Engineered bacterium for producing ansamitocin as well as construction method therefor and use thereof

Abstract

The present invention relates to an engineered bacterium for producing ansamitocin as well as a construction method therefor and the use thereof. The engineered bacterium overexpresses a FtsH protein. No study has reported the relationship between FtsH and ansamitocin. The present invention finds that the cell division protein FtsH is related to the synthesis of ansamitocin, and overexpressing FtsH can increase the yield of ansamitocin, thus saving the cost of industrial production of ansamitocin and increasing the fermentation efficiency. Therefore, the present invention is of great significance for realizing the industrial development of anti-tumor drugs in China, expanding the application range, and promoting clinical diagnosis and treatment of cancers.

Description

An engineered bacterium for producing ansamitocin, its construction method and application Technical Field

[0001] The present invention belongs to the technical field of genetic engineering and relates to an engineered bacterium for producing ansamitocin and a construction method and application thereof. Background Art

[0002] Ansamitocin is a macrolide antibiotic with a structural similarity to the plant-derived maytansine, demonstrating strong anti-tumor activity. Produced primarily by Actinosynnema pretiosum, its structure consists of a lactam ring formed by a specific 3-amino-5-hydroxybenzonic acid (AHBA) unit with fatty chains attached to either end. Ansamitocin contains four components: P1, P2, P3, and P4. Ansamitocin-P3 is the primary product, with activity similar to that of maytansine.

[0003] Researchers linked the C-3 ester chain of ansamitocin to a disulfide bond to form the DM1 molecule. After reduction with DTT2, it can be conjugated to various antibodies to form antibody-drug conjugates. By targeting tumor cells, ansamitocin's toxicity to normal cells is significantly reduced. In 2013, trastuzumab emtansine (T-DM1), an antibody-drug conjugate developed by Roche and featuring ansamitocin as the active moiety, received marketing authorization from the US FDA. As an effective treatment for breast cancer, it holds broad market potential.

[0004] Ning et al. systematically optimized the post-modification pathway of ansamitocin biosynthesis in Actinosynnema pretiosum ATCC31280 through metabolic engineering. By knocking out the gene ansa30, encoding a glycosyltransferase, they eliminated the accumulation of the byproduct carbamylated-glycosylated AP-3 (ACGP-3). Furthermore, overexpression of the gene asm10, encoding a methyltransferase, significantly reduced the accumulation of the byproduct N-demethyl-AP-3 (PND-3). Addition of 0.5 mM methionine and 40 mM valine successfully increased AP-3 production to 246 mg / L, a five-fold increase compared to the starting strain. Furthermore, Du et al. further increased AP-3 production by 60% by overexpressing the genes asmUdpg and asm13-17, encoding precursors for ansamitocin biosynthesis, in addition to traditional mutagenesis.

[0005] Summary of the Invention

[0006] In view of the shortcomings of the existing technology, the purpose of the present invention is to provide an engineered bacterium for producing ansamitocin and a construction method and application thereof.

[0007] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides an engineered bacterium for producing ansamitocin, wherein the engineered bacterium overexpresses FtsH protein.

[0009] The FtsH protein belongs to the AAA+ (ATPases Associated with diverse cellular Activities) protein family, is present in many bacteria and green plants, and contains one or more ATP-binding domains. The FtsH protein maintains homeostasis inside the cell by finding and degrading proteins that are marked as unnecessary or damaged, such as those that are misfolded or missynthesized. FtsH plays a vital role in the cell division process. By degrading unnecessary proteins, it ensures the normal growth of intracellular proteins and cell division function. No studies have yet reported on the relationship between FtsH and ansamitocin. The present invention reports that the cell division protein FtsH is related to the synthesis of ansamitocin. Overexpression of the FtsH gene can increase the production of ansamitocin, save the cost of industrial production of ansamitocin, and improve fermentation efficiency. This is of great significance for realizing the industrial development of anti-tumor drugs in my country, expanding the scope of use, and promoting the clinical diagnosis and treatment of cancer.

[0010] Preferably, the amino acid sequence of the FtsH protein is shown in SEQ ID NO: 1.

[0011] Preferably, the nucleotide sequence of the FtsH gene is shown in SEQ ID NO: 2.

[0012] In a second aspect, the present invention provides a method for constructing an engineered bacterium for producing ansamitocin, the method comprising:

[0013] (1) Perform PCR using the FtsH gene as a template to obtain a PCR product;

[0014] (2) The vector is digested with restriction endonucleases, ligated with the PCR product, and transferred into competent cells for sequencing and plasmid extraction to obtain the recombinant vector;

[0015] (3) transferring the recombinant vector into the conjugative transfer strain;

[0016] (4) The strain containing the recombinant vector is mixed with the mycelium of the recipient bacteria, cultured, and positive conjugates are screened.

[0017] Preferably, the source of the FtsH gene includes Actinosynnema pretiosum ATCC 31280.

[0018] Preferably, the vector is pLQ646 containing a strong promoter.

[0019] Preferably, the strong promoter comprises kasOp.

[0020] Preferably, the nucleotide sequence of the vector is shown as SEQ ID NO: 3.

[0021] Preferably, the conjugative transfer strain comprises Escherichia coli ET12567 / pUZ8002.

[0022] Preferably, the restriction endonucleases include NdeI and EcoRI.

[0023] Preferably, the recipient bacteria includes Actinosynnema pretiosum ATCC 31280.

[0024] Preferably, the culture time in step (4) is 3-7 days, for example, 3 days, 4 days, 5 days, 6 days, 7 days, etc. Other specific values ​​within the above numerical range can be selected and will not be repeated here.

[0025] In a third aspect, the present invention provides a use of the engineered bacteria for producing ansamitocin according to the first aspect in producing ansamitocin.

[0026] In a fourth aspect, the present invention provides a method for producing ansamitocin, comprising: fermenting using the engineered bacteria for producing ansamitocin described in the first aspect.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The FtsH protein belongs to the AAA+ (ATPases Associated with diverse cellular Activities) protein family, is present in many bacteria and green plants, and contains one or more ATP-binding domains. The FtsH protein maintains homeostasis within the cell by finding and degrading proteins that are marked as unnecessary or damaged, such as those that are misfolded or missynthesized. FtsH plays a vital role in the cell division process. By degrading unnecessary proteins, it ensures the normal growth of intracellular proteins and cell division function. No studies have yet reported on the relationship between FtsH and ansamitocin. The present invention reports that the cell division protein FtsH is related to the synthesis of ansamitocin. Overexpression of the FtsH gene can increase the production of ansamitocin by 60.5%, which can save the industrial production cost of ansamitocin and improve fermentation efficiency. This is of great significance for realizing the industrial development of anti-tumor drugs in my country, expanding the scope of use, and promoting the clinical diagnosis and treatment of cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] FIG1 is a map of the expression vector containing the gene for synthesizing the cell division protein FtsH.

[0030] FIG2 is a graph showing changes in ansamitocin production after expression of the cell division protein FtsH synthesis gene. DETAILED DESCRIPTION

[0031] The technical solution of the present invention is further described below by way of specific embodiments. It should be understood by those skilled in the art that the embodiments are merely to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0032] Example 1

[0033] Cloning of the gene fragment synthesizing the cell division protein FtsH

[0034] Total genomic DNA from Actinosynnema pretiosum ATCC 31280 was extracted from 1 mL of culture medium using a bacterial genome extraction kit (Tiangen). Primers FP-1 and RP-1 were used to amplify a fragment containing FtsH, approximately 2.2 kb in size, using Phanta Max Super-Fidelity DNA Polymerase (Norwegian).

[0035] The sequence of primer FP-1 is shown in SEQ ID NO: 4, and the sequence of primer RP-1 is shown in SEQ ID NO: 5.

[0036] The amplification system is as follows:

[0037] Table 1

[0038] PCR amplification conditions were as follows: 95°C pre-denaturation for 10 min, 95°C denaturation for 30 s, 65°C annealing for 30 s, 72°C extension for 3.5 min, 30 cycles, and 72°C extension for 10 min. Band accuracy was verified by agarose gel electrophoresis.

[0039] Example 2

[0040] Construction of recombinant vector by enzyme digestion and ligation

[0041] The Streptomyces expression vector pLQ646 and the amplified FtsH fragment were digested with NdeI and EcoRI, and the fragment was expressed by Linearized pLQ646 and the PCR fragment containing FtsH were recovered using an Extraction Kit (Omega). The fragments were ligated to the vector using a DNA Ligation Kit (Takara). The ligation system was configured according to the following ratios:

[0042] Table 2

[0043] The above system was placed at 16°C for 60 min and then immediately transferred to ice.

[0044] Example 3

[0045] Transformation of ligation products and screening of positive clones

[0046] Thaw competent E. coli DH5α cells stored at -80°C on ice. Add 10 μL of the assembly product to each tube and continue to incubate on ice for 10 minutes. Then, transfer the tubes to a 42°C water bath for a heat shock of 90 seconds. Return the tubes to ice for another 90 seconds. Add 500 μL of LB medium to each tube and incubate on a shaker at 37°C for 30 minutes. Then, spread the cells onto solid LB medium supplemented with apramycin and incubate them inverted at 37°C for 12 hours.

[0047] After single clones were grown, 8 clones were picked and placed in LB liquid medium supplemented with apramycin, cultured at 37°C for 8 hours, and then amplified using Vazyme, 2* Rapid Taq Master Mix (Novagen) with primers FP-2 and RP-2 to screen for positive clones that could amplify a 2.2 kb fragment.

[0048] The sequence of primer FP-2 is shown in SEQ ID NO: 6, and the sequence of primer RP-2 is shown in SEQ ID NO: 7.

[0049] The configuration method of the verification PCR system is as follows:

[0050] Table 3

[0051] PCR amplification conditions were as follows: pre-denaturation at 95°C for 10 min, 30 cycles of denaturation at 95°C for 30 s, annealing at 65°C for 30 s, and extension at 72°C for 2 min, followed by extension at 72°C for 10 min. Band accuracy was verified by agarose gel electrophoresis.

[0052] Example 4

[0053] The recombinant vector was transformed into Escherichia coli conjugative transfer strain ET12567

[0054] Thaw competent E. coli ET12567 / pUZ8002 cells stored at -80°C on ice. Add 5 μL of the constructed recombinant vector to each tube and continue to incubate on ice for 10 minutes. Then, transfer the tube to a 42°C water bath for 90 seconds. Return the tube to ice for 90 seconds. Add 500 μL of LB medium to each tube and incubate on a shaker at 37°C for 30 minutes. Then, spread the plate onto solid LB medium supplemented with apramycin and incubate inverted at 37°C for 12 hours.

[0055] After single clones were grown, 8 single clones were picked and placed in LB liquid medium supplemented with apramycin, cultured at 37°C for 8 hours, and then screened for positive clones that could amplify a 3.5 kb fragment using primers FP-2 and RP-2 using Vazyme, 2* Rapid Taq Master Mix (Novagen).

[0056] Example 5

[0057] Actinomyces pretitifolius-Escherichia coli indirect and transfer

[0058] Activate E. coli ET12567 / pUZ8002 containing the recombinant plasmid on LB plates containing kanamycin, chloramphenicol, and apramycin. After overnight culture at 37°C, pick a single colony and transfer it to 5 mL of LB (containing the above antibiotics at a 1:1000 ratio) and culture at 37°C. After 20 hours, collect 1 mL of cells by centrifugation, wash twice with LB, and resuspend in 500 μL of LB.

[0059] A. pretiosum ATCC31280 was spread on a solid YMG (0.4% yeast extract, 1% malt extract, 0.4% glucose, 1.5% agar) plate for activation and cultured at 30°C for 3 days. 2The mycelia were cultured in 10.3% TSBY medium (3% tryptone soy broth, 0.5% yeast extract, 10.3% sucrose) at 30° C. for 16 h. 1 mL of mycelia were collected by centrifugation, washed twice with antibiotic-free LB, and resuspended in 500 μL of LB.

[0060] The mycelia resuspended in LB were mixed with E. coli in a ratio of 1:1 and evenly spread on a plate containing 10 mM Mg 2+ Incubate the cells on YMG plates at 37°C, inverted. After 12 hours, cover with sterile water containing 2 mg of nalidixic acid and 2 mg of apramycin. Air dry the plates and incubate them inverted at 30°C until zygotes appear.

[0061] Example 6

[0062] Screening of positive clones

[0063] Streak the conjugate onto YMG plates containing nalidixic acid and apramycin. Once a single colony emerges, select it and culture it in TSBY medium supplemented with apramycin. Two days later, PCR verification is performed using this bacterial suspension, an ET strain transformed with the recombinant plasmid as a positive control, and the wild-type A. pretiosum ATCC31280 genome as a negative control. Positive clones capable of amplifying a 3.5 kb fragment are screened using Vazyme, 2* Rapid Taq Master Mix (Novozymes) and primers FP-2 and RP-2.

[0064] The configuration method of the verification PCR system is as follows:

[0065] Table 4

[0066] PCR amplification conditions were as follows: pre-denaturation at 95°C for 10 min, 30 cycles of denaturation at 95°C for 30 s, annealing at 65°C for 30 s, and extension at 72°C for 2 min, followed by extension at 72°C for 10 min. Band accuracy was verified by agarose gel electrophoresis.

[0067] Example 7

[0068] Ansamitocin fermentation

[0069] Under normal conditions, Actinomyces preciousi and its mutants are cultured in TSBY liquid medium or YMG solid medium at 30°C. During fermentation, the mycelium stored in a 20% glycerol tube is first activated in YMG solid medium supplemented with 10.3% sucrose and cultured at 30°C for 48 hours. Then, a 1 cm 2The mycelium was inoculated into S1 seed medium and cultured at 30°C, 220 rpm for 24 h; 1.5 mL of the culture was transferred into S2 seed medium and cultured at 30°C, 220 rpm for 24 h; 3 mL (10%) of the culture was transferred into fermentation medium and cultured at 25°C, 220 rpm for 10 days.

[0070] The formula of S1 seed medium is ( / L): 30 g tryptone soy broth, 5 g yeast extract, 103 g sucrose, pH 7.5. Dispense into 250 mL Erlenmeyer flasks, 30 mL per bottle.

[0071] The formula of S2 seed medium is ( / L): 30 g of tryptone soy broth, 8 g of yeast extract, 103 g of sucrose, 500 μL / L of isopropanol, 500 μL / L of isobutanol, pH 7.5. Dispense 30 mL into 250 mL Erlenmeyer flasks.

[0072] The fermentation medium formula is ( / L): yeast extract 16 g, malt extract 10 g, sucrose 103 g, isopropanol 12 mL / L, isobutanol 5 mL / L, MgCl2 2 mM, valine 40 mM, pH 7.5. Dispense into 250 mL Erlenmeyer flasks, 30 mL per bottle.

[0073] Example 8

[0074] Ansamitocin detection and quantification

[0075] The content of ansamitocin was determined as follows: ansamitocin fermentation broth was mixed with 2 volumes of methanol, ultrasonicated for 30 minutes, 1 mL was taken and centrifuged at 12,000 rpm for 1 minute, and the supernatant was filtered through a 0.22 μM filter membrane and directly analyzed by HPLC.

[0076] The HPLC method for determining ansamitocin content was as follows: an Agilent 1260 (Agilent, USA) instrument was used, using an Agilent Eclipse Plus C18 column (4.6×250 mm, 5 μm). The mobile phase ratio was 45% A (0.5% formic acid in water): 55% B (acetonitrile). The analysis time for each sample was 10 minutes. The detector was an ultraviolet detector set at a wavelength of 254 nm. The results are shown in Figure 2.

[0077] Ansamitocin production changes after expressing the gene encoding the cell division protein FtsH. The empty vector control represents the fermentation yield of an A. pretiosum strain transformed with a backbone vector devoid of genes. The FtsH gene represents the fermentation yield of an A. pretiosum strain transformed with the gene encoding the cell division protein FtsH. Figure 2 shows that the strain expressing the gene encoding the cell division protein FtsH exhibits higher ansamitocin production than the control.

[0078] The applicant declares that the present invention uses the above-mentioned embodiments to illustrate the engineered bacteria for producing ansamycin, its construction method, and its application. However, the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent replacements for various raw materials in the products of the present invention, addition of auxiliary ingredients, and selection of specific methods, etc., are all within the scope of protection and disclosure of the present invention.

[0079] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the scope of protection of the present invention.

[0080] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any appropriate manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. An engineered bacterium for producing ansamitocin, characterized in that, The FtsH protein is overexpressed in the engineered bacterium.

2. The engineered bacterium for producing ansamitocin according to claim 1, characterized in that, The amino acid sequence of the FtsH protein is as shown in SEQ ID NO:

1.

3. A method for constructing an engineered bacterium for producing ansamitocin, characterized in that, The construction method includes: (1) Performing PCR using the FtsH gene as a template to obtain a PCR product; (2) After the vector is digested with restriction endonucleases, ligating it with the PCR product, transferring it into competent cells, performing sequence determination and extracting the plasmid to obtain a recombinant vector; (3) Transferring the recombinant vector into a conjugation transfer strain; (4) Mixing the strain containing the recombinant vector with the mycelium of the recipient bacterium, culturing, and screening for positive conjugants to obtain the product.

4. The method for constructing an engineered bacterium for producing ansamitocin according to claim 3, characterized in that, The source of the FtsH gene includes Actinosynnema pretiosum ATCC 31280.

5. The method for constructing an engineered bacterium for producing ansamitocin according to claim 3 or 4, characterized in that, The vector is pLQ646 containing a strong promoter; Preferably, the strong promoter includes kasOp.

6. The method for constructing an engineered bacterium for producing ansamitocin according to any one of claims 3-5, characterized in that, The conjugation transfer strain includes Escherichia coli ET12567 / pUZ8002.

7. The method for constructing an engineered bacterium for producing ansamitocin according to any one of claims 3-6, characterized in that, The restriction endonucleases include NdeI and EcoRI.

8. The method for constructing an engineered bacterium for producing ansamitocin according to any one of claims 3-7, characterized in that, The recipient bacterium includes Actinosynnema pretiosum ATCC 31280; Preferably, the culturing time in step (4) is 3 - 7 days.

9. Use of the engineered bacterium for producing ansamitocin according to claim 1 or 2 in the production of ansamitocin.

10. A method for producing ansamitocin, characterized in that, The production method includes: fermenting using the engineered bacterium for producing ansamitocin described in claim 1 or 2.

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