Use of enterobacter hormaechei in the synthesis of dihydrovoacangine

Abstract

This invention discloses the application of *Enterobacter holmieae* in the synthesis of dihydroouabain, belonging to the field of microbial technology. The dihydroouabain synthesized by *Enterobacter holmieae* in this invention, due to its unique biological characteristics, has broad application prospects in cardiovascular disease treatment, neuroscience research, energy metabolism, and circadian rhythm regulation. The *Enterobacter holmieae* strain described in this invention is preserved under the name *Enterobacter holmieae*. Enterobacter hormaechei Deposited at Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou; Deposit date: April 16, 2026; Accession number: GDMCC No:68091.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to the application of Enterobacter holmie in the synthesis of dihydroouabain. Background Technology

[0002] Dihydroouabain (DHO) is a natural steroidal saponin in the cardiac glycoside family. Its structure is highly similar to that of the classic cardiac glycoside ouabain, the difference being the saturation modification of the C2-C3 double bond. This slight structural change endows it with a unique pharmacological activity spectrum. Unlike the widely studied digitalisin, dihydroouabain has differentiated binding affinity for different isoforms of Na⁺-K⁺-ATPase, making it an important tool molecule for studying the function of this enzyme. Current attention is mainly focused on its dual regulatory role: on the one hand, as a potential biomarker for cardiovascular diseases (endogenous digitalis-like factor hypothesis); on the other hand, it can activate cell proliferation signaling pathways at low concentrations, while inducing different forms of cell death at high concentrations. Notably, in recent years, its application in the field of tumor radiotherapy has made initial progress—by inhibiting key signals for DNA damage repair, dihydroouabain has been shown to improve the radiotherapy response rate of some solid tumors (such as cervical cancer).

[0003] However, the natural sources of this compound are extremely limited: it is only detected in trace amounts (ng / g level) in specific plants (such as the African arrow poison tree) and mammalian tissues. The total chemical synthesis route is long and the chiral control is difficult. Currently, it is still mainly based on semi-synthesis or biological extraction, which is difficult to meet the needs of drug development. Therefore, constructing efficient heterologous biosynthetic systems or developing analogs with simplified structures but retained activities have become the two main breakthrough directions of current research. Summary of the Invention

[0004] To solve or partially solve the problems existing in related technologies, the primary objective of this invention is to provide a *Enterobacter holmieae* bacterium, wherein the *Enterobacter holmieae* bacterium is *Enterobacter holmieae*. Enterobacter hormaechei It was deposited on April 16, 2026, at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC No: 68091), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. The proposed classification name is Enterobacter hopterii. Enterobacter hormaechei .

[0005] Another object of the present invention is to provide the aforementioned Enterobacter hominis. Enterobacter hormaechei Its application in the synthesis of dihydroouabain involves the following specific steps: (1) Inoculate Enterobacter holmie into the culture medium to obtain Enterobacter holmie seed liquid.

[0006] (2) The *Enterobacter holmiae* seed culture obtained in step (1) was inoculated into the culture medium and cultured by shaking to obtain a supernatant containing dihydroouabain.

[0007] Preferably, in step (1) of the present invention, the OD of the Enterobacter holmium seed culture is... 600 =0.8~1.0.

[0008] Preferably, in step (2) of the present invention, the amount of Enterobacter holmie seed culture in the culture medium is 1 to 5% of the culture medium volume.

[0009] Preferably, the culture medium in steps (1) and (2) of the present invention is liquid LB culture medium.

[0010] The beneficial effects of this invention are: (1) This invention is the first to isolate a strain of Enterobacter holmia that can produce dihydroouabain, opening up a new route for the synthesis of dihydroouabain.

[0011] (2) Enterobacter holmie strains are easy to culture, grow rapidly, and are easy to cultivate and reproduce in large quantities indoors. Therefore, the synthesis of dihydroouabain using Enterobacter holmie has the advantages of being convenient, efficient and simple. Attached Figure Description

[0012] Figure 1 The Enterobacter cholerae in Example 1 of this invention Enterobacter hormaechei Phylogenetic tree of L11.

[0013] Figure 2 The Enterobacter cholerae in Example 1 of this invention Enterobacter hormaechei Colony morphology diagram of L11.

[0014] Figure 3 This is the standard curve and mass spectrum of the content and area of ​​dihydroouabain standard in Example 2 of the present invention; wherein Figure 3 (a) is the standard curve of dihydroouabain standard content versus peak area. Figure 3 (b) is the mass spectrum of dihydroouabain standard.

[0015] Figure 4 This is a mass spectrum of the dihydroouabain content detection in the culture medium supernatant in Example 2 of the present invention, wherein... Figure 4 (a) Does not contain Enterobacter holmie Enterobacter hormaechei Mass spectrum of L11 medium supernatant Figure 4 (b) is Enterobacter hopterii. Enterobacter hormaechei Mass spectrum of L11 medium supernatant.

[0016] Figure 5 This is a comparison of the dihydroouabain content in the culture medium supernatant under different treatments in Example 2 of the present invention.

[0017] Figure 6 The common Enterobacter cholerae and Enterobacter cholerae in Example 5 of this invention Enterobacter hormaechei Comparison of dihydroouabain content in the supernatant of L11 culture medium. Detailed Implementation

[0018] The present invention will be further described in detail below with reference to specific embodiments and accompanying drawings, but the scope of protection of the present invention is not limited to the content described therein; unless otherwise specified, all reagents used in the present invention are commercially available analytical grade reagents, and all raw materials used can be purchased through commercial channels.

[0019] The culture medium used in this invention: Solid LB medium: 5g yeast extract, 10g peptone, 5g NaCl and 15-20g agar, bring the volume to 1L with water, adjust the pH to 7.4-7.6, and autoclave at 121℃ for 30 minutes.

[0020] Liquid LB medium: 5g yeast extract, 10g peptone and 5g NaCl, bring to a final volume of 1L with water, adjust pH to 7.4~7.6, and autoclave at 121℃ for 30 minutes. Example 1

[0021] Enterobacter hopterii Enterobacter hormaechei Isolation and identification of strains The Enterobacter holmium Enterobacter hormaechei It was isolated and purified from the midgut of tomato leafminer larvae, specifically including the following steps: (1) Collect healthy second-instar larvae of the tomato leafminer and freeze them immediately on ice.

[0022] (2) Disinfect the tomato leafminer larvae with 75% alcohol for 90 seconds, and finally rinse them with sterile water 3 times, 1 minute each time.

[0023] (3) After cleaning, place it in a sterile culture dish, use a sterile insect needle to remove the intestine from the tail of the tomato leafminer larva, and put the intestine into a 1.5 mL sterile centrifuge tube. The sterile centrifuge tube contains 1 mL of sterile phosphate buffered saline (1×PBS, pH 7.4). After mixing by pipetting, the intestinal flora is released into the PBS to obtain a PBS suspension.

[0024] (4) Use a sterile pipette tip to draw up the PBS suspension and drop it onto a plate containing solid LB medium. Inoculate the plate using the plate spreading method and incubate the plate in a 30°C incubator for 72 hours.

[0025] (5) Use a sterile pipette tip to pick up single colonies from the plates after 72 hours of incubation, streak them on fresh solid LB medium plates, and incubate them in a 30°C incubator for 72 hours for the first purification. Continue to use a sterile pipette tip to pick up single colonies from the plates after the first purification, streak them on fresh solid LB medium plates, and incubate them in a 30°C incubator for 72 hours for the second purification. After the second purification, all colonies on the same plate have the same morphology. Use the glycerol cryopreservation method to preserve the pure cultured bacterial strains for further molecular and morphological identification.

[0026] (6) Pick a single colony from the plate after the second purification in step (5), place it in 1 mL of liquid LB medium, and culture at 30°C and 200 rpm for 12 h to obtain bacterial culture. Send the culture to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The upstream primer used for sequencing is shown in SEQ ID NO: 1, and the downstream primer is shown in SEQ ID NO: 2. The 16S rRNA gene was amplified and sequenced. The sequencing results were spliced ​​using Contigexpress software to obtain the sequence of the isolated strain. The sequence was compared with the GenBank database to identify the isolated strain. A phylogenetic tree was constructed based on the sequence of the isolated strain using MEGA6.5 and the neighbor-joining method, as shown in the figure. Figure 1 As shown, a strain of Enterobacter hopterii was isolated. Enterobacter hormaechei The strain, numbered Enterobacter hopterii Enterobacter hormaechei L11.

[0027] (7) The Enterobacter holmium isolated and preserved with glycerol in step (5) Enterobacter hormaechei L11 strain was streaked on solid LB medium and incubated at 30°C for 24 hours. The results are as follows: Figure 2 As shown, the colony is round or oval, milky white, with a smooth, matte surface and opaque edges. Example 2

[0028] Enterobacter hopterii Enterobacter hormaechei Application of L11 in the synthesis of dihydroouabain (1) Take 50 μL of Enterobacter holmium preserved with glycerol in step (5) of Example 1. Enterobacter hormaechei L11 strain was inoculated into 1 mL of liquid LB medium and then placed in a shaker at 30℃ and 200 rpm for 12 h to produce OD. 600 =0.6 seed liquid.

[0029] (2) The seed culture was added to liquid LB medium at an inoculation amount of 5% of the medium volume (experimental group). The uninoculated LB medium was used as the control group. After shaking at 200 rpm and 30°C for 12 h, the medium inoculated with the seed culture became turbid and a mixed bacterial culture sample was obtained. 0.1 mL of the mixed bacterial culture sample was accurately transferred to a 2 mL centrifuge tube, and an equal volume of mass spectrometry grade methanol was added. The mixture was shaken at room temperature for 1 min and centrifuged at 13000 rpm / min for 20 min at 4°C. An appropriate amount of supernatant was filtered through a 0.22 μm filter membrane to obtain the sample test solution.

[0030] (3) Take 1 mL of the sample solution to detect the dihydroouabain content. The sample was analyzed by ultra-high performance liquid chromatography-tandem mass spectrometry (LC-MS) using a UPLC column (2.1×100 mm, 1.7 μm). The HPLC conditions were as follows: Analysis was performed in isocratic mode using a mobile phase consisting of mobile phase A (0.1% formic acid aqueous solution) and mobile phase B (mass spectrometry grade acetonitrile solution) in a volume ratio of 30:70; flow rate: 0.3 mL / min; time: 5 min; temperature: 40 °C; sample volume: 20 μL.

[0031] Under these conditions of liquid chromatography and tandem mass spectrometry, standard curves were plotted using dihydroouabain standards (98% purity) at different concentrations. The dihydroouabain concentration was plotted on the x-axis, and the peak area on the mass spectrum was plotted on the y-axis. The results are shown below. Figure 3 As shown in (a), the linear relationship is y = 8.65258 × x - 0.800439, R 2 =0.9919; Figure 3 (b) is the mass spectrum of dihydroouabain standard.

[0032] The sample solution was analyzed under the conditions of liquid chromatography and tandem mass spectrometry described above. The retention position of the target peak was confirmed by quality control experiments, and the peak area data was obtained. The content of dihydroouabain in the sample was calculated based on the standard curve and sample dilution factor. Each group was performed in triplicate. Results are shown below. Figure 4 (a) and Figure 4 As shown in (b), dihydroouabain was detected in the supernatant of the experimental group, and the peak area was significantly higher than that of the control group.

[0033] To prove that it is indeed Enterobacter hopterus. Enterobacter hormaechei L11 played a role in the synthesis of dihydroouabain. The content of dihydroouabain in the control group and the experimental group was compared, and the results are as follows: Figure 5 As shown, after 12 hours of culture, LC-MS detected a dihydroouabain content of 1466.57 ± 16.60 ng / mL in the supernatant of the experimental group, while no dihydroouabain was detected in the supernatant of the control group, demonstrating that the dihydroouabain synthesis capacity of Enterobacter holmie is limited. Enterobacter hormaechei The performance of L11.

[0034] The results showed that Enterobacter cholerae Enterobacter hormaechei After L11 seed culture was inoculated into LB liquid medium and cultured for 12 hours until it became turbid, Enterobacter holmie was detected. Enterobacter hormaechei The culture supernatant of L11 contained dihydroouabain. Example 3

[0035] (1) Take 50 μL of Enterobacter holmium preserved with glycerol in step (5) of Example 1. Enterobacter hormaechei L11 strain was inoculated into 1 mL of liquid LB medium and then placed in a shaker at 30℃ and 200 rpm for 12 h to produce OD. 600 =0.8 seed liquid.

[0036] (2) The seed culture was added to the liquid LB medium at an inoculation amount of 1% of the medium volume (experimental group). The uninoculated LB medium was used as the control group. After shaking at 200 rpm and 30°C for 12 h, the medium inoculated with the seed culture became turbid and a mixed bacterial culture sample was obtained. 0.1 mL of the mixed bacterial culture sample was accurately transferred to a 2 mL centrifuge tube, and an equal volume of mass spectrometry grade methanol was added. The mixture was shaken at room temperature for 1 min and centrifuged at 13000 rpm / min for 20 min at 4°C. An appropriate amount of supernatant was filtered through a 0.22 μm filter membrane to obtain the sample test solution.

[0037] (3) Take 1 mL of the sample solution to detect the dihydroouabain content. The sample was analyzed by ultra-high performance liquid chromatography-tandem mass spectrometry (LC-MS) using a UPLC column (2.1×100 mm, 1.7 μm). The HPLC conditions were as follows: Analysis was performed in isocratic mode using a mobile phase consisting of mobile phase A (0.1% formic acid aqueous solution) and mobile phase B (mass spectrometry grade acetonitrile solution) in a volume ratio of 30:70; flow rate: 0.3 mL / min; time: 5 min; temperature: 40 °C; sample volume: 20 μL.

[0038] The results showed that Enterobacter cholerae Enterobacter hormaechei After L11 seed culture was inoculated into LB liquid medium and cultured for 12 hours until it became turbid, Enterobacter holmie was detected. Enterobacter hormaechei The culture supernatant of L11 contained dihydroouabain. Example 4

[0039] (1) Take 50 μL of Enterobacter holmium preserved with glycerol in step (5) of Example 1. Enterobacter hormaechei L11 strain was inoculated into 1 mL of liquid LB medium and then placed in a shaker at 30℃ and 200 rpm for 12 h to produce OD.600 =1.0 seed liquid.

[0040] (2) The seed culture was added to liquid LB medium at an inoculation amount of 3% of the medium volume (experimental group). The uninoculated LB medium was used as the control group. After shaking at 200 rpm and 30°C for 12 h, the medium inoculated with the seed culture became turbid and a mixed bacterial culture sample was obtained. 0.1 mL of the mixed bacterial culture sample was accurately transferred to a 2 mL centrifuge tube, and an equal volume of mass spectrometry grade methanol was added. The mixture was shaken at room temperature for 1 min and centrifuged at 4°C and 13000 rpm / min for 20 min. An appropriate amount of supernatant was filtered through a 0.22 μm filter membrane to obtain the sample test solution.

[0041] (3) Take 1 mL of the sample solution to detect the dihydroouabain content. The sample was analyzed by ultra-high performance liquid chromatography-tandem mass spectrometry (LC-MS) using a UPLC column (2.1×100 mm, 1.7 μm). The HPLC conditions were as follows: Analysis was performed in isocratic mode using a mobile phase consisting of mobile phase A (0.1% formic acid aqueous solution) and mobile phase B (mass spectrometry grade acetonitrile solution) in a volume ratio of 30:70; flow rate: 0.3 mL / min; time: 5 min; temperature: 40 °C; sample volume: 20 μL.

[0042] The results showed that Enterobacter cholerae Enterobacter hormaechei After L11 seed culture was inoculated into LB liquid medium and cultured for 12 hours until it became turbid, Enterobacter holmie was detected. Enterobacter hormaechei The culture supernatant of L11 contained dihydroouabain. Example 5

[0043] Common Enterobacter holmium and Enterobacter holmium described in this invention Enterobacter hormaechei The comparison of L11 in the synthesis of dihydroouabain is as follows: (1) The method described in Example 2 was used to test common Enterobacter holmie (control group) and Enterobacter holmie described in this invention. Enterobacter hormaechei L11 (experimental group) was cultured to obtain culture medium.

[0044] (2) Centrifuge the obtained culture medium at 13000 rpm for 20 min, take the supernatant, and pass it through a 0.22 pm microporous membrane to obtain the sample test solution; perform three biological replicates for each type of bacteria.

[0045] (3) Take 1 mL of the sample solution to detect the dihydroouabain content. The sample was analyzed by ultra-high performance liquid chromatography-tandem mass spectrometry (LC-MS) using a UPLC column (2.1×100 mm, 1.7 μm). The HPLC conditions were as follows: The analysis was performed in isocratic mode using a mobile phase consisting of mobile phase A (0.1% formic acid aqueous solution) and mobile phase B (chromatographic grade acetonitrile solution) in a volume ratio of 30:70; flow rate: 0.3 mL / min; time: 5 min; temperature: 40 °C; sample volume: 20 μL.

[0046] Under these chromatographic conditions, the concentration of dihydroouabain in the test solution is quantified by comparing the peak area of ​​the test solution with that of the dihydroouabain standard. To demonstrate that this function is absent in common *Enterobacter holmieae*, a model strain of *Enterobacter holmieae* is used as a control. The content of dihydroouabain in the culture medium under different strain treatments is compared, such as... Figure 6 As shown, the results indicate that *Enterobacter holmium* Enterobacter hormaechei Strain L11 can synthesize dihydroouabain, while common Enterobacter hominis cannot.

[0047] 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 type of Enterobacter cholerae ( Enterobacter hormaechei The application of L11 in the synthesis of dihydroouabain is characterized by, The specific steps are as follows: (1) The Enterobacter holmie L11 was inoculated into a culture medium to obtain Enterobacter holmie L11 seed culture; (2) The Enterobacter holmium L11 seed culture obtained in step (1) was inoculated into the culture medium and cultured by shaking to obtain the supernatant containing dihydroouabain. The preservation number of the Enterobacter holmium L11 is GDMCC No: 68091.

2. The Enterobacter cholerae according to claim 1 ( Enterobacter hormaechei The application of L11 in the synthesis of dihydroouabain is characterized by, In step (1), the OD of the Enterobacter holmium L11 seed culture 600 =0.8~1.

0.

3. The Enterobacter cholerae according to claim 1 ( Enterobacter hormaechei The application of L11 in the synthesis of dihydroouabain is characterized by, In step (2), the amount of Enterobacter holmie L11 seed culture in the culture medium is 1 to 5% of the culture medium volume.

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