Highly active isopentenyl phosphate kinase mutants and uses thereof

Abstract

The application belongs to the technical field of enzyme engineering, and discloses a high-activity isopentenyl phosphate kinase mutant and application thereof. H274P The high-activity isopentenyl phosphate kinase mutant is AtIPK H274P , wherein the amino acid at position 274 of the wild-type AtIPK is mutated from histidine H to proline P. S47A,L124A SmDAGK S47A,L124A can constitute an efficient isopentenol utilization pathway which can effectively convert prenol into DMAPP. By using the pathway, an engineering strain with improved isopentenyl modification ability can be constructed, and the strain has more advantages compared with a traditional engineering strain based on a mevalonate pathway.

Description

Technical Field

[0001] This invention belongs to the field of enzyme engineering technology, and specifically relates to a highly active isopentenyl phosphokinase mutant and its applications. Background Technology

[0002] Isopentenylation is a ubiquitous modification found in almost all life forms, commonly occurring in aromatic compounds such as flavonoids and certain alkaloids. In nature, these enzymatic reactions primarily rely on dimethyl allyl pyrophosphate (DMAPP) as the isopentenyl donor. Isopentenylated flavonoids are derivatives formed under the action of isopentenyltransferases, which catalyze the binding of DMAPP to the flavonoid backbone. The introduction of the isopentenyl group enhances the lipophilicity of flavonoids, thereby promoting their crossing of cell membrane barriers and improving cellular uptake efficiency. These compounds possess a variety of pharmacological activities, including anti-inflammatory, analgesic, and anticancer effects.

[0003] Microbial cell factories have become a potential platform for the biosynthesis of isopentenylated flavonoids. Saccharomyces cerevisiae is widely used in the biosynthesis of flavonoids and terpenes and is an important host for isopentenylated flavonoids. However, the supply of DMAPP remains a major bottleneck for isopentenylated compounds. Figure 1 As shown, the mevalonic acid (MVA) pathway exists in most eukaryotic microorganisms and is responsible for the biosynthesis of terpenoids. Through this pathway, isopentenyl pyrophosphate (IPP) is converted to DMAPP by an isomerase encoded by IDI1. However, this reaction is inefficient, and IPP and DMAPP are rapidly condensed into GPP by ERG20, flowing downstream and reducing the DMAPP donor. The isopentenol utilization pathway is an artificial synthesis pathway for terpenoids, using 3-methyl-3-buten-1-ol or 3-methyl-2-buten-1-ol as substrates. The former can be converted to IPP via a two-step phosphorylation process, while the latter yields DMAPP. Similar to the MVA pathway, the method using isopentenol as a substrate is still subject to the conversion of IPP to DMAPP. Using prenol as a substrate, the method of directly converting to DMAPP via the isopentenol utilization pathway can bypass the corresponding steps, increasing the isopentenyl supply and enhancing the synthesis of isopentenylated flavonoids. However, existing research mainly focuses on phosphokinases (such as AtIPK). S270P, A272R Modifications can be made to improve the conversion of isoprenol through artificial synthesis pathways. For example, Chinese invention patent CN118530865A discloses an engineered Saccharomyces cerevisiae strain dependent on the isoprenol utilization pathway, its construction method, and its application. The diacylglycerol kinase SmDAGK mutant used in this study is SmDAGK. S47A , L124A The AtIPK mutant of isopentenyl phosphokinase is AtIPK.S270P, A272R The strain containing the two mutants has isoprenol as a substrate, and the content of squalene is 1.5 times higher than that of the wild type. At present, there is a lack of effective mutants for promoting the conversion of prenol in the artificial pathway. SUMMARY

[0004] The primary object of the present application is to overcome the defects and shortcomings of the prior art, and provide a high-activity isopentenyl phosphate kinase mutant.

[0005] Another object of the present application is to provide the application of the high-activity isopentenyl phosphate kinase mutant.

[0006] The object of the present application is achieved by the following technical solutions:

[0007] A high-activity isopentenyl phosphate kinase mutant, AtIPK H274P , refers to the mutation of the 274th amino acid of AtIPK from histidine H to proline P.

[0008] The amino acid sequence of AtIPK is shown in the accession number Genbank NP_173986.2, and there are 332 amino acids in total.

[0009] The sequence of the coding nucleic acid of the high-activity isopentenyl phosphate kinase mutant is shown in SEQ ID NO. 1.

[0010] The high-activity isopentenyl phosphate kinase mutant can improve the utilization efficiency of 3-methyl-2-butene-1-ol in the isopentenol utilization pathway, and is applied in the preparation of dimethylallyl pyrophosphate, terpenoids and isopentenyl flavonoids with 3-methyl-2-butene-1-ol as a substrate.

[0011] An enzyme combination of an isopentenol utilization pathway with 3-methyl-2-butene-1-ol as a substrate, comprising SmDAGK S47A, L124A and the high-activity isopentenyl phosphate kinase mutant.

[0012] The SmDAGK S47A, L124A refers to the mutation of the 47th amino acid of SmDAGK from serine S to alanine A, and the 124th from leucine L to alanine A.

[0013] The amino acid sequence of SmDAGK is shown in the accession number Genbank AAA26867.1, and there are 137 amino acids in total.

[0014] The sequence of the coding nucleic acid of SmDAGK S47A, L124A is shown in SEQ ID NO. 2.

[0015] The isoprenol utilization pathway is a pathway that can convert 3-methyl-2-butene-1-ol (CAS No. 556-82-1, English name prenol) into dimethylallyl pyrophosphate (DMAPP).

[0016] An enzyme combination for realizing isoprenylation of flavonoids with 3-methyl-2-butene-1-ol as a substrate, comprising an isoprenyltransferase or an isoprenyltransferase mutant, and the enzyme combination of the above-mentioned isoprenol utilization pathway with 3-methyl-2-butene-1-ol as a substrate.

[0017] The isoprenyltransferase mutant is tSfN8DT-1 Q12E, N305M ; the amino acid sequence of which is shown in SEQ ID NO. 3.

[0018] The sequence of the coding nucleic acid of the isoprenyltransferase mutant is shown in SEQ ID NO. 4.

[0019] The flavonoid is preferably naringenin.

[0020] A recombinant expression vector containing the coding nucleic acid of the enzyme combination of the above-mentioned isoprenol utilization pathway with 3-methyl-2-butene-1-ol as a substrate or the coding nucleic acid of the enzyme combination for realizing isoprenylation of flavonoids with 3-methyl-2-butene-1-ol as a substrate, which can express the enzyme combination of the above-mentioned isoprenol utilization pathway with 3-methyl-2-butene-1-ol as a substrate or the enzyme combination for realizing isoprenylation of flavonoids with 3-methyl-2-butene-1-ol as a substrate.

[0021] The sequence of the coding nucleic acid of the high-activity isoprenyl phosphate kinase mutant is shown in SEQ ID NO. 1.

[0022] The sequence of the coding nucleic acid of the SmDAGK S47A, L124A is shown in SEQ ID NO. 2.

[0023] The sequence of the coding nucleic acid of the isoprenyltransferase mutant is shown in SEQ ID NO. 4.

[0024] The vector backbone of the recombinant expression vector is from a P426 GAL vector or a pY26-GPD-TEF vector.

[0025] The two enzymes in the coding nucleic acid of the enzyme combination of the above-mentioned isoprenol utilization pathway with 3-methyl-2-butene-1-ol as a substrate are independently expressed, that is, each enzyme gene is provided with a promoter and a terminator; the structure is preferably as follows: T CYC1 -SmDAGK S47A, L124A -P GPD -P TEF1-AtIPK H274P -T ADH1 .

[0026] The three enzymes in the nucleic acid encoding the enzyme combination for realizing the isopentenylization of flavonoids with 3-methyl-2-buten-1-ol as the substrate are independently expressed, that is, each enzyme gene contains a promoter and a terminator; the structure is preferably as follows: T CYC1 -SmDAGK S47A, L124A -P GAL1,10 -AtIPK H274P -T GAL10 -P GAL7 -tSfN8DT- 1Q12E, N305M -T ADH1 .

[0027] A recombinant Saccharomyces cerevisiae cell containing the recombinant expression vector described above.

[0028] The starting strain of the recombinant Saccharomyces cerevisiae cell has the following characteristics: the mevalonate pathway is blocked, and the 156th amino acid in the PRM10 gene is mutated from leucine to glutamine.

[0029] The mevalonate pathway is blocked by deleting or generating a frameshift mutation in the ERG13 gene in the mevalonate pathway through gene editing technology.

[0030] The chassis cell of the starting strain is preferably a Saccharomyces cerevisiae of the CEN.PK series; more preferably, it is Saccharomyces cerevisiae CEN.PK2-1C.

[0031] When the recombinant expression vector contains nucleic acids encoding an enzyme combination for realizing the isopentenylization of flavonoids with 3-methyl-2-buten-1-ol as the substrate, the recombinant Saccharomyces cerevisiae cell preferably has the following characteristics: nucleic acids encoding high-activity isopentenyl phosphate kinase mutants, nucleic acids encoding SmDAGK S47A,L124A , and nucleic acids encoding isopentenyl transferase mutants are integrated into the chromosome of the cell through chromosomal gene sites.

[0032] The chromosomal gene site is preferably the GAL80 site.

[0033] The application of the recombinant Saccharomyces cerevisiae cell in preparing dimethylallyl pyrophosphate, terpenoids, and isopentenylated flavonoids.

[0034] The present application is based on further research on the basis of the previous stage. In the previous stage, a Saccharomyces cerevisiae engineering strain dependent on isoprenol utilization pathway was disclosed in Chinese invention patent CN118530865A. The natural mevalonate pathway of the Saccharomyces cerevisiae engineering strain was inactivated, the 156th amino acid in the PRM10 gene was mutated from leucine to glutamine, and the phosphokinase SmDAGK mutant SmDAGK S47A, L124A and the phosphokinase AtIPK mutant AtIPK S270P, A272R were contained. When the Saccharomyces cerevisiae engineering strain used prenol as a substrate, the yield of squalene was not high, that is, the utilization rate of prenol by the Saccharomyces cerevisiae engineering strain was not high, and thus further improvement was needed. Compared with the existing technology, the present application has the following advantages and beneficial effects:

[0035] (1) The AtIPK H274P mutant and the SmDAGK S47A, L124A mutant provided by the present application can constitute an efficient isoprenol utilization pathway.

[0036] (2) The efficient isoprenol utilization pathway provided by the present application can effectively convert prenol into DMAPP.

[0037] (3) The engineering strain provided by the present application has strong isoprenyl modification ability, and is more advantageous compared with traditional engineering strains based on the MVA pathway. BRIEF DESCRIPTION OF DRAWINGS

[0038] Figure 1 is a schematic diagram of the DMAPP supply strategy of the prior art and the present application.

[0039] Figure 2 is a schematic diagram of the high-throughput screening method.

[0040] Figure 3 is a graph of the activity results of different mutants.

[0041] Figure 4 is a graph of the effect results of different isoprenol utilization pathway gene combinations.

[0042] Figure 5 is a schematic diagram of the integration of the chromosomes of the NP01 strain and the AP01 strain.

[0043] Figure 6 is a graph of the comparison results of the synthesis of 8-isoprenyl naringenin of the NP01 strain and the AP01 strain. DETAILED DESCRIPTION

[0044] The present application will be further described in detail below in combination with the embodiments and the drawings, but the implementation manner of the present application is not limited thereto.

[0045] Unless otherwise noted, the following embodiments were carried out under conventional experimental conditions or as recommended by the reagent manufacturer. The materials, reagents, etc. used were commercially available reagents and materials unless otherwise specified.

[0046] Gibson assembly: The procedure was followed according to the instructions of the 2X MultiF Seamless Assembly Mix from Eppendorf.

[0047] Yeast strain transformation: Transformation for gene editing was performed using the Frozen-EZ Yeast Transformation II™ kit (purchased from ZYMO RESEARCH, USA) according to the product instructions unless otherwise specified.

[0048] PCR amplification included conventional denaturation, annealing, and extension steps, and was performed according to the product instructions of the PrimeSTAR® Max DNA Polymerase.

[0049] The following biological materials were used in the present application: plasmid p426-T CYC1 -SmDAGK-P GPD -P TEF1 -AtIPK-T ADH1 , plasmid p426-T CYC1 -SmDAGK S47A, L124A -P GPD -P TEF1 -AtIPK-T ADH1 , plasmid p426-T CYC1 -SmDAGK S47A, L124A -P GPD -P TEF1 -AtIPK S270P, A272R -T ADH1 The IUP1 strain has been disclosed in the literature “Li GJ, et al. Yeast metabolism adaptation for efficient terpenoids synthesis via isopentenol utilization. Nature Communications, 2024, Vol 15, Issue 1” and its supplementary files.

[0050] The primers used in the present application are shown in Table 1:

[0051] Table 1 Primers

[0052]

[0053] Example 1: Identification of amino acids near the active site of AtIPK

[0054] Using the protein sequence of AtIPK as a seed sequence, a homologous sequence alignment of prenylpyrophosphatase was performed on the Protein Data Bank website, and protein-ligand complex crystal structures with sequence similarities of 32% (PDB: 3lkk), 32% (PDB: 3ll5), and 27% (PDB: 7lnu) were screened. Analysis showed that the target enzyme was a homodimer, in which the active sites of the two monomers in the 3ll5 and 7lnu crystals were bound to ADP+IPP and ATP+IP, respectively, and the two monomers in the 3lkk crystal were bound to ATP and IP. Five protein monomer conformations were obtained by splitting and extracting using PyMOL software, namely 3lkk (ATP, IP), 3ll5 (ATP, IP), 3ll5 (ADP, IPP), 7lnu (ATP, IP), and 7lnu (ADP, IPP), which were pairwise paired with the five AtIPK models (Model_1-Model_5) predicted by AlphaFold3 for three-dimensional structure alignment. The results showed that the RMSD values of 3lkk (ATP, IP) and 3ll5 (ADP, IPP) crystals with Model_3 were the lowest. The ligands of 3lkk and 3ll5 were combined with Model_3 to form new protein-ligand complexes, and the amino acid residues within a range of 4 Å were extracted, and the key site combinations were determined by molecular docking, resulting in 37 candidate modification sites (with amino acid residue numbers 18, 20, 21, 22, 27, 95, 96, 97, 101, 126, 187, 198, 199, 201, 202, 203, 223, 224, 226, 227, 228, 229, 230, 231, 232, 267, 268, 269, 270, 272, 274, 275, 276, 277, 279, 280, 283, respectively). Based on the NNK method, the codon preference sequence of the Saccharomyces cerevisiae for the saturated mutation of the amino acid was A (GCT), G (GGT), V (GTT), L (TTG), I (ATT), E (GAA), Q (CAA), D (GAT), N (AAT), M (ATG), S (TCT), T (ACT), F (TTT), W (TGG), Y (TAT), R (AGA), H (CAT), C (TGT), P (CCA), K (AAA). The p426-T CYC1 -SmDAGK-P GPD -P TEF1 -AtIPK-TADH1 Plasmid as PCR template, and using the method disclosed in the literature with appropriate modification: that is, the 0.5 g / L isoprenol substrate added in the screening plate is replaced by 0.7 g / L prenol substrate. In addition, the OD 600 As a screening index.

[0055] The results are shown in Figure 2 By high-throughput screening system, the catalytic efficiency of AtIPK saturation mutant library for unnatural substrate DMAP was rapidly evaluated. Sequencing results showed that 39 mutants were preliminarily screened out, including S201A, G202A, G202S, D223R, D223E, D223Q, V224A, Y228H, D229R, D229W, D229G, R230F, P231R, P231H, P231Q, P232Y, P232K, P232N, P232G, P232M, P232A, P232R, P232S, P232F, Y269A, H274S, H274R, H274P, H274A, H274T, H274G, H274Y, H274L, H274C, D275G, T277S, G279A, M280R and M280V.

[0056] Example 2: Screening of optimal mutants

[0057] The corresponding mutants were obtained by site-directed mutagenesis technology for secondary verification. The mutant vectors of the above 39 AtIPKs were constructed. Taking the construction of AtIPK H274P mutant vector as an example, p426-T CYC1 -SmDAGK-P GPD -P TEF1 -AtIPK-T ADH1 Plasmid as template, using AtIPK H274P -F and AtIPK H274P -R as primer pairs, PrimeSTAR ® Max DNA polymerase as high-fidelity enzyme for polymerase chain reaction (PCR) amplification. After purification and recovery of the PCR product, 100 ng of the product was added to E. coli DH5α competent cells, and after transformation, it was coated on LB agar medium containing 100 mg / L ampicillin (Ampicillin) and incubated at 37°C overnight. Randomly selected 6 single colonies were inoculated in LB liquid medium containing Ampicillin and cultured at 37°C, 220 rpm for 10-12 h. After extracting the plasmid, it was sent to the company for sequencing verification, and p426-T CYC1 -SmDAGK-P GPD -P TEF1-AtIPK H274P -T ADH1 Recombinant plasmids. Other mutants are constructed using similar methods.

[0058] The above 39 mutants and p426-T respectively CYC1 -SmDAGK-P GPD -P TEF1 -AtIPK-T ADH1 (Control group) Plasmid transformed into IUP1 strain (knockout) ERG13 The transformation method for the IUP1 gene is as follows: IUP1 cells are seeded in 3 mL of YPD medium containing 5 mM mevalonic acid and cultured at 30℃ and 220 rpm for 36-48 h to obtain cell culture medium. 250 μL of cell culture medium is then diluted at 6000 × 10⁻⁶. g Centrifuge for 3 min and discard the supernatant. Then add 100 μL of transformation buffer, 0.5 μg of purified plasmid, and 3 μL of 10 mg / mL ssDNA. Mix well and incubate at 37°C for 30 min. Then incubate at 6000 × 10⁻⁶ for 30 min. g Centrifuge for 3 min and discard the supernatant. Resuspend the cell pellet in 100 μL of sterile deionized water and plate it onto a YPD agar plate containing 2 g / L prenol. Incubate at 30°C for 4–5 days. Pick a single colony from the transformation plate and inoculate it into 3 mL of YPD medium containing 4 g / L prenol. Incubate at 30°C and 220 rpm for 24 h. Then, use the initial OD value... 600 = 0.1 was inoculated into 48-well plates, with each well containing 1 mL of YPD medium supplemented with 4 g / L prenol or isoprenol, and cultured at 30℃ and 220 rpm for 4 days to obtain the fermentation broth.

[0059] 0.2 mL of fermentation broth was added to a 2 mL homogenization tube containing 0.7 g of 0.5 mm diameter glass beads and 1 mL of ethyl acetate. Cell disruption was performed using a biological sample homogenizer, followed by 10000 × 10⁻⁶ steps. g Centrifuge for 1 min, and filter the upper ethyl acetate phase through a 0.22 μm filter membrane. Analyze the results using an LC-16 high-performance liquid chromatography (HPLC) system equipped with an SPD-16 dual-wavelength UV detector (Shimadzu Corporation, Japan). The chromatographic column was an Agilent Poroshell 120 EC-C18 2.1 × 100 mm (Agilent Technologies, USA). The mobile phase was 100% acetonitrile with isocratic elution, the flow rate was 0.5 mL / min, the injection volume was 2 μL, and the elution time was 7.5 min.

[0060] The activity of the mutant is characterized by the accumulation amount of squalene, from which the activity of the mutant is calculated according to the following formula: Figure 3 It can be seen that the mutants in the secondary verification can basically improve the conversion of prenol by the isoprenol utilization pathway. Among them, AtIPK H274P The mutants can greatly improve the conversion of isoprenol in addition to improving the conversion of prenol.

[0061] AtIPK, AtIPK S270P, A272R , and AtIPK H274P are respectively combined with SmDAGK S47A, L124A to test the influence of different combinations on the conversion of prenol, wherein the plasmids used are p426-T CYC1 -SmDAGK S47A, L124A -P GPD -P TEF1 -AtIPK-T ADH1 , p426-T CYC1 -SmDAGK S47A, L124A -P GPD -P TEF1 -AtIPK S270P, A272R -T ADH1 , p426-T CYC1 -SmDAGK S47A , L124A -P GPD -P TEF1 -AtIPK H274P -T ADH1 are constructed based on p426-T CYC1 -SmDAGK S47A, L124A -P GPD -P TEF1 -AtIPK-T ADH1 according to the method of Example 2. The results are shown in Table 1. Figure 4 As shown in Table 1, the conversion rate of prenol by the isoprenol utilization pathway composed of wild-type AtIPK is poor, and the accumulation of squalene is only 4.9 mg / L; the mutant AtIPK S270P, A272R corresponding to the accumulation of squalene is 31.9 mg / L; and the conversion rate of prenol by the isoprenol utilization pathway composed of the mutant AtIPK H274P of the application is greatly improved, and the accumulation of squalene is 68.3 mg / L, which is 13.8 times and 2.1 times that of wild-type AtIPK and AtIPK S270P, A272R , respectively.

[0062] Example 3: Construction of isoprenylated flavonoid synthesis strain

[0063] To compare the DMAPP supply capacity of the natural biosynthetic pathway and the artificial biosynthetic pathway by the yield of 8-prenylnaringenin as a model product of prenylated flavonoids, a mutant tSfN8DT-1 of isoprenyltransferase from Sophora flavescens Sophora flavescens was introduced. Q12E, N305M The 8-prenylation of naringenin was achieved by tSfN8DT-1 (disclosed in the annex "Supplementary Data 5" of the document "Guo CJ, et al. De novo biosynthesis of 8-prenylnaringenin in Saccharomyces cerevisiae Saccharomyces cerevisiae improved by screening and engineering of prenyltransferases and precursor pathway. Systems Microbiology and Biomanufacturing, 2023, Vol 3, Issue 4"). Q12E, N305M The nucleotide sequence of the gene tSfN8DT-1 is shown as SEQ ID NO. 4.

[0064] The pYZ463 (obtained from the addgene global plasmid sharing platform, Plasmid # 187971, TEF1p-Cas9-CYC1t and SNR52p-Not1-SUP4t) was used as a template, and the primers GAL80sgRNA-F and GAL80sgRNA-R were used for amplification, followed by E. coli DH5a transformation, plasmid extraction and sequencing verification. Finally, the Crispr-Cas9 plasmid with 5'-ACGATAGTTGCAGTATGGCG-3' as the specific 20nt sgRNA was obtained, which was pYZ463-GAL80 plasmid targeting the GAL80 gene. Overexpression of the endogenous IDI1 (NCBI-GeneID: 855986) and tHMG1 (NCBI-GeneID: 854900, whose N-terminal is truncated) of Saccharomyces cerevisiae to realize the strengthening of the endogenous MVA pathway of Saccharomyces cerevisiae, the nucleotide sequence of IDI1 is shown in SEQ ID NO. 5, and the nucleotide sequence of tHMG1 is shown in SEQ ID NO. 6, the specific steps are as follows: using pY26-GPD-TEF plasmid (also named as pY26TEF-GPD) (obtained from BioVector NTCC plasmid vector strain cell gene preservation center) as a template, using primers pY26-F and pY26-R for amplification to obtain pY26-GPD-TEF plasmid backbone; using Saccharomyces cerevisiae CEN.PK2-1C genome as a template, using primer pairs IDI1-F / IDI1-R, P GAL1,10 -F / P GAL1,10 -R, tHMG1-F / tHMG1-R, T GAL10 -P GAL7 -F / T GAL10 -P GAL7 -R primers for amplification to obtain IDI1 gene, P GAL1,10 bidirectional promoter, tHMG1 gene and T GAL10 -P GAL7 terminator and promoter tandem fragment; using SEQ ID NO. 2 as a template, using tSfN8DT- 1Q12E, N305M -F / tSfN8DT- 1Q12E, N305M -R primers for amplification to obtain tSfN8DT- 1Q12E, N305M gene, using Gibson assembly method to construct pY26-T CYC1 -IDI1-P GAL1,10 -tHMG1-T GAL10 -P GAL7 -tSfN8DT- 1Q12E, N305M -T ADH1 plasmid. The p426-T CYC1 -SmDAGKS47A, L124A -P GPD -P TEF1 -AtIPK H274P -T ADH1 Recombinant plasmid as a template, using primer pair SK-F / SK-R and AI-F / AI-R primer amplification, respectively, to obtain p426 skeleton and SmDAGK S47A, L124A and AtIPK H274P gene tandem fragment, combined with the above P GAL1,10 bidirectional promoter and tSfN8DT- 1Q12E, N305M gene, using Gibson assembly method to construct pY26-T CYC1 -SmDAGK S47A, L124A -P GAL1,10 -AtIPK H274P -T GAL10 -P GAL7 -tSfN8DT- 1Q12E, N305M -T ADH1 plasmid.

[0065] Construction of 8-prenyl naringenin synthesis strain based on natural synthesis pathway: using gene editing technology to integrate IDI1 gene, tHMG1 gene and tSfN8DT- 1Q12E, N305M gene into the GAL80 site of wild type Saccharomyces cerevisiae strain CEN.PK2-1C. pY26-T CYC1 -IDI1-P GAL1,10 -tHMG1-T GAL10 -P GAL7 -tSfN8DT- 1Q12E, N305M -T ADH1 plasmid as a template, using 8PN-F / 8PN-R primer to obtain the Donor DNA used for transformation. Using Frozen-EZ Yeast Transformation Kit, 1 μg of pYZ463-GAL80 plasmid and 1 μg of Donor DNA were transformed into Saccharomyces cerevisiae CEN.PK2-1C to complete the gene integration, and obtain NP01 strain.

[0066] Construction of 8-prenyl naringenin synthesis strain based on artificial synthesis pathway: using gene editing technology to integrate SmDAGK S47A, L124A , AtIPK H274P and tSfN8DT- 1Q12E, N305MIntegration into the GAL80 locus of IUP1 strain dependent on isopentenol utilization pathway in Saccharomyces cerevisiae, wherein the starting strain of IUP1 is CEN.PK2-1C (see the strain genotype description in the Supplementary Data 6 of the article “Li GJ, et al. Yeast metabolism adaptation for efficient terpenoids synthesis via isopentenol utilization. Nature Communications, 2024, Vol 15, Issue 1”). The pY26-T CYC1 -SmDAGK S47A, L124A -P GAL1,10 -AtIPK H274P -T GAL10 -P GAL7 -tSfN8DT- 1Q12E, N305M -T ADH1 The plasmid was used as a template, and the 8PN-F / 8PN-R primers were used for amplification to obtain the Donor DNA used for transformation. Using the Frozen-EZ Yeast Transformation Kit, 1 μg of pYZ463-GAL80 plasmid and 1 μg of Donor DNA were transformed into the IUP1 strain to complete the gene integration, and the AP01 strain was obtained.

[0067] The chromosomal integration of the NP01 strain and the AP01 strain is shown in Figure 5 .

[0068] Example 4: Comparison of isoprenylated flavonoid synthesis efficiency

[0069] The following experimental groups were set up:

[0070] NP01 strain YPD: single colony of NP01 strain was picked from the plate and inoculated into 3 mL YPD medium, which was cultured at 30°C, 220 rpm for 24 h, and then inoculated into 25 mL YPD medium containing 0.5 g / L naringin with initial OD 600 = 0.1, and cultured at 30°C, 220 rpm for 3 days.

[0071] NP01 strain YPD + prenol: single colony of NP01 strain was picked from the plate and inoculated into 3 mL YPD medium containing 1 g / L prenol, which was cultured at 30°C, 220 rpm for 24 h, and then inoculated into 25 mL YPD medium containing 0.5 g / L naringin with initial OD 600= 0.1 was inoculated into 25 mL YPD medium containing 0.5 g / L naringenin and 1 g / L prenol, and cultured at 30°C, 220 rpm for 3 days.

[0072] AP01 strain YPD + prenol: the culture step was the same as NP01 strain YPD + prenol, except that AP01 strain was used to replace NP01 strain.

[0073] 0.7 mL of the fermentation broth was taken, and an equal volume of methanol was added. The mixture was shaken in a biological sample homogenizer for 2 cycles (without adding glass beads), and then centrifuged at 8000 x g, 4°C for 1 min. The supernatant was filtered through an organic system 0.22 μm nylon filter, and then subjected to HPLC detection. A Titan C18 (250 x 4.6 mm 5 μm) C18 column chromatographic column was used for separation of flavonoids, and a gradient elution procedure was used, with A phase being ultrapure water (0.1% v / v formic acid) and B phase being pure acetonitrile: 0 - 1 min, B phase 10%; 1 - 21 min, B phase gradually increased from 10% to 60%; 21 - 23 min, B phase returned to 10%; 23 - 28 min, B phase maintained at 10%. The detection wavelength for naringenin was 290 nm, and the detection wavelength for 8-prenyl naringenin was 350 nm. g The fermentation results are shown in Table 1.

[0074] Figure 6 The yield of 8-prenyl naringenin of NP01 strain was 1.27 mg / L, and the addition of prenol had no effect on the yield; the yield of 8-prenyl naringenin of AP01 strain was 5.25 mg / L. It can be seen that the AtIPK H274P mutant-containing isoprenol utilization pathway has higher DMAPP supply capacity compared with the strengthened MVA pathway, and the synthesis efficiency of isoprenylated flavonoids is 4 times that of the existing method.

[0075] The above examples are preferred embodiments of the present application, but the embodiments of the present application are not limited to the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application are equivalent replacement methods, and are all included in the protection scope of the present application.​

Claims

1. A highly active isopentenyl phosphokinase mutant, characterized in that: The highly active isopentenyl phosphokinase mutant is AtIPK. H274P ; The AtIPK mentioned H274P This refers to the mutation of the 274th amino acid in AtIPK from histidine H to proline P; The amino acid sequence of AtIPK is shown in Genbank NP_173986.

2.

2. An enzyme assembly for the isopentenol utilization pathway using 3-methyl-2-buten-1-ol as a substrate, characterized in that: By SmDAGK S47A, L124A It consists of the highly active isopentenyl phosphokinase mutant as described in claim 1; The SmDAGK S47A, L124A This refers to the mutation of the 47th amino acid in SmDAGK from serine S to alanine A and the 124th amino acid from leucine L to alanine A; The amino acid sequence of the SmDAGK is shown in the accession number Genbank AAA26867.

1.

3. An enzyme assembly for the isopentenylation of flavonoids using 3-methyl-2-buten-1-ol as a substrate, characterized in that: It consists of an isopentenyltransferase mutant and the enzyme combination of the isopentenol utilization pathway with 3-methyl-2-buten-1-ol as a substrate as described in claim 2; The isopentenyltransferase mutant is tSfN8DT-1 Q12E, N305M Its sequence encoding nucleic acid is shown in SEQ ID NO.

4.

4. A recombinant expression vector, characterized in that: The nucleic acid encoding the enzyme combination of the isopentenol utilization pathway based on 3-methyl-2-buten-1-ol as described in claim 2, or the nucleic acid encoding the enzyme combination of the isopentenylation of flavonoids based on 3-methyl-2-buten-1-ol as described in claim 3.

5. The recombinant expression vector according to claim 4, characterized in that: The sequence of the nucleic acid encoding the highly active isopentenyl phosphokinase mutant is shown in SEQ ID NO.1; The SmDAGK S47A, L124A The sequence encoding the nucleic acid is shown in SEQ ID NO.2; The sequence of the nucleic acid encoding the isopentenyltransferase mutant is shown in SEQ ID NO.

4.

6. A recombinant cell of *Saccharomyces cerevisiae*, characterized in that: Contains the recombinant expression vector as described in claim 4 or 5; The starting strain of the recombinant Saccharomyces cerevisiae cells described above has the following characteristics: the mevalonate pathway is blocked, and the 156th amino acid in the PRM10 gene is mutated from leucine to glutamine; The mevalonate pathway is blocked by gene editing technology to delete or generate a frameshift mutation in the ERG13 gene in the mevalonate pathway.

7. The recombinant Saccharomyces cerevisiae cell according to claim 6, characterized in that: When the recombinant expression vector contains nucleic acid encoding an enzyme combination that achieves isopentenylation of flavonoids using 3-methyl-2-buten-1-ol as a substrate, the recombinant Saccharomyces cerevisiae cells have the following characteristics: nucleic acid encoding a highly active isopentenyl phosphokinase mutant, SmDAGK... S47A, L124A The coding nucleic acid of the mutant and the coding nucleic acid of the isopentenyltransferase are integrated into the chromosome of the cell through chromosomal gene loci.

8. The use of the recombinant Saccharomyces cerevisiae cells according to claim 6 or 7 in the preparation of squalene or 8-isopentenyl naringenin.

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