An abf3h enzyme and uses thereof

Abstract

The application belongs to the technical field of bioengineering, and particularly relates to an AbF3H enzyme and application thereof. Bioinformatics analysis finds that the enzyme belongs to an alpha-ketoglutarate-dependent dioxygenase family, has the potential of catalyzing synthesis of dihydromorin, and an amino acid sequence thereof is shown as SEQ ID No.: 1. Escherichia coli BL21 (DE3) is used as a host bacterium, a plasmid pACYCDuet-1 is used to construct an expression system of the enzyme, the recombinant strain can use naringenin as a substrate, and dihydromorin is synthesized by using a whole cell catalysis method with the aid of vitamin C, ferrous sulfate and alpha-ketoglutarate. The preservation number of the recombinant strain is CCTCC M 20251244. The content of the application enriches a flavanone 3-hydroxylase resource library, and lays a foundation for industrialized production of dihydromorin and other flavonoid compounds.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, and in particular relates to an AbF3H enzyme and its applications. Background Technology

[0002] Dihydrokaempferol, also known as aromadendrin, belongs to the dihydroflavonol class of compounds and is widely found in citrus fruits, Scutellaria baicalensis, and other plants. It possesses important physiological functions such as antioxidant activity, antibacterial activity, and improvement of insulin resistance induced by high glucose levels. Given the increasing market demand for dihydrokaempferol, there is an urgent need to establish efficient synthetic technologies. Currently, the synthesis of dihydrokaempferol mainly involves plant extraction and chemical synthesis. While plant extraction of dihydrokaempferol is simple and easy, it is time-consuming and prone to product loss. Methods for synthesizing dihydrokaempferol using methods such as Orwell's synthesis, chalcone oxidation cyclization, the Baker-Venkataraman method, and flavonoid DMDO (3,3-dimethyldiethylene oxide) oxidation suffer from poor stability, poor selectivity, and high preparation difficulty. Therefore, it is imperative to improve the synthetic efficiency of dihydrokaempferol by changing the reaction strategy. Compared with the two methods mentioned above, biosynthesis, such as whole-cell catalysis, has shown great potential in the field of fine chemical synthesis due to its advantages such as environmental friendliness, mild reaction conditions, and high catalytic efficiency.

[0003] The whole-cell catalytic synthesis of hesperidin can be achieved through the hydroxylation of naringenin catalyzed by flavanone-3-hydroxylase (F3H). Although F3H from different species belongs to the same α-ketoglutarate-dependent dioxygenase superfamily and shares a common evolutionary origin, their catalytic activities differ significantly. Currently, the F3H used for hesperidin synthesis is generally derived from plants, and its heterologous expression in microorganisms faces many limitations. For example, the commonly used industrial chassis cell *Escherichia coli* lacks a post-translational modification system specific to eukaryotes, which may lead to a decrease in the heterologous expression level of F3H in *E. coli*, thus affecting the synthesis efficiency of hesperidin. In addition to plant-derived enzymes, some proteins derived from plant endophytes can also be used for the synthesis of flavonoids. Yin et al. identified the NRPS-PKS hybrid enzyme FnsA from the plant endophyte *Pestalotiopsis fici* and demonstrated that it has the function of catalyzing the synthesis of naringenin, a key precursor of flavonoids. This may be a result of long-term evolution caused by plants and microorganisms living in similar environments. AbF3H originates from α-proteobacteria. Alphaproteobacteria This bacterium, sp., is commonly found in soil and may have acquired similar functions to plant-derived F3H through long-term evolution. Summary of the Invention

[0004] The purpose of this invention is to provide an AbF3H enzyme and its applications. Through bioinformatics analysis, this invention discovered that AbF3H, derived from α-proteobacteria sp., belongs to the α-ketoglutarate-dependent dioxygenase superfamily and has the potential to catalyze the synthesis of hesperidin from naringenin. Subsequently, a whole-cell catalytic system was established based on *Escherichia coli*, using naringenin as a substrate, and the catalytic function of AbF3H was tested under different temperature conditions. This study, combining bioinformatics and experimental verification, revealed the role of bacterial AbF3H in hesperidin synthesis, providing a new approach for establishing an efficient and stable whole-cell catalytic production process for hesperidin.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] An AbF3H enzyme derived from α-Proteus Alphaproteobacteria This enzyme belongs to the 2-ketoglutarate-dependent dioxygenase family, and its amino acid sequence is shown in SEQ ID No.1. The amino acid sequence contains conserved 2-ODD sites HxD (sites: 227-229) and RxS (sites: 294-296).

[0007] Furthermore, the AbF3H enzyme has an intrachain predominantly intra-chain interaction among its amino acid residues, with 223 hydrogen bonds, 18 π-π stacks, 6 π-cations, 8 ionic bonds, and 190 van der Waals forces.

[0008] Furthermore, the AbF3H enzyme has a docking free energy of -5.71 kJ / mol with naringenin molecules. This enzyme specifically binds to naringenin through hydrophobic interactions, hydrogen bonds, and π-cation interactions. Specifically, Ile224A, Leu236A, and Phe300A form hydrophobic interactions with naringenin molecules; Glu120A, Tyr210A, Asp229A, Asn226A, and Ser340A form hydrogen bonds with naringenin molecules; and Arg206A binds to naringenin through π-cation interactions.

[0009] A recombinant vector pACYCDuet-AbF3H is obtained by codon optimization and nucleic acid sequence synthesis using the amino acid sequence of the AbF3H enzyme and ligating it into the protein expression vector pACYCDuet-1.

[0010] A recombinant strain (Escherichia coli AbF3H) was used to introduce a recombinant vector into host cells. E. coliBL21 was obtained and deposited at the China Center for Type Culture Collection (CCTCC) in Wuhan, China on June 4, 2025, with accession number CCTCM 20251244.

[0011] A method for whole-cell catalytic synthesis of dihydrokaempferol involves inoculating a recombinant bacterial strain into a culture medium, inducing protein expression at 16°C, centrifuging to collect the bacteria, resuspending the cells in a buffer solution, and then adding the cells to a reaction system for whole-cell catalytic reaction at a reaction temperature of 30°C–50°C. The reaction system comprises: phosphate buffer (pH 7), 0.1–20 mM naringenin, 0.1–10 mM vitamin C, 0.1–10 mM ferrous sulfate, and 0.1–10 mM α-ketoglutarate. More preferably, the reaction system comprises: phosphate buffer (pH 7), naringenin (7 mM), vitamin C (5 mM), ferrous sulfate (1 mM), and α-ketoglutarate (5 mM).

[0012] The advantages of this invention are:

[0013] 1. It breaks the previous dependence of flavonoid biosynthesis on plant biosynthetic pathways, broadens the selection range of flavanone 3-hydroxylases, and makes them more suitable for expression in microbial chassis cells;

[0014] 2. The whole-cell catalytic method for synthesizing dihydrokaempferol avoids the insufficient synthesis efficiency caused by inadequate balance control of complex synthetic pathways compared with de novo synthesis, reduces the occurrence of side reactions, and greatly shortens the construction cycle of recombinant strains and simplifies the operation. Attached Figure Description

[0015] Figure 1 Phylogenetic analysis of amino acid sequences of flavanone 3-hydroxylases from different sources

[0016] Figure 2 This is the result of the alignment of AbF3H with the soybean-derived GmF3H sequence.

[0017] Figure 3 This study analyzes the docking and interaction between AbF3H and the substrate naringenin.

[0018] Figure 4 yes E. coli Changes in the concentrations of naringenin and hesperidin in whole-cell catalysis by BL21(pACYCDuet-AbF3H) at 30℃ for 0 h and 24 h.

[0019] Figure 5 This is a comparison of the relative concentrations of dihydrokaempferol during catalysis at different temperatures. Detailed Implementation

[0020] Example 1 Proteobacteria Alphaproteobacteria Phylogenetic analysis of AbF3H from sp. source with F3H from other sources

[0021] 1) The amino acid sequence of soybean-derived flavanone 3-hydroxylase GmF3H was compared with the database using NCBI (https: / / www.ncbi.nlm.nih.gov / ), and phylogenetic analysis was performed on the amino acid sequences of 16 proteins, including AbF3H. These proteins originated from bacteria, fungi, and cyanobacteria. The results of the phylogenetic analysis are as follows: Figure 1 As shown, three flavanone 3-hydroxylases derived from fungi (from *Emersonia rossata*) Rasamsonia emersonii Orange thermophilic ascomycetes Themoascus themophilus Fishy thermophilic ascomycetes Themoascus crustaceus The flavanone 3-hydroxylase derived from soybean is most closely related to soybean flavanone 3-hydroxylase in terms of evolution, which may be related to the fact that they both originated from eukaryotes. Of the 13 flavanone 3-hydroxylases derived from bacteria, only 5 are evolutionarily closely related to soybean flavanone 3-hydroxylase, namely thermophilic bacteria... Caldovatus sedimis Proteobacteria Alphaproteobacteria sp., Acetobacter Acetobacteraceae sp2., Acetobacter Acetobacteraceae sp. and red thermophilic bacteria Candidatus rokubacteria The remaining 8 flavanone 3-hydroxylases are not closely related to flavanone 3-hydroxylases derived from soybean.

[0022] Example 2 Proteobacteria Alphaproteobacteria Physicochemical properties analysis of AbF3H protein from sp.

[0023] 1) The three-dimensional structure of the protein predicted by AlphaFold3 was analyzed using RING 3.0 Server (https: / / ring.biocomputingup.it / ) to examine the interactions between protein residues. The results showed that the interactions between amino acid residues in AbF3H were mainly intrachain, with 223 hydrogen bonds (H-Bonds), 18 π-π stacks, 6 π-cations, 8 ionic bonds, and 190 van der Waals.

[0024] 2) The soluble expression level of protein in E. coli was predicted using the Protein-Sol online tool (http: / / protein-sol.manchester.ac.uk); the results showed that the soluble expression level of AbF3H in E. coli was 0.454, which was higher than the average level of soluble protein expression in E. coli (0.45).

[0025] 3) Using the Expasy online website, the protein size of AbF3H was calculated to be 39.3 kDa, the isoelectric point was pH 5.5, and the hydrophilicity index was -0.412.

[0026] Example 3 Proteobacteria Alphaproteobacteria Amino acid analysis of key sites on AbF3H from sp.

[0027] 1) The AbF3H protein consists of 350 amino acids and its GenBank Accession Number is RMD48349.1. The reported flavanone-3-hydroxylase (GmF3H) from soybean Glycine I max consists of 375 amino acids and its protein sequence accession number in UniProt is Q53B69. The amino acid sequence similarity between the two is 45%.

[0028] 2) The amino acid sequences of AbF3H and GmF3H were aligned using MEGA 7.0 software based on the ClustalW method. The results were visualized using ESPript 3.0. The results are shown below. Figure 2 As shown, AbF3H, like GmF3H, contains the conserved domains HxD (sites: 227-229) and RxS (sites: 294-296) of the α-ketoglutarate-dependent dioxygenase family, to which flavanone-3-hydroxylase belongs. This indicates that AbF3H belongs to the ketoglutarate-dependent dioxygenase family and has the potential to catalyze the synthesis of dihydrokaempferol from naringenin.

[0029] Example 4 Proteobacteria Alphaproteobacteria Analysis of the binding affinity of AbF3H from sp. to naringenin

[0030] 1) The three-dimensional structure of AbF3H was obtained using AlphaFold3, and the molecular structure of naringenin was obtained using the ZINC15 database.

[0031] 2) Import the ligand naringenin molecule into PyMOL software, use the GexBox plugin in PyMOL to obtain the docking box information, and save the coordinate file.

[0032] 3) The protein was preprocessed in LeDock software to obtain pre.pdb and dock.in files. The ligand naringenin molecule was input, and docking was performed to obtain the molecular docking results. The results showed that the binding free energy of AbF3H with naringenin was -5.71 kJ / mol.

[0033] 4) Based on the molecular docking results, the interaction between AbF3H and naringenin was analyzed using the PLIP online tool. The results are as follows: Figure 3As shown, AbF3H specifically binds to the substrate naringenin through hydrophobic interactions, hydrogen bonds, and π-cation interactions. Ile224A, Leu236A, and Phe300A bind to naringenin through hydrophobic interactions; these nonpolar amino acids have strong hydrophobicity and approach the hydrophobic portion of naringenin, allowing naringenin to bind to the protein. Glu120A, Tyr210A, Asp229A, Asn226A, and Ser340A form hydrogen bonds with the naringenin molecule, achieving specific binding. Arg206A binds to naringenin through π-cation interactions.

[0034] Example 5 E. coli Construction of the BL21 (pACYCDuet-AbF3H) recombinant strain

[0035] 1) The amino acid sequence of AbF3H was codon-optimized, and then its corresponding nucleic acid sequence was synthesized by Beijing Qingke Biotechnology Co., Ltd. The synthesized sequence was then processed... Bam HI and Eco RI was ligated into the expression vector pACYCDuet-1 to obtain the recombinant vector pACYCDuet-AbF3H.

[0036] 2) Centrifuge the EP tube containing 4 μg of the above plasmid powder at 12000 rpm for 2 min, then add 20 μL of distilled water to dilute the plasmid concentration to 200 μg / mL.

[0037] 3) Take Escherichia coli BL21(DE3) competent cells were thawed on ice for 5 min, 2 μL of plasmid was added and gently mixed by aspiration, and then incubated on ice for 20 min.

[0038] 4) Heat shock at 42℃ for 1 min, then immediately place on ice for 2 min. Add 500 μL of sterile LB liquid medium to a clean bench and incubate at 37℃ and 200 rpm for 1 h.

[0039] 5) Centrifuge at 5000 rpm for 2 min, discard most of the supernatant, and resuspend the cells in about 100 μL. Spread the bacterial suspension on LB solid medium containing 25 μg / mL chloramphenicol and incubate overnight at 37°C.

[0040] 6) Pick a single colony from the plate and transfer it to a 50 mL Erlenmeyer flask containing 30 mL of LB medium (chloramphenicol concentration of 25 μg / mL). Incubate overnight at 37°C and 200 rpm.

[0041] 7) Take 1 mL of bacterial culture into a 1.5 mL sterile EP tube, centrifuge at 5000 rpm for 2 min, discard the supernatant, add an equal volume of 15% glycerol, mix well and store at -20℃.

[0042] Example 6 Whole-cell catalytic synthesis of dihydrokaempferol by recombinant strain

[0043] 1) Preserved at an inoculation rate of 1% E. coli BL21 (pACYCDuet-AbF3H) glycerol bacteria were transferred to 30 mL of sterile LB medium and incubated overnight at 37°C and 200 rpm.

[0044] 2) Transfer the activated bacterial culture at a 1% inoculation rate to a 500 mL Erlenmeyer flask containing 200 mL of sterile LB medium (chloramphenicol concentration 25 μg / mL), and incubate at 37℃ and 200 rpm until the logarithmic growth phase (OD50). 600nm (≈0.6-0.8), add IPTG solution to a final concentration of 0.5 mM, and incubate overnight at 16℃ and 200 rpm to induce protein expression.

[0045] 3) Centrifuge at 5000 rpm for 10 min, discard the supernatant, resuspend the cells in 15 mL of phosphate buffer, centrifuge at 5000 rpm for 10 min, discard the supernatant, and repeat the washing process twice.

[0046] 4) The reaction system is as follows: the total cell concentration is OD 600 = 30, 5 mM l-ascorbic acid, 7 mM naringenin, 1 mM ferrous sulfate and 5 mM α-ketoglutarate, with phosphate buffer as the reaction buffer and pH 7. The reaction was carried out in 50 mL Erlenmeyer flasks, with a reaction volume of 10 mL (set up as three replicates), and incubated at 30℃ and 200 rpm with shaking for 24 h.

[0047] 5) Take 500 μL of the final sample after the reaction is complete, add an equal volume of methanol, mix thoroughly, let stand for 10 min, centrifuge at 12,000 rpm for 5 min, and filter the supernatant into a sample bottle for testing.

[0048] HPLC parameters:

[0049] Instrument: Thermo Fisher U3000 liquid chromatograph; Column: 4.6 mm × 150 mm, 4.0 μm.

[0050] Mobile phase: Phase A (methanol), Phase B (1% phosphoric acid); gradient elution (0-20 min: Phase A concentration increases from 15% to 60%; 20-26 min: Phase A concentration decreases from 60% to 0%; 26-30 min: Phase A concentration increases from 0% to 15%).

[0051] Flow rate: 0.8 mL / min; column temperature: 35℃; injection volume: 10 μL; detection wavelength: 290 nm and 360 nm.

[0052] The results are as follows Figure 4 As shown, E. coli BL21 (pACYCDuet-AbF3H) reacted at 30℃. The initial concentration of naringenin was 1.84 g / L at 0 h, decreasing to 20.83 mg / L at 24 h. The initial concentration of dihydrokaempferol was 0 mg / L at 0 h, reaching 82.23 mg / L at the end of 24 hours, representing an increase of approximately 60.2% compared to the production level reported by Huang et al. These results indicate that AbF3H possesses the function of flavanone-3-hydroxylase, catalyzing the conversion of the substrate naringenin to hesperidin. The conversion rate of naringenin during the reaction was approximately 4.52%.

[0053] Example 7 Determination of the optimal reaction temperature for whole-cell catalytic synthesis of dihydrokaempferol by recombinant strain

[0054] 1) Preserved at an inoculation rate of 1% E. coli BL21 (pACYCDuet-AbF3H) glycerol bacteria were transferred to 30 mL of sterile LB medium and incubated overnight at 37°C and 200 rpm.

[0055] 2) Transfer the activated bacterial culture at a 1% inoculation rate to a 500 mL Erlenmeyer flask containing 200 mL of sterile LB medium (chloramphenicol concentration 25 μg / mL), and incubate at 37℃ and 200 rpm until the logarithmic growth phase (OD50). 600 nm (≈0.6-0.8), add IPTG solution to a final concentration of 0.5 mM, and incubate overnight at 16℃ and 200 rpm to induce protein expression.

[0056] 3) Centrifuge at 5000 rpm for 10 min, discard the supernatant, resuspend the cells in 15 mL of phosphate buffer, centrifuge at 5000 rpm for 10 min, discard the supernatant, and repeat the washing process twice.

[0057] 4) The reaction system is as follows: the total cell concentration is OD 600 = 30, 5 mM l-ascorbic acid, 7 mM naringenin, 1 mM ferrous sulfate and 5 mM α-ketoglutarate, with phosphate buffer as the reaction buffer and pH 7. The reaction was carried out in 50 mL Erlenmeyer flasks with a reaction volume of 10 mL (set up in triplicate), and the reaction was carried out at 30℃, 42℃ and 50℃ with shaking at 200 rpm for 24 h.

[0058] 5) Take 500 μL of the final sample after the reaction is complete, add an equal volume of methanol, mix thoroughly, let stand for 10 min, centrifuge at 12,000 rpm for 5 min, and filter the supernatant into a sample bottle for testing.

[0059] HPLC parameters:

[0060] Instrument: Thermo Fisher U3000 liquid chromatograph; Column: 4.6 mm × 150 mm, 4.0 μm.

[0061] Mobile phase: Phase A (methanol), Phase B (1% phosphoric acid); gradient elution (0-20 min: Phase A concentration increases from 15% to 60%; 20-26 min: Phase A concentration decreases from 60% to 0%; 26-30 min: Phase A concentration increases from 0% to 15%).

[0062] Flow rate: 0.8 mL / min; column temperature: 35℃; injection volume: 10 μL; detection wavelength: 290 nm and 360 nm.

[0063] When the reaction temperature was increased from 30℃, the concentration of dihydrokaempferol rapidly decreased to an unquantifiable level. The relative proportions of hesperidin concentration under different temperature conditions were compared by the peak area of ​​dihydrokaempferol in the sample after 24 hours of reaction. The reaction results are as follows: Figure 5 As shown, after reacting at 30℃ for 24 hours, the peak area of ​​the product dihydrokaempferol was 58.95 mAU*min; after reacting at 42℃ for 24 hours, the peak area of ​​the product dihydrokaempferol was 0.96 mAU*min, indicating that the relative concentration of hesperidin decreased by approximately 98.37% compared to 30℃. Further increasing the temperature to 50℃ did not result in the accumulation of dihydrokaempferol in the reaction system. The results indicate that 30℃ is the optimal reaction temperature for strain E. coli BL21 (pACYCDuet-AbF3H), and that increasing the temperature may reduce the activity of AbF3H, leading to a decrease in hesperidin synthesis capacity.

Claims

1. A method for the whole-cell catalytic synthesis of dihydrokaempferol, characterized in that: The recombinant strain was inoculated into a culture medium and cultured. Protein expression was induced at 16°C. The cells were collected by centrifugation, resuspended in buffer, and then added to the reaction system for whole-cell catalytic reaction at a temperature of 30°C–50°C. The reaction system consisted of phosphate buffer, 0.1–20 mM naringenin, 0.1–10 mM vitamin C, 0.1–10 mM ferrous sulfate, and 0.1–10 mM α-ketoglutarate. The recombinant strain was Escherichia coli AbF3H, which was deposited at the China Center for Type Culture Collection (CCTCC) in Wuhan, China on June 4, 2025, with accession number CCTCCNO: M 20251244.

Images