly improved. Moreover, the antitumor activity of the engineered strain EcN-23 was close to the antitumor activity of the positive drug FK228.

[0119] The mouse transplant tumor model was constructed as follows: mouse breast cancer 4T1 cells were cultured in RPMI 1640 medium containing 10% serum, 100 U / mL penicillin and 100 μg / mL streptomycin at 37°C in an incubator containing 5% carbon dioxide until the logarithmic growth phase. The cells were trypsinized, washed and resuspended in PBS, and diluted to 10 7cells / mL. Take 8-week-old female Balb / c mice, select the back or axillary area of ​​the mouse as the tumor-bearing area, and prepare the skin. Inject the prepared cell suspension subcutaneously into the same area of ​​each mouse, and the injection volume is generally 100uL / mouse. When the tumor grows to 60-100mm 3 When the tumor is of moderate size, tumor-bearing mice are selected and randomly divided into groups with 3-5 mice in each group for subsequent experiments. They are usually divided into experimental group, wild-type strain control group, blank control group, and positive drug control group.

[0120] The evaluation process of antitumor activity is as follows: take the engineered strain or the wild-type strain of the corresponding group, activate it on a plate, pick a single clone and culture it overnight, transfer the bacterial solution to the logarithmic growth phase the next day, collect the bacterial solution, centrifuge, resuspend, and configure the OD 600 The bacterial solution was homogenized to 0.2. Then the bacterial solution was injected into the tumor-bearing mice through the tail vein. The other control groups were injected with an equal volume of PBS or saline through the tail vein. The experimental group was given an inducer solution (containing 100 mg of arabinose) ip 2-3 times a week, the positive control group was given a romidepsin solution (at a dose of 1.5 mg / kg) ip 2-3 times a week, and the other groups were given PBS or saline at the same time points. The experimental group was gavaged with chloramphenicol or kanamycin every day. The tumor size was measured every two days. The time lasted for about 3 weeks. At the end of the experiment, the mice were killed by pulling the neck, dissected, and the tumor was separated and weighed. Tumor volume = length × width 2 / 2The tumor volume was calculated to evaluate the inhibitory effect of the strain on tumor growth.

[0121] The process of in situ tumor detection of the target compound is as follows: the construction of the mouse transplant tumor model and the preparation of the bacterial solution are the same as above. The bacterial solution was injected into the tumor-bearing mice in the experimental group through the tail vein. After 48 hours, the inducer (100 mg of arabinose inducer for EcN-23, EcN-40 and EcN (△araC)-23) was injected ip. After 6 hours and 24 hours, the mice were killed by pulling the neck, dissected, and the tumors were separated and weighed. The positive drug group was injected with a solution containing 6ug of romidepsin intratumorally or a solution containing 30ug of romidepsin intraperitoneally. After 1 hour or 2 hours, the mice were killed by pulling the neck, dissected, and the tumors were separated and weighed. The obtained tumor tissue was ground, homogenized, extracted, and the extract was concentrated and dried, fully dissolved with chromatographic pure methanol, filtered, and detected by high-performance liquid chromatography-mass spectrometry. At the same time, a romidepsin standard solution was prepared for sample testing as a reference.

[0122] Example 4: Application of TET series of tumor-targeted drug-producing strains

[0123] The tumor-targeted drug-producing strain TET series in this embodiment refers to Escherichia coli Nissle 1917 containing optimized, Tet promoter-regulated romidepsin biosynthesis pathway plasmid 24 or 44, specifically EcN-24 strain and EcN-44 strain.

[0124] (1) Tumor-targeted drug-producing strains 24 and 44 can ferment to produce romidepsin. The fermentation and separation analysis process is similar to (1) in Example 3. The inducer can be anhydrotetracycline or doxycycline. In this embodiment, the working concentration of anhydrotetracycline can be between 0.2 μg / mL and 3 μg / mL.

[0125] As attached Figure 6 As shown, LC-MS / MS-HDMS showed that, compared with the retention time of the FK228 standard and the secondary mass spectrometry, tumor-targeted drug-producing bacteria 24 and 44 can produce FK228 molecules after fermentation.

[0126] As attached Figure 2 As shown, through fermentation quantitative analysis, the average production of tumor-targeted drug-producing strain 24 was 0.69 mg / L, and the average production of 44 was 0.714 mg / L.

[0127] As attached Figure 3 As shown, most of the FK228 molecules produced by the tumor-targeted drug-producing strains 24 and 44 can be secreted outside the cells and exist in the fermentation supernatant.

[0128] (2) Antitumor activity of tumor-targeted drug-producing strain 24 in a mouse transplanted tumor model

[0129] The tumor-targeted drug-producing strain 24 can produce FK228 molecules in situ in the tumor of the mouse model, and its anti-tumor activity is significantly improved compared with the wild-type Escherichia coli Nissle 1917.

[0130] Mouse breast cancer cells were used to construct a mouse breast cancer xenograft model, and the anti-tumor activity of tumor-targeted drug-producing strain 24 was evaluated in the mouse xenograft model. After mice were given the strain and the corresponding inducer, romidepsin (FK228) could be detected in the tumor tissue, as shown in the attached Figure 7 The engineered strain was injected into the tail vein 48 hours later, and the inducer was injected into the abdomen. The mice were killed 6 hours and 24 hours later, and the samples were taken and processed. The contents of the compounds were tested as shown in the attached Figure 7 Therefore, the 24 engineered strains described in this patent can produce FK228 molecules in situ in the tumor.

[0131] As attached Figure 8As shown, the engineered strain 24 series showed anti-tumor activity in the small animal transplant tumor model. Compared with the blank group without drug administration, the engineered strain had a significant inhibitory effect on tumor growth; compared with the wild-type strain EcN, the inhibitory effect on tumor growth was significantly improved; compared with the positive control drug group, the tumor inhibition activity of group 24 gradually exceeded that of the positive control drug group in the later stage.

[0132] The construction process of the mouse transplant tumor model is the same as that in Example 3. The anti-tumor activity experiment operation process is the same as that in Example 3, and the inducer is a solution containing 120 μg of dehydrated tetracycline. The process of tumor in situ detection of the target compound is the same as that in Example 3, and the inducer is a solution containing 150 μg of dehydrated tetracycline.

[0133] (3) Antitumor activity of tumor-targeted drug-producing strain 44 in a mouse transplanted tumor model

[0134] The tumor-targeted drug-producing strain 44 can produce FK228 molecules in situ in the tumor of the mouse model, and its anti-tumor activity is significantly improved compared with the wild-type Escherichia coli Nissle 1917.

[0135] Mouse breast cancer cells were used to construct a mouse breast cancer xenograft model, and the anti-tumor activity of tumor-targeted drug-producing strain 44 was evaluated in the mouse xenograft model. After mice were given the strain and the corresponding inducer, romidepsin (FK228) could be detected in the tumor tissue, as shown in the attached Fig. 9 As shown. 48 hours after the tail vein injection of the engineered strain, the inducer was injected intraperitoneally. 24 hours later, the mice were killed, and after sampling and processing, FK228 molecules were detected by testing. Therefore, the 44 engineered strains described in this patent can produce FK228 molecules in situ in the tumor.

[0136] As attached Fig.10 As shown, the engineered strain 44 showed anti-tumor activity in the small animal transplant tumor model. Compared with the blank group without drug administration, the engineered strain had a significant inhibitory effect on tumor growth; compared with the wild-type strain EcN, the inhibitory effect on tumor growth was significantly improved; compared with the positive control drug group, the tumor inhibition activity of group 44 was basically consistent with that of the positive control drug group.

[0137] The construction process of the mouse transplant tumor model is the same as that in Example 3. The anti-tumor activity experiment operation process is the same as that in Example 3, and the inducer is a solution containing 120 μg of dehydrated tetracycline. The process of tumor in situ detection of the target compound is the same as that in Example 3, and the inducer is a solution containing 150 μg of dehydrated tetracycline.

[0138] Example 5: Application of tumor-targeted drug-producing strain 25

[0139] The tumor-targeted drug-producing strain 25 of this embodiment, Escherichia coli Nissle 1917, contains an optimized, fnrs promoter-regulated romidepsin biosynthesis pathway.

[0140] (1) Tumor-targeted drug-producing strain 25 can ferment and produce romidepsin. LC-MS / MS high-resolution mass spectrometry analysis is shown in the attached figure. Fig.11 As shown, compared with the retention time and secondary mass spectrum of FK228 standard, engineered strain 25 can produce FK228 molecules after fermentation. The fermentation and separation analysis process is similar to (1) in Example 3, but no inducer is added, and the fermentation broth is cultured in an anaerobic incubator.

[0141] (2) Antitumor activity of tumor-targeted drug-producing strain 25 in a mouse transplanted tumor model

[0142] A mouse breast cancer xenograft model was established using mouse breast cancer cells, and the anti-tumor activity of the tumor-targeted drug-producing strain 25 was evaluated in the mouse xenograft model.

[0143] As attached Fig.12 As shown, FK228 could be detected in tumor tissues of mice administered strain 25 at 1 day, 2 days, 3 days, and 4 days, respectively.

[0144] As attached Fig.13 As shown in the figure, in the mouse transplant tumor model, the engineered strain 25 series showed anti-tumor activity. Compared with the blank group without drug administration, the engineered strain had a significant effect of inhibiting tumor growth; compared with the wild-type strain EcN, the effect of inhibiting tumor growth was improved.

[0145] The construction process of the mouse transplant tumor model is the same as that in Example 3. The anti-tumor activity evaluation process is the same as that in Example 3, and no inducer is required during the experiment. The process of in situ tumor detection of the target compound is the same as that in Example 3, and no inducer is required during the experiment.

[0146] Matters not covered by the present invention are known technologies.

[0147] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A tumor-targeted drug-producing engineered strain, characterized in that: The tumor-targeted drug-producing engineering strain is obtained by using Escherichia coli Nissle 1917 as the starting strain and introducing an exogenous romidepsin biosynthetic gene cluster; wherein the specific genes of the romidepsin biosynthetic gene cluster include depA, depB, depC, depD, depE, depF, depG, depH, depI, depJ, depK, depL, depM, depN, fabD1, and sfp.

2. The tumor-targeted drug-producing engineered strain according to claim 1, characterized in that: The exogenous romidepsin biosynthesis gene cluster is regulated by a promoter, and the promoter includes one or more of a BAD promoter, a Tet promoter and a Pfnrs promoter.

3. The tumor-targeted drug-producing engineered strain according to claim 1, characterized in that: The Escherichia coli Nissle 1917 is a wild-type Escherichia coli Nissle 1917 after knocking out its own cryptic plasmids pMUT1 and pMUT2, or an Escherichia coli Nissle 1917 obtained after knocking out its own cryptic plasmids pMUT1 and pMUT2 and the araC gene in the genome.

4. The method for constructing the tumor-targeted drug-producing engineered strain according to any one of claims 1 to 3, characterized in that: The construction method comprises: The romidepsin biosynthetic gene cluster plasmid was constructed by Red / ET homologous recombination technology and introduced into Escherichia coli Nissle 1917; Among them, the specific genes of the romidepsin biosynthetic gene cluster include depA, depB, depC, depD, depE, depF, depG, depH, depI, depJ, depK, depL, depM, depN, fabD1, and sfp.

5. The construction method according to claim 4, characterized in that: The Escherichia coli Nissle 1917 is a wild-type Escherichia coli Nissle 1917 after knocking out its own cryptic plasmids pMUT1 and pMUT2, or an Escherichia coli Nissle 1917 obtained after knocking out its own cryptic plasmids pMUT1 and pMUT2 and the araC gene in the genome.

6. The construction method according to claim 4, characterized in that: The construction method comprises: introducing plasmids 23-p15A-km-pBAD-FK228, 24-p15A-km-pTet-FK228, 25-p15A-km-pFnrS-FK228, 40-p15A-km-△depK-pBAD-FK228 and 44-p15A-km-△depK-pTet-FK228 into Escherichia coli Nissle 1917 respectively to construct an engineering strain.

7. Use of the tumor-targeted drug-producing engineered strain according to any one of claims 1 to 3 in the preparation of anti-tumor drugs.

8. The use according to claim 7, characterized in that The tumors include blood tumors and solid tumors, and further include cutaneous T-cell lymphoma, peripheral T-cell lymphoma, breast cancer, pancreatic cancer and lung cancer.

9. An anti-tumor drug, characterized in that: The anti-tumor drug at least comprises the tumor-targeted drug-producing engineered strain according to any one of claims 1-3.

10. A method for producing romidepsin, characterized in that: The method comprises: culturing the tumor-targeted drug-producing engineered strain according to any one of claims 1 to 3.

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