A mutant of prenyltransferase and its application in producing icariin

Abstract

The application provides a prenyltransferase mutant and application thereof in production of icariin, and belongs to the technical field of bioengineering. The application solves the problem of low icariin yield in the prior art. The prenyltransferase mutant is obtained by mutating any one of the 52th, 131th, 205th and 303th positions of the amino acid shown in SEQ ID NO. 2. The prenyltransferase mutant is mainly used for efficient prenyltransferase and application thereof in synthesis of icariin.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, and in particular relates to an isopentenyltransferase mutant and its application in the production of icariin. Background Technology

[0002] Icaritin is an active ingredient extracted from the traditional Chinese medicine epimedium, belonging to the flavonoid class of compounds. In traditional Chinese medicine, it is believed to have kidney-tonifying and aphrodisiac effects, as well as strengthening muscles and bones. Modern pharmacological studies have found that it also possesses various biological activities, including anti-tumor, anti-inflammatory, neuroprotective, and osteoporosis-improving effects. Its research in the field of tumor treatment (such as liver cancer) has been particularly in-depth, and some related drugs have already entered the clinical application stage.

[0003] There are three possible methods for the industrial production of icariin: traditional plant extraction, chemical synthesis, and biosynthesis. Natural plant extraction is the traditional and widely used method, using the dried aerial parts of Epimedium plants (such as Epimedium brevicornu and Epimedium sagittatum) as raw materials, and obtaining icariin through extraction, separation, and purification. Although natural plant extraction aligns with the traditional Chinese medicine concept of "natural products" and has relatively mature technology suitable for large-scale production, it suffers from drawbacks such as reliance on plant resources, unstable raw material quality, low extraction efficiency, high cost, potential for the presence of other flavonoid impurities during extraction, and significant purification difficulties. Chemical synthesis uses structurally simple compounds as raw materials to synthesize icariin through a series of chemical reactions. Currently, commonly used synthetic routes often use isopentenyl-substituted flavonoids as precursors, constructing the target molecular structure through cyclization and oxidation steps. Although chemical synthesis does not rely on natural plant resources and can be produced on a large scale with high product purity using chemical raw materials, its synthetic route is relatively long (usually requiring 5-10 reaction steps), the reaction conditions are complex (such as high temperature, high pressure or special catalysts), the technical threshold is high, and some reaction steps may produce toxic byproducts, resulting in greater environmental pressure.

[0004] Compared to the methods mentioned above, biosynthesis is a relatively economical and environmentally friendly approach to realizing greater market value for icariin. Therefore, exploring components that can efficiently produce icariin is of great research significance. Summary of the Invention

[0005] In view of this, the present invention aims to propose an isopentenyltransferase mutant and its application in the production of icariin, and to provide an application of a highly efficient isopentenyltransferase in the synthesis of icariin, so as to solve the problem of low icariin yield in the prior art.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] The present invention provides an isopentenyltransferase mutant, which is obtained by mutating any one of the following positions: position 52, position 131, position 205, and position 303, starting from the amino acid sequence shown in SEQ ID NO. 2.

[0008] This invention provides an isopentenyltransferase mutant, wherein the isopentenyltransferase mutant is any one of the following:

[0009] (1) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace glycine at position 52 with aspartic acid;

[0010] (2) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace the threonine at position 131 with serine;

[0011] (3) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace valine at position 205 with phenylalanine;

[0012] (4) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace the aspartic acid at position 303 with glutamic acid.

[0013] The present invention provides a gene encoding the above-mentioned isopentenyltransferase mutant.

[0014] The present invention provides a recombinant vector containing the above-mentioned genes.

[0015] To further specify, the launch vehicle is the pACYCDuet series.

[0016] The present invention provides a host cell containing the above-mentioned genes.

[0017] Further specifying, this includes genes containing truncated and optimized site-directed mutant isopentenyltransferase mutants. EpPTtru60 Mut Isopentenyl pyrophosphate isomerase gene idi Methyltransferase gene MpOMT4 Isopentenyl kinase gene AtIPK and choline kinase gene ScCK The overexpression plasmid was transferred into Escherichia coli BL21(DE3) competent cells to obtain genetically engineered bacteria.

[0018] The present invention provides the application of the above-mentioned isopentenyltransferase mutant, the above-mentioned gene, the above-mentioned recombinant vector, or the above-mentioned host cell in the production of icariin.

[0019] This invention provides a method for producing epimedium, the steps of which are as follows: fermenting the above-mentioned genetically engineered bacteria at 25°C for 48 hours.

[0020] Further specifying, the fermentation medium consists of 20 g / L glucose, 4 mL / L glycerol, 12 g / L peptone, 16.4 g / L K2HPO4·3H2O, and 2.3 g / L KH2PO4.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows: The present invention provides an isopentenyltransferase mutant for efficient synthesis of icariin. Through co-evolutionary analysis and substrate binding analysis of the catalytic cavity modification, site-directed mutagenesis was performed on the truncated and optimized isopentenyltransferase mutant EpPTtru60 at positions 52 (Gly), 74 (Lys), 131 (Thr), 187 (Leu), 205 (Val), 271 (Phe), and 303 (Asp), respectively, yielding seven isopentenyltransferase mutants, namely EpPTtru60. G52D EpPTtru60 K74E EpPTtru60 T131S EpPTtru60 L187P EpPTtru60 V205F EpPTtru60 F271A EpPTtru60 D303E Icariin was obtained by shake-flask fermentation of genetically engineered bacteria overexpressing truncated and optimized isopentenyltransferase containing unmutated isopentenyltransferase and seven genetically engineered bacteria overexpressing truncated and optimized isopentenyltransferase containing site-directed mutation. The truncated EpPTtru60 containing site-directed mutation... G52D As the optimal mutant, after 48 h of shake-flask fermentation, the icariin yield was 9.35 mg / L, which was 23% higher than that of the control strain, providing a valuable component for improving icariin yield. Attached Figure Description

[0022] Figure 1 A is the liquid chromatography chromatogram of icariin standard, with a retention time of 22.079 min.

[0023] Figure 1 B contains EpPTtru60 G52D The liquid phase diagram of the fermentation broth of the mutant strain shows that the retention time of icariin in the fermentation broth is 22.093 min, which is consistent with the retention time of the icariin standard. Detailed Implementation

[0024] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. It should be noted that, unless otherwise specified, the embodiments and features in the embodiments of the present invention can be combined with each other, and the described embodiments are only some embodiments of the present invention, not all embodiments.

[0025] The seed culture system consisted of 10 mL of LB medium in a 100 mL Erlenmeyer flask containing 10 mL of culture medium, which was composed of 5 g / L yeast extract, 10 g / L NaCl, and 10 g / L peptone.

[0026] The fermentation system for shake-flask fermentation consisted of 250 mL Erlenmeyer flasks containing 50 mL of culture medium, the composition of which was 20 g / L glucose, 4 mL / L glycerol, 12 g / L peptone, 16.4 g / L K2HPO4·3H2O, and 2.3 g / L KH2PO4.

[0027] Example 1. Obtaining the isopentenyltransferase mutant

[0028] This invention provides an isopentenyltransferase mutant for efficient synthesis of icariin. Through co-evolutionary analysis and substrate binding analysis of the catalytic cavity modification, site-directed mutagenesis was performed on the truncated and optimized isopentenyltransferase mutant EpPTtru60 at positions 52 (Gly), 74 (Lys), 131 (Thr), 187 (Leu), 205 (Val), 271 (Phe), and 303 (Asp), yielding seven isopentenyltransferase mutants, namely EpPTtru60. G52D EpPTtru60 K74E EpPTtru60 T131S EpPTtru60 L187P EpPTtru60 V205F EpPTtru60 F271A EpPTtru60 D303EThe site-directed mutagenesis parent sequence is the truncated and optimized isopentenyl transferase EpPTtru60 derived from Epimedium, with its nucleotide sequence shown in SEQ ID NO: 1 and its amino acid sequence shown in SEQ ID NO: 2; the isopentenyl pyrophosphate isomerase IDI, with its nucleotide sequence shown in SEQ ID NO: 3 and its amino acid sequence shown in SEQ ID NO: 4; the methyltransferase MpOMT4, with its nucleotide sequence shown in SEQ ID NO: 5 and its amino acid sequence shown in SEQ ID NO: 6; the isopentenyl kinase AtIPK, with its nucleotide sequence shown in SEQ ID NO: 7 and its amino acid sequence shown in SEQ ID NO: 8; and the choline kinase ScCK, with its nucleotide sequence shown in SEQ ID NO: 9 and its amino acid sequence shown in SEQ ID NO: 10. (Reference: Biotransformation of Kaempferol to Icaritinin Engineered) Saccharomy ces cerevisiae )

[0029] SEQ ID NO: 1 (The truncated and optimized nucleotide sequence of isopentenyltransferase EpPTtru60):

[0030] ATGCACACCCATGAAAAAGAACTGCTGTTTAAAGACAAGAACCCGACCCGCGAAAATCCGTGCCCGTCAGCAACCAGCAGCGAAAATGCACCGCTGAGTTTTAGCACCAAACTGGATATGTTTATCAAGTTCGTGCGTCCGTATGCAACCATTGGTATTATTGGTAATACCATCTGCATGTGCATCCTGCCGGTTCAGACAATGGCTGATCTGAGCCCGAAATTTTTCATTGGTGTGGCCCAGGCAATTGCCAGTATGGTTCTGATGAATCTGTTTAATGTGGCAGTGAATCAGGTTTATGACGTTGAACTGGATAAGGTTAACAAGCCGTATCTGCCGCTGGCAAGCGGTGGTGTTAGCATGACCAGTGCAACCCTGTTTACCATTCTGACCGCCGCCCTGAGTATTGCACTGGGTTATTTTAGCAGCCCGGCACTGTTTTATGGCAGTATTGCCTTTTTCCTGAGCGCAAGCGCATATAGTGTTAATTTTCCGCTGCTGCGCTGGAAAAATAATGCCCTGGGCGCAATTATTAGTCTGATGCTGTGGGGTATTAGCCTGCAGACCGGTGTGTTTTTCCATATTCAGCAGTATGTGCTGGGTAAACCGATGGTTCTGAAAAATAGTTTCATCTACGCGATCATCTTCCAGAGCCTGTTTAGCATTGTTGTTGCCACCCTGAAAGATCTGCCGGATGTTGAAGGCGATAAAGCCAATGGCAGTACCAATCTGACCATTCTGATTGGCAAAGAAAAGGTGTTTTGGGGTTGCACCAGCCTGATGCTGGCCACATATATTGGCACCGCAGCATTTGGCGCAACCCTGCCTATTCTGAAAAATAAGCTGGTTACAATGGTTGCCCATAGCGCCCTGGCCGTTTTTCTGTGGCTGCAGGCTAAACAGATTGATCTGGCAGATGATGCAAGCACCCAGAGCTATTATCTGCTGATGTGGAAACTGTGCAATATTGAATACCTGCTGATCCCGTTTGTTGGC;

[0031] SEQ ID NO: 2 (Truncation of the optimized amino acid sequence of isopentenyltransferase EpPTtru60):

[0032] MHTHEKELLFKDKNPTRENPCPSATSSSENAPLSFSTKLDMFIKFVRPYATIGIIGNTICMCILPVQTMADLSPKFFIGVAQAIASMVLMNLFNVAVNQVYDVELDKVNKPYLPLASGGVSMTSATLFTILTAALSIALGYFSSPALFYGSIAFFLSASAYSVNFPLL RWKNNALGAIISLMLWGISLQTGVFFHIQQYVLGKPMVLKNSFIYAIIFQSLFSIVVATLKDLPDVEGDKANGSTNLTILIGKEKVFWGCTSLMLATYIGTAAFGATLPILKNKLVTMVAHSALAVFLWLQAKQIDLADDASTQSYYLLMWKLCNIEYLLIPFVG*;

[0033] SEQ ID NO: 3 (nucleotide sequence of isopentenyl pyrophosphate isomerase IDI):

[0034] ATGCAAACGGAACACGTCATTTTATTGAATGCACAGGGAGTTCCCACGGGTACGCTGGAAAAGTATGCCGCACACACGGCAGACACCCGCTTACATCTCGCGTTCTCCAGTTGGCTGTTTAATGCCAAAGGACAATTATTAGTTACCCGCCGCGCACTGAGCAAAAAAGCATGGCCTGGCGTGTGGACTAACTCGGTTTGTGGGCACCCACAACTGGGAGAAAGCAACGAAGACGCAGTGATCCGCCGTTGCCGTTATGAGCTTGGCGTGGAAATTACGCCTCCTGAATCTATCTATCCTGACTTTCGCTACCGCGCCACCGATCCGAGTGGCATTGTGGAAAATGAAGTGTGTCCGGTATTTGCCGCACGCACCACTAGTGCGTTACAGATCAATGATGATGAAGTGATGGATTATCAATGGTGTGATTTAGCAGATGTATTACACGGTATTGATGCCACGCCGTGGGCGTTCAGTCCGTGGATGGTGATGCAGGCGACAAATCGCGAAGCCAGAAAACGATTATCTGCATTTACCCAGCTTAAATAA;

[0035] SEQ ID NO: 4 (Amino acid sequence of isopentenyl pyrophosphate isomerase IDI):

[0036] MQTEHVILLNAQGVPTGTLEKYAAHTADTRLHLAFSSWLFNAKGQLLVTRRALSKKAWPGVWTNSVCGHPQLGESNEDAVIRRCRYELGVEITPPESIYPDFRYRATDPSGIVENEVCPVFAARTTSALQINDDEVMDYQWCDLADVLHGIDATPWAFSPWMVMQATNREARKRLSAFTQLK*;

[0037] SEQ ID NO: 5 (Nucleotide sequence of methyltransferase gene MpOMT4):

[0038]

[0039] SEQ ID NO: 6 (Amino acid sequence of methyltransferase gene MpOMT4):

[0040] MVADEEVRVRAEAWNNAFGYIKPTAVATAVELGLPDILENHDGPMSLLELSAATDCPAEPLHRLMRFLVFHGIFKKTAKPPLSNEAVYYARTALSRLFTRDELGDFMLLQTGPLSQHPAGLTASSLRTGKPQFIRSVNGEDSWTDPVNGYHMKVFSDAMAAHARETTAAIVR YCPAAFEGIGTVVDVGGRHGVALEKLVAAFPWVRGISFDLPEIVAKAPPRPGIEFVGGSFFESVPKGDLVLLMWILHDWSDESCIEIMKKCKEAIPTSGKVMIVDAIVDEDGEGDDFAGARLSLLDLIMMAVLARGKERTYREWEYLLREAGFTKFVVKNINTVEFVIEAYP*;

[0041] SEQ ID NO: 7 (nucleotide sequence of isopentenyl kinase AtIPK):

[0042] ATGGAACTGAATATTAGCGAAAGTCGCAGCCGCAGCATTCGCTGTATTGTTAAACTGGGTGGCGCAGCCATTACCTGCAAAAATGAACTGGAAAAAATCCACGACGAGAATCTGGAAGTTGTTGCATGTCAGCTGCGCCAGGCCATGCTGGAAGGTTCAGCACCTAGTAAAGTGATTGGCATGGATTGGAGTAAACGCCCGGGTAGCAGTGAAATTAGTTGTGATGTTGATGACATCGGCGATCAGAAAAGCAGCGAATTTTCAAAATTCGTGGTGGTTCATGGTGCAGGTAGCTTTGGTCATTTTCAGGCCAGCCGTAGCGGTGTGCATAAAGGCGGTCTGGAAAAACCGATTGTGAAAGCCGGCTTTGTTGCCACCCGTATTAGCGTTACCAATCTGAATCTGGAAATCGTTCGCGCACTGGCCCGTGAAGGTATTCCGACAATTGGTATGAGTCCGTTTAGCTGCGGTTGGAGTACCAGTAAACGCGATGTGGCCAGCGCCGATCTGGCAACCGTTGCAAAAACCATTGATAGTGGTTTTGTGCCGGTGCTGCATGGTGACGCAGTTCTGGATAATATTCTGGGCTGTACCATTCTGAGCGGCGATGTTATTATTCGCCATCTGGCCGATCATCTGAAACCGGAATATGTGGTGTTTCTGACCGATGTGCTGGGTGTTTATGATCGTCCGCCGAGCCCGAGCGAACCTGACGCAGTGTTACTGAAAGAAATTGCAGTGGGTGAAGATGGTAGCTGGAAAGTTGTTAATCCGCTGCTGGAACATACCGATAAAAAAGTTGATTACAGCGTGGCAGCCCATGATACCACCGGTGGTATGGAAACCAAAATTAGCGAAGCAGCCATGATTGCAAAACTGGGTGTGGATGTGTATATTGTGAAAGCAGCAACCACCCATAGCCAGCGCGCCTTAAATGGTGACCTGCGCGATAGCGTGCCGGAAGATTGGCTGGGTACCATTATTCGTTTTAGTAAATAA;。

[0043] SEQ ID NO: 8 (Amino acid sequence of isopentenyl kinase AtIPK):

[0044] MELNISESRSRSIRCIVKLGGAAITCKNELEKIHDENLEVVACQLRQAMLEGSAPSKVIGMDWSKRPGSSEISCDVDDIGDQKSSEFSKFVVVHGAGSFGHFQASRSGVHKGGLEKPIVKAGFVATRISVTNLNLEIVRALAREGIPTIGMSPFSCGWSTSKRDVAS ADLATVAKTIDSGFVPVLHGDAVLDNILGCTILSGDVIIRHLADHLKPEYVVFLTDVLGVYDRPPSPSEPDAVLLKEIAVGEDGSWKVVNPLLEHTDKKVDYSVAAHDTTGGMETKISEAAMIAKLGVDVYIVKAATTHSQRALNGDLRDSVPEDWLGTIIRFSK*;

[0045] SEQ ID NO: 9 (nucleotide sequence of choline kinase ScCK):

[0046]

[0047] SEQ ID NO: 10 (Amino acid sequence of choline kinase ScCK):

[0048] *

[0049] Example 2: Construction of genetically engineered bacteria containing different site-directed mutagenesis isopentenyltransferase mutants

[0050] Overexpression originates from Epimedium ( Epimedium The gene containing a truncated and optimized site-directed mutant isopentenyltransferase (STT) EpPTtru60 Mut Derived from Escherichia coli ( Escherichia coli Isopentenyl pyrophosphate isomerase gene idi Derived from mint ( Mentha methyltransferase gene MpOMT4 Derived from Arabidopsis thaliana ( Arabidopsis thaliana Isopentenyl kinase gene AtIPK and derived from brewer's yeast ( Saccharomyces cerevisiae choline kinase geneScCK overexpression plasmid pACYCDuet- EpPTtru60 Mut -idi-MpOMT4-AtIPK-ScCK (The reference for plasmid construction is Biotransformation of Kaempferol to Icaritinin Engineered) Saccharomy cescerevisiae The genetically engineered bacteria were obtained by transferring the gene into *E. coli* BL21(DE3) competent cells. The above-mentioned truncated and optimized genes containing different site-directed mutant isopentenyltransferases... EpPTtru60 Mut overexpression plasmid pACYCDuet- EpPTtru60- idi-MpOMT4-AtIPK-ScCK、 pACYCDuet- EpPTtru60 G52D -idi-MpOMT4-AtIPK-ScCK、 pACYCDuet- EpPTtru60 K74E -idi-MpOMT4-AtIPK- ScCK、 pACYCDuet- EpPTtru60 T131S - idi-MpOMT4-AtIPK-ScCK、 pACYCDuet- EpPTtru60 L187P - idi-MpOMT4-AtIPK-ScCK、 pACYCDuet- EpPTtru60 V205F - idi-MpOMT4-AtIPK-ScCK、 pACYCDuet- EpPTtru60 F271A - idi

[0051] -MpOMT4-AtIPK-ScCK、 pACYCDuet- EpPTtru60 D303E - idi-MpOMT4-AtIPK-ScCK All plasmids were synthesized by Suzhou Anshengda Company. The eight engineered strains containing different overexpression plasmids are shown in Table 1.

[0052] Table 1 Genetically engineered bacteria containing different site-directed mutagenesis isopentenyltransferase mutants

[0053]

[0054] Example 3: Fermentation experiment of icariin by genetically engineered bacteria containing different site-directed mutagenesis isopentenyltransferase mutants

[0055] Eight genetically engineered bacteria were inoculated into LB broth containing resistant culture medium. The culture system consisted of 10 mL aliquots of the medium in 100 mL Erlenmeyer flasks, with 20 µg / mL chloramphenicol added. The culture conditions were constant temperature shaking incubator at 37°C and 220 rpm overnight to obtain seed culture. This seed culture was then inoculated into fresh fermentation medium at a 1% volume inoculation rate. The culture system consisted of 50 mL aliquots of the medium in 250 mL Erlenmeyer flasks, with 20 μg / mL chloramphenicol added. The culture conditions were constant temperature shaking incubator at 37°C and 220 rpm. 600 Add 0.3 mM IPTG to a final concentration of 0.8-1.0, induce overnight at 25°C and 220 rpm, add 250 mg / L Kae and 25 mmol / L 3-methyl-3-buten-1-ol to the culture medium and continue fermentation for 48 h to prepare samples.

[0056] Example 4: Extraction and liquid chromatography of icariin

[0057] HPLC was performed using a Shimadzu LC20A system (equipped with an LC20ADXR pump, autosampler, and diode array detector). Flavonoid products were eluted on a Shim-pack XR-ODS column (75 mm x 2.0 mm, 2.2 lm, Shimadzu, Kyoto, Japan). The gradient elution system consisted of 0.01% acetic acid (A) and acetonitrile (B). Separation was performed using the following gradients: 0–2 min (15% B), 2–16 min (15%–70% B), 16–18 min (95% B), with the flow rate maintained at 0.45 mL / min. Kaempferol and icariin were detected at 350 nm. Conversions were calculated as the molar ratio of product to total substrate.

[0058] Using the strain Control containing a site-directed mutation of isopentenyltransferase as a control (shake-flask fermentation yield of 7.77 mg / L), the yield of icariin in shake-flask fermentation of seven genetically engineered bacteria containing different site-directed mutation isopentenyltransferase mutants was statistically analyzed. The results are shown in Table 2. Among the seven genetically engineered bacteria containing different site-directed mutation isopentenyltransferase mutants, the truncated EpPTtru60 containing the site-directed mutation showed the highest yield. G52D As the optimal mutant, after 48 h of shake-flask fermentation, the icariin yield was 9.35 mg / L, which was 20% higher than that of the control strain.

[0059] Table 2. Comparison of icariin production by isopentenyltransferase mutant strains and control strains.

[0060]

[0061] Note: - indicates no improvement.

[0062] From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects: The present invention provides a solution for the effects of epimedium (… Epimedium Site-directed mutagenesis was performed on the truncated and optimized isopentenyltransferase EpPTtru60, with the isopentenyltransferase mutant EpPTtru60 being the most effective mutant. G52D Overexpression of icariin in genetically engineered bacteria significantly increased the yield of icariin, as shown in the following results. Figure 1 As shown, this provides valuable components for constructing high-yield icariin strains.

[0063] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A prenyltransferase mutant, characterized in that, The isopentenyltransferase mutant is based on the amino acid sequence shown in SEQ ID NO.2, with glycine at position 52 replaced by aspartic acid.

2. The gene encoding the isopentenyltransferase mutant of claim 1.

3. A recombinant vector containing the gene described in claim 2.

4. A host cell containing the gene of claim 2.

5. The host cell of claim 4, wherein, The gene containing the gene described in claim 2, and the isopentenyl pyrophosphoisomerase gene. idi Methyltransferase gene MpOMT4 Isopentenyl kinase gene AtIPK and choline kinase gene ScCK The overexpression plasmid was transferred into E. coli BL21(DE3) competent cells to obtain genetically engineered bacteria; isopentenyl pyrophosphate isomerase gene. idi As shown in SEQ ID NO.3, the methyltransferase gene MpOMT4; as shown in SEQ ID NO.5, the isopentenyl kinase gene. AtIPK As shown in SEQ ID NO.7, the choline kinase gene ScCK As shown in SEQ ID NO.

9.

6. The use of the isopentenyltransferase mutant of claim 1, the gene of claim 2, the recombinant vector of claim 3, or the host cell of claim 4 or 5 in the production of icariin.

7. A method of producing icariin, characterized by, The method involves the following steps: fermenting the host cell described in claim 5 at 25°C for 48 hours.

8. The method of claim 7, wherein, The fermentation medium consisted of 20 g / L glucose, 4 mL / L glycerol, 12 g / L peptone, 16.4 g / L K2HPO4·3H2O, and 2.3 g / L KH2PO4.

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