Method for increasing production of oxidosqualene, triterpenes, and / or triterpenoids, and host cells therefor

Abstract

To provide: a method of increasing the yield of at least one of oxidosqualene, triterpenes and / or triterpenoids in a modified host cell; each host cell; and use of the host cell for manufacturing the at least one of oxidosqualene, triterpenes and / or triterpenoids.SOLUTION: A method of increasing the yield comprises: modifying a host cell to overexpress a 3-hydroxy-3-methylglutaryl-coenzyme A reductase as well as to overexpress a protein capable of producing at least one of acetoacetyl-CoA, 3-hydroxy-3-methylglutaryl-CoA, mevalonate-5-phosphate, mevalonate-5-pyrophosphate, isopentenyl-5-pyrophosphate, farnesyl-pyrophosphate or dimethylallyl-pyrophosphate; and culturing the same.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This application contains a sequence listing in computer readable form, which is incorporated herein by reference. It is cited.

[0002] The present invention is in the field of recombinant biotechnology, and in particular in the field of metabolic engineering. The invention generally relates to methods for producing oxidosqualene, triterpenes, and / or triterpenes in a host cell. A method for increasing the production of at least one terpenoid and a method for purifying the same. The present invention also provides oxidosqualene, triterpene, and / or triterpenoid. and improving the ability of a host cell to produce at least one of oxidosqualene, trimethylsilane, and hydroxybenzoates. and / or triterpenoids. The present invention also provides oxidosqualene, triterpene, and / or triterpenoid. The present invention relates to the use of a host cell for producing at least one of the [Background technology]

[0003] Five-membered triterpenes are a group of plant secondary metabolites derived from the mevalonate (MVA) pathway. This shows great potential in economics and medicine. Because it is desirable to produce these substances in seed systems, recent studies have focused on the use of such systems. Research and designs have been proposed to address this issue.

[0004] Isoprenoids are the largest group of naturally occurring compounds found in all living organisms and are currently The variety of structures known to date represents at least 50,000 different structures (Hemmerlin et al., 201 2; Liao et al., 2016). These structures are mainly composed of acetyl-CoA as a pre-isoprenoid. The highly regulated mevalonate pathway (MVA pathway) converts it into the precursor isopentenyl diphosphate (IPP). It is derived from acetyl-CoA. Six enzymes are required to convert acetyl-CoA to IPP, but 3- Hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) is known as the rate-limiting step of the pathway. (Demierre et al., 2005). After isomerization to dimethylallyl pyrophosphate (DMAPP), This allows IPP and DMAPP to enter into another isoprenoid pathway. After the reaction of a P molecule with one DMAPP molecule, farnesyl pyrophosphate (FPP) is generated. , which is converted to squalene via squalene synthase. Squalene is then converted to its oxidized form 2 In 3-oxidosqualene, sterol precursors (e.g., in fungi and animals) lanosterol in plants, or cycloartenol in plants), or Taraxacum officinalis cinale lupeol synthase or Artemisia annua β-amyrin synthase It has been used to synthesize five-membered triterpenes via various oxidosqualene cyclases. (Shibuya et al., 1999; Kirby et al., 2008). Furthermore, other FPP modifications have been shown to Lunesene, or amorpha-4,11-diamine, a precursor to the antimalarial drug artemisinin This results in the synthesis of sesquiterpenes such as ene (Martin et al., 2003).

[0005] Due to its economic and medicinal potential, much research has been done on yeasts such as Saccharomyces cerevisiae. The production of isoprenoids in heterologous systems has been addressed (Liao et al., 2016 and Vickers et al., 2017). (Reviewed in I., 2017). In 1997, the feasibility of the yeast MVA pathway for isoprenoid production was investigated. Starting with a closer look at the catalytic mechanism of HMGR in yeast, Donald et al. Overexpression of the MVA pathway resulted in increased production of the triterpene squalene. Further reports on deregulation show that when knocked out, mevalonate and trimethoprim inhibit the This was done in a CRISPR / Cas9 experiment to provide a series of loci that induce terpene accumulation. (Jakociunas et al., 2015; Ahrend et al., 2017). This series of events is related to the MVA pathway. and the transcriptional regulator ROX1, which is reported to repress genes in sterol biosynthesis. (Henry et al., 2002; Montanes et al., 2011; Ozaydin et al., 2013; Jakociunas et al., 2015). Furthermore, some studies have shown that endogenous but competitive isoprenaline Genes involved in the steroid pathway (e.g., sterol biosynthesis vs. five-membered triterpene biosynthesis) To regulate the metabolic flux in a precise and desired way, specific promoters reported the insertion of the vector into the yeast genome.

[0006] Therefore, Kirby et al. catalyzed the first step of late sterol biosynthesis to produce β-amyrin. Methionine-induced downregulation of the expression of yeast lanosterol synthase (ERG7) The MET3 promoter was used in the yeast strain. It was used for artemisinin production due to the suppression of the qualene synthase gene (ERG9) (Ro et al. l., 2006; Westfall et al., 2012).

[0007] Patent Document 1 discloses a yeast strain and a five-membered ring triterpene and / or triterpene in the yeast. In particular, the modified yeast strains are capable of producing oxidosinoids. At least one copy of the gene encoding qualene cyclase, NADPH-cytochrome c at least one copy of a gene encoding a P450 reductase and / or a cytochrome P450 reductase five-membered nucleotide sequence containing at least one copy of a gene encoding a P450 monooxygenase for the production of cyclic triterpenoids.

[0008] Patent Document 2 discloses mutant strains having one or two defects in the expression of sterol biosynthesis enzymes. Constructs encoding polypeptides with HMG-CoA reductase activity in mutant yeast and increasing the expression levels of squalene and specific steroid hormones in yeast. A method for increasing roll accumulation is described.

[0009] Five-membered triterpenes and triterpenoids have great potential for industrial and pharmaceutical applications. However, the total amount in the plant is small, Biotechnological production in heterologous hosts can be hindered by the low efficiency of the corresponding enzymes or the lack of post-translational modifications. extraction is often economically unfeasible due to several constraints It is known (Moses and Pollier, 2013; Arendt et al., 2016).

[0010] Therefore, oxidosqualene, trisqualene, and squalene obtained by organic extraction from plants and subsequent purification are The production and extraction of terpenes and / or triterpenoids is possible, but this is currently Since the concentration and composition of triterpenes in plants can vary widely, This leads to high costs to obtain the final product and a large variability in the quality of the final product. are. [Prior art documents] [Patent documents]

[0011] [Patent Document 1] US Patent Application Publication No. 2017 / 0130233 [Patent Document 2] U.S. Patent No. 5,460,948 Summary of the Invention [Problem to be solved by the invention]

[0012] In this way, it is possible to reduce costs and ensure the production of high amounts of oxidosqualene, trimethylsilyl methyl ... There is a need for methods for obtaining triterpenes and / or triterpenoids. [Means for solving the problem]

[0013] In the present invention, the inventors have developed a cost-effective, reliable, and highly pure oxidosqualene. Oxidosqualene, which allows for the production of benzoquinones, triterpenes, and / or triterpenoids A novel heterologous platform for the production of benzophenones, triterpenes, and / or triterpenoids. Designed the form.

[0014] The present invention relates to a method for producing oxidosqualene, triterpenes, and / or triterpenes in a host cell. The present invention provides a method for increasing the production of at least one penoid, the method comprising: 3-hydroxy-3-methylglutaryl coenzyme A containing the amino acid sequence shown in SEQ ID NO: 1 overexpressing the reductase or a gene having at least 44% sequence identity with the amino acid sequence shown in SEQ ID NO: 1; Overexpression of a protein with sequence identity that is capable of producing mevalonate So that, and, At least one amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, and 8 or selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, and 8. At least one amino acid sequence having at least 44% sequence identity with the amino acid sequence A protein containing a sequence containing acetoacetyl-CoA, 3-hydroxy-3-methylglucose Glutaryl-CoA, mevalonate-5-phosphate, mevalonate-5-pyrophosphate phosphate, isopentenyl-5-pyrophosphate, farnesyl-pyrophosphate, or a protein capable of generating at least one of dimethylallyl-pyrophosphate. modifying the host cell to overexpress the gene; At least one of oxidosqualene, triterpene, and / or triterpenoid modifying the host cell to express at least one heterologous protein to be produced; At least one of oxidosqualene, triterpene, and / or triterpenoid culturing said host cells under appropriate conditions to allow expression; and at least one of oxidosqualene, triterpene, and / or triterpenoid. By refining one more Compared to the host cell before modification, oxidosqualene, triterpene, and / or triterpene The present invention also includes increasing the production of at least one of the penoids.

[0015] In a preferred embodiment, in the method of the present invention, the host cell is or an amino acid sequence having at least 44% sequence identity with SEQ ID NO:3. A protein containing a 3-hydroxy-3-methylglutaryl-CoA sequence capable of producing 3-hydroxy-3-methylglutaryl-CoA. The cells are engineered to overexpress proteins that are capable of expressing the protein.

[0016] In a preferred embodiment, the method of the present invention comprises the steps of: S1 (SEQ ID NO: 54), YPL062W (SEQ ID NO: 55), DOS2 (SEQ ID NO: 56), YER134C (SEQ ID NO: 57), VBA5 (SEQ ID NO: 58), YNR063W (SEQ ID NO: No. 59), YJL064W (SEQ ID NO: 60), and YGR259C (SEQ ID NO: 61) and modifying a host cell to knock out at least one locus selected from the group consisting of: It further includes:

[0017] In a further embodiment, the method of the present invention comprises the step of: Inhibits lanosterol synthase (ERG7) containing SEQ ID NO: 9 or at least 34% A protein containing an amino acid sequence having sequence identity and capable of producing lanosterol. It further includes modifying the host cell to inhibit a certain protein. The repression was achieved by inserting the CTR3 promoter and / or by adding copper sulfate, CuSO4. It will be held.

[0018] In this preferred embodiment, the amount of copper sulfate added is at least 10, 20, 30, 4 0, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150 , 160, 170, 180, 190, 200, 210, 220, 230, 240, 250 , 260, 270, 280, 290, 300, 310, 320, 330, 340, 350 , 360, 370, or 375 mM CuSO4, preferably at least 150 mM C It may also be uSO4.

[0019] In the method of the present invention, oxidosqualene, triterpene, and / or triterpenoid At least one heterologous protein that produces at least one of the amides is lupeol synthase. enzymes, preferably lupeol synthase and oxidosqualene cyclase from Taraxacum koksaghyz Oxidosqualene cyclase (OSC), preferably Taraxacum koksaghyz oxidosqualene cyclase TkOSC1- 6. β-amyrin synthase, preferably β-amyrin synthase from Arabidopsis thaliana or Artemisia annua - amylin synthase, terpene cyclase, preferably terpenes from Glycyrrhiza uralensis The enzyme can be selected from the group consisting of cyclases (GuLUP1).

[0020] In the method of the present invention, oxidosqualene, triterpene, and / or triterpenoid At least one of the ides is extracted from a plant.

[0021] In a further embodiment of the method of the present invention, oxidosqualene, triterpenes, and and / or at least one purification of the triterpenoids by at least two chromatographic methods. Preferably, a C18 column is used for the first chromatography step, and a C18 column for the second chromatography step. The two chromatography steps are carried out using a biphenyl column.

[0022] In one embodiment of the method of the present invention, two or more of oxidosqualene, triterpene, and / or the production of triterpenoids can be increased.

[0023] The present invention relates to a method for producing a compound comprising at least one of oxidosqualene, triterpene, and / or triterpenoid. Further provided is a recombinant host cell for producing one of the above-mentioned polypeptides, the host cell comprising a polypeptide having a sequence similar to that of SEQ ID NO: 1. 3-hydroxy-3-methylglutaryl coenzyme A reductase containing the amino acid sequence shown or having at least 44% sequence identity with the amino acid sequence shown in SEQ ID NO: 1. The protein capable of producing mevalonate was then overexpressed. and at least one selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, and 8. or from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, and 8. At least one amino acid sequence having at least 44% sequence identity with the selected amino acid sequence. A protein containing the amino acid sequence acetoacetyl-CoA, 3-hydroxy-3-methyl- Glutaryl-CoA, mevalonate-5-phosphate, mevalonate-5-pyrophosphate phosphate, isopentenyl-5-pyrophosphate, farnesyl-pyrophosphate or dimethylallyl-pyrophosphate. The cells are engineered to overexpress the protein.

[0024] In the present invention, the host cell is selected from the group consisting of Saccharomyces cerevisiae, Nicotiana benthamiana, P. ichia pastoris, Pichia methanolica, Schizosaccharomyces pombe, Kluyveromyces lac tis, Kluyveromyces marxianus, Pichia stipitis, Candida albicans, Candida utilis and BY2 cells.

[0025] The host cell comprises a gene encoding a gene encoding a polypeptide having at least 44% sequence identity with SEQ ID NO:3. a protein comprising an amino acid sequence having 3-hydroxy-3-methylglutaryl- It can be further modified to overexpress proteins capable of producing CoA. .

[0026] The host cells of the present invention preferably contain a CTR3 promoter insertion and / or copper sulfate, Cu The addition of SO4 resulted in the production of lanosterol synthase (E) comprising the amino acid sequence shown in SEQ ID NO:9. RG7) or an amino acid sequence having at least 34% sequence identity with SEQ ID NO:9. The gene is further modified to inhibit the production of proteins capable of producing lanosterol. Preferably, the lanosterol synthase (ERG7) that is inhibited in the present invention is are Saccharomyces cerevisiae, Pichia pastoris, Pichia methanolica, Schizosacchar omyces pombe, Kluyveromyces lactis, Kluyveromyces marxianus, Pichia stipitis, Ca Candida albicans or Candida utilis.

[0027] The host cell is a recombinant vector encoding ROX1 (SEQ ID NO: 25), BTS1 (SEQ ID NO: 54), YPL062W (SEQ ID NO: 55), DOS2 (SEQ ID NO: 56), YER134C (SEQ ID NO: 57), VB A5 (SEQ ID NO: 58), YNR063W (SEQ ID NO: 59), YJL064W (SEQ ID NO: 6 0), and at least one selected from the group consisting of YGR259C (SEQ ID NO: 61). Preferably, it is further modified to knock out the locus.

[0028] At least one of oxidosqualene, triterpene, and / or triterpenoid , oxidosqualene, preferably 2,3-oxidosqualene, sterols, preferably is sigmasterol or sitosterol, a triterpene, preferably a five-membered ring triterpene and more preferably, but not limited to, lup-19(21)-en-3-ol and lup- Lupeol, β-amyrin, α-amyrin, taraki, etc., such as 20(29)-en-3-ol Susterol, triterpene acetate, acylated triterpenes, saponins, sapogenins , lup-19(21)-en-3-one, lup-20(29)-en-3-one , Taraxerol, Taraxerone, α-amyron, β-amyron, Taraxasterone, Leadin, betulin, betulinic acid, cholesterol, ergosterol, lanosterol Glucocorticoids, mineralocorticoids, estrogens, progesterone, and steroids Nolides, bufadienolides, steroid alkaloids, saponins, saponins Genin or an acylated triterpene is preferred for the host cell of the present invention.

[0029] The present invention also provides a method for producing oxidosqualene, triterpenes, and / or triterpenoids. The present invention resides in the use of a host cell of the present invention for producing at least one

[0030] Thus, the inventors have demonstrated that overexpression of mevalonate pathway genes and / or proteins Furthermore, in one embodiment, the method of the present invention comprises: , including knockout of negative regulators and knockdown of competing pathways of the mevalonate pathway . [Brief explanation of the drawings]

[0031] [Figure 1] FIG. 1 shows the amino acid sequence of 3-hydroxy-3-methylglutaryl coenzyme A reductase having the amino acid sequence shown in SEQ ID NO:1. [Figure 2] FIG. 2 shows a schematic representation of the Perg7::PCTR3-construct (SEQ ID NO: 10) and the rox1::PGal1-tHMGR;PGAL 19-ERG13-construct (SEQ ID NO: 11) used in one embodiment of the present invention. [Figure 3] FIG. 3 shows the nucleotide sequence of the Perg7::PCTR3-construct (SEQ ID NO: 10). [Figure 4] FIG. 4 shows the nucleotide sequence of the rox1::PGal1-tHMGR;PGAL10-ERG13 construct (SEQ ID NO: 11). [Figure 5] FIG. 5 shows the nucleotide sequence (SEQ ID NO: 12) of the Taraxacum koksaghyz enzyme LUP (TkLUP). [Figure 6]Figure 6 shows triterpene accumulation in TkLUP-expressing yeast. Figure 6a shows a schematic diagram of the TkLUP coding sequence (SEQ ID NO: 12) under the control of the GAL1 promoter (GAL1-P) and CYC1 terminator (CYC1-T). Figure 6b shows two additional peaks in the GC-MS spectrum of yeast cells harboring the TkLUP coding sequence (SEQ ID NO: 12) (arrows; m / z = 218), which likely represent β-amyrin (retention time 17.95 min) and lupeol (retention time 18.25 min), since they have the same retention time as the corresponding standards. Figure 6c shows that yeast harboring the TkLUP coding sequence (SEQ ID NO: 12) accumulated an estimated lupeol concentration of 0.16 mg / g CDW, while the β-amyrin peak could not be quantified. Untransformed (WT) and pAG424GAL1-ccdb-transformed (vector control) CEN.PK2-1C cells served as controls. Standard deviation was calculated from n=3 individual transformants. CDW=cell dry weight. [Figure 7]Figure 7 shows the accumulation of squalene and lupeol after deletion of ROX1 (SEQ ID NO:25) and overexpression of MVA pathway genes. Figure 7a shows a schematic diagram of the construct for deletion of ROX1 (SEQ ID NO:25) and overexpression of tHMGR (SEQ ID NO:32) and ERG13 (SEQ ID NO:14). The coding sequences for tHMGR and ERG13 were cloned under the control of the GAL1 / GAL10 bidirectional promoter (GAL1-P; GAL10-P). KlUra3 was used to complement uracil auxotrophy upon integration of the construct into the yeast genome. The integration locus was defined by sequences flanking the construct (target up, target down) and homologous to the genomic target site (ROX1, SEQ ID NO:25). Transformation of the NotI-linearized construct resulted in knockout of ROX1 (SEQ ID NO:25) by homologous recombination. Figures 7b and 7c show that a yeast strain containing the integrated construct (rox1::PGAL1-tHMGR PGAL10-ERG13 TkLUP) in addition to the TkLUP (SEQ ID NO: 12) coding sequence exhibited increased accumulation of the lupeol precursor squalene, in contrast to yeast containing only the TkLUP-containing plasmid (TkLUP; p = 0.0137). Cells containing the empty vector pAG424GALl_ccdB served as a control (rox1::PGAL1-tHMGR PGAL10-ERG13 vector control). Furthermore, deletion of ROX1 (SEQ ID NO: 25) and overexpression of tHMGR (SEQ ID NO: 32) and ERG13 (SEQ ID NO: 14) resulted in a 16.5-fold increase in lupeol accumulation (p = 0.00893). Figure 7d shows the MS spectrum of the identified lupeol peak, and Figure 7e shows the MS spectrum of the measured lupeol external standard. Standard deviations were calculated from n=3 individual transformants. CDW=cell dry weight, *=p≦0.05, **=p≦0.01. [Figure 8]Figure 8 shows that repression of ERG7 (SEQ ID NO: 20) results in 2,3-oxidosqualene accumulation. Figure 8a is a schematic diagram of a construct for integration of the copper-sensitive CTR3 promoter (CTR3-P) (SEQ ID NO: 42). To introduce the promoter into the yeast genome, leucine auxotrophy was complemented with the KlLeu2 gene. To target the CTR3 promoter (SEQ ID NO: 42) in front of the endogenous ERG7 coding sequence (SEQ ID NO: 20) for repression of sterol biosynthesis, the construct was flanked by sequences homologous to the ERG7 promoter (target up; ERG7-P) and the ERG7 coding sequence (target down; ERG7, SEQ ID NO: 42). Figures 8b and 8c show that yeast strains harboring the CTR3 promoter construct (SEQ ID NO:42) exhibited reduced squalene levels (Figure 8b) and accumulated 2,3-oxidosqualene (Figure 8c) without exposure to CuSO (rox1::PGAL1-tHMGR PGAL10-ERG13 PERG7Δ::PCTR3, 0 μM CuSO). This effect could be significantly enhanced by adding 150 μM CuSO to the growth medium (rox1::PGAL1-tHMGR PGAL10-ERG13 PERG7Δ::PCTR3, 150 μM CuSO; p = 0.00613 for squalene reduction; p = 0.00507 for 2,3-oxidosqualene accumulation). Addition of 2.5-fold higher copper concentration further significantly reduced squalene levels and further increased 2,3-oxidosqualene content (rox1::PGAL1-tHMGR PGAL10-ERG13 PERG7Δ::PCTR3, 375 μM CuSO). Standard deviations were calculated from n=3 individual transformants. CDW = cell dry weight; *=p≦0.05, **=p≦0.001, ***=p≦0.001. [Figure 9]Figure 9 shows that suppression of ERG7 (SEQ ID NO: 20) results in increased lupeol accumulation (Fig. 9e) and decreased sterol levels (Figs. 9c and 9d). Accumulation of 2,3-oxidosqualene (Fig. 9b) and decreased squalene (Fig. 9a) could be observed in the yeast strain harboring the rox1::PGAL1-tHMGR PGAL10-ERG13 and PERG7Δ::PCTR3 modifications plus the TkLUP sequence (SEQ ID NO: 12) after exposure to 150 μM CuSO4 during growth, compared to its parent strain (rox1::PGAL1-tHMGR PGAL10-ERG13 PERG7Δ::PCTR3 TkLUP; p = 0.04422 for squalene). As expected, sterol content was reduced, as indicated by the amounts of lanosterol and ergosterol, which are representative of sterol biosynthesis. Suppression of ERG7 (SEQ ID NO: 20) resulted in a 6.5-fold decrease in lanosterol (p = 0.02384) and a 3.9-fold decrease in ergosterol content (p = 0.00941). Suppression of sterol biosynthesis increased lupeol production by 7.6-fold (p = 0.00637), suggesting a shift in metabolic flux from sterol biosynthesis to lupeol production. Standard deviations were calculated from n = 3 individual transformants. CDW = cell dry weight; * = p ≤ 0.05; ** = p ≤ 0.001. [Figure 10] Figure 10 shows the identification of β-amyrin in yeast after suppression of ERG7 (SEQ ID NO: 20). Figure 10a shows the MS spectrum of the identified β-amyrin peak in the engineered yeast strain (rox1::PGAL1-tHMGR PGAL10-ERG13 Perg7Δ::PCTR3 TkLUP), and Figure 10b shows the MS spectrum of the measured β-amyrin external standard. [Figure 11] FIG. 11 shows a schematic excerpt of the mevalonate pathway. [Figure 12]Figure 12 shows triterpene purification by HPLC. Single triterpenes were separated using an Ultra C18 column (Figure 12a) followed by an Ultra Biphenyl column (Figure 12b). Figure 12c shows the MS spectra of β-amyrin and taraxasterol, and their ketone derivatives, purified from fractions 4 (F4) and 5 (F5), respectively (see Example 7). Figures 12c-12f show the molecular structures and MS spectra of the newly identified lup-19(21)-en-3-ol and its ketone derivative lup-19(21)-en-3-one, purified from F4 and F5, respectively (see Example 7). [Figure 13] FIG. 13 shows the cycloartenol synthase-like enzymes of Nicotiana tabacum (SEQ ID NO: 21, NP_001311688.1), Schizosaccharomyces pombe lanosterol synthase (SEQ ID NO: 63, AAA92502.1), Pichia pastoris lanosterol synthase (SEQ ID NO: 64, CCA38589.2), Kluyveromyces lactis lanosterol synthase (SEQ ID NO: 65, CAH02375.1), Kluyveromyces marxianus lanosterol synthase (SEQ ID NO: 66, BAP71121.1), Candida albicans SC5314 lanosterol synthase (SEQ ID NO: 67, XP_722612.2), and Pichia stipitis lanosterol synthase (oxidosqualene-lanosterol cyclase) (2,3-epoxysqualene-lanosterol cyclase) (OSC) (SEQ ID NO: 62, XP_001384446.2). The conditions for this alignment are further described below. [Figure 14] FIG. 14 shows the respective percent identity matrix generated from the alignment of FIG. DETAILED DESCRIPTION OF THE INVENTION

[0032] The present invention relates to a method for producing oxidosqualene, triterpenes, and / or triterpenes in a host cell. The present invention provides a method for increasing the production of at least one penoid, the method comprising: The amino acid sequence shown in SEQ ID NO: 1 can be used as a protein, provided that the protein is capable of producing mevalonate. overexpressing 3-hydroxy-3-methylglutaryl coenzyme A reductase containing the 3-hydroxy-3-methylglutaryl coenzyme A reductase sequence, or , a protein having at least 44% sequence identity with the amino acid sequence shown in SEQ ID NO: 1. To overexpress and, Proteins contain acetoacetyl-CoA and 3-hydroxy-3-methylglutaryl-CoA , mevalonate-5-phosphate, mevalonate-5-pyrophosphate, isopentene farnesyl-5-pyrophosphate, farnesyl-pyrophosphate, or dimethylallyl SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, , 4, 5, 6, 7, and 8. or an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, and 8. The protein contains at least one amino acid sequence having at least 44% sequence identity with the sequence. modifying the host cell to overexpress the protein; At least one of oxidosqualene, triterpene, and / or triterpenoid modifying the host cell to express at least one heterologous protein to be produced; At least one of oxidosqualene, triterpene, and / or triterpenoid culturing said host cells under appropriate conditions to allow expression; and at least one of oxidosqualene, triterpene, and / or triterpenoid. By refining one more Compared to the host cell before modification, oxidosqualene, triterpene, and / or triterpene The present invention also includes increasing the production of at least one of the penoids.

[0033] In the present invention, oxidosqualene, triterpene, and / or triterpenoid At least one of the oxidosqualenes is preferably 2,3-oxidosqualene, stearyl alcohol, or methyl squalene. sterols, preferably sigmasterol or sitosterol, triterpenes, preferably 5 19(21)-en-3-(1-methyl-2-phenyl-2-methyl-1,2-di ... lupeol, β-amyrin, α- Amyrin, taraxasterol, triterpene acetate, acylated triterpene, saponins nin, sapogenin, lup-19(21)-en-3-one, lup-20(29)- en-3-one, taraxerol, taraxerone, α-amyron, β-amyron, tara Xasterone, friedelin, betulin, betulinic acid, cholesterol, ergosterol Glucocorticoids, mineralocorticoids, estrogen, guanine, lanosterol Targen, cardenolides, bufadienolides, steroidal alkaloids , saponin, sapogenin, or acylated triterpene.

[0034] This means that in one embodiment, the present invention is directed to the production of at least one oxidoreductase in a host cell. A method for increasing the production of qualene, preferably 2,3-oxidosqualene, is provided, The method comprises: The amino acid sequence shown in SEQ ID NO: 1 can be used as a protein, provided that the protein is capable of producing mevalonate. overexpressing 3-hydroxy-3-methylglutaryl coenzyme A reductase containing the 3-hydroxy-3-methylglutaryl coenzyme A reductase sequence, or , a protein having at least 44% sequence identity with the amino acid sequence shown in SEQ ID NO: 1. To overexpress and, Proteins contain acetoacetyl-CoA and 3-hydroxy-3-methylglutaryl-CoA , mevalonate-5-phosphate, mevalonate-5-pyrophosphate, isopentene farnesyl-5-pyrophosphate, farnesyl-pyrophosphate, or dimethylallyl SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, , 4, 5, 6, 7, and 8. or an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, and 8. The protein contains at least one amino acid sequence having at least 44% sequence identity with the sequence. modifying the host cell to overexpress the protein; Producing at least one oxidosqualene, preferably 2,3-oxidosqualene modifying the host cell to express at least one heterologous protein; Expressing at least one oxidosqualene, preferably 2,3-oxidosqualene culturing said host cells under appropriate conditions so as to and at least one oxidosqualene, preferably 2,3-oxidosqualene. By refining the At least one oxidosqualene, preferably 2,3 - This means that it includes increasing the production of oxidosqualene.

[0035] This means that in one embodiment, the present invention is capable of producing 2,3-oxidosqualene in a host cell. The present invention provides a method for increasing the production of The amino acid sequence shown in SEQ ID NO: 1 can be used as a protein, provided that the protein is capable of producing mevalonate. overexpressing 3-hydroxy-3-methylglutaryl coenzyme A reductase containing the 3-hydroxy-3-methylglutaryl coenzyme A reductase sequence, or , a protein having at least 44% sequence identity with the amino acid sequence shown in SEQ ID NO: 1. To overexpress and, Proteins contain acetoacetyl-CoA and 3-hydroxy-3-methylglutaryl-CoA , mevalonate-5-phosphate, mevalonate-5-pyrophosphate, isopentene farnesyl-5-pyrophosphate, farnesyl-pyrophosphate, or dimethylallyl SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, , 4, 5, 6, 7, and 8. or an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, and 8. The protein contains at least one amino acid sequence having at least 44% sequence identity with the sequence. modifying the host cell to overexpress the protein; The method comprises: expressing at least one heterologous protein that produces 2,3-oxidosqualene; modifying the host cell, Culturing the host cells under appropriate conditions to express 2,3-oxidosqualene. thing, Furthermore, by purifying 2,3-oxidosqualene, The amount of 2,3-oxidosqualene produced is increased compared to the host cell before modification. It means to include.

[0036] In one embodiment, the present invention provides a method for increasing the production of lupeol in a host cell. A method is provided, the method comprising: The amino acid sequence shown in SEQ ID NO: 1 can be used as a protein, provided that the protein is capable of producing mevalonate. overexpressing 3-hydroxy-3-methylglutaryl coenzyme A reductase containing the 3-hydroxy-3-methylglutaryl coenzyme A reductase sequence, or , a protein having at least 44% sequence identity with the amino acid sequence shown in SEQ ID NO: 1. To overexpress and, Proteins contain acetoacetyl-CoA and 3-hydroxy-3-methylglutaryl-CoA , mevalonate-5-phosphate, mevalonate-5-pyrophosphate, isopentene farnesyl-5-pyrophosphate, farnesyl-pyrophosphate, or dimethylallyl SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, , 4, 5, 6, 7, and 8. or an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, and 8. The protein contains at least one amino acid sequence having at least 44% sequence identity with the sequence. modifying the host cell to overexpress the protein; and causing the host cell to express at least one heterologous protein to produce lupeol. To modify, Culturing the host cell under appropriate conditions to express lupeol; In addition, by refining lupeol, The present invention includes increasing the amount of lupeol produced compared to the host cell before modification.

[0037] This is because, in one embodiment, the present invention is capable of producing squalene, lanosterol, or the like in a host cell. and / or ergosterol, The method is: The amino acid sequence shown in SEQ ID NO: 1 can be used as a protein, provided that the protein is capable of producing mevalonate. overexpressing 3-hydroxy-3-methylglutaryl coenzyme A reductase containing the 3-hydroxy-3-methylglutaryl coenzyme A reductase sequence, or , a protein having at least 44% sequence identity with the amino acid sequence shown in SEQ ID NO: 1. To overexpress and, Proteins contain acetoacetyl-CoA and 3-hydroxy-3-methylglutaryl-CoA , mevalonate-5-phosphate, mevalonate-5-pyrophosphate, isopentene farnesyl-5-pyrophosphate, farnesyl-pyrophosphate, or dimethylallyl SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, , 4, 5, 6, 7, and 8. or an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, and 8. The protein contains at least one amino acid sequence having at least 44% sequence identity with the sequence. modifying the host cell to overexpress the protein; Produces at least one of squalene, lanosterol, and / or ergosterol. modifying the host cell to express at least one heterologous protein; expressing at least one of squalene, lanosterol, and / or ergosterol culturing the host cells under appropriate conditions so as to and at least one of squalene, lanosterol, and / or ergosterol. By refining the Compared to the host cell before modification, squalene, lanosterol, and / or ergosterol The term "increasing the production of at least one of the following compounds" means that the compound increases the production of at least one of the following compounds:

[0038] The present invention relates to a method for producing oxidosqualene, triterpenes, and / or triterpenes in a host cell. The present invention provides a method for increasing the production of at least one penoid, the method comprising: The amino acid sequence shown in SEQ ID NO: 1 can be used as a protein, provided that the protein is capable of producing mevalonate. overexpressing 3-hydroxy-3-methylglutaryl coenzyme A reductase containing the 3-hydroxy-3-methylglutaryl coenzyme A reductase sequence, or , a protein having at least 44% sequence identity with the amino acid sequence shown in SEQ ID NO: 1. To overexpress and, Proteins contain acetoacetyl-CoA and 3-hydroxy-3-methylglutaryl-CoA , mevalonate-5-phosphate, mevalonate-5-pyrophosphate, isopentene farnesyl-5-pyrophosphate, farnesyl-pyrophosphate, or dimethylallyl SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, , 4, 5, 6, 7, and 8. or an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, and 8. The protein contains at least one amino acid sequence having at least 44% sequence identity with the sequence. modifying the host cell to overexpress the protein; At least one of oxidosqualene, triterpene, and / or triterpenoid modifying the host cell to express at least one heterologous protein to be produced; At least one of oxidosqualene, triterpene, and / or triterpenoid culturing said host cells under appropriate conditions to allow expression; and at least one of oxidosqualene, triterpene, and / or triterpenoid. By refining one more Compared to the host cell before modification, oxidosqualene, triterpene, and / or triterpene increasing the production of at least one of the penoids; At least one of the oxidosqualene, triterpene, and / or triterpenoid , not glycyrrhetinic acid. Glycyrrhetinic acid is glycyrrhetinic acid. Also known as glycyrrhetic acid and enoxolone, it is a hydrolyzed form of glycyrrhizic acid. It is a five-membered triterpenoid derivative of the β-amyrin type obtained by decomposition.

[0039] Terpenoids, sometimes called isoprenoids, are five-carbon amino acids similar to terpenes. They are a large and diverse group of naturally occurring organic compounds derived from isoprene units.

[0040] Plant terpenoids are widely used for their aromatic properties and are used in traditional ha Terpenoids are found in the aroma of eucalyptus, cinnamon, cloves, and It contributes the flavor of ginger, the yellow of sunflowers, and the red of tomatoes. The ingredients are citral, menthol, camphor, salvinorin A from the plant Salvia divinorum, Cannabinoids found in cannabis, ginkgolides found in ginkgo biloba, and Bilobalide, and curcuminoids found in turmeric and mustard seeds include.

[0041] Animal steroids and sterols are biologically produced from terpenoid precursors Terpenoids are added to proteins, for example, to improve their adhesion to cell membranes. This process is known as isoprenylation.

[0042] Additionally, triterpenoids include, but are not limited to, saponins, sapogenins, Acylated triterpenes, such as α-amyron, β-amyron, and taraxasterone Keto derivatives of penicillin, as well as betulinic acid, glycyrrhetinic acid and boswellic acid carboxylic acid derivatives such as carboxylic acid derivatives (e.g., benzoyl ...

[0043] Triterpenes have the molecular formula C 30 H 48 A chemical compound consisting of three terpene units with They are also thought to consist of six isoprene units. Terpenes are subdivided into linear, four-membered ring, and five-membered ring triterpenes. All fungi and bacteria produce triterpenes, but perhaps the most important example is squalene. This is because squalene forms the basis of almost all steroids. Further examples are certain steroids and cardiac glycosides. Terpenes include, but are not limited to, lupeol, lup-19(21)-en- 3-ol, lup-20(29)-en-3-ol, β-amyrin, α-amyrin, and Furthermore, for example, triterpenoid saponins are compounds of this group. It is a triterpene that belongs to the saponin group of chemical substances.

[0044] Oxidosqualene is, for example, but not limited to, 2,3-oxidosqualene. do.

[0045] In the present invention, oxidosqualene, triterpene, and / or triterpenoid Increasing the production of at least one protein is a protein capable of producing mevalonate. 3-hydroxy-3-methylglutamate containing the amino acid sequence shown in SEQ ID NO: 1 under the condition that The enzyme does not overexpress aryl-coenzyme A reductase or has an amino acid sequence at least as similar to that shown in SEQ ID NO: 1. The protein, which has 44% sequence identity with the host cell, is not overexpressed. The host cell is adapted to produce a protein containing acetoacetyl-CoA, 3-hydroxy-3-methyl Glutaryl-CoA, mevalonate-5-phosphate, mevalonate-5-pyrophos phosphate, isopentenyl-5-pyrophosphate, farnesyl-pyrophosphate, or dimethylallyl-pyrophosphate. and wherein at least one selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, and 8 or from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, and 8. At least one amino acid sequence having at least 44% sequence identity with the selected amino acid sequence. The protein containing the amino acid sequence is not overexpressed.

[0046] In a preferred embodiment of the present invention, the method of the present invention comprises the step of synthesizing the amino acid sequence shown in SEQ ID NO: 1. To overexpress 3-hydroxy-3-methylglutaryl coenzyme A reductase, including The method further comprises modifying a host cell to produce a 3-hydroxy-3-methylglutaryl coenzyme A reducing agent. The enzyme can be abbreviated as tHMGR, which is the catalytic domain of the enzyme HMGR. In a preferred embodiment, the enzyme tHMGR is the amino acid sequence shown in SEQ ID NO: 1. It consists of a sequence of amino acids.

[0047] In a preferred embodiment, the method of the present invention is carried out in a host cell in which the protein is 3- SEQ ID NO: 3, provided that hydroxy-3-methylglutaryl-CoA can be produced. or an amino acid sequence having at least 44% sequence identity with SEQ ID NO:3. The vector is engineered to overexpress the protein containing the amino acid sequence.

[0048] In a preferred embodiment, in the method of the present invention, the host cell is the amino acid sequence of SEQ ID NO: 2, provided that the enzyme is capable of producing triacetyl-CoA; or the protein comprising an amino acid sequence having at least 44% sequence identity with SEQ ID NO: 2. The cells are engineered to overexpress the protein.

[0049] In a preferred embodiment, in the method of the present invention, the host cell is the amino acid sequence of SEQ ID NO: 4, provided that it is possible to produce thiamin-5-phosphate or an amino acid sequence having at least 44% sequence identity with SEQ ID NO: 4. It is engineered to overexpress the protein.

[0050] In a preferred embodiment, in the method of the present invention, the host cell is amino acid sequence of SEQ ID NO: 5, provided that it is possible to generate 5-pyrophosphate or an amino acid sequence having at least 44% sequence identity with SEQ ID NO:5. The gene is engineered to overexpress the protein comprising the gene.

[0051] In a preferred embodiment, in the method of the present invention, the host cell is Amino acid sequence of SEQ ID NO: 6, provided that pentenyl-5-pyrophosphate can be produced. an amino acid sequence comprising or having at least 44% sequence identity with SEQ ID NO:6 The gene is modified to overexpress the protein comprising:

[0052] In a preferred embodiment, in the method of the present invention, the host cell is Amino acid sequence of SEQ ID NO: 7, provided that runesyl-5-pyrophosphate can be produced or an amino acid sequence having at least 44% sequence identity with SEQ ID NO: 7. The gene is engineered to overexpress the protein comprising the gene.

[0053] In a preferred embodiment, in the method of the present invention, the host cell is methylallyl-5-pyrophosphate can be obtained by the addition of the amino acid sequence of SEQ ID NO: 8. an amino acid sequence comprising or having at least 44% sequence identity with SEQ ID NO: 8 The gene is modified to overexpress the protein comprising:

[0054] The present invention also provides a method for producing oxidosqualene, triterpenes, and / or triterpenes in a host cell. A method for increasing the production of at least one terpenoid is provided, the method comprising: 3-hydroxy-3-methylglutaryl coenzyme A containing the amino acid sequence shown in SEQ ID NO: 1 To overexpress the reductase, and, Proteins contain acetoacetyl-CoA and 3-hydroxy-3-methylglutaryl-CoA , mevalonate-5-phosphate, mevalonate-5-pyrophosphate, isopentene farnesyl-5-pyrophosphate, farnesyl-pyrophosphate, or dimethylallyl SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, , 4, 5, 6, 7, and 8. or an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, and 8. The protein contains at least one amino acid sequence having at least 44% sequence identity with the sequence. modifying the host cell to overexpress the protein; At least one of oxidosqualene, triterpene, and / or triterpenoid modifying the host cell to express at least one heterologous protein to be produced; At least one of oxidosqualene, triterpene, and / or triterpenoid culturing said host cells under appropriate conditions to allow expression; and at least one of oxidosqualene, triterpene, and / or triterpenoid. By refining one more Compared to the host cell before modification, oxidosqualene, triterpene, and / or triterpene The present invention also includes increasing the production of at least one of the penoids.

[0055] SEQ ID NO:1: MVLTNKTVISGSKVKSLSSAQSSSSGPSSSSEEDDSRDIESLDKKIRPLEELEALLSSGNTKQLKNKEVAALVIHGKLPL YALEKKLGDTTRAVAVRRKALSILAEAPVLASDRLPYKNYDYDRVFGACCENVIGYMPLPVGVIGPLVIDGTSYHIPMAT TEGCLVASAMRGCKAINAGGGATTVLTKDGMTRGPVVRFPTLKRSGACKIWLDSEEGQNAIKKAFNSTSRFARLQHIQTC LAGDLLFMRFRTTTGDAMGMNMISKGVEYSLKQMVEEYGWEDMEVVSVSGNYCTDKKPAAINWIEGRGKSVVAEATIPGD VVRKVLKSDVSALVELNIAKNLVGSAMAGSVGGFNAHAANLVTAVFLALGQDPAQNVESSNCITLMKEVDGDLRISVSMP SIEVGTIGGGTVLEPQGAMLDLLGVRGPHATAPGTNARQLARIVACAVLAGELSLCAALAAGHLVQSHMTHNRKPAEPTK PNNLDATDINRLKDGSVTCIKS

[0056] 3-Hydroxy-3-methyl-glutaryl coenzyme A reductase degrades cholesterol and It is the rate-limiting enzyme in the mevalonate pathway, which is a metabolic pathway that produces other isoprenoids. Normally, in mammalian cells, this enzyme acts as a receptor for the cleavage of low-density lipoprotein (LDL). Cholesterol derived from the internalization and degradation of cholesterol and oxidized species of cholesterol are inhibited. Competitive inhibitors of reductase induce the expression of LDL receptors in the liver, which Increases plasma LDL catabolism and promotes cholesterol, an important determinant of atherosclerosis This enzyme reduces the plasma concentration of sterols. It is the target of widely available cholesterol-lowering drugs known as

[0057] In the present invention, overexpression can be achieved by any method known to those skilled in the art. Generally, this is achieved by increasing gene transcription / translation, e.g., increasing gene copy numbers. Increase the number or alter the regulatory sequences or sites involved in gene expression For example, overexpression can be achieved by modifying the promoters encoding each protein. one or more copies of a nucleotide, or its respective regulatory sequence (e.g., promoter) This can be achieved by introducing functional homologs that are operably linked. The gene may be designed to use a strong constitutive promoter and / or a strong ubiquitous promoter to obtain high expression levels. The promoter may be operably linked to an endogenous promoter. Alternatively, the promoter may be regulated so that expression is constitutive. The natural promoter of a given gene can be modified to increase expression of that gene. or can be replaced with a heterologous promoter that provides constitutive expression of the gene. For example, tHMGR (SEQ ID NO: 32), and / or each of the proteins comprising the amino acid sequence of SEQ ID NO: 1. The protein tHMGR is expressed in the present invention in comparison with the host cells cultured under the same conditions before modification. Depending on the host cell, the percentages are 10%, 20%, 30%, 40%, 50%, 60%, 70%, and 80%. %, 90%, 100%, 200%, or 300% overexpression. For example, at least one selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, and 8 can be used. At least one protein containing at least one amino acid sequence is purified under the same conditions before modification. Depending on the host cell, the Over 50%, 60%, 70%, 80%, 90%, 100%, 200%, or 300% The use of an inducible promoter further improves the efficiency of host cell culture. Furthermore, overexpression can also be used to increase the expression of a particular gene, e.g. Modifying chromosomal locations, identifying ribosome binding sites or transcription terminators, etc. Altering the nucleic acid sequences flanking the gene, the transcription of the gene and / or the gene product Proteins involved in the translation of (e.g., regulatory proteins, suppressors, enhancers, modifying a transcriptional activator or inhibiting the expression of a repressor protein, for example using antisense nucleic acid molecules to block expression, or, in general, overexpression Deleting or mutating the gene for a transcription factor that suppresses the expression of the gene you want to express Methods for detecting specific gene expression conventional in the art, including but not limited to: This can also be achieved by any other conventional means of regulating mRNA. Increasing lifespan can also improve expression levels. For example, certain terminator regions , which can be used to extend the half-life of mRNA (Yamanishi et al., Biosci. B Biotechnol. Biochem. (2011) 75:2234 and US 2013 / 0244243). In some cases, the gene is located on a plasmid with various copy numbers or integrated into the chromosome. The host cell can then amplify the genes encoding each protein for overexpression. If the gene does not contain the nucleotide sequence, it can be introduced into a host cell for expression. "Overexpression" means expressing a gene product using any method known to those skilled in the art. Taste.

[0058] In the present invention, "ERG10" is a steroid and non-sterol isoprenoid biosynthetic enzyme. Acetoacetyl-Co in the biosynthesis of mevalonate, a required intermediate for its synthesis The protein acetyl-CoA-acetyltransferase (A) catalyzes the formation of The gene encoding acetoacetyl-CoA thiolase, also known as ERG10 (sequence Number 2). This means that an acetyl group is transferred from one acetyl-CoA molecule to another. transfer to form acetoacetyl-CoA and is the first step in mevalonate biosynthesis It encodes a cytosolic enzyme involved in the step. The sequence is set forth herein as SEQ ID NO:13.

[0059] In the present invention, the term "ERG13" refers to the acetoacetyl- It catalyzes the condensation of CoA with 3-hydroxy-3-(HMG-CoA) The enzyme 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) forms It refers to the gene encoding tharyl-CoA (HMG-CoA) synthase (ERG13) (SEQ ID NO: 3). Thus, it is involved in the second step of mevalonate biosynthesis. The nucleotide sequence of is set forth herein as SEQ ID NO:14.

[0060] In the context of the present invention, the term "ERG12" refers to the enzyme that regulates the reaction of mevalonate with ATP. The protein mevalonate kinase catalyzes the production of mevalonate-5-phosphate. It refers to the gene (ERG12) (SEQ ID NO: 4) encoding the enzyme. The nucleotide sequence is set forth herein as SEQ ID NO:15.

[0061] In the present invention, the term "ERG8" refers to an isoprenoid containing ergosterol. and is an essential cytosolic enzyme that functions in the biosynthesis of sterols from mevalonate. The gene encoding the protein phosphomevalonate kinase (ERG8) (SEQ ID NO: 5) This protein catalyzes the reaction of mevalonate-5-phosphate with ATP. The nucleotide sequence of "ERG8" is then catalyzed to produce mevalonate-5-pyrophosphate. The peptide sequence is set forth herein as SEQ ID NO:16.

[0062] In the context of the present invention, the term "ERG19" refers to an isoprenoid containing ergosterol. It is an essential enzyme involved in the biosynthesis of steroids and steroids, and also functions as a homodimer. It contains the protein mevalonate-5-pyrophosphate decarboxylase, which acts as a This refers to the gene (ERG19) (SEQ ID NO: 6) that encodes mevalonate-5-pyrophosphate. The reaction catalyzed by isopentenyl-5-pyrophosphate The "ERG19" gene is also known as "mvd1." Additionally, the nucleotide sequence of "ERG19" is set forth herein as SEQ ID NO: 17. do.

[0063] In the present invention, the term "ERG20" refers to the farnesyl pyrophosphate This refers to the gene encoding the enzyme phosphate synthase (ERG20) (SEQ ID NO: 7). Methylaryltransferase activity and geranyltransferase activity It has both hydroxyl and C15 phospholipids for isoprenoid and sterol biosynthesis. The "ERG20" gene catalyzes the formation of runesyl pyrophosphate units. It is also known as "bot3", "fds1", and "fpp1". The nucleotide sequence of "G20" is set forth herein as SEQ ID NO:18.

[0064] As used herein, the term "IDI1" refers to a relatively unreactive isopentane. The more reactive electrophilic dimethylallyl pyrophosphate (IPP) isopentenyl pyrophosphate (DMAPP), an isomerase that catalyzes the conversion of isopentenyl pyrophosphate (DMAPP) to isopentenyl pyrophosphate (DMAPP). The gene encoding the isomerase (IDI1) (SEQ ID NO: 8) is the isomerization reaction of It is a key step in the biosynthesis of isoprenoids via the valonate pathway. The nucleotide sequence of "IDI1" is set forth herein as SEQ ID NO:19.

[0065] In the present invention, genes are always written in capital letters herein, while mutations / Knockout alleles are written in lowercase.

[0066] In a preferred embodiment of the present invention, the method comprises the step of isolating 3-hydroxybenzoates as set forth in SEQ ID NO:1. -3-methylglutaryl coenzyme A reductase amino acid sequence and at least 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, Proteins with 96%, 97%, 98%, 99%, or even 100% sequence identity The method includes modifying a host cell to overexpress the protein, which produces mevalonate. It is possible.

[0067] "Sequence identity" or "% identity" as used in the context of the present invention is determined using standard algorithms At least two polypeptide or polynucleotide sequences aligned using These algorithms are used to align two sequences. To optimize alignment, gaps are introduced into the sequences being compared in a standardized and reproducible manner. may be inserted, thus allowing for a more meaningful comparison of the two sequences. For purposes of clarity, sequence identity between two amino acid or nucleotide sequences is considered to be a cluster. The default settings of Clustal Omega (https: / / www.ebi.ac.uk / Tools / msa / clustalo / ) The input parameters are as follows: program: clustalo; version 1.2 .4; output guide tree: false; output distance matrix: false; dealign input seque nces: false; mBed-like clustering guide tree: true; mBed-like clustering iterati on: true; number of iterations: 0; maximum guide tree iterations: -1; maximum HM M iterations: -1; output alignment format: clustal_num; output order: aligned; s Sequence type: protein.

[0068] Thus, in a further aspect of the present invention, there is provided a method for producing oxidosqualene in a host cell, Method for increasing the production of at least one triterpene and / or triterpenoid The method comprises: The amino acid sequence shown in SEQ ID NO: 1 can be used, provided that the protein is capable of producing mevalonate. Acid sequence and at least 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51% , 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61% , 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71% ,72%,73%,74%,75%,76%,77%,78%,79%,80%,81% ,82%,83%,84%,85%,86%,87%,88%,89%,90%,91% , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 10 so as to overexpress the protein with 0% sequence identity, and, Proteins contain acetoacetyl-CoA and 3-hydroxy-3-methylglutaryl-CoA , mevalonate-5-phosphate, mevalonate-5-pyrophosphate, isopentene farnesyl-5-pyrophosphate, farnesyl-pyrophosphate, or dimethylallyl SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, , 4, 5, 6, 7, and 8. or an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, and 8. The protein contains at least one amino acid sequence having at least 44% sequence identity with the sequence. modifying the host cell to overexpress the protein; At least one of oxidosqualene, triterpene, and / or triterpenoid modifying the host cell to express at least one heterologous protein to be produced; At least one of oxidosqualene, triterpene, and / or triterpenoid culturing said host cells under appropriate conditions to allow expression; and at least one of oxidosqualene, triterpene, and / or triterpenoid. By refining one more Compared to the host cell before modification, oxidosqualene, triterpene, and / or triterpene The present invention also includes increasing the production of at least one of the penoids.

[0069] At least one of oxidosqualene, triterpene, and / or triterpenoid Increasing the amount of production means herein that the protein is capable of producing mevalonate. The amino acid sequence shown in SEQ ID NO: 1 is at least 44%, 45%, 46%, or %, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56 %, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66 %, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76 %, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86 %, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96 %, 97%, 98%, 99%, or even 100% sequence identity. It is not overexpressed, and the protein is acetoacetyl-CoA, 3-hydroxy-3-methyl- Glutaryl-CoA, mevalonate-5-phosphate, mevalonate-5-pyrophosphate phosphate, isopentenyl-5-pyrophosphate, farnesyl-pyrophosphate or dimethylallyl-pyrophosphate. at least one selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, and 8, or comprising one amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, and 8. At least one amino acid sequence having at least 44% sequence identity with the selected amino acid sequence This means that the expression is compared to a host cell that does not overexpress the protein containing the amino acid sequence.

[0070] In a preferred embodiment of the invention, the method further comprises the step of: , 3-hydroxy-3-methylglutaryl-CoA, mevalonate-5-phosphate, Mevalonate-5-pyrophosphate, Isopentenyl-5-pyrophosphate, Fal At least one of dimethylallyl-pyrophosphate, dimethylallyl-pyrophosphate, The group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, and 8, provided that one of the and an amino acid sequence selected from the group consisting of at least 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 7 9%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 5 9%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 6 9%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 7 9%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 8 9%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 9 9%, or even 100% sequence identity. This involves modifying the host cell to overexpress the protein.

[0071] This means that a further aspect of the invention is to produce oxidosqualene, triterpene, and a method for increasing the production of at least one of terpenes, benzoates, and / or triterpenoids, The method comprises: The amino acid sequence shown in SEQ ID NO: 1 can be used as a protein, provided that the protein is capable of producing mevalonate. overexpressing 3-hydroxy-3-methylglutaryl coenzyme A reductase containing the 3-hydroxy-3-methylglutaryl coenzyme A reductase sequence, or , a protein having at least 44% sequence identity with the amino acid sequence shown in SEQ ID NO: 1. To overexpress and, Proteins contain acetoacetyl-CoA and 3-hydroxy-3-methylglutaryl-CoA , mevalonate-5-phosphate, mevalonate-5-pyrophosphate, isopentene farnesyl-5-pyrophosphate, farnesyl-pyrophosphate, or dimethylallyl SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, , 4, 5, 6, 7, and 8, and at least 44% of an amino acid sequence selected from the group consisting of: 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, A small number of sequences with 95%, 96%, 97%, 98%, 99%, or even 100% sequence identity. and modifying the host cell to overexpress the protein comprising at least one amino acid sequence. To do, At least one of oxidosqualene, triterpene, and / or triterpenoid modifying the host cell to express at least one heterologous protein to be produced; At least one of oxidosqualene, triterpene, and / or triterpenoid culturing said host cells under appropriate conditions to allow expression; and at least one of oxidosqualene, triterpene, and / or triterpenoid. By refining one more Compared to the host cell before modification, oxidosqualene, triterpene, and / or triterpene The term "enhancing the production of at least one of the penoids" means enhancing the production of at least one of the penoids.

[0072] At least one of oxidosqualene, triterpene, and / or triterpenoid In this embodiment, increasing the amount of production means that the protein is capable of producing mevalonate. 3-hydroxy-3-methyl 3-hydroxybenzoates having the amino acid sequence shown in SEQ ID NO: 1, glutaryl coenzyme A reductase is not overexpressed or the amino acid sequence shown in SEQ ID NO: 1 is Do not overexpress the protein with at least 44% sequence identity, and The enzymes are acetoacetyl-CoA, 3-hydroxy-3-methylglutaryl-CoA, and mevalonate. mevalonate-5-pyrophosphate, isopentenyl-5 -pyrophosphate, farnesyl-pyrophosphate, or dimethylallyl-pyrophosphate SEQ ID NOs: 2, 3, 4, 5, provided that at least one of the phosphates can be produced. , 6, 7, and 8 and at least 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, having at least 96%, 97%, 98%, 99%, or even 100% sequence identity This is in comparison to host cells that do not overexpress the protein containing a single amino acid sequence. .

[0073] In a further preferred embodiment, the present invention provides a method for producing oxidosqualene in a host cell. a method for increasing the production of at least one of a triterpene and / or a triterpenoid; A method is provided, the method comprising: The 3-hydroxybenzoate shown in SEQ ID NO: 1 can be used, provided that the protein is capable of producing mevalonate. The amino acid sequence of hydroxy-3-methylglutaryl coenzyme A reductase and at least 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% sequence identity. To overexpress proteins, and, Proteins contain acetoacetyl-CoA and 3-hydroxy-3-methylglutaryl-CoA , mevalonate-5-phosphate, mevalonate-5-pyrophosphate, isopentene farnesyl-5-pyrophosphate, farnesyl-pyrophosphate, or dimethylallyl SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, , 4, 5, 6, 7, and 8, and at least 44% of an amino acid sequence selected from the group consisting of: 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, A small number of sequences with 95%, 96%, 97%, 98%, 99%, or even 100% sequence identity. and modifying the host cell to overexpress the protein comprising at least one amino acid sequence. To do, At least one of oxidosqualene, triterpene, and / or triterpenoid modifying the host cell to express at least one heterologous protein to be produced; At least one of oxidosqualene, triterpene, and / or triterpenoid culturing said host cells under appropriate conditions to allow expression; and at least one of oxidosqualene, triterpene, and / or triterpenoid. By refining one more Compared to the host cell before modification, oxidosqualene, triterpene, and / or triterpene The present invention also includes increasing the production of at least one of the penoids.

[0074] At least one of oxidosqualene, triterpene, and / or triterpenoid Increasing production means that in this particular embodiment, the protein produces mevalonate. 3-hydroxy-3-methylglutarial compound shown in SEQ ID NO: 1, provided that it can be synthesized. The amino acid sequence of coenzyme A reductase and at least 44%, 45%, 46%, 47%, 48% %, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58 %, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68 %, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78 %, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88 %, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 %, 99%, or even 100% sequence identity to the protein, and proteins are acetoacetyl-CoA, 3-hydroxy-3-methylglutaryl- CoA, mevalonate-5-phosphate, mevalonate-5-pyrophosphate, iso Pentenyl-5-pyrophosphate, farnesyl-pyrophosphate, or dimethicone provided that at least one of the following can be produced: an amino acid sequence selected from the group consisting of 2, 3, 4, 5, 6, 7, and 8 and at least 4 4%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 5 4%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 6 4%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 7 4%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 8 4%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 9 4%, 95%, 96%, 97%, 98%, 99%, or even 100% sequence identity. and a host cell that does not overexpress the protein comprising at least one amino acid sequence It means an increase in comparison.

[0075] In a preferred embodiment of the method of the present invention, the host cell is 3, provided that the enzyme is capable of producing hydroxy-3-methyl-glutaryl-CoA. an amino acid sequence having at least 44% sequence identity with SEQ ID NO:3; The vector is engineered to overexpress the protein containing the nucleic acid sequence.

[0076] In a preferred embodiment of the method of the present invention, the method comprises the step of: ), BTS1 (SEQ ID NO: 54), YPL062W (SEQ ID NO: 55), DOS2 (SEQ ID NO: 56), YER134C (SEQ ID NO: 57), VBA5 (SEQ ID NO: 58), YNR063W (SEQ ID NO: 59), YJL064W (SEQ ID NO: 60), and YGR259C (SEQ ID NO: 6 1) in a host cell so as to knock out at least one locus selected from the group consisting of It further includes modifying the

[0077] In a preferred embodiment of the method of the present invention, the method comprises the step of: ), YPL062W (SEQ ID NO: 55), DOS2 (SEQ ID NO: 56), YER134C (SEQ ID NO: 57), Sequence number 57), VBA5 (sequence number 58), YNR063W (sequence number 59), YJL0 64W (SEQ ID NO: 60), and YGR259C (SEQ ID NO: 61). and further comprising modifying the host cell to knock out at least one locus in which the gene is expressed. .

[0078] In particular, the method involves modifying a host cell to knock out the ROX1 (SEQ ID NO: 25) locus. Even more preferably, the method further comprises changing the

[0079] A particularly preferred method according to the present invention is The amino acid sequence shown in SEQ ID NO: 1 can be used as a protein, provided that the protein is capable of producing mevalonate. overexpressing 3-hydroxy-3-methylglutaryl coenzyme A reductase containing the 3-hydroxy-3-methylglutaryl coenzyme A reductase sequence, or , a protein having at least 44% sequence identity with the amino acid sequence shown in SEQ ID NO: 1. To overexpress and, Proteins contain acetoacetyl-CoA and 3-hydroxy-3-methylglutaryl-CoA , mevalonate-5-phosphate, mevalonate-5-pyrophosphate, isopentene farnesyl-5-pyrophosphate, farnesyl-pyrophosphate, or dimethylallyl SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, , 4, 5, 6, 7, and 8. or an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, and 8. The protein contains at least one amino acid sequence having at least 44% sequence identity with the sequence. modifying the host cell to overexpress the protein; and, modifying the host cell to knock out the ROX1 (SEQ ID NO: 25) locus; At least one of oxidosqualene, triterpene, and / or triterpenoid modifying the host cell to express at least one heterologous protein to be produced; At least one of oxidosqualene, triterpene, and / or triterpenoid culturing said host cells under appropriate conditions to allow expression; and at least one of oxidosqualene, triterpene, and / or triterpenoid. By refining one more Compared to the host cell before modification, oxidosqualene, triterpene, and / or triterpene The present invention also includes increasing the production of at least one of the penoids.

[0080] At least one of oxidosqualene, triterpene, and / or triterpenoid Increasing production means that in this particular embodiment, the protein produces mevalonate. 3-hydroxy-3- Do not overexpress methylglutaryl coenzyme A reductase or the amino acid sequence shown in SEQ ID NO: 1 a host cell that does not overexpress a protein having at least 44% sequence identity with the sequence and the host cell is adapted to produce proteins that are acetoacetyl-CoA, 3-hydroxy-3-methylglutaryl-CoA, mevalonate-5-phosphate, mevalonate Valonate-5-pyrophosphate, Isopentenyl-5-pyrophosphate, Farne at least one of dimethylallyl-pyrophosphate, dimethylallyl-pyrophosphate, or dimethylallyl-pyrophosphate and a sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, and 8, provided that the sequence can be generated. or comprising at least one amino acid sequence selected from SEQ ID NOs: 2, 3, 4, 5, 6, 7, and 8, having at least 44% sequence identity with the amino acid sequence selected from the group consisting of and the host does not overexpress the protein comprising at least one amino acid sequence corresponding to the The cells do not knock out the ROX1 (SEQ ID NO: 25) locus.

[0081] In one embodiment, the present invention relates to a method for producing squalene, lanosterol, and / or ergosterol, The law is The amino acid sequence shown in SEQ ID NO: 1 can be used as a protein, provided that the protein is capable of producing mevalonate. overexpressing 3-hydroxy-3-methylglutaryl coenzyme A reductase containing the 3-hydroxy-3-methylglutaryl coenzyme A reductase sequence, or , a protein having at least 44% sequence identity with the amino acid sequence shown in SEQ ID NO: 1. To overexpress and, Proteins contain acetoacetyl-CoA and 3-hydroxy-3-methylglutaryl-CoA , mevalonate-5-phosphate, mevalonate-5-pyrophosphate, isopentene farnesyl-5-pyrophosphate, farnesyl-pyrophosphate, or dimethylallyl SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, , 4, 5, 6, 7, and 8. or an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, and 8. The protein contains at least one amino acid sequence having at least 44% sequence identity with the sequence. modifying the host cell to overexpress the protein; and, modifying the host cell to knock out the ROX1 (SEQ ID NO: 25) locus; Produces at least one of squalene, lanosterol, and / or ergosterol. modifying the host cell to express at least one heterologous protein; expressing at least one of squalene, lanosterol, and / or ergosterol culturing the host cells under appropriate conditions so as to and at least one of squalene, lanosterol, and / or ergosterol. By refining the Less squalene, lanosterol, or ergosterol than the host cell before modification This includes increasing the production of at least one of the

[0082] Production of at least one of squalene, lanosterol, and / or ergosterol Increasing the amount of mevalonate produced by the protein in this particular embodiment. 3-hydroxy-3-methyl 3-hydroxybenzoates having the amino acid sequence shown in SEQ ID NO: 1, glutaryl coenzyme A reductase is not overexpressed or the amino acid sequence shown in SEQ ID NO: 1 is a host cell that does not overexpress the protein having at least 44% sequence identity. The host cell is a cell that converts proteins into acetoacetyl-CoA, 3-hydroxybenzoates, and Hydroxy-3-methylglutaryl-CoA, mevalonate-5-phosphate, mevalonate Isopentenyl-5-pyrophosphate, Farnesyl- pyrophosphate, or dimethylallyl-pyrophosphate. Selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, and 8, provided that it is possible or comprising at least one amino acid sequence selected from SEQ ID NOs: 2, 3, 4, 5, 6, 7, and and 8. A small number of amino acid sequences having at least 44% sequence identity with an amino acid sequence selected from the group consisting of and the host cell does not overexpress the protein comprising at least one amino acid sequence. , does not knock out the ROX1 (SEQ ID NO: 25) locus.

[0083] In a preferred embodiment, the method of the present invention comprises the step of synthesizing the amino acid sequence set forth in SEQ ID NO:9. Column and at least 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 5 2%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 6 2%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 7 2%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 8 2%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 9 2%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% Lanosterol synthase comprising the amino acid sequence set forth in SEQ ID NO: 9, which has sequence identity with The host cell may further be modified to suppress the lanosterol synthase (ERG7). The nucleotide sequence of each of the elements is further set forth herein as set forth in SEQ ID NO:20. Preferably, said inhibition of a polynucleotide encoding lanosterol synthase (ERG7), Alternatively, the repression of the protein lanosterol synthase itself is achieved by the CTR3 promoter (SEQ ID NO: No. 42) and / or by the addition of copper sulfate CuSO4.

[0084] In this preferred embodiment, sulfate to inhibit lanosterol synthase (ERG7) The amount of copper added should be at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 1 00, 110, 120, 130, 140, 150, 160, 170, 180, 190, 2 00, 210, 220, 230, 240, 250, 260, 270, 280, 290, 3 00, 310, 320, 330, 340, 350, 360, 370, or 375 mM C It may be uSO4, preferably at least 150 mM CuSO4.

[0085] Lanosterol synthase converts (S)-2,3-oxidosqualene to protosterol cation and then to lanosterol by the enzyme oxidosqualene cyclase (OSC). Lanosterol is a key tetracyclic intermediate in cholesterol biosynthesis. In B. lanosterol synthase is encoded by the LSS gene.

[0086] Preferably, the lanosterol synthase (or its equivalent in plants) to be inhibited in the present invention Cycloartenol synthase (Saccharomyces cerevisiae, Nicotiana benthamiana) ana, Pichia pastoris, Pichia methanolica, Schizosaccharomyces pombe, Kluyveromyc es lactis, Kluyveromyces marxianus, Pichia stipitis, Candida albicans, Candida u tilis, and from BY2 cells.

[0087] In a preferred embodiment, the method of the present invention comprises the step of cloning the amino acid sequence shown in SEQ ID NO: 21. or the amino acid sequence set forth in SEQ ID NO: 21, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, Containing amino acid sequences with 97%, 98%, 99%, or even 100% sequence identity The host cell may further be modified to inhibit cycloartenol synthase. The nucleotide sequence of cycloartenol synthase is shown in SEQ ID NO:22 and is described herein. This is further shown in

[0088] That is, a particularly preferred method in the present invention is The amino acid sequence shown in SEQ ID NO: 1 can be used as a protein, provided that the protein is capable of producing mevalonate. overexpressing 3-hydroxy-3-methylglutaryl coenzyme A reductase containing the 3-hydroxy-3-methylglutaryl coenzyme A reductase sequence, or , a protein having at least 44% sequence identity with the amino acid sequence shown in SEQ ID NO: 1. To overexpress and, Proteins contain acetoacetyl-CoA and 3-hydroxy-3-methylglutaryl-CoA , mevalonate-5-phosphate, mevalonate-5-pyrophosphate, isopentene farnesyl-5-pyrophosphate, farnesyl-pyrophosphate, or dimethylallyl SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, , 4, 5, 6, 7, and 8. or an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, and 8. The protein contains at least one amino acid sequence having at least 44% sequence identity with the sequence. modifying the host cell to overexpress the protein; and, modifying the host cell to knock out the ROX1 (SEQ ID NO: 25) locus; and, the amino acid sequence shown in SEQ ID NO: 9, provided that the protein is capable of producing lanosterol. or an amino acid sequence having at least 34% sequence identity with SEQ ID NO: 9. modifying the host cell to inhibit lanosterol synthase (ERG7); At least one of oxidosqualene, triterpene, and / or triterpenoid modifying the host cell to express at least one heterologous protein to be produced; At least one of oxidosqualene, triterpene, and / or triterpenoid culturing said host cells under appropriate conditions to allow expression; and at least one of oxidosqualene, triterpene, and / or triterpenoid. By refining one more Compared to the host cell before modification, oxidosqualene, triterpene, and / or triterpene The present invention also includes increasing the production of at least one of the penoids.

[0089] Increasing the production of a protein in the mevalonate pathway is in this particular embodiment and the amino acid sequence shown in SEQ ID NO: 1, provided that the protein is capable of producing mevalonate. Do not overexpress 3-hydroxy-3-methylglutaryl coenzyme A reductase containing the carboxylic acid sequence or a sequence having at least 44% sequence identity with the amino acid sequence shown in SEQ ID NO: 1. The protein is not overexpressed, and the protein is synthesized from acetoacetyl-CoA, 3-hydroxybenzoates. -3-Methylglutaryl-CoA, mevalonate-5-phosphate, mevalonate-5 -pyrophosphate, isopentenyl-5-pyrophosphate, farnesyl-pyrophos phosphate, or dimethylallyl-pyrophosphate. a minimum of one or more sequences selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, and 8, provided that: or comprising at least one amino acid sequence selected from SEQ ID NOs: 2, 3, 4, 5, 6, 7, and 8. At least one amino acid sequence having at least 44% sequence identity with an amino acid sequence selected from the group consisting of: The protein containing one amino acid sequence is not overexpressed, and ROX1 (SEQ ID NO: 2) 5) The locus is not knocked out, and the protein is capable of producing lanosterol. 9 or at least 34 amino acid sequences different from SEQ ID NO: 9, provided that % sequence identity with lanosterol synthase (ERG7) By "enhancement," we mean an increase in the expression of the gene relative to the host cell.

[0090] This means that in one embodiment, the present invention is capable of producing 2,3-oxidosqualene in a host cell. The present invention provides a method for increasing the production of The amino acid sequence shown in SEQ ID NO: 1 can be used as a protein, provided that the protein is capable of producing mevalonate. overexpressing 3-hydroxy-3-methylglutaryl coenzyme A reductase containing the 3-hydroxy-3-methylglutaryl coenzyme A reductase sequence, or , a protein having at least 44% sequence identity with the amino acid sequence shown in SEQ ID NO: 1. To overexpress and, Proteins contain acetoacetyl-CoA and 3-hydroxy-3-methylglutaryl-CoA , mevalonate-5-phosphate, mevalonate-5-pyrophosphate, isopentene farnesyl-5-pyrophosphate, farnesyl-pyrophosphate, or dimethylallyl SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, , 4, 5, 6, 7, and 8. or an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, and 8. The protein contains at least one amino acid sequence having at least 44% sequence identity with the sequence. modifying the host cell to overexpress the protein; and, modifying the host cell to knock out the ROX1 (SEQ ID NO: 25) locus; and, the amino acid sequence shown in SEQ ID NO: 9, provided that the protein is capable of producing lanosterol. or an amino acid sequence having at least 34% sequence identity with SEQ ID NO: 9. modifying the host cell to inhibit lanosterol synthase (ERG7); The method comprises: expressing at least one heterologous protein that produces 2,3-oxidosqualene; modifying the host cell, Culturing the host cells under appropriate conditions to express 2,3-oxidosqualene. thing, Furthermore, by purifying 2,3-oxidosqualene, The amount of 2,3-oxidosqualene produced is increased compared to the host cell before modification. It means to include.

[0091] Increasing the production of 2,3-oxidosqualene in this particular embodiment and the amino acid sequence shown in SEQ ID NO: 1, provided that the protein is capable of producing mevalonate. Do not overexpress 3-hydroxy-3-methylglutaryl coenzyme A reductase containing the sequence or the protein having at least 44% sequence identity with the amino acid sequence shown in SEQ ID NO: 1. The protein is not overexpressed, and the protein is acetoacetyl-CoA, 3-hydroxy- 3-Methylglutaryl-CoA, mevalonate-5-phosphate, mevalonate-5- Pyrophosphate, Isopentenyl-5-pyrophosphate, Farnesyl-pyrophosphate phosphate, or dimethylallyl-pyrophosphate at least one selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, and 8, comprising at least one amino acid sequence, or consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, and 8 At least one amino acid sequence having at least 44% sequence identity with an amino acid sequence selected from the group consisting of: The protein comprising the two amino acid sequences is not overexpressed, and ROX1 (SEQ ID NO: 25) ) locus is not knocked out, and the protein is capable of producing lanosterol. 9, or has an amino acid sequence at least 34% identical to SEQ ID NO: 9, provided that does not inhibit lanosterol synthase (ERG7) with an amino acid sequence that shares sequence identity with , refers to an increase compared to the host cell.

[0092] In the method of the present invention, oxidosqualene, triterpene, and / or triterpenoid At least one heterologous protein encoding at least one of the ids is involved in lupeol synthesis. Enzyme, preferably lupeol synthase of Taraxacum koksaghyz, oxidosqualene cyclization Enzyme (OSC), preferably the oxidosqualene cyclase TkOSC1 of Taraxacum koksaghyz -6, β-amyrin synthase, preferably from Arabidopsis thaliana or Artemisia annua β-amyrin synthase, terpene cyclase, preferably terpene cyclase from Glycyrrhiza uralensis The enzyme can be selected from the group consisting of guanidine cyclase (GuLUP1).

[0093] In the method of the present invention, culturing the host cells is carried out under appropriate conditions. The conditions are preferably at least 50°C, 60°C, 70°C, 80°C, more preferably at least 50°C, 60°C, 70°C, 80°C. The freeze-dried yeast cells are preferably incubated at about 80°C for 2 to 6 minutes, more preferably for about 5 minutes. Preferably, KOH in methanol and cholesterol as an internal standard are used. Add oxidosqualene, triterpenes, and / or triterpenes to each sample. For at least one extraction of terpenoids, n-hexane is preferably added. The upper extraction phase is preferably used. The sample is then redissolved in, for example, acetone. The GC-MS analysis is preferably carried out using a GC-MS-QP2010U Ultra is performed with a temperature gradient.

[0094] In another embodiment of the method of the present invention, oxidosqualene, triterpenes, and / or At least one of the triterpenoids is extracted from a plant. , oxidosqualene, triterpene, and / or triterpenoid In each embodiment described herein, the host cell may be extracted from the host cell as described herein. , or can be extracted from plants.

[0095] In other embodiments, oxidosqualene, triterpene, and / or triterpenoid At least one of the amides is preferably prepared on a C18 column in the first chromatography step. The second chromatography step was performed using a biphenyl column to purify the compound from the plant extract. It is manufactured.

[0096] The present invention also provides a method for producing oxidosqualene, triterpenes, and / or triterpenes from plants. The present invention provides a method for increasing the production of at least one penoid, the method comprising: The amino acid sequence shown in SEQ ID NO: 1 can be used as a protein, provided that the protein is capable of producing mevalonate. overexpressing 3-hydroxy-3-methylglutaryl coenzyme A reductase containing the 3-hydroxy-3-methylglutaryl coenzyme A reductase sequence, or , a protein having at least 44% sequence identity with the amino acid sequence shown in SEQ ID NO: 1. To overexpress and, Proteins contain acetoacetyl-CoA and 3-hydroxy-3-methylglutaryl-CoA , mevalonate-5-phosphate, mevalonate-5-pyrophosphate, isopentene farnesyl-5-pyrophosphate, farnesyl-pyrophosphate, or dimethylallyl SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, , 4, 5, 6, 7, and 8. or an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, and 8. The protein contains at least one amino acid sequence having at least 44% sequence identity with the sequence. modifying the plant to overexpress the protein; In addition, under appropriate conditions, oxidosqualene, triterpenes, and / or purifying at least one of the triterpenoids, Compared to the plant before modification, oxidosqualene, triterpene, and / or triterpenoid The method includes increasing the production of at least one of the steroid hormones.

[0097] In the present specification, the plant is preferably Taraxacum koksaghyz, Adiantum capillus-v eneris, Ajuga reptans, Aquilegia coerulea, Arabidopsis thaliana, Arachis hypogae a, Artemisia annua, Aster sedifolius, Aster tataricus, Avena strigose, Barbarea vulgaris, Betula platyphylla, Bruguiera gymnorhiza, Bupleurum falcatum, Catharan thus roseus, Cicer arietinum, Centella asiatica, Chenopodium quinoa, Citrullus l anatus, Citrullus colocynthis, Costus speciosus, Cucumis melo, Cucumis sativus, Cucurbita pepo, Eleutherococcus senticosus, Euphorbia tirucalli, Gentiana strami nea、Glycine max、Glycyrrhiza glabra、Glycyrrhiza uralensis、Ilex asprella、Kala nchoe daigremontiana、Kandelia candel、Laurencia dendroidea、Lens culinaris、Luf fa cylindrica, Lotus japonicus, Maesa lanceolata, Malus domestica, Maytenus ilic ifolia、Medicago truncatula、Nicotiana benthamiana、Nicotiana tabacum、Nigella s ativa, Ocimum basilicum, Olea europaea, Oryza sativa, Phaeodactylum tricornutum 、Phaseolus vulgaris、Pisum sativum、Platycodon grandifloras、Polygala tenuifoli a、Polypodiodes niponica、Panax ginseng、Pisum sativum、Rhizophora stylosa、Rici common nut、Saponaria vaccaria、Siraitia grosvenorii、Stevia rebaudiana、Solan um aculeatissimum, Solanum chacoense, Solanum tuberosum, Solanum lycopersicum, S orghum bicolor, Taraxacum officinale, Taraxacum brevicorniculatum, Taraxacum mon golicum, Taraxacum platycarpum, Triticum aestivum, Vaccaria hispanica, Veratrum californicum, Vitis vinifera, Withania somnifera, and Zea mays. will be done.

[0098] Further, in the method of the present invention, oxidosqualene, triterpene, and / or triterpene may be used. At least one purification of the terpenoids involves at least two chromatography steps. In this embodiment, the first chromatography step In this embodiment, a C18 column is preferably used for the second chromatographic It is more preferred to use a biphenyl column for the chromatography step. A C18 column was used for the first chromatography step and a bifurcation column for the second chromatography step. Even more preferably, an phenyl column is used.

[0099] C18 columns are used in HPLC (High Performance Liquid Chromatography) where C18 material is used as the stationary phase. C18 HPLC columns are used in environmental science and chemical analysis, as well as chemical analysis. It is used in industries such as pharmaceuticals and environmental science to analyze the individual components of chemical mixtures. The C18 stationary phase is not identical from one C18 HPLC column to another. C18 simply means that the molecule contains 18 carbon atoms, so the other atoms in the molecule are However, those skilled in the art will appreciate that the C18 column Knowing the properties of the compounds flowing through it allows you to select a column to achieve the desired results. C18 columns can be of various sizes and may or may not be end-capped. The hydrophobicity may be varied, and may have various particle and pore sizes, and may have various degrees of hydrophobicity. C1 may have different capacities to separate acidic and / or basic components. Such a chromatography step in the present invention using 8 columns preferably comprises: This is done using methanol as the solvent phase.

[0100] The biphenyl column as used in the present invention has biphenyl as the ligand type. Such a chromatography step using a biphenyl column is preferred. Alternatively, the method may be carried out in accordance with the present invention by using a gradient of methanol and water as the solvent phase. .

[0101] By applying the method of the present invention, oxidosqualene, triterpenes, and / or triterpenes can be obtained. At least one of each of the terpenoids is at least 70%, 71%, 72%, 73%, 74% %, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84 %, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94 %, 95%, 96%, 97%, 98%, 99%, or even 100% purity can be done.

[0102] In the method of the present invention, two or more of oxidosqualene, triterpene, and / or Alternatively, the amount of triterpenoid produced can be increased.

[0103] The term "yield" refers to the amount of a substance obtained, for example, from an engineered host cell or plant extract, respectively. Oxidosqualene, triterpenes, and / or triterpenoids described herein and increased production refers to the amount of at least one of oxidosqualene by the host cell. , triterpenes, and / or triterpenoids. The production rate may be due to the large amount of mevalonate pathway protein / g of host cell biosynthesis. The increase in production may be measured as dry cell weight or wet cell weight. In the present invention, the amount of production obtained from the modified host cell is generally compared to the amount of production obtained from the host cell before modification, i.e. This can be measured when compared to the amount produced from unmodified host cells.

[0104] The present invention in a further embodiment provides a method for producing a compound obtained by any of the methods described herein. Further provided is an extract, preferably a plant extract, capable of:

[0105] The present invention relates to a method for producing a compound comprising at least one of oxidosqualene, triterpene, and / or triterpenoid. Further provided is a recombinant host cell for producing one of the proteins, the host cell comprising: 3-amino acid having the amino acid sequence shown in SEQ ID NO: 1, provided that it is capable of producing mevalonic acid. overexpressing hydroxy-3-methylglutaryl coenzyme A reductase or Overexpressing the protein having at least 44% sequence identity with the amino acid sequence shown. and proteins are acetoacetyl-CoA, 3-hydroxy-3-methylglucose Taryl-CoA, mevalonate-5-phosphate, mevalonate-5-pyrophosphate phosphate, isopentenyl-5-pyrophosphate, farnesyl-pyrophosphate, or is capable of producing at least one of dimethylallyl-pyrophosphate, At least one amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, and 8 or selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, and 8. At least one amino acid sequence having at least 44% sequence identity with the amino acid sequence The gene is engineered to overexpress the protein containing the sequence.

[0106] As used herein, a "host cell" refers to a cell in which protein expression, and optionally protein secretion, is carried out. Such host cells are applicable to the methods of the present invention. such that the host cell overexpresses or underexpresses a polypeptide involved in the mevalonate pathway. The nucleotide sequence encoding the polypeptide is present in or introduced into a host cell. The host cells provided by the present invention may be eukaryotic or prokaryotic host cells. The preferred host cells of the present invention are eukaryotic cells. Non-mammalian eukaryotic host cells are also preferred. Also preferred are fungal host cells or yeast cells. As will be appreciated by those skilled in the art, prokaryotic cells do not have a membrane-bound nucleus, while eukaryotic cells Biological cells have a membrane-bound nucleus. Examples of eukaryotic cells are vertebrate cells, mammalian cells, human cells, and so on. Human cells, animal cells, invertebrate cells, plant cells, nematode cells, insect cells, stem cells, fungi Cells, or yeast cells.

[0107] As used herein, an "modified" host cell is one that has been genetically engineered to transform the host cell without human intervention. The host cell is engineered to "overexpress" a given protein. When "altered," the host cell is preferably a host cell that has the ability to regulate gene expression. or mevalonate pathway genes and / or proteins or their functional homologs. engineered to overexpress a given protein, thereby increasing the expression of that protein under the same conditions as before the engineered The increase in the number of host cells is compared to that of the control cells.

[0108] The host cells of the present invention include Saccharomyces cerevisiae, Nicotiana benthamiana, Pichia pastoris, storis, Pichia methanolica, Schizosaccharomyces pombe, Kluyveromyces lactis, Klu yveromyces marxianus, Pichia stipitis, Candida albicans, Candida utilis, and BY2 The host cell is selected from the group consisting of cells.

[0109] The host cells of the present invention are capable of converting proteins into 3-hydroxy-3-methylglutaryl-CoA. Contains SEQ ID NO: 3 or is at least 44% identical to SEQ ID NO: 3, provided that it can be produced. The protein is further modified to overexpress the protein containing an amino acid sequence having sequence identity with It can be changed.

[0110] The host cell of the present invention can be used in any suitable manner, provided that the protein is capable of producing lanosterol. Preferably, the sequence is enhanced by inserting a CTR3 promoter and / or adding copper sulfate (CuSO4). Inhibiting lanosterol synthase (ERG7) containing the amino acid sequence shown in SEQ ID NO: 9, or Inhibits proteins containing an amino acid sequence with at least 34% sequence identity to sequence number 9 In a preferred embodiment, the sequence can be further modified to include a palindrome. The sequence containing TTTGCTC(A / G) ... (T / C)GAGCAAA is required for copper transcriptional regulation (Yamaguchi- Iwai et al., 1997; Jamison McDaniels et al., 1999; Labbe et al., 1997).

[0111] The host cell is a recombinant vector encoding ROX1 (SEQ ID NO: 25), BTS1 (SEQ ID NO: 54), YPL062W (SEQ ID NO: 55), DOS2 (SEQ ID NO: 56), YER134C (SEQ ID NO: 57), VB A5 (SEQ ID NO: 58), YNR063W (SEQ ID NO: 59), YJL064W (SEQ ID NO: 6 0), and at least one selected from the group consisting of YGR259C (SEQ ID NO: 61). Preferably, the host cell is further modified to knock out the ROX1 locus. (SEQ ID NO: 25), YPL062W (SEQ ID NO: 55), DOS2 (SEQ ID NO: 56), YE R134C (SEQ ID NO: 57), VBA5 (SEQ ID NO: 58), YNR063W (SEQ ID NO: 5 9), YJL064W (SEQ ID NO: 60), and YGR259C (SEQ ID NO: 61) Further modified to knock out at least one locus selected from the group is more preferable.

[0112] At least one of oxidosqualene, triterpene, and / or triterpenoid , oxidosqualene, preferably 2,3-oxidosqualene, sterols, preferably is sigmasterol or sitosterol, a triterpene, preferably a five-membered ring triterpene and more preferably, but not limited to, lup-19(21)-en-3-ol and lup- Lupeol, β-amyrin, α-amyrin, taraki, etc., such as 20(29)-en-3-ol Susterol, triterpene acetate, acylated triterpenes, saponins, sapogenins , lup-19(21)-en-3-one, lup-20(29)-en-3-one , Taraxerol, Taraxerone, α-amyron, β-amyron, Taraxasterone, Leadin, betulin, betulinic acid, cholesterol, ergosterol, lanosterol Glucocorticoids, mineralocorticoids, estrogens, progesterone, and steroids Nolides, bufadienolides, steroid alkaloids, saponins, saponins Genin or an acylated triterpene is preferred for the host cell of the present invention. In one particular embodiment of the host cell, oxidosqualene, triterpenes, and / or At least one of the triterpenoids is not glycyrrhetinic acid as described above.

[0113] Additionally, all embodiments described herein regarding the methods of the present invention are suitable for host cells. is the embodiment used, and vice versa.

[0114] The present invention also provides a method for producing oxidosqualene, triterpenes, and / or triterpenoids. The present invention resides in the use of a host cell of the present invention for producing at least one

[0115] For CuSO4-mediated transcriptional repression to inhibit lanosterol synthase (ERG7) By using the CTR3 promoter (SEQ ID NO: 42), the inventors were able to This resulted in lower costs compared to the conventional process (Paddon et al., 2013).

[0116] That is, the inventors herein provide oxidosqualene, triterpenes, and / or provide a new platform for the synthesis of at least one triterpenoid For example, the inventors have demonstrated that overexpression of the proteins encoded by these genes The MVA pathway genes ERG13 (SEQ ID NO: 14) and tHMGR (SEQ ID NO: 32) Overexpression, disruption of ROX1 (SEQ ID NO: 25), and ER via the CTR3 promoter This platform combines the copper-regulated regulation of G7 (SEQ ID NO: 20). The inventors of the present invention aim to increase the productivity of the MVA pathway while also improving the metabolism of the late sterol biosynthesis pathway. to change the flow of oxidosqualene, triterpenes, and / or triterpenoids At least one production could be increased by up to 127-fold.

[0117] For example, the inventors have demonstrated that ERG13 (sequence no. 13131313), a key enzyme in the MVA pathway, is expressed in yeast. The enzymes used in the experiments were overexpressed: HMG-CoA reductase 1 (tHMGR) (SEQ ID NO: 14) and HMG-CoA reductase 1 (tHMGR) (SEQ ID NO: 1). In the modification step, for example, the inventors have deleted the negative regulators of the pathway and sterol biosynthesis. (ROX1), creating a push-pull mechanism to increase metabolic flow in the system. For example, the inventors have investigated the increased metabolic flow of this late stage sterol biosynthesis. This leads to the formation of 2,3-oxidosqualene, the direct precursor of five-membered triterpenes. By this method, the inventors were able to produce up to 127 times the amount of five-membered triterpenes. The lupeol synthase of the Russian dandelion, Taraxacum koksaghyz, is a second enzyme that can synthesize lupeol. The model enzyme TkLUP was detected in the product.

[0118] This means that, in one embodiment, the present invention increases the amount of lupeol produced in a host cell. The method includes the steps of: The amino acid sequence shown in SEQ ID NO: 1 can be used as a protein, provided that the protein is capable of producing mevalonate. overexpressing 3-hydroxy-3-methylglutaryl coenzyme A reductase containing the 3-hydroxy-3-methylglutaryl coenzyme A reductase sequence, or , a protein having at least 44% sequence identity with the amino acid sequence shown in SEQ ID NO: 1. To overexpress and, Proteins contain acetoacetyl-CoA and 3-hydroxy-3-methylglutaryl-CoA , mevalonate-5-phosphate, mevalonate-5-pyrophosphate, isopentene farnesyl-5-pyrophosphate, farnesyl-pyrophosphate, or dimethylallyl SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, , 4, 5, 6, 7, and 8. or an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, and 8. The protein contains at least one amino acid sequence having at least 44% sequence identity with the sequence. modifying the host cell to overexpress the protein; and, modifying the host cell to knock out the ROX1 (SEQ ID NO: 25) locus; and, the amino acid sequence shown in SEQ ID NO: 9, provided that the protein is capable of producing lanosterol. or an amino acid sequence having at least 34% sequence identity with SEQ ID NO: 9. modifying the host cell to inhibit lanosterol synthase (ERG7); and, The host cells were transformed to express the T. koksaghyz lupeol synthase TkLUP (SEQ ID NO: 12). modifying the cells, and causing the host cell to express at least one heterologous protein to produce lupeol. To modify, Culturing the host cell under appropriate conditions to express lupeol; In addition, by refining lupeol, The present invention also includes increasing the amount of lupeol produced compared to the host cell before modification. do.

[0119] That is, in an embodiment of the present invention, for example, in order to enhance the production of five-membered ring triterpenes, Modified platforms are provided, for example, tHMGR (SEQ ID NO: 32) and ERG1 A yeast platform for overexpression of 3 (SEQ ID NO: 14) is provided. For example, knocking out ROX1 (SEQ ID NO: 25) can be used to inhibit oxidosqualene, triterpenoids, and Production of at least one of pens, and / or triterpenoids and late sterol biosynthesis In certain embodiments, T. koksaghyz lupeol synthase (TkLUP, synthase) Substance 12 induces a metabolic shift from late sterol biosynthesis to five-membered triterpene synthesis. Thus, for example, using the CTR3 promoter (CTR3-P) (SEQ ID NO: 42), Copper (Cu 2+ Upon addition of 2,3-oxidosulfonyl-2-(2,3-dihydro- ... A further effect on qualene accumulation is seen in the sterol content (lanosin) in this embodiment. ERG9 (SEQ ID NO: 49) and ERG1 due to the reduction of steroid hormones (steroids and ergosterol) (SEQ ID NO: 51).

[0120] As used herein, the singular forms "a," "an," and "the" are used interchangeably where the context requires. It is noted that the plural is also included unless expressly indicated otherwise. Thus, for example, Reference to a "reagent" includes one or more of such various reagents, and reference to a "method" includes reference to a method of the present invention. Equivalent steps and methods known to those skilled in the art that may be modified or substituted in the methods described herein. Furthermore, for example, when referring to a "host cell," it includes one or more Similarly, for example, when referring to a "method" or "host cells," , a host cell or a method, respectively.

[0121] Unless otherwise indicated, the term "at least" before a series of elements refers to the number of elements in the series. It should be understood that the present invention is not limited to the specific embodiments described herein. Many equivalents to the embodiments of the present invention will be recognized, or can be ascertained using no more than routine experimentation. Such equivalents are intended to be encompassed by this invention.

[0122] The term "and / or" as used anywhere in this specification means "and," "or," "or" " and "all or any other combination of the components connected by the term" For example, A, B, and / or C includes A, B, C, A+B, A+C, B+C, and A. +B+C.

[0123] In this specification and the claims that follow, unless the context requires, " The words "including" and variations such as "including (third person)" and "including" are used interchangeably. means to include any component or step or group of components or steps that does not exclude any component or step or group of components or steps It should be understood that, as used herein, the term "including" means "including" The term "having" or "including" is interchangeable with the term "having" or "including" and is used herein. As used herein, "from" can be used interchangeably with "having." "Consisting of" excludes any unspecified component, step, or ingredient.

[0124] The term "including" means "including but not limited to." and "including but not limited to" are used interchangeably.

[0125] This invention relates to the specific methodology, protocols, materials, reagents, and substances described herein. It should be understood that the present invention is not limited to the above and may therefore vary. The terminology used is used only for the purpose of describing particular embodiments and is intended to be used only in the claims. It is not intended to limit the scope of the invention, which is defined by the appended claims.

[0126] All publications cited in the text of this specification (including all patents, patent applications, scientific publications, All such materials, whether set forth above or below, including instruction manuals, are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the present invention has antedated such disclosure by virtue of prior invention. Nothing in this document shall be construed as an admission that no right to exercise any right whatsoever is granted in connection with any provision of this document. To the extent that the cited material contradicts or is inconsistent with this specification, this specification supersedes any such material. This document will replace the previously published material.

[0127] As used herein, the terms "about" or "approximately" refer to an inaccuracy of 20% of a given value or range. %, preferably 10% or less, more preferably 5% or less. Typical values, for example, about 20, include 20.

[0128] Furthermore, in describing exemplary embodiments of the present invention, the present specification refers to the methods and / or methods of the present invention. Or they may present a process as a sequence of specific steps. However, the method or process may not be construed as relying on the particular order of steps set forth herein. To the extent possible, the method or process is not limited to the particular sequence of steps described. As one skilled in the art would understand, other sequences of steps are possible. Therefore, the particular order of steps set forth in the specification may not limit the scope of the claims. Furthermore, nothing in this document should be construed as constituting a limitation on the scope of the present invention. The claims should be limited to performing those steps in the order recited. The sequence may be changed and still fall within the spirit and scope of the present invention. It is easy to understand what it means to stay within.

[0129] A better understanding of the present invention and its advantages may be obtained by reference to the following examples, which are provided for illustrative purposes only. The examples are not intended to limit the scope of the invention in any way. stomach. [Example]

[0130] The present invention will now be described in detail with reference to examples. The following examples are provided to illustrate the present invention. These are intended only as examples and are not intended to limit the scope of the present invention.

[0131] Materials and Methods Example 1: Cloning of constructs To generate pAG424Gal-TkLUP, TkLUP (SEQ ID NO: 12) (GenBa The coding sequence of the NK MG646375 was amplified using the fw-primer 5´-AAA (GTC GAC) TAA AAA AAT GTG GAA G CT GAA AAT AGC-3' (SEQ ID NO: 23) and bw-primer 5'-AAA (CTC GAG) ATA TAT TTT PCR amplification of T. koksaghyz cDNA with GAA CAA TAC GA-3' (SEQ ID NO: 24) Purification, digestion, and ligation into pENTR3c (Invitrogen, Carlsbad, USA) were performed. The TkLUP coding sequence (SEQ ID NO: 12) was then recombined into pAG42 4Gal1-ccdB (Alberti et al. 2007; Addgene, Cambridge, USA) .

[0132] To generate pESC-rox1-KlUra3_tHMGR / ERG13, ROX 1 coding sequence (SEQ ID NO: 25) was amplified from a yeast DNA template using the fw-primer 5´-AAA ( GCG GCC GC)A TGA ATC CTA AAT CCT CTAC-3' (SEQ ID NO: 26) and bw-primer 5'-AA A (GCG GCC GC)T CAT TTC GGA GAA ACT AGG-3´ (SEQ ID NO: 27) (The restriction site is in parentheses. PCR amplification was performed using pESC-Ura (Agilent Technologies, San Diego, CA). Using the DNA fragment (Taklara, USA) as a template, the fw-primer 5´- AAA (GCG GCC GC)C CAG CTG CAT TAA TGA ATC G (SEQ ID NO: 28), bw-primer 5´- AAA (GCG GCC GC)G pESC containing a NotI restriction site using AAG TTC CTA TTC TCT AGA AA (SEQ ID NO: 29) The -Ura vector backbone was amplified. Both NotI digested fragments were ligated. This vector was digested with BglII and cloned into pESC-rox1. Marker cassette (SEQ ID NO: 30) (Gueldener et al., 2002) and AsiSI / Nb.B Synthetic DNA consisting of the smI USER cassette (SEQ ID NO: 31) (Hansen et al., 2012) The fragment (Invitrogen, Carlsbad, USA) was inserted into pESC-rox1-Kl Ura3 was obtained. In parallel, tHMGR (SEQ ID NO: 32) and ERG13 (SEQ ID NO: 14) ) coding sequence from a yeast DNA template, and for tHMGR, fw-primer 5'-AAA (GGA TCC) AAA AAA ATG GTT TTA ACC AAT AAA AC-3 (SEQ ID NO: 33) and bw-primer -5'- AAA (GTC GAC) TTA GGA TTT AAT GCA GGT GAC (SEQ ID NO: 34), and ERG1 3 fw-Primer 5`- AAA (GAA TTC) AAA AAA ATG AAA CTC TCA ACT AAA CTT TG-3´ (SEQ ID NO: 35) and bw-primer5´- AAA (GCG GCC GC)T TAT TTT TTA ACA TCG TAA GAT C-3' (SEQ ID NO: 36) was used for PCR amplification. The resulting fragment was then amplified with BamH Digested with I / SalI and EcoRI / NotI and ligated into pESC-Ura pESC-Ura_tHMGR / ERG13 was generated according to the manufacturer's protocol. The NotI restriction site was then inserted using the QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, USA). GR (SEQ ID NO: 32) and ERG13 (SEQ ID NO: 14) coding sequences, and the bidirectional Ga Expression containing the l1 / Gal10 promoter and ADH-1 / CYC-1 terminator The cassette was synthesized using the fw-primer 5´-CGT GCG A{U}T CAG AGC GAC CTC ATG CTA TAC -3´ (sequence Sequence number 37) and bw-primer 5'-CAC GCG A{U}C TTC GAG CGT CCC AAA ACC -3' (sequence Column 38) was used for PCR amplification (uracil-based cloning for users, wave (bracketed part), uracil-specific excision reaction (USER)-based cloning was used to remove AsiSI. The vector was cloned into pESC-rox1-KlUra3.

[0133] To generate pESC_ERG7-P_KlLeu2_CTR3-P_ERG7, In one step, the ERG7 fragment (SEQ ID NO: 39) was amplified by PCR with the fw-primer 5'- [CAC ATT TAA GGG CTA TAC AAA G]AT GAC AGA ATT TTA TTC TGA CA-3´ (SEQ ID NO: 40) (The brackets indicate the overlapping region) and bw-primer 5´-AAA (GCG GCC GC)C CCA ATA AAC GTA AGA TTA CA-3' (SEQ ID NO: 41) was amplified from yeast DNA and used as the CTR3 promoter. The target fragment (SEQ ID NO: 42) was synthesized using the fw-primer 5'- AAA (GCG GCC GCC AGC TG)A A(GG ATC C)GG TAT TCC AAT GAG AAT CGC-3' (SEQ ID NO: 43) and bw-primer 5'-[T GT CAG AAT AAA ATT CTG TCA T]CT TTG TAT AGC CCT TAA ATG T-3´ (SEQ ID NO: 44) Both fragments were amplified using the CTR3 promoter by overlap PCR. -fw-primer (SEQ ID NO: 43) and ERG7-bw primer (SEQ ID NO: 41) Fuse the fragment into the NotI-digested pESC-Ura vector backbone. The resulting vector was ligated to obtain pESC_CTR3-P_ERG7. The ERG7-promoter fragment (SEQ ID NO: 45) was amplified using the fw-primer 5'-AAA (CA G CTG) AAT CTG CTG CTA TTC GTG-3' (SEQ ID NO: 46) and bw-primer 5'-AAA (GGA TCC CCT GCA GG)C GCT GCA GGT CGA CAA C-3' (SEQ ID NO: 47) was used to PCR amplify the sequence in yeast DNA. pESC_ ERG7-P_CTR3-P_ERG7 was obtained. A synthetic DNA fragment containing a tropism cassette (SEQ ID NO: 48) (Gueldener et al., 2002) (Invitrogen, Carlsbad, USA) was ligated into the SbfI / BamHI restriction sites. This resulted in pESC_ERG7-P_KlLeu2_CTR3-P_ERG7.

[0134] All constructs were analyzed using an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems). The yeast strain CE was verified by sequencing in the CE10 ... N.PK2-1C was obtained from EUROSCARF (Oberursel, Germany). New England Biolabs Restriction enzymes were obtained from GmbH (Frankfurt, Germany).

[0135] Example 2: Strain construction and culture conditions S. cerevisiae strain CEN-PK2-1C was transformed with uracil (pCFB255_rox1 _KlUra3_tHMGR / ERG13, SEQ ID NO: 11), leucine (pCFB255_ERG7-P_KlLeu2_CTR3-P_ERG7, SEQ ID NO: 10), and tryptophan (pAG424Gal-TkLup) were used to transfect the plasmid pAG424Gal-TkLup using the lithium acetate method (Gietz 2 007). For stable integration into the yeast genome, pCFB255_ro x1_KlUra3_tHMGR / ERG13 (SEQ ID NO: 11) and pCFB255_E RG7-P_KlLeu2_CTR3-P_ERG7 (SEQ ID NO: 10) was digested with NotI. The plasmid backbone was removed by cloning the cells in minimal synthetic medium (SD medium, Clontech, Mouse The cells were plated on a plate (Centenvue, USA) and grown at 30°C. Clones were identified as healthy by colony PCR using primers on either side of the construct. I checked it out.

[0136] For galactose-inducible gene expression, a single colony was picked and cultured in 5 mL of SD medium. The culture was inoculated into 50 mL of fresh medium and grown overnight at 30°C with rotation. SD medium (containing 150 μM CuSO4 when suppressing the expression of ERG7 (SEQ ID NO: 20)) was 0 5 Add 100 ml of PBS to a final cell density of 100 cells / mL in a 250 mL Erlenmeyer flask. The culture was grown at 30°C and 140 rpm. 6 cells / m When the cell density reached 100 μg, the medium was replaced with SD medium containing galactose instead of glucose. In turn, gene expression was induced. 6 The yeast was incubated until a cell density of 100 cells / mL was reached. The mother was grown and harvested by centrifugation (10 min, 1000 xg).

[0137] Example 3: Extraction and measurement of squalene and triterpenes Extraction of yeast metabolites was performed according to the protocol of Rodriguez et al. (2014). The freeze-dried yeast cells were then frozen in 1 mL of 6% [w / v] KOH (Roth, Carlsbad, CA) in methanol. -He, Germany) and 100 μg cholesterol (as an internal standard, Sigma, St. Louis, MO). After adding HCl (Bio-Rad, USA) to each sample, the sample was incubated in a water bath at 80°C for 5 minutes. To extract metabolites from the mixture, 2 ml of n-hexane (Roth, Karlsruhe, Germany) was added. The upper phase was transferred to a new vial and n-hexane was added in three steps after vortexing. The solvent was removed by evaporation. The sample was then resuspended in 1 mL of acetone (Roth, Karlsruhe, Germany). The sample was redissolved in 30 ml of Rtx-5M (Germany) and subjected to GC-MS. GC-MS-QP 2010 Ultra equipped with an S column (Shimadzu, Duisburg) , Germany) with a temperature gradient (120-330°C; 21°C per min; pressure: 58.8 kPa). A 1 minute hold time at 120°C followed by a 10 minute hold time at 330°C was used. Amount 43m / z, 55m / z, 69m / z, 95m / z, 109m / z and 189m / z, 204m / z, 207m / z, 218m / z, 271m / z, The molecules were detected at 285 m / z and 411 m / z using LabSolution software (Shimadzu, Peak integration and identification were performed by the NIST library (Duisburg, Germany). The total ion current (TIC) of the extracted substance was calculated based on the cholesterol derived from the internal standard. The data were normalized to the dry weight of the samples used. Two-sample t-tests were performed. The statistical significance of the results was confirmed using p<0.05.

[0138] Example 4: Identification of TkLUP as the lupeol synthase in T. koksaghyz As a model enzyme for the proposed yeast system, we used a hydroxylase enzyme derived from the rubber-producing dandelion, Taraxacum koksaghyz. The lupeol synthase from T. officinale was selected (see Figure 6). The primers used to amplify a certain TRX (Shibuya et al., 1999) produced a 759 amino acid fragment. 22 of the cDNA of T. koksaghyz (GenBank accession number MG646375) encoding (SEQ ID NO: 12) A 77-bp ORF was amplified. This sequence is consistent with the previously described lupeol synthase. Since it has 9.3% amino acid sequence similarity, it was cloned into pE The vector was cloned into NTR3c and expressed in the CEN.PK2-1C yeast strain (WT). Advanced Gateway Destination Vector System (pAG-Vector System) The resulting sequence was then transformed into pAG4, which allows expression under the control of the GAL1 promoter. The construct was recombined into 24GAL1-ccdB. A schematic diagram of the construct is shown in Figure 6a. The vector pAG424GAL1-ccdB was used as a vector control in the expression experiments. After cultivation and triterpene extraction, two additional m / z 218 fragments were identified in the putative Extracts from three individual transformants containing the peol synthase sequence (SEQ ID NO: 12) These peaks were identified in GC-MS analysis (see Figure 6b). - It could not be identified even in the control sample. Since the retention times match those measured in phosphorus and lupeol standards (Extrasynthetic , Genet, France; Figure 1b), the resulting fragments contained traces of unquantifiable amounts of β-amylimidazole. The accumulation of 0.16 mg / gCDW of lupeol and 0.16 mg / gCDW of methicone was estimated (Fig. 6c). Based on sequence homology and a major identifying peak that likely represents lupeol, the resulting sequence The lupeol synthase from T. koksaghyz, designated TkLUP (SEQ ID NO: 12), was cloned. They concluded that it would be better to

[0139] Example 5: Deletion of ROX1 and overexpression of tHMGR and ERG13 inhibits lupeol storage Productivity increases by 16.5 times To further enhance the production of putative lupeol in yeast, we previously investigated the effect of lupeol production in a heterologous yeast system. Overexpression of yeast mevalonate pathway genes was shown to enhance isoprenoid production in (Kirby et al., 2008; Asadollahi et al., 2010; Paddon et al., 201 3) HMG1 is a key enzyme in the pathway and is the basis for strict control by a feedback mechanism. Therefore, the lack of ubiquitination signaling leads to truncation that does not provide a basis for a feedback mechanism. The recombinant form (termed tHMGR) (SEQ ID NO: 32) was overexpressed (DeBose-Boyd, 2008). and ERG13 (SEQ ID NO: 1), which has also been shown to have a positive effect on isoprenoid biosynthesis. We chose to overexpress 4) (Yuan et al., 2014). To upregulate / increase the mevalonate pathway and late sterol synthesis, the inventors knocking out ROX1 (SEQ ID NO: 25), which has been described as a negative regulator of the synthesis (Henry et al., 2002; Montanes et al., 2011; Ozaydin et al., 2013; Jakociun as et al., 2015), down-regulation of squalene by enhancing late sterol biosynthesis Regarding the improvement of mevalonate metabolism, the mevalonate pathway itself is deregulated. The aim was to enhance the flow in the transport route. tHMGR (SEQ ID NO: 32) and ER under the control of the tropic GAL1 / GAL10 promoter G13 (SEQ ID NO: 14) was selected to express the overexpression cassette, rox1 (SEQ ID NO: 15) Therefore, NotI-digested pCFB 255_rox1_KlUra3_tHMGR / ERG13 (SEQ ID NO: 11) The resulting fragment was transformed into CEN.PK2-1C yeast cells (Figure 7a). As shown, after expression of the integrated gene and knockout of rox1 (SEQ ID NO: 25), T Compared to cells with only the kLUP sequence (SEQ ID NO: 12), the construct and TkL In yeast containing the UP sequence (SEQ ID NO: 12), a significant 8.2-fold increase in squalene accumulation was observed. Furthermore, a 16.5-fold increase in the accumulation of the designated lupeol was detected (Fig. 7b). An increase in lanosterol content was observed (Fig. 7b), with a 3.6-fold increase in lanosterol content (see below). See Table 1 below).

[0140] As per the data previously described, yeast cells were grown at a cell density (material) after induction and before cell harvest. The results were slightly lower because it took longer to reach the target concentration (described in Materials and Methods). proliferation, which occurs as a result of high levels of squalene, which is toxic to cells. (Donald et al., 1997; Asadollahi et al., 2010). The modification step was performed to compare the estimated lupeol mass spectrum with that of the measured lupeol standard (Figure 7d). The mass spectra of the peol peaks (Figure 7c) allowed for comparison. Equivalent peak masses were The observed coding sequence encodes the lupeol synthase of T. koksaghyz. The predicted β-amyrin peaks are not yet fully identified by detailed mass spectrometry. It was very weak.

[0141] The five-membered ring is not 2,3-oxidosqualene, which is the substrate of TkLUP (SEQ ID NO: 12). At this point in the pathway, accumulation of the triterpene precursor squalene could be observed. A bottleneck was observed in ERG1 compared with ERG9. (Asadohalli et al., 2010) and the 2,3-oxidase activity of TkLUP itself. It is thought to be caused by its high activity towards squalene or by its activity in endogenous late sterol biosynthesis. To overcome this limiting factor, ERG1 (squalene to 2,3-oxidoreductase) Overexpression of the gene encoding yeast squalene epoxidase, which catalyzes the reaction to qualene Although the accumulation of 2,3-oxidosqualene in yeast strains overexpressing ERG1 is thought to be a factor, Therefore, it is not suitable for improving the production of five-membered triterpenes (Veen et al. l., 2003). Therefore, we aim to investigate the effect of endogenous but competitive late sterol biosynthesis on the Downregulation of ERG7 (SEQ ID NO: 20), the initiation site of late sterol biosynthesis I installed it.

[0142] Example 6: CTR3 promoter-mediated repression of ERG7 (SEQ ID NO: 20) -Oxidosqualene accumulation, resulting in 7.6 times increased lupeol accumulation Knockouts in essential late sterol biosynthesis lead to lethal yeast strains and ER Overexpression of G1 increased 2,3-oxidosqualene accumulation in the production of five-membered triterpenes. Therefore, the inventors have identified a bottleneck in the synthesis of 2,3-oxidosqualene. To overcome this block, ERG7 (SEQ ID NO: 20) was inhibited to remove the loop from late sterol biosynthesis. The metabolic flux to peol production was altered. Therefore, the endogenous ERG7 coding sequence (SEQ ID NO: 1) 20) preceded by a 735 bp promoter fragment of the copper transporter (CTR3) (SEQ ID NO: 42) In the same step, a 196 bp fragment of the endogenous core promoter was deleted. The inserted CTR3 promoter fragment (SEQ ID NO: 4) In 2), two cis-elements (TTTGCTC, copper response element, abbreviated as CuRE) are involved in the regulation of CuSO It has been shown to cause decreased gene expression in the presence of 4 ( Labbe et al., 1997 ). Furthermore, the CTR3 promoter fragment (SEQ ID NO: 42) inhibits artemisinin production. This industrial process can be appropriately used to inhibit ERG9 (SEQ ID NO: 49) to enhance The MET3 promoter (sequence number 10 ... column number 50) (Paddon et al., 2013).

[0143] To test the efficacy of the constructs, KlLeu2 and CTR were used as selectable markers. pCFB255_ERG7-P_K containing the 3 promoter fragment (SEQ ID NO: 42) The NotI-digested fragment of lLeu2_CTR3-P_ERG7 (SEQ ID NO: 10) was pCFB255_rox1_KlUra3_tHMGR / ERG13 (SEQ ID NO: 11) Positive transformants were selected from the 0 μM, 150 μM, and 375 μM tHMGR (SEQ ID NO: 32) and ERG13 (SEQ ID NO: 33) were cultured in the presence of 1000 mg of CuSO4. 14) gene expression was induced by galactose to convert squalene to 2,3-octadecane in yeast. This resulted in a shift to the accumulation of hydroxydosqualene (Fig. 8b). A slight accumulation of 2,3-oxidosqualene was detected in the GC-MS samples. , a weaker CTR3 fragment compared to the endogenous ERG7 promoter (SEQ ID NO: 45). The promoter activity of the nucleotide sequence ... This effect was even stronger in yeast that had been treated with 2,3-oxidosqualene. A 4.7-fold increase in squalene accumulation (p=0.00507) was associated with a significant decrease in squalene (p=0.00613). This is due to the ergosterol-induced ERG9 (SEQ ID NO: 49) and ERG1 (SEQ ID NO: 51) Lack of repression of ERG1 (SEQ ID NO: 51) and induction of ERG1 under limiting lanosterol conditions This may be due to improved conduction (Table 1; M´Baya et al., 1989). After yeast incubation, a further significant decrease in squalene levels (p=0.00061) was observed. However, no significant change in 2,3-oxidosqualene was observed. However, the yeast showed a reduced growth rate, indicating a bottleneck in 2,3-oxidosqualene synthesis. To overcome the toxicity of high levels of CuSO4 and in the presence of 150 μM CuSO4 Thus, the inventors demonstrated that rox1 significantly suppressed ERG7 (SEQ ID NO: 20) expression. ::P GAL1 -tHMGR P GAL10 -ERG13 (SEQ ID NO: 11)P erg7 Δ ::P CTR3 (SEQ ID NO: 10) and the coding sequence of TkLup (SEQ ID NO: 12) or In the strains in which the control vector pAG424GAL1_ccdB was combined with the empty plasmid To enhance the production of lupeol in the sucrose solution, a CuSO4 concentration of 150 μM was used. was selected.

[0144] As expected, yeast carrying the three constructs and grown in the presence of copper The strain was compared to its parent strain lacking the CTR3 promoter fragment (SEQ ID NO: 42). The levels of squalene and 2,3-oxidosqualene were altered by the diet. Accumulation of 2,3-oxidosqualene was 2.6-fold less (p=0.04422), and accumulation of 2,3-oxidosqualene was observed. Furthermore, lanosterol (6.5 times; p = 0.02384) and ergosterol (3 A decrease in the amount of sterols was detected, as indicated by the expression of TkLUP (SEQ ID NO: 12) (0.9-fold; p=0.00941). ), as well as the function of copper repression by these compounds in strains expressing ERG9 (SEQ ID NO: 49) and ERG1 (SEQ ID NO: 51). Furthermore, the use of copper-repressible promoters Further enhancement of lupeol accumulation by the use of β- Comparison of the MS spectra of the external amylin standard (Figure 10b) indicates that β-amylin Lupeol levels increased 7.6-fold (p=0.00637), while β -Quantitation of amylin was not yet possible. However, 2,3-oxidosqualene The accumulation of lupeol and lupeol, and the decrease in the amount of sterols, were due to the sterol-rich phenotype in this modified yeast strain. This indicates that the metabolic flow has changed from lupeol biosynthesis to lupeol production.

[0145] Table 1 shows the metabolites of the WT and engineered yeast strains in g / g CDW quantified by GC-MS. Indicates the level. [Table 1] TIFF0007824920000002.tif104165

[0146] g / gCDW (± standard deviation). Standard deviation is calculated by Student's t-test using n=3 data. Calculated from individual transformants. nd = not detectable. CDW = cell dry weight.

[0147] Example 7: Triterpene purification by HPLC Shimadzu LC connected to a UV detector (SPD-M20A) and a fraction collector (FRC-10A) Semi-preparative HPLC was performed using a 20A HPLC system (Shimadzu, Duisburg, Germany). UltraC18 column (250 x 21.2 mm, particle size: 5 μm, Restek GmbH, Bad Homburg) Triterpene was used with methanol as the solvent at a flow rate of 10 ml / min. The pen was separated. The column oven temperature was set to 40°C. Detection was performed at 205 nm. The terpene fraction was collected and evaporated using a Rocket evaporator system (Thermo Fisher Scientific). The samples were dried using a HPLC Scientific, dissolved in acetone, and analyzed by GC-MS. As the second stationary phase, an Ultra biphenyl column (250 x 21.2 mm, particle size: 5 The column oven temperature was set to 40 °C. The triterpenes were eluted with methanol at a flow rate of 8 ml / min using the following elution profile: The separation was performed in a gradient of ethanol (A) and water (B): 0-25 min, isocratic 90% A: 25-71 min, linear 90%-100% A; 71-75 min, isocratic 100% A; Post-column re-equilibration: 75–76 min, linear 100%–90% A; 76–85 min, isocratic 90% A.

[0148] [Chemical analysis] Triterpenes were quantified and identified by CG-MS as previously described (Putter et al., 2017).

[0149] By the above-mentioned method, the inventors have found that the natural rubber in the acetone extract of the roots of T. koksaghyz A detailed analysis of the five-membered triterpenoid composition was performed. HPLC-based purification of the compound revealed the novel pentacyclic triterpene lup-19(21)-e n-3-ol and its corresponding five-membered triterpenoid ketone lup-19 (21) -en-3-one was identified.

[0150] Taraxacum koksaghyz natural gum acetone extract reveals triterpene composition With the exception of the main component poly(cis-1,4-isoprene), natural rubber is a polymer It contains additional substances such as proteins, fatty acids, and triterpenes that affect sexual function (X u et al., 2017). To obtain a detailed overview of the single triterpenes involved in NR properties, The lipid fraction was extracted from T. koksaghyz NR using acetone as the solvent. The extract was separated by HPLC on a C18 column and analyzed using UV detection at 205 nm. Three major fractions were observed (Fig. 12a). The fractions were then analyzed by GC-MS. F1 lupeol (lup-20(29)-en-3-ol), F4 taraxasterol and β-amyrin, as well as F6 α-amyrin, which is described as being highly abundant in the roots of Taraxacum species (Po (St et al. 2012) Various five-membered triterpenes were detected. In addition to alcohols, these Four ketone derivatives of five-membered triterpenes, namely lupenone (lup-20(29)-en-3-o ne), taraxasterone and β-amyron in F5, and α-amyron in F7 also have three forms. Furthermore, in two fractions (F3 and F5), Taraxacum Two sterol compounds previously described as present in the root wood of the species, stigmaste ol and sitosterol were detected (Post et al. 2012).

[0151] Using a second stationary phase (biphenyl column) in HPLC, the inventors of the present invention obtained the following results: The single triterpenes can be further separated from each other as shown in F4 and F5 in Fig. 2b. In addition to taraxasterol and β-amyrin, a third, currently unknown triterpenoid was also identified. The pen was detected in F4 and the corresponding ketone in F5 (GC-MS data In Figure 12c, β-amyrin, taraxasterol, and their ketones are shown as examples. Approximately 1.5 mg of the pure triterpene and 0.3 mg of the corresponding ketone were analyzed by NMR. Purified for analysis, the currently unknown five-membered triterpene lup-19(21)-en-3-o l and its corresponding five-membered triterpenoid ketone lup-19(21)-en-3 -one was identified (Figure 12d).

[0152] All triterpenoids found in the acetone extract are summarized in Table 2 below.

[0153] Additional trace compounds were detected in fractions F2, F3, F5, F6, and F7. and their GC-MS profiles allowed them to be classified as triterpenoids. However, the detailed molecular structures of eight of these compounds are still unknown. Due to the limited amount of triterpenes, NMR analysis was not possible.

[0154] Table 2 shows the compounds identified in the NR acetone extract and their corresponding concentrations in the C18 HPLC fraction. The triterpenoids are classified according to their occurrence. [Table 2]

[0155] Herein, the inventors of the present invention demonstrate that in heterologous systems such as yeast, At least one of squalene, triterpene, and / or triterpenoid, e.g., a five-membered We established a new platform for producing cyclic triterpenes. The lupeol synthase of ksaghyz (SEQ ID NO: 12) was used as a model enzyme.

[0156] This platform allows for, for example, overexpression of mevalonate pathway genes and preferably It is based on a push-pull approach, involving deletion of negative regulators of the pathway and late sterol biosynthesis. Furthermore, the inventors have demonstrated that a copper-regulated promoter induces a post-transcriptional pathway that initiates the formation of lanosterol. Direct five-membered triterpene precursor 2,3-oxidosqua from primary sterol biosynthesis By shifting the metabolic flow towards the production of benzophenones, for example, increasing the production of five-membered triterpenes. was created.

[0157] In one embodiment, the inventors have identified the MVA pathway gene ERG13 (SEQ ID NO: 14) and We chose to overexpress tHMGR (SEQ ID NO: 32). HMGR is a key step in the pathway. tHMGR catalyzes the synthesis of tHMGR and its deregulated form, tHMGR, on various yeast platforms. It has been used to enhance squalene accumulation and isoprenoid production in mice (K irby et al., 2008; Asadohalli et al., 2010; Westfall et al., 2011; Scalcinati et al. al., 2012; Paddon et al., 2013; Lv et al., 2014; Yuan et al., 2014; etc.). description In one embodiment of the present invention, rox1 (SEQ ID NO: (No. 25) locus and this negative regulation in the mevalonate pathway and late sterol biosynthesis. knockout of nodal factors ( Henry et al., 2002 ; Montanes et al., 2011 ; Ozaydin et al., 2013; Jakociunas et al., 2015). Enhanced squalene consumption in terol biosynthesis leads to subsequent mevalonate pathway Regarding squalene downgrade, the upregulation of the mevalonate pathway in general This method results in the production of squalene, lanosterol, ergosterol, and lecithin. This enhanced the accumulation of peol (Figure 7b and Table 1), demonstrating the functionality of this method and construct. I was able to prove that.

[0158] 2,3-oxidosqualene, a direct precursor molecule for the synthesis of five-membered triterpenes Since no accumulation was observed, the inventors determined that the endogenous ERG7 promoter (SEQ ID NO: 45 ) with the copper-repressible CTR3 promoter (SEQ ID NO: 42), The metabolic pathway shifted from ethanol biosynthesis to the production of pentacyclic triterpenes (Labbe et al., 1997). Thus, there are two reasons for the lack of accumulation of 2,3-oxidosqualene. First, E The lower potency of ERG1 (SEQ ID NO: 52) compared to RG9 (SEQ ID NO: 53) (Asadohalli et al. al., 2010), and secondly, lanosterol (Ve en et al., 2003), and / or in this case the five-membered ring at the expense of TkLUP (SEQ ID NO: 12). It is a rapid response to triterpenes.

[0159] In one embodiment of the present invention, the inventors of the present invention have used squalene and five-membered ring triglycerides. Suppression of endogenous ERG7 (SEQ ID NO: 20) in a litterpene-accumulating yeast strain resulted in squalene production. A decrease in squalene levels and an accumulation of 2,3-oxidosqualene were detected. This accumulation may be due to decreased expression of ERG7 (SEQ ID NO: 20) itself, but not due to decreased expression of ERG1 ( The regulatory mechanisms of ERG9 (SEQ ID NO: 51) and ERG9 (SEQ ID NO: 49) may also contribute to this finding. In this particular embodiment, the authors found that low levels of ergosterol and lanosterol Deregulation of the described negative feedback loop also contributes to the development of the enzyme ERG9 (SEQ ID NO: 4) encoding the parent squalene synthase and squalene epoxidase 9) and ERG1 (SEQ ID NO: 51) expression. In a small number of cases, ERG9 (SEQ ID NO: 49) and ERG1 are regulated by limiting ergosterol levels. (SEQ ID NO: 51) and the lack of inhibition from lanosterol 1 (SEQ ID NO: 51) (M'Baya et al., 1989). However, The present inventors have demonstrated that TkLUP (SEQ ID NO: 12) can be synthesized by providing a number of direct substrates. For example, the accumulation of lupeol can be further increased, and furthermore, its production as β-amyrin can be enhanced. In each CG-MS spectrum, currently unknown peaks could be identified. Ta.

[0160] That is, the inventors of the present invention have been studying the synthesis of oxidosquas, for example, five-membered triterpenes. The productivity of at least one of methionine, triterpene, and / or triterpenoid is up to 12 Furthermore, the present inventors have demonstrated that the enzyme activity of the model enzyme used in the present invention can be increased by 7 times. The second five-membered triterpene (β-amylidene triterpene) synthesized by the enzyme TkLUP (SEQ ID NO: 12) We were able to characterize the

[0161] Additionally, the inventors of the present invention have discovered a triterpene mixture from, for example, T. koksaghyz plant material. a C18 column in the first chromatography step, as exemplarily shown for the product, and Single triterpenes were isolated using a biphenyl column in the second chromatography step. could be purified.

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Claims

1. 1. A method for increasing production of at least one five-membered triterpene in a host yeast cell, comprising: - to overexpress a 3-hydroxy-3-methylglutaryl coenzyme A reductase comprising the amino acid sequence shown in SEQ ID NO: 1, or to overexpress a protein having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, and capable of producing mevalonate, and, modifying the host yeast cell to overexpress a protein comprising an amino acid sequence set forth in SEQ ID NO:3 or an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO:3, wherein the protein is capable of producing 3-hydroxy-3-methylglutaryl-CoA; -CTR3-promoter insertion and / or copper sulfate CuSO ４ modifying the host yeast cell to repress lanosterol synthase (EGR7) comprising the amino acid sequence set forth in SEQ ID NO:9 by adding - modifying said host yeast cell to express lupeol synthase; - culturing said host yeast cells under suitable conditions so as to express at least one of said five-membered triterpenes; and purifying at least one of said five-membered triterpenes, The method comprises increasing the production of at least one five-membered triterpene compared to the host yeast cell before modification.

2. 2. The method of claim 1, further comprising modifying the host yeast cell to overexpress a protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 5, 6, 7, and 8, or at least one amino acid sequence having at least 95% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 5, 6, 7, and 8, wherein the protein is capable of producing at least one of acetoacetyl-CoA, mevalonate-5-phosphate, mevalonate-5-pyrophosphate, isopentenyl-5-pyrophosphate, farnesyl-pyrophosphate, or dimethylallyl-pyrophosphate.

3. 3. The method of claim 1 or 2, further comprising modifying the host yeast cell to knock out at least one locus selected from the group consisting of ROX1 (SEQ ID NO:25), BTS1 (SEQ ID NO:54), YPL062W (SEQ ID NO:55), DOS2 (SEQ ID NO:56), YER134C (SEQ ID NO:57), VBA5 (SEQ ID NO:58), YNR063W (SEQ ID NO:59), YJL064W (SEQ ID NO:60), and YGR259C (SEQ ID NO:61).

4. The method according to any one of claims 1 to 3, wherein the purification of at least one of the five-membered triterpenes is carried out by at least two chromatography steps.

5. The method according to any one of claims 1 to 4, wherein the production amount of two or more five-membered triterpenes is increased.

6. A recombinant host yeast cell for producing at least one five-membered ring triterpene, wherein the host yeast cell has been modified to overexpress 3-hydroxy-3-methylglutaryl-coenzyme A reductase comprising the amino acid sequence set forth in SEQ ID NO:1, or to overexpress a protein having at least 95% sequence identity with the amino acid sequence set forth in SEQ ID NO:1, and capable of producing mevalonate, and to overexpress a protein comprising the amino acid sequence set forth in SEQ ID NO:3, or an amino acid sequence having at least 95% sequence identity with the amino acid sequence set forth in SEQ ID NO:3, and capable of producing 3-hydroxy-3-methylglutaryl-CoA; The host cell may be modified by the insertion of a CTR3 promoter and / or copper sulfate (CuSO). ４ is further modified to inhibit lanosterol synthase (ERG7) comprising the amino acid sequence set forth in SEQ ID NO: 9 by the addition of A host yeast cell, wherein the host cell is further modified to express lupeol synthase.

7. 7. The host yeast cell of claim 6, wherein the host yeast cell is selected from the group consisting of Saccharomyces cerevisiae, Nicotiana benthamiana, Pichia pastoris, Pichia methanolica, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces marxianus, Pichia stipitis, Candida albicans, and Candida utilis.

8. 8. The host yeast cell of claim 6 or 7, wherein the host yeast cell is further modified to overexpress a protein comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 5, 6, 7, and 8, or at least one amino acid sequence having at least 95% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 5, 6, 7, and 8, wherein the protein is capable of producing at least one of acetoacetyl-CoA, mevalonate-5-phosphate, mevalonate-5-pyrophosphate, isopentenyl-5-pyrophosphate, farnesyl-pyrophosphate, or dimethylallyl-pyrophosphate.

9. 9. The host yeast cell of claim 6, wherein the host yeast cell has been further modified to knock out at least one locus selected from the group consisting of ROX1 (SEQ ID NO:25), BTS1 (SEQ ID NO:54), YPL062W (SEQ ID NO:55), DOS2 (SEQ ID NO:56), YER134C (SEQ ID NO:57), VBA5 (SEQ ID NO:58), YNR063W (SEQ ID NO:59), YJL064W (SEQ ID NO:60), and YGR259C (SEQ ID NO:61).

10. The host yeast cell of any one of claims 6 to 9, wherein at least one of the five-membered ring triterpenes is β-amyrin, α-amyrin, taraxasterol, five-membered ring saponin, five-membered ring sapogenin, lup-19(21)-en-3-one, lup-20(29)-en-3-one, taraxerol, taraxerone, α-amyron, β-amyron, taraxasterone, friedelin, betulin, or betulinic acid.

11. Use of a host yeast cell according to any one of claims 6 to 10 for producing at least one five-membered triterpene.

12. The amount of copper sulfate added is at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, or 375 mM CuSO ４ The method of claim 1, wherein

13. 5. The method of claim 4, wherein the purification of at least one of the five-membered triterpenes is carried out by at least two chromatography steps using a C18 column in the first chromatography step and a biphenyl column in the second chromatography step.

14. 11. The host yeast cell of claim 10, wherein at least one of the five-membered triterpenes is lupeol or β-amyrin.

15. The method of claim 1, wherein the lupeol synthase is T. koksaghyz lupeol synthase TkLUP.

16. The host yeast cell of claim 6 , wherein the lupeol synthase is the T. koksaghyz lupeol synthase TkLUP.

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