Preparation method and application of hyperforin

Abstract

The invention provides a novel preparation method and application of hyperforin. According to the invention, all metabolism genes in the biosynthesis process of hypericin are analyzed for the first time in the field. The analysis process is pioneering work and comprises discovery of the gene and the enzyme coded by the gene, functional demonstration and analysis of the hyperforin produced by using yeast and tobacco as biological chassis. The invention lays a foundation for industrial production of the hypericin and the intermediate thereof.

Description

Technical Field

[0001] This invention belongs to the fields of biotechnology and botany, and more specifically, this invention relates to a novel method for preparing hypericin and its application. Background Technology

[0002] St. John's wort (Hypericum perforatum L.) is a high-value medicinal plant with global annual sales exceeding $6 billion. In ancient times, extracts of St. John's wort were used to treat skin diseases due to their antiseptic and anti-inflammatory properties. Today, it is used to treat mild to moderate depression, with fewer side effects compared to common antidepressants.

[0003] Hypericin, the first isolated polycyclic polyisoprene-based phloroglucinol (PPAP), is a class of natural products with diverse biological activities. Many of its compounds possess anti-tumor and weight-loss properties and it also serves as a taxonomic marker for plants in the Hypericaceae family. The unique structure of hypericin presents a challenging task for enantioselective synthesis, inspiring chemists to pursue novel organic synthetic strategies. This chemical synthetic route involves multiple steps, but the overall yield is very low.

[0004] With the development of pathway reconstruction in microbial or plant systems, obtaining hypericin from these sources is a sustainable and environmentally friendly strategy for isolating this valuable pharmacological agent. However, despite the importance of hypericin, its de novo biosynthesis remains unresolved, and there is an urgent need in the field to develop efficient biosynthetic protocols for hypericin. Summary of the Invention

[0005] The purpose of this invention is to provide a new method for preparing hypericin and its application.

[0006] In a first aspect of the present invention, a method for preparing hypericin or an intermediate thereof is provided, the method comprising:

[0007] (1) Provide an expression system (such as a host cell or plant) in which an enzyme or enzyme expression cassette from the following group is introduced: (i) a short-chain fatty acid CoA ligase: CCL2; (ii) a polyketide synthase: PKS2; (iii) an isopentenyl transferase, including those selected from the following group: PIBP-GT, G-PIBP-PT, PG-PIBP-PT, PPG-PIBP-PT or combinations thereof;

[0008] (2) Produce hypericin or its intermediates using the expression system of (1).

[0009] In one or more embodiments, the short-chain fatty acid CoA ligase CCL2 is HpCCL2.

[0010] In one or more embodiments, the polyketide synthase is HpPKS2.

[0011] In one or more embodiments, in step (1), the enzymes or enzyme expression cassettes of (i), (ii) and (iii) are introduced into the expression system; thereby in step (2), isobutyryl coenzyme A is formed by the enzyme of (i), Phloroisobutyrophenone (PIBP) intermediate is formed by the enzyme of (ii), G-PIBP is formed by the PIBP-GT enzyme of (iii), PG-PIBP is formed by the G-PIBP-PT enzyme of (iv), PPG-PIBP is formed by the PG-PIBP-PT enzyme of (iv), and Hypericin is formed by the PPG-PIBP-PT enzyme of (iv).

[0012] In one or more embodiments, in step (2), the catalysis is carried out within the cell using an endogenous substrate.

[0013] In one or more embodiments, the endogenous substrate is valine.

[0014] In one or more embodiments, in step (2), the catalysis is carried out using valine as a substrate in an extracellular or in vitro system.

[0015] In one or more embodiments, the PIBP-GT is a nucleic acid sequence as shown in SEQ ID NO:1, or a conserved variant thereof.

[0016] In one or more embodiments, the G-PIBP-PT is a nucleic acid sequence as shown in SEQ ID NO:2, or a conserved variant thereof.

[0017] In one or more embodiments, the PG-PIBP-PT is a nucleic acid sequence as shown in SEQ ID NO:3, or a conserved variant thereof.

[0018] In one or more embodiments, the PPG-PIBP-PT is a nucleic acid sequence as shown in SEQ ID NO:4, or a conserved variant thereof.

[0019] In one or more embodiments, the PIBP-GT is a polypeptide with an amino acid sequence as shown in SEQ ID NO:5, or a conserved variant thereof.

[0020] In one or more embodiments, the G-PIBP-PT is a polypeptide with an amino acid sequence as shown in SEQ ID NO:6, or a conserved variant thereof.

[0021] In one or more embodiments, the PG-PIBP-PT is a polypeptide with an amino acid sequence as shown in SEQ ID NO:7, or a conserved variant thereof.

[0022] In one or more embodiments, the PPG-PIBP-PT is a polypeptide with an amino acid sequence as shown in SEQ ID NO:8, or a conserved variant thereof.

[0023] In one or more embodiments, the host cell includes a eukaryotic cell or a prokaryotic cell; preferably, the eukaryotic cell includes yeast, plant cells, fungal cells, insect cells or mammalian cells, or the prokaryotic cell includes Escherichia coli, Bacillus subtilis or Streptomyces; more preferably, the host cell is yeast; even more preferably, the yeast is Saccharomyces cerevisiae.

[0024] In one or more embodiments, the plant includes angiosperms or gymnosperms; preferably, the plant is a dicotyledonous plant or a monocotyledonous plant; more preferably, the plant includes plants of the Solanaceae family; even more preferably, the Solanaceae family includes tobacco.

[0025] In one or more embodiments, in step (2), the host cell is yeast, which is cultured using a yeast culture medium, preferably containing D-glucose as a carbon source; preferably, after culturing with D-glucose as a carbon source for 1-3 days, the supernatant is discarded, and then cultured with a culture medium containing D-galactose as a carbon source.

[0026] In one or more embodiments, in step (2), the plant is Nicotiana benthamiana; preferably, it is cultivated using nutrient soil.

[0027] In one or more embodiments, the yeast is Saccharomyces cerevisiae DD104 strain.

[0028] In one or more embodiments, the tobacco is the wild type of Nicotiana benthamiana.

[0029] In one or more embodiments, the conserved variant polypeptide of any of the aforementioned enzymes includes: (1) a polypeptide formed by substitution, deletion or addition of one or more (e.g., 1-20, preferably 1-10; more preferably 1-5; more preferably 1-3) amino acid residues of the polypeptide of the enzyme sequence, and having the function of the wild-type enzyme; (2) a polypeptide that has 50% or more (preferably 60% or more; more preferably 70% or more; more preferably 80% or more; more preferably 85% or more; more preferably 90% or more; more preferably 95% or more; more preferably 98% or more; more preferably 99% or more) similarity to the polypeptide of the enzyme sequence, and having the function of the wild-type enzyme; or (3) a polypeptide formed by adding a tag sequence to the N or C end of the polypeptide of the enzyme sequence, or by adding a signal peptide sequence to its N end.

[0030] In one or more embodiments, the respective enzyme expression cassettes are placed in one or more expression constructs (expression vectors).

[0031] In one or more embodiments, the production of hypericin or its intermediates using the expression system of (1) comprises carrying out the reaction under suitable reaction conditions, including appropriate temperature and appropriate pH (e.g., setting an appropriate buffer).

[0032] In another aspect of the invention, an expression system (e.g., a host cell or plant) for producing hypericin is provided, comprising an enzyme or enzyme expression cassette from the group consisting of: (i) a short-chain fatty acid CoA ligase: CCL2; (ii) a polyketide synthase: PKS2; and (iii) an isopentenyltransferase, comprising an enzyme selected from the group consisting of: PIBP-GT, G-PIBP-PT, PG-PIBP-PT, PPG-PIBP-PT, or a combination thereof.

[0033] In another aspect of the invention, a kit is provided for preparing hypericin or its intermediates, comprising: the expression system (such as host cells or plants) for producing hypericin.

[0034] In one or more embodiments, the kit further includes materials selected from: a culture medium for the host cells, a culture medium for the plant, and an instruction manual explaining the preparation method.

[0035] In another aspect of the invention, the application of PIBP-GT is provided as an isopentenyltransferase; preferably, it is used to catalyze the formation of hyperoside intermediate Phloroisobutyrophenone (PIBP) into hyperoside intermediate G-PIBP.

[0036] In another aspect of the invention, the application of G-PIBP-PT is provided as an isopentenyltransferase; preferably, it is used to catalyze the formation of the hypericin intermediate PG-PIBP from Translucin I.

[0037] In another aspect of the invention, the application of PG-PIBP-PT is provided as an isopentenyltransferase; preferably, it is used to catalyze the formation of hypericin intermediate PPG-PIBP from Translucin II.

[0038] In another aspect of the invention, the application of PPG-PIBP-PT is provided as an isopentenyltransferase; preferably, it is used to catalyze the formation of hypericin from the hypericin intermediate Translucin III.

[0039] In another aspect of the invention, the application of a combination of PIBP-GT, G-PIBP-PT, PG-PIBP-PT and PPG-PIBP-PT is provided for the production of hypericin.

[0040] In one or more embodiments, the combination of PIBP-GT, G-PIBP-PT, PG-PIBP-PT and PPG-PIBP-PT produces hypericin from a host-endogenous substrate.

[0041] In one or more embodiments, the combination of PIBP-GT, G-PIBP-PT, PG-PIBP-PT and PPG-PIBP-PT uses endogenous valine as a substrate.

[0042] In one or more embodiments, when produced in vitro, the combination of PIBP-GT, G-PIBP-PT, PG-PIBP-PT and PPG-PIBP-PT uses exogenously added valine as a substrate.

[0043] Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein. Attached Figure Description

[0044] Figure 1 Chemical synthesis of standards for intermediates of hypericin.

[0045] Figure 2 The activity of enzymes in yeast that catalyze the synthesis of hypericin and its intermediates.

[0046] Figure 3 The activity of enzymes in tobacco that catalyze the synthesis of hypericin. Detailed Implementation

[0047] Based on in-depth research, the inventors have, for the first time in the field, elucidated the biosynthetic process of hypericin and its intermediates. This elucidation is pioneering work, revealing new functions of the gene and its encoded enzymes, including the elucidation of intracellular biosynthesis and synthesis within the plant. This invention provides new ideas for the industrial production of hypericin and its intermediates.

[0048] the term

[0049] As used herein, an "expression cassette" or "gene expression cassette" refers to a gene expression system containing all the necessary elements required to express a target polypeptide, typically including the following elements: a promoter, a gene sequence encoding the polypeptide, and a terminator; additionally, it may optionally include a signal peptide encoding sequence, etc.; these elements are operatively linked.

[0050] As used herein, an "expression construct" or "expression building block" refers to a recombinant DNA molecule containing a desired nucleic acid coding sequence, which may contain one or more gene expression cassettes. These "constructs" are typically contained within an expression vector.

[0051] As used herein, "exogenous" or "heterogeneous" refers to the relationship between two or more nucleic acid or protein sequences from different sources, or the relationship between a protein (or nucleic acid) from different sources and a host cell. For example, if the combination of nucleic acid and host cell is not normally naturally occurring, then the nucleic acid is exogenous to that host cell. A particular sequence is "exogenous" to the cell or organism in which it is inserted.

[0052] As used herein, “operationally linked” or “operationally connected” refers to a functional spatial arrangement of two or more nucleic acid regions or sequences. For example, a promoter region is placed at a specific position relative to the nucleic acid sequence of a target gene, such that transcription of the nucleic acid sequence is guided by the promoter region, thereby “operationally linked” to the nucleic acid sequence.

[0053] As used herein, “isolated” means that the gene (including gene clusters and combinations of genes) or polypeptide has been isolated from the genome or cell and is substantially free of other genes, proteins, lipids, sugars or other substances naturally associated with it; the isolation process involves an artificial process that distinguishes it from its naturally occurring state.

[0054] As used herein, the term "conservative variant polypeptide" refers to a polypeptide that substantially retains the same biological function or activity as the polypeptide. A "conservative variant polypeptide" may be (i) a polypeptide in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code; or (ii) a polypeptide having substituent groups in one or more amino acid residues; or (iii) a polypeptide formed by fusing a mature polypeptide with another compound (e.g., a compound that extends the half-life of the polypeptide, such as polyethylene glycol); or (iv) a polypeptide formed by fusing an additional amino acid sequence to the sequence of this polypeptide (e.g., a leader sequence or secretion sequence or a sequence used to purify this polypeptide or a proteogen sequence, or a fusion protein formed with an antigen IgG fragment). In accordance with the teachings herein, such fragments, derivatives, and analogs are within the scope well known to those skilled in the art.

[0055] As used herein, the term "variant" or "mutant" refers to a peptide or polypeptide whose amino acid sequence has been altered compared to a reference sequence by the insertion, deletion, or substitution of one or more amino acids, but which retains at least one biological activity. Mutants described in any embodiment herein include amino acid sequences having at least 50%, 60%, or 70%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 97% sequence identity with a reference sequence (such as the native sequence of any enzyme described herein) and retaining the biological activity of the reference sequence (such as activity as a CoA ligase, etc.). Sequence identity between two aligned sequences can be calculated using, for example, NCBI's BLASTp. Mutants also include amino acid sequences having one or more mutations (insertion, deletion, or substitution) in the amino acid sequence of a reference sequence while still retaining the biological activity of the reference sequence. The plurality of mutations typically refers to 1-20, for example 1-15, 1-10, 1-8, 1-5, or 1-3. Substitutions are preferably conserved substitutions. For example, in the art, conservative substitution with amino acids of similar or analogous properties generally does not alter the function of a protein or peptide. "Amino acids of similar or analogous properties" includes, for example, families of amino acid residues with similar side chains, including amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, replacing one or more sites in the peptides of the present invention with another amino acid residue from the same side chain class will not substantially affect their activity.

[0056] Genes and their expression systems

[0057] In this invention, on one hand, the inventors successfully achieved the co-expression of these genes and the highly active production of the compound by introducing genes into host cells to produce hypericin or its intermediates in host cells.

[0058] The genes or polypeptides (proteins) described in this invention can be naturally occurring, for example, they can be isolated or purified from autotrophic plants or microorganisms. Furthermore, the genes or polypeptides can also be artificially prepared, for example, by obtaining the genes using conventional genetic engineering recombination techniques, or by obtaining the genes through artificial synthesis methods, and having them encode polypeptides.

[0059] The amino acid sequences of the various polypeptides (enzymes) described in this invention are preferably those listed in Table 2, and also include "conserved variant polypeptides" having the same function as the enzymes shown, and the sequences listed in Table 2. This invention also includes fragments, derivatives, and analogs of the said polypeptides. As used herein, the terms "fragment," "derivative," and "analyte" refer to polypeptides that substantially retain the same biological function or activity as the said polypeptide.

[0060] The “conserved variant polypeptide” may include (but is not limited to): deletions, insertions, and / or substitutions of one or more amino acids (typically 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10), and additions or deletions of one or more amino acids (e.g., up to 50, more preferably up to 20 or 10, more preferably up to 5) at the C-terminus and / or N-terminus. For example, in the art, substitution with amino acids of similar or analogous properties generally does not alter the function of the protein. Similarly, adding one or more amino acids at the C-terminus and / or N-terminus generally does not alter the function of the protein. The present invention also provides analogs of the said polypeptide. These analogs may differ from the natural polypeptide in amino acid sequence, in the form of modifications that do not affect the sequence, or both. These polypeptides include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis by radiation or exposure to a mutagen, site-directed mutagenesis, or other known molecular biology techniques. Analogs also include those having residues different from naturally occurring L-amino acids (such as D-amino acids), and those having non-naturally occurring or synthetic amino acids (such as β- or γ-amino acids). It should be understood that the polypeptides of the present invention are not limited to the representative polypeptides exemplified above.

[0061] The amino or carboxyl terminus of the polypeptide of the present invention may also contain one or more polypeptide fragments as protein tags. Any suitable tag can be used in the present invention. For example, the tag may be FLAG, HA, HA1, c-Myc, Poly–His, Poly-Arg, Strep-TagII, AU1, EE, T7, 4A6, ε, B, gE, and Ty1. These tags can be used for protein purification.

[0062] When used for the production of the enzymes of the present invention or other enzymes (e.g., enzymes used in host cells to form / promote the formation (e.g., increasing metabolic flux) of specific substrates, or enzymes involved in any step of the synthetic pathway of the product of the present invention), a signal peptide sequence may be added to the amino terminus of the polypeptide of the present invention to enable secretory expression of the translated protein (e.g., secretion outside the cell). The signal peptide may be cleaved during the secretion of the polypeptide from the cell.

[0063] The active polypeptides of the present invention can be recombinant polypeptides, natural polypeptides, or synthetic polypeptides. The polypeptides of the present invention can be naturally purified products, chemically synthesized products, or produced from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, higher plants) using recombinant technology. Depending on the host used in the recombinant production scheme, the polypeptides of the present invention can be glycosylated or non-glycosylated. The polypeptides of the present invention may or may not include an initial methionine residue.

[0064] The polynucleotide encoding the polypeptide of the present invention can be in DNA or RNA form. DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. DNA can be single-stranded or double-stranded. DNA can be a coding strand or a non-coding strand. The term "polynucleotide encoding a polypeptide" can include a polynucleotide encoding the polypeptide, or it can include polynucleotides with additional coding and / or non-coding sequences.

[0065] The present invention also relates to vectors containing the polynucleotides of the present invention, host cells generated by genetic engineering using the vectors or polypeptide coding sequences of the present invention, and methods for generating the polypeptides of the present invention via recombinant technology.

[0066] This invention relates to nucleic acid constructs containing the polynucleotides described herein, and one or more regulatory sequences or sequences required for genomic homologous recombination operatively linked to these sequences. The polynucleotides described herein can be manipulated in various ways to ensure the expression of the polypeptide or protein. The nucleic acid constructs can be manipulated according to the expression vector or requirements before insertion into a vector. Techniques for altering polynucleotide sequences using recombinant DNA methods are known in the art.

[0067] In some embodiments, the nucleic acid construct is a vector. The vector can be a cloning vector, an expression vector, or a gene knock-in vector. The polynucleotides of the present invention can be cloned into many types of vectors, such as plasmids, phage particles, phage derivatives, animal viruses, and granules. Cloning vectors can be used to provide the coding sequence of the protein or polypeptide of the present invention. Expression vectors can be provided to cells in the form of bacterial or viral vectors. Expression of the polynucleotides of the present invention is typically achieved by operably linking the polynucleotides of the present invention to a promoter and incorporating the construct into an expression vector. This vector is suitable for replication and integration into eukaryotic cells. A typical expression vector contains expression control sequences that can be used to regulate the expression of the desired nucleic acid sequence.

[0068] Gene knock-in vectors can be used to integrate the polynucleotide sequences described herein into regions of interest in the genome. Typically, gene knock-in vectors contain, in addition to the polynucleotide sequences described herein, 5' and 3' homologous arms required for genomic homologous recombination. In some embodiments, the nucleic acid constructs described herein contain 5' homologous arms, the polynucleotide sequences described herein, and 3' homologous arms. When using gene knock-in vectors, CRISPR / Cas9 technology can be used simultaneously to homologously recombine the polynucleotide sequences into the sites of interest. CRISPR / Cas9 technology guides the Cas9 nuclease to modify the genome at the insertion site by designing guide RNAs targeting the target gene, resulting in increased homologous recombination efficiency in the modified region, thus homologously recombinating the target fragment contained in the gene knock-in vector into the target site. The steps of CRISPR / Cas9 technology and the reagents used, such as the Cas9 nuclease, are well known in the art.

[0069] Methods well known to those skilled in the art can be used to construct nucleic acid constructs. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombination techniques, etc. The DNA sequence can be efficiently ligated to an appropriate promoter in the expression vector to direct mRNA synthesis. Representative examples of these promoters include: the lac or trp promoter of *E. coli*; the PL promoter of λ phage; eukaryotic promoters including the CMV immediate early promoter, the HSV thymidine kinase promoter, early and late SV40 promoters, LTRs of retroviruses, and other known promoters that control gene expression in prokaryotic or eukaryotic cells or their viruses. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. Furthermore, the expression vector preferably contains one or more selective marker genes to provide phenotypic traits for selecting host cells for transformation, such as dihydrofolate reductase, neomycin resistance, and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for *E. coli*.

[0070] In expression regulatory sequences or expression cassettes, inducible or constitutive promoters can be applied according to different needs. Inducible promoters can achieve more controllable protein expression and compound production, which is beneficial for industrial applications.

[0071] When the polynucleotides of this invention are expressed in higher eukaryotic cells, the insertion of an enhancer sequence into the vector will enhance transcription. Enhancers are cis-acting factors of DNA, typically approximately 10 to 300 base pairs, that act on the promoter to enhance gene transcription. Examples include the SV40 enhancer (100 to 270 base pairs) located late on the replication origin side, the polyoma enhancer located late on the replication origin side, and adenovirus enhancers.

[0072] Vectors containing appropriate DNA sequences and appropriate promoters or control sequences can be used to transform appropriate host cells so that they can express proteins.

[0073] This invention also provides host cells for the biosynthesis of target products. Host cells can be prokaryotic cells, such as bacterial cells; lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples include: fungal cells such as yeast; prokaryotic cells such as *Escherichia coli* and *Streptomyces*; bacterial cells of *Salmonella typhimurium*; plant cells; insect cells of *Drosophila* S2 or Sf9; animal cells such as CHO, COS, 293 cells, or Bowes melanoma cells. As a preferred embodiment of this invention, the host cell is yeast. It should be understood that a cell host is a production tool, and those skilled in the art can modify various host cells using various technical means to achieve biosynthesis as described in this invention. The host cells and production methods thus constructed should also be included in this invention.

[0074] In a preferred embodiment of the present invention, the recombinant expression vector may include, but is not limited to, the following (I) to (IV):

[0075] (I) The recombinant plasmid obtained by inserting CCL2 (preferably HpCCL2) and PKS2 (preferably HpPKS2) into the pESC-LEU vector;

[0076] (II) The recombinant plasmids obtained by inserting PIBP-GT and G-PIBP-PT into the pESC-HIS vector;

[0077] (III) The recombinant plasmids obtained by inserting PG-PIBP-PT and PPG-PIBP-PT into the pESC-URA vector.

[0078] (IV) The recombinant plasmids obtained by inserting CCL2, PKS2, PIBP-GT, G-PIBP-PT, PG-PIBP-PT, and PPG-PIBP-PT into the pEAQ-HT-DEST1 vector were obtained.

[0079] In a preferred embodiment of the present invention, the recombinant bacteria is as follows (V) or (VI):

[0080] (V) The recombinant bacteria obtained by introducing the recombinant plasmids described in (I)(II)(III)(IV) above into Escherichia coli;

[0081] (VI) The recombinant bacteria obtained by introducing the recombinant plasmids described in (I), (II) and (III) above into yeast.

[0082] (VII) The recombinant bacteria obtained by introducing the recombinant plasmid described in (IV) above into Agrobacterium.

[0083] In a preferred embodiment of the present invention, the yeast may be brewer's yeast, specifically yeast strain DD104.

[0084] In a preferred embodiment of the present invention, the Agrobacterium may specifically be Agrobacterium GV3101.

[0085] The polypeptide (enzyme) of the present invention can be expressed or produced using conventional recombinant DNA technology. Generally, the steps are as follows: (1) transforming or transducing a suitable host cell with a polynucleotide (or variant) encoding the polypeptide, or with an expression vector containing the polynucleotide; (2) culturing the host cell in a suitable culture medium; and (3) isolating and purifying the protein from the culture medium or the cell.

[0086] Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. The recombinant peptides used in the methods described above can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If desired, the recombinant proteins can be isolated and purified using various separation methods based on their physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitants (salting out), centrifugation, permeation, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques, as well as combinations of these methods.

[0087] After obtaining the fermentation product, hypericin or its intermediates can be extracted from the fermentation product or the Agrobacterium-infected plant using techniques known in this invention. For example, high-performance liquid chromatography can be used to analyze and identify the product to confirm that the desired compound has been obtained.

[0088] Preparation methods and applications

[0089] The structural formulas of the hypericin intermediate are shown in formulas (I), (II), and (III) below, and the structural formula of hypericin is shown in formula (IV) below.

[0090]

[0091] This invention discloses a method for heterologous preparation of hypericin or its intermediates using microorganisms and plants. The method comprises: (1) providing an expression system (such as host cells or plants) in which an enzyme or enzyme expression cassette selected from the group consisting of: (i) a short-chain fatty acid CoA ligase: CCL2 (preferably HpCCL2, NCBI number: OM160649); (ii) a polyketide synthase: PKS2 (preferably HpPKS2, NCBI number: OM160656); (iii) an isopentenyl transferase, including the group consisting of: PIBP-GT, G-PIBP-PT, PG-PIBP-PT, PPG-PIBP-PT or combinations thereof; and (2) culturing the host cells or plants of (1) to react, thereby preparing hypericin or its intermediates.

[0092] In a preferred embodiment, in step (1), enzymes or enzyme expression cassettes of (i), (ii), and (iii) are introduced into the host cell or plant; thereby, in step (2), isobutyryl coenzyme A is formed by enzyme (i) using endogenous valine in the host cell or plant as a substrate, then Phlorisobutyrophenone is formed by enzyme (ii), then G-Phlorisobutyrophenone is formed by PIBP-GT enzyme (iii), then PG-Phlorisobutyrophenone is formed by G-PIBP-PT enzyme (iv), then PPG-Phlorisobutyrophenone is formed by PG-PIBP-PT enzyme (iv), and finally Hypericin is formed by PPG-PIBP-PT enzyme (iv).

[0093] The isopentenyltransferases provided by this invention, namely PIBP-GT, G-PIBP-PT, PG-PIBP-PT, and PPG-PIBP-PT proteins, are key factors essential for the synthesis pathway of hypericin. Hypericin and its intermediates can be produced by co-expressing short-chain fatty acid CoA ligase, polyketide synthase, and isopentenyltransferase genes in yeast and tobacco.

[0094] This invention has broad application prospects in the pharmaceutical, food, and cosmetic industries of hypericin.

[0095] Reagent test kit

[0096] The present invention also provides a kit for preparing hypericin or intermediates thereof.

[0097] In one embodiment, the kit may include the encoding genes of the following enzymes: CCL2, PKS2, PIBP-GT, G-PIBP-PT, PG-PIBP-PT, and PPG-PIBP-PT.

[0098] In one embodiment, the kit may include isolated polypeptides (enzymes), including the various functional enzymes of the present invention.

[0099] In one embodiment, the kit may include: the host cells or plants described above for producing hypericin according to the present invention.

[0100] As a preferred embodiment of the present invention, the kit also includes other reagents required for cell culture or plant growth.

[0101] As a preferred embodiment of the present invention, the kit also includes an instruction manual describing the method for performing biosynthesis.

[0102] The main advantages of this invention are:

[0103] This invention marks the first time that microorganisms and plants have achieved de novo synthesis of hypericin or its intermediates using basic carbon sources. The recombinant host cells constructed in this invention exhibit good compatibility with plant-derived enzymes.

[0104] This invention utilizes recombinant host cells and plant heterologous production of hypericin or its intermediates, which not only solves the problems of plant extraction methods such as consuming large amounts of plant raw materials, being limited by seasonal and regional factors, and having low extraction efficiency, but also minimizes the adverse factors of chemical synthesis methods such as numerous by-products, low activity of the target product, and significant environmental pollution.

[0105] This invention represents a technological breakthrough in the whole-cell de novo biosynthesis of hypericin or its intermediates from tobacco infected with recombinant yeast and Agrobacterium, without the need for additional amino acid precursors; the basic carbon source is sufficient to meet production requirements. This invention opens up a new pathway for the industrial production of hypericin or its intermediates.

[0106] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed according to conventional conditions such as those described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Edition, or according to the manufacturer's recommendations.

[0107] Materials and methods

[0108] In the embodiments, the gene nucleic acid sequences used are shown in Table 1.

[0109] Table 1

[0110]

[0111]

[0112]

[0113] In the examples, the protein sequences of the enzymes used are shown in Table 2.

[0114] Table 2

[0115]

[0116] In subsequent embodiments, the primers required for constructing the recombinant expression plasmid are shown in Table 3.

[0117] Table 3

[0118]

[0119]

[0120] In subsequent embodiments, the backbone plasmids on which the recombinant plasmids are constructed are as follows:

[0121] The pESC-URA vector, pESC-HIS vector, and pESC-LEU vector were all purchased from Agilent Technologies.

[0122] The pEAQ-HT-DEST1 dual carrier was obtained from PBL in the UK via the MTA protocol.

[0123] Example 1: Chemical Synthesis of Standards

[0124] See synthesis route diagram Figure 1 .

[0125] Phlorisobutyrophenone (2.55 mmol) and geraniol (2.55 mmol) were dissolved in anhydrous dichloromethane (5 mL), and then BF3·Et2O (0.25 mmol) was added at 35 °C. The reaction mixture was stirred at the same temperature for 1 hour. After the reaction, the mixture was quenched with saturated sodium bicarbonate solution (10 mL) and extracted with dichloromethane (3 × 20 mL). The dried organic phase (sodium persulfate) was then purified by silica gel column chromatography with ethyl acetate-hexane to give G-PIBP.

[0126] G-PIBP (0.2 mmol) was dissolved in water (2 mL) and tetrahydrofuran (1 mL) and treated at 0 °C under a nitrogen stream. Potassium hydroxide (0.6 mmol) was added, followed by isopentenyl bromide (0.8 mmol). After stirring at 0 °C for 1 hour, the reaction was quenched with saturated sodium bicarbonate solution (10 mL) and extracted with dichloromethane (3 × 10 mL). The combined organic phases were dried over sodium sulfate and purified by silica gel column chromatography with ethyl acetate and hexane to obtain PG-PIBP and PPG-PIBP.

[0127] Example 2: Construction and production of hypericin-producing strain in yeast

[0128] I. Construction of Recombinant Expression Vectors

[0129] Primers were designed (Table 3), and the relevant genes were synthesized by polymerase chain reaction (PCR) amplification using Hypericum perforatum genomic DNA as a template.

[0130] The sequences of the nucleic acids and proteins encoded by the genes are shown in Tables 1 and 2.

[0131] The amplified gene sequence was recombinated with pESC-HIS, pESC-URA, and pESC-LEU vectors using homologous recombinase (ClonExpress II One Step Cloning kit, Novizan) to obtain plasmids:

[0132] pESC-HIS-HpCCL2-HpPKS2;

[0133] pESC-URA-PIBP-GT;

[0134] pESC-URA-PIBP-GT-G-PIBP-PT;

[0135] pESC-LEU-PG-PIBP-PT;

[0136] pESC-LEU-PG-PIBP-PT-PPG-PIBP-PT.

[0137] II. Construction of Recombinant Bacteria

[0138] The constructed plasmids pESC-HIS-HpCCL2-HpPKS2, pESC-URA-PIBP-GT, pESC-URA-PIBP-GT-G-PIBP-PT, pESC-LEU-PG-PIBP-PT, and pESC-LEU-PG-PIBP-PT-PPG-PIBP-PT were selected and transformed into Saccharomyces cerevisiae DD104 strain (obtained from Wang Guodong's laboratory at the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences) using the lithium acetate method. The plasmid combinations introduced into the strain are shown in Table 4.

[0139] Table 4

[0140]

[0141]

[0142] III. Production Analysis of Recombinant Microorganisms

[0143] Three days after transformation, colonies were grown for two days in 5 mL of yeast auxotrophic medium (His, Leu, and Ura excluding 2% (w / v) glucose). Cells were then harvested and resuspended in 15 mL of yeast auxotrophic medium (His, Leu, and Ura excluding 2% (w / v) d-galactose) and cultured for another three days. After induction, the culture was extracted with an equal volume of ethyl acetate (EtOAc). The resulting organic layer was separated and dried under reduced pressure. The dried extract was then resuspended in 400 μL of methanol and filtered using a syringe filter with a 0.22-micron pore size. In this production, endogenous valine was used as the substrate.

[0144] Metabolites were analyzed using the Q Exactive system in conjunction with a Dionex Ultimate 3000U HPLC system. Three microliters (3 μL) of each treated sample were injected into a UPLC (Kinetex 2.6 μm C18) microscope. The dimensions are 100 x 2.1 mm (Phenomenex). This process was carried out at a flow rate of 0.35 mL per minute. Using a gradient of solvent A (2 mM NH4Ac) and solvent B (MeCN), 10% B was maintained from 0 to 0.5 minutes, and then B was linearly increased from 10% to 98% between 0.5 and 11 minutes. From 11 to 13 minutes, it was maintained at 98% B.

[0145] Mass spectrometry acquisition was performed in positive mode with the following parameters: spray voltage 3500V, capillary temperature 320℃, shear gas 40 and auxiliary gas 10.

[0146] The products obtained from each combination were compared with standards of hypericin and its intermediates. The results are as follows: Figure 2 As shown in Table 5, the desired intermediates and products can be obtained.

[0147] Table 5

[0148]

[0149] Example 3: Biosynthesis of Hypericin in Tobacco

[0150] I. Construction of Recombinant Expression Vectors

[0151] Primers were designed (Table 3), and the recombinant plasmid from Example 2 was used as a template. The synthesis-related gene was amplified by polymerase chain reaction (PCR) and inserted into pEAQ-HT-DEST1 using the Golden Gate Cloning method.

[0152] pEAQ-HT-DEST1-HpCCL2;

[0153] pEAQ-HT-DEST1-HpPKS2;

[0154] pEAQ-HT-DEST1-PIBP-GT;

[0155] pEAQ-HT-DEST1-G-PIBP-PT;

[0156] pEAQ-HT-DEST1-PG-PIBP-PT;

[0157] pEAQ-HT-DEST1-PPG-PIBP-PT.

[0158] II. Construction of Recombinant Bacteria

[0159] The constructed plasmids were selected and transformed into Agrobacterium GV3101 strain using liquid nitrogen quick-freezing and heating method.

[0160] III. Verification of the function of isopentenyltransferase by injecting recombinant bacteria into tobacco.

[0161] For transient expression of Agrobacterium-mediated gene in tobacco, PCR-validated Agrobacterium colonies were cultured overnight in 15 mL LB medium with relevant selection criteria.

[0162] After centrifugation, the cells were resuspended in MMA buffer (containing 10 mM MgCl2, 10 mM MES and 150 μM acetylsylcholine) and the concentration was adjusted to OD600 nm = 0.2. The cells were then incubated in the dark for 2 hours.

[0163] Leaves of tobacco plants aged 4-6 weeks were injected with a mixed bacterial strain. The mixed bacterial strain consisted of Agrobacterium bacteria: pEAQ-HT-DEST1-HpCCL2, pEAQ-HT-DEST1-HpPKS2, pEAQ-HT-DEST1-PIBP-GT, pEAQ-HT-DEST1-G-PIBP-PT, pEAQ-HT-DEST1-PG-PIBP-PT, and pEAQ-HT-DEST1-PPG-PIBP-PT.

[0164] Five days later, the leaves were collected, rapidly frozen, and stored at -80°C. The frozen leaves were then freeze-dried and ground into a fine powder, which was subsequently extracted in 10 mL of methanol. The extract was concentrated under vacuum, resuspended in 500 μL of methanol, filtered through a 0.22 μm organic injection filter, and stored at -20°C.

[0165] Metabolite analysis was performed using the same liquid chromatography-mass spectrometry method as in Example 2. The obtained products were compared with hyperoside standards. In this reaction, valine, an endogenous substance from tobacco, was used as the substrate.

[0166] The results are as follows Figure 3 This shows that hypericin can be biosynthesized in tobacco.

[0167] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A method for preparing hypericin or its intermediates, characterized in that, The method includes: (1) Provide an expression system in which the enzymes or enzyme expression cassettes of the following group are introduced: (i) Short-chain fatty acid CoA ligase: CCL2; (ii) Polyketide synthase: PKS2; (iii) Isopentenyltransferases, including those selected from the group consisting of PIBP-GT, G-PIBP-PT, PG-PIBP-PT, PPG-PIBP-PT or combinations thereof; (2) Produce hypericin or its intermediates using the expression system of (1).

2. The method as described in claim 1, characterized in that, In step (1), enzymes or enzyme expression cassettes of (i), (ii) and (iii) are introduced into the expression system; thereby, in step (2), isobutyryl coenzyme A is formed by enzyme of (i), Phloroisobutyrophenone intermediate is formed by enzyme of (ii), G-PIBP is formed by PIBP-GT enzyme of (iii), PG-PIBP is formed by G-PIBP-PT enzyme of (iv), PPG-PIBP is formed by PG-PIBP-PT enzyme of (iv), and Hypericin is formed by PPG-PIBP-PT enzyme of (iv).

3. The method as described in claim 2, characterized in that, The PIBP-GT is a nucleic acid sequence as shown in SEQ ID NO:1, or a conserved variant thereof; The G-PIBP-PT is a nucleic acid sequence as shown in SEQ ID NO:2, or a conserved variant thereof; The PG-PIBP-PT is a nucleic acid sequence as shown in SEQ ID NO:3, or a conserved variant thereof; The PPG-PIBP-PT is a nucleic acid sequence as shown in SEQ ID NO:4, or a conserved variant thereof; The PIBP-GT is a polypeptide with an amino acid sequence as shown in SEQ ID NO:5, or a conserved variant thereof; The G-PIBP-PT is a polypeptide with an amino acid sequence as shown in SEQ ID NO:6, or a conserved variant thereof; The PG-PIBP-PT is a polypeptide with an amino acid sequence as shown in SEQ ID NO:7, or a conserved variant thereof; The PPG-PIBP-PT is a polypeptide with an amino acid sequence as shown in SEQ ID NO:8, or a conserved variant thereof.

4. The method according to any one of claims 1-3, characterized in that, The host cell includes eukaryotic cells or prokaryotic cells; preferably, the eukaryotic cell includes yeast, plant cells, fungal cells, insect cells, or mammalian cells, or the prokaryotic cell includes *Escherichia coli*, *Bacillus subtilis*, or *Streptomyces*; more preferably, the host cell is yeast; even more preferably, the yeast is *Saccharomyces cerevisiae*; or The plant includes angiosperms or gymnosperms; preferably, the plant is a dicotyledonous plant or a monocotyledonous plant; more preferably, the plant includes plants of the Solanaceae family; even more preferably, the Solanaceae family includes tobacco.

5. An expression system for producing hypericin, characterized in that, This includes the following groups of enzymes or enzyme expression cassettes: (i) Short-chain fatty acid CoA ligase: CCL2; (ii) Polyketide synthase: PKS2; (iii) Isopentenyltransferases, including those selected from the group consisting of PIBP-GT, G-PIBP-PT, PG-PIBP-PT, PPG-PIBP-PT or combinations thereof.

6. A kit for preparing hypericin or its intermediates, characterized in that, These include: The expression system for producing hypericin as described in claim 5; Preferably, it also includes materials selected from: a culture medium for the host cells, a culture medium for the plant, and an instruction manual explaining the preparation method.

7. Application of PIBP-GT as an isopentenyltransferase; preferably, for catalyzing the formation of hyperoside intermediate Phloroisobutyrophenone from hyperoside intermediate G-PIBP.

8. Application of G-PIBP-PT as an isopentenyltransferase; preferably, for catalyzing the formation of hypericin intermediate PG-PIBP from TranslucinI.

9. Application of PG-PIBP-PT as an isopentenyltransferase; preferably, for catalyzing the formation of hypericin intermediate PPG-PIBP from TranslucinII.

10. The application of PPG-PIBP-PT as an isopentenyltransferase; preferably, for catalyzing the formation of hypericin from the hypericin intermediate TranslucinIII.

11. The application of combinations of PIBP-GT, G-PIBP-PT, PG-PIBP-PT and PPG-PIBP-PT for the production of hypericin.

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