A method for increasing the yield of a production strain

Abstract

The present application relates to a kind of host cells of producing gimalen A, can realize the efficient synthesis of gimalen A, construction, fermentation mode is simple and effective, easy to realize industrialization.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering and relates to a method for increasing the yield of β-elemene. Background Technology

[0002] β-elemene is a Class II antitumor drug extracted from Curcuma longa, a plant in the ginger family of traditional Chinese medicine, and has broad-spectrum antitumor activity. However, due to the low content of elemene in Curcuma longa and the presence of multiple isomers such as α-elemene and γ-elemene in the extract, it is difficult to separate and has high cost, which affects the large-scale production and supply of β-elemene.

[0003] Researchers are currently attempting to achieve efficient production of elemol by constructing in vivo synthetic pathways, such as optimizing the mevalonate pathway (MVA pathway), identifying and modifying key enzymes, and modifying cell tolerance. However, current research has not focused on product yield, and further breakthroughs in production volume have been achieved.

[0004] Further research is needed on genes in the side metabolic pathways that affect its synthesis. Summary of the Invention

[0005] The present invention provides a host cell for producing gemmaene A, the host cell containing one or more enzymes, wherein the activity of endogenous malate dehydrogenase MDH2 is reduced compared with the endogenous activity of the MDH2 enzyme in the corresponding wild-type host cell, wherein the activity of the endogenous MDH2 is reduced by one or more genetic modifications, the genetic modifications including disruption, substitution, deletion or knockout.

[0006] Furthermore, the host cell contains an overexpressed malic acid oxidase. In some embodiments, the malic acid oxidase is derived from *Rhodosporidium toruloides*, in some embodiments from *Corynebacterium glutamicum*, and in some embodiments from *Escherichia coli*.

[0007] Furthermore, the malic enzyme is selected from malE, RtME or malB, preferably malE or RtME, and more preferably malE.

[0008] Furthermore, the amino acid sequence of the endogenous MDH2 protein is as shown in SEQ ID NO: 1, and the nucleotide coding sequence is as shown in SEQ ID NO: 2; the amino acid sequence of the malic acid enzyme malE is as shown in SEQ ID NO: 3 or a sequence having 80%, 90%, 95%, 98%, or 99% homology with SEQ ID NO: 3, and the nucleotide coding sequence is as shown in SEQ ID NO: 4 or a sequence having 80%, 90%, 95%, 98%, or 99% homology with SEQ ID NO: 4; the amino acid sequence of the malic acid enzyme RtME is as shown in SEQ ID NO: 5 or a sequence having 80%, 90%, 95%, 98%, or 99% homology with SEQ ID NO: 5, and the nucleotide coding sequence is as shown in SEQ ID NO: 6 or a sequence having 80%, 90%, 95%, 98%, or 99% homology with SEQ ID NO: 6.

[0009] Furthermore, the host cell comprises any one or more of the following: overexpressed acetyl-CoA-acetyltransferase / 3-hydroxy-3-methylglutaryl-CoA reductase EfmvaE, isopentenyl pyrophosphate isomerase IDI1, 3-hydroxy-3-methylglutaryl-CoA synthase ERG13, mevalonate kinase ERG12, mevalonate phosphate kinase ERG8, mevalonate pyrophosphate decarboxylase ERG19, preferably the host cell comprises inactivated or inhibited LPP1 responsible for farnesol synthesis and GAL80 responsible for transcriptional regulation.

[0010] Furthermore, the host cell contains an expression cassette encoding farnesyl diphosphate synthase ERG20, preferably the expression cassette containing farnesyl diphosphate synthase ERG20 and gamma-aluminum A synthase (GAS) and / or terpene synthase (TPS), wherein farnesyl diphosphate synthase ERG20 and gamma-aluminum A synthase and / or terpene synthase (TPS) are linked by a linker peptide. The gamma-aluminum A synthase gene of this invention can be cloned from plants or microorganisms known to contain gamma-aluminum A synthase, such as sunflower (Helianthus annuus L.), chrysanthemum (Tanacetum parthenium), lettuce (Lactucasativa Linn.), artemisia carvifolia, cyanobacteria, etc. The farnesyl diphosphate synthase gene can be cloned from plants or microorganisms known to contain gamma-aluminum A synthase, such as tanshinone (Salvia miltiorrhiza) and yeast (Saccharomyces). Cerevisiae, Acanthopanax senticosus (Rupr. Maxim.) Harms, Eucommia m. Mia ulmoides Oliv., etc. In some embodiments, the gemathene A synthase and / or terpene synthase (TPS) are derived from lettuce and salvia miltiorrhiza. The linker peptide is selected from GGGS, YGQ, PGGH, YRSQI, VIPFIS, FLYLKF, WRFSPKLQ, or HHVQESQCISTV; preferably GGGS. In a preferred embodiment, ERG20 may be in a different expression cassette from GAS and / or TPS.

[0011] In some embodiments, the endogenous MDH2 gene in the host cell is knocked out. In some embodiments, the malicase gene malE or RtME is inserted into the site of the MDH2 gene and replaces the MDH2 gene. In some embodiments, a LEU2-malE expression cassette or a LEU2-RtME expression cassette is inserted into the site of the MDH2 gene.

[0012] In some embodiments, the yeast host cell further includes one or more of the following characteristics:

[0013] Increase the activity of acetyl-CoA C-acetyltransferase / 3-hydroxy-3-methylglutaryl-CoA reductase EfmvaE, for example, by increasing its expression level;

[0014] Increased activity of mevalonate kinase ERG12, such as increased expression levels;

[0015] Increased activity of the phosphovalerate kinase ERG8, such as increased expression levels;

[0016] Increased activity of mevalonate pyrophosphate decarboxylase ERG19, for example, increased expression levels;

[0017] Increased activity of 3-hydroxy-3-methylglutaryl-CoA synthase ERG13, for example, increased expression levels;

[0018] Reduced activity of GAL80, which is responsible for regulating GAL promoter expression, such as reduced expression levels;

[0019] Increased surface activity of isopentenyl pyrophosphate isomerase IDI1, for example, by increasing the level of [something]; or

[0020] Reduced expression levels of LPP1, the gene responsible for farnesol synthesis;

[0021] In some embodiments, the yeast host cell further includes one or more of the following characteristics:

[0022] Reduced expression levels of LPP1, the gene responsible for farnesol synthesis;

[0023] Increased activity of acetyl-CoA C-acetyltransferase / 3-hydroxy-3-methylglutaryl-CoA reductase EfmvaE;

[0024] Reduced activity of GAL80, which is responsible for regulating GAL promoter expression;

[0025] Increased activity of isopentenyl pyrophosphate isomerase IDI1;

[0026] Increased activity of mevalonate kinase ERG12;

[0027] Increased activity of mevalonate kinase ERG8;

[0028] Increased activity of mevalonate pyrophosphate decarboxylase ERG19; or

[0029] Increased activity of 3-hydroxy-3-methylglutaryl-CoA synthase ERG13.

[0030] The increased activity can be achieved by changing its promoter, modifying the enzyme, or increasing its copy number.

[0031] In some embodiments, the host cell overexpresses EfmvaE by inserting a gene expression cassette encoding acetyl-CoA-acetyltransferase / 3-hydroxy-3-methylglutaryl-CoA reductase EfmvaE.

[0032] In some embodiments, the host cell overexpresses IDI1 by inserting a gene expression cassette encoding isopentenyl pyrophosphate isomerase IDI1;

[0033] In some embodiments, the host cell overexpresses ERG13 by inserting a gene expression cassette encoding 3-hydroxy-3-methylglutaryl-CoA synthase ERG13;

[0034] In some embodiments, the host cell overexpresses ERG12 by inserting a gene expression cassette encoding mevalonate kinase ERG12;

[0035] In some embodiments, the host cell overexpresses ERG8 by inserting a gene expression cassette encoding mevalonate phosphokinase ERG8;

[0036] In some embodiments, the host cell overexpresses ERG19 by inserting a gene expression cassette encoding mevalonate pyrophosphate decarboxylase ERG19;

[0037] GAL80 and / or LPP1 can be inactivated or inhibited by disrupting, substituting, deleting or knocking out GAL80 and / or LPP1 in yeast, preferably by knocking out GAL80 and / or LPP1 in yeast.

[0038] In some embodiments, the expression box includes a promoter and a terminator, the promoter including but not limited to P TEF1 P TEF2 P MF1 or P PGK1 The preferred promoter is P. TEF1 or P TEF2 The terminator includes, but is not limited to, T ADH2 T CYC1 T ADH1 etc., preferably T ADH2 .

[0039] For example: the expression box is P TEF1 -EfmvaE-T ADH2 P TEF2 -IDI1-T GAL80 ,

[0040] P TEF1 -ERG12-T ERG12 -P FBA1 -ERG8-T ERG8 -P TEF2 -ERG19-T ERG19 T ERG13 -ERG13-P GAL1 -P GAL10-EfmvaE-T ADH2 P TEF1 -RtME-T CYC1 P TEF1 -malE-T CYC1 wait.

[0041] The available selection markers for Saccharomyces cerevisiae gene integration can be any selection marker known to those skilled in the art, as long as the selection markers used when integrating different fragments into the same Saccharomyces cerevisiae strain are different from each other. Common selection markers include auxotrophic selection markers and resistance selection markers. Among them, auxotrophic selection markers can be selected from LEU2, HIS3, URA3, or TRP1. Resistance selection markers can be G418 or HYG. In a further embodiment, the host cell is obtained by knocking out LPP1 in Saccharomyces cerevisiae CEN.PK2-1D and then inserting P at the LPP1 site. TEF1 -EfmvaE-T ADH2 Knock out GAL80 and insert P at the GAL80 site. TEF2 -IDI1-T GAL80 URA3-P was inserted at the YPRC15C site on chromosome 1. TEF1 -ERG12-T ERG12 -P FBA1 -ERG8-T ERG8 -P TEF2 -ERG19-T ERG19

[0042] HIS3-T was inserted at site 1622b. ERG13 -ERG13-P GAL1 -P GAL10 -EfmvaE-T ADH2 Insert P at site XI-5 TEF1 -EfmvaE-T ADH2 The host cells are obtained by further knocking out mdh2 and inserting LEU2 at the mdh2 site. In some embodiments, the host cells are obtained by knocking out mdh2 and inserting LEU2-RtME at the mdh2 site. In some embodiments, the host cells are obtained by knocking out mdh2 and inserting LEU2-malE at the mdh2 site.

[0043] In a preferred embodiment, the host cell is yeast or Escherichia coli, preferably yeast, and more preferably Saccharomyces cerevisiae. The yeast of the present invention can be any Saccharomyces cerevisiae available in the art. For example, commercially available Saccharomyces cerevisiae CEN.PK2-1D, Saccharomyces cerevisiae INVSC1, Saccharomyces cerevisiae BY4742, Saccharomyces cerevisiae YPH499, or Saccharomyces cerevisiae W303-1B, etc.

[0044] In a more preferred embodiment, the host cell may be derived from wild-type yeast or Escherichia coli, or from mutant yeast or Escherichia coli induced by mutagenesis or engineering. In an even more preferred embodiment, the host cell may be derived from wild-type yeast or Escherichia coli capable of providing farnesyl pyrophosphate (FPP), or from mutant yeast or Escherichia coli induced by mutagenesis or engineering.

[0045] Furthermore, the host cells described in this invention have higher gemmaene production and / or higher product yield compared to wild-type host cells.

[0046] Furthermore, the host cell provided by this invention has the function of producing gemmaene A.

[0047] The present invention provides a method for producing gemmaene A, the method comprising growing and fermenting the aforementioned host cells.

[0048] This invention provides a method for producing β-elemene, comprising the following steps:

[0049] 1) Ferment the above-mentioned host cells or host cells to obtain fermentation broth;

[0050] 2) Extract the fermentation broth with an organic solvent and collect the organic phase;

[0051] 3) The organic phase is heated to obtain β-elemene.

[0052] In a further embodiment, the fermentation comprises yeast fermentation or Escherichia coli fermentation. Yeast-containing or Escherichia coli-containing fermentation can be carried out using methods known in the art.

[0053] In some embodiments, the fermentation includes biphasic fermentation or monophasic fermentation.

[0054] In some embodiments, the fermentation temperature is 16-40°C. In some embodiments, the fermentation temperature is 16-37°C; in some embodiments, the fermentation temperature is 37°C; and in some embodiments, the fermentation temperature is 30°C. In some embodiments, the fermentation temperature may be 16-30°C. In some embodiments, the fermentation temperature may be 20-30°C. In some embodiments, the fermentation temperature is 25°C; and in some embodiments, the fermentation temperature is 30°C.

[0055] In some embodiments, the fermentation speed is 100-500 rpm. In some embodiments, the fermentation speed is 250 rpm.

[0056] In some embodiments, the fermentation time is 40-144 hours, and in some embodiments, the fermentation time is 40-48 hours.

[0057] In some embodiments, the fermentation is a single-phase yeast fermentation. For example, the single-phase yeast fermentation includes inoculating a single clone of the host cell into a fermentation medium, fermenting at 30°C and 250 rpm for 72 hours. Prior to inoculation into the fermentation medium, the cell may undergo amplification culture, including single-stage or multi-stage amplification culture. In some embodiments, the fermentation is a single-phase yeast fermentation. For example, the single-phase yeast fermentation includes inoculating a single clone of the host cell into a fermentation medium, fermenting at 30°C and 250 rpm for 40-48 hours. Prior to inoculation into the fermentation medium, the cell may undergo amplification culture, including single-stage or multi-stage amplification culture.

[0058] In some embodiments, the fermentation is a yeast biphasic fermentation. For example, the yeast biphasic fermentation includes inoculating a single clone of the host cell into an amplification medium, fermenting at 30°C with shaking at 250 rpm for 120-168 h; and transferring 5 vol%-10 vol% of the cultured bacterial solution into Erlenmeyer flasks containing fermentation medium and n-dodecane, fermenting at 30°C with shaking at 250 rpm for 120-168 h. In some embodiments, the fermentation is an Escherichia coli biphasic fermentation, including inoculating a single clone of the host cell into an amplification medium, fermenting at 37°C with shaking at 250 rpm overnight; and transferring 5 vol%-10 vol% of the overnight cultured bacterial solution into Erlenmeyer flasks containing fermentation medium and n-dodecane, fermenting at 37°C with shaking at 250 rpm for 24-48 h. In biphasic fermentation, one or more stages of amplification culture may be performed.

[0059] In some embodiments, the gemmaene A is extracted by extraction or chromatography. In some embodiments, the organic solvent used for extraction includes, but is not limited to, n-dodecane, isopropyl myristate, n-hexane, and n-heptane; in some embodiments, the organic solvent used for extraction is n-dodecane.

[0060] In some embodiments, the gemmaene A can be converted into β-elemene by heating.

[0061] The beneficial effects of this invention are:

[0062] The method of this invention can achieve efficient synthesis of gemmaene A. The construction and fermentation methods are simple and effective, and it is easy to industrialize. Detailed Implementation

[0063] The technical solution of the present invention will be further described in detail below with reference to the specification and specific implementation examples, but the implementation of the present invention is not limited thereto. Equivalent substitutions, combinations, improvements, or modifications made by those skilled in the art based on the description of the present invention should all be covered within the protection scope of the present invention.

[0064] Unless otherwise stated, the experimental methods used below are conventional methods well known to those skilled in the art, and can be performed using standard procedures described in the following works: Sambrook et al., *Molecular Cloning: A Laboratory Manual* (3rd edition) (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2001); Davis et al., *Basic Methods in Molecular Biology* (Elsevier Science Publishing, Inc., New York, USA, 1995); and Juan S. Bonifacino et al., *Current Protocols in Cell Biology* (John Wiley and Sons, Inc.).

[0065] Unless otherwise stated, all reagents and materials used in the following examples are commercially available.

[0066] The gene and primer information used in this invention is shown in Tables 1 and 2:

[0067] Table 1 Information on recombinant yeast chassis bacteria

[0068]

[0069] Table 2 Primer Information

[0070]

[0071]

[0072]

[0073] Main reagents:

[0074] Frozen-EZ YeastTransformation II Kit™ (Zymo Research Biotechnology Co., Ltd.);

[0075] SD-Trp liquid medium: SD-Trp yeast culture medium powder (Beijing Pankino Technology Co., Ltd.) + 2% glucose;

[0076] Calcium chloride mother liquor: prepared according to Example 3 of CN108060092B;

[0077] Trace metal salt mother liquor: prepared according to Example 3 of CN108060092B;

[0078] Vitamin stock solution: 0.2g / L biotin, 0.8g / L sodium para-aminobenzoate, 4g / L niacin, 4g / L calcium pantothenate, 4g / L pyridoxine hydrochloride, 4g / L thiamine hydrochloride, 100g / L inositol;

[0079] Succinic acid stock solution: 60 g / L succinic acid, adjusted to pH 5.05 with NaOH;

[0080] F2 fermentation medium: 25 g / L glucose, 15 g / L ammonium sulfate, 6.15 g / L magnesium sulfate heptahydrate, 0.72 g / L zinc sulfate heptahydrate, 8 g / L potassium dihydrogen phosphate, 2 mL / L calcium chloride mother liquor, 10 mL / L trace metal salt mother liquor, 3 mL / L vitamin mother liquor, 100 mL / L succinic acid mother liquor;

[0081] Reagent kits: Gel Extraction Kit (Omega); KOD FX (TOYOBO); Max DNA Polymerase (TAKARA); Frozen-EZ Yeast Transformation II Kit™ (ZYMO); YeaStar Genomic DNA Kit (ZYMO); MiniBEST DNA Fragment Purification Kit (TAKARA); Seamless Cloning Kit Seamless Cloning and Assembly Kit: Full Gold, China;

[0082] Escherichia coli Trans 5α competent cells: TransGold, China;

[0083] Anhydrous ethanol: Sigma WXBC939V

[0084] Dodecane: Chengdu Kelong Chemical Co., Ltd.

[0085] Soda lime glass beads: biospec, 0.5mm

[0086] Example 1: Strain Construction

[0087] 1. Strain construction

[0088] The whole genome sequence of CEN.pK2-1D was extracted according to the instructions of the YeaStar Genomic DNA Kit (ZYMO).

[0089] Information on the recombinant yeast chassis strain of this invention is shown in Table 1. The chassis strain used in this study is CEN.pK2-1D. All endogenous genes of the MVA pathway, ERG13, ERG12, ERG8, ERG19, IDI1, and ERG20, were derived from the genomic DNA of CEN.pK2-1D. The strain construction method is described in CN108060092B. The gene EfmvaE (GenBank-KX064239) was synthesized by Beijing Liuhe Huada, and its nucleotide sequence is shown in SEQ ID NO: 7. The promoter P was selected. TEF1 Select the termination child T ADH2 The homologous arms L1 and L2 of the LPP1 gene were knocked out. The PCR amplification template and primers are shown in Table 3. After obtaining the fragments, L1 and P were assembled using overlap PCR. TEF1-1 EfmvaE-1, T ADH2-1 L2 fragment forms expression box lpp1△::P TEF1 -EfmvaE-T ADH2 Its nucleotide sequence is shown in SEQ ID NO: 8. Using gene editing technology (Amanda ReiderApel et al., 2016), the EfmvaE gene was inserted after knocking out the LPP1 gene in the CEN1 strain. The IDI1 gene was inserted after knocking out the GAL80 gene, and the promoter P was selected. TEF2 Select the termination child T GAL80 The homologous arms L3 and L4 of the GAL80 gene were knocked out. The PCR amplification template and primers are shown in Table 3. After obtaining the fragments, L3 and P were assembled using overlap PCR. TEF2-1 IDI1 and L4 fragments form expression boxes gal80△::P TEF2 -IDI1-T GAL80 Its nucleotide sequence is shown in SEQ ID NO: 9. ERG12, ERG8, and ERG19 genes were inserted at the YPRC15C site, and the promoter P was selected. TEF1 P FBA1 P TEF2 Select the termination child T ERG12 T ERG8 T ERG19 Homologous arms L5 and L6 of the YPRC15C site were designed. PCR amplification templates and primers are shown in Table 3. After obtaining the fragments, L5, URA3, and P were assembled using overlap PCR. TEF1-2 ERG12, PFBA1 ERG8, P TEF2-2 ERG19 and L6 fragments form expression cassettes YPRC15C::URA3-P TEF1 -ERG12-T ERG12 -P FBA1 -ERG8-T ERG8 -P TEF2 -ERG19-T ERG19 Its nucleotide sequence is shown in SEQ ID NO: 10. The ERG13 and EfmvaE genes were inserted at site 1622b, and the promoter P was selected. GAL1 P GAL10 Select the termination child T ERG13 T ADH2 Homologous arms L7 and L8 at the 1622b site were designed. PCR amplification templates and primers are shown in Table 3. After obtaining the fragments, L7, HIS3, ERG13, and P were assembled using overlap PCR. GAL1-GAL10 EfmvaE-2, T ADH2-1 L8 fragment forms expression cassette 1622b::HIS3-T ERG13 -ERG13-P GAL1 -P GAL10 -EfmvaE-T ADH2 Its nucleotide sequence is shown in SEQ ID NO: 11. Select promoter P TEF1 Select the termination child T ADH2 Homologous arms L9 and L10 at the XI-5 site were designed. PCR amplification templates and primers are shown in Table 3. After obtaining the fragments, L9 and P were assembled using overlap PCR. TEF1-3 EfmvaE-1, T ADH2-2 L10 fragment forms expression box XI-5△::P TEF1 -EfmvaE-T ADH2 Its nucleotide sequence is shown in SEQ ID NO: 12. The EfmvaE gene was inserted into the neutral site XI-5 to construct strain CEN2.

[0090] The LEU2 fragment was amplified from the PESC-LEU vector (Agilent Technologies), and the homologous arms L3 and L4 of the MDH2 gene were knocked out at both ends. The PCR amplification template and primers are shown in Table 3. The amino acid sequence of the endogenous MDH2 protein is shown in SEQ ID NO: 1, and the nucleotide coding sequence is shown in SEQ ID NO: 2. After obtaining the fragment, it was assembled into an expression cassette mdh2△::LEU2 using overlap PCR. The nucleotide sequence of mdh2△::LEU2 is shown in SEQ ID NO: 13. This expression cassette sequence was transformed and integrated into the genome of strain CEN2 to construct strain CEN3.

[0091] RtME was synthesized by Beijing Liuhe Huada. The amino acid sequence is shown in SEQ ID NO: 5, and the nucleotide sequence is shown in SEQ ID NO: 6. The promoter P was selected. TEF1 Select the termination child T CYC1 The PCR amplification template and primers are shown in Table 3. Overlap PCR was used to amplify L11, LEU2, and P. TEF1-3 、RtME、T CYC1 The L12 fragment is assembled into the expression box mdh2△:LEU2-P TEF1 -RtME-T CYC1 Its nucleotide sequence is shown in SEQ ID NO: 14. This expression cassette sequence was transformed and integrated into the genome of strain CEN2 to construct strain CEN4.

[0092] malE was synthesized by Beijing Liuhe Huada. Its amino acid sequence is shown in SEQ ID NO: 3, and its nucleotide sequence is shown in SEQ ID NO: 4. The promoter P was selected. TEF1 Select the termination child T CYC1 The PCR amplification template and primers are shown in Table 3. Overlap PCR was used to amplify L11, LEU2, and P... TEF1-3 ,malE,T CYC1 The L12 fragment is assembled into the expression box mdh2△:LEU2-P TEF1 -malE-T CYC1， Its nucleotide sequence is shown in SEQ ID NO: 15. This expression cassette sequence was transformed and integrated into the genome of strain CEN2 to construct strain CEN5.

[0093] 2. Plasmid construction

[0094] Fragment P was amplified from the CEN.pK2-1D genome using conventional methods. SSA1 (SEQ ID NO: 17). Extraction of ELE-020

[0095] (CN108060092B) Whole genome sequence (achieved according to the instructions of the YeaStar Genomic DNA Kit (ZYMO)). Fragment ERG20-GGGS-LsLTC2-T amplified from the ELE-020 genome. ADH1 (SEQ ID NO: 18). From plasmid

[0096] pRS425-LEU2-P MF1- SynSmFPS-GGGS-STpGMAS-T CYC1 The backbone fragment pRS425-Line (SEQ ID NO: 19) was amplified on (CN108060092B). After agarose gel electrophoresis, the fragments were excised and recovered. A full-gold seamless cloning kit was used. The seamless cloning and assembly kit (Quanshijin, China) is used for connection. The connection system is: pRS425-Line 130ng, SSA1p 70ng, ERG20-GGGS-LsLTC2-T ADH1 115 ng, 2×Assemblymix 6.35 μL. Conditions: 50℃, 30 min. Transfected *E. coli* Trans 5α competent cells (TransGold, China), plated on LB agar plates containing ampicillin, cultured at 37℃ for 16 h, single clones were picked and cultured overnight at 37℃, plasmids were extracted and sequenced to obtain yeast expression vector P2. pRS425-LsLTC2-Line was amplified from P2, and fragment TRP1 (SEQ ID NO: 20) was amplified from the CEN.pK2-1D genome. The ligation system was: pRS425-LsLTC2-Line 100 ng, TRP1 100 ng, 2×Assemblymix 5 μL. Conditions: 50℃, 30 min. Transformed Escherichia coli Trans5α competent cells (TransGold, China) were plated on LB agar plates containing ampicillin and cultured at 37°C for 16 h. Single clones were picked and cultured overnight at 37°C. Plasmids were extracted and sequenced to verify the results, yielding the yeast expression vector P3 (its nucleotide sequence is shown in SEQ ID NO: 16).

[0097] Plasmid P3 was transformed into CEN2, CEN3, CEN4, and CEN5 strains to construct expression strains CEN2+P3, CEN3+P3, CEN4+P3, and CEN5+P3.

[0098] Table 3 Primers and templates for amplifying the target fragment

[0099]

[0100]

[0101]

[0102] Example 2: Fermentation of strains

[0103] 1. Fermentation by bacterial strain

[0104] (1) Pick three single clones from each of the fresh plates of CEN2+P3, CEN3+P3, CEN4+P3 and CEN5+P3, and place them in 3 mL of SD-Trp medium and incubate at 30℃ and 250 rpm for 16 h.

[0105] (2) Transfer 1 mL of bacterial culture to 20 mL of F2 fermentation medium and incubate at 30 °C and 250 rpm for 40 h to obtain fermentation products.

[0106] 2. Extraction of fermentation products

[0107] (1) After fermentation, 750 mL of fermentation liquid was taken out, 300 mL of dodecane and 0.8 g of glass beads were added, and vortexed extraction was performed for 1 h.

[0108] (2) Centrifuge at 12000 rpm for 10 min, take 100 μL of the upper layer liquid, add 900 μL of anhydrous ethanol, mix well, transfer to a sample vial, and detect the yield of gemmaene A by liquid chromatography.

[0109] (3) Liquid chromatography analysis conditions: The chromatographic column was Elispe plus RRHD 1.8μm, 2.1×150mm. 1μL of sample was injected. The column temperature was 30℃. The mobile phases were A: water, B: acetonitrile-isopropanol (1:1), flow rate 0.2mL / min, detection wavelength 210nm, and gradient program as shown in Table 4.

[0110] Table 4 Gradient Procedure

[0111] Time (min) Solution A percentage B solution percentage 0.00 10％ 90％ 4.00 10％ 90％ 5.00 0 100％ 9.00 0 100％ 9.10 10％ 90％ 12.00 10％ 90％

[0112] 3. The test results are shown in Table 5.

[0113] Table 5. Gemmaene A yield from the fermentation broth of strain 5 (rounded to one decimal place)

[0114] strain name Gemmaene A yield (mg / L) over 40 hours CEN2+P3 237.5 CEN3+P3 305.0 CEN4+P3 286.4 CEN5+P3 345.7

[0115] Example 3: Fermentation of strain

[0116] 1. Fermentation by bacterial strain

[0117] (1) Pick three single clones from each of the fresh plates of CEN2+P3, CEN3+P3 and CEN5+P3, and place them in 3 mL of SD-Trp medium. Incubate at 30℃ and 250 rpm for 16 h.

[0118] (2) Transfer 1 mL of bacterial culture to 20 mL of F2 fermentation medium, culture at 30 °C and 250 rpm for 48 h, add ethanol once, and continue fermentation for 72 h to obtain fermentation product.

[0119] 2. Extraction of fermentation products

[0120] (1) Take 750 mL of fermentation broth from each sampling time point, add 300 mL of dodecane and 0.8 g of glass beads, and vortex extract for 1 h.

[0121] (2) Centrifuge at 12000 rpm for 10 min, take 100 μL of the upper layer liquid, add 900 μL of anhydrous ethanol, mix well, transfer to a sample vial, and detect the yield of gemmaene A by liquid chromatography (detection method is the same as in Example 2).

[0122] 3. The test results are shown in Table 6.

[0123] Table 6. Gemmaene A yield from fermentation broth of strain 6 (rounded to one decimal place)

[0124] CEN2+P3 CEN3+P3 CEN5+P3 Gemmaene A yield over 72 hours (mg / L) 505.3 579.1 823.5

[0125] 4. Residual ethanol detection

[0126] (1) During the fermentation period of 20-68h, a sampling point was taken every 4h. 100μL of fermentation product was taken from each point, centrifuged at 12000rpm for 5min, 50μL of the upper liquid was taken and added to 950μL of pure water and mixed. 25μL of the sample was injected using a biosensor analyzer to detect the ethanol content.

[0127] (2) Ethanol residue is shown in Table 7.

[0128] Table 7. Ethanol residue in fermentation broth of strains (rounded to one decimal place)

[0129] CEN2+P3 CEN3+P3 CEN5+P3 Ethanol residue after 20 hours (g / L) 7.4 6.8 7.2 24-hour ethanol residue (g / L) 6.6 6.8 5.8 Ethanol residue after 28 hours (g / L) 5.2 6.0 5.6 Ethanol residue at 32h (g / L) 4.4 4.6 4.6 Ethanol residue at 36 hours (g / L) 4.6 5.0 4.2 Ethanol residue after 40 hours (g / L) 4.2 4.4 3.4 Ethanol residue after 44 hours (g / L) 3.6 3.8 2.2 Ethanol residue after 48 hours (g / L) 7.0 7.4 5.6 Ethanol residue at 52h (g / L) 6.4 6.8 4.6 Ethanol residue at 56 hours (g / L) 5.8 6.0 3.4 Ethanol residue at 60h (g / L) 5.2 5.4 2.4 Ethanol residue after 64 hours (g / L) 4.8 4.6 1.2 Ethanol residue at 68h (g / L) 4.2 4.2 0.2 Ethanol residue after 72 hours (g / L) 3.2 3.2 0.0

[0130] Example 4: Preparation of β-elemene

[0131] The fermentation broth of (-)-gemmaene A obtained in Example 3 was mixed with n-dodecane at a ratio of 1:2 (V n-dodecane: V fermentation broth), and the mixture was stirred and extracted for 16 h. The organic phase was then heated at 150 °C for 2 h to obtain the conversion broth. The conversion broth was subjected to silver nitrate silica gel column chromatography and silica gel column chromatography to prepare β-elemene.

Claims

1. A host cell for producing gemmaene, said host cell comprising one or more enzymes, characterized in that... The activity of endogenous malate dehydrogenase MDH2 is reduced compared to the endogenous activity of MDH2 in the corresponding wild-type host cells, wherein the activity of endogenous MDH2 is reduced by one or more genetic modifications, including disruption, substitution, deletion or knockout.

2. The host cell according to claim 1, characterized in that... The host cell overexpresses malic acid enzyme, preferably derived from one or more of Rhodosporidium toruloides, Corynebacterium glutamicum, and Escherichia coli, preferably selected from malE, RtME, or malB, more preferably malE or RtME, and even more preferably malE.

3. The host cell according to claim 2, characterized in that... The amino acid sequence of the endogenous MDH2 protein is as shown in SEQ ID NO: 1, and the nucleotide coding sequence is as shown in SEQ ID NO: 2; the amino acid sequence of the malic enzyme malE is as shown in SEQ ID NO: 3 or a sequence having 80%, 90%, 95%, 98%, or 99% homology with SEQ ID NO: 3, and the nucleotide coding sequence is as shown in SEQ ID NO: 4 or a sequence having 80%, 90%, 95%, 98%, or 99% homology with SEQ ID NO: 4; the amino acid sequence of the malic enzyme RtME is as shown in SEQ ID NO: 5 or a sequence having 80%, 90%, 95%, 98%, or 99% homology with SEQ ID NO: 5, and the nucleotide coding sequence is as shown in SEQ ID NO: 6 or a sequence having 80%, 90%, 95%, 98%, or 99% homology with SEQ ID NO:

6.

4. The host cell according to claim 3, characterized in that... The host cell overexpresses one or more of the following: acetyl-CoA acetyltransferase / 3-hydroxy-3-methylglutaryl-CoA reductase EfmvaE, isopentenyl pyrophosphate isomerase IDI1, 3-hydroxy-3-methylglutaryl-CoA synthase ERG13, mevalonate kinase ERG12, mevalonate phosphokinase ERG8, and mevalonate pyrophosphate decarboxylase ERG19. Preferably, the host cell contains an inactivated or inhibited lipid phosphophosphatase LPP1 and GAL80 responsible for transcriptional regulation. The cell contains an expression cassette encoding farnesyl pyrophosphate synthase ERG20, preferably the expression cassette containing farnesyl pyrophosphate synthase ERG20 and gemmaene A synthase and / or terpene synthase (TPS), the farnesyl pyrophosphate synthase ERG20 and gemmaene A synthase and / or terpene synthase (TPS) linked by a linker peptide selected from GGGS, YGQ, PGGH, YRSQI, VIPFIS, FLYLKF, WRFSPKLQ or HHVQESQCISTV; preferably GGGS.

5. The host cell according to claim 1, characterized in that... The host cell endogenous MDH2 gene is knocked out, preferably the malic enzyme gene malE or RtME is inserted into the site of the MDH2 gene and replaces the MDH2 gene, preferably the LEU2-malE expression cassette or LEU2-RtME expression cassette is inserted into the site of the MDH2 gene.

6. The host cell according to claim 5, characterized in that... The host cell includes one or more of the following characteristics: Increase the activity of acetyl-CoA-acetyltransferase / 3-hydroxy-3-methylglutaryl-CoA reductase EfmvaE; Increased activity of mevalonate kinase ERG12; Increased activity of mevalonate kinase ERG8; Increased activity of mevalonate pyrophosphate decarboxylase ERG19; Increased activity of 3-hydroxy-3-methylglutaryl-CoA synthase ERG13; Reduced activity of GAL80, which is responsible for regulating GAL promoter expression; Increased activity of isopentenyl pyrophosphate isomerase IDI1; Reduced expression levels of LPP1, the gene responsible for farnesol synthesis; Preferably, it includes one or more of the following characteristics: The host cells overexpress EfmvaE by inserting the gene expression cassette encoding acetyl-CoA acetyltransferase / 3-hydroxy-3-methylglutaryl-CoA reductase EfmvaE. The host cells overexpress IDI1 by inserting a gene expression cassette encoding isopentenyl pyrophosphate isomerase IDI1; The host cells overexpress ERG13 by inserting a gene expression cassette encoding 3-hydroxy-3-methylglutaryl-CoA synthase ERG13. The host cells overexpress ERG12 by inserting a gene expression cassette encoding mevalonate kinase ERG12; The host cells overexpress ERG8 by inserting a gene expression cassette encoding mevalonate phosphokinase ERG8; The host cells overexpress ERG19 by inserting a gene expression cassette encoding mevalonate pyrophosphate decarboxylase ERG19. GAL80 and / or LPP1 can be inactivated or inhibited by disrupting, substituting, deleting or knocking out GAL80 and / or LPP1 in yeast, preferably by knocking out GAL80 and / or LPP1 in yeast.

7. The host cell according to claim 6, characterized in that... The expression box includes a promoter and a terminator, wherein the promoter is selected from P. TEF1 P TEF2 P MF1 P FBA1 or P PGK1 The preferred promoter is P. TEF1 or P TEF2 The terminator is selected from T ADH2 T CYC1 T ADH1 T is preferred. ADH2 .

8. The host cell according to claim 7, characterized in that... The host cell was created by knocking out LPP1 in CEN.PK2-1D Saccharomyces cerevisiae and inserting P at the LPP1 site. TEF1 -EfmvaE-T ADH2 Knock out GAL80 and insert P at the GAL80 site. TEF2 -IDI1-T GAL80 URA3-P was inserted at the YPRC15C site on chromosome 1. TEF1 -ERG12-T ERG12 -P FBA1 -ERG8-T ERG8 -P TEF2 -ERG19-T ERG19 HIS3-T was inserted at site 1622b. ERG13 -ERG13-P GAL1 -P GAL10 -EfmvaE-T ADH2 Insert P at site XI-5 TEF1 -EfmvaE-T ADH2 The host cells are preferably obtained by further knocking out mdh2 and inserting LEU2 at the mdh2 site, or more preferably by knocking out mdh2 and inserting LEU2-RtME at the mdh2 site, or more preferably by knocking out mdh2 and inserting LEU2-malE at the mdh2 site.

9. The host cell according to any one of claims 1-8, wherein, The host cell is yeast or Escherichia coli; yeast is preferred; Saccharomyces cerevisiae is more preferred.

10. The host cell according to claim 9, characterized in that... The host cells have higher gemmaene production and / or higher yield compared to wild-type host cells.

11. The host cell according to claim 1, characterized in that... The host cell specifically produces gemmaene A.

12. A method for producing gemmaene A, characterized in that... To enable the host cell according to any one of claims 1-11 to grow.

13. A method for producing β-elemene, comprising: 1) Fermenting the host cells according to any one of claims 1-12 to obtain a fermentation broth; 2) Extract the fermentation broth with an organic solvent and collect the organic phase; 3) The organic phase is heated to obtain β-elemene.

14. The method according to claim 13, wherein, The organic solvent is selected from one or more of n-dodecane, isopropyl myristate, n-hexane, and n-heptane.

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