Ketene reductase combined mutant for cascade biological catalysis and application thereof

Abstract

The invention relates to a ketene reductase combination mutant for cascade biological catalysis and application thereof, the ketene reductase combination mutant comprises a W380P / L124V combination mutant and a W380S / L124V combination mutant, and after the ketene reductase combination mutant and chalcone isomerase are prepared into a combined expression strain, the combined expression strain is subjected to high-speed amplification to obtain the ketene reductase. Naringin is catalytically converted into naringin dihydrochalcone through a cascade reaction, and the conversion efficiency of the naringin dihydrochalcone of a W380P / L124V combined mutant and the conversion efficiency of the naringin dihydrochalcone of a W380S / L124V combined mutant are 2.28 times and 1.44 times that of a wild type respectively. According to the ketene reductase combined mutant disclosed by the invention, by enhancing the affinity of a substrate and optimizing the structure of an active site, the catalytic efficiency is remarkably improved, so that the naringin is effectively promoted to be converted into naringin dihydrochalcone.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a combined mutant of enone reductases for cascade biocatalysis and its applications. Background Technology

[0002] Naringin is a natural flavonoid compound mainly found in the peel and pulp of citrus fruits such as grapefruit and pomelo. It belongs to the flavonoid group and has typical chemical structural features, including a benzene ring and a glycosidic moiety linked by a sugar group. Naringin typically has a bitter taste and possesses certain antioxidant, anti-inflammatory, and antitumor activities, making it widely used in food, health products, and traditional herbal medicines. Numerous studies support the health benefits of naringin. For example, it can effectively scavenge free radicals and slow down the aging process; it also has a protective effect on the cardiovascular system, lowering blood lipid levels and improving vascular endothelial function. Furthermore, naringin has been found to enhance liver detoxification, help lower cholesterol, and possess certain antibacterial, antiviral, and anti-inflammatory effects. However, naringin has low bioavailability, meaning its absorption rate in the body is poor, making it difficult to fully realize its effects. Therefore, its application still faces certain challenges, especially in improving bioavailability and safety.

[0003] Enoate reductases (EREDs) are a class of enzymes that catalyze the reduction of natural compounds containing olefinic bonds, playing a crucial role, particularly in the biotransformation of flavonoids. EREDs can reduce the olefinic double bond in naringin to a saturated structure, generating naringin dihydrochalcone. This transformation not only enhances the pharmacological activity of the product but also expands its application potential in the field of natural medicine. In practical applications, ERED-catalyzed reactions exhibit high selectivity and efficiency in the reduction and conversion of naringin, thus becoming an important pathway for producing high-value-added natural products. By optimizing reaction conditions, efficient conversion can be achieved, increasing the yield and purity of naringin dihydrochalcone. This type of conversion process utilizes mild reaction conditions (such as temperature and pH), reducing the side effects and environmental pollution associated with chemical catalysis. Compared to other enzymatic catalysis methods, ERED catalysis offers the advantage of milder reaction conditions and ease of control and optimization in large-scale applications. Further improvements in conversion efficiency and product purity can be achieved by using specialized enzymes and optimizing catalytic reaction conditions. However, natural enzymes also have some drawbacks in practical applications. First, natural enzymes exhibit poor stability, particularly under high temperatures or extreme pH conditions, which limits their widespread application in industrial production. Second, the acquisition cost of these enzymes is high, requiring complex purification processes that increase production costs. Furthermore, the strong substrate specificity of natural enzymes can sometimes lead to limited reaction selectivity, making them unsuitable for complex substrates or reaction conditions. Therefore, despite the significant potential of ERED in green biocatalysis, further improvements in performance through genetic engineering, enzyme engineering, and other technologies are needed to enhance the feasibility of their industrial applications. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide a combined mutant of enone reductase for cascade biocatalysis and its application. The combined mutant of enone reductase can selectively convert naringin chalcone into naringin dihydrochalcone. This mutant aims to solve the problems of insufficient activity and poor stability of traditional enone reductase, thereby improving its efficiency and stability in practical applications.

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

[0006] A combined mutant of enone reductase for cascade biocatalysis, wherein the combined mutant is formed by two mutations at positions 380 and 124 in the amino acid sequence ERED as shown in SEQ ID NO: 1, and the combined mutant of enone reductase is selected from any one of the following (I)-(II):

[0007] (I) The enone reductase combined mutant is the W380P / L124V combined mutant, whose amino acid sequence is as shown in SEQ ID NO:1. Tryptophan at position 380 is mutated to proline and leucine at position 124 is mutated to proline and valine, respectively. Its amino acid sequence is shown in SEQ ID NO:5.

[0008] (II) The enone reductase combined mutant is the W380S / L124V combined mutant, whose amino acid sequence is as shown in SEQ ID NO:1. Tryptophan at position 380 is mutated to serine and leucine at position 124 is mutated to proline and valine, respectively. Its amino acid sequence is shown in SEQ ID NO:7.

[0009] This invention utilizes bioinformatics methods such as protein three-dimensional structure prediction and substrate molecular docking to design three combined mutants of enone reductase. These mutants enhance enzyme activity and stability through specific mutations in the amino acid sequence (e.g., ERED as shown in SEQ ID NO:2). Specifically, the mutations include replacing tryptophan at position 380 with proline or serine, leucine at position 124 with valine, and leucine at position 277 with proline. These amino acid substitutions are based on their potential advantages in improving enzyme activity, enhancing substrate affinity, and improving molecular stability. The ERER enzymes resulting from these combined mutations all exhibit improved reduction efficiency compared to wild-type enone reductases.

[0010] Furthermore, the ERED is derived from an enone reductase (GenBank sequence number: KF154735; UniProt sequence number: V9P074) in the anaerobic bacterium *Eubacterium ramulus*, and its translated protein sequence is SEQ ID NO:1. The nucleotide sequence of the ERED gene is shown in SEQ ID NO:2.

[0011] Preferably, the nucleotide sequence of the gene encoding the mutant W380P / L124V is shown in SEQ ID NO:6; the nucleotide sequence of the gene encoding the mutant W380S / L124V is shown in SEQ ID NO:8.

[0012] In a second aspect, the present invention provides the use of the enone reductase combination mutant of the first aspect for cascade biocatalysis in the preparation of naringin dihydrochalcone, phlorizin or combinations thereof.

[0013] Thirdly, the present invention provides a strain co-expressing an enone reductase combined mutant and a chalcone isomerase, wherein the strain is any one of the enone reductase combined mutants described in the first aspect and a chalcone isomerase co-expressing strain; the co-expressing strain of the enone reductase combined mutant and the chalcone isomerase includes the CHI-W380P / L124V expression strain or the CHI-W380S / L124V expression strain; wherein the amino acid sequence of the chalcone isomerase is shown in SEQ ID NO:9, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO:10.

[0014] Specifically, the preparation method of the CHI-W380P / L124V expression strain or the CHI-W380S / L124V expression strain includes the following steps:

[0015] S1. The CHI and ERED encoding genes to be mutated are cloned into a plasmid to obtain a recombinant plasmid;

[0016] S2. Design and use site-directed mutagenesis primers to amplify the recombinant plasmid as a template, followed by enzyme digestion to obtain the mutant product, wherein the site-directed mutagenesis primers are used to mutate the leucine residue at position 124 of ERED.

[0017] S3. The mutant product obtained in S2 is transformed into the host cell and the mutant L124V is obtained by screening.

[0018] S4. Design and use site-directed mutagenesis primers to amplify the recombinant plasmid L124V as a template. After enzyme digestion, the mutant product is obtained. The site-directed mutagenesis primers are used to mutate the tryptophan residue at position 380 of ERED to proline or serine.

[0019] S5. The mutant product obtained in S4 is transformed into host cells, and the host cells are screened and induced to express it. Positive transformants are screened in the culture medium to obtain the CHI-W380P / L124V expression strain or the CHI-W380S / L124V expression strain.

[0020] Preferably, the site-directed mutagenesis primers for mutating leucine at position 124 of ERED to valine are upstream primer L124V-F and downstream primer L124V-R, the nucleotide sequence of the upstream primer L124V-F is shown in SEQ ID NO:15, and the nucleotide sequence of the downstream primer L124V-R is shown in SEQ ID NO:16.

[0021] The site-directed mutagenesis primers for mutating tryptophan at position 380 of ERED to proline are upstream primer W380P-F and downstream primer W380P-R. The nucleotide sequence of the upstream primer W380P-F is shown in SEQ ID NO:17, and the nucleotide sequence of the downstream primer W380P-R is shown in SEQ ID NO:18.

[0022] The site-directed mutagenesis primers for mutating tryptophan at position 380 of ERED to serine are upstream primer W380S-F and downstream primer W380S-R. The nucleotide sequence of the upstream primer W380S-F is shown in SEQ ID NO:19, and the nucleotide sequence of the downstream primer W380S-R is shown in SEQ ID NO:20. Mutating leucine at position 124 of ERED to valine optimizes the substrate binding ability and catalytic efficiency of ERED by altering the characteristics of its hydrophobic side chain. Specifically, leucine (Leu) itself has a large hydrophobic side chain; by mutating it to valine (Val), the hydrophobic environment of the substrate binding pocket can be fine-tuned, thereby improving substrate binding efficiency and enzyme activity. Therefore, using the mutant L124V (i.e., first mutating position 124 of ERED) as the initial template, we constructed the following mutants by iterative site-directed mutagenesis at position 380 of tryptophan: a combined mutant library of W380P / L124V and W380S / L124V.

[0023] It should be noted that the expression strain can be obtained by co-transforming CHI and the ERED strain to be mutated into a vector, followed by site mutation of ERED; alternatively, ERED mutation can be performed first, followed by co-transformation with CHI. The combination of CHI and ERED does not affect the effectiveness of ERED mutation.

[0024] Thirdly, the present invention provides the application of the co-expression strain of the enone reductase combined mutant and chalcone isomerase in the second aspect, wherein the co-expression strain of the enone reductase combined mutant and chalcone isomerase is used to catalyze the conversion of naringin chalcone to prepare naringin dihydrochalcone.

[0025] Preferably, the specific steps for using the co-expression strain of the enone reductase combined mutant and chalcone isomerase for the catalytic conversion of naringin chalcone to prepare naringin dihydrochalcone are as follows: the co-expression strain of the enone reductase combined mutant and chalcone isomerase is prepared into a whole-cell enzyme solution, then naringin and buffer are added, and the reaction is carried out at 27-33°C to obtain the naringin dihydrochalcone.

[0026] The cascade reaction of chalcone isomerase (CHI) with the conversion of flavanones to chalcones plays a crucial role in the biotransformation of natural products. CHI catalyzes the cleavage of the ring structure in flavanones to generate chalcone structures (i.e., ring-opening reaction). This transformation not only provides new possibilities for the structural diversity of flavanones but also plays an important role in the biosynthesis of many natural products. Specifically, CHI catalyzes the isomerization of flavanones to chalcone structures. This reaction not only alters the chemical structure of the substrate but also provides necessary intermediates for subsequent transformation steps. For example, chalcone, as an important intermediate, can be further converted by ERED to the biologically active dihydrochalcone structure. In this process, the concept of a cascade reaction is fully demonstrated: by constructing engineered strains containing CHI and ERED, the conversion of flavanones to chalcones can be achieved, and then ERED can reduce chalcones to dihydrochalcones. This reaction pathway is not only highly efficient and environmentally friendly, but also offers strong selectivity and mild reaction conditions, making it suitable for large-scale production. This invention achieves a two-step cascade reaction by fusing two enzymes (CHI and ERED) into the same strain: the first step involves the ring-opening reaction of flavanones catalyzed by CHI to generate chalcone, and the second step involves the reduction of chalcone to dihydrochalcone via the reduction reaction of ERED. This type of cascade reaction has significant advantages, effectively increasing the yield and purity of the target product while avoiding side reactions in chemical catalysis, reducing environmental pollution, and aligning with the principles of green chemistry.

[0027] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0028] (1) Both the W380P / L124V and W380S / L124V combined mutants of the enone reductase of the present invention can promote the reduction reaction of naringin and effectively improve the efficiency of naringin to naringin dihydrochalcone. The above mutants exhibit higher catalytic efficiency by enhancing the substrate affinity of the enzyme and changing the spatial structure of the active site.

[0029] (2) The preparation method of the enone reductase combinatorial mutant of the present invention uses computer-aided protein engineering enzyme molecular design to analyze the active pocket region of the enone reductase ERED from anaerobic bacteria (Eubacterium ramulus) for molecular docking, and at the same time predicts the three-dimensional structure of the mutant protein to simulate the pocket phenotypic structural changes. Then, a mutant library is constructed by site-directed mutagenesis technology. The preparation method is accurate and effective.

[0030] (3) The enone reductase combination mutant of the present invention can be used to catalyze the conversion of naringin chalcone and prepare compositions containing different concentrations of naringin dihydrochalcone. It has broad application prospects and is suitable for the industrial production of naringin.

[0031] (4) After the enone reductase hybrid mutant of the present invention was combined with chalcone isomerase to form a co-expression strain, it catalyzed the conversion of naringin to naringin dihydrochalcone through a cascade reaction. The conversion efficiencies of the W380P / L124V hybrid mutant and the W380S / L124V hybrid mutant to naringin dihydrochalcone were 2.28 times and 1.44 times that of the wild type, respectively. This indicates that the cascade biocatalysis of the enone reductase hybrid mutant and chalcone isomerase can not only effectively improve the yield and purity of the target product, but also avoid side reactions in the chemical catalysis process, reduce environmental pollution, and conform to the concept of green chemistry. This kind of enzyme-engineered conversion strategy provides a new way for the high-value-added conversion of natural products and shows great application potential in the production and optimization of natural drugs. With the continuous development of technology, the application of enzyme-catalyzed cascade reactions in the production of natural products will become more and more widespread, and will provide strong support for green chemistry and sustainable development. Attached Figure Description

[0032] Figure 1 A flowchart illustrating the steps involved in the conversion of naringin to naringin dihydrochalcone.

[0033] Figure 2 This is a simulation diagram of the docking of ERED with naringin chalcone molecules;

[0034] Figure 3 Three-dimensional structure prediction model for AlphaFold3 protein of ERED wild-type WT and its mutants L124V, W380P / L124V combined mutant, and W380S / L124V combined mutant;

[0035] Figure 4 The conversion efficiency results of CHI and ERED wild-type and mutant transformations to naringin dihydrochalcone are shown in the figure.

[0036] Figure 5 The HPLC results are shown for the reaction solution after whole-cell catalysis by the ERED wild-type WT and W380P / L124V combined mutant. Detailed Implementation

[0037] The present invention will now be further described with reference to the accompanying drawings and specific embodiments. It should be noted that, without conflict, the various embodiments or technical features described below can be arbitrarily combined to form new embodiments. The following are specific embodiments of the present invention. Unless otherwise specified, the raw materials, equipment, etc. used in the following embodiments can all be obtained by purchase.

[0038] Experimental methods in the following examples that do not specify specific conditions are generally performed under standard experimental conditions or as recommended by the manufacturer. Unless otherwise specified, all reaction reagents involved in the examples are commercially available. Molecular biology experimental methods not specifically described in these examples can be found in *Molecular Cloning: A Laboratory Manual*.

[0039] Example 1

[0040] The preparation method of the CHI-W380P / L124V expression strain includes the following steps:

[0041] S1. Construction of the BL21-pET28a-ERED-CHI plasmid: First, the BL21-pET28a plasmid was double-digested with restriction endonucleases EcoRI and XhoI to open the multiple cloning site (MCS). Then, the nucleotide sequence of erone reductase (ERED) (as shown in SEQ ID NO:2) was inserted into the plasmid using T4 DNA ligase to construct the BL21-pET28a-ERED recombinant plasmid. Subsequently, using this recombinant plasmid as a template, the 3' end of the ERED gene was digested again with XhoI, and homologous recombination technology was used to insert the nucleotide sequence of the chalcone isomerase (CHI) gene (as shown in SEQ ID NO:9) between the ERED and XhoI sites, thereby constructing the BL21-pET28a-ERED-CHI recombinant plasmid containing the fusion expression of ERED and CHI.

[0042] S2. Design and utilize site-directed mutagenesis primers to perform full plasmid PCR amplification using the BL21-pET28a-ERED-CHI recombinant plasmid as a template, followed by enzyme digestion to obtain the mutant product. The site-directed mutagenesis primers are used to mutate the leucine residue at position 124 of ERED to valine. The site-directed mutagenesis primers are upstream primer L124V-F and downstream primer L124V-R. The nucleotide sequence of the upstream primer L124V-F is shown in SEQ ID NO:13, and the nucleotide sequence of the downstream primer L124V-R is shown in SEQ ID NO:14.

[0043] S3. The mutant product obtained in S2 is transformed into the host cell and the mutant L124V is obtained by screening.

[0044] S4. Design and utilize site-directed mutagenesis primers to amplify the recombinant plasmid L124V as a template. After enzyme digestion, the mutant product is obtained. The site-directed mutagenesis primers are used to mutate the tryptophan residue at position 380 of ERED to proline. The site-directed mutagenesis primers are upstream primer W380P-F and downstream primer W380P-R. The nucleotide sequence of the upstream primer W380P-F is shown in SEQ ID NO:15, and the nucleotide sequence of the downstream primer W380P-R is shown in SEQ ID NO:16.

[0045] S5. The mutant product obtained in S4 is transformed into host cells, and the host cells are screened and induced to express the mutant. Positive transformants are screened in the culture medium to obtain the CHI-W380P / L124V expression strain.

[0046] Example 2

[0047] The preparation method of the CHI-W380S / L124V expression strain includes the following steps:

[0048] S1. The encoding genes of ERED (as shown in SEQ ID NO: 1) and CHI (as shown in SEQ ID NO: 21) were cloned into a plasmid to obtain the BL21-pET28a-ERED-CHI recombinant plasmid.

[0049] S2. Design and utilize site-directed mutagenesis primers to perform full plasmid PCR amplification using the BL21-pET28a-ERED-CHI recombinant plasmid as a template, followed by enzyme digestion to obtain the mutant product. The site-directed mutagenesis primers are used to mutate the leucine residue at position 124 of ERED to valine. The site-directed mutagenesis primers are upstream primer L124V-F and downstream primer L124V-R. The nucleotide sequence of the upstream primer L124V-F is shown in SEQ ID NO:15, and the nucleotide sequence of the downstream primer L124V-R is shown in SEQ ID NO:16.

[0050] S3. The mutant product obtained in S2 is transformed into the host cell and the mutant L124V is obtained by screening.

[0051] S4. Design and utilize site-directed mutagenesis primers to amplify the recombinant plasmid L124V as a template. After enzyme digestion, the mutant product is obtained. The site-directed mutagenesis primers are used to mutate the tryptophan residue at position 380 of ERED to serine. The site-directed mutagenesis primers are upstream primer W380S-F and downstream primer W380S-R. The nucleotide sequence of the upstream primer W380S-F is shown in SEQ ID NO:19, and the nucleotide sequence of the downstream primer W380S-R is shown in SEQ ID NO:20.

[0052] S5. The mutant product obtained in S4 is transformed into host cells, and the host cells are screened and induced to express it. Positive transformants are screened in the culture medium to obtain the CHI-W380S / L124V expression strain or the CHI-W380S / L124V expression strain.

[0053] Comparative Example 1

[0054] Comparative Example 1 is the CHI-ERED (wild-type WT) expression strain, i.e. ERED is not mutated (wild-type WT). Its preparation method is as follows: S1, the coding genes of ERED with amino acid sequences as shown in SEQ ID NO: 1 and CHI with amino acid sequences as shown in SEQ ID NO: 21 are cloned into a plasmid to obtain the BL21-pET28a-ERED-CHI recombinant plasmid (WT).

[0055] Comparative Example 2

[0056] Comparative Example 2 is the CHI-L124V expression strain, which is prepared by the following steps:

[0057] S1. Construct the BL21-pET28a-ERED-CHI recombinant plasmid using the same steps as in Example 1;

[0058] S2. Design and utilize site-directed mutagenesis primers to perform full plasmid PCR amplification using the BL21-pET28a-ERED-CHI recombinant plasmid as a template, followed by enzyme digestion to obtain the mutant product. The site-directed mutagenesis primers are used to mutate the leucine residue at position 124 of ERED to valine. The site-directed mutagenesis primers are upstream primer L124V-F and downstream primer L124V-R. The nucleotide sequence of the upstream primer L124V-F is shown in SEQ ID NO:15, and the nucleotide sequence of the downstream primer L124V-R is shown in SEQ ID NO:16.

[0059] S3. The mutant product obtained in S2 is transformed into host cells and the mutant L124V is obtained by screening. The amino acid sequence of the mutant enzyme L124V is SEQ ID NO:3, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO:4.

[0060] S4. The mutant L124V obtained in S3 is transformed into host cells, and host cells are screened and induced to express the mutant. Positive transformants are screened in the culture medium to obtain the CHI-L124V expression strain.

[0061] Comparative Example 3

[0062] Comparative Example 3 is the CHI-W380P expression strain, which is prepared by the following steps:

[0063] S1. The encoding genes of ERED (as shown in SEQ ID NO: 1) and CHI (as shown in SEQ ID NO: 21) were cloned into a plasmid to obtain the BL21-pET28a-ERED-CHI recombinant plasmid.

[0064] S2. Design and utilize site-directed mutagenesis primers to perform full plasmid PCR amplification using the BL21-pET28a-ERED-CHI recombinant plasmid as a template, followed by enzyme digestion to obtain the mutant product. The site-directed mutagenesis primers are used to mutate tryptophan at position 380 of ERED to proline. The site-directed mutagenesis primers are upstream primer W380P-F and downstream primer W380P-R. The nucleotide sequence of the upstream primer W380P-F is shown in SEQ ID NO:17, and the nucleotide sequence of the downstream primer W380P-R is shown in SEQ ID NO:18.

[0065] S3. The mutant product obtained in S2 is transformed into host cells and the mutant W380P is obtained by screening. The amino acid sequence of mutant W380P is SEQ ID NO:11, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO:12.

[0066] S5. The mutant W380P obtained in S4 is transformed into host cells, and the expression of host cells is screened and induced. Positive transformants are screened in the culture medium to obtain the CHI-W380P expression strain.

[0067] Comparative Example 4

[0068] Comparative Example 4 is the CHI-W380S expression strain, which was prepared as follows:

[0069] S1. Construct the BL21-pET28a-ERED-CHI recombinant plasmid in the same manner as in Example 1;

[0070] S2. Design and utilize site-directed mutagenesis primers to perform full plasmid PCR amplification using the BL21-pET28a-ERED-CHI recombinant plasmid as a template, followed by enzyme digestion to obtain the mutant product. The site-directed mutagenesis primers are used to mutate tryptophan at position 380 of ERED to proline. The site-directed mutagenesis primers are upstream primer W380S-F and downstream primer W38SP-R. The nucleotide sequence of the upstream primer W380S-F is shown in SEQ ID NO:19, and the nucleotide sequence of the downstream primer W380S-R is shown in SEQ ID NO:20.

[0071] S3. The mutant product obtained in S2 is transformed into host cells and the mutant W380S is obtained by screening. The amino acid sequence of mutant W380S is SEQ ID NO:13, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO:14.

[0072] S4. The mutant W380S obtained in S3 is transformed into host cells, and host cells are screened and induced to express the mutant. Positive transformants are screened in the culture medium to obtain the CHI-W380S expression strain.

[0073] In step S2 of the preparation methods for the expression strains in Examples 1-2 and Comparative Examples 1-4, the whole plasmid PCR amplification system is shown in Table 1.

[0074] Table 1. PCR amplification system of the whole plasmid

[0075]

[0076] All the enzyme digestion systems used in the preparation methods of the expression strains in Examples 1-2 and Comparative Examples 1-4 are shown in Table 2.

[0077] Table 2 Enzyme digestion system

[0078] QuickCutDpnⅠ 1μL PCR products ≤1μg 10×QuickCutBuffer 3μL <![CDATA[ddH2O]]> Add to 30μL

[0079] The above enzyme digestion system was placed in a metal bath at 37°C for 1 hour for digestion. After the reaction, the enzyme digestion products were recovered using a kit.

[0080] The PCR amplification programs for steps S4 and S5 in Examples 1-2 are as follows: pre-denaturation at 98℃ for 3 min; cycle settings: denaturation at 98℃ for 15 s, annealing at 60℃ for 30 s, extension at 72℃ for 5 min, 30 cycles; final extension at 72℃ for 10 min; after the reaction, the PCR products are recovered using the kit.

[0081] The PCR site-directed mutagenesis primers used in Examples 1-2 and Comparative Examples 2-4 are detailed in Table 3.

[0082] Table 3. Site-directed mutagenesis primers

[0083]

[0084] Application Example 1: Induction of Recombinant Strains and Preparation of Whole-Cell Enzyme Solutions

[0085] Step 1: Take the strains from Examples 1-2 and Comparative Examples 1-4 respectively, and streak them on LB agar plates containing Kan (100 μg / mL) for activation. After overnight incubation at 37°C with the plates inverted, pick a single colony and inoculate it into 1 mL of LB liquid medium containing Kan, and incubate at 37°C with shaking at 200 rpm for 4-6 h. Inoculate the seed culture into 25 mL of fresh LB liquid medium containing Kan at a 1% inoculation rate, and incubate at 37°C with shaking at 200 rpm for 3-4 h until the OD600 reaches 0.6-0.8. Then add IPTG to a final concentration of 0.4 mM, and incubate the culture at 20°C with shaking at 150 rpm for 18 h to induce protein expression.

[0086] Step 2: After protein induction, collect the cells by centrifugation at 5000 rpm for 10 min at 4°C. Wash the cells once with Tris-HCl buffer (pH 6.0), and then resuspend the cells in an appropriate amount of Tris-HCl buffer (pH 6.0) according to the wet weight of the cells to obtain a cell suspension with a total cell concentration of 100 mg / mL. All operations were performed on ice or at 4°C.

[0087] Application Example 2: Whole Cell Response

[0088] The whole-cell enzyme solutions containing strains from Examples 1-2 and Comparative Examples 1-4 obtained in the above experiments were used as enzyme solutions, and the reaction systems shown in Table 4 were prepared:

[0089] Table 4 Whole-cell reaction solution system

[0090] reagents volume Whole cell enzyme solution 100mg / mL Naringin solution 0.435 mg / mL Tris-HCl buffer (pH 6.0) Add to 250μL

[0091] The whole-cell reaction system was reacted at 30℃ for 5 h. After the reaction was completed, 1 volume of DMSO was added to terminate the reaction. After thorough vortexing, the cells were removed by centrifugation at 10000 rpm for 1 min. The reaction product was filtered through a 0.45 μm organic filter and then analyzed by HPLC to calculate the conversion rate (normalization method).

[0092] Application Example 3: Detection Method for Naringin Dihydrochalcone

[0093] The quantitative analysis of naringin dihydrochalcone was performed by HPLC, and the chromatographic conditions are as follows:

[0094] High Performance Liquid Chromatography (HPLC): Agilent 1100 Series

[0095] Chromatographic column: 5μm C18(250mm x 4.6mm x 5μm)

[0096] Detector: VWD detector, detection wavelength 270nm

[0097] Mobile phase ratio and elution conditions: flow rate 1 mL / min; column temperature 30 ℃; injection volume 10 μL; gradient elution system as shown in Table 5.

[0098] Table 5 HPLC gradient elution system

[0099] time A% (acetic acid solution) B% (acetonitrile) 0min 77 23 5min 77 23 10min 77 23 15min 77 23

[0100] Results Analysis

[0101] 1. Molecular docking of the ERED tertiary structure model with naringin chalcone

[0102] The mechanism by which enone reductase (ERED) binds to substrate molecules, such as Figure 1 As shown, after the constructed ERED three-dimensional structural model was molecularly docked with the substrate molecule, the results were as follows: Figure 2 As shown, leucine (Leu) at position 124 of ERED is located in the enzyme's active pocket, while the rest of the structure is located on the enzyme surface. Specifically, leucine at position 124 of ERED creates significant steric hindrance with the domains within the pocket. This steric hindrance affects the enzyme's ability to bind to substrates, including tryptophan (Trp) at position 380. Leucine at position 124 and its surrounding amino acid residues (such as tryptophan at position 380) create significant steric hindrance, thus affecting enzyme activity. In particular, the structure of tryptophan at position 380 interacts with the geometry of the enzyme pocket, resulting in steric hindrance during substrate binding. These steric hindrance factors work together to determine the activity and catalytic ability of ERED.

[0103] 2. Results of whole-cell catalytic reaction as follows Figure 4-5 As shown. Under reaction conditions of 30℃, pH 6.0, and a reaction time of 5 h, the efficiency of naringin dihydrochalcone production in Comparative Example 1 (using the wild-type WT ERED and CHI co-strains) was 16.067%. The efficiency of naringin dihydrochalcone production in Comparative Example 2 (CHI-L124V strain) increased to 19.197%, with a catalytic efficiency 1.19 times that of the wild type. The efficiency of naringin dihydrochalcone production in Comparative Example 3 (CHI-W380P expression strain) increased to 20.079%, with a catalytic efficiency 1.25 times that of the wild type. The efficiency of naringin dihydrochalcone production in Comparative Example 4 (CHI-W380S expression strain) increased to 22.569%, with a catalytic efficiency 1.40 times that of the wild type.

[0104] In Examples 1-2 (using CHI-W380P / L124V and CHI-W380S / L124V expression strains), the conversion efficiency of the product naringin dihydrochalcone from Examples 1-2 was increased by 2.28 and 1.44 times, respectively, compared to Comparative Example 1. Their cascade catalytic efficiencies were also higher than those of Comparative Examples 2-4. This indicates that the combination of CHI-W380P and L124V may have improved the conversion rate through a synergistic effect, even if their individual conversion rates were low. The L124V mutation may have altered the protein's structure or function, improving the stability and activity of CHI-W380P under certain conditions, thereby enhancing the complex's performance. The nonlinear effect of the mutants may have made the combined effect of the two exceed the sum of the individual mutants, further improving the conversion rate. These mutants may have improved the protein's expression level and stability, thus further enhancing the conversion rate.

[0105] In summary, the co-expression strains of the enone reductase hybrid mutant and chalcone isomerase of this invention catalyze the conversion of naringin to naringin dihydrochalcone via a cascade reaction. The conversion efficiencies of the W380P / L124V and W380S / L124V hybrid mutants to naringin dihydrochalcone are 2.28 times and 1.44 times that of the wild type, respectively. This demonstrates that the cascade biocatalysis of the enone reductase hybrid mutant and chalcone isomerase not only effectively improves the yield and purity of the target product but also avoids side reactions in chemical catalysis, reducing environmental pollution and aligning with the principles of green chemistry.

[0106] The above description is merely a preferred embodiment of the present invention. Those skilled in the art can make various improvements and modifications based on the technical solutions and concepts described above, without departing from the principles of the present invention. These improvements and modifications should also be considered within the scope of protection of the present invention, and all such changes should fall within the scope of protection of the claims of the present invention.

Claims

1. A combinatorial mutant of enone reductase for cascade biocatalysis, characterized in that, The enone reductase combination mutant is formed by two mutations at positions 380 and 124 in the ERED amino acid sequence as shown in SEQ ID NO: 1, and the enone reductase combination mutant is selected from any one of the following (I)-(II): (I) The enone reductase combined mutant is the W380P / L124V combined mutant, whose amino acid sequence is as shown in SEQ ID NO: 1, where tryptophan at position 380 is mutated to proline and leucine at position 124 is mutated to proline and valine, respectively. Its amino acid sequence is shown in SEQ ID NO:

5. (II) The enone reductase combined mutant is the W380S / L124V combined mutant, whose amino acid sequence is shown in SEQ ID NO:

1. Tryptophan at position 380 is mutated to serine and leucine at position 124 is mutated to proline and valine, respectively. Its amino acid sequence is shown in SEQ ID NO:

7.

2. The enone reductase combinatorial mutant for cascade biocatalysis as described in claim 1, characterized in that, The nucleotide sequence of the gene encoding the mutant W380P / L124V is shown in SEQ ID NO:6; the nucleotide sequence of the gene encoding the mutant W380S / L124V is shown in SEQ ID NO:

8.

3. The use of the enone reductase combination mutant of claim 1 or 2 for cascade biocatalysis in the preparation of naringin dihydrochalcone, phlorizin or combinations thereof.

4. A strain co-expressing an enone reductase hybrid mutant and a chalcone isomerase, characterized in that, The strain is any one of the enone reductase combined mutants according to claim 1 or 2, co-expressing a chalcone isomerase; the co-expressing strains of the enone reductase combined mutant and the chalcone isomerase include the CHI-W380P / L124V expression strain and the CHI-W380S / L124V expression strain; wherein, the amino acid sequence of the chalcone isomerase is shown in SEQ ID NO:9, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO:

10.

5. The application of the strain co-expressing the enone reductase combined mutant and chalcone isomerase as described in claim 4, characterized in that, The co-expression strain of the enone reductase combined mutant and chalcone isomerase was used to catalyze the conversion of naringin chalcone to prepare naringin dihydrochalcone.

6. The application of the co-expression strain of the enone reductase combined mutant and chalcone isomerase as described in claim 5, characterized in that, The specific steps for using the co-expression strain of the enone reductase combined mutant and chalcone isomerase for the catalytic conversion of naringin chalcone to prepare naringin dihydrochalcone are as follows: the co-expression strain of the enone reductase combined mutant and chalcone isomerase is prepared into a whole-cell enzyme solution, then naringin and buffer are added, and the reaction is carried out at 27-33℃ to obtain the naringin dihydrochalcone.

Images