Isopentene diphosphate isomerase mutant, method for improving lupeol yield of yarrowia lipolytica and application of isopentene diphosphate isomerase mutant

Abstract

The invention relates to an isopentene diphosphate isomerase mutant, a method for improving lupeol produced by yarrowia lipolytica and application of the isopentene diphosphate isomerase mutant, and belongs to the technical field of genetic engineering. The isopentene diphosphate isomerase mutant is obtained by rational design and screening on the basis of an amino acid sequence as shown in SEQ ID NO.1. According to the invention, a recombinant strain Po1FK01 for producing lupeol is constructed on the basis of a yarrowia lipolytica Po1f strain, the constructed isopentene diphosphate isomerase mutant is transformed into the recombinant strain for expression, and the activity of each mutant is characterized by the yield of lupeol. Compared with a control strain, namely a non-mutated strain, the yield of lupeol in the yarrowia lipolytica engineering strain of the overexpressed isopentene diphosphate isomerase mutant Y1IDI1 (K78R + S129D) is increased by about 86%. The invention provides powerful enzyme and bacterial strain for the biosynthesis of lupeol, and has important significance for the production of lupeol.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to an isopentenyl diphosphate isomerase mutant and a method and application for improving the production of lupeol by Yersinia lipolytica. Background Technology

[0002] Lupeol is a pentacyclic triterpenoid compound widely found in mango, olive, and birch bark. It possesses antioxidant, anti-inflammatory, antitumor, and hypoglycemic activities, attracting significant attention in the pharmaceutical field. The biosynthesis of lupeol primarily occurs through two pathways: the mevalonate (MVA) pathway and the methyl erythrose-4-phosphate (MEP) pathway. The MVA pathway is mainly found in the cell sap of eukaryotes and higher plants, while the MEP pathway is primarily found in plant plastids, bacteria, and algae. In *Yarrowia lipolytica* (…), it is also found in… Yarrowia lipolytica In the terpene synthesis pathway, isopentenyl diphosphate isomerase (YlIDI1) is one of the key regulatory enzymes in the intracellular terpene synthesis pathway. It catalyzes a crucial reaction in the mevalonate (MVA) pathway: the isomerization of isopentenyl pyrophosphate (IPP) with dimethyl allyl pyrophosphate (DMAPP). This reaction is essential for maintaining the balance of IPP and DMAPP in the cell. IPP and DMAPP are also precursors for the synthesis of many important natural products, such as terpenes and sterols. However, in its natural state, YlIDI1 is limited by low enzyme activity and weak substrate affinity. Therefore, improving YlIDI1 activity through protein engineering is of great significance. The activity of each YlIDI1 mutant was characterized by lupeol production.

[0003] Site-directed mutagenesis is a technique in molecular biology used to precisely and directionally modify specific DNA sequences. It involves introducing desired changes into the target DNA fragment using polymerase chain reaction (PCR), including base addition, deletion, and point mutations. Site-directed mutagenesis can efficiently and precisely improve the traits and characterization of the target protein expressed by DNA, making it a very useful tool in genetic engineering research and a powerful instrument for studying the complex relationships between protein structure and function. Summary of the Invention

[0004] To address the aforementioned problems, this invention provides an isopentenyl diphosphate isomerase mutant and a method and application for improving lupeol production in *Yarrowia lipolytica*. Through rational design of wild-type isopentenyl diphosphate isomerase from *Yarrowia lipolytica*, a highly efficient mutant was successfully obtained, resulting in a significant increase in lupeol production in the engineered strain.

[0005] One of the technical solutions provided by this invention is an isopentenyl diphosphate isomerase mutant. The isopentenyl diphosphate isomerase mutant is based on the wild-type isopentenyl diphosphate isomerase from Yersinia lipolytica shown in SEQ ID NO.1, which undergoes one of the following amino acid mutations: K78R, S129D, E161M, E192V, H93Y, Q111L, R112Y, Y197F, E194I, C128W, K78R+H93Y, K78R+C128W, K78R+S129D, S129D+E161M, C128W+S129D, C128W+E192V.

[0006] The second technical solution provided by this invention is a nucleic acid (i.e., a gene) encoding the above-mentioned isopentenyl diphosphate isomerase mutant. The third technical solution provided by the present invention is a recombinant vector containing the above-mentioned nucleic acid (i.e., a recombinant plasmid carrying the above-mentioned gene). Furthermore, the recombinant vector uses pYLXP' as the expression vector.

[0007] The fourth technical solution provided by the present invention is a recombinant strain containing the above-mentioned nucleic acid (i.e., a recombinant strain expressing the above-mentioned mutant). Furthermore, the host cell used for the recombinant strain is Yersinia lipophila.

[0008] Furthermore, the recombinant strain originated from Yersinia lipophila Po1f, and the YlIDI1 mutant gene was expressed using the pYLXP' plasmid.

[0009] The fifth technical solution provided by the present invention provides a method for producing lupeol, comprising fermenting and culturing the above-mentioned recombinant Yersinia lipolyticis to obtain the fermentation product lupeol.

[0010] Preferably, the fermentation culture temperature is 25-35 ℃; and the culture is carried out under shaking conditions for 90-150 h at a shaking speed of 150-400 rpm. More preferably, the culture temperature is 30 ℃, the culture time is 120 h, and the culture is carried out under shaking conditions at a shaking speed of 250 rpm.

[0011] The sixth technical solution provided by this invention is the application of the above-mentioned recombinant strain in the production of lupeol.

[0012] The seventh technical solution provided by the present invention is the application of the above-mentioned isopentenyl diphosphate isomerase mutant in enhancing the mevalonate pathway or producing lupeol.

[0013] The beneficial effects of this invention are: This invention uses the endogenous isopentenyl diphosphate isomerase of *Yersinia lipolytica* as the research object, and obtains isopentenyl diphosphate isomerase mutants through rational design and enzyme evolution screening. By molecularly docking isopentenyl diphosphate isomerase (YlIDI1) and the substrate isopentenyl pyrophosphate (IPP), key amino acid residues are identified, and site-directed mutagenesis is used to alter amino acid residues near the protein molecule's binding site with the substrate, thereby improving the catalytic activity of isopentenyl diphosphate isomerase. The activity of each mutant is characterized by the production of lupeol. This invention first knocks out the KU70 gene of *Yersinia lipolytica* Po1f, then integrates the three genes OeLUP1, YlERG1, and YlERG9 into the *Yersinia lipolytica* genomic locus POT1. Using this recombinant strain as a chassis cell, the constructed isopentenyl diphosphate isomerase mutants are transformed into the chassis cells for expression. Compared with the control strain, i.e. the unmutated strain, the engineered strain that overexpressed the isopentenyl diphosphate isomerase mutant YlIDI1 (K78R+S129D) showed an approximately 86% increase in lupeol production. Attached Figure Description

[0014] Figure 1-4 These are the spectra of plasmids pYLXP', pYLTDH, pYLFBA, and pUrloxp-POT1 in this invention; Figure 5 This is a graph showing the effect of the isopentenyl diphosphate isomerase mutant on lupeol production. Figure 6 This is a graph showing the effect of combined mutations at key sites of isopentenyl diphosphate isomerase on lupeol production. Detailed Implementation

[0015] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the methods and equipment used in the present invention are conventional methods and equipment in this technical field.

[0016] Unless otherwise specified, all reagent kit materials used in the following examples are commercially available products.

[0017] Unless otherwise specified, the experimental methods described in this invention are conventional methods.

[0018] The DNA polymerases, SnaBI, KpnI, XbaI, BamHI, MluI, DpnⅠ, NheI, and AvrII used in these embodiments were purchased from Takara Biotech Inc., and the gel extraction and plasmid extraction kits were purchased from Axygen Biotech Ltd. Primer synthesis and gene sequencing were performed by General Biotech (Anhui) Co., Ltd.

[0019] The starting strain used was Yersinia lipophila Po1f.

[0020] Example 1: Construction of a recombinant strain Po1FK01 with lupeol production capability (1) Knockout of the KU70 gene in *Yarrowia lipolytica* Po1f: The KU70 gene (NCBI ID: XM_501610.1, nucleotide sequence as shown in SEQ ID NO.3) was knocked out in *Yarrowia lipolytica* strain used in this study. The knockout method is described in the literature: Iterative integration of multiple-copy pathway genes in Yarrowia lipolytica For heterologous β-carotene production. Metabolic Engineering, 2017, 41, 192-201.). Yersinia lipolytica was obtained by knocking out the KU70 gene, which is responsible for non-homologous recombination, from the starting strain Po1f of *Yersinia lipolytica*. Yarrowia lipolytica Po1f ΔKU70, named Po1FK.

[0021] (2) Find olives on NCBI ( Olea europaea The lupeol synthase gene sequence (OeLUP1, NCBI number AB025343.1) from the source was sent to General Biotechnology (Anhui) Co., Ltd. for codon optimization according to the codon preference of Yersinia lipolytica to synthesize the OeLUP1 gene sequence (the nucleotide sequence after codon optimization is shown in SEQ ID NO.4).

[0022] (3) Construction of plasmid pYLXP'-OeLUP1 using pYLXP' as expression vector: First, the linearized pYLXP' vector (digested with SnaBI and KpnI) recovered from gel using the Gibson Assembly method was ligated with the corresponding PCR amplified (using the synthesized gene OeLUP1 as a template, the nucleotide sequences of primers OeLUP1-F / OeLUP1-R are shown in SEQ ID NO.12 and SEQ ID NO.13) and purified OeLUP1 gene fragment using a seamless cloning kit (ClonExpress II One Step Cloning Kit). Ligation was carried out at 50 ℃ for 1 hour, and then transformed into E. coil DH5α competent cells were evenly spread on LB solid medium containing 100 μg / ml ampicillin and cultured overnight at 37 ℃. Positive transformants were confirmed by colony PCR and transferred to LB liquid medium containing ampicillin for overnight culture and sequencing verification. After successful verification, the plasmid pYLXP'-OeLUP1 was obtained.

[0023] The pYLXP' plasmid is a laboratory deposit and carries a leucine expression cassette for the auxotroph selection gene, the resistance marker gene Amp, the promoter TEF, intron sequences, and the terminator XPR2. The nucleotide sequence of plasmid pYLXP' is shown in SEQ ID NO. 8, and its map is shown below. Figure 1 As shown.

[0024] (4) Construction of plasmid pYLTDH-YlERG9 using pYLTDH as the expression vector: First, the linearized pYLTDH vector (double-digested with XbaI and KpnI) recovered from the gel was ligated with the corresponding PCR-amplified (using Yersinia lipolyticis genomic DNA as a template, the nucleotide sequences of primers YlERG9-F / YlERG9-R are shown in SEQ ID NO.14 and SEQ ID NO.15) and purified squalene synthase (YlERG9, NCBI number XM_499929.1, nucleotide sequence shown in SEQ ID NO.5) gene fragment using a seamless cloning kit using the Gibson Assembly method. Subsequent steps were the same as those for constructing plasmid pYLXP'-OeLUP1. After verification, plasmid pYLTDH-YlERG9 was obtained.

[0025] The pYLTDH plasmid was preserved in the laboratory and carries a leucine expression cassette for the auxotroph selection gene, the resistance marker gene Amp, the promoter TDH1, and the terminator MIG1. The nucleotide sequence of plasmid pYLTDH is shown in SEQ ID NO. 9, and its map is shown below. Figure 2 As shown.

[0026] (5) Construction of plasmid pYLFBA-YlERG1 using pYLFBA as the expression vector: First, the linearized pYLFBA vector (digested with BamHI and MluI) recovered from the gel was ligated with the corresponding PCR-amplified (using Yersinia lipolyticis genomic DNA as a template, the nucleotide sequences of primers YlERG1-F / YlERG1-R are shown in SEQ ID NO.16 and SEQ ID NO.17) and purified squalene monooxygenase (YlERG1, NCBI number XM_503994.2, nucleotide sequence shown in SEQ ID NO.6) gene fragment using a seamless cloning kit using the Gibson Assembly method. Subsequent steps were the same as those for constructing plasmid pYLXP'-OeLUP1. After verification, plasmid pYLFBA-YlERG1 was obtained.

[0027] The pYLFBA plasmid was preserved in the laboratory and carries a leucine expression cassette for the auxotroph selection gene, the resistance marker gene Amp, the promoter FBA1, and the terminator PEX20. The nucleotide sequence of plasmid pYLFBA is shown in SEQ ID NO. 10, and its map is shown below. Figure 3 As shown.

[0028] (6) Construction of the integration plasmid pUrloxp-POT1-OeLUP1-YlERG9-YlERG1: Using plasmid pYLXP'-OeLUP1 as a template, primer loxP-P TEF -F / T XPR2 -P TDH -R (nucleotide sequence as shown in SEQ ID NO.18 and SEQ ID NO.19) was amplified by PCR to obtain P TEF -OeLUP1-T XPR2 Gene expression cassette fragment. Using plasmid pYLTDH-YlERG9 as a template, primer P was used. TDH -F / T MIG1 -P FBA -R (nucleotide sequence as shown in SEQ ID NO. 20 and SEQ ID NO. 21) was amplified by PCR to obtain P TDH -YlERG9-T MIG1 Gene expression cassette fragment. Using plasmid pYLFBA-YlERG1 as a template, primer P was used. FBA -F / T PEX20 -Dw-R (nucleotide sequences shown in SEQ ID NO.22 and SEQ ID NO.23) was amplified by PCR to obtain P FBA -YlERG1-T PEX20 Gene expression cassette fragments. The three gene expression cassette fragments were ligated to the gel-recovered linearized vector pUrloxp-POT1 (digested with NheI) using the Gibson Assembly method and then ligated using a seamless cloning kit. Subsequent steps were the same as those used to construct plasmid pYLXP'-OeLUP1. After verification, the plasmid pUrloxp-POT1-OeLUP1-YlERG9-YlERG1 was obtained.

[0029] The pUrloxp-POT1 plasmid was preserved in the laboratory. This plasmid carries the auxotroph selection genes leucine and uracil expression cassette, the resistance marker gene Amp, a homologous recombination arm, and loxP. The nucleotide sequence of plasmid pUrloxp-POT1 is shown in SEQ ID NO.11, and its map is shown below. Figure 4 As shown.

[0030] (7) The integration plasmid pUrloxp-POT1-OeLUP1-YlERG9-YlERG1 was digested with AvrII restriction endonuclease, and the target fragment to be integrated (containing homologous recombination arms and three gene expression cassettes) was recovered by gel extraction. The fragment was then transformed using the lithium acetate method (see reference: One-step transformation of the dimorphic yeast). Yarrowia lipolytica (Applied Microbiology and Biotechnology, 1997, 48 (2), 232–235.) The strain was transformed into Po1FK and plated on a uracil-deficient plate (CSM-Ura). The recombinant strain Po1FK01 was obtained by screening for the integration of the three gene expression cassettes OeLUP1-YlERG9-YlERG1 into the genomic locus POT1 (NCBI number XM_504109.3, nucleotide sequence as shown in SEQ ID NO.7).

[0031] Table 1 Primers and sequences involved in Example 1

[0032] More detailed information about the plasmids pYLXP', pYLTDH, pYLFBA, and pUrloxp-POT1 involved in this embodiment can be found in the literature: High titer production of gastrodin enabled by systematic refactoring of yeast genome and an antisense-transcriptional regulation toolkit. Metabolic Engineering, 2024, 82, 250-261. Example 2 Cloning of the isopentenyl diphosphate isomerase gene (1) Using Yersinia lipolytica genomic DNA as a template, PCR amplification was performed using primers YllDI1-F / YllDI1-R (nucleotide sequences shown in SEQ ID NO.24 and SEQ ID NO.25) to obtain the isopentenyl diphosphate isomerase gene fragment (YllDI1, NCBI number XM_504974.3, nucleotide sequence shown in SEQ ID NO.2), whose encoded amino acid sequence is shown in SEQ ID NO.1. The gel-recovered linearized vector pYLXP' (double digested with SnaBI and KpnI) was ligated to the YllDI1 gene fragment using a seamless cloning kit via the Gibson Assembly method. Subsequent steps were the same as those for constructing plasmid pYLXP'-OeLUP1. After verification, the recombinant plasmid pYLXP'-YlIDI1 expressing the YlIDI1 gene was obtained.

[0033] The amino acid sequence encoded by gene YlIDI1 in SEQ ID NO.1: MTTSYSDKIKSISVSSVAQQFPEVAPIADVSKASRPSTESSDSSAKLFDGHDEEQIKLMDEICVVLDWDDKPIGGASKKCCHLMDNINDGLVHRAFSVFMFNDRGELLLQQRAAEKITFANMWTNTCCSHPLAVP SEMGGLDLESRIQGAKNAAVRKLEHELGIDPKAVPADKFHFLTRIHYAAPSSGPWGEHEIDYILFVRGDPELKVVANEVRDTVWVSQQGLKDMMADPKLVFTPWFRLICEQALFPWWDQLDNLPAGDDEIRRWIK Table 2 Primers used to clone the YlIDI1 gene

[0034] Example 3: Obtaining the isopentenyl diphosphate isomerase mutant (1) Molecular docking and virtual saturation mutagenesis of isopentenyl diphosphate isomerase derived from Yersinia lipophila.

[0035] The amino acid sequence of Y. lipolyticis isopentenyl diphosphate isomerase (YlIDI1) was retrieved from NCBI (NCBI ID: XP_504974.3). Homology modeling of YlIDI1 was performed using SWISS-MODEL (https: / / swissmodel.expasy.org) (template PDB ID: 2dho) to obtain the protein's three-dimensional structure. The IPP substrate model (PubChem CID: 1195) was obtained from PubChem (https: / / www.pubchem.ncbi.nlm.nih.gov). Molecular docking of the potential binding mode between the enzyme YlIDI1 and the ligand IPP was performed using AutoDock 4.2, with a docking box size of 40×40×40 and a grid spacing of 0.375 Å. Visualization analysis was then performed using PyMOL 3.1.1 software. Virtual saturation mutation analysis was performed on amino acids near the substrate binding pocket (hotspot residues such as E161, E192, H93, Q111, K78, R112, Y197, E194, S129, and C128) using FoldX 5.0. The binding free energy change (ΔΔG) of each mutant was calculated to assess the effect of the mutation on the enzyme. Mutations with negative ΔΔG values ​​enhance affinity and stabilize the favorable binding conformation of the enzyme-substrate complex. Finally, mutants E161M, E192V, H93Y, Q111L, K78R, R112Y, Y197F, E194I, S129D, and C128W were selected for experimental validation.

[0036] (2) The isopentenyl diphosphate isomerase mutant gene was obtained by site-directed mutagenesis of the whole plasmid. Using pYLXP'-YlIDI1 plasmid as a template, primers were designed using Primer Premier 5.0. The primer sequences are shown in the table below: Table 3. Site-directed mutagenesis primer sequences

[0037] (3) PCR amplification of the target gene Table 4 Reaction System

[0038] Table 5 Reaction Procedure

[0039] The PCR product was treated with 0.5 μL of DpnI to remove methylation, and then transformed. E. coliAfter overnight culture of DH5α strain, plasmids were extracted and sequenced to verify whether the mutation was successful, and finally plasmids pYLXP'-YlIDI1 (E161M, E192V, H93Y, Q111L, K78R, R112Y, Y197F, E194I, S129D or C128W) were obtained.

[0040] Example 4: Effect of overexpression of isopentenyl diphosphate isomerase mutant on lupeol yield (1) The original plasmid pYLXP'-YlIDI1 and the correctly sequenced mutant plasmid were transformed into strain Po1FK01, respectively. After the colonies grew on the leucine-deficient plate, single colonies from the solid medium were picked and placed into a shaker tube containing 2 mL of CSM-Leu liquid seed medium. The tubes were then cultured in a shaker at 30 °C and 250 rpm for 2 days to obtain the fermentation seed liquid. The seed liquid was inoculated at a rate of 2% into a 250 mL Erlenmeyer flask containing 30 mL of CSM-Leu liquid fermentation medium. The flasks were then cultured in a shaker at 30 °C and 250 rpm for 5 days.

[0041] Yeast culture medium formulation: Each liter of CSM-Leu liquid seed medium contains 20 g glucose, 1.7 g amino-free yeast nitrogen source, 5 g ammonium sulfate, and 0.7 g CSM-Leu. Solid medium requires the addition of 20 g agar powder. Each liter of CSM-Leu liquid fermentation medium contains 40 g glucose, 1.7 g amino-free yeast nitrogen source, 1.1 g ammonium sulfate, and 0.7 g CSM-Leu.

[0042] (2) After fermentation, take 1 mL of fermentation broth, centrifuge at 12000 rpm for 5 min, discard the supernatant, add 100 μL of 2000 U / mL lysozyme, and shake in a metal bath at 30 ℃ and 1800 rpm for 2 h. Then add 0.25 g of acid-washed glass beads and 1 mL of acetone, and grind and shake in a metal bath for 2 h. Centrifuge to collect the supernatant, and filter it through a 0.22 μm organic nylon membrane for later use.

[0043] (3) The extract was analyzed by high performance liquid chromatography (HPLC). A C18 reversed-phase column (EclipsePlus C18, 4.6 mm × 150 mm, diameter 5 μm) was used. The chromatographic conditions were as follows: column temperature: 35 ℃; mobile phase: methanol; total flow rate: 1 mL / min; detection wavelength: 205 nm; isocratic elution; injection volume: 10 μL.

[0044] (4) The yield of lupeol synthesized by the engineered bacteria overexpressing the isopentenyl diphosphate isomerase mutant was detected by high performance liquid chromatography as shown in Table 6 below. Figure 5 As shown: Table 6. Yield of lupeol synthesized by engineered bacteria overexpressing isopentenyl diphosphate isomerase mutant

[0045] Example 5 Functional Validation of Key Site Combination Mutants of Isoprene Diphosphate Isomerase The mutants obtained from the above screening were subjected to combined mutations and further screening to obtain mutants with enhanced enzyme activity. For example, the construction process of the K78R+S129D combined mutant plasmid was as follows: using the pYLXP'-YlIDI1 (K78R) plasmid as a template, the F and R primers of S129D in Example 3 were used for PCR amplification according to Example 3. DpnI was added to the PCR product to remove methylation, and then the product was transformed. E. coli After overnight culture of the DH5α strain, plasmids were extracted and sequenced to verify whether the mutation was successful, ultimately yielding the K78R+S129D combined mutant plasmid.

[0046] The combined mutant plasmids were transformed into the Po1FK01 strain. After colonies grew on leucine-deficient plates, single colonies from the solid medium were picked and transferred to shake tubes containing 2 mL of CSM-Leu liquid seed medium. The culture was then incubated in a shaker at 30 °C and 250 rpm for 2 days to obtain the fermentation seed culture. The seed culture was inoculated at a 2% inoculation rate into 250 mL Erlenmeyer flasks containing 30 mL of CSM-Leu liquid fermentation medium, and then incubated in a shaker at 30 °C and 250 rpm for 5 days. After fermentation, 1 mL of the fermentation broth was transferred to a centrifuge tube and centrifuged at 12000 rpm for 5 min to collect the bacterial cells for later use.

[0047] Cells were broken and fermentation products were extracted according to the method in Example 4 (2), and then analyzed by HPLC according to the method in Example 4 (3). Figure 6 The results showed that after five days of shake-flask fermentation, the lupeol production of the K78R+S129D combined mutant was approximately 1.86 times that of the control (unmutated wild-type WT), indicating that the K78R+S129D combined mutant can increase lupeol production. However, the lupeol production of the K78R+H93Y, K78R+C128W, and S129D+E161M combined mutants was lower than that of the control (unmutated wild-type WT), and the lupeol production of the C128W+S129D and C128W+E192V combined mutants increased only slightly compared to the control (unmutated wild-type WT).

[0048] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the patent. It should be noted that those skilled in the art can make various changes, combinations, and improvements to the above embodiments without departing from the concept of this patent, and these all fall within the scope of protection of this patent. Therefore, the scope of protection of this patent is determined by the claims.

Claims

1. An isopentenyl diphosphate isomerase mutant, characterized in that, The isopentenyl diphosphate isomerase mutant described herein is based on the wild-type isopentenyl diphosphate isomerase from Yersinia lipolytica shown in SEQ ID NO.1, with one of the following amino acid mutations: K78R, S129D, E161M, E192V, H93Y, Q111L, R112Y, Y197F, E194I, C128W, K78R+H93Y, K78R+C128W, K78R+S129D, S129D+E161M, C128W+S129D, C128W+E192V.

2. A nucleic acid, characterized in that, The isopentenyl diphosphate isomerase mutant of claim 1 is encoded.

3. A recombinant vector or recombinant strain containing the nucleic acid of claim 2.

4. The recombinant vector as described in claim 3, characterized in that, The expression plasmid used was pYLXP'.

5. The recombinant strain according to claim 3, characterized in that, The host cell used was Yeast lipolytica.

6. The recombinant strain according to claim 5, characterized in that, The recombinant strain originated from Yersinia lipophila Po1f, and the isopentenyl diphosphate isomerase mutant of claim 1 was expressed using the pYLXP' plasmid.

7. A method for producing lupeol, characterized in that: This includes fermenting the recombinant strain as described in claim 3 or 6 to produce lupeol.

8. The method according to claim 7, characterized in that: The fermentation culture was carried out at a temperature of 25-35 ℃ and under shaking conditions for 90-150 h at a shaking speed of 150-400 rpm.

9. The use of the recombinant vector or recombinant strain according to claim 3 in the production of lupeol.

10. The use of the isopentenyl diphosphate isomerase mutant of claim 1 in enhancing the mevalonate pathway or in the production of lupeol.

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