Hlmo18 protein and its use in the production of 6s-humulone

Abstract

The application discloses HlMO18 protein and application thereof in production of 6 S -Humulone, and belongs to the technical field of biotechnology.The technical problem solved by the application is how to specifically prepare 6 S -Humulone in Humulone.The HlMO18 protein disclosed by the application is a protein with an amino acid sequence of SEQ ID No.1.Experiments prove that after a coding gene of the HlMO18 protein of the application is introduced into yeast together with CCL2, CCL4, AtPT1LTP, AtPT2TP and VPS genes, not only can Humulone be prepared, but also the proportion of 6 S -Humulone in the Humulone product can be greatly improved.It is proved that the HlMO18 protein of the application can specifically catalyze the generation of 6 S -Humulone, and has potential application value in the beer brewing industry.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the HlMO18 protein and its application in the production of 6 S Applications in Humulone. Background Technology

[0002] Hops ( Humulus lupulus Hops (L., 2n=2X=20) are perennial climbing herbs belonging to the genus *Humulus* in the family Cannabaceae. The female flowers are primarily used in the beer brewing industry, being the main source of beer's characteristic aroma and bitterness. The quality of beer largely depends on hops. As a distinctive component that differentiates beer from other alcoholic beverages, hops are even hailed as the "soul of beer," highlighting their crucial role in beer brewing.

[0003] Bitter acids are unique secondary metabolites of hops, accounting for approximately 30% of the total compounds in glandular trichomes (VanCleemput). et al. (Zanoli and Zavatti, 2009). Structurally, picric acids consist of a benzene ring skeleton, an acyl side chain, and an isopentenyl group. Based on the number of isopentenyl side chains and hydroxyl groups on the benzene ring, they can be classified into α-picric acids and β-picric acids. Besides imparting a distinctive flavor (primarily bitterness) to beer, picric acids also help stabilize beer foam and extend its shelf life (Zanoli and Zavatti, 2008, Abram). et al. (Chen and Lin, 2004, Yajima, 2015). Furthermore, picric acid shows promising application prospects in areas such as antibacterial, antioxidant, anti-inflammatory, anticancer, and antitumor activity (Chen and Lin, 2004, Yajima). et al. , 2005, Van Cleemput et al. , 2009).

[0004] Given the significant economic and medicinal value of hop plants, researchers began exploring the biosynthetic pathway of picric acid in hops as early as the 1960s (Wright and Howard, 1961; Mori, 1961). After more than half a century of research, the biosynthetic pathway of picric acid has been largely elucidated. In mitochondria, branched-chain amino acid transferases BCAT and ketoacid dehydrogenases BCKHD catalyze the degradation of amino acids to generate branched-chain acyl-CoA. Branched-chain acyl-CoA does not directly participate in the construction of the picric acid backbone; it must first be hydrolyzed by thiolipase TE to produce the corresponding branched-chain fatty acids. These fatty acids are then transported across the mitochondrial membrane to the cytoplasm via free diffusion or transport proteins. There, the cytoplasmic carboxyl-CoA ligases CCL2 or CCL4 catalyze the regeneration of the corresponding branched-chain acyl-CoA. Finally, under the catalysis of phenylpentanone synthase VPS, it condenses with malonyl-CoA to form the picric acid backbone structure—acylated phloroglucinol compounds (PIBP / PIVP / PMBP). The isopentenyltransferase PT1L / PT2 complex on the plastid membrane catalyzes the acylation of phloroglucinol compounds into three isopentenylations, thereby generating β-picric acid.

[0005] Studies have found that α-picric acids in hops (mainly including Humulone, Cohumulone, and Adhumulone) have 6... S Chiral conformation (Urban) et al. (2013). However, 6, which are the main sources of bitterness in beer... S The enzymatic mechanism by which α-picric acid is catalyzed for synthesis remains unclear. Therefore, analysis of 6 S The synthesis pathway of α-picric acid remains a very challenging task.

[0006] Monooxygenases involve numerous gene families, including the cytochrome P450 family, the iron / ketoglutarate-dependent oxidase family, and the flavin-dependent monooxygenase family. Furthermore, each gene family has many members in plants, such as Arabidopsis thaliana (…). Arabidopsis thaliana The genome encodes 246 P450 proteins, including approximately 130 iron / ketoglutarate-dependent oxidases (Hagel and Facchini, 2018, Mitchell and Weng, 2019). Summary of the Invention

[0007] The technical problem to be solved by this invention is how to specifically prepare 6 in Humulone products. S -Humulone, that is, how to increase the content of 6 in Humulone products. S -Humulone and 6 R - The proportion of Humulone.

[0008] To address the aforementioned technical problems, the present invention first provides a protein derived from hops ( Humulus lupulus L.), its name is HlMO18 protein, HlMO18 protein is A1), A2) or A3). A1) A protein whose amino acid sequence is SEQ ID No. 1; A2) A protein that has more than 98% identity with and has the same function as the protein described in A1) by substitution and / or deletion and / or addition of amino acid residues of the amino acid sequence shown in SEQ ID No. 1 in the sequence listing; A3) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of A1) or A2).

[0009] In this invention, "more than 98% identity" means 98% or 99% identity. Identity refers to the identity of the amino acid sequences. The identity of amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, using blastp as the program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, and performing an identity search on a pair of amino acid sequences to calculate the identity value (%), then the identity value can be obtained.

[0010] The proteins mentioned in A2 above can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0011] The gene encoding the protein in A2) above can be obtained by deleting one or more amino acid residues from the codons in the DNA sequence shown in SEQ ID No. 2, and / or by performing a missense mutation of one or more base pairs, and / or by attaching a tag coding sequence to its 5′ end and / or 3′ end. The DNA molecule shown in SEQ ID No. 2 encodes the HlMO18 protein shown in SEQ ID No. 1.

[0012] In this invention, the tag can be a polypeptide or protein fused with the target protein using in vitro DNA recombination technology, to facilitate the expression, detection, tracing, and / or purification of the target protein. The tag can be Poly-Arg, Poly-His, FLAG, Strep-tag II, c-myc, MBP tag, HA tag, GST tag, and / or SUMO tag, etc.

[0013] To address the aforementioned technical problems, this invention also provides a substance for regulating the HlMO18 protein.

[0014] The aforementioned substance may be any one of the following B1) to B7): B1) a nucleic acid molecule encoding the HlMO18 protein; B2) an expression cassette containing the nucleic acid molecule described in B1); B3) a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4) a recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3); B5) a transgenic plant cell line containing the nucleic acid molecule described in B1), or a transgenic plant cell line containing the expression cassette described in B2); B6) a transgenic plant tissue containing the nucleic acid molecule described in B1), or a transgenic plant tissue containing the expression cassette described in B2); B7) a transgenic plant organ containing the nucleic acid molecule described in B1), or a transgenic plant organ containing the expression cassette described in B2).

[0015] The nucleic acid molecule can be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecule can also be RNA, such as mRNA or hnRNA.

[0016] Those skilled in the art can readily mutate the nucleotide sequence encoding the HlMO18 protein of this invention using known methods, such as directed evolution and point mutation. Artificially modified nucleotides that possess 75% or higher identity to the nucleotide sequence of the HlMO18 protein isolated in this invention, as long as they encode and function the HlMO18 protein, are derived from and equivalent to the nucleotide sequence of this invention.

[0017] In the above substances, the nucleic acid molecule described in B1) may be a DNA molecule whose coding sequence is SEQ ID No. 2 in the sequence listing.

[0018] Specifically, the nucleic acid molecule described in B1) may be the DNA molecule shown in SEQ ID No. 2.

[0019] In the aforementioned substances, the expression cassette (HlMO18 gene expression cassette) containing a nucleic acid molecule encoding the HlMO18 protein described in B2) refers to DNA capable of expressing the HlMO18 protein in host cells. This DNA may include not only a promoter to initiate HlMO18 gene transcription but also a terminator to terminate HlMO18 gene transcription. Furthermore, the expression cassette may also include an enhancer sequence.

[0020] Recombinant vectors containing the HlMO18 gene expression cassette can be constructed using existing expression vectors.

[0021] In the above-mentioned substances, the vector can be a plasmid, a phage, or a viral vector. Specifically, the plasmid can be the pESC-His vector or the pESC-His-VPS vector. The pESC-His-VPS vector is formed by replacing the small fragment between the EcoRI and NotI recognition sequences of the pESC-His vector with the one shown in SEQ ID No. 12. VPS Recombinant vectors from which genes are obtained.

[0022] B3) The recombinant vector may specifically be pESC-His-VPS-MO18. pESC-His-VPS-MO18 is formed by replacing the small fragment between the BamHI and SalI recognition sequences of the pESC-His-VPS vector with the sequence shown in SEQ ID No. 2. HlMO18 Recombinant vectors from which genes are obtained.

[0023] The present invention also provides at least one of the following applications of the HlMO18 protein or the substance that regulates the HlMO18 protein: X1. Produce Humulone; X2. Prepare products for producing Humulone; X3. (Specifically) produce 6S-Humulone; X4. Prepare (specifically) products for producing 6S-Humulone; X5. Increase the proportion of 6S-Humulone in Humulone; X6. Prepare products with increased proportion of 6S-Humulone in Humulone.

[0024] The present invention also provides a product comprising M1) and M3), or M2) and M3), or M1) and M4), or M2) and M4): M1) HlMO18 protein; M2) substances that regulate HlMO18 protein; M3) CCL2 protein, CCL4 protein, AtPT1LTP protein, AtPT2TP protein and / or VPS protein; M4) substances that regulate CCL2 protein, CCL4 protein, AtPT1LTP protein, AtPT2TP protein and / or VPS protein.

[0025] In the above products, the CCL2 protein may be C1), C2), or C3: C1) is a protein whose amino acid sequence is SEQ ID No. 3; C2) is a protein whose amino acid sequence shown in SEQ ID No. 3 in the sequence listing has been modified by substitution and / or deletion and / or addition of amino acid residues, and has more than 98% identity with the protein in C1) and has the same function. C3) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of C1) or C2); the CCL4 protein may be D1), D2), or D3): D1) a protein whose amino acid sequence is SEQ ID No. 5; D2) a protein that has more than 98% identity with and has the same function as the protein described in D1) by substituting and / or deleting and / or adding amino acid residues of the amino acid sequence shown in SEQ ID No. 5 in the sequence listing; D3) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of D1) or D2); The AtPT1LTP protein may be E1), E2), or E3: E1) is a protein whose amino acid sequence is SEQ ID No. 7; E2) is a protein with more than 98% identity and the same function as the protein described in E1) by substitution and / or deletion and / or addition of amino acid residues of the amino acid sequence shown in SEQ ID No. 7 in the sequence listing; E3) is a fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of E1) or E2); The AtPT2TP protein may be F1), F2), or F3: F1) is a protein whose amino acid sequence is SEQ ID No. 9; F2) is a protein that has more than 98% identity with and has the same function as the protein described in F1) by substituting and / or deleting and / or adding amino acid residues of the amino acid sequence shown in SEQ ID No. 9 in the sequence listing; F3) is a fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of F1) or F2); The VPS protein may be G1), G2), or G3: G1) is a protein whose amino acid sequence is SEQ ID No. 11; G2) is a protein that has more than 98% identity with and has the same function as the protein described in G1) by substitution and / or deletion and / or addition of amino acid residues of the amino acid sequence shown in SEQ ID No. 11 in the sequence listing; G3) is a fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of G1) or G2); The substance regulating the CCL2 protein may be any one of the following H1) to H7): H1) a nucleic acid molecule encoding the CCL2 protein; H2) an expression cassette containing the nucleic acid molecule H1); H3) a recombinant vector containing the nucleic acid molecule H1) or a recombinant vector containing the expression cassette H2); H4) a recombinant microorganism containing the nucleic acid molecule H1) or a recombinant microorganism containing the expression cassette H2) or a recombinant microorganism containing the recombinant vector H3); H5) a transgenic plant cell line containing the nucleic acid molecule H1) or a transgenic plant cell line containing the expression cassette H2); H6) a transgenic plant tissue containing the nucleic acid molecule H1) or a transgenic plant tissue containing the expression cassette H2); H7) a transgenic plant organ containing the nucleic acid molecule H1) or a transgenic plant organ containing the expression cassette H2. The substance regulating the CCL4 protein may be any one of the following I1) to I7): I1) a nucleic acid molecule encoding the CCL4 protein; I2) an expression cassette containing the nucleic acid molecule I1); I3) a recombinant vector containing the nucleic acid molecule I1), or a recombinant vector containing the expression cassette I2); I4) a recombinant microorganism containing the nucleic acid molecule I1), or a recombinant microorganism containing the expression cassette I2), or a recombinant microorganism containing the recombinant vector I3); I5) a transgenic plant cell line containing the nucleic acid molecule I1), or a transgenic plant cell line containing the expression cassette I2); I6) a transgenic plant tissue containing the nucleic acid molecule I1), or a transgenic plant tissue containing the expression cassette I2); I7) a transgenic plant organ containing the nucleic acid molecule I1), or a transgenic plant organ containing the expression cassette I2; The substance regulating the AtPT1LTP protein may be any one of the following J1) to J7): J1) a nucleic acid molecule encoding the AtPT1LTP protein; J2) an expression cassette containing the nucleic acid molecule of J1); J3) a recombinant vector containing the nucleic acid molecule of J1), or a recombinant vector containing the expression cassette of J2); J4) a recombinant microorganism containing the nucleic acid molecule of J1), or a recombinant microorganism containing the expression cassette of J2), or a recombinant microorganism containing the recombinant vector of J3); J5) a transgenic plant cell line containing the nucleic acid molecule of J1), or a transgenic plant cell line containing the expression cassette of J2); J6) a transgenic plant tissue containing the nucleic acid molecule of J1), or a transgenic plant tissue containing the expression cassette of J2); J7) a transgenic plant organ containing the nucleic acid molecule of J1), or a transgenic plant organ containing the expression cassette of J2; The substance regulating the AtPT2TP protein may be any one of the following K1) to K7): K1) a nucleic acid molecule encoding the AtPT2TP protein; K2) an expression cassette containing the nucleic acid molecule of K1); K3) a recombinant vector containing the nucleic acid molecule of K1), or a recombinant vector containing the expression cassette of K2); K4) a recombinant microorganism containing the nucleic acid molecule of K1), or a recombinant microorganism containing the expression cassette of K2), or a recombinant microorganism containing the recombinant vector of K3); K5) a transgenic plant cell line containing the nucleic acid molecule of K1), or a transgenic plant cell line containing the expression cassette of K2); K6) a transgenic plant tissue containing the nucleic acid molecule of K1), or a transgenic plant tissue containing the expression cassette of K2); K7) a transgenic plant organ containing the nucleic acid molecule of K1), or a transgenic plant organ containing the expression cassette of K2; The substance regulating the VPS protein may be any one of the following L1) to L7): L1) a nucleic acid molecule encoding the VPS protein; L2) an expression cassette containing the nucleic acid molecule of L1); L3) a recombinant vector containing the nucleic acid molecule of L1) or a recombinant vector containing the expression cassette of L2); L4) a recombinant microorganism containing the nucleic acid molecule of L1) or a recombinant microorganism containing the expression cassette of L2) or a recombinant microorganism containing the recombinant vector of L3); L5) a transgenic plant cell line containing the nucleic acid molecule of L1) or a transgenic plant cell line containing the expression cassette of L2); L6) a transgenic plant tissue containing the nucleic acid molecule of L1) or a transgenic plant tissue containing the expression cassette of L2); L7) a transgenic plant organ containing the nucleic acid molecule of L1) or a transgenic plant organ containing the expression cassette of L2).

[0026] The proteins in C2), D2), E2), F2) and G2) mentioned above can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0027] The coding genes for the proteins in C2), D2), E2), F2), and G2) mentioned above can be obtained by deleting one or more amino acid residues from the DNA sequences shown in SEQ ID No. 4, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10, and SEQ ID No. 12, and / or by performing a missense mutation of one or more base pairs, and / or by attaching a tag coding sequence to its 5′ and / or 3′ ends. Specifically, the DNA molecules shown in SEQ ID No. 4, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10, and SEQ ID No. 12 encode the CCL2 protein shown in SEQ ID No. 3, the CCL4 protein shown in SEQ ID No. 5, the AtPT1LTP protein shown in SEQ ID No. 7, the AtPT2TP protein shown in SEQ ID No. 9, and the VPS protein shown in SEQ ID No. 11, respectively.

[0028] In the above products, the nucleic acid molecule described in H1) may be a DNA molecule whose coding sequence is SEQ ID No. 4 in the sequence listing; I1) The nucleic acid molecule described may be a DNA molecule whose coding sequence is SEQ ID No. 6 in the sequence listing; J1) The nucleic acid molecule described may be a DNA molecule whose coding sequence is SEQ ID No. 8 in the sequence listing; K1) The nucleic acid molecule described may be a DNA molecule whose coding sequence is SEQ ID No. 10 in the sequence listing; L1) The nucleic acid molecule described may be a DNA molecule whose coding sequence is SEQ ID No. 12 in the sequence listing.

[0029] Specifically, the nucleic acid molecule described in H1) may be the DNA molecule shown in SEQ ID No. 4 of the sequence listing; I1) The nucleic acid molecule may be the DNA molecule shown in SEQ ID No. 6 of the sequence listing; J1) The nucleic acid molecule may be the DNA molecule shown in SEQ ID No. 8 of the sequence listing; K1) The nucleic acid molecule described may be the DNA molecule shown in SEQ ID No. 10 of the sequence listing; L1) The nucleic acid molecule may be the DNA molecule shown in SEQ ID No. 12 in the sequence listing.

[0030] Recombinant vectors containing the CCL2 gene expression cassette, CCL4 gene expression cassette, AtPT1LTP gene expression cassette, AtPT2TP gene expression cassette and / or VPS gene expression cassette can be constructed using existing expression vectors.

[0031] In the above products, the vector can be a plasmid, a granule, a bacteriophage, or a viral vector. Specifically, the plasmid can be the pESC-His vector, the pESC-Leu vector, or the pESC-Ura vector.

[0032] The recombinant vector may specifically be pESC-His-VPS-MO18, pESC-Leu-CCL2-CCL4, and / or pESC-Ura-AtPT1LTP-AtPT2TP. pESC-Leu-CCL2-CCL4 is created by replacing the small DNA fragment between the Not I and Pac I recognition sequences of the pESC-Leu vector with the one shown in SEQ ID No. 4. CCL2 The gene, replacing the small DNA fragment between the BamHI and XhoI recognition sequences of pESC-Leu-CCL2 with the one shown in SEQ ID No. 6. CCL4 The recombinant vector obtained from the gene. pESC-Ura-AtPT1LTP-AtPT2TP is a vector in which a small fragment between the BamHI and SalI recognition sequences of the pESC-Ura vector is replaced with the sequence shown in SEQ ID No. 8. AtPT1LTP Replace the small fragment between the Spe I and Pac I recognition sequences of the pESC-Ura-AtPT1LTP vector with the one shown in SEQ ID No. 10. AtPT2TP Recombinant vectors from which genes are obtained.

[0033] The aforementioned product may specifically be a recombinant yeast containing the encoding genes for HlMO18 protein, CCL2 protein, CCL4 protein, AtPT1LTP protein, AtPT2TP protein, and VPS protein.

[0034] In one embodiment of the present invention, the recombinant yeast was obtained by introducing the above-mentioned pESC-His-VPS-MO18, pESC-Leu-CCL2-CCL4 and pESC-Ura-AtPT1LTP-AtPT2TP into yeast strain DD104.

[0035] In this invention, the microorganism may be yeast, bacteria, algae, or fungi. In one embodiment of this invention, the yeast is yeast strain DD104.

[0036] In this invention, the transgenic plant cell lines, transgenic plant tissues, and transgenic plant organs do not include propagation material.

[0037] The above products can be used to produce Humulone, (specifically) to produce 6S-Humulone, and / or to increase the proportion of 6S-Humulone in Humulone products.

[0038] At least one of the following applications of the above-mentioned products also falls within the scope of protection of this invention: X1. Produce Humulone; X2. Prepare products for producing Humulone; X3. (Specifically) produce 6S-Humulone; X4. Prepare (specifically) products for producing 6S-Humulone; X5. Increase the proportion of 6S-Humulone in Humulone; X6. Prepare products with increased proportion of 6S-Humulone in Humulone.

[0039] In this invention, the method for producing Humulone may include: culturing recombinant yeast containing the coding genes for HlMO18 protein, CCL2 protein, CCL4 protein, AtPT1LTP protein, AtPT2TP protein, and VPS protein to obtain a culture medium; and extracting Humulone from the bacterial cells and / or the extracellular fluid in the culture medium to obtain 6 R -Humulone content is lower than 6S-Humulone content.

[0040] Experiments have shown that introducing the HlMO18 protein coding gene of this invention, along with the coding genes for CCL2, CCL4, AtPT1LTP, AtPT2TP, and VPS proteins, into yeast not only produces Humulone but also significantly increases the 6% concentration of the Humulone product. S - The proportion of Humulone. This indicates that the HlMO18 protein of this invention can specifically catalyze 6... S The generation of Humulone has potential applications in the beer brewing industry.

[0041] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way. Attached Figure Description

[0042] Figure 1 for HlMO18 Tissue-specific gene expression in hop plants. (Using hops as an example)GAPDH gene as the reference gene.

[0043] Figure 2 is 6 S type and 6 R type chemical structural formulas of Humulone and Deoxyhumulone. Figure 3 is the resolution of 6 S type and 6 R type Humulone using a chiral chromatographic column. The mixed standard refers to a Humulone mixture standard. A chiral chromatographic column-equipped LC-MS system is used to resolve chiral compounds. In the negative ion mode, the mass-to-charge ratio is selected as m / z 361.2020 for extracting Humulone.

[0044] Figure 4 is the detection of Deoxyhumulone production in engineered yeast strains. No Deoxyhumulone can be detected in the wild-type DD104 strain. The control (engineered yeast strain expressing CCL2 / CCL4 / VPS / AtPT1LTP / AtPT2TP ) and "control + HlMO18 " (engineered yeast strain expressing CCL2 / CCL4 / VPS / AtPT1LTP / AtPT2TP / HlMO18 ) can produce Deoxyhumulone. The compound is detected in the negative ion mode, and the mass-to-charge ratio is selected as m / z 345.2071 for extracting Deoxyhumulone.

[0045] Figure 5 is the detection of Humulone production in engineered yeast strains. No Humulone can be detected in the wild-type DD104 strain, and the control (engineered yeast strain expressing CCL2 / CCL4 / VPS / AtPT1LTP / AtPT2TP ) and "control + HlMO18 " (engineered yeast strain expressing CCL2 / CCL4 / VPS / AtPT1LTP / AtPT2TP / HlMO18 ) can produce Humulone. However, the ratio of 6 HlMO18 -Humulone to 6 S -Humulone in R " is significantly higher than that in the control. In the negative ion mode, the mass-to-charge ratio is selected as m / z 361.2020 for extracting Humulone.

[0046] Figure 6 is the ratio and content of 6 S type and 6 R type Humulone in the engineered yeast strain. (a), the ratio of 6 S -Humulone to 6 R -Humulone in the engineered yeast strain. (b), 6 in the engineered yeast strainS -Humulone and 6 R - The content of Humulone. (To express...) CCL2 / CCL4 / VPS / AtPT1LTP / AtPT2TP The engineered yeast strain was used as a control, "control + HlMO18 "Indicates expression" CCL2 / CCL4 / VPS / AtPT1LTP / AtPT2TP / HlMO18 Engineered yeast strains. Detailed Implementation

[0047] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials, reagents, instruments, etc., used in the following examples are commercially available.

[0048] In the quantitative experiments described below, at least three replicates were performed. The data were statistically analyzed using the two-tailed Student's T-Test. P < 0.05 (*) indicates a significant difference, P < 0.01 (**) indicates a highly significant difference, and P < 0.001 (***) indicates a highly significant difference.

[0049] The yeast strain DD104 (genotype) in the following examples MATα erg9-1 erg20-2 ura3 his3 leu2 ade2 (Polyprenol formation in the yeast Saccharomyces cerevisiae: effect of farnesyl diphosphate synthase overexpression. Anna Szkopihska et al., Journal of Lipid Research, 38: 962-668, 1997) This biological material is available to the public from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences (i.e., the applicant). It is intended solely for the purpose of replicating experiments related to this invention and may not be used for any other purpose.

[0050] SD-Ura-Leu-His liquid medium: 6.7 g / L amino acid-free yeast nitrogen source, 20 g / L dextrorotatory glucose, 1.3 g / L amino acid-deficient medium powder (deficient in L-uracil, L-leucine, and L-histidine), with the balance being water. Autoclave at 121°C for 15 min.

[0051] SG-Ura-Leu-His liquid medium: 6.7 g / L amino acid-free yeast nitrogen source, 20 g / L dextrose, 1.3 g / L amino acid-deficient medium powder (deficient in L-uracil, L-leucine, and L-histidine), with the balance being water. Autoclave at 121°C for 15 min.

[0052] Example 1 HlMO18 Encoding sequence acquisition and its expression features Using glandular trichome cDNA from the hop cultivar "Nugget" as a template, PCR amplification was performed using primers HlMO18-F: 5′-ATGGCCACAGCAACAGCTTATTC-3′ and HlMO18-R: 5′-TTATTGTTTGGTGTGTAAGCCTAAGGGC-3′, yielding a PCR product of approximately 1.4 Kb (i.e., HlMO18 (The coding region of the gene). After sequencing, the PCR product was 1368 bp. HlMO18 The gene's coding sequence is shown in SEQ ID No. 2, and the gene encodes the HlMO18 protein with the amino acid sequence shown in SEQ ID No. 1.

[0053] Hop materials from different developmental stages and different tissues / organs were collected from field-grown hops. After collection, materials at the corresponding stages were flash-frozen in liquid nitrogen and then stored at -80℃. RNA was extracted from each material and reverse transcribed to obtain cDNA for further real-time quantitative PCR experiments. The primers used in the experiment, including the primers for the internal control gene (GAPDH), are as follows: HlMO18-RT-F: 5′-AGGGTGGCCTGCCGAGACAC-3′; HlMO18-RT-R: 5′-GTCTTCCAATGCAACACTAGCTCC-3′; GAPDH-RT-F: 5′-GGACTGGAGAGGTGGAAGAGC-3′; GAPDH-RT-R: 5′-GCAAGCCTGACTGTGAGGTCA-3′.

[0054] See results Figure 1 . HlMO18 The gene was most highly expressed in glandular hairs four weeks after flowering, while its expression was low or absent in other tissues.

[0055] Examples 2 and 6 S Type and 6 R Splitting of type Humulone Humulone mixture standard (containing 6 in equal proportions) S -Humulone and 6 R -Humulone was synthesized by Shenzhen Feisi Pharmaceutical Co., Ltd. The chemical structural formulas of the two compounds are as follows: Figure 2As shown. Preparation of hop glandular trichome extract: Weigh 0.1g of hop glandular trichomes, grind them in liquid nitrogen, add 1mL of methanol, extract by ultrasonication at room temperature for 20 min, centrifuge at 12000 rpm for 10 min, collect the supernatant and pass it through a 0.22 µm nylon membrane. The filtrate is the hop glandular trichome extract. According to literature reports, the absolute configuration of Humulone in the hop extract is 6. S -Humulone can be used to identify corresponding isomers in mixed standards (Urban, J., Dahlberg, CJ, Carroll, BJ, and Kaminsky, W. (2013). Absolute configuration of beer's bitter compounds. Angew Chem Int Ed Engl 52:1553-1555. 10.1002 / anie.201208450.). The chiral column was used to analyze the chiral conformation of the 6... S Type and 6 R The Humulone type is split. Specific analysis conditions are as follows: The liquid chromatograph is an Agilent 1290, and the mass spectrometer is an Agilent 6550 QTOF.

[0056] The chromatographic column was a Daicel CHIRALPAK IC-U (3.0 × 100 mm, 1.6 µm).

[0057] Mobile phase: Phase A is water (containing 10 mM NH4HCO3), and Phase B is pure acetonitrile.

[0058] The elution procedure is as follows: From 0 min to 10 min, the mobile phase consisted of 50% (volume percentage) phase B and 50% (volume percentage) phase A. From 10 to 11 minutes, the mobile phase changed uniformly from 50% (volume percentage) phase B and 50% (volume percentage) phase A to 90% (volume percentage) phase B and 10% (volume percentage) phase A. From 11 to 15 minutes, the mobile phase consisted of 90% (volume percentage) phase B and 10% (volume percentage) phase A. From 15 to 16 minutes, the mobile phase changed uniformly from 90% (volume percentage) phase B and 10% (volume percentage) phase A to 50% (volume percentage) phase B and 50% (volume percentage) phase A. 16 min to 25 min, the mobile phase is 50% (volume percentage) phase B and 50% (volume percentage) phase A.

[0059] Flow rate 0.5 mL / min; column temperature 30°C. Capillary voltage 4000 V; carrier gas temperature 225°C; drying gas flow rate 13 L / min; sheath gas temperature 350°C; sheath gas flow rate 12 L / min. Injection volume 4 µL, data acquisition in negative ion mode (…). m / z Performed at 50–1700).

[0060] The results are as follows Figure 3 As shown, the Humulone mixture standard contains two chiral compounds (6... S -Humulone and 6 R -Humulone) was well separated and identified.

[0061] Example 3: Obtaining recombinant plasmids and preparing engineered yeast strains 1. Obtaining the recombinant plasmid pESC-Leu-CCL2-CCL4 Using glandular trichome cDNA from the hop cultivar "Nugget" as a template, PCR amplification was performed using primers CCL2-For: 5′-ATGGATAACTATAGAAGGCTCCACACTCCGG-3′ and CCL2-Rev: 5′-TCAAGAAAGGCTGCCCATGGCCATTGC-3′, yielding a PCR product of approximately 1.7 Kb. CCL2 The gene, whose sequence is shown in SEQ ID No. 4, encodes the CCL2 protein shown in SEQ ID No. 3. The obtained... CCL2 The full-length gene sequence was ligated to the pESC-Leu vector backbone to obtain the recombinant plasmid pESC-Leu-CCL2. Based on the sequencing results, the structure of the correctly sequenced recombinant plasmid is described as follows: pESC-Leu-CCL2 is formed by replacing the small DNA fragment between the Not I and Pac I recognition sequences of the pESC-Leu vector with the sequence shown in SEQ ID No. 4. CCL2 Recombinant vectors from which genes are obtained.

[0062] Using glandular trichome cDNA from the hop cultivar "Nugget" as a template, PCR amplification was performed using primers CCL4-For: 5′-ATGGAAGATCTGAAGCCGAGAC-3′ and CCL4-Rev: 5′-TCACATTCGGCTCCGGCTC-3′, yielding a PCR product of approximately 1.7 Kb. CCL4 The gene, whose sequence is shown in SEQ ID No. 6, encodes the CCL4 protein shown in SEQ ID No. 5. The obtained... CCL4The full-length gene sequence was ligated to the pESC-Leu-CCL2 vector backbone to obtain the recombinant plasmid pESC-Leu-CCL2-CCL4. Based on the sequencing results, the structure of the correctly sequenced recombinant plasmid is described as follows: pESC-Leu-CCL2-CCL4 is formed by replacing the small DNA fragment between the BamHI and XhoI recognition sequences of pESC-Leu-CCL2 with the sequence shown in SEQ ID No. 6. CCL4 Recombinant vectors from which genes are obtained.

[0063] 2. Obtaining the recombinant plasmid pESC-Ura-AtPT1LTP-AtPT2TP Genes derived from hops, codon-optimized from Arabidopsis thaliana, and with the signal peptide removed were obtained using a synthetic gene approach. PT1LTP Gene (denoted as) AtPT1LTP The gene, whose sequence is shown in SEQ ID No. 8, encodes the AtPT1LTP protein shown in SEQ ID No. 7. AtPT1LTP The gene sequence and the pESC-Ura vector backbone were ligated to obtain the recombinant plasmid pESC-Ura-AtPT1LTP. Based on the sequencing results, the structure of the correctly sequenced recombinant plasmid is described as follows: pESC-Ura-AtPT1LTP is formed by replacing the small fragment between the BamHI and SalI recognition sequences of the pESC-Ura vector with the sequence shown in SEQ ID No. 8. AtPT1LTP Recombinant vectors from which genes are obtained.

[0064] Genes derived from hops, codon-optimized from Arabidopsis thaliana, and with the signal peptide removed were obtained using a synthetic gene approach. PT2TP Gene (denoted as) AtPT2TP The gene, whose sequence is shown in SEQ ID No. 10, encodes the AtPT2TP protein shown in SEQ ID No. 9. AtPT2TP The full-length sequence was ligated to the pESC-Ura-AtPT1LTP vector backbone to obtain the recombinant plasmid pESC-Ura-AtPT1LTP-AtPT2TP. Based on the sequencing results, the structure of the correctly sequenced recombinant plasmid is described as follows: pESC-Ura-AtPT1LTP-AtPT2TP is formed by replacing the small fragment between the Spe I and Pac I recognition sequences of the pESC-Ura-AtPT1LTP vector with the sequence shown in SEQ ID No. 10. AtPT2TP Recombinant vectors from which genes are obtained.

[0065] 3. Obtaining the recombinant plasmid pESC-His-VPS Using glandular trichome cDNA from the hop cultivar "Nugget" as a template, PCR amplification was performed using primers VPS-For: 5′-ATGGCGTCCGTAACTGTAGAGCAAATC-3′ and VPS-Rev: 5′-TTAGACGTTTGTGGGCACGCTGT-3′, yielding a PCR product of approximately 1.2 Kb. VPS The gene, whose sequence is shown in SEQ ID No. 12, encodes the VPS protein shown in SEQ ID No. 11. The obtained... VPS The full-length gene sequence was ligated to the pESC-His vector backbone to obtain the recombinant plasmid pESC-His-VPS. Based on the sequencing results, the structure of the correctly sequenced recombinant plasmid is described as follows: pESC-His-VPS is formed by replacing the small fragment between the EcoRI and NotI recognition sequences of the pESC-His vector with the sequence shown in SEQ ID No. 12. VPS Recombinant vectors from which genes are obtained.

[0066] 4. Obtaining the recombinant plasmid pESC-His-VPS-MO18 Using glandular trichome cDNA from the hop cultivar "Nugget" as a template, PCR amplification was performed using primers HlMO18-F: 5′-ATGGCCACAGCAACAGCTTATTC-3′ and HlMO18-R: 5′-TTATTGTTTGGTGTGTAAGCCTAAGGGC-3′, yielding a PCR product of approximately 1.4 Kb, as shown in SEQ ID NO.2. HlMO18 The gene encodes the HlMO18 protein shown in SEQ ID NO.1. The obtained... HlMO18 The full-length gene sequence was ligated to the pESC-His-VPS vector backbone obtained in step 3 to obtain the recombinant plasmid pESC-His-VPS-MO18. Based on the sequencing results, the structure of the correctly sequenced recombinant plasmid is described as follows: pESC-His-VPS-MO18 is formed by replacing the small fragment between the BamHI and SalI recognition sequences of the pESC-His-VPS vector with the sequence shown in SEQ ID No. 2. HlMO18 Recombinant vectors from which genes are obtained.

[0067] 5. Obtaining engineered yeast strains The recombinant plasmids pESC-His-VPS, pESC-Leu-CCL2-CCL4, and pESC-Ura-AtPT1LTP-AtPT2TP were co-introduced into the DD104 yeast strain to obtain expression. CCL2 / CCL4 / VPS / AtPT1LTP / AtPT2TPFive gene-producing yeast strains were engineered; pESC-His-VPS-MO18, pESC-Leu-CCL2-CCL4, and pESC-Ura-AtPT1LTP-AtPT2TP were co-introduced into the DD104 strain to obtain strains expressing these genes. CCL2 / CCL4 / VPS / AtPT1LTP / AtPT2TP / HlMO18 A yeast strain with six genes.

[0068] Example 4: Production and Detection of Compounds by Yeast Engineered Strains Test strain: The expression strain obtained in Example 3 CCL2 / CCL4 / VPS / AtPT1LTP / AtPT2TP Yeast engineered strains, expressing CCL2 / CCL4 / VPS / AtPT1LTP / AtPT2TP / HlMO18 The engineered yeast strain, DD104.

[0069] 1. Yeast engineered strains produce compounds The steps for producing compounds using engineered yeast strains are as follows: a) Select a single yeast clone and inoculate it into 5 mL of SD-Ura-Leu-His liquid medium. Incubate at 28°C and 230 rpm for 48 h with shaking. Detect the yeast concentration by OD600 and ensure that OD600 is greater than 2.0. b) After step a) is completed, collect the yeast cells, wash away excess SD-Ura-Leu-His liquid medium with sterile water; then resuspend the yeast cells in 5 mL of SG-Ura-Leu-His liquid medium, and culture at 30°C and 230 rpm for 48 h with an OD600 of 0.4. c) After step b) is completed, detect and record the concentration of yeast cells; then take 4 mL of yeast culture, collect the cells and retain the supernatant, break the cells with acid-washed glass beads, and combine the supernatant and broken cells. d) After step c) is completed, add an equal volume of ethyl acetate (with 1.25 μM naringenin added as an internal standard); rotate on a shaker for 20 min, let stand for 15 min, centrifuge at 3,000 g for 10 min, and collect the supernatant; e) After step d) is completed, take 4 mL of the supernatant into a clean glass tube and dry it with nitrogen gas at room temperature; f) After step e), redissolve the solution in 400 μL of methanol, filter it through a 0.22 μm microporous membrane (organic nylon membrane), and then analyze the resulting filtrate by LC-Q-TOF-MS.

[0070] 2. Detection of Deoxyhumulone Production in Engineered Yeast Strains Deoxyhumulone produced in engineered yeast strains was detected using a C18 reverse-phase chromatography column. The sample was the filtrate obtained in step 1. Deoxyhumulone (directly synthesized by Shenzhen Feisi Pharmaceutical Co., Ltd., chemical structure as shown) was used.Figure 2 As shown, CAS NO.4374-93-0 is a standard product (referred to as standard product), and the specific analysis conditions are as follows: The liquid chromatograph is an Agilent 1290, and the mass spectrometer is an Agilent 6550 QTOF.

[0071] The chromatographic column was an Agilent ZORBAX Extend-C18 (2.1 × 50 mm, 1.8 µm).

[0072] Mobile phase: Phase A is a 10% (volume percentage) aqueous solution of acetonitrile (containing 5 mM ammonium formate and 0.05% (volume percentage) formic acid), and Phase B is a 90% aqueous solution of acetonitrile (containing 5 mM ammonium formate and 0.05% (volume percentage) formic acid).

[0073] The elution procedure is as follows: From 0 min to 10 min, the mobile phase changed uniformly from 20% (volume percentage) phase B and 80% (volume percentage) phase A to 90% (volume percentage) phase B and 10% (volume percentage) phase A; 10 min to 15 min, the mobile phase is 90% (volume percentage) phase B and 10% (volume percentage) phase A; From 15 min to 15.1 min, the mobile phase changed uniformly from 90% (volume percentage) B phase and 10% (volume percentage) A phase to 20% (volume percentage) B phase and 80% (volume percentage) A phase; 15.1 min to 20 min, the mobile phase is 20% (volume percentage) phase B and 80% (volume percentage) phase A.

[0074] Flow rate 0.4 mL / min; column temperature 35°C. Capillary voltage 4000 V; carrier gas temperature 225°C; drying gas flow rate 13 L / min; sheath gas temperature 350°C; sheath gas flow rate 12 L / min. Injection volume 2 µL, data acquisition in negative ion mode (…). m / z Performed at 50–1700).

[0075] The results are as follows Figure 4 As shown, Deoxyhumulone was not detected in the wild-type DD104 strain. The control strain (expressing...) CCL2 / CCL4 / VPS / AtPT1LTP / AtPT2TP Yeast engineered strains) and expression "control + HlMO18 "After (i.e., expressing)" CCL2 / CCL4 / VPS / AtPT1LTP / AtPT2TP / HlMO18 Deoxyhumulone was detected in engineered yeast strains.

[0076] 3. Yeast engineered strains produce 6 S-Humulone detection Using the 6 established in Example 2 S Type and 6 R The Humulone assay was used to detect Humulone produced by the above-mentioned engineered yeast strain. The sample to be tested was the filtrate obtained in step 1. The Humulone containing a chiral conformation was analyzed using a 6-type Humulone assay. S Type and 6 R Type Humulone is the standard product.

[0077] The results are as follows Figure 5 As shown, less than 6% of the wild-type DD104 strain was detected. S Type and 6 R Humulone type. Compared with control strain (expressing...) CCL2 / CCL4 / VPS / AtPT1LTP / AtPT2TP Compared to the engineered yeast strain, "control + HlMO18 (i.e., expression) CCL2 / CCL4 / VPS / AtPT1LTP / AtPT2TP / HlMO18 6 engineered yeast strains S -The Humulone product peak is significantly higher than 6. R -Humulone product peak. Further quantitative analysis results showed ( Figure 6 ), "comparison + HlMO18 6 S -Humulone and 6 R The ratio of -Humulone was 7.97 ± 1.53 (n=4), while this ratio was only 1.17 ± 0.11 (n=4) in the control strain. In the "control + HlMO18 "Among engineered yeast strains, 6" S -Humulone and 6 R The concentrations of Humulone were 0.088 ± 0.074 µmol / L / OD (n=4) and 0.010 ± 0.007 µmol / L / OD (n=4), respectively; in the control strain, 6 S -Humulone and 6 R The concentrations of -Humulone were 0.096 ± 0.012 µmol / L / OD (n=4) and 0.083 ± 0.017 µmol / L / OD (n=4), respectively.

[0078] The above results indicate that HlMO18 It is to participate in 6 S -Humulone synthesis is a key monooxygenase that specifically catalyzes 6-hydroxylamine (HLA) synthesis. S The generation of Humulone has potential applications in the beer brewing industry.

[0079] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. Protein, in the form of A1), A2), or A3). A1) The amino acid sequence of this protein is that of SEQ ID No. 1; A2) A protein having more than 98% identity with and the same function as the protein described in A1) by substitution and / or deletion and / or addition of amino acid residues of the amino acid sequence shown in SEQ ID No. 1 in the sequence listing; A3) is a fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of A1) or A2).

2. A substance that regulates the protein of claim 1.

3. The substance according to claim 2, characterized in that: The substance is any one of B1) to B7) below: B1) The nucleic acid molecule that encodes the protein; B2) An expression cassette containing the nucleic acid molecule described in B1); B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4) Recombinant microorganisms containing the nucleic acid molecules described in B1), or recombinant microorganisms containing the expression cassette described in B2), or recombinant microorganisms containing the recombinant vector described in B3); B5) A transgenic plant cell line containing the nucleic acid molecule described in B1), or a transgenic plant cell line containing the expression cassette described in B2); B6) Transgenic plant tissue containing the nucleic acid molecules described in B1), or transgenic plant tissue containing the expression cassette described in B2); B7) A transgenic plant organ containing the nucleic acid molecule described in B1), or a transgenic plant organ containing the expression cassette described in B2).

4. The substance according to claim 3, characterized in that: B1) The nucleic acid molecule described is a DNA molecule whose coding sequence is SEQ ID No. 2 in the sequence listing.

5. At least one of the following applications of the protein of claim 1 or any of the substances of claims 2-4: X1. Production of Humulone; X2. Prepare products for the production of Humulone; X3, Production of 6S-Humulone; X4. Prepare and produce 6S-Humulone products; X5. Increase the proportion of 6S-Humulone in Humulone; X6. Prepare products that increase the proportion of 6S-Humulone in Humulone.

6. A product composed of M1) and M3), or M2) and M3), or M1) and M4), or M2) and M4): M1) The protein according to claim 1; M2) The substance described in any of claims 2-4; M3) CCL2 protein, CCL4 protein, AtPT1LTP protein, AtPT2TP protein and / or VPS protein; M4) Substances that regulate CCL2 protein, CCL4 protein, AtPT1LTP protein, AtPT2TP protein, and / or VPS protein.

7. The product according to claim 6, characterized in that: The CCL2 protein is C1), C2), or C3). C1) The amino acid sequence of this protein is that of SEQ ID No. 3; C2) A protein having more than 98% identity with and the same function as the protein described in C1) by substitution and / or deletion and / or addition of amino acid residues of the amino acid sequence shown in SEQ ID No. 3 in the sequence listing; C3) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of C1) or C2); The CCL4 protein is D1), D2), or D3). D1) The amino acid sequence of this protein is that of SEQ ID No. 5; D2) A protein having more than 98% identity with and the same function as the protein described in D1) by substitution and / or deletion and / or addition of amino acid residues of the amino acid sequence shown in SEQ ID No. 5 in the sequence listing; D3) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of D1) or D2); The AtPT1LTP protein is E1, E2, or E3. E1) The amino acid sequence of this protein is that of SEQ ID No. 7; E2) A protein having more than 98% identity with and the same function as the protein described in E1) by substitution and / or deletion and / or addition of amino acid residues of the amino acid sequence shown in SEQ ID No. 7 in the sequence listing; E3) is a fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of E1) or E2); The AtPT2TP protein is F1, F2, or F3. F1) The amino acid sequence of this protein is that of SEQ ID No. 9; F2) A protein having more than 98% identity with and the same function as the protein described in F1) by substitution and / or deletion and / or addition of amino acid residues of the amino acid sequence shown in SEQ ID No. 9 in the sequence listing; F3) is a fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of F1) or F2); The VPS protein is G1, G2, or G3. The amino acid sequence of G1 is that of the protein SEQ ID No. 11; G2) A protein having more than 98% identity with and the same function as the protein described in G1) by substitution and / or deletion and / or addition of amino acid residues of the amino acid sequence shown in SEQ ID No. 11 in the sequence listing; G3) is a fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of G1) or G2); The substance regulating the CCL2 protein is any one of the following (H1) to (H7): H1) The nucleic acid molecule encoding the CCL2 protein; H2) contains an expression cassette containing the nucleic acid molecule described in H1); H3) a recombinant vector containing the nucleic acid molecule described in H1), or a recombinant vector containing the expression cassette described in H2); H4) Recombinant microorganisms containing the nucleic acid molecules described in H1), or recombinant microorganisms containing the expression cassette described in H2), or recombinant microorganisms containing the recombinant vector described in H3); H5) A transgenic plant cell line containing the nucleic acid molecule described in H1), or a transgenic plant cell line containing the expression cassette described in H2); H6) Transgenic plant tissue containing the nucleic acid molecules described in H1), or transgenic plant tissue containing the expression cassette described in H2); H7) Transgenic plant organs containing the nucleic acid molecules described in H1), or transgenic plant organs containing the expression cassette described in H2; The substance regulating the CCL4 protein is any one of the following I1) to I7): I1) The nucleic acid molecule encoding the CCL4 protein; I2) An expression cassette containing the nucleic acid molecule described in I1); I3) A recombinant vector containing the nucleic acid molecule described in I1), or a recombinant vector containing the expression cassette described in I2); I4) Recombinant microorganisms containing the nucleic acid molecules described in I1), or recombinant microorganisms containing the expression cassette described in I2), or recombinant microorganisms containing the recombinant vector described in I3); I5) A transgenic plant cell line containing the nucleic acid molecule described in I1), or a transgenic plant cell line containing the expression cassette described in I2); I6) Transgenic plant tissue containing the nucleic acid molecules described in I1), or transgenic plant tissue containing the expression cassette described in I2); I7) Transgenic plant organs containing the nucleic acid molecules described in I1), or transgenic plant organs containing the expression cassette described in I2); The substance regulating the AtPT1LTP protein is any one of the following J1) to J7): J1) The nucleic acid molecule encoding the AtPT1LTP protein; J2) An expression cassette containing the nucleic acid molecule described in J1); J3) A recombinant vector containing the nucleic acid molecule described in J1), or a recombinant vector containing the expression cassette described in J2); J4) Recombinant microorganisms containing the nucleic acid molecules described in J1), or recombinant microorganisms containing the expression cassette described in J2), or recombinant microorganisms containing the recombinant vector described in J3); J5) A transgenic plant cell line containing the nucleic acid molecule described in J1), or a transgenic plant cell line containing the expression cassette described in J2); J6) Transgenic plant tissue containing the nucleic acid molecules described in J1), or transgenic plant tissue containing the expression cassette described in J2); J7) A transgenic plant organ containing the nucleic acid molecule described in J1), or a transgenic plant organ containing the expression cassette described in J2); The substance regulating the AtPT2TP protein is any one of the following K1) to K7): K1) is a nucleic acid molecule encoding the AtPT2TP protein; K2) contains an expression cassette containing the nucleic acid molecules described in K1); K3) A recombinant vector containing the nucleic acid molecule described in K1), or a recombinant vector containing the expression cassette described in K2); K4) Recombinant microorganisms containing the nucleic acid molecules described in K1), or recombinant microorganisms containing the expression cassette described in K2), or recombinant microorganisms containing the recombinant vector described in K3); K5) A transgenic plant cell line containing the nucleic acid molecule described in K1), or a transgenic plant cell line containing the expression cassette described in K2); K6) Transgenic plant tissue containing the nucleic acid molecules described in K1), or transgenic plant tissue containing the expression cassette described in K2); K7) Transgenic plant organs containing the nucleic acid molecules described in K1), or transgenic plant organs containing the expression cassette described in K2); The substance regulating the VPS protein is any one of the following L1) to L7): L1) The nucleic acid molecule encoding the VPS protein; L2) contains an expression cassette containing the nucleic acid molecules described in L1); L3) A recombinant vector containing the nucleic acid molecule described in L1), or a recombinant vector containing the expression cassette described in L2); L4) Recombinant microorganisms containing the nucleic acid molecules described in L1), or recombinant microorganisms containing the expression cassette described in L2), or recombinant microorganisms containing the recombinant vector described in L3); L5) A transgenic plant cell line containing the nucleic acid molecule described in L1), or a transgenic plant cell line containing the expression cassette described in L2); L6) Transgenic plant tissue containing the nucleic acid molecules described in L1), or transgenic plant tissue containing the expression cassette described in L2); L7) A transgenic plant organ containing the nucleic acid molecule described in L1) or a transgenic plant organ containing the expression cassette described in L2).

8. The product according to claim 7, characterized in that: H1) The nucleic acid molecule described is a DNA molecule whose coding sequence is SEQ ID No. 4 in the sequence listing; I1) The nucleic acid molecule described is a DNA molecule whose coding sequence is SEQ ID No. 6 in the sequence listing; J1) The nucleic acid molecule described is a DNA molecule whose coding sequence is SEQ ID No. 8 in the sequence listing; K1) The nucleic acid molecule described is a DNA molecule whose coding sequence is SEQ ID No. 10 in the sequence listing; L1) The nucleic acid molecule described is a DNA molecule whose coding sequence is SEQ ID No. 12 in the sequence listing.

9. The product according to any one of claims 6-8, characterized in that: The product is yeast containing the coding genes for the protein of claim 1, the coding genes for the CCL2 protein, the coding genes for the CCL4 protein, the coding genes for the AtPT1LTP protein, the coding genes for the AtPT2TP protein, and the coding genes for the VPS protein.

10. At least one of the following applications of the product of any one of claims 6-8: X1. Production of Humulone; X2. Prepare products for the production of Humulone; X3, Production of 6S-Humulone; X4. Prepare and produce 6S-Humulone products; X5. Increase the proportion of 6S-Humulone in Humulone; X6. Prepare products that increase the proportion of 6S-Humulone in Humulone.

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