Recombinant expression vector, recombinant bacterium, synthetic method of cannabidiol analogue O-1821 and application of cannabidiol analogue O-1821

Abstract

The invention discloses a recombinant expression vector, a recombinant bacterium, a cannabidiol analogue O-1821 synthesis method and application thereof, and relates to the technical field of bioengineering. According to the method, the synthesis efficiency is remarkably improved through multiple innovative strategies. Firstly, the catalytic efficiency of a key enzyme can be enhanced by optimizing the metabolic flux of endogenous malonyl-coenzyme A and geranyl diphosphate; and secondly, a condensed phase vesicular enzyme active concentrate-diisopentenyl transferase synergistic expression system is introduced, and diisopentenyl transferase is synergistically expressed, so that the efficiency of converting the serotinic acid into the cannabis rhizopus acid is remarkably improved, and the efficient synthesis of the O-1821 and the precursor compound thereof is realized. The invention has wide application potential in the fields of drug research and development, natural product biosynthesis and biotechnology, and provides a new way for sustainable production and industrial development of cannabinoid compounds.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology. Specifically, it relates to a recombinant expression vector, a recombinant bacterium, a method for synthesizing cannabidiol analog O-1821, and its applications. Background Art

[0002] Cannabinoids are a general term for a class of secondary metabolites derived from the plant Cannabis sativa, and their potential in the medical field has been widely studied. Due to their significant effects in relieving pain, inhibiting inflammation, reducing anxiety, treating epilepsy and metabolic diseases, cannabinoids have gradually become a hot spot in drug research and development. Among them, O-1821 (also known as Cannabidiorcol, CBDO or CBD-C1) is a structural analog of cannabidiol (CBD). O-1821 exhibits a low affinity for cannabinoid receptors (CB), but as an agonist of transient receptor potential channels (TRP channels), it can exert anti-inflammatory effects through multiple signaling pathways. In addition, O-1821 also shows potential efficacy in neuroprotection and antioxidant aspects. However, existing studies have shown that high concentrations of O-1821 may promote tumorigenesis, indicating the need for more precise dose regulation and application exploration.

[0003] Cannabigerorcinic acid (CBGOA) and Orsellinic acid (OSA) are two key precursor compounds of O-1821, and both have important pharmacological properties. As a natural compound, Orsellinic acid has been proven to have biological activities such as antioxidant and neuroprotection. Cannabigerorcinic acid has potential application value in cannabinoid synthesis and drug research and development due to its unique chemical structure and function. However, the traditional process of obtaining these cannabinoids and their precursors through plant extraction is restricted by high costs, limited resource supply, and the uncertainty of periodic production, which greatly limits its industrial production and extensive medicinal development.

[0004] The development of microbial metabolic engineering provides a new solution for the sustainable production of cannabinoids. By designing and synthesizing specific metabolic pathways, microbial cell factories can efficiently produce target compounds under controlled conditions. Especially the application of non-traditional microbial platforms provides new possibilities for breaking through the bottlenecks of traditional production.

[0005] Yarrowia lipolytica is a non-traditional yeast with high fatty acid metabolism capacity and genetic engineering flexibility. Compared with traditional model microorganisms (such as Saccharomyces cerevisiae), Yarrowia lipolytica has significant advantages in malonyl-CoA and isoprene pyrophosphate (IPP) flux, making it an ideal platform for the biosynthesis of complex natural products (such as cannabinoids). However, the research on the synthesis of cannabinoids and their analogs using Yarrowia lipolytica is still in its infancy, especially the research on the efficient production of O-1821 and its precursor compounds (such as cannabidiol and rutinic acid) is still blank, which hinders the development and application of these high value-added compounds in the pharmaceutical field.

[0006] In view of this, the present invention is proposed. Summary of the invention

[0007] The purpose of the present invention is to provide a recombinant expression vector, a recombinant bacterium, a method for synthesizing a cannabidiol analogue O-1821 and its application, so as to promote the industrialization process and clinical application prospects of cannabinoid drugs.

[0008] The present invention is achieved in that:

[0009] In a first aspect, the present invention provides a recombinant expression vector, comprising: a first expression vector, a second expression vector and a third expression vector;

[0010] The first expression vector includes the ArmB gene of the cannabinoid synthase; the second expression vector includes the tCsPT4 gene of the isopentenyl transferase derived from the plant Cannabis sativa and the NphB (Y288A / G286S) mutant gene of the isopentenyl transferase derived from Streptomyces; the third expression vector includes the CBDAS (G183V / N482W) gene of the cannabidiol synthase mutant derived from the plant Cannabis sativa.

[0011] In a second aspect, the present invention further provides a recombinant bacterium or a recombinant cell, which comprises the above-mentioned recombinant expression vector.

[0012] In a third aspect, the present invention also provides the use of the recombinant expression vector or the above-mentioned recombinant bacteria or recombinant cells in the synthesis of the cannabidiol analog O-1821, cannabiprofen acid or rutin acid.

[0013] In a fourth aspect, the present invention also provides a method for synthesizing a cannabidiol analogue O-1821, which comprises the following steps: transforming the above-mentioned recombinant expression vector into a chassis strain, eliminating the selection marker in the transformed bacteria, and screening to obtain recombinant bacteria; culturing the recombinant bacteria or culturing the above-mentioned recombinant bacteria.

[0014] In a fifth aspect, the present invention further provides a method for constructing an engineered strain of Yarrowia lipolytica, the method comprising:

[0015] The above recombinant expression vector was transfected into competent cells of Yarrowia lipolytica strain, and positive transformants were obtained by culture.

[0016] The present invention has the following beneficial effects:

[0017] The present invention reconstructs the de novo synthesis pathway of cannabidiol analog O-1821 and its precursors cannabidiol acid and moss acid in Yarrowia lipolytica through modular metabolic engineering and gene optimization design, and realizes the biosynthesis of the above products in yeast. The recombinant expression vector provided by the present invention can realize the co-expression of moss acid synthase gene, isopentenyl transferase tCsPT4 gene derived from plant cannabis, isopentenyl transferase NphB (Y288A / G286S) mutant gene derived from Streptomyces, and cannabidiol acid synthase mutant CBDAS (G183V / N482W) gene from plant cannabis after transfection into competent cells of chassis strains, and realizes the de novo synthesis of O-1821 and its precursors cannabidiol acid and moss acid. First, the catalytic efficiency of key enzymes can be enhanced by optimizing the metabolic flux of endogenous malonyl-CoA and geranyl diphosphate (GPP). Secondly, the co-expression system of condensed phase vesicle-like enzyme activity concentrate-diisopentenyl transferase was introduced to co-express diisopentenyl transferase (tCsPT4 and NphB), which significantly improved the efficiency of converting rutin acid to cannabidiol acid, and achieved the efficient synthesis of O-1821 and its precursor compounds. Compared with the chassis strain before the design, the Yarrowia lipolytica engineered strain provided by the present invention achieves the efficient synthesis of rutin acid, cannabidiol acid and cannabidiol analog O-1821 from scratch, which helps the Yarrowia lipolytica engineered strain to become an ideal platform for the biosynthesis of complex natural products (such as cannabinoids), and promotes the development and application of these high value-added compounds in the field of medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0019] Figure 1 The artificial synthesis route diagram of cannabinoid O-1821 and its precursor provided by the present invention;

[0020] Figure 2 The pYLXP' plasmid vector map provided in Example 1 of the present invention;

[0021] Figure 3 The pUrlp plasmid vector map provided in Example 1 of the present invention;

[0022] Figure 4 The pYLXP'-ArmB plasmid vector map provided in Example 1;

[0023] Figure 5 The pYLXP'-tCsPT4-NphB (Y288A / G286S) plasmid vector map provided in Example 1;

[0024] Figure 6 The pUrlp'-ArmB plasmid vector map provided in Example 1;

[0025] Figure 7 The pUrlp'-tCsPT4-NphB (Y288A / G286S) plasmid vector map provided in Example 1;

[0026] Figure 8 The pUrlp'-CBDAS (G183V / N482W) plasmid vector map provided in Example 1;

[0027] Fig. 9 is the MS spectrum of moss acid;

[0028] Fig.10 This is the MS spectrum of cannabinoid acid;

[0029] Fig.11 This is the mass spectrum of O-1821MS. DETAILED DESCRIPTION

[0030] References to embodiments of the present invention will now be provided in detail, one or more examples of which are described below. Each example is provided as an explanation rather than a limitation of the present invention. In fact, it will be apparent to those skilled in the art that various modifications and variations may be made to the present invention without departing from the scope or spirit of the present invention. For example, a feature illustrated or described as part of one embodiment may be used in another embodiment to produce a further embodiment.

[0031] The term "vector" is used herein in its most general sense and includes any intermediate medium for nucleic acid, which enables the nucleic acid to be introduced, for example, into prokaryotic and / or eukaryotic cells and, where appropriate, integrated into the genome. This type of vector is preferably replicated and / or expressed in cells. The term "vector" refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses or other vectors well known in the art. The term "plasmid" as used herein generally relates to a construct of extrachromosomal genetic material, typically a circular DNA double strand, which can replicate independently of chromosomal DNA. Any plasmid and vector can be used as long as they can replicate and be stable in the host.

[0032] In an optional embodiment, the above-mentioned vector is an expression vector. An important feature of an expression vector is that it usually contains a replication origin, a promoter, a marker gene and a translation control element.

[0033] In a first aspect, the present invention provides a recombinant expression vector, comprising: a first expression vector, a second expression vector and a third expression vector;

[0034] The first expression vector includes the ArmB gene of the mossic acid synthase; the second expression vector includes the tCsPT4 gene of the isopentenyl transferase derived from the plant Cannabis sativa and the NphB (Y288A / G286S) mutant gene of the isopentenyl transferase derived from Streptomyces; the third expression vector includes the CBDAS (G183V / N482W) gene of the cannabidiol synthase mutant derived from the plant Cannabis sativa.

[0035] The recombinant expression vector provided by the present invention can achieve co-expression of the cannabiquinone synthase gene (ArmB gene), the condensed phase vesicular enzyme activity concentrate-diisopentenyl transferase gene (tCsPT4 gene, NphB (Y288A / G286S) gene) and the cannabiquinone synthase gene (CBDAS (G183V / N482W) gene) after being transfected into the competent cells of the chassis strain, and through fermentation, the de novo synthesis of O-1821 and its precursors cannabiquinone acid and cannabiquinone acid is achieved.

[0036] The amino acid sequence of tricholonic acid synthase is shown in SEQ ID NO.5, the amino acid sequence of isopentenyl transferase tCsPT4 derived from the plant cannabis is shown in SEQ ID NO.6, the amino acid sequence of the isopentenyl transferase NphB (Y288A / G286S) mutant derived from Streptomyces is shown in SEQ ID NO.7, and the amino acid sequence of the cannabidiolic acid synthase mutant CBDAS (G183V / N482W) derived from the plant cannabis is shown in SEQ ID NO.8.

[0037] In a preferred embodiment of the present invention, the nucleotide sequence of the orsellinic acid synthase ArmB gene is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence shown in SEQ ID NO.1, the nucleotide sequence of the isopentenyltransferase tCsPT4 gene is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence shown in SEQ ID NO.2, the nucleotide sequence of the isopentenyltransferase NphB (Y288A / G286S) mutant gene is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence shown in SEQ ID NO. The sequence shown in NO.3 has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity, and the nucleotide sequence of the cannabidiol acid synthase mutant CBDAS (G183V / N482W) gene has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the sequence shown in SEQ ID NO.4. Those skilled in the art can set the corresponding gene coding sequence according to the preference of the target host cell for codons, not limited to the nucleotide sequences listed above.

[0038] In a preferred embodiment of the present invention, the 5' end of the orsellinic acid synthase ArmB gene on the first expression vector has a promoter, and the 3' end has a terminator corresponding to the promoter; the 5' end of the isopentenyl transferase tCsPT4 gene has a promoter, and the 3' end has a terminator corresponding to the promoter; the 5' end of the isopentenyl transferase NphB (Y288A / G286S) mutant gene has a promoter, and the 3' end has a terminator corresponding to the promoter, and the 5' end of the cannabidiol synthase mutant CBDAS (G183V / N482W) gene has a promoter, and the 3' end has a terminator corresponding to the promoter.

[0039] In a preferred embodiment of the present invention, the promoter is selected from a constitutive promoter, an inducible promoter, a universal promoter or a cell type specific promoter. Those skilled in the art can adjust the type of promoter according to the type of host cell.

[0040] In a preferred embodiment of the present invention, the constitutive promoter is selected from TEF, RPS7, ADH1, GAPDH, PGK1, TP1, ENO, PYK1, GAP, and YPT1.

[0041] Among them, TEF and RPS7 are promoters of Yarrowia lipolytica, ADH1, GAPDH, PGK1, TP1, ENO, and PYK1 are promoters of Saccharomyces cerevisiae, and GAP and YPT1 are promoters of Pichia pastoris.

[0042] The inducible promoter is selected from POX2, POT1, ICL1, LAC4, ADH4, GAL1-10, CUP1, ADH2, PLD1 or AOX1.

[0043] In a preferred embodiment of the present invention, the promoters of the cannabidiol synthase ArmB gene, the isopentenyl transferase tCsPT4 gene, the isopentenyl transferase NphB (Y288A / G286S) mutant gene and the cannabidiol synthase mutant CBDAS (G183V / N482W) gene on the expression vector are all TEF promoters with intron sequences.

[0044] In a preferred embodiment of the present invention, the terminator is selected from XPR2, CYC1, TDH1, SUC2, ADH1, RBL41B or PGK1, preferably XPR2.

[0045] In a preferred embodiment of the present invention, the backbone vector of the expression vector is pYLXP or pUrlp, wherein pYLXP is used as the basic vector for synthesizing the target gene, and pUrlp is used as the intermediate vector for introducing into the host cell.

[0046] In a second aspect, the present invention further provides a recombinant bacterium or a recombinant cell, which comprises the above-mentioned recombinant expression vector.

[0047] The pathway for the synthesis of cannabinoid O-1821 by recombinant bacteria is as follows:

[0048] S1: After glucose enters the Yarrowia lipolytica cell, it is decomposed into pyruvate through the intracellular glycolysis pathway. Pyruvate is oxidatively decarboxylated to generate acetyl-CoA by the catalysis of pyruvate dehydrogenase complex. Acetyl-CoA is carboxylated to generate malonyl-CoA by the catalysis of acetyl-CoA carboxylase (ACC). The above pathway is the endogenous pathway of Yarrowia lipolytica cells and can be enhanced by genetic engineering.

[0049] S2: expression and integration of Armillaria mellea lysine synthase gene ArmB, catalyzing acetyl-CoA and malonyl-CoA to produce lysine;

[0050] S3: Co-expression system of enzyme activity concentrate in vesicle-like form-di-isopentenyl transferase: by simultaneously expressing the truncated isopentenyl transferase tCsPT4 from the plant Cannabis sativa and the isopentenyl transferase NphB (Y288A / G286S) mutant from Streptomyces, the two heterologous isopentenyl transferases form a condensed phase vesicle-like enzyme activity concentrate, catalyzing the binding of rutin acid to endogenous geranyl diphosphate (GPP), thereby achieving the synthesis of rutin acid;

[0051] S4: The cannabidiolic acid synthase mutant CBDAS (G183V / N482W) from the plant cannabis catalyzes cannabidiol acid to form the O-1821 precursor CBDOA, and finally CBDOA is decarboxylated to form O-1821 under heating (150-200℃).

[0052] In a preferred embodiment of the present invention, the base strain of the recombinant bacteria is yeast. In other embodiments, the base strain of the recombinant bacteria can also be bacteria, such as Escherichia coli and Streptomyces.

[0053] Recombinant cells are competent cells of recombinant bacteria.

[0054] In a preferred embodiment of the present invention, the yeast is selected from Yarrowia lipolytica, Saccharomyces cerevisiae or Pichia pastoris.

[0055] In a preferred embodiment of the present invention, the chassis strain of the recombinant bacteria is a Yarrowia lipolytica strain that is double-deficient in leucine LEU and uracil URA.

[0056] In a third aspect, the present invention also provides the use of the recombinant expression vector or the above-mentioned recombinant bacteria or recombinant cells in the synthesis of the cannabidiol analog O-1821, cannabiprofen acid or rutin acid.

[0057] In a preferred embodiment of the present invention, the recombinant bacteria refers to: Yarrowia lipolytica or a culture thereof;

[0058] In an optional embodiment, the culture refers to the fermentation broth, fermentation supernatant, bacterial sludge or fermentation broth extract of Yarrowia lipolytica.

[0059] The bacterial sludge can be obtained by centrifugation or static sedimentation.

[0060] In an optional embodiment, the Yarrowia lipolytica includes but is not limited to at least one of a concentrate, a gelatinized product, a dried product, a liquid product, a diluted product, and a crushed product of the Yarrowia lipolytica. The dried product includes but is not limited to a spray-dried product, a freeze-dried product, a vacuum-dried product, a drum-dried product, and the like.

[0061] In a preferred embodiment of the present invention, the bacterial agent is solid, liquid or semi-solid. Semi-solid is, for example, an ointment.

[0062] In a preferred embodiment of the present invention, the bacterial agent further comprises a carrier and / or auxiliary materials;

[0063] In a preferred embodiment of the present invention, the carrier or auxiliary material is selected from:

[0064] At least one of a protective agent, an excipient, a binder, a disintegrant, a lubricant, a flavor, a preservative, a stabilizer, a suspending agent, a dispersing agent and a diluent.

[0065] Examples include: excipients such as sucrose, starch, mannitol, sorbitol, lactose, glucose, cellulose, talc, calcium phosphate, calcium carbonate; binders such as cellulose, methylcellulose, hydroxypropyl cellulose, polypropyl pyrrolidone, gelatin, gum arabic, polyethylene glycol, sucrose, starch; disintegrants such as starch, hydrolyzed starch, carboxymethyl cellulose, carboxymethyl cellulose calcium salt, hydroxypropyl starch, sodium starch glycolate, sodium bicarbonate, calcium phosphate, calcium citrate; lubricants such as magnesium stearate, talc, sodium lauryl sulfate; flavors such as citric acid, menthol, glycine , orange powder; preservatives, such as sodium benzoate, sodium bisulfite, parabens (such as methyl paraben, ethyl paraben, propyl paraben, butyl paraben); stabilizers, such as citric acid, sodium citrate, acetic acid and polycarboxylic acids from the titriplex series, such as diethylenetriaminepentaacetic acid (DTPA); suspending agents, such as methylcellulose, polyvinylpyrrolidone, aluminum stearate; dispersants; diluents, such as water, organic solvents; waxes, fats and oils, such as beeswax, cocoa butter; polyethylene glycol; white petrolatum, etc.

[0066] In a preferred embodiment of the present invention, the bacterial agent is prepared from the culture precipitate and / or supernatant of Yarrowia lipolytica.

[0067] The culture includes but is not limited to that obtained by solid fermentation or liquid fermentation.

[0068] The culture of Yarrowia lipolytica includes but is not limited to at least one of a concentrate, a gelatinized product, a dried product, a liquid product, a diluted product, and a crushed product of Yarrowia lipolytica. The dried product includes but is not limited to a spray-dried product, a freeze-dried product, a vacuum-dried product, a drum-dried product, and the like.

[0069] In an optional embodiment, the culture is in the form of a bacterial agent, which is obtained by diluting a fermentation product, for example.

[0070] In a fourth aspect, the present invention also provides a method for synthesizing a cannabidiol analogue O-1821, which comprises the following steps: transforming the above-mentioned recombinant expression vector into a double-deficient chassis strain of leucine LEU and uracil URA, and eliminating the selection markers in the transformed bacteria to obtain recombinant bacteria by screening; culturing the recombinant bacteria or culturing the above-mentioned recombinant bacteria.

[0071] In a preferred embodiment of the present invention, the culture conditions are culture at a temperature of 28-32°C, pH: 4.5-6.0, and a rotation speed of 180-250 rpm;

[0072] In a preferred embodiment of the present invention, the culture is carried out in a YPD medium or in a fermentation medium, and the fermentation medium comprises: a nitrogen source, a carbon source, an inorganic salt and a TRACE component, wherein the nitrogen source comprises yeast extract powder with a final concentration of 2.0-5.0 g / L, peptone with a final concentration of 5.0-10.0 g / L and urea with a final concentration of 1.0-5.0 g / L, the carbon source comprises glucose with a final concentration of 50-100 g / L, and the inorganic salt comprises magnesium sulfate with a final concentration of 0.2-1.0 g / L, potassium dihydrogen phosphate with a final concentration of 2-6 g / L, potassium hydrogen phosphate with a final concentration of 2-6 g / L, and potassium phosphate with a final concentration of 0.1-1.0 mg / L. Biotin, 5-20 mg / L calcium pantothenate, 5-20 mg / L niacin, 100-500 mg / L inositol, 1-5 mg / L p-aminobenzoic acid, 5-20 mg / L VB1 and 5-20 mg / L VB6, the TRACE component includes EDTA with a final concentration of 100-200 mg / L, 50-150 mg / L zinc sulfate heptahydrate, 1-10 mg / L manganese chloride tetrahydrate, 1-10 mg / L copper sulfate, 5-15 mg / L cobalt chloride hexahydrate, 1-10 mg / L sodium molybdate dihydrate, 10-50 mg / L calcium chloride and 30-100 mg / L ferrous sulfate heptahydrate.

[0073] In a preferred embodiment, the nitrogen source includes yeast extract powder with a final concentration of 3.4 g / L, peptone with a final concentration of 6.6 g / L, and urea with a final concentration of 2.67 g / L; the carbon source includes glucose with a final concentration of 80 g / L; the inorganic salts include magnesium sulfate with a final concentration of 0.49 g / L, potassium dihydrogen phosphate with a final concentration of 4 g / L, potassium hydrogen phosphate with a final concentration of 4 g / L, biotin with a final concentration of 0.6 mg / L, calcium pantothenate with a final concentration of 12 mg / L, nicotinic acid with a final concentration of 300 mg / L. The TRACE ingredients include EDTA with a final concentration of 150 mg / L, zinc sulfate heptahydrate of 102 mg / L, manganese chloride tetrahydrate of 5 mg / L, copper sulfate of 5 mg / L, cobalt chloride hexahydrate of 8.6 mg / L, sodium molybdate dihydrate of 5.6 mg / L, calcium chloride of 29 mg / L, and ferrous sulfate heptahydrate of 54.7 mg / L. Cultivation under the above optimized culture medium can greatly increase the yield of O-1821 and its precursors cannabinoid acid and rutin acid.

[0074] In a fifth aspect, the present invention further provides a method for constructing an engineered strain of Yarrowia lipolytica, the method comprising:

[0075] The above recombinant expression vector was transfected into competent cells of Yarrowia lipolytica strain, and positive transformants were obtained by culture.

[0076] Preferably, the first expression vector is first transformed into a competent cell of the chassis strain to obtain a first strain, and then the competent cell of the first strain is prepared, and the Cre enzyme expression vector is transformed into the competent cell of the first strain to obtain a strain containing the ArmB gene with the screening tag sequence removed, and then the second expression vector is transformed into the strain containing the ArmB gene with the screening tag sequence removed to obtain a second strain; then the competent cell of the second strain is prepared, and the Cre enzyme expression vector is transformed into the competent cell of the second strain to obtain a strain containing the ArmB gene and tCsPT4-Nph with the screening tag sequence removed. B (Y288A / G286S) gene strain; then the third expression vector is transformed into the strain containing the ArmB gene and tCsPT4-NphB (Y288A / G286S) gene without the screening tag sequence to obtain the third strain, and then the competent state of the third strain is prepared, and the Cre enzyme expression vector is transformed into the competent state of the third strain to obtain the strain containing the ArmB gene, tCsPT4-NphB (Y288A / G286S) gene and CBDAS (G183V / N482W) gene without the screening tag sequence;

[0077] The strains containing the ArmB gene, the tCsPT4-NphB (Y288A / G286S) gene and the CBDAS (G183V / N482W) gene were knocked out in sequence, and the key genes in the competitive metabolic pathway included: Ku70, ARO8, ACE, LDH, DGA1, GGPPS, POX4 and LIP1 to obtain engineered strains.

[0078] By knocking out key genes in the competing metabolic pathway and improving the effectiveness of substrate supply, the efficient synthesis of O-1821 and its precursor compounds was further achieved.

[0079] Ku70: ATP-dependent DNA helicase II subunit 1, ARO8: aromatic amino acid transaminase 8, ACE: acetyl-CoA carboxylase, LDH: lactate dehydrogenase, DGA1: diacylglycerol acyltransferase 1, GGPPS: geranylgeranyl pyrophosphate synthetase, POX4: peroxisomal acyl-CoA oxidase 4 and LIP1: lipase 1, to obtain the final engineered strain with the fermentation advantage of the target product.

[0080] In a preferred embodiment of the present invention, the Yarrowia lipolytica strain is double-deficient in uracil Ura and leucine Leu.

[0081] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0082] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0083] Example 1

[0084] Construction of recombinant expression vector.

[0085] (1) Obtain the pYLXP' and pUrlp plasmid vectors. Both pYLXP' and pUrlp vectors were obtained from the Laboratory of Synthetic Biology and Intelligent Control, Guangdong Technion-Israel Institute of Technology.

[0086] Figure 2 This is the pYLXP' plasmid vector map.

[0087] Depend on Figure 2It can be seen that the pYLXP' vector is a circular plasmid vector containing a leucine Leu2 tag used for screening in Yarrowia lipolytica, as well as a strong promoter TEF and terminator XPR2 that function in Yarrowia lipolytica, and about 1000bp sequences upstream and downstream of the NotI restriction site are homologous arms of the pBR322 docking platform, which can integrate the carried gene into the pBR322 platform of the chassis strain.

[0088] Figure 3 This is the map of the pUrlp plasmid vector.

[0089] Depend on Figure 3 It can be seen that the pUrlp vector is a circular plasmid vector containing leucine Leu2 and uracil ylUra3 tags used for screening in Yarrowia lipolytica, wherein uracil ylUra3 is controlled by the TEF promoter and the XPR2 terminator, and contains LoxP sites before TEF and after XPR2, which can realize the Cre-LoxP technology to recover the Ura gene for nutritional screening. pUrlp can be embedded in the upstream and downstream homologous arms of the site-specific insertion chromosome site for site-specific gene recombination.

[0090] (2) Double enzyme digestion of pYLXP' and pUrlp plasmid vectors was performed to obtain linearized vectors of pYLXP' and pUrlp with sticky ends.

[0091] In this example, the pYLXP' plasmid vector was double-digested with KpnI (endonuclease 1)-SnaBI (endonuclease 2), and the pUrlp plasmid vector was double-digested with NheI (endonuclease 3)-SalI (endonuclease 4).

[0092] The relevant reaction system refers to Table 1 (double enzyme digestion reaction system), wherein the reaction conditions are: 37° C., 60 min.

[0093] Table 1: Double enzyme digestion reaction system

[0094] Reagents Usage amount (μL) Carrier 8 10X Buffer 2 Endonuclease 1 / 3 1 Endonuclease 2 / 4 1 Double distilled water 8

[0095] When the enzyme digestion reaction is completed, gel electrophoresis is used to determine whether the digestion is successful.

[0096] The linearized pYLXP' vector fragments were separated by agarose gel electrophoresis and the test kit was recovered by DNA gel extraction ( The target vector fragments in the linearized pYLXP' vector fragments were recovered by using an agarose gel extraction kit (Cat. No.: 19101ES70) to obtain the purified pYLXP' linearized vector fragments.

[0097] Agarose gel electrophoresis is a method used to separate DNA fragments based on their length.

[0098] The DNA gel recovery test kit is a kit for recovering the required gene fragments from DNA agarose gel. The DNA gel recovery test kit can be used to recover the DNA fragments cut out from the pYLXP' vector. That is, the pYLXP' fragment is a gene fragment formed after double enzyme cutting.

[0099] (3) Assemble the linearized pYXLP vector and the target gene. Figure 2 The Incomplete Intron and XPR2_terminator of the pYLXP' vector were obtained. Figure 4 ), pYLXP'-tCsPT4, pYLXP'-NphB(Y288A / G286S), pYLXP'-CBDAS(G183V / N482W)(single gene) recombinant plasmids and pYLXP'-tCsPT4-NphB(Y288A / G286S)(multi-gene) recombinant plasmids ( Figure 5 ).

[0100] Linearize the pYLXP' vector and PCR amplify the DNA fragments of ArmB, tCsPT4, NphB (Y288A / G286S) and CBDAS (G183V / N482W), PCR reaction (conditions refer to Table 3), and Gibson assembly through seamless cloning (Biyuntian Seamless Cloning Kit, Catalog No.: D7010M) shown in Table 2 to construct pYLXP'-ArmB, pYLXP'-tCsPT4, pYLXP'-NphB (Y288A / G286S), pYLXP'-CBDAS (G183V / N482W) (single gene) recombinant plasmids, respectively.

[0101] Table 2: List of main primers for seamless cloning Gibson construction, where Genes_Fw and Genes_Rvs are universal primers for amplifying genes, and the amplified PCR product fragments are seamlessly cloned and assembled with the pYLXP linearized vector, and TEF_Fwd and XPR2_Rvs are universal primers used to verify the correctness of the assembly after assembly:

[0102]

[0103] Table 3: PCR reaction conditions:

[0104]

[0105] The pYLXP'-tCsPT4 and pYLXP'-NphB (Y288A / G286S) (single gene) recombinant plasmids with different genes were digested by two combinations of NheI (endonuclease 3) + SalI (endonuclease 4) and AvrII (endonuclease 5) + SalI (endonuclease 4), respectively, and the target single gene band was recovered by agarose gel electrophoresis and DNA gel again. Since NheI and AvrII have the same enzyme-cut sticky ends, the recovered DNA fragments with the target genes can be assembled by multi-gene subcloning by T4 ligase (T4 ligase reaction system refers to Table 4), and finally constructed into pYLXP'-tCsPT4-NphB (Y288A / G286S) (multi-gene) recombinant plasmid.

[0106] Table 4: T4 ligase reaction system:

[0107] Reagents Usage amount (μL) Vector gene fragment 2 Insert gene fragment 2 T4 Ligase 1 10X Buffer 1 Double distilled water 4

[0108] (4) The linearized pUrlp vector was site-specifically integrated with the pYLXP'-ArmB recombinant plasmid, pYLXP'-tCsPT4-NphB (Y288A / G286S), and pYLXP'-CBDAS (G183V / N482W) recombinant plasmids by restriction digestion and ligation.

[0109] Specifically, the steps include: firstly, double-digesting the pYLXP'-ArmB recombinant plasmid, pYLXP'-tCsPT4-NphB (Y288A / G286S), and pYLXP'-CBDAS (G183V / N482W) recombinant plasmid with NheI and SalI endonucleases, and then connecting the linearized pUrlp vector with the linearized pYLXP'-ArmB recombinant plasmid, pYLXP'-tCsPT4-NphB (Y288A / G286S), and pYLXP'-CBDAS (G183V / N482W) recombinant plasmid with T4 ligase to obtain the site-specific integration pUrlp'-ArmB ( Figure 6 ), pUrlp'-tCsPT4-NphB(Y288A / G286S)( Figure 7 ), pUrlp'-CBDAS(G183V / N482W)( Figure 8 )Gene site-directed recombination recombinant plasmid.

[0110] Example 2

[0111] Construction of an engineered strain of Yarrowia lipolytica for synthesizing the cannabidiol analog O-1821 and its precursors cannabiprole acid and rutinic acid.

[0112] (i) Preparation of competent cells of Yarrowia lipolytica chassis strain.

[0113] The chassis strain was cultured in non-selective yeast medium YPD, where the chassis strain was a po1fk Yarrowia lipolytica strain that was double deficient in leucine LEU and uracil URA.

[0114] Note: The above-mentioned leucine LEU and uracil URA double-deficient po1fk Yarrowia lipolytica strain is stored and backed up in the Synthetic Biology and Intelligent Control Laboratory of Guangdong Israel Institute of Technology and can be sold to the outside.

[0115] Specifically:

[0116] Take 1 mL of overnight bacterial liquid and centrifuge at low speed, discard the supernatant, and thoroughly mix and shake the precipitate with 95 μL 50% PEG4000 and 5 μL 2M lithium acetate solution to form competent cells.

[0117] The culture conditions are as follows: the chassis strain is inoculated into a test tube containing 3 mL YPD medium, and cultured in a shaking incubator at 30° C. and a rotation speed of 250 rpm.

[0118] The non-selective yeast culture medium was YPD (Yeast extract peptone dextrose), which included 10 g / L yeast extract, 20 g / L glucose and 20 g / L peptone.

[0119] (ii) The recombinant expression vectors obtained in Example 1 (pUrlp'-ArmB, pUrlp'-tCsPT4-NphB (Y288A / G286S), pUrlp'-CBDAS (G183V / N482W)) were linearized and transformed into the competent cells prepared in the previous step by enzyme digestion.

[0120] Specific methods include:

[0121] 1. pUrlp'-ArmB, pUrlp'-tCsPT4-NphB (Y288A / G286S), and pUrlp'-CBDAS (G183V / N482W) were digested with AvrII (endonuclease 5), and then linearized vectors were obtained by agarose gel electrophoresis and DNA gel recovery;

[0122] 2. First, thoroughly mix the obtained pUrlp'-ArmB linearized vector (0.25-0.5 μg or 5 μL) and 5 μL of boiled ssDNA (deformed fish sperm DNA), and then add it to the prepared leucine LEU and uracil URA double-deficient po1fk Yarrowia lipolytica strain competent cells;

[0123] Incubate in a 30°C water bath for 30-45 minutes, vortexing for 15 seconds every 10 minutes;

[0124] Heat shock at 39°C for 10 min;

[0125] 3. Spread the corresponding screening plates. The transformed pUrlp'-ArmB contains the URA3 marker and is spread on CSM-ura plates.

[0126] The CSM-ura medium formula is as follows: glucose 20 g / L, yeast nitrogen matrix without ammonium sulfate 1.7 g / L, ammonium sulfate (NH 4 ) 2 SO 4 )5g / L, uracil Ura defective amino acid 1.29g / L.

[0127] 4. Recover the URA gene through nutritional screening using Cre-LoxP technology.

[0128] The genome-integrated strain with the target ArmB gene obtained in the previous step is used to prepare a competent strain with the target ArmB gene, and then the Cre enzyme expression vector is transformed into the competent strain. The URA gene (tag) in the integrated strain can be removed by Cre, and the uracil Ura-deficient strain can be obtained again, so that the pUrlp vector plasmid can be used again to carry out multi-gene multi-copy number integration transformation of the target strain.

[0129] 5. Prepare the competent cells of the integrated strain containing the ArmB gene and the URA gene removed. Mix the pUrlp'-tCsPT4-NphB (Y288A / G286S) linearized vector (0.25-0.5 μg or 5 μL) and 5 μL of boiled ssDNA (deformed fish sperm DNA) thoroughly, and then add them to the competent cells of the integrated strain containing the ArmB gene and the URA gene removed;

[0130] Incubate in a 30°C water bath for 30-45 minutes, vortexing for 15 seconds every 10 minutes;

[0131] Heat shock at 39°C for 10 min;

[0132] 6. Spread the corresponding screening plates. The transformed pUrlp'-tCsPT4-NphB (Y288A / G286S) contains the URA3 marker and is spread on CSM-ura plates.

[0133] 7. Recover nutrition and screen URA genes through Cre-LoxP technology.

[0134] The genome-integrated strain with the target ArmB and tCsPT4-NphB (Y288A / G286S) genes obtained in the previous step is used to prepare a competent state of the integrated strain with the ArmB and tCsPT4-NphB (Y288A / G286S) genes, and then the Cre enzyme expression vector is transformed into the competent state. The URA gene (tag) in the integrated strain can be removed by Cre, and the uracil Ura-deficient strain can be obtained again, so that the pUrlp vector plasmid can be used again to carry out multi-gene multi-copy number integration transformation of the target strain.

[0135] 8. Prepare a competent cell of the integration strain containing ArmB and tCsPT4-NphB (Y288A / G286S) genes and removing the URA gene. Fully mix the pUrlp'-CBDAS (G183V / N482W) linearized vector (0.25-0.5 μg or 5 μL) and 5 μL of boiled ssDNA (deformed fish sperm DNA), and then add it to the competent cells of the integration strain containing ArmB and tCsPT4-NphB (Y288A / G286S) genes and removing the URA gene.

[0136] Incubate in a 30°C water bath for 30-45 minutes, vortexing for 15 seconds every 10 minutes;

[0137] Heat shock at 39°C for 10 min;

[0138] 9. Spread the corresponding screening plates. The transformed pUrlp'-CBDAS (G183V / N482W) contains the URA3 marker and is spread on CSM-ura plates.

[0139] 10. Recover the URA gene through nutritional screening using Cre-LoxP technology.

[0140] The genome-integrated strain with the target ArmB, tCsPT4-NphB (Y288A / G286S), and CBDAS (G183V / N482W) genes obtained in the previous step is used to prepare a competent state of the integrated strain with the ArmB, tCsPT4-NphB (Y288A / G286S), and CBDAS (G183V / N482W) genes, and then the Cre enzyme expression vector is transformed into the competent state. The URA gene (tag) in the integrated strain can be removed by Cre, and the uracil Ura-deficient strain (i.e., the engineered Yarrowia lipolytica) is obtained again.

[0141] (III) Screening and obtaining engineered Yarrowia lipolytica strains that have the ability to synthesize O-1821 and its precursors cannabidiol and rutinic acid from scratch.

[0142] Pathway diagram for the de novo synthesis of O-1821 and its precursors cannabidiol acid and rutin acid Figure 1 shown.

[0143] The Yarrowia lipolytica engineered bacteria capable of synthesizing O-1821 and its precursors were fermented in YPD liquid medium at 30° C. for 4 to 5 days.

[0144] The non-selective yeast culture medium was YPD (Yeast extract peptone dextrose), which included 10 g / L yeast extract, 80 g / L glucose and 20 g / L peptone.

[0145] The fermentation conditions were as follows: the transformed Yarrowia lipolytica was placed in a 250 mL baffled flask with a liquid volume of 30 mL.

[0146] Example 3

[0147] Product Identification:

[0148] To quantify the concentration of the product, 400 μL of the whole cell culture was taken and treated with a three-dimensional centrifugal cryogenic sample grinder, 400 μL of ethyl acetate (0.05% formic acid) and glass beads (0.5 mm) at -20°C. The grinding speed was 21 m / s for 60 seconds, with an interval of 15 seconds, and repeated 30 cycles.

[0149] After the initial extraction, 300 μL of the ethyl acetate organic layer was transferred to a new microcentrifuge tube. Two more extractions were performed, each with 400 μL of ethyl acetate (0.05% formic acid), and then 300 μL of the upper extract was mixed with the previous extraction. The organic portion was then evaporated using an Eppendorf concentrator, and the dried extract was resuspended in methanol / HO (80 / 20) and vortexed for 1 minute.

[0150] The extracts were then subjected to HPLC analysis using a 1260infinity liquid chromatograph and a reversed-phase C18 column (ZORBAX Eclipse Plus C18, 4.6 × 100 mm, 3.5 μm, Agilent) connected to a diode array detector (210, 270 nm).

[0151] The mobile phase was set to solvent A (water containing 0.05% formic acid) and solvent B (methanol). The compounds were separated by gradient elution, and the proportion of solvent B was linearly increased from 35% to 100% in 15 minutes, maintained at 100% for 5.0 minutes, decreased from 100% to 35% in 4 minutes, and maintained at 35% for 4 minutes.

[0152] LC-MS analysis was performed using a Thermo Scientific Orbitrap Exploris 120 mass spectrometer equipped with a heated electrospray ionization source (HESI).

[0153] Chromatographic separation was performed on an Accucore C18 HPLC column (2.6 μm, 2.1 x 150 mm). The mobile phase was 0.1% formic acid in water (phase A) and methanol (phase B), with a gradient of: 0 min, 5% B; 0.5 min, 5% B; 13 min, 95% B; 16.9 min, 95% B; 17 min, 5% B; 20 min, 5% B. The mass range of mass spectrometry detection was 100-1000 m / z, with collision energies of 20, 50, and 80 eV. Compound identification was performed using multiple database and spectral library automatic search tools (including mzCloud, Chemspider, KEGG, and BioCyc) as well as local database search tools (such as mzVault spectral library and mass list).

[0154] Chromatographic identification results refer to Figure 9-11 As shown, the results showed that O-1821 ( Fig.11 ) and its precursor cannabinoid acid ( Fig.10 ) and lysine ( Fig. 9 ).

[0155] Example 4

[0156] This example optimizes the fermentation medium.

[0157] Take the Yarrowia lipolytica engineered strain (5*10 7 CFU / mL) 600 μL was added to 30 ml fermentation medium and shaken for 1 week;

[0158] At the same time, the blank vector pUrlp' transformed with the po1fk Yarrowia lipolytica chassis strain in step (I) was cultured as a control.

[0159] The fermentation medium was a non-selective yeast medium, YPD (Yeast extract peptone dextrose), which included 10 g / L yeast extract, 80 g / L glucose and 20 g / L peptone.

[0160] The conditions for shake flask culture were: 500 mL flat-bottom flask, 30° C., and 250 rpm.

[0161] After one week of shake flask culture, it was finally verified that the strain had the ability to synthesize O-1821 and its precursors cannabinoid acid and lysine acid. The shake flask yields were:

[0162]

[0163] Experimental Example 1

[0164] This experimental example provides an optimization scheme for the fermentation medium.

[0165] The fermentation medium formula of the optimized solution is as follows;

[0166]

[0167] The conditions for shake flask culture were: 500 mL flat-bottom flask, 30° C., and 250 rpm.

[0168] After 1 week of shake flask culture, the production capacity of the strain in YPD and optimized medium was compared and verified. The shake flask yields were:

[0169]

[0170] The experimental results showed that the yields of orsellinic acid OSA, cannabinoids CBGOA and O-1821 obtained using the optimized culture medium were much greater than the yields of each substance before optimization.

[0171] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A recombinant expression vector, characterized in that: It includes: a first expression vector, a second expression vector, and a third expression vector; The first expression vector includes the ArmB gene of the cannabinoid synthase; the second expression vector includes the tCsPT4 gene of the isopentenyl transferase derived from the plant Cannabis sativa and the NphB (Y288A / G286S) mutant gene of the isopentenyl transferase derived from Streptomyces; the third expression vector includes the CBDAS (G183V / N482W) gene of the cannabidiol synthase mutant derived from the plant Cannabis sativa.

2. The recombinant expression vector according to claim 1, characterized in that The amino acid sequence of the tricholonic acid synthase is shown in SEQ ID NO.5, the amino acid sequence of the isopentenyl transferase tCsPT4 derived from the plant cannabis is shown in SEQ ID NO.6, the amino acid sequence of the isopentenyl transferase NphB (Y288A / G286S) mutant derived from Streptomyces is shown in SEQ ID NO.7, and the amino acid sequence of the cannabidiol acid synthase mutant CBDAS (G183V / N482W) derived from the plant cannabis is shown in SEQ ID NO.8; Preferably, the nucleotide sequence of the mossic acid synthase ArmB gene has at least 85% identity with the sequence shown in SEQ ID NO.1, the nucleotide sequence of the isopentenyltransferase tCsPT4 gene has at least 85% identity with the sequence shown in SEQ ID NO.2, the nucleotide sequence of the isopentenyltransferase NphB (Y288A / G286S) mutant gene has at least 85% identity with the sequence shown in SEQ ID NO.3, and the nucleotide sequence of the cannabidiol acid synthase mutant CBDAS (G183V / N482W) gene has at least 85% identity with the sequence shown in SEQ ID NO.

4.

3. The recombinant expression vector according to claim 2, characterized in that The 5' end of the orsellinic acid synthase ArmB gene on the first expression vector has a promoter, and the 3' end has a terminator corresponding to the promoter; the 5' end of the isopentenyl transferase tCsPT4 gene has a promoter, and the 3' end has a terminator corresponding to the promoter; the 5' end of the isopentenyl transferase NphB (Y288A / G286S) mutant gene has a promoter, and the 3' end has a terminator corresponding to the promoter; and the 5' end of the cannabidiol acid synthase mutant CBDAS (G183V / N482W) gene has a promoter, and the 3' end has a terminator corresponding to the promoter; Preferably, the promoter is selected from a constitutive promoter, an inducible promoter, a ubiquitous promoter or a cell type specific promoter; Preferably, the constitutive promoter is selected from TEF, RPS7, ADH1, GAPDH, PGK1, TP1, ENO, PYK1, GAP or YPT1; The inducible promoter is selected from POX2, POT1, ICL1, LAC4, ADH4, GAL1-10, CUP1, ADH2, PLD1 or AOX1; Preferably, the promoters of the orsellinic acid synthase ArmB gene, the isopentenyl transferase tCsPT4 gene, the isopentenyl transferase NphB (Y288A / G286S) mutant gene and the cannabidiol acid synthase mutant CBDAS (G183V / N482W) gene on the expression vector are all TEF promoters with intron sequences; Preferably, the terminator is selected from XPR2, CYC1, TDH1, SUC2, ADH1, RBL41B or PGK1.

4. The recombinant expression vector according to claim 3, characterized in that The backbone vector of the expression vector is pYLXP or pUrlp.

5. A recombinant bacterium or recombinant cell, characterized in that: It comprises the recombinant expression vector according to any one of claims 1 to 4.

6. The recombinant bacterium or recombinant cell according to claim 5, characterized in that: The chassis strain of the recombinant bacteria is yeast; the recombinant cells are competent cells of the recombinant bacteria; Preferably, the yeast is selected from a strain of Yarrowia lipolytica, Saccharomyces cerevisiae or Pichia pastoris; Preferably, the chassis strain of the recombinant bacteria is a Yarrowia lipolytica strain that is double-deficient in leucine LEU and uracil URA.

7. Use of the recombinant expression vector according to any one of claims 1 to 4 or the recombinant bacteria or recombinant cells according to any one of claims 5 to 6 in the synthesis of cannabidiol analog O-1821, cannabiprofen acid or rutin acid.

8. A method for synthesizing a cannabidiol analogue O-1821, characterized in that: The method comprises the following steps: transforming the recombinant expression vector described in any one of claims 1 to 4 into a double-deficient chassis strain of leucine LEU and uracil URA, and eliminating the selection marker in the transformed bacteria to obtain recombinant bacteria by screening; culturing the recombinant bacteria or culturing the recombinant bacteria or recombinant cells described in any one of claims 5 to 6; Preferably, the culture conditions are culture at a temperature of 28-32°C, a pH of 4.5-6.0, and a rotation speed of 180-250 rpm; Preferably, the culture medium is YPD medium or fermentation medium, wherein the fermentation medium comprises: a nitrogen source, a carbon source, an inorganic salt and a TRACE component, wherein the nitrogen source comprises a final concentration of 2.0 – 5.0 g / L yeast extract, 5.0-10.0 g / L peptone and 1.0-5.0 g / L urea, the carbon source includes glucose with a final concentration of 50-100 g / L, the inorganic salts include magnesium sulfate with a final concentration of 0.2-1.0 g / L, potassium dihydrogen phosphate with a final concentration of 2-6 g / L, potassium hydrogen phosphate with a final concentration of 2-6 g / L, biotin with a final concentration of 0.1-1.0 mg / L, calcium pantothenate with a final concentration of 5-20 mg / L, nicotinic acid with a final concentration of 5-20 mg / L, inositol with a final concentration of 100-500 mg / L, and 1-5 The TRACE component comprises EDTA with a final concentration of 100-200 mg / L, zinc sulfate heptahydrate of 50-150 mg / L, manganese chloride tetrahydrate of 1-10 mg / L, copper sulfate of 1-10 mg / L, cobalt chloride hexahydrate of 5-15 mg / L, sodium molybdate dihydrate of 1-10 mg / L, calcium chloride of 10-50 mg / L and ferrous sulfate heptahydrate of 30-100 mg / L.

9. A method for constructing an engineered strain, characterized in that: The construction method comprises: Transfecting the recombinant expression vector according to any one of claims 1 to 4 into competent cells of the chassis strain, and culturing to obtain positive transformants; Preferably, the first expression vector is first transformed into a competent cell of the chassis strain to obtain a first strain, and then the competent cell of the first strain is prepared, and the Cre enzyme expression vector is transformed into the competent cell of the first strain to obtain an ArmB gene-containing strain with the screening tag sequence removed, and then the second expression vector is transformed into the ArmB gene-containing strain with the screening tag sequence removed to obtain a second strain; then the competent cell of the second strain is prepared, and the Cre enzyme expression vector is transformed into the competent cell of the second strain to obtain an ArmB gene-containing strain with the screening tag sequence removed and tCsPT4- NphB (Y288A / G286S) gene strain; then transforming the third expression vector into the strain containing the ArmB gene and tCsPT4-NphB (Y288A / G286S) gene without the screening tag sequence to obtain the third strain, and then preparing the competent state of the third strain, transforming the Cre enzyme expression vector into the competent state of the third strain to obtain the strain containing the ArmB gene, tCsPT4-NphB (Y288A / G286S) gene without the screening tag sequence, and CBDAS (G183V / N482W) gene; The strain containing the ArmB gene, tCsPT4-NphB (Y288A / G286S) gene and CBDAS (G183V / N482W) gene is knocked out in sequence, and the key genes in the competitive metabolic pathway include: Ku70, ARO8, ACE, LDH, DGA1, GGPPS, POX4 and LIP1 to obtain an engineered strain.

10. The method for constructing an engineered strain of Yarrowia lipolytica according to claim 9, characterized in that: The Yarrowia lipolytica strain is a double-deficient type of uracil Ura and leucine Leu.

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