A ge002724 gene and its use in the synthesis of dihydro-ubiquinone

Abstract

This invention relates to the field of biotechnology, and discloses a... GE002724 Genes and their application in the synthesis of dihydroouabain. This invention utilizes homologous recombination technology to knock out Enterobacter hominis (…). Enterobacter hormaechei of GE002724 Gene analysis yielded the *Enterobacter holmie* mutant ge002724. Compared to wild-type *Enterobacter holmie*, the mutant ge002724 showed a significantly reduced synthesis of dihydroouabain, indicating... GE002724 The gene positively regulates the ability of *Enterobacter holmide* to synthesize dihydroouabain; expression of this gene in *E. coli* showed that *E. coli* transformed with this gene could synthesize dihydroouabain, further demonstrating... GE002724 GE002724 This invention mediates the synthesis of dihydroouabain. It facilitates the screening of strains capable of synthesizing dihydroouabain and the preparation of engineered bacteria that synthesize dihydroouabain.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a... GE002724 Genes and their application in the synthesis of dihydroouabain. Background Technology

[0002] Dihydroouabain (DHO) is a cardiac glycoside that enhances myocardial contractility and affects myocardial electrophysiological properties. It is primarily found in mammals and plants in nature. Traditional theory suggests that the positive inotropic effect of cardiac glycosides is closely related to their inhibition of the Na / K pump, thus cardiac glycosides are generally recognized as Na / K pump inhibitors. Studies have shown that nanomolar concentrations of DHO, during excitotoxic injury in rats, can inhibit Na+ / K+ pump activity. + -Ca 2+ Ion exchangers (NCX) accelerate the Ca2+ of neurons. 2+ Excretion, thereby maintaining intracellular calcium 2+ Concentration stability.

[0003] Currently, the synthesis of dihydroouabain is mainly achieved through chemical reduction. Specifically, it involves selective hydrogenation of the lactone ring double bond in the molecular structure of naturally extracted ouabain as a precursor. In laboratory or industrial production, this process typically uses palladium on carbon (Pd / C) as a catalyst, and the catalytic hydrogenation reaction is carried out under mild hydrogen pressure to obtain dihydroouabain. Although this compound has significant scientific research value, its source is highly dependent on the semi-synthetic modification of the natural product ouabain. Because the synthetic steps involve complex stereoselectivity control, and enzyme catalysis has extremely high regioselectivity and stereoselectivity, it can precisely convert ouabain into the target product dihydroouabain, greatly reducing side reactions and simplifying subsequent purification.

[0004] Current research has found that bacteria can synthesize DHO, and E. hormaecheiThese bacteria are commonly found in the intestines of lepidopteran pests and also exhibit DHO synthesis capabilities. Investigating key genes involved in DHO synthesis in these bacteria can provide a foundation for subsequent DHO production through bacterial fermentation. Homologous recombination technology is one of the earliest and most important methods applied in gene editing. Its advantage lies in its ability to achieve precise gene modification, particularly suitable for the insertion, replacement, or introduction of large DNA fragments or specific point mutations. The principle of this technology is based on the intracellular homologous recombination repair mechanism. Using homologous arms carried on a vector and the inserted fragment in the middle as a repair template, when cellular DNA breaks, the cell's own homologous recombination repair mechanism uses the introduced exogenous homologous fragment as a template to initiate a precise repair process. This integrates pre-designed mutations, marker genes, or exogenous sequences into specific sites in the genome, achieving the knockout, knock-in, or replacement of the target gene. Homologous recombination technology can not only be used for gene knockout but also for the precise insertion of target genes into the genome, achieving stable and heritable overexpression. Homologous recombination technology can rapidly produce safe and stable heritable overexpression mutants, which can be applied to optimize conditions in the bacterial fermentation process for DHO synthesis. Summary of the Invention

[0005] The purpose of this invention is to solve the technical difficulties in the traditional synthesis of dihydroouabain, and to provide... GE002724 Genes, the ones mentioned GE002724 The nucleotide sequence of the gene is as shown in SEQ ID NO:1 or a nucleotide sequence that encodes a protein as shown in SEQ ID NO:2. GE00272 Application of 4 genes in the synthesis of dihydroouabain.

[0006] To achieve the above objectives, the present invention provides the following technical solution: First, a knockout strain was constructed. GE00272 The four-gene Enterobacter holmie mutant was named Enterobacter holmie mutant ge002724, and then a strain was constructed. GE002724 Two strains of *E. coli* expressing the gene were cultured, and the results showed that dihydroouabain was not detected in the fermentation broth of the *Enterobacter holmie* mutant strain ge002724. The *Enterobacter holmie* mutant strain ge002724 and... GE002724 Dihydroouabain was detected in the co-culture fermentation broth of gene-expressing *E. coli* strains, but not in strains that were not transformed with the gene. GE002724 Dihydroouabain was also not detected in the fermentation broth of *E. coli* containing the gene, thus proving... GE002724 Application of genes in the synthesis of dihydroouabain.

[0007] Beneficial effects: (1) First discovery GE002724The application of the gene in the synthesis of dihydroouabain: By transferring the gene into bacteria, engineered bacteria capable of synthesizing dihydroouabain can be obtained, providing new ideas and directions for the synthesis of dihydroouabain.

[0008] (2) This invention is helpful for screening strains that can synthesize dihydroouabain, providing a basis for screening high-yielding dihydroouabain strains in the later stage. Attached Figure Description

[0009] Figure 1 Enterobacter hopterus GE002724 The results of 1% agarose gel electrophoresis detection of upstream and downstream homologous arms of the gene, where M is a 2000bp DNA marker.

[0010] Figure 2 This is an agarose gel electrophoresis image of the fragment used for amplification and knockout of the 2724-UD-pUC19 recombinant plasmid, where M is a 2000bp DNA marker.

[0011] Figure 3 for GE002724 PCR identification of gene knockout strains, where M is a 2000bp DNA marker, and 1-5 are randomly selected positive clones.

[0012] Figure 4 for GE002724 The identification results of gene knockout strains and wild-type strains, where 1 and 3 in the figure indicate... GE002724 Gene knockout Enterobacter holmie amplification products; 2 and 4 refer to the amplification products of the original Enterobacter holmie strain; 1 and 2 are amplification products using 2724-JD-F / R; 3 and 4 are amplification products using 2724-ter-F / R.

[0013] Figure 5 In Example 2 GE002724 Agarose gel electrophoresis image of the PCR product of the gene, where M is a 5000bp DNA marker.

[0014] Figure 6 The results of bacterial culture PCR identification in Example 2 are shown, where M is a 5000bp DNA Marker and 1-8 are randomly selected positive clones.

[0015] Figure 7 The image shows the double enzyme digestion electrophoresis detection of the recombinant plasmid obtained in Example 2, where M is a 12000bp DNA marker.

[0016] Figure 8Figure 1 shows the detection results of dihydroouabain in the four fermentation broths in Example 3; Figure 2 shows the detection results of dihydroouabain in the fermentation broth of wild-type Enterobacter holmie; Figure 3 shows the detection results of dihydroouabain in the fermentation broth of Enterobacter holmie mutant ge002724; Figure 4 shows the detection results of dihydroouabain in the fermentation broth of BL21(DE3) competent cells; Figure 5 shows the detection results of dihydroouabain in the fermentation broth of the in vitro expression strain and Enterobacter holmie mutant ge002724 co-cultured. Detailed Implementation

[0017] The specific embodiments of the invention will be further described below with reference to the accompanying drawings, but the invention is not limited to the following embodiments. Modifications, combinations, or substitutions made to the invention within its scope or without departing from its content, spirit, and scope are obvious to those skilled in the art and are included within the scope of the invention. Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by those skilled in the art to which this application pertains.

[0018] The Enterobacter hominis used in the examples ( Enterobacter hormaechei This laboratory obtained information from the tomato leafminer ( Tuta absoluta A strain capable of synthesizing dihydroouabain was isolated from the intestine and deposited on April 16, 2026, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC); the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou; the accession number is GDMCC No: 68091; the taxonomic name is... Enterobacter hormaechei .

[0019] Example 1 GE002724 The specific steps for gene knockout are as follows: (1) Enterobacter cholerae ( Enterobacter hormaechei Using DNA as a template, the gene was amplified using primers 2724-19-F (primer sequence shown in SEQ ID NO:3) / 2724-up-R (primer sequence shown in SEQ ID NO:4) and 2724-down-F (primer sequence shown in SEQ ID NO:5) / 2724-19-R (primer sequence shown in SEQ ID NO:6). GE002724 The upstream and downstream homologous arm sequences used for knockout. The reaction system was prepared using 2×Super pfu PCR mix (Hangzhou Baosai Biotechnology Co., Ltd.) as shown in Table 1, and the reaction procedure is shown in Table 2.

[0020] Table 1 Amplification System Table 2 PCR reaction procedure The amplification products were detected by 1% agarose gel electrophoresis, and the results are as follows: Figure 1 As shown in the figure, the amplification yielded approximately 800 bp. GE002724 Upstream and downstream homologous arms of a gene.

[0021] (2) The amplified products were purified using a PCR product purification kit (Hangzhou Baosai Company). For specific operation steps, refer to the kit instructions. The purified upstream and downstream homologous arms were seamlessly cloned and ligated according to the volumes in Table 3. The upstream and downstream homologous arms were then ligated to the linearized pUC19 vector digested with EcoRI and HindIII.

[0022] Table 3 Seamless Cloning Connection System After mixing the seamless cloning ligation system, incubate it on ice for 30 minutes to obtain the recombinant plasmid. Transform the recombinant plasmid. E. coli DH5α competent cells (Chongqing Xuanzun Biotechnology Co., Ltd.) were plated on Amp-IPTG-Xgal plates and cultured at 37°C. White spot clones were then picked and sequenced at Chongqing Life Knowledge Biotechnology Co., Ltd. Sequencing results confirmed that the full-length inserted fragment was correct and that the upstream and downstream ligations were consistent with the design. The clone was determined to be correct and the recombinant plasmid with correct sequencing was named 2724-UD-pUC19.

[0023] (3) Using primers 2724-H73sacb-F (sequence shown in SEQ ID NO:7) / R (sequence shown in SEQ ID NO:8) as a template, the correctly sequenced 2724-UD-pUC19 plasmid was amplified and constructed. GE002724 The gene knockout fragments and amplification systems are shown in Table 4, and the amplification procedures are shown in Table 5.

[0024] Table 4 Amplification System Table 5 Amplification Procedure The amplification products were detected by 1% agarose gel electrophoresis, and the results are as follows: Figure 2 As shown in the figure, the amplification yielded a product of approximately 1500 bp.

[0025] The amplified products were purified using a PCR product purification kit (Hangzhou Baosai Company). For specific operating procedures, please refer to the kit instructions. After purification, the products were ligated into a linearized vector of pH73sacb digested with smaI according to the volume in Table 6.

[0026] Table 6 Seamless Cloning Connection System The seamless cloning ligation system was mixed thoroughly and placed on ice for 30 minutes to obtain the ligation product. The ligation product was then chemically transformed. E. coli S17 λpir competent cells (Chongqing Xuanzun Biotechnology Co., Ltd.) were plated on chloramphenicol resistance plates (34 μg / mL) and cultured at 37℃. Single clones were picked for identification. The single clones were mixed into 10 μL of sterile water and 0.5 μL was used as template for amplification. The amplification system is shown in Table 7, and the amplification program is shown in Table 8.

[0027] Table 7 Amplification System Table 8 Amplification Procedure The amplification products were detected by 1% agarose gel electrophoresis, and the results are as follows: Figure 3 As shown in the figure, a band of approximately 750 bp was amplified.

[0028] Positive clones were sent to Chongqing Life Source Biotechnology Co., Ltd. for sequencing. The sequencing results were compared with the expected theoretical spliced ​​sequence. The sequencing results should include the upstream and downstream homologous arms and the sequences flanking the vector insertion site. The correctly sequenced recombinant plasmid was named 2724-UD-H73sacb. *Enterobacter holmieae* strain and single clones of 2724-UD-H73sacb were picked and inoculated into LB liquid medium and LB (chloramphenicol resistant) liquid medium, respectively, until OD... 600When the bacterial culture reaches approximately 0.8 μg / mL, conjugation transfer experiments are performed: 700 μL each of *Enterobacter holmieae* and bacterial culture containing the 2724-UD-H73sacb plasmid are mixed, centrifuged, and the bacterial cells are spotted onto the center of a sterile nitrocellulose filter membrane. The membrane is then incubated overnight on LB agar. The filter membrane is removed with sterile forceps, washed with 1 mL of LB agar, diluted, and spread onto LB agar plates containing ampicillin and chloramphenicol (ampicillin concentration 100 μg / mL, chloramphenicol concentration 34 μg / mL). Single-exchange strains identified as positive by the double-antibiotic plate assay are inoculated into LB agar containing 10% sucrose. Patching is performed on plates with low chloramphenicol resistance for identification. Finally, positive clones and wild-type strains are screened. Primers 2724-JD-F / R (primer sequences shown in SEQ ID NO:9 and SEQ ID NO:10) and 2724-ter-F / R (primer sequences shown in SEQ ID NO:11 and SEQ ID NO:10) are used. (as shown in NO:12) Bacterial culture PCR amplification and identification were performed. The amplification system is shown in Table 7, the amplification program is shown in Table 8, and the identification was performed as follows: Figure 4 As shown in the figure, it can be seen that when amplifying with 2724-JD-F / R, knockout GE002724 The PCR product of the gene-infected *Enterobacter holmieae* was 761 bp, while the PCR product of the original *Enterobacter holmieae* was 1766 bp; when amplified using 2724-ter-F / R, the knockout gene... GE002724 The gene of Enterobacter holmie could not be amplified. The PCR product of the original Enterobacter holmie was 962bp. The successfully knocked-out strain was named Enterobacter holmie mutant ge002724.

[0029] Example 2 GE002724 The construction steps for gene expression strains are as follows: (1) Total RNA extraction kit (Guangzhou Meiji Biotechnology Co., Ltd., R4130-02) was used to extract Enterobacter holmium (Cholobacterium hominis). Enterobacter hormaechei Total RNA in ), of which Enterobacter hopterii ( Enterobacter hormaechei This laboratory obtained information from the tomato leafminer ( Tuta absoluta The total RNA was isolated from the intestine and reverse transcribed into cDNA according to the instructions of HiScript II Q RTSuperMix for qPCR (+gDNA wiper) (Novizan, R223-01). (2) Using the cDNA prepared in step (1) as a template, a PCR reaction was performed. The upstream and downstream primers were respectively equipped with... Nde I and Xho I restriction site, where GE002724 -F (upstream primer) is shown in SEQ ID NO:13. GE002724-R (downstream primer) is shown in SEQ ID NO:14, the PCR reaction system is shown in Table 9, the PCR reaction procedure is shown in Table 10, and the synthesized product is obtained after PCR.

[0030] Table 9 PCR Reaction System Table 10 PCR reaction procedure After the reaction, the product size was determined by 1% (w / v) agarose gel electrophoresis. The electrophoresis image is shown below. Figure 5 As shown, gene bands of the appropriate size (approximately 1000 bp) were recovered according to the instructions of the gel recovery kit (Anhui Global Gene Technology Co., Ltd., TRS0701-100) to obtain the gel recovery product of the target gene.

[0031] (3) The pET-22b(+) vector was double-digested. The digestion system is shown in Table 11.

[0032] Table 11 (4) The gel-recovered product of the target gene obtained by PCR amplification was assembled into the NdeI--XhoI site of the pET-22b(+) vector by recombination ligation to obtain a product containing the target gene. GE002724 The full-length recombinant plasmid and its specific ligation system are shown in Table 12 below.

[0033] Table 12 Connection System (5) Transformation method of ligation product: Take 10 μL of the recombinant plasmid obtained in step (4) and add it to 100 μL of Top10 competent cells (General Biotechnology (Anhui) Co., Ltd.). Gently shake and rotate to ensure full contact between the product and the cells. Place on ice for 10 min; incubate in a 42℃ water bath for 90 s without shaking; place on ice for 3 min; add 500 μL of LB medium pre-warmed to 37℃ to each tube, and incubate in a constant temperature shaker at 37℃ and 200 rpm for 40 min. After recovery, centrifuge at 6000 rpm for 2 min, discard the supernatant, mix the remaining bacterial solution in the centrifuge tube thoroughly, and spread it all onto LB plates resistant to Amp (100 μg / mL). Incubate overnight at 37℃; remove the plates the next day and observe to confirm the colony growth; pick 8 single colonies from the plate and perform colony PCR identification while shaking the bacteria at 37℃ and 250 rpm; the identification primer sequences were synthesized by General Biotechnology (Anhui) Co., Ltd., such as SEQ ID NO:15 and SEQ ID NO:15. As shown in NO:16; the colony PCR reaction used a 30μL system: 1μL each primer, 6μL polymerase buffer, 0.6μL 10mM dNTP, 0.6μL polymerase, and 21.8μL ddH2O; cycling parameters: 96℃ pre-denaturation for 3 min; 95℃ for 30 s; 58℃ for 30 s; 72℃ for 50 s; 23 cycles, with a final extension at 72℃ for 1 min; the amplified products were subjected to electrophoresis, and after PCR amplification, a fragment of approximately 1000bp was obtained, consistent with the predicted fragment size of 1071bp. The amplification results of the sample are shown in [Figure number missing]. Figure 6 .

[0034] (6) Plasmids were extracted from the obtained positive bacterial culture using a plasmid extraction kit (Anhui Global Gene Technology Co., Ltd.), and sent to Anhui Global Gene Technology Co., Ltd. for sequencing. The recombinant plasmids that were correctly sequenced were digested with ApaI-XhoI. The digestion system is shown in Table 13. The digestion products were subjected to gel electrophoresis, and the results are shown in Table 13. Figure 7 As shown, the size is consistent with the expected 2043 / 4326, and the correct enzyme digestion result indicates the presence of... GE002724 Successful acquisition of recombinant plasmids for the gene.

[0035] Table 13 shows the enzyme digestion system as follows: Utilizing GE002724 The recombinant plasmid of the gene and the transformation method of the ligation product were described. The recombinant plasmid was transformed into BL21(DE3) competent cells, plated, and incubated overnight at 37°C inverted position. Single clones were selected and cultured in LB medium at 37°C until the bacterial OD600 reached 0.6-0.8, obtaining cells containing the gene. GE002724 BL21 (DE3) cells of the gene.

[0036] Example 3 The specific steps for detecting the synthetic capacity of dihydroouabain are as follows: (1) The *Enterobacter holmieae* mutant ge002724 obtained in Example 1, the in vitro expression strain obtained in Example 2, BL21(DE3) competent cells, and wild-type *Enterobacter holmieae* (not knocked out) were combined. GE002724 Enterobacter hominis (genes) Enterobacter hormaechei The cells were plated, and single colonies of wild-type *Enterobacter holmieae*, *Enterobacter holmieae* mutant ge002724, and BL21(DE3) competent cells were picked and added to LB medium. The in vitro expression strain and *Enterobacter holmieae* mutant ge002724 were co-cultured at 37°C and 200 rpm for 16 h to obtain a stock solution. The stock solution was inoculated into LB medium at a 1:100 volume ratio and cultured at 37°C until OD500. 600 =0.8, and centrifuged at 4℃ and 14000rpm for 4 min. The supernatant was filtered through a 0.22μm filter membrane and analyzed by ultra-high performance liquid chromatography-tandem mass spectrometry. Each supernatant was performed in triplicate. The specific UPLC-MS conditions were as follows: Chromatographic separation conditions: Column temperature: 40°C, flow rate: 0.3 mL / min; Sample inlet temperature: 10℃; Mobile phase composition: A - 0.1% formic acid in water; B - acetonitrile; Elution procedure: isocratic elution.

[0037] MSMS conditions: Curtain gas: 35 psi; Collision gas: 7 psi; IonSpray voltage: +5500V; Ion mode: ESI+ ion mode; Ion source temperature: 450°C; (Ion Source Gas) 1: 40 psi; (Ion Source Gas) 2: 50 psi.

[0038] Test results: See results Figure 8 Dihydroouabain can be detected in wild-type Enterobacter hopterii. Figure 8 In (a) to (c) of the *Enterobacter holmie* mutant ge002724, dihydroouabain was not detected in the fermentation broth. Figure 8 Dihydroouabain was not detected in the fermentation broth of BL21(DE3) competent cells (d~f); Figure 8 Dihydroouabain was detected in the fermentation broth of the in vitro expression strain and the Enterobacter holmium mutant ge002724. Figure 8 (j) ~ (l)).

[0039] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A kind GE002724 Genes are characterized by: The GE002724 The nucleotide sequence of the gene is as shown in SEQ ID NO.1 or the nucleotide sequence that encodes the protein shown in SEQ ID NO.

2.

2. The claim 1 GE002724 Application of genes in the synthesis of dihydroouabain.

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