Yeast strain with high yield of cannabinoid synthesis precursor, construction method and application thereof

Abstract

The application discloses a yeast strain with high yield of cannabinoid synthesis precursors, a construction method and application thereof, and belongs to the technical field of synthetic biology and microorganisms. The application takes Saccharomyces cerevisiae as a host, screens genes participating in protein folding proteins to obtain an engineering strain for improving the yield of OA / OLO, further down-regulates the expression of FAS1 and MvaE enzymes, and then inserts a gene expression cassette CsAAE1-TKS-OA cassette into the rDNA site of the Saccharomyces cerevisiae genome in multiple copies to obtain a genetic engineering strain with improved OA / OLO yield. The application further improves the yield of OA / OLO by optimizing the substrate in the fermentation system, i.e. using a mixed substrate of sodium caproate and ethyl caproate, and the yield of OA / OLO in a 10L fermenter is about 10 mM OA and 10 mM OLO, and the equivalent yield is 2.2 g / L and 1.8 g / L respectively.

Description

Technical Field

[0001] This invention relates to a yeast strain that produces high-yield cannabinoid synthesis precursors, its construction method, and its application. Specifically, it involves specific modifications to the yeast strain and corresponding substrate ratio optimization to make it suitable for producing oleic acid and oleuropein, intermediate products of the high-yield cannabinoid biosynthesis pathway. This invention belongs to the fields of synthetic biology and microbiology. Background Technology

[0002] A common precursor in the biosynthetic pathway of cannabinoids is cannabigerolic acid (CBGA). CBGA synthesis originates from two precursors: geranyl diphosphate (GPP) and olivetolic acid (OA). GPP is derived from either the 2-methyl-D-erythritol-4-phosphate biosynthesis pathway (MEP pathway) or the mevalonate pathway (MVA pathway). OA synthesis begins with hexanoyl-CoA and proceeds via the polyketide synthase (PKS) biosynthesis pathway to obtain three molecules of malonyl-CoA. Hexanoyl-CoA is derived from de novo fatty acid synthesis or exogenously added hexanoic acid. CBGA can be converted into cannabinoids such as cannabidiol (CBDA) and tetrahydrocannabinol (THCA). These carboxylic acid forms of cannabinoids can be decarboxylated by heating and converted into common cannabinoids such as cannabidiol (CBG), cannabidiol (CBD), and tetrahydrocannabinol (THC).

[0003] Olivetolic acid (OA) and olivetol (OLO) are type III polyketide compounds derived from plants, possessing antibacterial, antitumor, and anti-UV activities. They provide the polyketide core for cannabinoid biosynthesis, representing the first key step in this process. Studies have found that cannabinoid phenolic acid synthase (CBGAS) uses GPP as a donor and OA as a specific isopentenyl acceptor; therefore, OA yield affects CBGA yield, and consequently, cannabinoid yield. OL, the decarboxylation product of OA, while not a substrate for CBBAS, can be recognized by the isopentenyltransferase NphB for the direct biocatalytic synthesis of CBG; or it can be used in chemical synthesis, using nerol as a starting substrate to synthesize CBD.

[0004] Currently, there are few reports on the natural extraction of oleyl alcohol and oleolic acid from plants. Furthermore, the extraction and preparation of oleyl alcohol (acid) from plants is affected by various factors, such as the plant's sensitivity to climate and disease, lack of GAP standardization, low content, large land area requirements, and long production cycles. However, chemical synthesis processes are complex, costly, and have very low yields, and also involve environmental pollution and demanding conditions.

[0005] Biosynthesis primarily uses *E. coli* and *Saccharomyces cerevisiae* as substrate bacteria. In 2022, patent publication CN114703171A reported an OA yield of 349.87 mg / L achieved using *E. coli* as the substrate bacteria. Also in 2022, patent publication CN114262695A reported a method using *Saccharomyces cerevisiae* as the substrate bacteria, inhibiting hexanoyl-CoA hydrolysis by knocking out three thioester hydrolases to promote OA production. However, the OA yield from fermentation remained low, falling short of industrial-scale production. Existing strain modifications offer limited improvement in OA / OLO yield and hinder industrial-scale production. Therefore, further development of new genes, enzymes, strains, and substrates is needed to enhance OA / OLO yield. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides an engineered strain that produces high levels of oleuropein and / or oleuropein, which can be further used for the production of cannabinoids.

[0007] The first objective of this invention is to provide an engineered strain that produces high levels of oleuropein and / or oleyl alcohol, using *Saccharomyces cerevisiae*, a yeast that produces cannabinoid phenolic acids, as a host, and integrating and expressing protein folding-related genes, a gene expression cassette CsAAE1-TKS-OAC cassette; the gene expression cassette CsAAE1-TKS-OAC cassette includes tandemly expressed acyl activator gene CsAAE1, polyketide synthase gene TKS, oleate cyclase gene OAC, and oleoyl ester geranyyl transferase gene CsPT.

[0008] In one embodiment, the protein folding-related gene is selected from any two of PDI1, SEC22, KAR2, ERO1, SED5, or BET1.

[0009] In one embodiment, the protein folding protein-related genes are PDI1 and SEC22.

[0010] In one embodiment, the genes PDI1 and SEC22 are integrated into the 1414a or 106a sites of the Saccharomyces cerevisiae genome, respectively.

[0011] In one embodiment, the engineered bacteria also overexpress Fad1, Fmn1, Hac1, and Gal4, knock out Pep4 and Gal80, downregulate the expression of the FAS1 gene and MvaE enzyme, and express OAC in multiple copies.

[0012] In one embodiment, the downregulation of FAS1 gene and MvaE enzyme expression is achieved by using the promoter pHXT1 to initiate the expression of FAS1 gene and MvaE enzyme.

[0013] In one embodiment, the gene expression cassette CsAAE1-TKS-OAC cassette is integrated in multiple copies into the rDNA site of the Saccharomyces cerevisiae genome.

[0014] In one embodiment, the multiple copy expression of OAC is the overexpression of TKS and OAC at site 1622b, the overexpression of OAC at site YPRCd15c, and the overexpression of OAC, OAC3, OAC5, and OAC4 at site HO.

[0015] In one embodiment, the amino acid sequences encoded by the genes PDI1, SEC22, KAR2, SED5, ERO1, or BET1 are shown in SEQ ID NO. 1 to 6, respectively.

[0016] In one embodiment, the amino acid sequence encoded by the gene FAS1 is shown in SEQ ID NO.7.

[0017] In one embodiment, the nucleotide sequence of the promoter pHXT1 is shown in SEQ ID NO.8.

[0018] In one embodiment, the amino acid sequences of the acyl activator gene CsAAE1, the polyketide synthase gene TKS, the oleate cyclase gene OAC, and the olive oil ester geraniyl transferase gene CsPT are shown in SEQ ID NO. 9–12.

[0019] In one embodiment, the Fad1 nucleotide sequence may be NC_001136.10(372688..373608,complement).

[0020] In one embodiment, the Fmn1 nucleotide sequence may be NC_001136.10(935236..935892,complement).

[0021] In one embodiment, the Hac1 nucleotide sequence may be NC_001138.5(75179..76147).

[0022] In one embodiment, the Gal4 nucleotide sequence may be NC_001148.4(79711..82356,complement).

[0023] In one embodiment, the Pep4 nucleotide sequence may be NC_001148.4(259714..260931,complement).

[0024] In one embodiment, the Gal80 nucleotide sequence may be NC_001145.3(171594..172901).

[0025] In one embodiment, the amino acid sequences of OAC3, OAC5, and OAC4 are shown in SEQ ID NO.18-20.

[0026] A second objective of this invention is a method for producing cannabinoid synthesis precursors, wherein the method involves inoculating the aforementioned engineered strain into a reaction system and fermenting to prepare cannabinoid synthesis precursors, wherein the cannabinoid synthesis precursors are oleuropein and / or oleuropein.

[0027] In one embodiment, the method uses a mixture of ethyl hexanoate and sodium hexanoate as a substrate.

[0028] In one embodiment, the mixing ratio of ethyl hexanoate and sodium hexanoate is (1:1) to (50:1).

[0029] In one embodiment, the mixing ratio of ethyl hexanoate and sodium hexanoate is 3:1.

[0030] In one embodiment, the reaction system uses glucose as a carbon source.

[0031] In one embodiment, the method involves activating the aforementioned engineered strain to obtain a seed liquid, inoculating the seed liquid into a reaction system containing ethyl hexanoate and sodium hexanoate, and fermenting and culturing cannabinoid synthesis precursors.

[0032] The present invention also provides the application of the above-described engineered strains or methods in the preparation of cannabinoid precursors and / or cannabinoids.

[0033] Beneficial effects:

[0034] This invention uses *Saccharomyces cerevisiae*, a yeast that produces cannabinoid phenolic acids, as the host. Based on overexpression of Fad1, Fmn1, Hac1, and Gal4, knockout of Pep4 and Gal80, and multiple copies of OAC, genes involved in protein folding were screened to obtain engineered strains with increased OA / OLO yields. Furthermore, the original promoters of FAS1 and MvaE enzymes were replaced with the pHXT1 promoter, and multiple copies of the gene expression cassette CsAAE1-TKS-OAC were inserted into the *Saccharomyces cerevisiae* genomic rDNA site to obtain genetically engineered strains with increased OA / OLO yields. This invention further improves OA / OLO yields by optimizing the substrate in the fermentation system, using a mixture of sodium hexanoate and ethyl hexanoate as the substrate. The yield in a 10L fermenter is approximately 10mM OA and 10mM OLO, equivalent to yields of 2.2g / L and 1.8g / L, respectively. Attached Figure Description

[0035] Figure 1 A schematic diagram of the MG36 plasmid CsAAE1-TKS-OAC expression cassette;

[0036] Figure 2 Horizontal fermentation test of modified protein overlap-related recombinant yeast strains in well plates;

[0037] Figure 3 Shake-flask horizontal fermentation test of multi-copy inserted recombinant yeast strain;

[0038] Figure 4 Shake-flask horizontal substrate fermentation test of recombinant yeast strain;

[0039] Figure 5 Recombinant brewing yeast is used in the production of OA / OLO. Detailed Implementation

[0040] the term

[0041] CBGA: cannabigerolic acid;

[0042] CsAAE1: Acyl activator enzyme that converts hexanoic acid or sodium hexanoate into hexanoyl-CoA;

[0043] TKS: Polyketide synthase;

[0044] OAC: Olilate cyclase;

[0045] GFP: Green fluorescent protein;

[0046] Leu2: Leucine;

[0047] pHXT1: Promoter, a promoter that responds to glucose concentration;

[0048] PDI1: Protein disulfide isomerase;

[0049] KAR2: An ATPase involved in protein translocation to the ER;

[0050] ERO1: Thiol oxidase required for oxidative protein folding in the ER;

[0051] SED5: a cis-Golgi t-SNARE synthetic protein;

[0052] BET1: A type II membrane protein required for vesicle transport;

[0053] SEC22: R-SNARE protein;

[0054] FAS1: Endogenous fatty acid synthase;

[0055] Pep4: Encodes vacuolar protease;

[0056] Fad1: Encodes flavin adenine dinucleotide synthase;

[0057] Fmn1: Encodes riboflavin kinase;

[0058] pGal1: The promoter is the 600bp upstream portion of the Gal1 gene, which encodes galactokinase. The nucleotide sequence of the Gal1 gene is NC_001134.8(279021..280607).

[0059] Gal80: A transcriptional regulator involved in repressing the Gal gene;

[0060] Gal4: Encodes the transcriptional activator protein GAL4

[0061] Experimental methods:

[0062] The PCR amplification methods, fusion methods of different fragments, gene knockout and overexpression methods used in the following examples can employ techniques commonly used in the art, such as fusion PCR, homologous recombination, and CRISPR-Cas9 technology. The enzymes and kits used are all commercially available products.

[0063] Overexpression refers to the upregulation of gene expression, that is, the gene is excessively transcribed and translated, and the final gene expression product exceeds the normal level.

[0064] Transformation was performed using the lithium acetate method. First, the host strain was activated in 1×YPD medium and cultured overnight at 30°C and 200 rpm. Then, it was inoculated into fresh 2×YPD medium with an initial OD value of 0.2 and cultured at 30°C for 4-4.5 hours. A 5OD bacterial suspension was collected, centrifuged at 3000 rcf for 5 minutes at room temperature, the supernatant was discarded, and the cells were washed twice with sterile ultrapure water to obtain yeast cells. For DNA preparation, 5OD of bacterial suspension was collected from each construct to obtain cells, which were then mixed with 50 μL of the DNA mixture to resuspend the cells. The 50 μL DNA mixture consisted of 2 μg of the inserted fragment, 250 ng of the tool plasmid, and sufficient ddH2O. The lithium acetate transformation mixture was added to the suspended cells, and after culturing, cells were obtained and plated onto selection plates. Single colonies were obtained, representing recombinant Saccharomyces cerevisiae. The presence of fluorescence was observed using a blue light plate, and positive colonies were selected.

[0065] Colony PCR and sequencing verification: After single colonies grew on the screening plates, colony PCR and sequencing verification were performed. The specific steps were as follows: A small amount of cells was picked up with a pipette tip and placed in 20 μL of 20 mmol / L NaOH solution, vortexed to mix, and incubated in a metal bath at 95°C for 20 min. After vortexing to mix, 1 μL of the bacterial solution was used as a template for colony PCR. The primers for the reaction varied depending on the verification sequence. The size of the clonal bands was compared with that of the negative clonal bands. The bacterial solutions of positive colony PCR clones were sent to Genewiz for sequencing verification. Stranded strains with correct sequencing were streaked and cryopreserved in glycerol.

[0066] The modification of the yeast genome, including gene insertion, knockout, and promoter replacement, is based on the CRISPR / Cas9 yeast homologous recombination technology. The relevant technology is based on the SOP that our company has previously developed and used, and can be found in patents CN114657078A and CN114591923B.

[0067] 2×YPD medium formulation: yeast extract 20.0 g / L, peptone 40.0 g / L, glucose 40.0 g / L.

[0068] Semi-synthetic culture medium: (NH)₂SO₄: 5 g / L, KH₂PO₄: 3 g / L, MgSO₄: 0.32 g / L, Leu: 0.2 g / L, Ura: 0.2 g / L, Trp: 0.2 g / L, succinic acid: 5.9 g / L, yeast extract 10.0 g / L, glucose 20.0 g / L.

[0069] Lithium acetate conversion mixture: 50% W / V PEG3350 260 μL, 1 mol / L LiOAc 36 μL, denatured salmon sperm DNA 10 μL (denatured salmon sperm DNA was placed in a 95℃ metal bath for 5 min before use), ddH2O 4 μL.

[0070] Screening plates lacking uracil: yeast nitrogen source stock solution 1.7 g / L, ammonium sulfate 5 g / L, various amino acids as shown in Table 1, agar 20 g / L, glucose 20 g / L. Note: glucose was sterilized separately.

[0071] Table 1. Content of various amino acids in the screening plates

[0072] amino acids (mg / L) amino acids (mg / L) adenine hemisulfate 18 L-phenylalanine 76 L-alanine 76 L-proline 76 L-arginine 76 L-threonine 76 L-Aspartic acid 76 L-serine 76 L-Asparagine 76 L-Tryptophan 76 L-cysteine 76 L-tyrosine 76 L-glutamic acid 76 L-valine 76 L-glycine 76 L-methionine 76 L-Isoleucine 76 L-Lysine 76 L-glutamine 76 L-Leucine 360 L-histidine 76

[0073] Method for detecting the amount of OA or OLO produced by recombinant Saccharomyces cerevisiae: After sample collection, based on the sample OD600, the sample was first incubated with 2U / OD of cell wall-breaking enzyme at 30℃ and 200rpm for 60min on a shaker. Then, 0.2mL of 0.5mm glass beads and 0.4mL of ethyl acetate:formic acid (0.05%) were added. The mixture was then processed in a high-speed tissue homogenizer at 65Hz for 180s, with a 30s interval, and repeated three times. After each treatment, the homogenizing tray was placed on ice to cool for 1min, shaken for 15-30s, and then briefly centrifuged. 0.28mL of the upper organic layer was transferred to a 1.5mL centrifuge tube. This process was repeated twice, and the collected upper organic phases were combined. The organic layers extracted three times were evaporated in V-AL mode at 45℃ for 1 hour until no solvent residue remained. The resuspended layers were then resuspended in 140 μL of AHF (acetonitrile:H₂O:formic acid = 80:20:0.05%, containing 15 μM internal standard PHB), filtered through a 0.22 μm PVDF membrane, and transferred to the inner tube of the HPLC detection vial as the test sample. Two replicates were performed for each sample. After preparation, the samples were analyzed by HPLC under the conditions shown in Table 2.

[0074] Table 2 HPLC detection conditions

[0075]

[0076] Table 3 lists the strains involved in the following examples.

[0077]

[0078]

[0079] Example 1: Construction of a promoter strain for replacing the MvaE enzyme in the genome

[0080] (1) Construction of strain ySC461

[0081] The integrated fragment was amplified by PCR using 2×Phanta Max Master Mix (Phanta DNA polymerase). Using *Saccharomyces cerevisiae* yG278 (disclosed in Chinese patent application CN115927029A) as a template, the 1622b-up-TKS fragment was amplified with primers 1 and 2 in Table 4, and the OAC-1622b-dn fragment was amplified with primers 3 and 4 in Table 4. These fragments were then transformed into the host *Saccharomyces cerevisiae* ySC340 to obtain strain ySC461. PCR verification was performed using primers 5 and 6 in Table 4, yielding PCR-positive clones for gene sequencing. This achieved overexpression of TKS and OAC at the 1622b site.

[0082] Table 4 Primer sequences

[0083]

[0084] (2) Construction of strain ySC471

[0085] The integrated fragment was amplified by PCR using 2×Phanta Max Master Mix (Phanta DNA polymerase). Using *Saccharomyces cerevisiae* CEN.PK2-1C as a template, the YPRcd15c-up fragment was amplified with primers 1 and 2 in Table 5, and the YPRcd15c-dn fragment with primers 3 and 4 in Table 5. Using *Saccharomyces cerevisiae* yG278 as a template, the OAC fragment was amplified with primers 5 and 6 in Table 5. These fragments were then transformed into the host *Saccharomyces cerevisiae* ySC461 constructed in step (1), resulting in strain ySC471. PCR verification was performed using primers 1 and 4 in Table 5, yielding a PCR-positive clone for gene sequencing. This achieved overexpression of OAC at the YPRCd15c site.

[0086] Table 5 Primer sequences

[0087]

[0088] (3) Construction of strain ySC476

[0089] The integrated fragment was amplified by PCR using 2×Phanta Max Master Mix (Phanta DNA polymerase). Using *Saccharomyces cerevisiae* CEN.PK2-1C as a template, the HO-up fragment was amplified using primers 1 and 2 in Table 6, and the HO-dn fragment was amplified using primers 3 and 4 in Table 6. Using *Saccharomyces cerevisiae* yG278 as a template, the OAC-OAC3-OAC5-OAC4 fragment was amplified using primers 5 and 6 in Table 6. These fragments were then transformed into the host *Saccharomyces cerevisiae* ySC471 constructed in step (2), resulting in strain ySC476. PCR verification was performed using primers 7 and 8 in Table 6, yielding a PCR-positive clone for gene sequencing. This achieved overexpression of OAC, OAC3, OAC5, and OAC4 at the HO site.

[0090] Table 6 Primer sequences

[0091]

[0092] (4) Construction of ySC481 strain

[0093] The integrated fragment was amplified by PCR using 2×Phanta Max Master Mix (Phanta DNA polymerase). Using yG024 (disclosed in Chinese patent application CN114369541A) as a template, the MvaE-up-pHXK1 fragment was amplified with primers 1 and 2 in Table 7, and the MvaE-dn fragment was amplified with primers 3 and 4 in Table 7. This fragment was then transformed into the host *Saccharomyces cerevisiae* ySC476 constructed in step (3), resulting in strain ySC481. PCR verification was performed using primers 5 and 6 in Table 7, yielding a PCR-positive clone for gene sequencing. This resulted in recombinant *Saccharomyces cerevisiae* ySC481 with the promoter pHXK1 regulating MvaE.

[0094] Table 7 Primer sequences

[0095]

[0096]

[0097] Example 2: Construction of a Saccharomyces cerevisiae strain overexpressing proteins involved in protein folding

[0098] The integrated fragment was amplified by PCR using 2×Phanta Max Master Mix (Phanta DNA polymerase). Using the CEN.PK2-1C genome as a template, primers 1 and 2 (Table 8) were used to amplify the 1414a-up fragment. Using the ySC222 genome as a template, primers 3 and 4 (Table 8) were used to amplify the promoter pPGK1 fragment. Using the CEN.PK2-1C genome as a template, primers 5 and 6 (Table 8) were used to amplify the 1414a-dn fragment. Using the CEN.PK2-1C genome as a template, primers 7 and 8 (Table 8) were used to amplify the 106a-up fragment. Using the ySC222 genome as a template, primers 9 and 10 (Table 8) were used to amplify the promoter pPGK1 fragment. Using the CEN.PK2-1C genome as a template, primers 11 and 12 (Table 8) were used to amplify the 106a-dn fragment. Using the PK2-1C genome as a template, the PDI1-tPDI1 fragment was amplified with primers 13 and 14 in Table 8; the KAR2-tKAR2 fragment was amplified with primers 15 and 16 in Table 8; the ERO1-tERO1 fragment was amplified with primers 17 and 18 in Table 8; the SED5-tSED5 fragment was amplified with primers 19 and 20 in Table 8; the BET1-tBET1 fragment was amplified with primers 21 and 22 in Table 8; and the SEC22-tSEC22 fragment was amplified with primers 23 and 24 in Table 8. These fragments were then combined and transformed into the 1414a and 106a sites of the host *Saccharomyces cerevisiae* ySC481, respectively, to obtain strains yAW1, yAW2, yAW3, yAW4, yAW5, yAW6, yAW7, yAW8, and ySC812 (gene information is shown in Table 3). Primers 25 and 26, and primers 27 and 28 in Table 8 were used to perform PCR reactions on strains yAW1, yAW2, yAW3, yAW4, yAW5, yAW6, yAW7, yAW8, and ySC812 to obtain bacterial solutions of colony PCR-positive clones for gene sequencing.

[0099] Table 8 Primer sequences

[0100]

[0101]

[0102] Example 3: Synthesis of olivine acid (alcohol) using sodium hexanoate as a substrate.

[0103] Using sodium hexanoate as a substrate, the engineered yeast strains yAW1, yAW2, yAW3, yAW4, yAW5, yAW6, yAW7, yAW8, and ySC812 prepared in Example 2 were cultured in shake flasks. Two to three single colonies were picked from each strain and inoculated into 1×YPD medium. After 16 hours of culture, the culture was transferred to 20 mL of semi-synthetic medium at 0.2 OD / mL and cultured at 30°C for 24 hours. Sodium hexanoate was then added twice daily (morning and evening) for a total of four times to achieve a final concentration of 3 mM in the fermentation broth. Following inoculation, 2% glucose was added every 24 hours until fermentation was complete. Fermentation time was calculated from the start of inoculation, and samples were taken at 72 and 96 hours for analysis. The results showed that ySC812 had the highest yield, with an OA yield of 0.26 mM and an OD of 14.87 (…). Figure 2 It is evident that, compared to other combinations, the engineered bacteria overexpressing PDI1 and SEC22 have certain advantages in the preparation and production of OA and OLO, and can be used for further modification.

[0104] Example 4: Construction of strain ySC815 integrating expression of the FAS1 gene

[0105] Using the genome of Saccharomyces cerevisiae yG011 as a template, P was amplified using 2×Phanta Max Master Mix (Phanta DNA polymerase) and primers 1 and 2 listed in Table 9. HXT1 The promoter was used to transform the amplified fragment into the *Saccharomyces cerevisiae* host strain ySC812 constructed in Example 2. PCR verification was performed using primers 3 and 4 in Table 9, yielding PCR-positive clones for gene sequencing. Sequencing confirmed the successful construction of the promoter-replaced strain ySC815.

[0106] Table 9 Primer sequences

[0107]

[0108]

[0109] Example 5: Construction of strain ySC1821 integrating expression of CsAAE1-TKS-OAC cassette

[0110] (1) Construction of plasmid MG36

[0111] The integrated fragment was amplified by PCR using 2×Phanta Max Master Mix (Phanta DNA polymerase). Using pUASR (disclosed in DOI:10.1038 / s41467-020-15977-4, named pUASR in this application, nucleotide sequence as shown in SEQ ID NO.13) as a template, the promoter pUASR was amplified using primers 1 and 2 in Table 10. Using the yCAN31 genome as a template, the CsAAE1 fragment was amplified using primers 3 and 4 in Table 10. Finally, using tSyn1 (disclosed in DOI:10.1038 / s41467-020-15977-4, named tSyn1 in this application, nucleotide sequence as shown in SEQ ID NO.13), the integrated fragment was further amplified. Using NO.14 as a template, the tSyn1 fragment was amplified with primers 5 and 6 in Table 10. Using pGPD (disclosed in DOI:10.1038 / s41467-020-15977-4, nucleotide sequence as shown in SEQ ID NO.15) as a template, the pGPD fragment was amplified with primers 7 and 8 in Table 10. Using the yCAN31 genome as a template, the TKS-OAC fragment was amplified with primers 9 and 10 in Table 10. The tSyn3 fragment (which is Tsynth9 disclosed in DOI:10.1038 / s41467-020-15977-4, named tSyn3 in this application, nucleotide sequence as shown in SEQ ID NO.15) was amplified. Using NO.16 as a template, the tSyn3 fragment was amplified with primers 11 and 12 in Table 10. Using the yCAN31 genome as a template, the pGal1-CsPT fragment was amplified with primers 13 and 14 in Table 10. tSyn5 (which is Tsynth18 disclosed in the literature DOI:10.1038 / s41467-020-15977-4, named tSyn5 in this application, nucleotide sequence as shown in SEQ ID) was used. Using NO.17 as a template, the tSyn5 fragment was amplified with primers 15 and 16 in Table 10. The plasmid backbone (disclosed in Chinese patent application document CN113999783A) was double-digested with restriction endonucleases PacI and KpnI. The above fragment and the double-digested linearized vector were mixed and transformed into E. coli DH5α (derived from TransGold). Transformants were screened on LB plates containing ampicillin (50ug / ml). Transformants were identified by colony PCR using primers 17 and 18 in Table 10. Plasmids were extracted from correctly identified transformants and sent for sequencing. The correctly sequenced plasmid was named MG36. Figure 1 ).

[0112] Table 10 Primer sequences

[0113]

[0114]

[0115] (2) Construction of strain ySC1821

[0116] Using the Leu2 gene as a selection marker, the CsAAE1-TKS-OAC cassette was overexpressed at the rDNA site. The recombinant Saccharomyces cerevisiae strain overexpressed optimized acyl activator genes, polyketide synthase genes, oleic acid cyclase genes, and oleoyl geraniyl transferase genes at multiple rDNA sites. To facilitate multiple-copy insertion of the rDNA site, an rDNA integration cassette was constructed, comprising: 1) upstream and downstream homologous arms of the rDNA site; 2) the leu2 gene or loxL-Ura3-Cre-LoxR cassette used as a selection marker; 3) CsAAE1 / TKS / OAC expressed in tandem with individual promoters and terminators; 4) constitutively expressed GFP; and 5) CsPT expressed with individual promoters and terminators. Among them, loxL-Ura3-Cre-LoxR cassette is designed to eliminate the cassette itself through homologous recombination between LoxL / R mediated by Cre under induction conditions, and GFP is used to determine the relative strength of expression intensity of expression cassettes among different single colonies by fluorescence intensity.

[0117] The integrated fragment was amplified by PCR using 2×Phanta Max Master Mix (Phanta DNA polymerase). Using the genome of *Saccharomyces cerevisiae* CEN.PK2-1C as a template, the rDNA-up fragment was amplified using primers 1 and 2 in Table 11. Using plasmid MG36 constructed in step (1) as a template, the CsAAE1-TKS fragment was amplified using primers 3 and 4 in Table 11. Using plasmid MG36 as a template, the OAC fragment was amplified using primers 5 and 6 in Table 11. Using the GFP gene as a template, the GFP fragment was amplified using primers 7 and 8 in Table 11. Using Leu2 as a template, the Leu2 fragment was amplified using primers 9 and 10 in Table 11. Using the genome of CEN.PK2-1C as a template, the rDNA-dn fragment was amplified using primers 11 and 12 in Table 11. The above fragments were mixed and transformed into the host yeast strain ySC815 constructed in Example 4. After single colony culture of the transformants, the GFP fluorescence intensity of each strain was detected by a microplate reader. Strains with different fluorescence intensity ranges were selected for initial screening via well plate fermentation, and the results are as follows: Figure 3As shown, strain ySC1821 exhibited the highest OA and OLO yields. Sequencing revealed that strain ySC1821 integrated a 5-copy expression cassette, CsAAE1-TKS-OAC.

[0118] Table 11 Primer sequences

[0119]

[0120]

[0121] Example 6: Synthesis of oleic acid (alcohol) using different substrates

[0122] Using the engineered yeast ySC1821 obtained in Example 5 as the fermentation strain, the mixing ratio of ethyl hexanoate and sodium hexanoate in the substrate solution was set to 3:1, with only ethyl hexanoate or only sodium hexanoate added, to explore the effect of different substrate solutions on the synthesis of oleic acid (alcohol).

[0123] Pick 2-3 single colonies and inoculate them into 1×YPD medium. Incubate for 16 hours, then transfer to 20 mL of semi-synthetic medium at 0.2 OD / mL. After inoculation, incubate at 30℃ for 24 hours, then supplement with a mixture of ethyl hexanoate / sodium hexanoate substrate in different proportions. Substrate supplementation uses 1M ethyl hexanoate (dissolved in isopropyl myristate, IPM) / 1M sodium hexanoate, twice daily (morning and evening), for a total of 4 times, to achieve a final sodium hexanoate concentration of 3 mM and a final ethyl hexanoate concentration of 9 mM in the fermentation broth. After inoculation, add 2% glucose every 24 hours until fermentation ends. Fermentation time is calculated from the start of inoculation, and samples are taken at 72 and 96 hours for analysis. Results show that when the substrate contains ethyl hexanoate, the OA yield of ySC1821 is 1.501 mM, and the OD is 8.73 (…). Figure 4 When the substrate does not contain sodium hexanoate, the OA yield of ySC1821 is 0.846 mM and the OD yield is 6.43.

[0124] And from Figure 4 It can be seen that, although the OD values ​​of the engineered yeast ySC812 obtained in Example 3 and the engineered yeast ySC815 obtained in Example 4 were higher than those of the engineered strain ySC1821 in the oleic acid (alcohol) synthesis reaction, the yield was significantly reduced.

[0125] Example 7: Shake-flask fermentation test of ySC1821 and other strains synthesizing oleic acid (alcohol) with different substrates.

[0126] This example uses a 10L stainless steel fermenter from Bailun Biotechnology (4-unit system). The ySC1821 glycerol tubes obtained in Example 5 were inoculated into a 250ml shake flask containing 20mL of 1×YPD medium to prepare a primary seed culture. After overnight incubation at 30℃ / 200rpm, the culture was transferred to a 2000ml shake flask containing 500mL of 1×YPD medium to prepare a secondary seed culture. This secondary seed culture was then incubated at 30℃ / 200rpm before inoculation into the fermenter. The initial OD600 in the fermenter was controlled to be approximately 0.2. A semi-synthetic culture medium was used, with the following fermentation parameters: 1.5 vvm / 0.5 MPa / DO ≥ 50% / pH 5.0. Glucose (carbon source) was added via a fed-batch method during fermentation, maintaining residual sugar < 5g / L and ethanol concentration < 10g / L. Starting 18 hours into fermentation, the substrate was added in six batches using a feed-in method. The ethyl hexanoate feed used an IPM stock solution with a 2.5% ethyl hexanoate content (ethyl hexanoate / IPM (V / V) = 2.5%), and the sodium hexanoate feed used a 1M sodium hexanoate aqueous solution, resulting in final concentrations of 18mM ethyl hexanoate and 6mM sodium hexanoate, respectively. During fermentation, samples were taken every 24 hours to analyze the OD and products. After fermentation, the fermentation broth was sterilized in situ at high temperature and then treated as fermentation waste. Results showed that after 144 hours of fermentation, the strain accumulated approximately 10mM OA and 10mM OLO, equivalent to yields of 2.2 g / L and 1.8 g / L, respectively. Figure 5 ).

[0127] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. An engineered bacterial strain producing oleolic acid and / or oleanol, characterized in that, Using Saccharomyces cerevisiae as a host, a protein folding protein-related gene and a gene expression cassette CsAAE1-TKS-OAC cassette are integrated into the genome of Saccharomyces cerevisiae. The gene expression cassette CsAAE1-TKS-OAC cassette includes tandemly expressed CsAAE1, TKS and OAC genes, and the expression cassette is integrated into the rDNA site of the Saccharomyces cerevisiae genome in 5 copies. The protein folding protein-related genes are PDI1 and SEC22; The engineered bacteria also overexpressed Fad1, Fmn1, Hac1 and Gal4, knocked out Pep4 and Gal80, downregulated the expression of FAS1 and MvaE, and expressed multiple copies of OAC. The multiple copy expression of OAC is achieved by overexpressing TKS and OAC at the 1622b site of the genome, overexpressing OAC at the YPRCd15c site of the genome, and overexpressing OAC, OAC3, OAC5, and OAC4 at the HO site of the genome. The amino acid sequences encoded by the acyl activator gene CsAAE1, the polyketide synthase gene TKS, and the oleic acid cyclase gene OAC are shown in SEQ ID NO. 9-11. The amino acid sequences encoded by OAC3, OAC5, and OAC4 are shown in SEQ ID NO.18-20; The amino acid sequences encoded by the PDI1 and SEC22 genes are shown in SEQ ID NO.1-2.

2. The engineered strain according to claim 1, characterized in that, The downregulation of FAS1 and MvaE expression was achieved by using the promoter pHXT1 to initiate the expression of genes FAS1 and MvaE.

3. A method for producing cannabinoid synthesis precursors, characterized in that, The cannabinoid synthesis precursor is oleic acid and / or oleanol, and the method is to inoculate the engineered strain described in claim 1 or 2 into the reaction system for fermentation to prepare the cannabinoid synthesis precursor.

4. The method according to claim 3, characterized in that, The method uses a mixture of ethyl hexanoate and sodium hexanoate as a substrate.

5. The method according to claim 4, characterized in that, The mixing ratio of ethyl hexanoate and sodium hexanoate is (1:1) to (50:1).

6. The use of any of the engineered strains of claims 1-2 or any of the methods of claims 3-5 in the preparation of cannabinoid precursors and / or cannabinoids, wherein the cannabinoid synthesis precursor is oleic acid and / or oleuropein.

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