High-activity isopentenyl phosphokinase mutant and application thereof

Abstract

The invention belongs to the technical field of enzyme engineering, and discloses a high-activity isopentenyl phosphokinase mutant and application thereof. The high-activity isopentenyl phosphokinase mutant is AtIPKH274P, and the amino acid at the 274 site of wild type AtIPK is mutated from histidine H to proline P. The invention further discloses a preparation method of the high-activity isopentenyl phosphokinase mutant. According to the present invention, the AtIPKH274P, the SmDAGKS47A and the L124A can form the efficient isopentenol utilization pathway so as to effectively convert the prenol into the DMAPP; an engineering strain with improved isopentenyl modification capability can be constructed by utilizing the approach, and the strain has more advantages compared with a traditional engineering strain based on a mevalonic acid approach.

Description

Technical Field

[0001] The invention belongs to the technical field of enzyme engineering, and particularly relates to a highly active isopentenyl phosphate kinase mutant and application thereof. Background Art

[0002] Prenylation is a ubiquitous modification found in nearly all forms of life, often occurring in aromatic compounds such as flavonoids and certain alkaloids. In nature, these enzymatic reactions primarily rely on dimethylallyl pyrophosphate (DMAPP) as the prenyl donor. Prenylated flavonoids are derivatives produced by the enzyme prenyltransferase, which catalyzes the conjugation of DMAPP to the flavonoid backbone. The introduction of the prenyl group enhances the lipophilicity of flavonoids, thereby promoting their ability to cross cell membranes and improve cellular uptake. These compounds exhibit diverse pharmacological activities, including anti-inflammatory, analgesic, and anti-cancer effects.

[0003] Microbial cell factories have become a potential platform for the biosynthesis of prenylated flavonoids. Saccharomyces cerevisiae is widely used in the biosynthesis of flavonoids and terpenoids and is an important host for prenylated flavonoids. However, DMAPP supply remains a major bottleneck for the production of prenylated compounds. Figure 1 As shown, the mevalonate (MVA) pathway, present in most eukaryotic microorganisms, is responsible for the biosynthesis of terpenoids. Through this pathway, isopentenyl pyrophosphate (IPP) is converted to DMAPP by the isomerase encoded by IDI1. However, this reaction is inefficient, and IPP and DMAPP are rapidly condensed to GPP by ERG20, which then flows downstream, reducing the availability of DMAPP. The prenol utilization pathway is an artificial pathway for the synthesis of terpenoids. It uses 3-methyl-3-buten-1-ol (isoprenol) or 3-methyl-2-buten-1-ol (prenol) as substrates. The former can be converted to IPP and the latter to DMAPP through a two-step phosphorylation process. Like the MVA pathway, methods using isoprenol as a substrate are still limited by the conversion of IPP to DMAPP. Using prenol as a substrate, methods that directly convert prenol to DMAPP via the prenol utilization pathway can circumvent this step, increasing the availability of isopentenyl groups and thus enhancing the synthesis of prenylated flavonoids. However, existing studies have mainly focused on the phosphorylation of kinases such as AtIPK S270P, A272R ) modification to improve the conversion of isoprenol by artificial synthesis pathways, such as Chinese invention patent CN118530865A discloses an isoprenol utilization pathway-dependent Saccharomyces cerevisiae engineered bacteria and its construction method and application, wherein the diacylglycerol kinase SmDAGK mutant used is SmDAGK S47A , L124A , isopentenyl phosphate kinase AtIPK mutant is AtIPKS270P, A272R The strain containing these two mutants produced squalene 1.5 times higher than the wild type using isoprenol as a substrate. Currently, there is a lack of effective mutants that can promote the artificial conversion of prenol. Summary of the Invention

[0004] The primary purpose of the present invention is to overcome the defects and shortcomings of the prior art and provide a highly active isopentenyl phosphate kinase mutant.

[0005] Another object of the present invention is to provide the application of the highly active isopentenyl phosphate kinase mutant.

[0006] The purpose of the present invention is achieved through the following technical solutions: A highly active isopentenyl phosphate kinase mutant, AtIPK H274P , refers to the mutation of amino acid 274 of AtIPK from histidine H to proline P.

[0007] The amino acid sequence of AtIPK is shown in the accession number of Genbank NP_173986.2, which contains 332 amino acids in total.

[0008] The sequence of the nucleic acid encoding the highly active isopentenyl phosphate kinase mutant is shown in SEQ ID NO.1.

[0009] The highly active isopentenyl phosphate kinase mutant can improve the utilization efficiency of 3-methyl-2-butene-1-ol in the isopentenol utilization pathway, and is used in the preparation of dimethylallyl pyrophosphate, terpenoids and isopentenylated flavonoids using 3-methyl-2-butene-1-ol as a substrate.

[0010] An enzyme combination for an isopentenol utilization pathway using 3-methyl-2-buten-1-ol as a substrate, including SmDAGK S47A, L124A and the aforementioned hyperactive isopentenyl phosphate kinase mutants.

[0011] The SmDAGK S47A, L124A It refers to the mutation of the 47th amino acid of SmDAGK from serine S to alanine A, and the mutation of the 124th amino acid from leucine L to alanine A.

[0012] The amino acid sequence of SmDAGK is shown in Genbank accession number AAA26867.1, which contains 137 amino acids in total.

[0013] The SmDAGK S47A, L124A The sequence of the encoding nucleic acid is shown in SEQ ID NO.2.

[0014] The isopentenol 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).

[0015] An enzyme combination for realizing the prenylation of flavonoids using 3-methyl-2-butene-1-ol as a substrate comprises an isopentenyltransferase or an isopentenyltransferase mutant and an enzyme combination of the isopentenol utilization pathway using 3-methyl-2-butene-1-ol as a substrate.

[0016] The isopentenyl transferase mutant is tSfN8DT-1 Q12E, N305M ; Its amino acid sequence is shown in SEQ ID NO.3.

[0017] The sequence of the nucleic acid encoding the isopentenyl transferase mutant is shown in SEQ ID NO.4.

[0018] The flavonoid is preferably naringenin.

[0019] A recombinant expression vector containing a nucleic acid encoding the enzyme combination for the isopentenol utilization pathway using 3-methyl-2-buten-1-ol as a substrate or the nucleic acid encoding the enzyme combination for achieving isopentenylation of flavonoids using 3-methyl-2-buten-1-ol as a substrate, capable of expressing the enzyme combination for the isopentenol utilization pathway using 3-methyl-2-buten-1-ol as a substrate or the enzyme combination for achieving isopentenylation of flavonoids using 3-methyl-2-buten-1-ol as a substrate.

[0020] The sequence of the nucleic acid encoding the highly active isopentenyl phosphate kinase mutant is shown in SEQ ID NO.1.

[0021] The SmDAGK S47A, L124A The sequence of the encoding nucleic acid is shown in SEQ ID NO.2.

[0022] The sequence of the nucleic acid encoding the isopentenyl transferase mutant is shown in SEQ ID NO.4.

[0023] The vector skeleton of the recombinant expression vector comes from the P426 GAL vector or the pY26-GPD-TEF vector.

[0024] The two enzymes in the encoding nucleic acid of the enzyme combination for the isopentenol utilization pathway using 3-methyl-2-butene-1-ol as a substrate are expressed independently, that is, each enzyme gene is provided with a promoter and a terminator; the structure is preferably as follows: CYC1 -SmDAGK S47A, L124A -P GPD -P TEF1-AtIPK H274P -T ADH1 .

[0025] The three enzymes in the nucleic acid encoding the enzyme combination for achieving prenylation of flavonoids using 3-methyl-2-butene-1-ol as a substrate are independently expressed, that is, each enzyme gene contains a promoter and a terminator; its structure is preferably as follows: CYC1 -SmDAGK S47A, L124A -P GAL1,10 -AtIPK H274P -T GAL10 -P GAL7 -tSfN8DT- 1Q12E, N305M -T ADH1 .

[0026] A recombinant cell of Saccharomyces cerevisiae contains the above-mentioned recombinant expression vector.

[0027] The starting strain of the recombinant yeast 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.

[0028] The mevalonate pathway is blocked by gene editing technology to delete the ERG13 gene in the mevalonate pathway or generate a frameshift mutation.

[0029] The chassis cells of the starting strain are preferably Saccharomyces cerevisiae of the CEN.PK series; more preferably Saccharomyces cerevisiae CEN.PK2-1C.

[0030] When the recombinant expression vector contains a nucleic acid encoding an enzyme combination for realizing the isopentenylation of flavonoids using 3-methyl-2-butene-1-ol as a substrate, the recombinant yeast cell preferably has the following characteristics: a nucleic acid encoding a highly active isopentenyl phosphate kinase mutant, SmDAGK S47A,L124A The encoding nucleic acid of the isopentenyl transferase mutant is integrated into the chromosome of the cell through a chromosomal gene site.

[0031] The chromosomal gene locus is preferably the GAL80 locus.

[0032] The invention relates to an application of the recombinant yeast cell of Saccharomyces cerevisiae in the preparation of dimethylallyl pyrophosphate, terpenoid compounds and prenylated flavonoids.

[0033] The present invention is a further study based on the previous research. In the previous research, for example, Chinese invention patent CN118530865A disclosed an engineered yeast strain of isopentenol-dependent Saccharomyces cerevisiae, in which the natural mevalonate pathway is inactivated, the 156th amino acid in the PRM10 gene is mutated from leucine to glutamine, and the strain contains a phosphokinase mutant SmDAGK. S47A, L124A , and the AtIPK kinase mutant is AtIPK S270P, A272R When the engineered yeast Saccharomyces cerevisiae uses prenol as a substrate, the yield of squalene is not high, that is, the utilization rate of prenol by the engineered yeast Saccharomyces cerevisiae is not high, thus requiring further improvement. Compared with the existing technology, the present invention has the following advantages and beneficial effects: (1) AtIPK provided by the present invention H274P Mutants and SmDAGK S47A, L124A It can form an efficient isopentenol utilization pathway.

[0034] (2) The efficient isopentenol utilization pathway provided by the present invention can effectively convert prenol into DMAPP.

[0035] (3) The engineered strain provided by the present invention has a strong ability to modify isopentenyl groups, which is more advantageous than the traditional engineered strain based on the MVA pathway. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 Schematic diagram of DMAPP supply strategy in the prior art and the present invention.

[0037] Figure 2 Schematic diagram of high-throughput screening method.

[0038] Figure 3 The graph shows the activity results of different mutants.

[0039] Figure 4 This figure shows the effect of different gene combinations on the isopentenol utilization pathway.

[0040] Figure 5 Schematic diagram of chromosomal gene integration in NP01 and AP01 strains.

[0041] Figure 6 This is a comparison of the synthesis results of 8-isopentenylnaringenin by the NP01 strain and the AP01 strain. DETAILED DESCRIPTION

[0042] The present invention will be described in further detail below with reference to the embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0043] If specific experimental conditions are not specified in the following embodiments, conventional experimental conditions or those recommended by the reagent company will generally be used. Materials and reagents used were commercially available unless otherwise specified.

[0044] Gibson assembly: The specific operations are in accordance with the instructions of 2X MultiF Seamless Assembly Mix of Abotec Biotech.

[0045] Yeast strain transformation: Unless otherwise specified, gene editing transformation was performed using the Frozen-EZ Yeast Transformation II™ Kit (purchased from ZYMO RESEARCH, USA) according to the product instructions.

[0046] PCR amplification included conventional denaturation, annealing, and extension steps, and was performed according to the PrimeSTAR® Max DNA Polymerase product manual.

[0047] The present invention uses the following biological materials: 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 , IUP1 strains have been disclosed in the literature “Li GJ, et al. Yeast metabolismadaptation for efficient terpenoids synthesis via isopentenol utilization. Nature Communications, 2024, Vol 15, Issue 1” and its supplementary documents.

[0048] The primers used in the present invention are shown in Table 1: Table 1 Primers

[0049] Example 1: Identification of amino acids near the active site of AtIPK Using the AtIPK protein sequence as a seed, a sequence alignment of prenyl phosphate kinase homologs was performed on the Protein Data Bank website. Crystal structures of protein-ligand complexes with sequence similarities of 32% (PDB: 3lkk), 32% (PDB: 3ll5), and 27% (PDB: 7lnu) were identified. Analysis revealed that the target enzyme is a homodimer. The active sites of the two monomers in the 3ll5 and 7lnu crystals are bound to ADP+IPP and ATP+IP, respectively, while both monomers in the 3lkk crystal are bound to ATP and IP. Five protein monomer conformations were extracted using PyMOL software: 3lkk (ATP, IP), 3ll5 (ATP, IP), 3ll5 (ADP, IPP), 7lnu (ATP, IP), and 7lnu (ADP, IPP). These conformations were then paired with five AtIPK models (Model_1-Model_5) predicted by AlphaFold3 for three-dimensional structural alignment. The all-atom root mean square deviation (RMSD) of these crystals was compared with the five AtIPK models predicted by AlphaFold3. The results showed that the 31kk (ATP, IP) and 3115 (ADP, IPP) crystals had the lowest RMSD values ​​with Model_3. The ligands of 3lkk and 3ll5 were respectively combined with Model_3 to form new protein-ligand complexes, and the amino acid residues within 4 Å were extracted. The key site combinations were determined by molecular docking, and a total of 37 candidate modification sites were obtained (their amino acid residue numbers were 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, and 283). Saturation mutagenesis was performed based on the NNK method. The codon preference sequence of the saturated mutant amino acids in Saccharomyces cerevisiae is: A (GCT), G (GGT), V (GTT), L (GTT), and L (GTT). (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). Take p426-T CYC1 -SmDAGK-P GPD -P TEF1 -AtIPK-T ADH1The plasmid was used as a PCR template and the method disclosed in the literature was modified appropriately: the 0.5 g / L Lisoprenol substrate added to the screening plate was replaced with 0.7 g / L prenol substrate. In addition, the OD value of the strain was directly used for the initial screening. 600 as a screening indicator.

[0050] The results are as follows Figure 2 As shown in the figure, a high-throughput screening system was used to rapidly evaluate mutants with improved catalytic efficiency for the non-natural substrate DMAP in the AtIPK saturation mutant library. Sequencing results showed that the initially screened mutants S201A, G202A, G202S, D223R, D223E, D223Q, V224A, Y228H, D229R, D229W, D229G, R230F, P231R, P231H, P231Q, P232Y, P232K, P232N, P232G, A total of 39 mutants include P232M, P232A, P232R, P232S, P232F, Y269A, H274S, H274R, H274P, H274A, H274T, H274G, H274Y, H274L, H274C, D275G, T277S, G279A, M280R and M280V.

[0051] Example 2: Screening of optimal mutants The corresponding mutants were obtained by site-directed mutagenesis for secondary verification, and the mutants of the above 39 AtIPKs were constructed. H274P For example, the mutant vector p426-T CYC1 -SmDAGK-P GPD -P TEF1 -AtIPK-T ADH1 Plasmid as template, using AtIPK H274P -F and AtIPK H274P -R is a primer pair, PrimeSTAR ® Max DNA polymerase was used as a 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 Escherichia coli DH5α competent cells. After transformation, the cells were plated on LB agar medium containing 100 mg / L ampicillin and cultured overnight at 37°C. Six single colonies were randomly selected and inoculated into LB liquid medium containing ampicillin and cultured at 37°C and 220 rpm for 10-12 hours. After plasmid extraction, the plasmid was sent to the company for sequencing verification, and p426-T was obtained. CYC1 -SmDAGK-P GPD -P TEF1 -AtIPK H274P -TADH1 The construction methods of other mutants are similar.

[0052] The above 39 mutants and p426-T CYC1 -SmDAGK-P GPD -P TEF1 -AtIPK-T ADH1 (Control group) Plasmid was transformed into IUP1 strain (knockout ERG13 The specific transformation method is as follows: IUP1 was inoculated into 3 mL of YPD medium containing 5 mM mevalonic acid, and cultured at 30°C and 220 rpm for 36-48 h to obtain cell culture fluid. 250 μL of cell culture fluid was taken and 6000 × g Centrifuge for 3 minutes and discard the supernatant. Then add 100 μL of transformation solution, 0.5 μg of pure plasmid and 3 μL of 10 mg / mL ssDNA. Mix well and incubate in a 37°C water bath for 30 minutes. g Centrifuge for 3 minutes and discard the supernatant. Resuspend the cell pellet in 100 μL of sterile deionized water, spread on a YPD agar plate supplemented with 2 g / L prenol, and culture at 30°C for 4-5 days. Pick a single colony from the transformation plate and inoculate it into 3 mL of YPD medium supplemented with 4 g / L prenol. Culture at 30°C and 220 rpm for 24 hours. 600 = 0.1 was inoculated into a 48-well plate, where each well contained 1 mL of YPD medium supplemented with 4 g / L of isoprenol or isoprenol, and cultured at 30°C and 220 rpm for 4 days to obtain fermentation broth.

[0053] 0.2 mL of fermentation broth was added to a 2 mL homogenizer tube containing 0.7 g of glass beads with a diameter of 0.5 mm and 1 mL of ethyl acetate. The cells were disrupted using a biological sample homogenizer and then subjected to 10000 × g After centrifugation for 1 min, the upper ethyl acetate phase was filtered through a 0.22 μm filter membrane. Analysis was performed using an LC-16 high-performance liquid chromatography (HPLC) instrument 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 column (Agilent Technologies, USA). The mobile phase was 100% acetonitrile, and the elution time was 7.5 min with an injection volume of 2 μL and an isocratic elution rate of 0.5 mL / min.

[0054] The activity of the mutant was characterized by the accumulation of squalene. Figure 3 It can be seen that the mutants verified in the second round can basically improve the conversion of prenol to prenol by the isopentenol utilization pathway. H274P In addition to improving the conversion of prenol, the mutant also significantly improved the conversion of isoprenol.

[0055] AtIPK, AtIPK S270P, A272R 、AtIPK H274P SmDAGK S47A, L124A Combination, test the effect of different combinations on prenol conversion, the plasmid used is 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 (According to the method of Example 2, p426-T CYC1 -SmDAGK S47A, L124A -P GPD -P TEF1 -AtIPK-T ADH1 The result is as follows. Figure 4 As shown in the figure, the conversion rate of prenol by the isopentenol utilization pathway composed of wild-type AtIPK was poor, and the accumulation of squalene was only 4.9 mg / L; the mutant AtIPK S270P, A272R The corresponding squalene accumulation was 31.9 mg / L; while the mutant AtIPK H274P The conversion rate of prenol to prenol by the prenol utilization pathway was greatly improved, and the accumulation of squalene was 68.3 mg / L, which was significantly higher than that of wild-type AtIPK and AtIPK S270P, A272R 13.8 times and 2.1 times.

[0056] Example 3: Construction of a prenylated flavonoid-synthesizing strain 8-Isopentenylnaringenin was used as the prenylated flavonoid model product, and the DMAPP supply capacity based on the natural synthesis pathway and the artificial synthesis pathway was compared by the yield of 8-Isopentenylnaringenin. Sophora flavescens )-derived isopentenyltransferase mutant tSfN8DT-1 Q12E, N305M (Already in the literature "Guo CJ, et al. De novo biosynthesis of 8-prenylnaringenin in Saccharomyces cerevisiae Improved screening and engineering of prenyltransferases and precursor pathways. (Disclosed in "Supplementary Data 5" of "Systems Microbiology and Biomanufacturing, 2023, Vol 3, Issue 4") achieves 8-position prenylation of naringenin. tSfN8DT-1 Q12E, N305M The gene is codon-optimized based on Saccharomyces cerevisiae, and its nucleotide sequence is shown in SEQ ID NO.4.

[0057] Using pYZ463 (obtained from the Addgene global plasmid sharing platform, Plasmid # 187971, TEF1p-Cas9-CYC1t and SNR52p-Not1-SUP4t) as a template, primers GAL80sgRNA-F and GAL80sgRNA-R were used for amplification. After Escherichia coli DH5α transformation, plasmid extraction and sequencing verification, the Crispr-Cas9 plasmid with 5'-ACGATAGTTGCAGTATGGCG-3' as the specific 20nt sgRNA was finally obtained, which is the pYZ463-GAL80 plasmid, which targets the GAL80 gene. The endogenous MVA pathway of Saccharomyces cerevisiae was enhanced by overexpressing IDI1 (NCBI-GeneID: 855986) and tHMG1 (NCBI-GeneID: 854900, with its N-terminus truncated). 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 the pY26-GPD-TEF (also named pY26TEF-GPD) plasmid (obtained from the BioVector NTCC Plasmid Vector Culture Cell Gene Collection Center) as a template, primers pY26-F and pY26-R were used to amplify the pY26-GPD-TEF plasmid backbone; using the Saccharomyces cerevisiae CEN.PK2-1C genome as a template, primer pairs IDI1-F / IDI1-R and P GAL1,10 -F / P GAL1,10 -R、tHMG1-F / tHMG1-R、T GAL10 -P GAL7 -F / T GAL10 -P GAL7 -R primers were used to amplify the 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 were used for amplification to obtain tSfN8DT- 1Q12E, N305M Gene, pY26-T was constructed using the Gibson assembly method CYC1 -IDI1-P GAL1,10 -tHMG1-T GAL10 -P GAL7 -tSfN8DT- 1Q12E, N305M -T ADH1 Plasmid. Plasmid p426-T obtained in Example 2 CYC1 -SmDAGKS47A, L124A -P GPD -P TEF1 -AtIPK H274P -T ADH1 The recombinant plasmid was used as a template and primer pairs SK-F / SK-R and AI-F / AI-R were used to amplify the p426 backbone and SmDAGK S47A, L124A With AtIPK H274P Gene tandem fragment, and the above P GAL1,10 Bidirectional promoter and tSfN8DT- 1Q12E, N305M Gene assembly, using the 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.

[0058] Construction of 8-isopentenylnaringenin synthesis strain based on natural synthesis pathway: Gene editing technology was used to modify IDI1 gene, tHMG1 gene and tSfN8DT- 1Q12E, N305M The gene was integrated into the GAL80 site of wild-type Saccharomyces cerevisiae strain CEN.PK2-1C. CYC1 -IDI1-P GAL1,10 -tHMG1-T GAL10 -P GAL7 -tSfN8DT- 1Q12E, N305M -T ADH1 Using the plasmid as a template, 8PN-F / 8PN-R primers were used to amplify the Donor DNA used for transformation. Using the Frozen-EZ Yeast Transformation Kit, 1 μg of the pYZ463-GAL80 plasmid and 1 μg of the Donor DNA were transformed into Saccharomyces cerevisiae CEN.PK2-1C for gene integration, generating the NP01 strain.

[0059] Construction of 8-isopentenylnaringenin synthesis strain based on artificial synthesis pathway: SmDAGK was transformed into 8-isopentenylnaringenin by gene editing technology. S47A, L124A 、AtIPK H274P and tSfN8DT- 1Q12E, N305MIntegrate into the GAL80 locus of the isopentenol utilization pathway-dependent Saccharomyces cerevisiae IUP1 strain, where the starting strain of IUP1 is CEN.PK2-1C (see the strain genotype description in the appendix "Supplementary Data 6" of the reference "Li GJ, et al. Yeast metabolism adaptation for efficient terpenoids synthesis viaisopentenol utilization. Nature Communications, 2024, Vol 15, Issue 1" for details). 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 for amplification using 8PN-F / 8PN-R primers to obtain donor DNA 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 for gene integration, generating the AP01 strain.

[0060] Chromosomal integration of NP01 and AP01 strains Figure 5 shown.

[0061] Example 4: Comparison of prenylated flavonoid synthesis efficiency Set up the following experimental groups: NP01 strain YPD: Pick a single colony of NP01 strain from the plate and inoculate it into 3 mL YPD medium. Incubate at 30°C and 220 rpm for 24 h. 600 = 0.1 was inoculated into 25 mL of YPD medium containing 0.5 g / L naringenin and cultured at 30°C and 220 rpm for 3 days.

[0062] NP01 strain YPD + prenol: Pick a single colony of NP01 strain from the plate and inoculate it into 3 mL of YPD medium containing 1 g / L prenol. Cultivate it at 30°C and 220 rpm for 24 h. 600 = 0.1 was inoculated into 25 mL of YPD medium containing 0.5 g / L naringenin and 1 g / L prenol and cultured at 30°C and 220 rpm for 3 days.

[0063] AP01 strain YPD + prenol: The culture steps are the same as those for NP01 strain YPD + prenol, except that the AP01 strain is used instead of the NP01 strain.

[0064] Take 0.7 mL of fermentation broth, add an equal volume of methanol, shake for 2 cycles in a biological sample homogenizer (without adding glass beads), and then g The mixture was centrifuged for 1 minute under the appropriate conditions and filtered through a 0.22 μm nylon membrane for organic filtration before HPLC analysis. Flavonoids were separated using a Titank C18 column (250 × 4.6 mm, 5 μm) using a gradient elution procedure with ultrapure water (0.1% v / v formic acid) in phase A and pure acetonitrile in phase B: 10% phase B from 0 to 1 minute; from 1 to 21 minutes, the phase B percentage was gradually increased from 10% to 60%; from 21 to 23 minutes, the phase B percentage was returned to 10%; and from 23 to 28 minutes, the phase B percentage was maintained at 10%. The detection wavelengths for naringenin were 290 nm, and for 8-isopentenylnaringenin, 350 nm.

[0065] Fermentation results such as Figure 6 As shown in the figure, the yield of 8-isopentenylnaringenin of NP01 strain is 1.27 mg / L, and the addition of prenol has no effect on the yield; the yield of 8-isopentenylnaringenin of AP01 strain is 5.25 mg / L. H274P Compared with the enhanced MVA pathway, the mutant prenol utilization pathway has a higher DMAPP supply capacity and its synthesis efficiency of prenylated flavonoids is four times that of existing methods.

[0066] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

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

2.

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

1.

3. An enzyme combination for achieving prenylation of flavonoids using 3-methyl-2-butene-1-ol as a substrate, characterized in that: The invention relates to an enzyme combination comprising an isopentenyl transferase or an isopentenyl transferase mutant and the isopentenol utilization pathway with 3-methyl-2-butene-1-ol as a substrate according to claim 2.

4. The enzyme combination for achieving prenylation of flavonoids using 3-methyl-2-buten-1-ol as a substrate according to claim 3, characterized in that: The isopentenyl transferase mutant is tSfN8DT-1 Q12E, N305M , and its amino acid sequence is shown in SEQ ID NO.

3.

5. A recombinant expression vector, characterized in that: A nucleic acid encoding an enzyme combination for the isopentenol utilization pathway using 3-methyl-2-buten-1-ol as a substrate according to claim 2, or a nucleic acid encoding an enzyme combination for achieving isopentenylation of flavonoids using 3-methyl-2-buten-1-ol as a substrate according to any one of claims 3-4.

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

4.

7. The recombinant expression vector according to claim 5, characterized in that: The structure of the nucleic acid encoding the enzyme combination of the isopentenol utilization pathway with 3-methyl-2-butene-1-ol as a substrate is as follows: CYC1 -SmDAGK S47A, L124A -P GPD -P TEF1 -AtIPK H274P -T ADH1 ; The structure of the nucleic acid encoding the enzyme combination for achieving prenylation of flavonoids using 3-methyl-2-butene-1-ol as a substrate is as follows: CYC1 -SmDAGK S47A, L124A -P GAL1,10 -AtIPK H274P -T GAL10 -P GAL7 -tSfN8DT- 1Q12E, N305M -T ADH1 .

8. A recombinant cell of Saccharomyces cerevisiae, characterized in that: Containing the recombinant expression vector according to any one of claims 5 to 7; The starting strain of the recombinant yeast 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.

9. The recombinant cell of Saccharomyces cerevisiae according to claim 8, characterized in that: The mevalonate pathway is blocked by gene editing technology to delete the ERG13 gene in the mevalonate pathway or generate a frameshift mutation; When the recombinant expression vector contains a nucleic acid encoding an enzyme combination for realizing isopentenylation of flavonoids using 3-methyl-2-butene-1-ol as a substrate, the recombinant yeast cell has the following characteristics: a nucleic acid encoding a highly active isopentenyl phosphate kinase mutant, SmDAGK S47A, L124A The encoding nucleic acid of the isopentenyl transferase mutant is integrated into the chromosome of the cell through a chromosomal gene site.

10. Use of the recombinant Saccharomyces cerevisiae cell according to claim 8 or 9 in the preparation of dimethylallyl pyrophosphate, terpenoids and prenylated flavonoids.

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