Naringenin-producing genetically engineered escherichia coli and application thereof

Abstract

This invention discloses a genetically engineered *E. coli* strain that produces naringin. This strain co-expresses α-L-rhamnosidase and β-D-glucosidase and is deposited at the China General Microbiological Culture Collection Center (CGMCC) under accession number CGMCC No. 37964. The invention also discloses the application of this genetically engineered *E. coli* strain in the preparation of naringin, wherein the strain converts naringin to naringin in a single step intracellularly. By constructing a genetically engineered strain that simultaneously expresses α-L-rhamnosidase and β-D-glucosidase, this invention enables a novel one-step continuous biotransformation method from naringin to naringin within a single engineered strain, utilizing the inexpensive substrate naringin. This method achieves direct and efficient conversion of naringin, thereby simplifying the process, improving efficiency, and reducing costs.

Description

Technical Field

[0001] This invention belongs to the field of microbial metabolic engineering and synthetic biology technology, specifically relating to a genetically engineered Escherichia coli that produces naringenin and its applications. Background Technology

[0002] Naringin is a class of abundant and inexpensive flavanone glycosides widely found in citrus fruits such as grapefruit and oranges, belonging to the Rutaceae family. Its chemical structure consists of a naringenin core linked to a disaccharide group composed of L-rhamnose and D-glucose. Naringenin, a hydrolysis product of naringin, is chemically named 4',5,7-trihydroxyflavanone and possesses anti-inflammatory properties. ] It possesses a variety of biological activities, including antioxidant, anticancer, antidiabetic, antifibrotic, antiviral, neuroprotective, liver-protective, and DNA-protective properties, making it an important compound with high added value.

[0003] Currently, the preparation of naringin mainly relies on chemical hydrolysis, in vitro enzyme catalysis, co-immobilized enzyme methods, and microbial fermentation, but all of these methods have limitations to varying degrees. 1. Chemical hydrolysis: This method uses strong acids or bases, involves violent reaction conditions, has poor selectivity, causes significant environmental pollution, and does not conform to the principles of green chemistry. 2. In vitro enzymatic catalysis: Existing research mainly focuses on using naringinase (a complex enzyme with both α-L-rhamnosidase and β-D-glucosidase activities) to convert naringin into naringenin. Although it has high efficiency and environmental friendliness, it relies on soluble enzyme preparations, which has problems such as high enzyme preparation and purification costs, easy inactivation in the reaction system, poor stability, and difficulty in recycling and reuse (e.g., Vila-Real H, Alfaia JA, Bronze RM, et al. Enzymatic Synthesis of the Flavon Glucosides, Prunin and Isoquercetin, and the Aglycones, Naringenin and Quercetin, with Selective α-L-Rhamnosidase and β-D-Glucosidase Activities of Naringinase[J]. Enzyme Research, 2011, 2011692618. DOI:10.4061 / 2011 / 692618. and Mengfan L. ,Simin L ,Linguo Z , et al.Screening β-glucosidase and α-rhamnosidase for biotransformation of naringin to naringenin by the one-potenzymatic cascade[J].Enzyme and Microbial Technology, 2023, 167110239-110239. DOI: 10.1016 / J.ENZMICTEC.2023.110239. And Carceller MJ, Galán MPJ, Monti R, et al. Selective synthesis of citrus flavonoids prunin andnaringenin using heterogeneized biocatalyst on graphene oxide[J].GreenChemistry,2019.DOI:10.1039 / C8GC03661F.); 3. Co-immobilized enzyme method: By co-immobilizing two glycosidases on a carrier to construct an artificial naringinase system, the stability of the enzyme is improved to some extent. However, the carrier material is expensive, the preparation process is complex, and the transfer efficiency is limited, making it difficult to achieve efficient integration (e.g., Chi W, Pei-Xu C, Qiong X, et al. Artificial naringinase system for cooperative enzymatic synthesis of naringenin[J]. Biochemical Engineering Journal, 2022, 178. DOI:10.1016 / J.BEJ.2021.108277. and You Hongyan. Study on immobilized naringinase and its enzymatic properties and enzymatic preparation of naringenin[D]. Jimei University, 2014.). 4. Microbial fermentation: Microbial co-culture systems developed in recent years can achieve multi-step transformation and byproduct removal, but involve multi-species synergy, complex process control, potential competition among microbial cells, and the need to separate intermediate products. The overall process is long, and integration and scale-up face challenges (e.g., Lu M, Liu S, Liu J, et al. Asusable and efficient strategy for bioconverting naringin to L-rhamnose, 2R-naringenin, and kaempferol[J]. Food Chemistry, 2024, 447138942-.DOI:10.1016 / J.FOODCHEM.2024.138942. Thorsten S, Eckhard B. Competition between pentoses and glucose during uptake and catabolism in recombinant Saccharomyces cerevisiae.[J]. Biotechnology for biofuels,2012,5(1):14.DOI:10.1186 / 1754-6834-5-14.).

[0004] In summary, existing technologies generally suffer from core defects such as cumbersome process steps, high enzyme usage costs, insufficient stability and reusability, low process integration, and difficulty in adapting to continuous industrial production. There is an urgent need to develop a new biomanufacturing strategy that is highly integrated, has stable catalytic efficiency, is low in cost, and is easy to scale up. Summary of the Invention

[0005] The purpose of this invention is to provide a genetically engineered Escherichia coli that produces naringin. By constructing a genetically engineered strain that simultaneously expresses α-L-rhamnosidase and β-D-glucosidase, a novel method can be used to achieve one-step continuous biotransformation from naringin to naringin within a single engineered strain using the inexpensive substrate naringin. This method enables direct and efficient conversion of naringin, thereby simplifying the process, improving efficiency, and reducing costs.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: A genetically engineered *E. coli* strain that produces naringenin, co-expressing α-L-rhamnosidase and β-D-glucosidase, is named *E. coli* BL21(DE3) / pETDuet- RhmA - Cell B, It is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 37964, and the deposit date is March 18, 2026.

[0007] To address the problems of insufficient stability and reusability, low process integration, and difficulty in adapting to continuous industrial production in existing microbial synthesis of naringenin, this invention provides a genetically engineered Escherichia coli strain for efficient biosynthesis of naringenin. This strain enables an intracellular biocatalytic method for the direct conversion of naringin to naringenin within a single microbial cell. The catalytic mechanism involves the strain sequentially catalyzing the derhamnosyl and deglucosylation reactions of naringin within the cell to directly generate naringenin.

[0008] The coding region nucleotide sequence of the α-L-rhamnosidase is shown in SEQ ID NO.1.

[0009] The coding region nucleotide sequence of the β-D-glucosidase is shown in SEQ ID NO.2.

[0010] The pETDuet series was used as the expression vector, and the host was Escherichia coli BL21(DE3). α-L-rhamnosidase was derived from thermophilic bacteria, and β-D-glucosidase was derived from extremely thermophilic archaea.

[0011] The method for constructing the genetically engineered Escherichia coli that produces naringenin includes: 1) Plasmid pET-22b- RhmA and pET-22b- CellB Using high-fidelity DNA polymerase and specific primers as templates, polymerase chain reaction (PCR) was performed to amplify the target gene fragments: CelB gene fragment and RhmA gene fragment. 2) The dual expression vector pETDuet-1 was selected as the basic backbone. The dual expression vector was digested with restriction endonucleases to open the multiple cloning sites: multiple cloning site 1 and multiple cloning site 2. 3) Using the empty vector pETDuet-1 as the vector backbone, restriction endonucleases are employed. BglⅡ and KpnⅠ The multiple cloning site 2 (MCS-2) was double-digested to prepare a linearized vector; the purified CelB gene fragment was incubated with the linearized vector to achieve homologous recombination, forming the recombinant plasmid pETDuet- CellB ; 4) Using recombinant plasmid pETDuet- CellB Using restriction endonucleases as the vector backbone BamHI and EcoRI The RhmA gene fragment was double-digested at its multiple cloning site 1 (MCS-1) to prepare a linearized vector; the purified RhmA gene fragment was seamlessly assembled with the linearized vector and transformed to obtain the dual-gene co-expression recombinant plasmid pETDuet- RhmA - CellB ; 5) The recombinant plasmid pETDuet-, which co-expresses the two genes, is used. RhmA-CelB By introducing the strain into competent Escherichia coli BL21(DE3) cells expressing host Escherichia coli using the heat shock method, a genetically engineered Escherichia coli strain capable of simultaneously expressing α-L-rhamnosidase and β-D-glucosidase activities was obtained.

[0012] The present invention also provides an application of the above-mentioned genetically engineered Escherichia coli strain in the preparation of naringenin.

[0013] Furthermore, the genetically engineered strain converts naringin into naringenin in one step within the cell.

[0014] Furthermore, the method for the genetically engineered strain to convert naringin into naringenin in one step intracellularly specifically involves: using naringin at a final concentration of 1 mM as a substrate, the genetically engineered strain is inoculated and cultured until OD... 600 After the value was 0.6-0.8, IPTG was added to a final concentration of 1 mM, and the strain was induced and cultured at 37℃ for 6 h to convert naringin into naringenin intracellularly.

[0015] Compared with the prior art, the present invention has the following outstanding advantages: 1. Significantly simplified process: By co-expressing α-L-rhamnosidase and β-D-glucosidase in a single engineered strain, a one-step intracellular conversion of naringin to naringenin is achieved, eliminating the need for multi-step reactions and intermediate product separation; 2. Reduced cost and improved stability: The process of in vitro enzyme preparation, separation and immobilization is avoided, which reduces the cost of enzyme use and improves the overall stability of the system; 3. Excellent process integration: Utilizing inexpensive and readily available naringin as a substrate, it is suitable for coupling with E. coli fermentation systems and has broad application prospects; 4. Great potential for large-scale production: Based on a mature E. coli expression system, it has advantages such as short fermentation cycle, mature genetic manipulation, and strong feasibility for scale-up, making it suitable for further research on large-scale production. Attached Figure Description

[0016] Figure 1 The structural formulas for naringin and naringenin are shown. Figure 2 Screening for positive clones by PCR (Note: A stands for pETDuet-) CellB Identification spectrum; B is pETDuet- RhmA - CellB Identification pattern; M: GoldBand 1kb DNA Leader; Lanes 1-5: Selected positive clones); Figure 3 Standard curves for naringin and naringenin; Figure 4 The results are as follows: HPLC detection results (naringin standard, naringenin standard, pETDuet- RhmA - CellB HPLC chromatograms of whole-cell reaction solution, culture medium control, and pETDuet empty control. Detailed Implementation

[0017] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto.

[0018] (I) Construction of pETDuet-RhmA-CelB strain 1. Amplification of the target gene fragment: using plasmid pET-22b- RhmA and pET-22b- CellB Using pET-22b as a template (where pET-22b is used as the plasmid vector, and the RhmA and CelB insertion sites are NcoI / XhoI and BamHI / XhoI, respectively, the plasmid pET-22b is obtained). RhmA and pET-22b- CellB Using high-fidelity DNA polymerase and specific primers (sequences shown in Table 1) as templates, polymerase chain reaction (PCR) was performed to amplify the target gene fragments (RhmA gene fragment and CelB gene fragment). The PCR reaction system and procedure were optimized according to the instructions of the polymerase used. After the amplification products were verified by agarose gel electrophoresis, they were purified using a DNA purification and recovery kit.

[0019] 2. Preparation of linearized vector: The dual expression vector pETDuet-1 was selected as the basic backbone, and restriction endonucleases were used. BglⅡ , KpnⅠ , BamHI , EcoRI The vector was subjected to enzyme digestion to open its multiple cloning sites: multiple cloning site 2 (MCS-2) and multiple cloning site 1 (MCS-1); the digestion products were separated by agarose gel electrophoresis, and the linearized vector backbone fragments were recovered by gel extraction using a gel extraction kit.

[0020] 3. Stepwise assembly of recombinant expression vectors (GibsonAssembly, a seamless DNA assembly technology based on homologous recombination): (1) Using the empty vector pETDuet-1 as the vector backbone, restriction endonucleases were used. BglⅡ and KpnⅠ The multiple cloning site 2 (MCS-2) was double-digested to prepare a linearized vector; the purified CelB gene fragment was mixed with the linearized vector according to the seamless assembly reagent procedure, and incubated at 50°C to achieve homologous recombination, forming the recombinant plasmid pETDuet- CellB The assembly product was transformed into Escherichia coli DH5α competent cells, plated on LB solid plates containing ampicillin (Amp), and colony PCR and sequencing were performed using CelB-F / R identification primers to verify the colony (theoretical length 749 bp), thus obtaining the correct recombinant clone. (2) To verify the correct pETDuet- CellB The plasmid serves as the vector backbone, utilizing restriction endonucleases. BamHI and EcoRI The multiple cloning site 1 (MCS-1) was double-digested to prepare a linearized vector; the purified RhmA gene fragment was seamlessly assembled with this linearized vector and transformed. Colony PCR and sequencing verification were performed using RhmA-F identification / R identification primers (theoretical length 578 bp) to obtain the dual-gene recombinant plasmid pETDuet- RhmA - CellB ; in, Figure 2 Screening for positive clones by PCR (Note: A stands for pETDuet-) CellB Identification spectrum; B is pETDuet- RhmA - CellB Identification pattern; M: GoldBand 1kb DNA Leader; Lanes 1-5: Selected positive clones).

[0021] 4. Obtaining the engineered strain: The finally verified double-gene co-expression recombinant plasmid pETDuet- RhmA- CellBThe transformation medium was introduced into competent Escherichia coli BL21(DE3) cells via heat shock; the transformation medium was plated on LB selection plates containing ampicillin and incubated upside down overnight at 37°C; single clones were picked, and after colony PCR verification, an engineered strain capable of simultaneously expressing α-L-rhamnosidase and β-D-glucosidase activities was obtained and named E. coli BL21(DE3) / pETDuet- RhmA - CellB This strain can express the two functional enzymes mentioned above at 37℃ and under isopropyl-β-D-thiogalactoside (IPTG) induction. The nucleotide sequence of the coding region for α-L-rhamnosidase is shown in SEQ ID NO.1. The nucleotide sequence of the coding region for β-D-glucosidase is shown in SEQ ID NO.2.

[0022] Table 1 Primers used in the experiment (ii) Intracellular transformation reaction 1. Experimental setup: Correctly identified *E. coli* BL21(DE3) / pETDuet- were picked from LB agar plates containing 100 μg / mL ampicillin (AMP). RhmA - CellB Single colonies were inoculated into LB tubes containing the same concentration of AMP and cultured. When the OD600 of the culture medium reached 0.6-0.8, the colonies were transferred to shake flasks containing 30 mL of LB liquid medium containing the same concentration of antibiotics. Three parallel experimental groups were set up. Naringin and IPTG were added to 30 mL of bacterial culture to a final concentration of 1 mM, and expression was induced at 37℃ for 6 h. 2. Control group setup: (1) Background control group of culture medium: The same concentration of naringin and IPTG were added, and 30 mL of LB medium was used but no bacterial culture was inoculated; (2) Empty vector control group: Add the same concentration of naringin and IPTG, inoculate 30 mL of BL21 bacterial suspension transferred into empty vector, and induction conditions are the same as experimental group.

[0023] (III) Detection Methods 1. Preparation of test solutions: Take two 500 μL samples from each reaction solution bottle, labeled A and B; Add 1 mL of methanol to sample A, sonicate for 15 min, centrifuge at 12000 rpm for 10 min, filter the supernatant through a 0.22 μm filter membrane and place it into an HPLC vial for analysis; Centrifuge sample B at 1200 rpm for 5 min, transfer the supernatant to a new 1.5 mL centrifuge tube (labeled C) and add 1 mL of methanol, resuspend the bacterial precipitate in 500 μL of water and add 1 mL of methanol; Sonicate, centrifuge and filter the samples B and C (supernatant) together, and place them into HPLC vials for analysis. 2. Preparation of reference solutions: The reaction solutions for the culture medium background control group and the empty control group were prepared according to the post-treatment method of the test solution described above; 3. Preparation of standard solutions: Naringin and naringenin standards with concentration gradients of 0.01 mM, 0.05 mM, 0.1 mM, 0.2 mM, 0.4 mM, 0.6 mM, 0.8 mM and 1.0 mM were prepared, centrifuged and filtered, and then placed into HPLC sample vials for analysis. 4. HPLC Detection Conditions: HPLC detection was performed using an Agilent C18 column (150 × 4.6 nm, 5 μm) at 30 ℃. The sample loading volume was 10 μL. Formic acid water (A) and acetonitrile (B) were used as the mobile phase at a flow rate of 1.0 mL / min. Gradient elution was performed with the following program: 0–1 min: A (90%), B (10%); 1–10 min: A (60%), B (40%); 10–21 min: A (20%), B (80%); 23–25 min: A (90%), B (10%). The split flow rate after column split was 0.2 mL / min.

[0024] (iv) Results like Figure 4 As shown, HPLC detection results revealed a distinct characteristic peak at the retention time of the naringenin standard in the experimental reaction system, with a peak shape consistent with that of the standard. This indicates that the engineered strain can effectively catalyze the formation of the target product naringenin from naringin. The structural formulas of naringin and naringenin are shown below. Figure 1 As shown; calculated based on the standard curves of naringin and naringenin (e.g.) Figure 3 As shown in the figure, the naringenin production was 170.09 mg / L, and the product conversion rate was 62.48%.

[0025] Under the same culture and induction conditions, no obvious naringin characteristic peak was detected in either the empty vector control group or the negative control group, indicating that the host bacteria itself does not have the ability to convert naringin into naringin. The dual-enzyme co-expression system constructed in this invention plays a key catalytic role in the conversion of naringin.

[0026] In summary, the genetically engineered strain constructed in this invention can stably achieve the biotransformation of naringin to naringenin, providing a new technical approach for developing efficient and green biomanufacturing processes for naringenin.

Claims

1. A genetically engineered Escherichia coli that produces naringin, characterized in that, The genetically engineered Escherichia coli co-expresses α-L-rhamnosidase and β-D-glucosidase and is deposited at the China General Microbiological Culture Collection Center with accession number CGMCC No. 37964.

2. The genetically engineered Escherichia coli producing naringin according to claim 1, characterized in that, The coding region nucleotide sequence of the α-L-rhamnosidase is shown in SEQ ID NO.

1.

3. The naringenin-producing genetically engineered Escherichia coli according to claim 1, characterized by, The coding region nucleotide sequence of the β-D-glucosidase is shown in SEQ ID NO.

2.

4. The naringenin-producing genetically engineered Escherichia coli according to claim 1, characterized by, The pETDuet series was used as the expression vector, and the host was Escherichia coli BL21. α-L-rhamnosidase was derived from thermophilic bacteria, and β-D-glucosidase was derived from extremely thermophilic archaea.

5. The naringenin-producing genetically engineered Escherichia coli according to claim 1, characterized by, The method for constructing the genetically engineered Escherichia coli that produces naringenin includes: 1) Plasmid pET-22b- RhmA and pET-22b- CelB The target gene fragments, CelB gene fragment and RhmA gene fragment, were amplified by polymerase chain reaction (PCR) using high-fidelity DNA polymerase and specific primers with the plasmid pET-22b- 2) The dual expression vector pETDuet-1 was selected as the basic backbone. The dual expression vector was digested with restriction endonucleases to open the multiple cloning sites: multiple cloning site 1 and multiple cloning site 2. 3) Using restriction enzymes BglⅡ and KpnⅠ The linearized vector was prepared by double digestion of the polycloning site 2. The purified CelB gene fragment was incubated with the linearized vector to achieve homologous recombination, forming the recombinant plasmid pETDuet- CelB ; 4) Using recombinant plasmid pETDuet- CelB Using restriction endonucleases as the vector backbone BamHI and EcoRI The multiple cloning site 1 was double-digested to prepare a linearized vector; the purified RhmA gene fragment was seamlessly assembled with the linearized vector and transformed to obtain the dual-gene co-expression recombinant plasmid pETDuet- RhmA - CelB ; 5) The recombinant plasmid pETDuet-, which co-expresses the two genes, is used. RhmA-CelB A genetically engineered Escherichia coli strain capable of simultaneously expressing α-L-rhamnosidase and β-D-glucosidase activities was obtained by introducing the strain into competent E. coli BL21 cells expressed by heat shock.

6. The use of the genetically engineered Escherichia coli strain according to any one of claims 1-5 in the preparation of naringenin.

7. Use according to claim 6, characterized in that, The genetically engineered strain converts naringin into naringin in one step within the cell.

8. The application according to claim 7, characterized in that, The method for the genetically engineered strain to convert naringin into naringenin in one step intracellularly is as follows: using naringin at a final concentration of 1 mM as a substrate, the genetically engineered strain is inoculated and cultured until OD... 600 After the value was 0.6-0.8, IPTG was added to a final concentration of 1 mM, and the strain was induced and cultured at 37℃ for 6 h to convert naringin into naringenin intracellularly.

Images