Dendrobium huoshanense methyltransferase, mutants and applications thereof in synthesis of key intermediates of dendrophenol

Abstract

The application belongs to the technical field of enzyme engineering, and relates to a dendrobium huoshanense methyltransferase, a mutant and application of the methyltransferase and the mutant in synthesis of a key intermediate of dendrophenol. The dendrobium huoshanense methyltransferase or the mutant catalyzes dihydroconiferyl alcohol to generate 3,4,3'-trihydroxy-5'-methoxy-bibenzyl. The 128th serine in the amino acid sequence in SEQ ID NO. 1 is mutated into alanine; or the 314th threonine in the amino acid sequence shown in SEQ ID NO. 1 is mutated into alanine; or the 128th serine and the 314th threonine in the amino acid sequence shown in SEQ ID NO. 1 are simultaneously mutated into alanine, to obtain a methyltransferase mutant. The relative activity of the mutant is increased to 155.16%, 168.64% and 208.5% respectively compared with that of the wild type, and the yield is increased to 1.67 g / L, 1.81 g / L and 2.58 g / L respectively compared with 1.49 g / L of the wild type.

Description

Technical Field

[0001] This invention relates to the field of enzyme engineering technology, and in particular to a Dendrobium huoshanense methyltransferase, a mutant thereof, and its application in the synthesis of a key intermediate of Dendrobium phenols (catalytic preparation of 3,4,3'-trihydroxy-5'-methoxybibenzyl from dihydropiperidine). Background Technology

[0002] Dendrophenol (also known as gigantol) is a characteristic polymethoxylated bibenzyls (PMBs) active ingredient found in precious traditional Chinese medicines such as Dendrobium huoshanense and Dendrobium officinale. Its chemical structure is 3,3'-dihydroxy-5-methoxybibenzyl. In recent years, numerous pharmacological studies have demonstrated that dendrophenol possesses significant antitumor activity, exerting its effects through multiple mechanisms, including inducing tumor cell apoptosis, inhibiting angiogenesis, and blocking cell migration. It exhibits good inhibitory effects against various malignant tumors, including lung cancer, liver cancer, and breast cancer. Furthermore, dendrophenol also possesses a wide range of pharmacological functions, including anti-inflammatory, antioxidant, neuroprotective, and immunomodulatory effects, showing unique advantages, particularly in protecting retinal nerve cells and improving memory. Toxicological evaluations have confirmed that dendrobine has good safety within the effective dose range. Therefore, it can not only be developed as a lead compound for adjuvant anti-tumor drugs, but also as an active ingredient in functional health foods, anti-aging cosmetics, and special medical purpose formula foods, with extremely broad market application prospects.

[0003] Currently, the main methods for obtaining dendrobium phenols are plant extraction and chemical total synthesis. In terms of plant extraction, Dendrobium huoshanense is a perennial epiphytic herb belonging to the genus Dendrobium of the Orchidaceae family. It is naturally distributed on rocks or tree trunks under forests at an altitude of 300-900 meters in the Dabie Mountains. Its growth conditions are harsh, its natural reproduction rate is extremely low, and its growth cycle is long (usually 3-5 years before it can be harvested). In addition, wild Dendrobium huoshanense has been listed as a national second-class key protected wild plant, and its harvesting is strictly prohibited. Although artificial cultivation has made some progress, the content of dendrobium phenols in cultivated plants is extremely low (usually only 0.001%~0.01% of dry weight) (Khodzhaeva M, etal. Bibenzyls of Dendrobium huoshanense. Chemistry of Natural Compounds,2024, 60(4): 563-567). The extraction and separation steps are cumbersome, the solvent consumption is large, and the total yield is low, making it difficult to meet the needs of large-scale production. In terms of total chemical synthesis, since dendrobine contains an asymmetric substitution mode of two phenolic hydroxyl groups and one methoxyl group, total synthesis requires multi-step selective protection and deprotection strategies. Some steps involve noble metal catalysts (such as palladium-catalyzed cross-coupling reactions) or environmentally unfriendly reagents. The overall yield is generally less than 15%, the production cost is high and a large amount of chemical waste is generated, which is not in line with the concept of green chemistry and sustainable development.

[0004] With the rapid development of synthetic biology and metabolic engineering, the heterologous production of plant natural products using microbial cell factories has become an important means of overcoming resource bottlenecks. In Dendrobium species, the biosynthesis of bibenzyl compounds begins with the phenylpropane metabolic pathway: phenylalanine is deaminated by phenylalanine ammonia-lyase (PAL) to generate cinnamic acid, which is then hydroxylated by cinnamic acid-4-hydroxylase (C4H) to give 4-coumaric acid. 4-Coumaric acid is then catalyzed by 4-coumaric acid-coenzyme A ligase (4CL) to generate 4-coumaroyl-CoA. Subsequently, the key bibenzyl synthase (BBS), using 4-coumaryl-CoA as a starting substrate, undergoes iterative condensation with three molecules of malonyl-CoA, Claisen cyclization, and decarboxylation to generate the bibenzyl skeleton intermediate—dihydropinosylvin. The upstream step of this pathway—from phenylalanine to dihydropiperidine—has been well validated at both the genetic and enzymatic levels in species such as Dendrobium officinale (Zhou SG, et al. Functional characterization of a bibenzyl synthase DoBS1 involved in bibenzyl biosynthesis in Dendrobium officinale. Molecules, 2024,29(22): 5320). However, the biological activity and commercial application value of dihydropiperidine itself are limited, and it needs to be further catalyzed by downstream modifying enzymes (mainly O-methyltransferase, OMT) to introduce a methoxy functional group and convert it into polymethoxybibenzyl compounds with higher pharmacological activity.

[0005] The conversion of dihydrotaxyl alcohol to dendrobine requires a two-step sequential O-methylation reaction. For example... Figure 1As shown, the first step is catalyzed by methyltransferase OMT1 (DhOMT1 in this invention, derived from the Dendrobium huoshanense genome), using S-adenosylmethionine (SAM) as a methyl donor, selectively methylating dihydropiperidine at the 5'-OH position to generate the key intermediate 3,4,3'-trihydroxy-5'-methoxybibenzyl; the second step is catalyzed by methyltransferase OMT2 (DhOMT2), further methylating at the 3-OH position to finally generate dendrobin. It is worth noting that both OMT-catalyzed methylation reactions have strict regioselectivity: OMT1 only recognizes the 5'-OH site and does not act on the 3-OH site at all, while OMT2 specifically recognizes the 3-OH site (Ta H, et al. Catalytic divergence of O-methyltransferases shapes the chemo-diversity of polymethoxylated bibenzyls in Dendrobium catenatum. The Plant Journal, 2024, 110(3): e16962). Compared to the cumbersome protection and deprotection strategies in chemical synthesis, OMT-mediated methylation reactions have significant advantages such as high regioselectivity, mild reaction conditions (30~37°C, pH 7~8), and no need for toxic or harmful catalysts, making it an ideal green biocatalytic method.

[0006] However, the wild-type DhOMT1 enzyme has low catalytic efficiency for dihydrotaurine, so it is necessary to develop a wild-type DhOMT1 enzyme with higher catalytic efficiency. Summary of the Invention

[0007] This invention addresses the problem of low activity in existing methyltransferases by screening and developing a methyltransferase from *Dendrobium huoshanense* with high catalytic efficiency. To further improve the efficiency of the dendrobium phenol biosynthesis pathway, targeted modification was performed using protein engineering techniques. Specifically, by site-directed mutagenesis of amino acids at the substrate binding pocket, three *Dendrobium huoshanense* methyltransferase mutants with high catalytic activity for dihydropiperidine were obtained, significantly increasing the yield of 3,4,3'-trihydroxy-5'-methoxybibenzyl.

[0008] To solve the above-mentioned technical problems, the first objective of this invention is to disclose a Dendrobium huoshanense methyltransferase, the amino acid sequence of which is shown in SEQ ID NO.1.

[0009] A second objective of this invention is to provide a Dendrobium huoshanense methyltransferase mutant. Specifically, this enzyme mutant mutates serine at position 128 of the amino acid sequence shown in SEQ ID NO.1 to alanine, and the coding gene sequence of the S128A mutant is shown in SEQ ID NO.2; or mutates threonine at position 314 of the amino acid sequence shown in SEQ ID NO.1 to alanine, and the coding gene sequence of the T314A mutant is shown in SEQ ID NO.3; or mutates both serine at position 128 and threonine at position 314 of the amino acid sequence shown in SEQ ID NO.1 to alanine, and the coding gene sequence of the S128A / T314A mutant is shown in SEQ ID NO.4.

[0010] A third objective of this invention is to provide a gene encoding the aforementioned Dendrobium huoshanense methyltransferase or a mutant thereof.

[0011] A fourth objective of this invention is to provide an expression cassette or expression vector carrying the aforementioned genes.

[0012] The fifth objective of this invention is to provide a microorganism that expresses the above-mentioned Dendrobium huoshanense methyltransferase or its mutant, and carries the above-mentioned gene or the above-mentioned expression cassette or expression vector.

[0013] As a specific example, the microorganisms include, but are not limited to, genetically engineered bacteria.

[0014] A sixth objective of this invention is to disclose a catalyst comprising or expressing the aforementioned Dendrobium huoshanense methyltransferase or its mutant. The catalyst may be Dendrobium huoshanense methyltransferase or its mutant itself, or may be cells, genetically engineered bacteria, etc., expressing Dendrobium huoshanense methyltransferase or its mutant.

[0015] A seventh objective of this invention is to provide the application of the above-mentioned Dendrobium huoshanense methyltransferase or its mutant, the above-mentioned gene, the above-mentioned expression cassette or expression vector, the above-mentioned microorganism or the above-mentioned catalyst in the synthesis of the key intermediate 3,4,3'-trihydroxy-5'-methoxybibenzyl of Dendrobium phenols.

[0016] In a preferred embodiment, in the presence of SAM and DTT, the substrate dihydrotaxyl alcohol undergoes a methylation reaction catalyzed by Dendrobium huoshanense methyltransferase or its mutant to generate 3,4,3'-trihydroxy-5'-methoxybibenzyl. 3,4,3'-trihydroxy-5'-methoxybibenzyl is an important precursor for the synthesis of dendrobine.

[0017] Furthermore, the reaction temperature was 30-40℃, the concentration of the substrate dihydrotapertanol was 0.1-20 mM, the concentration of SAM was 0.2-30 mM, and the concentration of DTT was 2-250 mM.

[0018] The eighth objective of this invention is to disclose a method for the catalytic synthesis of 3,4,3'-trihydroxy-5'-methoxybibenzyl, a key intermediate of dendrobine, wherein, in the presence of SAM and DTT, the substrate dihydropiperidine undergoes a methylation reaction catalyzed by the aforementioned Dendrobium huoshanense methyltransferase or its mutant to generate 3,4,3'-trihydroxy-5'-methoxybibenzyl.

[0019] As a specific example, the synthesis process of 3,4,3'-trihydroxy-5'-methoxybibenzyl is as follows: In the presence of S-adenosylmethionine (SAM) and dithiothreitol (DTT), the substrate dihydropiperidine undergoes a methylation reaction to generate 3,4,3'-trihydroxy-5'-methoxybibenzyl under the catalysis of Dendrobium huoshanense methyltransferase or its mutant. Specifically, an enzyme solution of the methyltransferase or its mutant is first prepared, and then added to the substrate dihydropiperidine, mixed with SAM and DTT. The methylation reaction is carried out in the mixed system to obtain 3,4,3'-trihydroxy-5'-methoxybibenzyl.

[0020] The enzyme solution can be a crude enzyme solution of genetically engineered bacteria expressing the Dendrobium huoshanense methyltransferase or its mutant, or a purified enzyme solution.

[0021] The beneficial technical effects achieved by this invention are as follows: The Dendrobium huoshanense methyltransferase (WT) screened in this invention catalyzed the reaction of dihydropiperidine with 1,4,3'-trihydroxy-5'-methoxybibenzyl with 1.49 g / L after 6 h. This Dendrobium huoshanense methyltransferase has high catalytic activity and efficiency, and good reaction selectivity, generating a single methylated product, 3,4,3'-trihydroxy-5'-methoxybibenzyl.

[0022] The methyltransferase mutant of *Dendrobium huoshanense* obtained in this invention exhibits significantly improved catalytic efficiency for dihydropascualhin, with relative activities increasing by 155.16%, 168.64%, and 208.5% compared to the wild type, respectively. The yield also increased from 1.49 g / L in the wild type to 1.67, 1.81, and 2.58 g / L, respectively. It is capable of generating a single methylated product, 3,4,3'-trihydroxy-5'-methoxybibenzyl.

[0023] This invention successfully solves the key problems of difficult expression and low activity of existing methyltransferases in the preparation process of 3,4,3'-trihydroxy-5'-methoxybibenzyl. The wild-type enzyme and mutant obtained by this invention have high conversion rate, strong selectivity, mild reaction conditions and few by-products, showing great promise for industrial application. Attached Figure Description

[0024] Figure 1Schematic diagram of the reaction catalyzed by methyltransferase to produce dendrobine from dihydrotaxyl alcohol; Figure 2 The overexpression effect of methyltransferase DhOMT1 in Escherichia coli was analyzed by SDS-PAGE; where M is a marker; 1 is the lysate after induction; 2 is the supernatant after induction; and 3 is the whole cell lysate after induction. Figure 3 This refers to all amino acid residues within a 4A range near the substrate binding pocket of DhOMT1; Figure 4 The PCR band represents a site-directed mutation of the DhOMT1 methyltransferase recombinant plasmid. Figure 5 SDS-PAGE protein electrophoresis of purified WT and 12 mutant enzymes; Figure 6 The relative catalytic activities of WT and 12 mutants of DhOMT1; Figure 7 The relative activities of WT, S128A, T314A and S128A / T314A for DhOMT1; Figure 8 The HPLC chromatogram of dihydropiperidine, a substrate catalyzed by the DhOMT 1 double mutant methyltransferase; Figure 9 Time curves for the methyltransferase DhOMT 1 and its mutants to catalyze the production of 3,4,3'-trihydroxy-5'-methoxybibenzyl; Figure 10 The 1H NMR spectrum of 3,4,3'-trihydroxy-5'-methoxybibenzyl is shown. Figure 11 The 13C nuclear magnetic resonance spectrum of 3,4,3'-trihydroxy-5'-methoxybibenzyl is shown.

[0025] Figure 12 The HSQC (¹H-¹³C correlation spectrum) of 3,4,3'-trihydroxy-5'-methoxybibenzyl is shown.

[0026] Figure 13 HMBC (long-range ¹H-¹³C correlation spectrum) of 3,4,3'-trihydroxy-5'-methoxybibenzyl.

[0027] Figure 14 The ¹H-¹H COSY (hydrogen-hydrogen ortho-coupled spectrum) of 3,4,3'-trihydroxy-5'-methoxybibenzyl is shown.

[0028] Figure 15 NOESY (space hydrogen correlation spectrum) for 3,4,3'-trihydroxy-5'-methoxybibenzyl. Detailed Implementation

[0029] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0030] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0031] Unless otherwise specified, the experimental methods described in this invention are conventional methods. For specific gene cloning operations, please refer to "Molecular Cloning: A Laboratory Manual" edited by J. Sambrook et al.

[0032] The DNA polymerase (2×Phanta Max Master Mix), Dpn I enzyme, recombinant cloning kit, and plasmid extraction kit used in the embodiments of this invention were all purchased from Takara Biotechnology Co., Ltd.; gene and primer synthesis and gene sequencing were performed by General Biotechnology (Anhui) Co., Ltd., and the usage methods of the above reagents are as per the product instructions.

[0033] The expression vector involved in this invention is pET-28a(+), and the host used is Escherichia coli BL21(DE3), both of which are preserved in the laboratory of Dabieshan Institute of Traditional Chinese Medicine, Anhui University of Science and Technology, where the inventors are located.

[0034] All dihydropiperidine used in this invention was purchased from Anhui Xierrui Chemical Co., Ltd., SAM was purchased from Shanghai Yien Chemical Technology Co., Ltd., and DTT was purchased from Shanghai Maclean Biochemical Technology Co., Ltd.

[0035] The amino acid three-letter or single-letter expression used in this invention adopts the amino acid code specified by IUPAC (Eur.J.Biochem.,138:9-37,1984).

[0036] Example 1: Cloning of Dendrobium huoshanense methyltransferase

[0037] The Dendrobium huoshanense material used in this embodiment was collected from Huoshan County, Anhui Province, and identified as Dendrobium huoshanense, a plant belonging to the genus Dendrobium of the Orchidaceae family. Using the reported Dendrobium officinale methyltransferase gene sequence (GenBank accession number: XM 020829677.2) as a query probe, a comparative analysis was performed on the Dendrobium huoshanense genome sequencing and assembly data previously completed by the applicant team (Han BX, Yi J, Dai J, et al. A chromosome-level genome assembly of Dendrobium huoshanense using long reads and Hi-C data [J]. Genome Biol Evol,2020, evaa215.), from which a complete candidate gene sequence of Dendrobium huoshanense methyltransferase was obtained.

[0038] Based on the open reading frame of this sequence, specific amplification primers DhOMT1-F and DhOMT1-R (sequences shown in Table 1) were designed. Using Dendrobium huoshanense genomic DNA as a template, PCR amplification was performed using the above primers to obtain the complete coding region sequence of the Dendrobium huoshanense methyltransferase gene. Sequencing results showed that the gene sequence encoded by this gene is shown in SEQ ID NO.1 and is named WT-DhOMT1.

[0039] The amplified DhOMT1 gene was ligated between the BamHI / HindⅢ linearization sites of the pET-28a(+) plasmid using a one-step cloning kit (ClonExpress II One Step Cloning Kit), and a His-tag purification tag was added to the C-terminus to construct the recombinant plasmid pET28a-DhOMT1. To ensure correct insertion direction and accurate reading frames, the recombinant plasmid was identified by colony PCR and sequenced using pET-28a-F and pET-28a-R primers of the pET-28a vector. The sequence-verified recombinant plasmid was transformed into E. coli BL21(DE3) competent cells, and positive clones were screened and detected to obtain recombinant engineered bacteria containing the DhOMT1 gene.

[0040] Table 1 Primers used to clone methyltransferases

[0041] Note: The lowercase part of the primer is the homologous sequence to the vector. SEQ ID NO.1 MDSESHQIQEEIEACNHALKICSMAVLPMTLTAAIELGLFKLLSAAGPGAQLSAEEFAAKLINDSTSDYTALKLERILRLLASYSVLTWSTETDSHGRTVHRFGASPVCKFLAPNEDGVSLAALGLMSTDRVAIESWYHLKDAILHGGVAFNRAHGGLNEFEYHAKDPRFSKVFNEGMRSHSIIMKNILKIYKGFEDINVLVDVGGGTGGTLSMITAKHSHIKGINFDLPHVIAQAPSLPGVEHVGGDVFESIPSGEALLLKWVLHDWGDEDSLKILKNCWKSVPENGKVIVIDSFLPEAPDYTDLTHCVLNTDVIMLLESPSGKERTEKEFRELAKLSGFSGFNVICNFSNAWIMELSK SEQ ID NO.2 SEQ ID NO.3 SEQ ID NO.4

[0042] Example 2 Expression and purification of recombinant methyltransferase DhOMT1

[0043] The recombinant engineered bacterial strain obtained in Example 1 was inoculated into 2 mL of LB liquid medium containing 50 μg / mL kanamycin and cultured on a shaker at 37°C and 200 rpm for 12 hours. 1 mL of the culture was added to a 250 mL Erlenmeyer flask containing 50 mL of LB medium and cultured aerobically at 37°C and 200 rpm. When the OD600 reached approximately 0.6-0.8, cells were induced with 0.1 mM IPTG. Cells were harvested after induction at 16°C for 24 hours. The cells were centrifuged, resuspended in 50 mM pH 7.5 potassium phosphate buffer, and disrupted by sonication. The cell extract was centrifuged at 12000 rpm for 20 minutes, and the supernatant was the crude enzyme solution. The supernatant and precipitate were analyzed by SDS-PAGE to detect DhOMT1 expression. The results are as follows: Figure 2 As shown, the content of the target protein in the supernatant is comparable to the total target protein content, proving that DhOMT1 can be efficiently and solublely expressed in Escherichia coli.

[0044] The recombinant methyltransferase DhOMT1 was purified using nickel column affinity chromatography. 1) Sample pretreatment: The bacterial cells were resuspended in loading buffer (50mM pH 7.5 phosphate buffer, 150mM NaCl), sonicated in an ice bath, centrifuged at 4℃ and 12000rpm to remove cell debris, and the supernatant was filtered through a 0.22μm filter membrane. 2) Loading: Rinse the nickel column with 10 column volumes of loading buffer, and slowly load the pretreated sample from (1) onto the column to ensure that the protein is fully bound to the nickel column; 3) Washing: Wash the nickel column with 10 column volumes of washing buffer (20 mM pH 7.5 phosphate buffer, 50 mM imidazole, 100 mM imidazole); 4) Target protein elution: Elute the target protein with elution buffer (20 mM pH 7.5 phosphate buffer, 300 mM imidazole); 5) Ultrafiltration and preservation: Transfer the collected eluent to an ultrafiltration tube and desalt and concentrate the protein sample by centrifugation at 4°C; add glycerol to a final concentration of 20% to the ultrafiltration protein sample, flash freeze in liquid nitrogen and store at -80°C.

[0045] Example 3: Activity assay of recombinant methyltransferase DhOMT1

[0046] 0.2 mM dihydropiperidine, 0.5 mM SAM, 4 mM DTT, and 10 μg of purified methyltransferase DhOMT1 protein were added to a final volume of 100 μL with 100 mM Tris–HCl buffer (pH 7.5). The reaction was carried out at 37°C for 3 h, followed by two equal volumes of ethyl acetate extraction to terminate the enzyme production. The bound organic phase was evaporated to dryness, and the residue was dissolved in 100 μL of 80% methanol for product identification. HPLC detection was performed using a C18 column (4.6*250mm, Elite), with a detection wavelength of 230nm or 280nm, a column temperature of 40℃, and a mobile phase of 0.1% formic acid and water and methanol. The flow rate was 0.8mL / min. The reaction solution was analyzed using the following steps: 0-15min 40-80% methanol; 15.1-25min 80-90% methanol; 25.1-30min 100% methanol; 30.1-35min 40% methanol.

[0047] Example 4: Site-directed mutagenesis and activity screening of recombinant methyltransferase DhOMT1

[0048] This invention, through homology modeling and molecular docking, identifies all amino acid residues within the substrate 4A range based on the substrate binding pocket of DhOMT1, including L124, M127, S128, E160, F174, W264, H267, D268, T314, I317, M318, and E321, as shown below. Figure 3 As shown in Table 2, this invention designs specific primers (see Table 2) and uses Quickchange technology (An efficient one-step site-directed and site-saturation mutagenesis protocol[J]. Nucleic Acids Research, 2004, 32(14):e115) to introduce mutations into the wild-type methyltransferase DhOMT1 gene sequence, constructing L124A, M127A, S128A, E160A, F174A, W264A, H267A, D268A, T314A, I317A, M318A and E321A.

[0049] Specifically, the PCR reaction system was as follows: 3 μL ddH2O, 5 μL DNA polymerase, 0.5 μL upstream primer, 0.5 μL downstream primer, and 1 μL template, wherein the template was the recombinant plasmid pET28a-DhOMT1. PCR amplification conditions were: 1) pre-denaturation at 94℃ for 2 min; 2) denaturation at 98℃ for 10 s, annealing at 60℃ for 15 s, extension at 72℃ for 35 s (this step was repeated 30 times); and final extension at 72℃ for 5 min. The PCR results are shown below. Figure 4As shown. The PCR product was digested with Dpn I restriction enzyme at 37°C for 30 minutes. The digested product was then transformed into E. coli BL21(DE3) competent cells, plated on LB / Kan solid plates, and incubated overnight at 37°C.

[0050] Table 2 Primers used in the rational design of DhOMT1 methyltransferase

[0051] After mutation, the mutant was expressed and purified according to Example 2 (purification results are as follows). Figure 5 As shown in Example 3, the activity was determined, and the activities of WT and all mutants were compared to screen for single-point mutants with significantly improved activity. The results showed that the relative activities of S128A and T314A mutants were increased to 155.16% and 168.64% respectively compared to the wild type (relative catalytic activity results are shown in Example 3). Figure 6 (As shown). Therefore, according to Example 4, using the S128A primer as a template, the T314A mutant was mutated to construct the S128A / T314A co-mutant. After mutation, the S128A / T314A mutant was expressed and purified according to Example 2, and its activity was determined according to Example 3. The relative catalytic activity results of the co-mutant (S128A / T314A) are as follows. Figure 7 As shown in the figure. Ultimately, the relative activity of 3,4,3'-trihydroxy-5'-methoxybibenzyl was 208.5%. These results demonstrate that the engineered DhOMT1 has significant practical application value in the synthesis of 3,4,3'-trihydroxy-5'-methoxybibenzyl.

[0052] Example 5 Application of wild type and its mutants in the preparation of 3,4,3'-trihydroxy-5'-methoxybibenzyl

[0053] The reaction mixture consisted of 100 mM Tris-HCl (pH 7.5), 10 mM dihydrotapertanol, 25 mM SAM, 0.2 M DTT, and 2.0% induced *E. coli* wet cells (expressing wild-type or mutant DhOMT1 methyltransferase; 2% refers to the mass percentage of induced *E. coli* wet cells in the reaction system), with a total reaction volume of 10 mL. The reaction was carried out at 37°C and 200 rpm for 6 h. Samples were taken periodically during the reaction, and the reaction progress was monitored using the detection method described in Example 3. The results are as follows: Figure 9As shown, the conversion rates of wild-type and mutant S128A, T314A, and S128A / T314A after 6 h of reaction were 57.5%, 64.8%, 69.5%, and 99.5%, respectively, indicating that the catalytic efficiency of the mutant was significantly improved. Compared with the wild type, the yield of 3,4,3'-trihydroxy-5'-methoxybibenzyl also increased from 1.49 g / L in the wild type to 1.67, 1.81, and 2.58 g / L, respectively, with the highest space-time yield reaching 0.43 g / L / h.

[0054] For example, Figure 8 The top image shows the results of the catalytic reaction of the Dendrobium huoshanense methyltransferase double mutant S128A / T314A, and the bottom image shows the collected 3,4,3'-trihydroxy-5'-methoxybibenzyl, which was verified to have a purity >98%.

[0055] Example 6 Product Identification

[0056] The product obtained in Example 5 was purified by semi-preparative high-performance liquid chromatography (HPLC) using a C18 semi-preparative column. The column temperature was 40°C, the mobile phase was 0.1% formic acid, water, and methanol, and the flow rate was 2 mL / min. The flow rates were: 0-25 min 10-90% methanol; 25.1-30 min 90-50% methanol; 30.1-33 min 50-10% methanol; 33.1-35 min 10% methanol. The collected 3,4,3'-trihydroxy-5'-methoxybibenzyl was concentrated by rotary evaporation and freeze-dried. The resulting powder was dissolved in DMSO-d6 and subjected to further processing. 1 H and 13 ¹H NMR spectroscopy, HSQC, HMBC, ¹H-¹H COSY, and NOESY analyses showed that the products of wild-type and mutant S128A, T314A, and S128A / T314A were all 3,4,3'-trihydroxy-5'-methoxybibenzyl, and only the 5'-O-methylated product was generated. The product results for the S128A / T314A mutant are shown below. Figure 10-15 The product is 3,4,3'-trihydroxy-5'-methoxybibenzyl, with no 3-O-methylation byproducts produced.

[0057] The Dendrobium huoshanense methyltransferase (WT) screened by this invention achieved a yield of 1.49 g / L of 3,4,3'-trihydroxy-5'-methoxybibenzyl after 6 h of catalytic reaction with dihydropiperidine. This Dendrobium huoshanense methyltransferase has high catalytic activity and efficiency, and good reaction selectivity.

[0058] Furthermore, this invention uses the O-methyltransferase DhOMT1 from *Dendrobium huoshanense* as the target for modification. Through a site-directed mutagenesis strategy guided by structure analysis, three mutants with significantly enhanced catalytic efficiency were successfully obtained—S128A (single mutant), T314A (single mutant), and S128A / T314A (double mutant). A highly efficient enzymatic synthesis method for 3,4,3'-trihydroxy-5'-methoxybibenzyl was established based on these mutants. Experimental results show that, compared with the wild type, the relative catalytic activities of the S128A, T314A, and S128A / T314A mutants for dihydrotaxane increased to 155.16%, 168.64%, and 208.5%, respectively, and the yield increased from 1.49 g / L in the wild type to 1.67, 1.81, and 2.58 g / L, respectively. Moreover, the regioselectivity of the product remained unchanged, generating only the 5'-O-methylated product without the formation of any 3-O-methylated byproduct. This invention lays a key technological foundation for the green, efficient, and sustainable enzymatic synthesis of dendrobine and its downstream bibenzyl active ingredients, and has good prospects for industrial application.

[0059] The present invention has been disclosed above with reference to preferred embodiments, but it is not intended to limit the present invention. All technical solutions obtained by adopting equivalent substitutions or equivalent transformations fall within the protection scope of the present invention.

Claims

1. A methyltransferase from Dendrobium huoshanense, characterized in that, The amino acid sequence of this Dendrobium huoshanense methyltransferase is shown in SEQ ID NO.

1.

2. A methyltransferase mutant of Dendrobium huoshanense, characterized in that: The enzyme mutant mutates serine at position 128 of the amino acid sequence shown in SEQ ID NO.1 to alanine; or threonine at position 314 of the amino acid sequence shown in SEQ ID NO.1 to alanine; or mutates both serine at position 128 and threonine at position 314 of the amino acid sequence shown in SEQ ID NO.1 to alanine.

3. The gene encoding the Dendrobium huoshanense methyltransferase of claim 1 or the Dendrobium huoshanense methyltransferase mutant of claim 2.

4. An expression cassette or expression vector carrying the gene of claim 3.

5. Microorganisms expressing the Dendrobium huoshanense methyltransferase of claim 1 or the Dendrobium huoshanense methyltransferase mutant of claim 2, carrying the gene of claim 3 or the expression cassette or expression vector of claim 4.

6. The microorganism according to claim 5, characterized in that: The microorganisms mentioned are genetically engineered bacteria.

7. A catalyst, characterized in that: It contains or expresses the Dendrobium huoshanense methyltransferase of claim 1 or the Dendrobium huoshanense methyltransferase mutant of claim 2.

8. The use of the Dendrobium methyltransferase of claim 1 or the Dendrobium methyltransferase mutant of claim 2, the gene of claim 3, the expression cassette or expression vector of claim 4, the microorganism of any one of claims 5-6 or the catalyst of claim 7 in the synthesis of the key intermediate 3,4,3'-trihydroxy-5'-methoxybibenzyl of Dendrobium phenols.

9. The application according to claim 8, characterized in that: In the presence of SAM and DTT, the substrate dihydropiperidine undergoes methylation under the catalysis of Dendrobium huoshanense methyltransferase or a Dendrobium huoshanense methyltransferase mutant to generate 3,4,3'-trihydroxy-5'-methoxybibenzyl; the reaction temperature is 30-40℃, and the concentration of the substrate dihydropiperidine is 0.1-20 mM.

10. A method for the catalytic synthesis of 3,4,3'-trihydroxy-5'-methoxybibenzyl, a key intermediate in dendrobium phenols, characterized in that: In the presence of SAM and DTT, the substrate dihydropiperidine undergoes a methylation reaction to generate 3,4,3'-trihydroxy-5'-methoxybibenzyl under the catalysis of the Dendrobium huoshanense methyltransferase of claim 1 or the Dendrobium huoshanense methyltransferase mutant of claim 2.

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