A method for heterologous biosynthesis of gastrodin, a recombinant expression vector, a recombinant agrobacterium and an extract

Abstract

The application provides a method for biosynthesis of heterologous gastrodin, a rapid and efficient transient expression system of Nicotiana benthamiana is used, multiple key enzyme genes in a gastrodin biosynthesis pathway from gastrodia elata are introduced into plant cells at the same time, and the complete conversion from aromatic compounds to 4-hydroxybenzyl alcohol and then to gastrodin is realized by means of metabolized precursors, cofactors and organelle environment of the plant cells. Compared with traditional extraction, chemical synthesis or microbial fermentation, the method has the advantages of short cycle (4-6 days), simple operation, no need for stable transgene, complete eukaryotic modification environment, easy multi-gene coordinated expression and the like, so that the rapid, green and scalable production of gastrodin is realized. In addition, the application also provides a recombinant expression vector, a recombinant agrobacterium and an extract.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and specifically relates to a method for heterologous biosynthesis of gastrodin, a recombinant expression vector, recombinant Agrobacterium and its extract. Background Technology

[0002] Gastrodin (chemical name 4-hydroxybenzyl alcohol-4-O-β-D-glucopyranoside) is a representative phenolic glycoside active ingredient from the orchid Gastrodia elata. It has various pharmacological activities such as neuroprotection, sedation, anticonvulsant, and improvement of learning and memory, and has important application value in the fields of medicine and functional products.

[0003] Currently, the main methods for preparing gastrodin include plant extraction, chemical synthesis, and biosynthesis.

[0004] Plant extraction typically uses Gastrodia elata tubers as raw material, which are crushed, extracted with solvents, and purified to obtain gastrodin. While this method is relatively mature, it is highly dependent on natural or artificially cultivated Gastrodia elata resources, and suffers from problems such as long cultivation cycles, significant influence from the environment and origin, unstable raw material supply, and large batch-to-batch product variations.

[0005] Chemical synthesis typically uses p-hydroxybenzyl alcohol or related aromatic intermediates as starting materials to obtain gastrodin through glycosylation. While this method avoids dependence on plant resources, it usually involves multiple reaction steps, protection / deprotection, and complex post-processing, resulting in problems such as long process routes, insufficient greenness, and numerous byproducts.

[0006] Biosynthesis mainly includes microbial fermentation and enzymatic catalysis. Microbial fermentation generally introduces relevant metabolic pathways into hosts such as Escherichia coli and yeast to synthesize 4-hydroxybenzyl alcohol and its glycoside products. Enzymatic catalysis mainly utilizes glycosyltransferases to perform in vitro glycosylation reactions on substrates. Although biosynthesis has advantages such as mild conditions and high designability, microbial systems still have certain limitations in terms of the expression and activity retention of plant-derived enzymes, multi-enzyme synergistic efficiency, adaptation to eukaryotic metabolic environments, and compatibility with complex substrates.

[0007] In recent years, the production of recombinant proteins and natural products using the transient expression system of *Nicotiana benthamiana* has become an important direction in plant synthetic biology. *Nicotiana benthamiana* possesses advantages such as rapid growth, large leaf biomass, high Agrobacterium-mediated transformation efficiency, suitability for multi-gene co-expression, and a complete eukaryotic post-translational modification environment, making it an ideal host for reconstructing plant natural product pathways. Existing studies have utilized *Nicotiana benthamiana* to achieve heterologous synthesis of various plant natural products, but these mainly focus on terpenes, alkaloids, and flavonoids. For the synthesis of phenolic glycosides, especially gastrodin, a mature heterologous biosynthesis scheme is still lacking.

[0008] The prior art most similar to this invention includes: the traditional process of directly extracting gastrodin from Gastrodia elata; a biosynthetic scheme for introducing gastrodin synthesis-related enzymes into a microbial host to achieve the accumulation of 4-hydroxybenzyl alcohol and its glucosides; an enzymatic catalytic scheme for in vitro glycosylation of 4-hydroxybenzyl alcohol using glycosyltransferases; and a plant transient expression technology for reconstructing other natural product pathways using Nicotiana benthamiana. However, the prior art lacks a heterologous accumulation technology scheme that uses Nicotiana benthamiana as a host, introduces key genes from Gastrodia elata such as GeCNL, GeCHD, GeKAT, GeBSa, GeBSb, and GeUGT, and achieves complete transformation from upstream aromatic precursors to gastrodin in plant cells. Therefore, developing a rapid, efficient, green, and scalable heterologous biosynthesis method for gastrodin has significant scientific research value and industrial application prospects. Summary of the Invention

[0009] In view of this, the present invention provides a method for heterologous biosynthesis of gastrodin, as well as a recombinant expression vector, recombinant Agrobacterium, host plant tissue and their applications for implementing the method, thereby achieving rapid, green and scalable production of gastrodin.

[0010] To solve the above problems, this application adopts the following technical solution: One of the objectives of this application is to provide a method for heterologous biosynthesis of gastrodin, comprising the following steps: a) Obtain key genes related to gastrodin biosynthesis, including: the TAL gene encoding tyrosine ammonia-lyase, the GeCNL gene encoding the catalytic enzyme activating aromatic acid coenzyme A, the GeCHD gene encoding the chain shortening-related enzyme, the GeKAT gene encoding the β-oxidation-related chain shortening enzyme, the GeBSa and GeBSb genes encoding the key enzyme subunit for 4-hydroxybenzaldehyde formation, and the GeUGT gene encoding the uridine diphosphate-dependent glycosyltransferase that can catalyze the glycosylation of 4-hydroxybenzyl alcohol to gastrodin, or their functional homologs; b) Construct one or more genes obtained in step a) into vectors suitable for plant cell expression, either individually or in combination, to obtain recombinant plant expression vectors; c) The recombinant plant expression vector obtained in step b) is introduced into Agrobacterium to obtain a recombinant Agrobacterium strain; d) The recombinant Agrobacterium strain obtained in step c) is introduced into tobacco leaves by the infiltration method, so that the gene is transiently expressed in the plant cells; e) The *Nicotiana benthamiana* plants soaked in step d) are cultured under suitable conditions to allow the enzyme encoded by the gene to catalyze the conversion of the substrate into gastrodin and accumulate in the leaves; f) Extract and analyze the tobacco leaves cultured in step e) to determine the formation of gastrodin.

[0011] In some embodiments, the GeCNL, GeCHD, GeKAT, GeBSa, GeBSb, and GeUGT genes are derived from Gastrodia elata. Preferably, the encoding sequence of GeCNL is shown in SEQ ID NO:1, the encoding sequence of GeCHD is shown in SEQ ID NO:2, the encoding sequence of GeKAT is shown in SEQ ID NO:3, the encoding sequence of GeBSa is shown in SEQ ID NO:4, and the encoding sequence of GeBSb is shown in SEQ ID NO:5; Preferably, the GeUGT is selected from GeUGT85BT1 and / or GeUGT72BK5, and their encoding sequences are shown in SEQ ID NO:6 and SEQ ID NO:7, respectively; More preferably, the functional homolog is a protein-coding gene that satisfies the following conditions: (i) Derived from species in the genus Gastrodia or closely related species in the family Orchidaceae; (ii) Its coding sequence has at least 95% sequence homology with the corresponding gene sequence shown in SEQ ID NO:1-7; (iii) Verified by in vitro enzyme activity assay or transient expression system of Nicotiana benthamiana, it retains the same catalytic function as the protein encoded by the sequence shown in SEQ ID NO:1-7.

[0012] In some embodiments, the immersion method described in step d) is selected from leaf injection immersion, vacuum immersion, or spray-assisted immersion; and / or, the culture conditions described in step e) are: temperature 20–28°C, photoperiod of 12–16 hours light / 8–12 hours darkness, relative humidity 40%–80%, and culture time of 3–7 days; and / or, the extraction and detection methods described in step f) include: using methanol, ethanol, water, or acetonitrile as extraction solvents, and using high performance liquid chromatography or liquid chromatography-mass spectrometry for detection.

[0013] In some embodiments, the plant expression vector in step b) is a pEAQ-HT, pCAMBIA series, pBI series, pGreen series, or Gateway series vector; and / or, the vector contains an operatively linked promoter and terminator, wherein the promoter is preferably a CaMV 35S promoter and the terminator is preferably a Nos terminator.

[0014] In some embodiments, the Agrobacterium strain described in step c) is LBA4404, GV3101, or EHA105.

[0015] In some embodiments, the recombinant Agrobacterium strain described in step d) is resuspended in an infiltration buffer before infiltration, the infiltration buffer comprising: 5–20 mM MgCl2, 5–20 mM MES and 50–200 μM acetylsylgenone, pH 5.2–5.8; the total bacterial culture OD600 of the recombinant Agrobacterium strain mixture is 0.1–1.0, more preferably 0.3–0.8.

[0016] In some embodiments, the method further includes adding one or more precursors selected from 4-hydroxybenzaldehyde, 4-hydroxybenzyl alcohol, p-coumaric acid, p-hydroxybenzoic acid, or derivatives thereof to the leaves or culture system during the cultivation process or before extraction.

[0017] A second objective of this application is to provide a recombinant expression vector for use in any of the methods described herein, wherein the recombinant expression vector comprises one or more genes selected from GeCNL, GeCHD, GeKAT, GeBSa, GeBSb, GeUGT85BT1, and GeUGT72BK5, or their functional homologs.

[0018] In some embodiments, the recombinant expression vector is a single-gene expression vector, a dual-gene co-expression vector, or a multi-gene tandem expression vector; the vector backbone is pEAQ-HT.

[0019] The third objective of this application is to provide a recombinant Agrobacterium, wherein the recombinant Agrobacterium contains the aforementioned recombinant expression vector.

[0020] The fourth objective of this application is to provide a host plant tissue or cell, wherein the host plant tissue or cell is a leaf tissue of *Tobacco Bengal* after introduction of the recombinant *Agrobacterium* as described in claim 10, or a protoplast isolated therefrom; the host plant tissue or cell further comprises genes related to the gastrodin biosynthesis pathway derived from *Gastrodia elata* that are stably or transiently integrated.

[0021] Fifthly, this application provides a system for the heterologous biosynthesis of gastrodin in *Nicotiana benthamiana*, comprising: The gene acquisition module is used to acquire the GeCNL, GeCHD, GeKAT, GeBSa, GeBSb and GeUGT genes; A vector construction module is used to construct the gene into a plant expression vector; Agrobacterium-mediated transformation module, used to introduce recombinant plant expression vectors into Agrobacterium; Plant infiltration module for introducing recombinant Agrobacterium tumefaciens into tobacco leaves; The expression culture module is used to culture infiltrated plant tissues to express the target product. An extraction and detection module is used to extract and detect gastrodin from plant tissues; The purification module is used to purify gastrodin from plant extracts.

[0022] In some embodiments, the method, the recombinant expression vector, the recombinant Agrobacterium, the host plant tissue or cell, or the preparation system are used in the preparation of gastrodin, verification of the function of key enzymes in the gastrodin biosynthetic pathway, or screening of gastrodin glycosyltransferases.

[0023] In some embodiments, the method, the recombinant expression vector, the recombinant Agrobacterium, the host plant tissue or cell, or the preparation system are used in the production of gastrodin analogs or gastrodin glycosylation derivatives.

[0024] The sixth objective of this application is to provide an extract or purified product containing gastrodin, said extract or purified product being prepared by any of the methods or preparation systems described herein.

[0025] The present application adopts the above technical solution, and its beneficial effects are as follows: This application provides a method for heterologous biosynthesis of gastrodin, utilizing a rapid and efficient transient expression system from *Nicotiana benthamiana* to simultaneously introduce multiple key enzyme genes from the gastrodin biosynthetic pathway into plant cells. Leveraging the plant cell's own metabolic precursors, cofactors, and organelle environment, the complete transformation from aromatic compounds to 4-hydroxybenzyl alcohol, and then to gastrodin, is achieved. Compared to traditional extraction, chemical synthesis, or microbial fermentation methods, this method offers significant advantages such as a shorter cycle time (4-6 days), simpler operation, no need for stable transgenic structures, a complete eukaryotic modification environment, and easy multi-gene synergistic expression, thus enabling rapid, green, and scalable production of gastrodin. Attached Figure Description

[0026] To more clearly illustrate the technical solutions of the embodiments of this application, the drawings used in the description of the embodiments of this application or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1 A flowchart illustrating the steps of a method for heterologous biosynthesis of gastrodin provided in an embodiment of the present invention.

[0028] Figure 2 A technical roadmap for heterologous biosynthesis of gastrodin from Tobacco Benzoinus provided for embodiments of the present invention.

[0029] Figure 3 This is a schematic diagram of the construction of the GeCNL plant expression vector provided in Example 1 of the present invention.

[0030] Figure 4 This is a schematic diagram of the construction of the GeCHD plant expression vector provided in Example 1 of the present invention.

[0031] Figure 5 This is a schematic diagram of the construction of the GeKAT plant expression vector provided in Example 1 of the present invention. Figure 6 This is a schematic diagram of the construction of the GeBSa plant expression vector provided in Embodiment 1 of the present invention.

[0032] Figure 7 This is a schematic diagram of the construction of the GeBSb1 plant expression vector provided in Embodiment 1 of the present invention.

[0033] Figure 8 This is a schematic diagram of the construction of the GeBSb2 plant expression vector provided in Embodiment 1 of the present invention.

[0034] Figure 9 This is a schematic diagram of the construction of the GeUGT0131 plant expression vector provided in Example 1 of the present invention.

[0035] Figure 10 This is a schematic diagram of the construction of the GeUGT1156 plant expression vector provided in Embodiment 1 of the present invention.

[0036] Figure 11 This is a schematic diagram illustrating the operation of soaking tobacco leaves with recombinant Agrobacterium tumefaciens solution provided in Embodiment 2 of the present invention.

[0037] Figure 12 This is a schematic diagram illustrating the detection of gastrodin produced by recombinant Agrobacterium tumefaciens bacterial solution infiltrating Tobacco Benzoinus leaves, as provided in Example 3 of the present invention.

[0038] Figure 13 This is a schematic diagram illustrating the effect of recombinant Agrobacterium tumefaciens solution in soaking Tobacco Benzoenta leaves to produce gastrodin, as provided in Example 3 of the present invention. Detailed Implementation

[0039] The embodiments of this application are described in detail below. Examples of these embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain this application, and should not be construed as limiting this application. In the description of this application, it should be understood that the terms "upper", "lower", "horizontal", "inner", "outer", etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings, and are only for the convenience of describing this application and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of this application. Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of this application, "multiple" means two or more, unless otherwise explicitly specified. To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with the accompanying drawings and embodiments.

[0040] Please see Figure 1 and Figure 2 The present invention provides a method for heterologous biosynthesis of gastrodin, comprising the following steps a to f, and the specific implementation of each step is described in detail below.

[0041] Step a): Obtain key genes related to gastrodin biosynthesis, including: the TAL gene encoding tyrosine ammonia-lyase, the GeCNL gene encoding the catalytic aromatic acid coenzyme A activation enzyme, the GeCHD gene encoding the chain shortening-related enzyme, the GeKAT gene encoding the β-oxidation-related chain shortening enzyme, the GeBSa and GeBSb genes encoding the key enzyme subunit for 4-hydroxybenzaldehyde formation, and the GeUGT gene encoding the uridine diphosphate-dependent glycosyltransferase that can catalyze the glycosylation of 4-hydroxybenzyl alcohol to gastrodin, or their functional homologs.

[0042] In this embodiment, the GeCNL, GeCHD, GeKAT, GeBSa, GeBSb, and GeUGT genes are derived from Gastrodia elata. Preferably, the encoding sequence of GeCNL is shown in SEQ ID NO:1, the encoding sequence of GeCHD is shown in SEQ ID NO:2, the encoding sequence of GeKAT is shown in SEQ ID NO:3, the encoding sequence of GeBSa is shown in SEQ ID NO:4, and the encoding sequence of GeBSb is shown in SEQ ID NO:5; Preferably, the GeUGT is selected from GeUGT85BT1 and / or GeUGT72BK5, and their encoding sequences are shown in SEQ ID NO:6 and SEQ ID NO:7, respectively; More preferably, the functional homolog is a protein-coding gene that satisfies the following conditions: (i) Derived from species in the genus Gastrodia or closely related species in the family Orchidaceae; (ii) Its coding sequence has at least 95% sequence homology with the corresponding gene sequence shown in SEQ ID NO:1-7; (iii) Verified by in vitro enzyme activity assay or transient expression system of Nicotiana benthamiana, it retains the same catalytic function as the protein encoded by the sequence shown in SEQ ID NO:1-7.

[0043] SEQ ID NO:1 is GeCNL; SEQ ID NO:2 is GeCHD; SEQ ID NO:3 is GeKAT; SEQ ID NO:4 is GeBSa; SEQ ID NO:5 is GeBSb; SEQ ID NO:6 is GeUGT85BT1; SEQ ID NO:7 is GeUGT72BK5.

[0044] It should be noted that the sequences shown in SEQ ID NO:1-7 were amplified from *Gastrodia elata* cDNA via RT-PCR; or the genes were synthesized through a commissioned process. For homologs, the transcriptomes of closely related species of *Gastrodia elata* can be searched in databases to obtain sequences with ≥95% homology and functionally validated (e.g., in vitro enzyme activity or in vivo functional complementation in yeast) that retain catalytic activity. The sequences shown in SEQ ID NO:1-7 are the first genes cloned and functionally validated from *Gastrodia elata*. Using these sequences, the active enzyme can be definitively obtained. The 95% sequence homology requirement ensures functional reliability while maintaining a reasonable protection range for future gene mining and engineering modifications.

[0045] Step b): Construct one or more genes obtained in step a) into vectors suitable for plant cell expression, either individually or in combination, to obtain recombinant plant expression vectors.

[0046] In this embodiment, the plant expression vector in step b) is a pEAQ-HT, pCAMBIA series, pBI series, pGreen series, or Gateway series vector; and / or, the vector contains an operatively linked promoter and terminator, wherein the promoter is preferably a CaMV 35S promoter and the terminator is preferably a Nos terminator.

[0047] It should be noted that the pEAQ-HT vector was selected in this embodiment because it has dual 35S promoters, an expression-enhancing 5'UTR, and an NSs protein silencing repressor, making it particularly suitable for transient high expression of multiple genes. pCAMBIA1301 (containing 35S-GFP as a reporter gene) and pGreenII 0029 can also be used. During construction, the target gene is inserted into the multiple cloning site, ensuring it is downstream of the 35S promoter and upstream of the Nos terminator. pEAQ-HT can increase gene expression levels several times, making it more suitable for Agrobacterium-mediated transient systems than ordinary plant expression vectors, and is key to increasing gastrodin yield. The limitation of other vectors maintains the flexibility of the technical solution.

[0048] c) The recombinant plant expression vector obtained in step b) is introduced into Agrobacterium to obtain a recombinant Agrobacterium strain.

[0049] In this embodiment, the Agrobacterium strain mentioned in step c) is LBA4404, GV3101 or EHA105.

[0050] Specifically, the recombinant plasmid was transformed into LBA4404, GV3101, or EHA105 competent cells, respectively. LBA4404 is a commonly used strain, GV3101 has low virulence and high transformation efficiency, and the EHA105 strain has strong vir gene activity. Different strains show differences in infecting different plant varieties or different genes, providing multiple options can adapt to practical operational needs and expand the scope of application of the technology.

[0051] d) The recombinant Agrobacterium strain obtained in step c) is introduced into tobacco leaves by the infiltration method, so that the gene is transiently expressed in the plant cells.

[0052] Further, the immersion method described in step d) is selected from leaf injection immersion, vacuum immersion, or spray-assisted immersion; and / or, the culture conditions described in step e) are: temperature 20–28°C, photoperiod of 12–16 hours light / 8–12 hours darkness, relative humidity 40%–80%, and culture time 3–7 days; and / or, the extraction and detection methods described in step f) include: using methanol, ethanol, water, or acetonitrile as extraction solvents, and using high performance liquid chromatography or liquid chromatography-mass spectrometry for detection.

[0053] It is understandable that vacuum impregnation can treat dozens of tobacco plants at once, suitable for medium- to high-yield requirements; spraying is suitable for large-scale, low-cost field or greenhouse operations. Specific cultivation conditions (especially 22℃ and 5 days) are optimal for gastrodin accumulation; excessively high temperatures or prolonged periods can lead to leaf senescence or product degradation. The provided HPLC conditions can clearly separate gastrodin (approximately 6.2 min) from impurities, meeting quantitative requirements.

[0054] In this embodiment, the recombinant Agrobacterium strain described in step d) is resuspended in an infiltration buffer before infiltration. The infiltration buffer contains 5–20 mM MgCl2, 5–20 mM MES and 50–200 μM acetylsylgenone, with a pH of 5.2–5.8. The total bacterial culture OD600 of the mixed recombinant Agrobacterium strain is 0.1–1.0, more preferably 0.3–0.8.

[0055] It is understandable that Mg² + It increases cell membrane permeability; MES stabilizes pH; AS induces vir gene expression. An OD600 range of 0.3-0.8 ensures sufficient bacterial load without causing leaf necrosis. This formulation is key to achieving high infection efficiency and low toxicity, directly impacting gastrodin yield.

[0056] e) The *Nicotiana benthamiana* plants soaked in step d) are cultured under suitable conditions to allow the enzyme encoded by the gene to catalyze the conversion of the substrate into gastrodin and its accumulation in the leaves.

[0057] f) Extract and analyze the tobacco leaves cultured in step e) to determine the formation of gastrodin.

[0058] Furthermore, the method also includes adding one or more precursors selected from 4-hydroxybenzaldehyde, 4-hydroxybenzyl alcohol, p-coumaric acid, p-hydroxybenzoic acid or their derivatives to the leaves or culture system during the cultivation process or before extraction.

[0059] It is understandable that if the upstream pathway is inefficient, adding intermediates can bypass the bottleneck and allow UGT to act directly, increasing gastrodin production by 2-5 times. This approach provides an effective means to address pathway imbalances in industrialization.

[0060] This application provides a method for heterologous biosynthesis of gastrodin, which involves extracting total RNA from fresh Gastrodia elata tubers or other tissues and obtaining cDNA through reverse transcription. Specific primers are designed, and the full-length coding sequences of GeCNL, GeCHD, GeKAT, GeBSa, GeBSb, and GeUGT are obtained by PCR amplification. Alternatively, codon-optimized sequences can be obtained through artificial synthesis. Each of the above genes is cloned separately between the CaMV 35S promoter and the Nos terminator in a plant binary expression vector (such as pEAQ-HT) to construct a single-gene expression vector; or multiple genes are tandemly constructed into the same vector using multiple cloning sites or 2A peptide / IRES sequences. The recombinant plasmid is transformed into Agrobacterium competent cells (such as LBA4404) using a freeze-thaw method or electroporation, plated on LB agar plates containing the corresponding antibiotics, incubated at 28°C for 2 days, and single colonies are picked and verified by colony PCR. Positive single colonies were picked and cultured in LB liquid medium (containing antibiotics) at 28°C and 200 rpm with shaking until OD600 ≈ 0.8. The cells were collected by centrifugation, resuspended in infiltration buffer (10 mM MgCl2, 10 mM MES, 150 μM acetylsylphenone, pH 5.6), and OD600 was adjusted to 0.5. The cells were then incubated at room temperature for 2 hours. Agrobacterium suspensions carrying different genes were mixed in equal volumes or at an optimized ratio and injected into the underside of *Nicotiana benthamiana* leaves using a needle-free syringe, with approximately 0.5-1 mL of bacterial suspension injected per leaf. The infiltrated plants were placed in a plant culture chamber and cultured for 5 days at 22°C, 16 h light / 8 h dark, and 60% humidity. During this period, T-DNA transferred to the plant cell nucleus, the exogenous gene was transiently transcribed and translated, and the enzyme protein localized to the cytoplasm or corresponding organelles, sequentially catalyzing the conversion of upstream substrates (such as phenylalanine derivatives) to 4-hydroxybenzyl alcohol, which was then glycosylated by UGT to generate gastrodin. Collect the soaked leaves, grind them with liquid nitrogen, add 70% methanol, and extract by sonication for 30 min. After centrifugation and filtration, inject the extract into HPLC (C18 column, acetonitrile / water gradient elution, detection wavelength 220 nm). Compare the retention time and UV spectrum with gastrodin standard, and confirm the molecular ion peak by LC-MS / MS if necessary.

[0061] This application provides a method for heterologous biosynthesis of gastrodin, utilizing a rapid and efficient transient expression system from *Nicotiana benthamiana* to simultaneously introduce multiple key enzyme genes (GeCNL, GeCHD, GeKAT, GeBSa, GeBSb, GeUGT) from the gastrodin biosynthetic pathway into plant cells. Leveraging the plant cell's own metabolic precursors, cofactors, and organelle environment, the complete transformation from aromatic compounds to 4-hydroxybenzyl alcohol, and then to gastrodin, is achieved. Compared to traditional extraction, chemical synthesis, or microbial fermentation methods, this method offers significant advantages, including a shorter cycle time (4-6 days), simpler operation, no need for stable transgenic structures, a complete eukaryotic modification environment, and easy multi-gene synergistic expression.

[0062] This application also provides a recombinant expression vector for the method described above, wherein the recombinant expression vector comprises one or more genes selected from GeCNL, GeCHD, GeKAT, GeBSa, GeBSb, GeUGT85BT1, GeUGT72BK5, or their functional homologs.

[0063] In this embodiment, the recombinant expression vector is a single-gene expression vector, a dual-gene co-expression vector, or a multi-gene tandem expression vector; the vector backbone is pEAQ-HT.

[0064] It is understood that this embodiment constructs single-gene vectors (such as pEAQT-GeUGT85BT1), dual-gene vectors (such as using a bidirectional promoter or IRES to link GeBSa and GeBSb), and multi-gene tandem vectors (linking multiple ORFs via a 2A peptide). After construction using conventional molecular cloning methods, sequencing verification is performed. Single-gene vectors facilitate combination optimization; dual / multi-gene vectors can reduce the types of Agrobacterium required for co-transformation, simplifying the operation. The pEAQ-HT backbone can significantly enhance expression levels.

[0065] This application also provides a recombinant Agrobacterium, which comprises the recombinant expression vector described above.

[0066] It is understandable that recombinant Agrobacterium acts as a bridge connecting the vector and the plant host, and the purity and activity of the strain directly affect the subsequent infiltration effect. Providing stable recombinant strains facilitates technology promotion and replication.

[0067] This application also provides a host plant tissue or cell, wherein the host plant tissue or cell is a leaf tissue of *Nicotiana benthamiana* after the introduction of the recombinant *Agrobacterium* or a protoplast isolated therefrom; the host plant tissue or cell further comprises genes related to the gastrodin biosynthesis pathway derived from *Gastrodia elata* that are stably or transiently integrated.

[0068] It is understood that the tissue or cells have already expressed all pathway enzymes and accumulated gastrodin, which can be used for subsequent metabolic flux analysis, enzyme activity assays, or as extraction raw materials. The protoplast system can be further used for single-cell analysis or transient transfection optimization.

[0069] This application also provides a system for the preparation of gastrodin through heterologous biosynthesis in *Nicotiana benthamiana*, comprising: The gene acquisition module is used to acquire the GeCNL, GeCHD, GeKAT, GeBSa, GeBSb and GeUGT genes; A vector construction module is used to construct the gene into a plant expression vector; Agrobacterium-mediated transformation module, used to introduce recombinant plant expression vectors into Agrobacterium; Plant infiltration module for introducing recombinant Agrobacterium tumefaciens into tobacco leaves; The expression culture module is used to culture infiltrated plant tissues to express the target product. An extraction and detection module is used to extract and detect gastrodin from plant tissues; The purification module is used to purify gastrodin from plant extracts.

[0070] This application provides a system for the heterologous biosynthesis of gastrodin from *Nicotiana benthamiana*. Utilizing a rapid and efficient transient expression system from *Nicotiana benthamiana*, multiple key enzyme genes (GeCNL, GeCHD, GeKAT, GeBSa, GeBSb, GeUGT) from the gastrodin biosynthetic pathway are simultaneously introduced into plant cells. Leveraging the plant cells' own metabolic precursors, cofactors, and organelle environment, the complete transformation from aromatic compounds to 4-hydroxybenzyl alcohol, and then to gastrodin, is achieved. Compared to traditional extraction, chemical synthesis, or microbial fermentation methods, this approach offers significant advantages, including a shorter cycle time (4-6 days), simpler operation, no need for stable transgenic structures, a complete eukaryotic modification environment, and easy multi-gene synergistic expression.

[0071] Furthermore, the above embodiments provide the application of a method for heterologous biosynthesis of gastrodin, recombinant expression vector, recombinant Agrobacterium, host plant tissue or cell, or preparation system in the preparation of gastrodin, verification of the function of key enzymes in the gastrodin biosynthesis pathway, or screening of gastrodin glycosyltransferases.

[0072] Furthermore, the above embodiments provide the application of a method for heterologous biosynthesis of gastrodin, a recombinant expression vector, recombinant Agrobacterium, host plant tissue or cell, or preparation system in the production of gastrodin analogs or gastrodin glycosylation derivatives.

[0073] It is understood that the natural gastrodin configuration obtained in the above embodiments can be used in pharmaceuticals or as a reference product. The *Nicotiana benthamiana* system allows for rapid (within one week) parallel screening of multiple UGT candidate enzymes, reflecting the true in vivo situation more accurately than in vitro enzyme activity assays. Compared to traditional yeast screening, the plant system more closely resembles the natural metabolic environment, making the results more valuable.

[0074] This application also provides an extract or purified product containing gastrodin, which is obtained by the method or the preparation system described herein.

[0075] It is understood that the extracts or purified products obtained in this embodiment can be directly used for bioactivity testing, formulation development, or as a control for chemical synthesis. This invention provides the only technical route for directly obtaining gastrodin from engineered plant tissues, with the product having a natural source and a green, environmentally friendly label.

[0076] To further understand the present invention, the present invention will be further described in detail below with reference to specific embodiments, but the present invention is not limited to the following embodiments.

[0077] Example 1: Cloning of Key Genes and Construction of Plant Expression Vectors. In this example, specific primers were designed based on the transcriptome or genome information of *Gastrodia elata*. Using *Gastrodia elata* cDNA as a template, the full-length coding sequences of GeCNL, GeCHD, GeKAT, GeBSa, GeBSb, and GeUGT were amplified. The PCR products were detected by agarose gel electrophoresis, purified, and ligated into pEAQ-HT or other plant expression vectors, placed between the 35S promoter and the Nos terminator, respectively. Colony PCR, enzyme digestion analysis, or sequencing confirmed the correctness of the recombinant vectors. Figure 3 A schematic diagram of the construction of the GeCNL plant expression vector. Figure 4 A schematic diagram of the construction of the GeCHD plant expression vector. Figure 5 A schematic diagram of the construction of the GeKAT plant expression vector. Figure 6 A schematic diagram of the construction of the GeBSa plant expression vector. Figure 7 This is a schematic diagram of the construction of the GeBSb1 plant expression vector. Figure 8 This is a schematic diagram of the construction of the GeBSb2 plant expression vector. Figure 9 A schematic diagram of the construction of the GeUGT0131 plant expression vector. Figure 10 A schematic diagram of the construction of the GeUGT1156 plant expression vector.

[0078] Example 2: Preparation of recombinant Agrobacterium and infiltration expression in Nicotiana benthamiana. Please refer to [link / reference]. Figure 11The recombinant expression vector obtained in Example 1 was introduced into Agrobacterium competent cells, and positive single colonies were screened. Positive single colonies were picked and inoculated into liquid culture medium containing antibiotics, and cultured at 28°C with shaking until the OD600 was approximately 0.8. The cells were collected by centrifugation, resuspended in infiltration buffer, and the OD600 of each bacterial solution was adjusted to 0.2–0.8, preferably 0.5. The bacterial solutions corresponding to each gene were mixed in a predetermined ratio, and after induction at room temperature, the mixture was infiltrated from the underside of 4–6 week old Nicotiana benthamiana leaves using a needle-free syringe. An empty vector control, a GeUGT deletion control, a upstream key module deletion control, and an uninfiltrated control were also included. After infiltration, the plants were cultured at 24°C under 16 h light / 8 h dark conditions for 3–7 days.

[0079] Example 3: Extraction and detection of gastrodin in leaves. Leaves from the soaked area were collected on the 5th day after soaking, flash-frozen in liquid nitrogen, and ground into powder. 100 mg of leaf powder was weighed, and 1 mL of 70% methanol or methanol was added. After vortexing and ultrasonic extraction for 30 min, the mixture was centrifuged at 12000 rpm for 10 min. The supernatant was collected and filtered through a 0.22 μm filter membrane for HPLC analysis. Preferred HPLC conditions were: C18 reversed-phase column; mobile phase A: 0.1% formic acid water; mobile phase B: methanol; flow rate: 0.3–0.8 mL / min, preferably 0.5 mL / min; column temperature: 30℃; detection wavelength: 220 nm or 270 nm; injection volume: 5–10 μL.

[0080] Please see Figure 12 and Figure 13 The results showed that the target peak with the same retention time as the gastrodin standard was detected in tobacco leaves co-expressing GeCNL, GeCHD, GeKAT, GeBSa, GeBSb, and GeUGT, indicating that de novo synthesis of gastrodin was achieved in tobacco leaves. No corresponding target peak was detected in the empty vector control and the uninfiltrated control, or the signal was significantly lower than that in the experimental group. Preferably, the target product reached a high accumulation level on days 4–6 after infiltration, with the strongest detection signal on day 5.

[0081] The above are merely preferred embodiments of this application, and only specifically describe the technical principles of this application. These descriptions are only for explaining the principles of this application and should not be construed as limiting the scope of protection of this application in any way. Based on this explanation, any modifications, equivalent substitutions, and improvements made within the spirit and principles of this application, as well as other specific embodiments of this application that can be conceived by those skilled in the art without creative effort, should be included within the scope of protection of this application.

Claims

1. A method for heterologous biosynthesis of gastrodin, characterized in that, Includes the following steps: a) Obtain key genes related to gastrodin biosynthesis, including: the TAL gene encoding tyrosine ammonia-lyase, the GeCNL gene encoding the catalytic enzyme activating aromatic acid coenzyme A, the GeCHD gene encoding the chain shortening-related enzyme, the GeKAT gene encoding the β-oxidation-related chain shortening enzyme, the GeBSa and GeBSb genes encoding the key enzyme subunit for 4-hydroxybenzaldehyde formation, and the GeUGT gene encoding the uridine diphosphate-dependent glycosyltransferase that can catalyze the glycosylation of 4-hydroxybenzyl alcohol to gastrodin, or their functional homologs; b) Construct one or more genes obtained in step a) into vectors suitable for plant cell expression, either individually or in combination, to obtain recombinant plant expression vectors; c) The recombinant plant expression vector obtained in step b) is introduced into Agrobacterium to obtain a recombinant Agrobacterium strain; d) The recombinant Agrobacterium strain obtained in step c) is introduced into tobacco leaves by the infiltration method, so that the gene is transiently expressed in the plant cells; e) The *Nicotiana benthamiana* plants soaked in step d) are cultured under suitable conditions to allow the enzyme encoded by the gene to catalyze the conversion of the substrate into gastrodin and accumulate in the leaves; f) Extract and analyze the tobacco leaves cultured in step e) to determine the formation of gastrodin.

2. The method according to claim 1, characterized in that, The GeCNL, GeCHD, GeKAT, GeBSa, GeBSb, and GeUGT genes are derived from Gastrodia elata. The encoding sequence of GeCNL is shown in SEQ ID NO:1, the encoding sequence of GeCHD is shown in SEQ ID NO:2, the encoding sequence of GeKAT is shown in SEQ ID NO:3, the encoding sequence of GeBSa is shown in SEQ ID NO:4, and the encoding sequence of GeBSb is shown in SEQ ID NO:

5. The GeUGT is selected from GeUGT85BT1 and / or GeUGT72BK5, and their encoding sequences are shown in SEQ ID NO:6 and SEQ ID NO:7, respectively. The functional homologs are protein-coding genes that meet the following conditions: (i) Derived from species of the genus Gastrodia or closely related species of the Orchidaceae family; (ii) Its coding sequence has at least 95% sequence homology with the corresponding gene sequence shown in SEQ ID NO:1-7; (iii) Verified by in vitro enzyme activity assay or transient expression system of Nicotiana benthamiana, it retains the same catalytic function as the protein encoded by the sequence shown in SEQ ID NO:1-7.

3. The method according to claim 1 or 2, characterized in that, The immersion method described in step d) is selected from leaf injection immersion, vacuum immersion, or spray-assisted immersion; and / or, the culture conditions described in step e) are: temperature 20-28℃, photoperiod of 12-16 hours light / 8-12 hours darkness, relative humidity 40%-80%, and culture time of 3-7 days; and / or, the extraction and detection methods described in step f) include: using methanol, ethanol, water, or acetonitrile as extraction solvents, and using high performance liquid chromatography or liquid chromatography-mass spectrometry for detection.

4. The method according to any one of claims 1-3, characterized in that, The plant expression vector mentioned in step b) is a pEAQ-HT, pCAMBIA series, pBI series, pGreen series, or Gateway series vector; and / or, the vector contains an operatively linked promoter and terminator, wherein the promoter is preferably a CaMV 35S promoter and the terminator is preferably a Nos terminator.

5. The method according to any one of claims 1-4, characterized in that, The Agrobacterium strain mentioned in step c) is LBA4404, GV3101 or EHA105.

6. The method according to any one of claims 1-5, characterized in that, The recombinant Agrobacterium strain described in step d) is resuspended in an infiltration buffer before infiltration. The infiltration buffer contains 5–20 mM MgCl2, 5–20 mM MES and 50–200 μM acetylsylgenone, with a pH of 5.2–5.

8. The total bacterial culture OD600 of the recombinant Agrobacterium strain mixture is 0.1–1.0, more preferably 0.3–0.

8.

7. The method according to any one of claims 1-6, characterized in that, The method further includes adding one or more precursors selected from 4-hydroxybenzaldehyde, 4-hydroxybenzyl alcohol, p-coumaric acid, p-hydroxybenzoic acid or their derivatives to the leaves or culture system during the cultivation process or before extraction.

8. A recombinant expression vector for implementing the method according to any one of claims 1-7, characterized in that, The recombinant expression vector contains one or more genes selected from GeCNL, GeCHD, GeKAT, GeBSa, GeBSb, GeUGT85BT1, and GeUGT72BK5, or their functional homologs.

9. The recombinant expression vector according to claim 8, characterized in that, The recombinant expression vector is a single-gene expression vector, a dual-gene co-expression vector, or a multi-gene tandem expression vector; the vector backbone is pEAQ-HT.

10. A recombinant Agrobacterium, characterized in that, The recombinant Agrobacterium comprises the recombinant expression vector as described in claim 8 or 9.

11. A host plant tissue or cell, characterized in that, The host plant tissue or cell is a leaf tissue of *Tobacco Bengal* after introduction of the recombinant *Agrobacterium* as described in claim 10, or a protoplast isolated therefrom; the host plant tissue or cell also contains genes related to the gastrodin biosynthesis pathway derived from *Gastrodia elata* that are stably or transiently integrated.

12. A system for the preparation of gastrodin through heterologous biosynthesis in *Nicotiana benthamiana*, characterized in that, include: A gene acquisition module for acquiring the GeCNL, GeCHD, GeKAT, GeBSa, GeBSb and GeUGT genes as described in claim 1; A vector construction module is used to construct the gene into a plant expression vector; Agrobacterium-mediated transformation module, used to introduce recombinant plant expression vectors into Agrobacterium; Plant infiltration module for introducing recombinant Agrobacterium tumefaciens into tobacco leaves; The expression culture module is used to culture infiltrated plant tissues to express the target product. An extraction and detection module is used to extract and detect gastrodin from plant tissues; The purification module is used to purify gastrodin from plant extracts.

13. The application of the method according to any one of claims 1-7, the recombinant expression vector according to claim 8 or 9, the recombinant Agrobacterium according to claim 10, the host plant tissue or cell according to claim 11, or the preparation system according to claim 12 in the preparation of gastrodin, verification of the function of key enzymes in the gastrodin biosynthesis pathway, or screening of gastrodin glycosyltransferases.

14. The use of the method according to any one of claims 1-7, the recombinant expression vector according to claim 8 or 9, the recombinant Agrobacterium according to claim 10, the host plant tissue or cell according to claim 11, or the preparation system according to claim 12 in the production of gastrodin analogs or gastrodin glycosylated derivatives.

15. An extract or purified product containing gastrodin, characterized in that, The extract or purified product is prepared by the method according to any one of claims 1-7 or by the preparation system according to claim 12.

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