Coding gene in lonicera macranthoides, expression vector containing coding gene and application thereof

Abstract

The invention relates to the technical field of plant genetic engineering, and particularly discloses a coding gene in lonicera macranthoides, an expression vector containing the coding gene and application of the coding gene. According to the invention, a coding gene is found in lonicera macranthoides, and an expression vector containing the coding gene can catalyze campsis grandiflora saponin A to synthesize akebia stem saponin D; therefore, the coding gene and the expression vector containing the coding gene can be used for synthesis and preparation of akebiaquinata saponin D, the yield of akebiaquinata saponin D can be increased, and economic values such as medicinal values and the like are high; biosynthesis of akebiaquinata saponin D has remarkable advantages in the aspects of sustainability, purity and environmental protection, is an ideal direction for replacing traditional extraction, and is particularly suitable for the requirement for pharmaceutical grade high purity.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, and specifically discloses a gene encoding Lonicera grayi, an expression vector containing the gene, and its uses. Background Technology

[0002] Mountain honeysuckle refers to the dried flower buds or newly opened flowers of Lonicera macranthoides Hand.-Mazz., Lonicera hypoglauca Miq., Lonicera confusa DC., or Lonicera fulvotomentosa Hsu et SCCheng, all belonging to the Caprifoliaceae family.

[0003] Lonicera macranthoides contains abundant hederagenin-type saponins, with the total content in flower buds ranging from approximately 0.5% to 2.5%. These include akebia saponin D, Lonicera macranthoides saponin A, and Dipsacus saponin B. Akebia saponin D is a type of oleanane-type saponin. Akebia saponin D (ASD) has a glycosyl substitution at the C-28 position, forming different sugar chains through different glycosylations, and thus different glycosides. Therefore, glycosylation is crucial for the biosynthesis of Akebia saponin D. The sugar chain structure of natural products significantly influences their activity and also aids in storage and transport. Therefore, elucidating the glycosylation modification of saponins is of great significance. Studying the enzymes that catalyze glycosylation at specific sites in Akebia saponin D not only helps elucidate the glycosylation mechanism in the biosynthesis of this compound but also further helps clarify the synthetic pathways of other active components of Lonicera macranthoides. Although the pharmacological effects of Akebia saponin D have broad application prospects, its poor bioavailability greatly limits its development and promotion.

[0004] Biosynthesis of akebia saponin D offers significant advantages in terms of sustainability, purity, and environmental friendliness, making it an ideal alternative to traditional extraction methods, particularly suitable for pharmaceutical-grade high-purity applications. Researching the enzymes and encoding genes involved in the synthesis of akebia saponin D in plants can help increase its yield and bioavailability.

[0005] While tobacco systems present greater challenges for extracting and purifying products compared to microbial systems in synthetic biology, they are more suitable for identifying functional genes and expressing genes from related plants, offering a simpler and more economical approach. Numerous studies have utilized tobacco transient expression systems to elucidate natural product synthetic pathways. The function of glycosyltransferases is related to glycosylation modification; verifying the function of multiple candidate genes in vitro requires large amounts of sugar donors. Since sugar donors are expensive, the tobacco transient expression system can utilize readily available sugar donors within the tobacco plant, effectively reducing experimental costs. Summary of the Invention

[0006] To address the problems mentioned in the background art, this invention involves screening the glycosyltransferase gene for the catalytic synthesis of Akebia saponin D from *Lonicera japonica* A, constructing the vector, infecting *Tobacco Benzoinus* leaves, extracting compounds from the infected leaves, and determining the products. The purpose of this invention is to provide a gene encoding *Lonicera japonica*, an expression vector containing this gene, and its uses, thereby increasing the yield of Akebia saponin D through biosynthesis.

[0007] Screening of glycosyltransferase genes for the synthesis of akebia saponin D from catalytic oleanolic acid A: The NCBI database was accessed to search for published UGT families (UGT73 and UGT74) used to modify oleanane-type saponins. Based on the previously screened 64 glycosyltransferase genes related to the biosynthesis of ivy-type saponins, a phylogenetic tree was constructed to further identify key genes.

[0008] EVM0009182 gene vector construction and infection of Tobacco Benzoenta leaf: First, primers were designed to amplify the cDNA template to obtain the PCR fragment of the EVM0009182 gene. Then, the vector was constructed by homologous recombination and transformed into Agrobacterium to infect Tobacco Benzoenta leaves to obtain transiently transfected plants.

[0009] Compounds were extracted from infected tobacco leaves for product determination: the components of transient expression products in tobacco were determined using LC / MS coupled with an Agilent 1290 ultra-high performance liquid chromatography system.

[0010] To achieve the above objectives, the present invention provides the following technical solution:

[0011] This invention provides a glycosyltransferase gene from Lonicera macrantha, wherein the glycosyltransferase gene is EVM0009182 and the amino acid sequence is SEQ ID NO.1.

[0012] The present invention provides a gene encoding the Lonicera japonica glycosyltransferase gene of claim 1, wherein the gene encoding the Lonicera japonica glycosyltransferase gene described above.

[0013] Furthermore, the nucleotide sequence of the encoding gene is shown in SEQ ID NO.2.

[0014] The present invention also provides an expression vector containing the coding gene as described above.

[0015] Furthermore, the expression vector is a plasmid.

[0016] The present invention also provides a method for constructing the expression vector as described above, comprising the following steps: according to the two restriction sites added by the designed primers, the selected expression vector plasmid is subjected to a double restriction reaction, and the gene shown in SEQ ID NO.2 is recombined with the restriction-digested plasmid to obtain plasmid pHB-EVM0009182.

[0017] The present invention also provides the application of the expression vector described above in the catalytic synthesis of akebia saponin A into akebia saponin D.

[0018] Compared with the prior art, the beneficial effects of the present invention are:

[0019] This invention discovers a coding gene in *Lonicera japonica*, and an expression vector containing this gene can catalyze the synthesis of *Akebia trifoliata* saponin A into *Akebia trifoliata* saponin D. Therefore, this coding gene and its expression vector can be used for the synthesis of *Akebia trifoliata* saponin D, which helps to increase its yield and has high economic value, including medicinal applications. The biosynthesis of *Akebia trifoliata* saponin D offers significant advantages in terms of sustainability, purity, and environmental friendliness, making it an ideal alternative to traditional extraction methods, especially suitable for pharmaceutical-grade high-purity applications. Attached Figure Description

[0020] Figure 1 Flowchart of transient expression of gene EVM0009182 in tobacco and its catalytic formation of barrel saponin D compound;

[0021] Figure 2 Phylogenetic analysis of candidate genes and the UGT family;

[0022] Figure 3 Agarose gel electrophoresis image of EVM0009182 amplification;

[0023] Figure 4 The chromatogram shows the transient expression product of pHB-EVM0009182 in tobacco.

[0024] Figure 5 This is a mass spectrometry image of the transient expression product of pHB-EVM0009182 tobacco. Detailed Implementation

[0025] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0026] This invention provides a glycosyltransferase gene from Lonicera macrantha, wherein the glycosyltransferase gene is EVM0009182 and the amino acid sequence is SEQ ID NO.1.

[0027] The present invention also provides a gene encoding the above-mentioned honeysuckle glycosyltransferase gene, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0028] The present invention also provides an expression vector containing the above-mentioned coding gene.

[0029] The present invention also provides an expression vector containing the encoding gene for use in catalyzing the synthesis of akebia saponin A into akebia saponin D.

[0030] The flowchart of transient expression of gene EVM0009182 in tobacco and its LC-MS analysis of the catalytic formation of barrel saponin D is shown below. Figure 1 As shown. Figure 1 The experimental procedure and verification results of expressing the gene EVM0009182 described in this invention in a heterologous system (tobacco) and successfully catalyzing the synthesis of the target product are presented.

[0031] Specifically, it includes:

[0032] 1. Gene source: cDNA was obtained from gray honeysuckle.

[0033] 2. Gene cloning: The target gene EVM0009182 was amplified using PCR technology.

[0034] 3. Vector construction: The target gene was cloned into the plant expression vector pHB using homologous recombination technology.

[0035] 4. Plasmid amplification and transformation: The homologous recombination product was transformed into Escherichia coli DH5α competent cells, positive single clones were screened and plasmids were extracted; then the correctly constructed recombinant plasmid was transformed into Agrobacterium tumefaciens GV3101 competent cells.

[0036] 5. Transient transformation in plants: Agrobacterium carrying recombinant plasmids is used to infect leaves of Nicotiana benthamiana to achieve transient gene expression.

[0037] 6. Product extraction and detection: Metabolites were extracted from tobacco leaves after cultivation and analyzed by liquid chromatography-mass spectrometry (LC-MS). The results showed that the gene EVM0009182 described in this invention can utilize the substrate CaulosideA to catalyze the generation of akebia saponin D.

[0038] Example 1

[0039] Screening of glycosyltransferase genes catalyzing the synthesis of akebia saponin D from akebia saponin A.

[0040] Log in to the NCBI database to find published UGT families (UGT73 and UGT74) used for modifying oleanane-type saponins; then, based on the 64 glycosyltransferase genes previously screened that are related to the biosynthesis of ivy-type saponins, construct a phylogenetic tree (e.g., Figure 2 As shown), EVM0009182, EVM0015711, EVM0016228, EVM0014132, EVM0000721, EVM0000099, EVM0017857, EVM0003181, EVM0005876, EVM0018645, EVM0007233, EVM0010336, EVM0017550, EVM0001837, EVM0015063, EVM0004289, and EVM0001933 are grouped with JQ291614 and JQ291613, indicating a close kinship. JQ291614 and JQ291613 can simultaneously catalyze glycosylation at the C-3 and C-28 sites of oleanane-type saponins, and both belong to the UGT73C10 subfamily. These candidate genes were randomly grouped, transformed into Agrobacterium, and then infected with tobacco according to their respective groups. EVM0009182 was ultimately selected as the candidate glycosyltransferase gene for the final catalysis of the synthesis of akebia saponin D from oleanene saponin A.

[0041] The amino acid sequence of EVM0009182 is shown in SEQ ID NO.1:

[0042] MGHEFLHKFPVESRRIVWLIGVLFAMVLVVQYFELPYGNVISSLISSGKAQILSPRPESFSNTTLFDGLNSTNMNVADEVHEIANITEISEGHDLSLRNDSMV ERVKGKDGSELKKAKESEYEFSANNGTSERNSSSDENRSINVGPTGTPPLASPIKVDANLGTPPTSERNPSSEKNRSDDVGPKVKNSTKKRSYEIMEQTLKVY IYREGERPIFHQPVLIGIYASEGWFMKQIQADKKFVTKDPKKAHLFYLPFSSRMLEEVLYVPDSHNHKNLIKHLKNYLDVITARYPFWNRTGGADHFLVACHD WAASETRQYMAKCIRALCNADIKEGFEIGKDVSLPETYVRSAQNPLKAVGGKRPSQRPFLAFYAGNMHGYLRPILLKHWENKDPVMKIFGPMPNPKVTRTIYNT

[0043] Example 2

[0044] Cloning of the gene of Lonicera lyrata var. gravidae EVM0009182

[0045] 1. Experimental Materials

[0046] In this experiment, the experimental material used was *Lonicera macrantha*, cultivated in the Medicinal Botanical Garden of China Pharmaceutical University. The flower buds of *Lonicera macrantha* were used for DNA and RNA extraction. The tobacco material used for transient expression was cultivated in the Plant Culture Laboratory of the Medicinal Botanical Garden of China Pharmaceutical University.

[0047] 2. Cloning of the EVM0009182 gene

[0048] 2.1 Extraction and reverse transcription synthesis of cDNA from total RNA of Lonicera macrantha.

[0049] Clean the flower buds of *Lonicera japonica* and grind them in liquid nitrogen. Quickly add the powder to a 1.5 mL Eppendorf (EP) centrifuge tube containing lysis buffer. After thorough shaking, extract total RNA according to the instructions of the Plant Tissue Total RNA Extraction Kit from Novizan Biotechnology Co., Ltd. Determine the concentration and purity using Nanoparticles. Store at -80℃ for later use. Remove the extracted RNA solution and remove residual genomic DNA according to the system in Table 1.

[0050] Table 1 Reverse Transcription System

[0051]

[0052]

[0053] Incubate at 42℃ for 2 min, add 5 μL of 4×HifairⅢSuperMixplus, gently mix by pipetting, and then place in a reverse transcription apparatus for reverse transcription. The reaction program is shown in Table 2. The product cDNA is stored at -20℃.

[0054] Table 2 Reverse transcription procedures

[0055] temperature Time (min) 25℃ 5 55℃ 15 85℃ 5

[0056] 2.2 Full-length amplification of EVM0009182

[0057] Based on the full-length CDS sequence (SEQ ID NO. 2) of EVM0009182 from the genome of *Lonicera macrantha*, specific primers were designed, as shown in Table 4. The full-length EVM0009182 was amplified using *Lonicera macrantha* cDNA as a template. The PCR amplification system (total volume 50 μL) is shown in Table 3, and the PCR amplification program is shown in Table 5.

[0058] Table 3 PCR amplification system

[0059] reagents Usage DEPC water 17μL 2×PhantaMaxBuffer 25μL dNTPMix(10mMeach) 1μL PhantaMaxSuper-FidelityDNAPolymerase 1μL EVM0009182-F 2μL EVM0009182-R 2μL template cDNA 2μL

[0060] SEQ ID NO.2

[0061]

[0062] The specific primers used for constructing the pHB-EVM0009182 vector are shown in Table 4.

[0063] Table 4. Specific Primers

[0064]

[0065] Table 5 PCR amplification program

[0066]

[0067] Add 10× DNA Loading Buffer to the PCR amplification product and use an agarose gel electrophoresis system to detect the gel electrophoresis pattern of the amplification product (e.g., ...). Figure 3 The candidate gene was successfully cloned through sequencing analysis. After gel excision, the DNA gel was recovered according to the Axygen DNA Gel Recovery Kit method. The concentration and purity of the recovered product were measured, and the product was stored at -20°C for later use.

[0068] 1.3.3 Construction of EVM0009182 expression vector

[0069] The selected expression vector plasmid was subjected to double digestion reaction according to the two restriction enzyme sites added by the designed primers. The reaction system is shown in Table 6.

[0070] Table 6. pHB plasmid digestion system (40 μL)

[0071]

[0072]

[0073] The double enzyme digestion reaction was performed at 37°C for 40 min. After digestion, the plasmid was first subjected to agarose gel electrophoresis to extract the target band. The band was then recovered using an Axygen DNA gel recovery kit. The concentration and purity of the recovered product were measured, and the product was stored at -20°C for later use. The target gene was then recombined with the digested plasmid. The recombination system (20 μL) is shown in Table 7.

[0074] Table 7 Recombination Reaction System

[0075] Reagent Name Usage DEPC water up to 10 μL 5×CEIIBuffer 2μL Exnase II 1μL Target gene 1μL Enzyme digested vector 2μL

[0076] The ligation product was transformed into E. coli DH5α competent cells. The specific transformation steps are as follows:

[0077] (1) Take out Escherichia coli DH5α competent cells from -80℃ and thaw them on ice.

[0078] (2) Add the recombinant product (10 μL) to the competent cells, mix gently, and let stand on ice for 25 min.

[0079] (3) Heat shock in a 42℃ water bath for 90s, then stand on ice for 2min. Add 700μL of antibiotic-free LB liquid medium and incubate at 37℃ and 180rpm for 1h.

[0080] (4) Centrifuge at 6000 rpm for 5 min, discard part of the supernatant, leaving approximately 150 μL. Resuspend the bacterial culture and spread it onto LB agar plates containing kanamycin (Kan). Incubate upright at 37°C for 5 min, then invert for 12 h. Pick a single colony and transfer it to 800 μL of LB liquid medium containing Kan. Incubate at 37°C with a shaker at 180 rpm for 4 h until the bacterial culture becomes turbid. Perform PCR amplification to verify positive colonies. The specific reaction system is shown in Table 8.

[0081] Table 8 Bacterial PCR System

[0082] Reagent Name Usage DEPC water 3.2μL 2×RapidTaqMasterMix 5μL EVM0009182-F 0.4μL EVM0009182-R 0.4μL bacterial solution 1μL

[0083] The PCR amplification procedure was the same as the gene cloning procedure described above. After the procedure, the products were analyzed by agarose gel electrophoresis. A positive bacterial culture was selected and sent to Genewiz for sequencing. For the successfully sequenced bacterial culture, 600 μL of the culture was removed and 400 μL of 50% glycerol was added, and the culture was incubated at -80°C. Separately, approximately 60 μL of the culture was added to 6 mL of LB broth containing kanamycin and incubated overnight at 37°C with shaking. Following the instructions of the Axygen plasmid extraction kit, plasmids were extracted from the overnight culture. The concentration and purity of the obtained plasmids were determined using a micro-volume nucleic acid and protein analyzer, and the plasmids were stored at -20°C for later use.

[0084] Example 3

[0085] Tobacco transient expression verification experiment

[0086] 1. Transformation of GV3101 Agrobacterium

[0087] The extracted recombinant candidate glycosyltransferase gene pHB plasmid was transformed into Agrobacterium GV3101. The specific steps are as follows:

[0088] (1) Take out the competent Agrobacterium GV3101 cells from -80℃ and thaw them on ice.

[0089] (2) Add 5 μL of recombinant plasmid to competent cells, mix gently, and let stand on ice for 25 min. Freeze in liquid nitrogen for 8 min, remove immediately, heat shock in a 37°C water bath for 5 min, and let stand on ice for 5 min.

[0090] (3) Add 700 μL of antibiotic-free LB liquid medium and incubate at 28°C and 200 rpm for 3 h on a shaker.

[0091] (4) Centrifuge at 6000 rpm for 5 min, discard part of the supernatant, leaving about 150 μL.

[0092] (5) Resuspend the bacterial culture and spread it onto LB agar plates containing Kan and Rif. Incubate at 28°C upright for 5 min, then invert for 2–3 days. Pick a single colony and transfer it to 800 μL of LB liquid medium containing Kan and Rif. Incubate at 28°C and 200 rpm for 20 h until the culture becomes turbid. Perform PCR amplification to verify positive colonies. Take a small amount of the bacterial culture and boil it at 99°C for 2 min beforehand to break the cell wall. Use the boiled bacterial culture as a PCR template.

[0093] (6) The PCR amplification products of the bacterial culture were detected by agarose gel electrophoresis. Positive bacterial cultures were picked, preserved, and stored at -80℃ for subsequent tobacco transient conversion experiments.

[0094] 2. Instantaneous conversion of tobacco

[0095] (1) Take the preserved positive Agrobacterium GV3101 bacterial culture and incubate it in 800 μL of LB liquid medium containing Kan and Rif until turbidity is reached. Take 200 μL of activated bacterial culture and incubate it in an appropriate amount of LB liquid medium containing Kan and Rif at 28℃ and 200 rpm until OD is reached. 600 The value is 0.8–1.0. Centrifuge at 4000 rpm for 10 min, discard the supernatant, collect the bacterial precipitate, and resuspend it in MES infection buffer. If a bacterial culture is being transformed, adjust the OD value. 600 The value should not be less than 0.6; if multiple bacterial solutions are mixed for transformation, adjust the OD of each bacterial solution. 600 The value should not be less than 0.4, and it should be left to stand in the dark for 3 hours.

[0096] (2) Select tobacco leaves in good growth condition and use Agrobacterium GV3101 containing empty pHB as a negative control. Inject Agrobacterium into the back of the tobacco leaves using a 1 mL syringe and mark them.

[0097] (3) Incubate the tobacco that has been injected with bacterial solution in the dark for 3 days.

[0098] (4) Prepare the corresponding standard solution with double-distilled water containing 1% DMSO as solvent, so that the concentration is 100 μmol / L. Inject the tobacco leaves that have been injected with bacterial solution again with the standard solution.

[0099] (5) Incubate the tobacco leaves injected with the standard solution in the dark for 2 days. Dry the injected tobacco leaves in an oven at 37°C until constant weight.

[0100] (6) Place the dried tobacco leaves in a 2mL centrifuge tube, add methanol, and add two 4mm zirconium beads. Grind the leaves into powder using a high-throughput tissue homogenizer, extract by sonication for 30min, and invert the tube 2-3 times every 10min. Centrifuge at 12000rpm for 15min, collect the supernatant, evaporate excess solvent using a nitrogen blower, add 200μL methanol to redissolve the leaves, centrifuge again at 12000rpm for 10min, and transfer to a liquid chromatography vial for LC / MS analysis.

[0101] Example 4

[0102] Product determination

[0103] 1. Liquid Chromatography Analysis

[0104] The components of the transient expression product of tobacco were determined by LC / MS using an Agilent 1290 ultra-high performance liquid chromatography system. The chromatographic column was a ZORBAX SB-C18 (1.8 μm, 3.0 × 150 mm); the column temperature was 40℃; the mobile phase was 0.1% formic acid water (A)-0.1% formic acid acetonitrile (B). The flow rate was 0.4 mL / min; the injection volume was 3 μL; and the run time was 5 min. The chromatogram of the transient expression product of tobacco pHB-EVM0009182 is shown below. Figure 4 As shown.

[0105] 2. Mass spectrometry analysis conditions

[0106] An Agilent quadrupole-time-of-flight mass spectrometer (Agilent Q / TOF6545A) was used, with operating parameters set and negative ion mode employed for sample detection. Gradient elution conditions were set as shown in Table 9. The mass spectrometry chromatogram of the transient expression product of pHB-EVM0009182 tobacco is shown below. Figure 5 As shown.

[0107] Table 9 Gradient elution conditions

[0108] Time (min) A％ B％ 0.00 95.00 5.00 23.00 5.00 95.00 25.00 5.00 95.00

[0109] The LC-MS chromatographic analysis comparison directly demonstrates the function of the gene in this invention:

[0110] Alkebia saponin D standard: The peak position and time of the standard Alkebia saponin D are used as a reference.

[0111] pHB vector + Cauloside A: Chromatogram of the empty vector control group (with Cauloside A added only), serving as a negative control.

[0112] pHB vector+EVM0009182+CaulosideA: Chromatogram of the experimental group transformed with the gene EVM0009182 of this invention.

[0113] The experimental results clearly show that, compared with the empty vector control group, a significant characteristic chromatographic peak appeared in the experimental group transformed with EVM0009182 at approximately 8.3 min, near the same retention time as the akebia saponin D standard. Simultaneously, mass spectrometry results showed that, at 8.3 min, compared with the empty vector control group, the experimental group transformed with EVM0009182 also exhibited a characteristic fragment ion 927.4 near the same retention time as the akebia saponin D standard. These results indicate that the gene EVM0009182 described in this invention can utilize the substrate Cauloside A to catalyze the production of akebia saponin D.

[0114] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0115] It should be noted that the above content merely illustrates the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. For those skilled in the art, various improvements and modifications can be made without departing from the principle of the present invention, and all such improvements and modifications fall within the scope of protection of the claims of the present invention.

Claims

1. A glycosyltransferase gene from Lonicera macrantha, characterized in that, The glycosyltransferase gene is EVM0009182, and its amino acid sequence is SEQ ID NO.

1.

2. A gene encoding the Lonicera japonica glycosyltransferase gene of claim 1, wherein the gene encoding the Lonicera japonica glycosyltransferase gene of claim 1 is capable of encoding the Lonicera japonica glycosyltransferase gene of claim 1.

3. The coding motif of the *Lonicera japonica* glycosyltransferase gene as described in claim 2, characterized in that, The nucleotide sequence of the encoding gene is shown in SEQ ID NO.

2.

4. An expression vector containing the coding gene as described in claim 2 or 3.

5. The expression vector according to claim 4, wherein the expression vector is a plasmid.

6. The method for constructing the expression vector as described in claim 5, characterized in that, Includes the following steps: The selected expression vector plasmid was subjected to double digestion reaction according to the two restriction sites added by the designed primers. The gene shown in SEQ ID NO.2 was recombined with the digested plasmid to obtain plasmid pHB-EVM0009182.

7. The application of the expression vector as described in claim 4 in the catalytic synthesis of akebia saponin A into akebia saponin D.

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