Construction for preparing recombinant bacteria of enantio-kaurenoic acid and 16 alpha-hydroxyl enantio-kaurane-19-carboxylic acid and application of recombinant bacteria

Abstract

The invention discloses a method for synthesizing enantio-kaurenoic acid and 16 alpha-hydroxyl enantio-kaurane-19-carboxylic acid based on a recombinant microbiological method. The invention provides genes such as an enantiocopal pyrophosphate synthase gene, a similar enantiokaurene synthase gene, an enantiokaurene oxidase gene, a cytochrome P450 reductase gene, a cytochrome b5 gene, an FPP / GGPP synthase gene and the like which are expressed in a host saccharomyces cerevisiae. The copy number of genes such as an acetyl coenzyme A acyltransferase gene, an HMG coenzyme A synthase gene, a GGPP synthase gene, a glucose-6-phosphate dehydrogenase gene and a 6-phosphogluconate dehydrogenase gene in the saccharomyces cerevisiae is increased to improve the expression or activity; the obtained recombinant saccharomyces cerevisiae is used for producing enantio-kaurenoic acid and 16 alpha-hydroxyl enantio-kaurane-19-carboxylic acid, and a powerful strain and a research basis are provided for biosynthesis of active diterpenoid components.

Description

Technical Field

[0001] This invention relates to the field of biochemical engineering, and in particular to the construction and application of a recombinant yeast strain, specifically to the construction of a recombinant yeast strain for the fermentation production of enantio-kauronic acid and 16α-hydroxyenantio-kauronic acid-19-carboxylic acid. Background Technology

[0002] Enant-kaurenoic acid (KA) is a biosynthetic precursor of diterpenoids with important physiological activities in plants and microorganisms. It plays a key role in plant growth and development, microbial defense, and response to abiotic stress. It has pharmacological activities such as antibacterial (Ndoma JC, et al., 2010, Phytochemistry Letters, 3(4):201-206), antihypertensive, diuretic (Cavalcanti BC, et al., 2006, Food and Chemical Toxicology, 44:388-392), anti-inflammatory (Choi RJ, et al., 2011, Phytomedicine, 18(8-9):677-682), and vascular smooth muscle relaxation (Tirapelli CR, et al., 2004, Phytomedicine, 18(8-9):677-682). 16α-hydroxy-ent-kauran-19-oic acid (16-OH KOA) is another important class of diterpenoids with anti-inflammatory and analgesic pharmacological effects. It is also a key intermediate in the formation of triptolide, an active anti-HIV drug (Ping S, et al., 2017, Frontiers in Plant Science, 8:1756). Furthermore, both classes of compounds exhibit significant cytotoxicity, immunomodulatory effects, antibacterial activity, and antiviral activity, thus finding wide application in drug development and plant cultivation. Currently, both ( Figure 1 It is mainly obtained through isolation and extraction from plants, but this method has many drawbacks, including low content and large variability, difficulty in product purification, long plant growth cycle, and serious damage to biological resources, especially wild resources; or it can be obtained through chemical synthesis, but this method has many problems such as lengthy reaction steps and low yield. In addition, it is difficult to achieve stereoselective or site-specific oxidation modification in organic synthesis.

[0003] Using synthetic biology to build microbial cell factories for fermentation production of natural active ingredients is a novel green production model that can break free from dependence on medicinal plant resources and reduce environmental damage. It has been recognized internationally as one of the most promising methods. In recent years, research teams at home and abroad have successfully constructed a series of engineered yeast strains containing terpenes and alkaloids, achieving heterologous and efficient production of natural active ingredients or their precursors in microorganisms. For example, the production of taxadiene, a precursor of paclitaxel, in Escherichia coli has reached 1000 mg / L (Parayil KuMaranAjikuMar et al., 2010, Science, 330:70-74); the production of L-pinediene, a precursor of ginkgolide, in engineered Escherichia coli has reached 700 mg / L (Effendi Leonard et al., 2010, PNAS, 107(31):13654-13659); and the construction of a metabolic pathway in engineered yeast to efficiently produce artemisinin precursor artemisinic acid has reached a maximum yield of 25 g / L, which can be synthesized into artemisinin through a simple reaction. A fermentation workshop of less than 100 cubic meters has produced 35 tons of artemisinin (Chris J. Paddon et al.). Al., 2013, Nature, 496(7446):528-531); In engineered yeast, the biosynthetic pathways of hyoscyamine and scopolamine were integrated and heterologous production was achieved, with a yield of 30-80 μg / L (Prashanth Srinivasan et al., 2020, Nature, 585(7826):614-619); In engineered yeast, the biosynthetic pathways of key precursors of the marketed antitumor drug molecule vinblastine (vinblastine, vinblastine) were reconstructed and heterologous production was achieved, and then vinblastine was produced through semi-synthetic means, with a yield of 23.9 μg / L (Jie Zhang et al., 2022, Nature, 609(7926):341-347). Currently, there is relevant research in China on the biosynthesis of drug molecules such as artemisinin, paclitaxel, ginkgolide, triptolide, triptolide, and tanshinone.

[0004] KA and 16-OH KOA belong to the diterpenoid class of compounds. Diterpenoids are synthesized by the cyclization of four isoprene units. Their biosynthesis is divided into three stages: (1) through the mevalonic acid pathway in the cytoplasm. (1) The 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway in the plastid synthesizes two simple five-carbon units, namely isoprene pyrophosphate (IPP) and dimethyl isoprene pyrophosphate (DMAPP); (2) Under the catalysis of isoprene transferase, IPP and DMAPP are linked end to end to generate geraniol pyrophosphate (GPP), farnesyl pyrophosphate (FPP) and geraniol geraniol pyrophosphate (GGPP). GGPP, as a common precursor of diterpenoid compounds, is catalyzed by diterpenoid synthase to form a diterpenoid intermediate skeleton structure; (3) CYP450 modifies the structure of diterpenoid intermediate compounds until structurally diverse diterpenoid compounds are formed (Michel Rohmer et al., 1993, Biochemical Journal, 1993, 295: 517-524). In nature, GGPP can be sequentially reacted with enantio-copalyl diphosphate synthase (CPS), enantio-kaurene synthase (KS), or enantio-kaurene synthase-like (KSL) to generate enantio-kaurene (Kau) and 16α-hydroxy-ent-kaurane (16-OHKau); subsequently, under the catalysis of enantio-kaurene oxidase (KO), it synthesizes KA and 16-OH KOA (e.g., Kau). Figure 1-2 (Flowchart shown). Given its promising application prospects, there is an urgent need in industry for a highly efficient biosynthetic method that does not rely on plant extraction and chemical synthesis. That is, starting with a simple carbon source (glucose), and without adding complex precursor substances, using recombinant yeast to achieve de novo biosynthesis of KA and 16-OH KOA in vitro. Summary of the Invention

[0005] The purpose of this invention is to provide a method for constructing and applying a recombinant yeast strain that produces enantiocarboxylic acid and 16α-hydroxyenantiocarboxane-19-carboxylic acid.

[0006] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0007] The present invention discloses a method for constructing a recombinant yeast strain for producing enantiocarboxylic acid and 16α-hydroxyenantiocarboxane-19-carboxylic acid, comprising the following steps:

[0008] 1) Gene expression cassette P TEF1 -Cas9-T CYC1 The recombinant strain CEN.PK2-1D was obtained by importing it into the starting yeast CEN.PK2-1D;

[0009] 2) By increasing the expression or activity of the following genes in the recombinant Saccharomyces cerevisiae CEN.PK2-1D(a): acetyl-CoA acyltransferase gene ERG10, HMG-CoA synthase gene ERG13, HMG-CoA reductase N-terminal truncated gene tHMG1, mevalonate kinase gene ERG12, mevalonate phosphate kinase gene ERG8, mevalonate phosphate decarboxylase gene ERG19, IPP isomerase gene IDI1, FPP synthase gene ERG20, GGPP synthase gene BTS1, and acetyl-CoA acyltransferase / HMG-CoA reductase gene EfmvaE, recombinant strain C20 was obtained.

[0010] 3) In recombinant strain C20, the following enzymes were expressed: enantio-cobamic pyrophosphate synthase TwCPS3, enantio-kaurene synthase TwKSL2, bifunctional kaurene synthase PpCPS / KS, enantio-kaurene oxidases TwKO and AtKO, cytochrome P450 reductases TwCPR3, TwCPR5, and AtCPR2, cytochrome b5 AaCYB5, CrCYB5, RsCYB5, TwCYB5-A, and FPP / GGPP synthase GgFPS. F112A At least one of GGPP synthase XdGGPPS, glucose-6-phosphate dehydrogenase ScZWF1, and 6-phosphate gluconate dehydrogenase ScGND1 was used to obtain a recombinant yeast strain for the production of enantiocarboxylic acid and 16α-hydroxyenantiocarboxane-19-carboxylic acid.

[0011] Step 3) includes:

[0012] 31) The gene t1PpCPS / KS-MBP of the fusion protein was introduced into recombinant bacteria C20 to obtain recombinant bacteria 1;

[0013] 32) One of the AtKO and AtCPR2 genes, AtKO and TwCPR3 genes, AtKO and TwCPR5 genes, TwKO and AtCPR2 genes, TwKO and TwCPR3 genes, and TwKO and TwCPR5 genes was introduced into recombinant bacteria 1 to obtain recombinant bacteria 2.

[0014] 33) Gene GgFPS F112AXdGGPPS was introduced into recombinant strain 2 to obtain recombinant strain 3, which is a recombinant yeast strain that produces enantio-kauronic acid and 16α-hydroxyenantio-kaurane-19-carboxylic acid.

[0015] Furthermore, step 3) includes:

[0016] 34) One of the AtKO gene, AtKO and AaCYB5 gene, AtKO and CrCYB5 gene, AtKO and RsCYB5 gene, and AtKO and TwCYB5-A gene was introduced into recombinant strain 3 to obtain recombinant strain 4. Recombinant strain 4 is a recombinant yeast strain that produces enantio-kaurethane and 16α-hydroxyenantio-kaurethane-19-carboxylic acid.

[0017] Furthermore, step 3) includes:

[0018] 35) The ScZWF1 and ScGND1 genes were introduced into recombinant strain 4 to obtain recombinant strain 5. Recombinant strain 5 is a recombinant yeast strain that produces enantio-kauronic acid and 16α-hydroxyenantio-kauronic acid-19-carboxylic acid.

[0019] The method of gene introduction involves integrating a gene expression cassette containing the gene into the chromosome of the starting bacterium.

[0020] That is to say, steps 3) 31)-35) are respectively:

[0021] 31) Gene expression cassette P GAL2 -t1PpCPS / KS-MBP-T PDC1 and / or gene expression cassette P GAL2 -t2PpCPS / KS-MBP-T PDC1 Recombinant strain 1 was obtained by integrating into the XII-1 site of chromosome C20 of the recombinant strain.

[0022] 32) Gene expression cassette P GAL1 -AtKO-T CYC1 -P GAL10 -AtCPR2-T ADH1 P GAL1 -AtKO-T CYC1 -P GAL10 -TwCPR3-T ADH1 P GAL1 -AtKO-T CYC1 -P GAL10 -TwCPR5-T ADH1 P GAL1 -TwKO-T CYC1 -P GAL10 -AtCPR2-T ADH1 PGAL1 -TwKO-T CYC1 -P GAL10 -TwCPR3-T ADH1 P GAL1 -TwKO-T CYC1 -P GAL10 -TwCPR5-T ADH1 One of the strains was integrated into the XII-5 site of recombinant bacteria 1 chromosome to obtain recombinant bacteria 2;

[0023] 33) Gene expression cassette P GAL1 -GgFPS F112A -T CYC1 -P GAL10 -XdGGPPS-T ADH1 Recombinant strain 3 was obtained by integrating into the Int4 site of chromosome 2. Recombinant strain 3 is a recombinant yeast strain that produces enantio-kauronic acid and 16α-hydroxyenantio-kauronic acid-19-carboxylic acid.

[0024] 34) Gene expression cassette P GAL1 -AtKO-T CYC1 P GAL1 -AtKO-T CYC1 -P GAL10 -AaCYB5-T ADH1 P GAL1 -AtKO-T CYC1 -P GAL10 -CrCYB5-AT ADH1 P GAL1 -AtKO-T CYC1 -P GAL10 -RsCYB5-T ADH1 P GAL1 -AtKO-T CYC1 -P GAL10 -TwCYB5-AT ADH1 Recombinant strain 4, which is obtained by integrating into the Int14 site of chromosome 3 of recombinant strain 3, is a recombinant yeast strain that produces enantio-kauronic acid and 16α-hydroxyenantio-kauronic acid-19-carboxylic acid.

[0025] 35) Gene expression cassette P GAL1 -ScZWF1-T CYC1 -P GAL10 -ScGND1-T ADH1 Recombinant strain 5, which was integrated into the Int17 site of chromosome 4, is a recombinant yeast strain that produces enantio-kauridine and 16α-hydroxyenantio-kauridine-19-carboxylic acid.

[0026] The recombinant strain C20 is used to increase the expression or activity of the above-mentioned genes in Saccharomyces cerevisiae by increasing the copy number of the nucleic acids encoded by the following genes in the yeast strain CEN.PK2-1D(a): acetyl-CoA acyltransferase gene ERG10, HMG-CoA synthase gene ERG13, HMG-CoA reductase N-terminal truncated gene tHMG1, mevalonate kinase gene ERG12, mevalonate phosphate kinase gene ERG8, mevalonate phosphate decarboxylase gene ERG19, IPP isomerase gene IDI1, FPP synthase gene ERG20, GGPP synthase gene BTS1, and acetyl-CoA acyltransferase / HMG-CoA reductase gene EfmvaE.

[0027] The recombinant strain C20 increases the copy number of the above-mentioned gene-encoded nucleic acids in Saccharomyces cerevisiae by introducing the expression cassettes encoding the nucleic acids of acetyl-CoA acyltransferase ERG10, HMG-CoA synthase ERG13, HMG-CoA reductase N-terminal truncated form tHMG1, mevalonate kinase ERG12, mevalonate phosphate kinase ERG8, mevalonate phosphate decarboxylase ERG19, IPP isomerase IDI1, FPP synthase ERG20, GGPP synthase BTS1, and acetyl-CoA acyltransferase / HMG-CoA reductase EfmvaE into the recombinant yeast CEN.PK2-1D(a) using homologous recombination.

[0028] The nucleotide sequences of the ERG10 gene are shown in SEQ ID No. 1; the nucleotide sequences of the ERG13 gene are shown in SEQ ID No. 2; the nucleotide sequences of the tHMG1 gene are shown in SEQ ID No. 3; the nucleotide sequences of the ERG12 gene are shown in SEQ ID No. 4; the nucleotide sequences of the ERG8 gene are shown in SEQ ID No. 5; the nucleotide sequences of the ERG19 gene are shown in SEQ ID No. 6; the nucleotide sequences of the IDI1 gene are shown in SEQ ID No. 7; the nucleotide sequences of the ERG20 gene are shown in SEQ ID No. 8; the nucleotide sequences of the BTS1 gene are shown in SEQ ID No. 9; and the nucleotide sequences of the EfmvaE gene are shown in SEQ ID No. 10.

[0029] The recombinant strains C20-01 to C20-23 express enantiomeric cobamic pyrophosphate synthase TwCPS3, enantiomeric kauriene synthase TwKSL2, bifunctional kauriene synthase PpCPS / KS, enantiomeric kauriene oxidases TwKO and AtKO, cytochrome P450 reductases TwCPR3, TwCPR5, and AtCPR2, cytochrome b5 AaCYB5, CrCYB5, RsCYB5, TwCYB5-A, and FPP / GGPP synthase GgFPS in the host *Saccharomyces cerevisiae*. F112AGGPP synthase XdGGPPS, glucose-6-phosphate dehydrogenase ScZWF1 and 6-phosphate gluconate dehydrogenase ScGND1 are used to introduce the nucleic acid encoding the protein or fusion protein into yeast C20.

[0030] The recombinant bacteria C20-01 to C20-05 are used to produce the precursor compounds Kau and 16-OH Kau of enantio-kauronic acid and 16α-hydroxyenantio-kauronic acid-19-carboxylic acid; the recombinant bacteria C20-06 to C20-23 are used to produce KA and 16-OH KA.

[0031] The recombinant strains C20-01 to C20-23 express enantiomeric cobamic pyrophosphate synthase TwCPS3, enantiomeric kauriene synthase TwKSL2, bifunctional kauriene synthase PpCPS / KS, enantiomeric kauriene oxidases TwKO and AtKO, cytochrome P450 reductases TwCPR3, TwCPR5, and AtCPR2, cytochrome b5 AaCYB5, CrCYB5, RsCYB5, TwCYB5-A, and FPP / GGPP synthase GgFPS in the host *Saccharomyces cerevisiae*. F112A GGPP synthase XdGGPPS, glucose-6-phosphate dehydrogenase ScZWF1 and 6-phosphate gluconate dehydrogenase ScGND1 are used to introduce the nucleic acid expression cassette encoding the protein or fusion protein into yeast C20.

[0032] The recombinant bacteria C20-01 to C20-23, wherein the genes comprise: the nucleotide sequence of the enantio-cobamic pyrophosphate synthase gene TwCPS3 as shown in SEQ ID No. 11; the nucleotide sequence of the enantio-kaurene synthase gene TwKSL2 as shown in SEQ ID No. 12; the nucleotide sequence of the bifunctional kaurene synthase gene PpCPS / KS as shown in SEQ ID No. 13; the nucleotide sequence of the enantio-kaurene oxidase gene TwKO as shown in SEQ ID No. 14; the nucleotide sequence of the kaurene oxidase gene AtKO as shown in SEQ ID No. 15; the nucleotide sequence of the cytochrome P450 reductase gene AtCPR2 as shown in SEQ ID No. 16; the nucleotide sequence of the cytochrome P450 reductase gene TwCPR3 as shown in SEQ ID No. 17; the nucleotide sequence of the cytochrome P450 reductase gene TwCPR5 as shown in SEQ ID No. 18; and the nucleotide sequence of the cytochrome b5 AaCYB5 gene as shown in SEQ ID No. 19. As shown in No. 19; the nucleotide sequence of the cytochrome b5 CrCYB5 gene is shown in SEQ ID No. 20; the nucleotide sequence of the cytochrome b5 gene RsCYB5 is shown in SEQ ID No. 21; the nucleotide sequence of the cytochrome b5 TwCYB5-A gene is shown in SEQ ID No. 22; the FPP / GGPP synthase gene GgFPS F112A The nucleotide sequence of the gene is shown in SEQ ID No. 23; the nucleotide sequence of the GGPP synthase gene XdGGPPS is shown in SEQ ID No. 24; the nucleotide sequence of the glucose-6-phosphate dehydrogenase gene ScZWF1 is shown in SEQ ID No. 25; and the nucleotide sequence of the 6-phosphate gluconate dehydrogenase gene ScGND1 is shown in SEQ ID No. 26.

[0033] In the recombinant bacteria C20-01 to C20-23, the gene of the fusion protein is composed, sequentially, of the bifunctional kauriene synthase gene PpCPS / KS or its truncated variant 1 (removing the N-terminal 252 nucleotides, t1PpCPS / KS), truncated variant 2 (removing the N-terminal 363 nucleotides, t2PpCPS / KS), a linker peptide, and the maltose-binding protein gene MBP. The nucleotide sequence of the t1PpCPS / KS gene is shown in SEQ ID No. 27; the nucleotide sequence of the t2PpCPS / KS gene is shown in SEQ ID No. 28; the nucleotide sequence of the linker peptide is shown in SEQ ID No. 29; and the nucleotide sequence of the MBP gene is shown in SEQ ID No. 30.

[0034] In an embodiment of the present invention, the gene expression cassette required for gene editing is:

[0035] P TEF1 -Cas9-T CYC1 .

[0036] The nucleic acid expression cassette for the gene is selected from the following:

[0037] P GAL1 -ERG10-T TPI1 -P GAL10 -ERG13-T PGI ;

[0038] P GAL1 -tHMG1-T ADH1 -P GAL10 -ERG12-T PDC1 ;

[0039] P GAL1 -ERG8-T RPS2 -P GAL10 -ERG19-T TDH1 ;

[0040] P GAL1 -IDI1-T CCW12 -P GAL10 -BTS1-ERG20-T RPL9A ;

[0041] P GAL1 -tHMG1-T ADH1 -P GAL10 -EfmvaE-T PDC1 ;

[0042] P GAL1 -TwCPS3-T CYC1 -P GAL10 -TwKSL2-T ADH1 ;

[0043] P GAL2 -PpCPS / KS-T PDC1 ;

[0044] P GAL1 -AtKO-T CYC1 -P GAL10 -AtCPR2-T ADH1 ;

[0045] P GAL1 -AtKO-T CYC1 -P GAL10 -TwCPR3-T ADH1 ;

[0046] P GAL1 -AtKO-T CYC1-P GAL10 -TwCPR5-T ADH1 ;

[0047] P GAL1 -TwKO-T CYC1 -P GAL10 -AtCPR2-T ADH1 ;

[0048] P GAL1 -TwKO-T CYC1 -P GAL10 -TwCPR3-T ADH1 ;

[0049] P GAL1 -TwKO-T CYC1 -P GAL10 -TwCPR5-T ADH1 ;

[0050] P GAL1 -GgFPS F112A -T CYC1 -P GAL10 -XdGGPPS-T ADH1 ;

[0051] P GAL1 -AtKO-T CYC1 ;

[0052] P GAL1 -AtKO-T CYC1 -P GAL10 -AaCYB5-T ADH1 ;

[0053] P GAL1 -AtKO-T CYC1 -P GAL10 -CrCYB5-A-T ADH1 ;

[0054] P GAL1 -AtKO-T CYC1 -P GAL10 -RsCYB5-T ADH1 ;

[0055] P GAL1 -AtKO-T CYC1 -P GAL10 -TwCYB5-A-T ADH1 ;

[0056] P GAL1 -ScZWF1-T CYC1 -P GAL10 -ScGND1-T ADH1.

[0057] The gene expression cassettes for expressing fusion proteins were selected from the following:

[0058] P GAL2 -PpCPS / KS-MBP-T PDC1 ,

[0059] P GAL2 -t1PpCPS / KS-MBP-T PDC1 ,

[0060] P GAL2 -t2PpCPS / KS-MBP-T PDC1 .

[0061] In the aforementioned recombinant bacteria, the gene expression cassette for expressing the protein and fusion protein in vivo includes a promoter, the coding nucleic acid of the protein and fusion protein, and a terminator.

[0062] Alternatively, the promoter may be selected from TEF1, GAL1, GAL10, or GAL2; the terminator may be CYC1, RPS2, CCW12, RPL9A, PGI, ADH1, or PDC1.

[0063] Or the promoter is TEF1 and the terminator is CYC1;

[0064] Or the promoter is GAL1 and the terminator is TPI1;

[0065] Or the promoter is GAL1 and the terminator is ADH1;

[0066] Or the promoter is GAL1 and the terminator is CYC1;

[0067] Or the promoter is GAL1 and the terminator is RPS2;

[0068] Or the promoter is GAL1 and the terminator is CCW12;

[0069] Or the promoter is GAL1 and the terminator is PDC1;

[0070] Or the promoter is GAL2 and the terminator is PDC1;

[0071] Or the promoter is GAL10 and the terminator is PGI;

[0072] Or the promoter is GAL10 and the terminator is PDC1;

[0073] Or the promoter is GAL10 and the terminator is RPL9A;

[0074] Alternatively, the promoter may be GAL10 and the terminator may be ADH1.

[0075] The promoter sequences described above include TEF1, GAL1, GAL2, and GAL10. The nucleotide sequence of TEF1 is shown in SEQ ID No. 31; the nucleotide sequence of GAL1 is shown in SEQ ID No. 32; the nucleotide sequence of GAL2 is shown in SEQ ID No. 33; and the nucleotide sequence of GAL10 is shown in SEQ ID No. 34.

[0076] The aforementioned terminator sequences include CYC1, TPI1, PGI, ADH1, PDC1, RPS2, TDH1, CCW12, and RPL9A. The nucleotide sequence of CYC1 is shown in SEQ ID No. 35; the nucleotide sequence of TPI1 is shown in SEQ ID No. 36; the nucleotide sequence of PGI is shown in SEQ ID No. 37; the nucleotide sequence of ADH1 is shown in SEQ ID No. 38; the nucleotide sequence of PDC1 is shown in SEQ ID No. 39; the nucleotide sequence of RPS2 is shown in SEQ ID No. 40; the nucleotide sequence of TDH1 is shown in SEQ ID No. 41; the nucleotide sequence of CCW12 is shown in SEQ ID No. 42; and the nucleotide sequence of RPL9A is shown in SEQ ID No. 43.

[0077] The application of the aforementioned recombinant bacteria in the production of KA and 16-OH KOA is also within the scope of protection of this invention.

[0078] Another object of the present invention is a method and application for producing enantio-kauridine and 16α-hydroxyenantio-kauridine-19-carboxylic acid from recombinant Saccharomyces cerevisiae.

[0079] The method provided by this invention specifically includes the following steps:

[0080] 1) Ferment the above-mentioned recombinant bacteria to obtain the fermentation product;

[0081] 2) Extract the fermentation product with an organic solution, collect the organic phase, and obtain Kau and 16-OH Kau; or / and KA and 16-OH KOA.

[0082] In the above method, the fermentation involves first culturing the recombinant bacteria in a seed culture medium to obtain a seed liquid; then inoculating the seed liquid into a fermentation culture medium for fermentation, and the product of the fermentation is referred to as the fermentation system.

[0083] YPD medium was used for fermentation medium, seed medium, and solid medium.

[0084] In the above method, each L volume of the YPD culture medium formulation contains: 10g yeast extract; 20g peptone; 20g glucose, with the remainder being deionized water.

[0085] Prior to fermentation, the following steps are also included:

[0086] a) Activate the recombinant bacteria in a solid selective medium;

[0087] b) After shaking culture in liquid selective medium, transfer to seed culture medium to obtain seed solution;

[0088] The solid or liquid selective culture medium is YPD medium;

[0089] The culture conditions in step b) above are 30℃ and 200 rpm;

[0090] The specific method for culturing the seed culture in the above fermentation method is as follows: after the recombinant bacteria are activated, single clones on the plate are picked and transferred to test tubes containing YPD medium, and cultured overnight at 30°C and 200 rpm with shaking; 500 μL of bacterial culture is transferred to a 250 mL Erlenmeyer flask containing 50 mL of YPD medium, and cultured at 30°C and 200 rpm with shaking for 24 h; 2 mL of bacterial culture is transferred to a 1 L Erlenmeyer flask containing 200 mL of seed culture medium, and cultured at 30°C and 200 rpm with shaking for 72 h.

[0091] The experiments of this invention demonstrate that key genes for KA and 16-OH KOA biosynthesis are expressed in the host yeast *Saccharomyces cerevisiae*, including the acetyl-CoA acyltransferase gene ERG10, the HMG-CoA synthase gene ERG13, the HMG-CoA reductase N-terminal truncated variant gene tHMG1, the mevalonate kinase gene ERG12, the phosphate mevalonate kinase gene ERG8, the phosphate mevalonate decarboxylase gene ERG19, the IPP isomerase gene IDI1, the FPP synthase gene ERG20, the GGPP synthase gene BTS1, the acetyl-CoA acyltransferase / HMG-CoA reductase gene EfmvaE, and the enantio-cobamic pyrophosphate synthase gene TwCPS. 3. Enantio-kaurene synthase gene TwKSL2, bifunctional kaurene synthase gene PpCPS / KS or bifunctional kaurene synthase gene truncated version 1t1PpCPS / KS or bifunctional kaurene synthase gene truncated version 2t2PpCPS / KS, enantio-kaurene oxidase genes TwKO, AtKO, cytochrome P450 reductase genes TwCPR3, TwCPR5, AtCPR2, cytochrome b5 genes AaCYB5, CrCYB5, RsCYB5, TwCYB5-A, FPP / GGPP synthase gene GgFPS F112AThe recombinant Saccharomyces cerevisiae strain that produces KA and 16-OH KOA was constructed by using the GGPP synthase gene XdGGPPS, the glucose-6-phosphate dehydrogenase gene ScZWF1, the 6-phosphate gluconate dehydrogenase gene ScGND1 or their fusion genes, providing a strong strain and research foundation for drug development and plant cultivation. Attached Figure Description

[0092] Figure 1 The chemical structural formulas of KA and 16-OH KOA are given.

[0093] Figure 2 A schematic diagram of the building blocks for engineered bacteria that produce KA and 16-OH KOA.

[0094] Figure 3 The graphs show the detection and content of Kau and 16-OH Kau produced by recombinant bacteria (C20-01 to C20-05). A is the GC-MS detection graph, and B is the content graph of Kau and 16-OH Kau produced by recombinant bacteria.

[0095] Figure 4 The graphs show the detection and content of KA and 16-OH KOA produced by recombinant bacteria (C20-06 to C20-23). ​​A is the GC-MS detection graph, and B is the content graph of KA and 16-OH KOA produced by recombinant bacteria. Detailed Implementation

[0096] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0097] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available. Invention Overview

[0099] This invention provides a method for constructing an engineered strain of *Saccharomyces cerevisiae* for producing enantiosorbic acid and 16α-hydroxyenantiosorbane-19-carboxylic acid. The method uses *Saccharomyces cerevisiae* CEN.PK2-1D as the starting strain to construct the engineered strain, and includes the following steps:

[0100] 1) Prepare the gene expression cassette P required for gene editing. TEF1 -Cas9-T CYC1The recombinant strain CEN.PK2-1D(a) was obtained by integrating into the host Saccharomyces cerevisiae CEN.PK2-1D chromosome XI-5 site;

[0101] 2) The gene expression cassette P required for GGPP biosynthesis GAL1 -ERG10-T TPI1 -P GAL10 -ERG13-T PGI P GAL1 -tHMG1-T ADH1 -P GAL10 -ERG12-T PDC1 P GAL1 -ERG8-T RPS2 -P GAL10 -ERG19-T TDH1 P GAL1 -IDI1-T CCW12 -P GAL10 -BTS1-ERG20-T RPL9A P GAL1 -tHMG1-T ADH1 -P GAL10 -EfmvaE-T PDC1 The recombinant strain C20 was obtained by sequentially integrating into the gal80, ypl062w, XI-3, rox1, and YPRCΔ15 sites on the chromosome of the recombinant strain CEN.PK2-1D(a).

[0102] 3) Gene expression cassette P GAL1 -TwCPS3-T CYC1 -P GAL10 -TwKSL2-T ADH1 Integration into the XII-1 site of chromosome C20 of the recombinant bacteria yielded recombinant bacteria C20-01; and / or

[0103] Gene expression cassette P GAL2 -PpCPS / KS-T PDC1 Integration into the XII-1 site of chromosome C20 of the recombinant bacteria yielded recombinant bacteria C20-02; and / or

[0104] The gene expression cassette P of the fusion protein GAL2 -PpCPS / KS-MBP-T PDC1 Integration into the XII-1 site of chromosome C20 of the recombinant bacteria yielded recombinant bacteria C20-03; and / or

[0105] The gene expression cassette P of the fusion protein GAL2 -t1PpCPS / KS-MBP-T PDC1 Integration into the XII-1 site of chromosome C20 of the recombinant bacteria yielded recombinant bacteria C20-04; and / or

[0106] The gene expression cassette P of the fusion protein GAL2 -t2PpCPS / KS-MBP-T PDC1 Integration into the XII-1 site of chromosome C20 of the recombinant bacteria yielded recombinant bacteria C20-05; and / or

[0107] Gene expression cassette P GAL1 -AtKO-T CYC1 -P GAL10 -AtCPR2-T ADH1 Integration into the XII-5 site of chromosome C20-05 of recombinant bacteria yields recombinant bacteria C20-06; and / or

[0108] Gene expression cassette P GAL1 -AtKO-T CYC1 -P GAL10 -TwCPR3-T ADH1 Integration into the XII-5 site of chromosome C20-05 of the recombinant strain C20-07 yielded the recombinant strain C20-07; and / or

[0109] Gene expression cassette P GAL1 -AtKO-T CYC1 -P GAL10 -TwCPR5-T ADH1 Integration into the XII-5 site of chromosome C20-05 of the recombinant strain C20-05 yielded the recombinant strain C20-08; and / or

[0110] Gene expression cassette P GAL1 -TwKO-T CYC1 -P GAL10 -AtCPR2-T ADH1 Integration into the XII-5 site of chromosome C20-05 of the recombinant strain yielded recombinant strain C20-09; and / or

[0111] Gene expression cassette P GAL1 -TwKO-T CYC1 -P GAL10 -TwCPR3-T ADH1 Integration into the XII-5 site of chromosome C20-05 of the recombinant strain yielded the recombinant strain C20-10; and / or

[0112] Gene expression cassette P GAL1 -TwKO-T CYC1 -P GAL10 -TwCPR5-T ADH1 Integration into the XII-5 site of chromosome C20-05 of the recombinant strain yielded the recombinant strain C20-11; and / or

[0113] Gene expression cassette P GAL1-GgFPS F112A -T CYC1 -P GAL10 -XdGGPPS-T ADH1 Integration into the Int4 site of chromosome C20-06 of recombinant bacteria yields recombinant bacteria C20-12; and / or

[0114] Gene expression cassette P GAL1 -GgFPS F112A -T CYC1 -P GAL10 -XdGGPPS-T ADH1 Integration into the Int4 site of chromosome C20-07 of recombinant bacteria yields recombinant bacteria C20-13; and / or

[0115] Gene expression cassette P GAL1 -GgFPS F112A -T CYC1 -P GAL10 -XdGGPPS-T ADH1 Integration into the Int4 site of chromosome C20-08 of recombinant bacteria yields recombinant bacteria C20-14; and / or

[0116] Gene expression cassette P GAL1 -GgFPS F112A -T CYC1 -P GAL10 -XdGGPPS-T ADH1 Integration into the Int4 site of chromosome C20-09 of recombinant bacteria yields recombinant bacteria C20-15; and / or

[0117] Gene expression cassette P GAL1 -GgFPS F112A -T CYC1 -P GAL10 -XdGGPPS-T ADH1 Integration into the Int4 site of chromosome C20-10 of the recombinant bacteria yielded recombinant bacteria C20-16; and / or

[0118] Gene expression cassette P GAL1 -GgFPS F112A -T CYC1 -P GAL10 -XdGGPPS-T ADH1 Integration into the Int4 site of chromosome C20-11 of recombinant bacteria yields recombinant bacteria C20-17; and / or

[0119] Gene expression cassette P GAL1 -AtKO-T CYC1 Integration into the Int14 site of chromosome C20-13 of the recombinant bacteria yielded recombinant bacteria C20-18; and / or

[0120] Gene expression cassette P GAL1 -AtKO-T CYC1 -P GAL10 -AaCYB5-T ADH1 Integration into the Int14 site of chromosome C20-13 of the recombinant bacteria yielded recombinant bacteria C20-19; and / or

[0121] Gene expression cassette P GAL1 -AtKO-T CYC1 -P GAL10 -CrCYB5-AT ADH1 Integration into the Int14 site of chromosome C20-13 of the recombinant bacteria yielded recombinant bacteria C20-20; and / or

[0122] Gene expression cassette P GAL1 -AtKO-T CYC1 -P GAL10 -RsCYB5-T ADH1 Integration into the Int14 site of chromosome C20-13 of the recombinant bacteria yielded recombinant bacteria C20-21; and / or

[0123] Gene expression cassette P GAL1 -AtKO-T CYC1 -P GAL10 -TwCYB5-AT ADH1 Integration into the Int14 site of chromosome C20-13 of the recombinant bacteria yielded recombinant bacteria C20-22; and / or

[0124] Gene expression cassette P GAL1 -ScZWF1-T CYC1 -P GAL10 -ScGND1-T ADH1 The recombinant strain C20-23 was obtained by integrating into the Int17 site of chromosome C20-20.

[0125] The recombinant bacteria C20-01 to C20-05 are used to produce the precursor compounds Kau and 16-OH Kau of enantiosorbic acid and 16α-hydroxyenantiosorbane-19-carboxylic acid; the recombinant bacteria C20-06 to C20-23 are used to produce KA and 16-OHKA.

[0126] The following is a description of a portion of the sequences in the sequence list:

[0127]

[0128]

[0129] Example 1: Preparation of the target gene and plasmids used

[0130] I. Preparation of the target gene

[0131] 1. Obtaining ERG10, ERG13, tHMG1, ERG12, ERG8, ERG19, IDI1, ERG20, BTS1, GAL1, GAL2, GAL10, TPI1, PGI, ADH1, PDC1, RPS2, TDH1, CCW12 and RPL9A.

[0132] Genomic DNA was extracted from yeast strain CEN.PK2-1D (Rui Ma et al., 2021, Frontiers in Bioengineering and Biotechnology, 9:631863) as a template. The primers required for gene amplification were used for amplification in Table 1. The gene fragments, promoters and terminators of the expected size were obtained by gel recovery.

[0133] Gene JET Gel Extraction Kit Gel Recovery Steps:

[0134] (1) Take the PCR product and premix it with 6× loading buffer, and electrophoresis it on a 1.5% agarose gel at low voltage (about 5V / cm) for 15-20 minutes;

[0135] (2) Use a scalpel or razor blade to cut the gel containing DNA fragments, cutting as close to the DNA fragments as possible to reduce the gel content. Place the gel in a pre-weighed 1.5 mL centrifuge tube and weigh it. Record the weight of the gel. Avoid prolonged exposure to UV light, as this may damage the DNA and affect subsequent experiments.

[0136] (3) Add a 1:1 amount of Binding Buffer to the film (the amount is by weight, such as 100 μL of Binding Buffer for every 100 mg of agarose gel);

[0137] (4) Incubate the gel mixture at 65℃ for 10 min, inverting and mixing 2-3 times during the period to promote gel melting and ensure that the gel is completely dissolved. Before loading onto the column, quickly vortex the gel mixture once.

[0138] (5) Transfer up to 800 μL of gel dissolution solution to the gene recovery and purification column, centrifuge at 13000g for 1 min, discard the effluent, and then put the column back into the same collection tube;

[0139] (6) Add 700 μL Wash Buffer (diluted with ethanol) to the Gene JET purification column, centrifuge at 13000g for 1 min, discard the eluent, and then put the column back into the same collection tube;

[0140] (7) Centrifuge an empty GeneJET purification column at 13000g for 1 min to completely remove residual Wash Buffer;

[0141] (8) Transfer the Gene JET purification column to a clean 1.5 mL centrifuge tube, add 20-30 μL ddH2O (preheated at 65 °C) to the purification column membrane, and centrifuge at 13000 g for 1 min;

[0142] (9) Discard the Gene JET purification column and store the purified DNA at -20°C for later use.

[0143] Genomic DNA was extracted from yeast strain PS2 (described in Chinese patent ZL201911086944.4) as a template, and amplification was performed using the primers required for gene amplification in Table 1. The BTS1-ERG20 fusion gene fragment of the expected size was obtained by gel recovery.

[0144] DNA from pESC-LEU, T1-(TPI1-PGI), T2-(ADH1-PDC1), T4-(RPS2-TDH1), and T5-(CCW12-RPL9A) vectors (Rui Ma et al., 2021, Frontiers in Bioengineering and Biotechnology, 9:631863) was used as templates, and amplification was performed using the primers required for gene amplification listed in Table 1. Fragments of the expected size were obtained by gel recovery.

[0145] 2. EfmvaE, GgFPS F112A The acquisition of XdGGPPS, TwCPS3, TwKSL2, TwKO, AtKO, TwCPR3, TwCPR5, AtCPR2, TwCYB5-A, RsCYB5, AaCYB5, CrCYB5, ScZWF1 and ScGND1.

[0146] Shanghai Langjing Biotechnology Co., Ltd. has research on EfmvaE and GgFPS. F112A The genes XdGGPPS, TwCPS3, TwKSL2, TwKO, AtKO, TwCPR3, TwCPR5, AtCPR2, TwCYB5-A, RsCYB5, AaCYB5, CrCYB5, ScZWF1, and ScGND1 underwent codon optimization and total gene synthesis. Clonal plasmids containing the above target genes, namely pUC57-target gene, were then constructed.

[0147] Using the plasmid DNA described above as templates, amplification was performed using the primers required for gene amplification listed in Table 1. The gel recovered the amplified EfmvaE and GgFPS cells, which were of the expected size.F112A XdGGPPS, TwCPS3, TwKSL2, TwKO, AtKO, TwCPR3, TwCPR5, AtCPR2, TwCYB5-A, RsCYB5, AaCYB5, CrCYB5, ScZWF1 and ScGND1 gene fragments.

[0148] The amplification system described above consisted of: 25 μL of TAKARA PrimeSTAR Max DNA polymerase MIX, 2.5 μL of each primer, 1 μL of genomic DNA template, and distilled water to a total volume of 50 μL. The amplification conditions were: 98℃ pre-denaturation for 3 min (1 cycle); 98℃ denaturation for 10 s, 55℃ annealing for 15 s, and 72℃ extension at 3 kb / min (35 cycles); and 72℃ extension for 5 min (1 cycle).

[0149] 3. Obtaining PpCPS / KS, t1PpCPS / KS, t2PpCPS / KS, and MBP fusion genes

[0150] Shanghai Langjing Biotechnology Co., Ltd. performed codon optimization and total gene synthesis on the PpCPS / KS and MBP genes, and constructed clonal plasmids containing the above target genes, namely pUC57-target gene.

[0151] Using the plasmid DNA described above as templates, amplification was performed using the primers listed in Table 1 to obtain PpCPS / KS, t1PpCPS / KS, t2PpCPS / KS, and MBP gene fragments of the expected size. These fragments were then combined with the previously obtained promoter GAL2 and terminator PDC1 fragments, and the fusion gene P was obtained using overlap extension PCR (Yongjin J. Zhou et al., 2012, Journal of The American Chemical Society, 134:3234-3241). GAL2 -PpCPS / KS-MBP-T PDC1 P GAL2 -t1PpCPS / KS-MBP-T PDC1 and P GAL2 -t2PpCPS / KS-MBP-T PDC1 .

[0152] The amplification system described above is: (First round PCR) TAKARA HSDNA polymerase 5×PS Buffer 5 μL, dNTPMix 3 μL, and the above fragments (including gene fragments, promoter GAL2 fragment, and terminator PDC1 fragment). HS polymerase (2.5 U / μL) 0.5 μL, add distilled water to a total volume of 25 μL. Amplification conditions: 98℃ pre-denaturation for 3 min (1 cycle); 98℃ denaturation for 10 s, 58℃ annealing for 15 s, 72℃ extension at 1 kb / min (15 cycles); 72℃ extension for 5 min (1 cycle). (Second round PCR) TAKARA PrimeSTAR Max DNA polymerase MIX 25 μL, primers 2.5 μL each, first round PCR product 2 μL, add distilled water to a total volume of 50 μL. Amplification conditions: 98℃ pre-denaturation for 3 min (1 cycle); 98℃ denaturation for 10 s, 56℃ annealing for 15 s, 72℃ extension at 3 kb / min (35 cycles); 72℃ extension for 5 min (1 cycle).

[0153] Table 1 Primer sequences

[0154]

[0155]

[0156]

[0157]

[0158]

[0159] II. Construction of Recombinant Plasmids

[0160] 1. Plasmid pP GAL1 -ERG10-T TPI1 -P GAL10 -ERG13-T PGI ,pP GAL1 -tHMG1-T ADH1 -P GAL10 -ERG12-T PDC1 ,pP GAL1 -ERG8-T RPS2 -P GAL10 -ERG19-T TDH1 ,pP GAL1 -IDI1-T CCW12 -P GAL10 -BTS1-ERG20-T RPL9A ,pP GAL1 -tHMG1-T ADH1 -P GAL10 -EfmvaE-T PDC1 The construction.

[0161] This invention employs the Golden Gate cloning method (Siwei Li et al., 2016, Biotechnology for Biofuels, 9:232) to construct the aforementioned plasmids. The specific steps are as follows: The DNA fragments described in 'I. Preparation of the Target Gene' (gene fragments ERG10, ERG13, tHMG1, ERG12, ERG8, ERG19, IDI1, BTS1-ERG20, EfmvaE, promoters GAL10-GAL1, terminators TPI1, PGI, ADH1, PDC1, RPS2, TDH1, CCW12, RPL9A) are taken. Each plasmid includes two gene fragments, two promoters GAL10-GAL1, and two terminators. 0.1 pmol (pmol = mass ng / (fragment length bp × 0.65)) is added to each fragment for a Golden Gate reaction. The Golden Gate reaction system is as follows:

[0162]

[0163] PCR reaction conditions: 37℃ for 3 min, 16℃ for 4 min, 25 cycles; 50℃ for 5 min; 80℃ for 5 min; maintain at 4℃. The reaction product was transformed into Trans5α, and positive clones were identified by PCR.

[0164] 2. Plasmid pP GAL1 -TwCPS3-T CYC1 -P GAL10 -TwKSL2-T ADH1 ,pP GAL1 -AtKO-T CYC1 -P GAL10 -AtCPR2-T ADH1 ,pP GAL1 -AtKO-T CYC1 -P GAL10 -TwCPR3-T ADH1 ,pP GAL1 -AtKO-T CYC1 -P GAL10 -TwCPR5-T ADH1 ,pP GAL1 -TwKO-T CYC1 -P GAL10 -AtCPR2-T ADH1 ,pP GAL1 -TwKO-T CYC1 -P GAL10 -TwCPR3-T ADH1 ,pP GAL1 -TwKO-T CYC1 -P GAL10-TwCPR5-T ADH1 ,pP GAL1 -GgFPS F112A -T CYC1 -P GAL10 -XdGGPPS-T ADH1 ,pP GAL1 -AtKO-T CYC1 -P GAL10 -AaCYB5-T ADH1 ,pP GAL1 -AtKO-T CYC1 -P GAL10 -CrCYB5-T ADH1 ,pP GAL1 -AtKO-T CYC1 -P GAL10 -RsCYB5-T ADH1 ,pP GAL1 -AtKO-T CYC1 -P GAL10 -TwCYB5-AT ADH1 P GAL1 -ScZWF1-T CYC1 -P GAL10 -ScGND1-T ADH1 Construction

[0165] (1) The above plasmids were constructed using a seamless splicing method. The specific steps are as follows: Take the DNA fragments described in 'I. Preparation of the target gene' (gene fragments TwCPS3, AtKO, TwKO, GgFPS) F112A The enzymes were mixed with the BamHI-digested linear vector pESC-LEU and subjected to a seamless splicing reaction. The seamless splicing reaction system is as follows:

[0166]

[0167] PCR reaction conditions: 50℃ for 20 min; maintained at 4℃, reaction product was transformed into Trans5α, and positive clones were identified by PCR.

[0168] (2) Take the DNA fragments described in 'I. Preparation of Target Genes' (gene fragments TwKSL2, AtCPR2, TwCPR3, TwCPR5, XdGGPPS, RsCYB5, TwCYB5-A, AaCYB5, CrCYB5), and cut them with NotI single enzyme linear plasmids pESC-LEU::TwCPS3, pESC-LEU::AtKO, pESC-LEU::TwKO, pESC-LEU::GgFPS, respectively. F112A Mix and carry out a seamless splicing reaction. The seamless splicing reaction system and reaction conditions are the same as described in (1).

[0169] Example 2: Construction of recombinant bacteria

[0170] I. Genetic manipulation of yeast strains using gene editing methods

[0171] 1. Constructing gRNA plasmids targeting different chromosomal loci

[0172] Using p426-SNR52p-gRNA.CAN1 as the starting plasmid, two inverted AarI recognition sites (8NGCAGGTGNNNNCACCTGCN4) were inserted before its gRNA scaffold to generate a new gRNA vector. Based on the aforementioned plasmid, gRNA plasmids targeting different chromosomal sites were constructed. The specific steps are as follows:

[0173] (1) For different chromosomal loci, primers of 24 bp in length (consisting of a 20 bp target sequence and a 4 bp 5' overhang) were synthesized specifically. The primers are shown in Table 2. Annealing yielded double-stranded inserts with overhangs at both ends. The annealing reaction system is as follows:

[0174]

[0175] PCR reaction conditions: 95℃ for 5 min; 95℃ to 25℃ (-1℃ / min), maintained at 4℃, and the annealed product is ready for use.

[0176] (2) The above annealing products were used to construct a gRNA vector using the Golden Gate method to obtain a plasmid containing specific gRNA. The Golden Gate reaction system is as follows:

[0177]

[0178] PCR reaction conditions: 37℃ for 3 min, 16℃ for 4 min, 25 cycles; 50℃ for 5 min; 80℃ for 5 min; maintain at 4℃. The reaction product was transformed into Trans5α, and positive clones were identified by PCR.

[0179] Table 2 Primer sequences

[0180]

[0181]

[0182] 2. Obtaining the supplementary fragment

[0183] (1) Using the fusion gene P described in 'I. Preparation of the target gene' GAL2 -PpCPS / KS-MBP-T PDC1 P GAL2 -t1PpCPS / KS-MBP-TPDC1 and P GAL2 -t2PpCPS / KS-MBP-T PDC1 For the corresponding supplementary segments;

[0184] (2) Using the plasmid DNA described in 'II. Construction of Recombinant Plasmids' as a template, amplification was performed using the primers required for gene amplification in Table 3. The gel was then recovered to obtain P plasmids of the expected size. GAL1 -ERG10-T TPI1 -P GAL10 -ERG13-T PGI P GAL1 -tHMG1-T ADH1 -P GAL10 -ERG12-T PDC1 P GAL1 -ERG8-T RPS2 -P GAL10 -ERG19-T TDH1 P GAL1 -IDI1-T CCW12 -P GAL10 -BTS1-ERG20-T RPL9A P GAL1 -tHMG1-T ADH1 -P GAL10 -EfmvaE-T PDC1 P GAL1 -TwCPS3-T CYC1 -P GAL10 -TwKSL2-T ADH1 P GAL1 -AtKO-T CYC1 -P GAL10 -AtCPR2-T ADH1 P GAL1 -AtKO-T CYC1 -P GAL10 -TwCPR3-T ADH1 P GAL1 -AtKO-T CYC1 -P GAL10 -TwCPR5-T ADH1 P GAL1 -TwKO-T CYC1 -P GAL10 -AtCPR2-T ADH1 P GAL1 -TwKO-T CYC1 -P GAL10 -TwCPR3-T ADH1 P GAL1 -TwKO-T CYC1 -P GAL10-TwCPR5-T ADH1 P GAL1 -GgFPS F112A -T CYC1 -P GAL10 -XdGGPPS-T ADH1 P GAL1 -AtKO-T CYC1 ,PGAL1-AtKO-T CYC1 -P GAL10 -RsCYB5-T ADH1 P GAL1 -AtKO-T CYC1 -P GAL10 -TwCYB5-AT ADH1 P GAL1 -AtKO-T CYC1 -P GAL10 -AaCYB5-T ADH1 P GAL1 -AtKO-T CYC1 -P GAL10 -CrCYB5-T ADH1 The supplementary segment.

[0185] The amplification system described above consisted of: 25 μL of TAKARA PrimeSTAR Max DNA polymerase MIX, 2.5 μL of each primer, 1 μL of genomic DNA template, and distilled water to a total volume of 50 μL. The amplification conditions were: 98℃ pre-denaturation for 3 min (1 cycle); 98℃ denaturation for 10 s, 55℃ annealing for 15 s, and 72℃ extension at 3 kb / min (35 cycles); and 72℃ extension for 5 min (1 cycle).

[0186] Table 3 Primer sequences

[0187]

[0188]

[0189]

[0190] 3. Preparation and transformation of yeast competent cells

[0191] The ZYMO RESEARCH Frozen-EZ Yeast Transformation II kit was used to prepare competent yeast cells. The specific steps are as follows:

[0192] (1) Pick freshly activated single colonies from YPD solid medium (1% yeast extract, 2% peptone, 2% glucose, 2% agar), inoculate them into 3 mL of YPD liquid medium, and incubate at 30°C with shaking until OD. 600 = Approximately 0.8-1.0;

[0193] (2) Centrifuge at 500g for 4 minutes at room temperature, and discard the supernatant;

[0194] (3) Add 10 mL of Frozen-EZ Solution 1 to suspend the bacterial cells, centrifuge at 500 g for 4 min at room temperature, and discard the supernatant;

[0195] (4) Add 1 mL of Frozen-EZ Solution 2 suspension and dispense 50 μL into sterile 1.5 mL EP tubes;

[0196] (5) Take the specific gRNA plasmid (300 μg) and the corresponding complement fragment (300 μg), mix them thoroughly and then mix them with 50 μL of competent cells;

[0197] (6) Add 500 μL of Frozen-EZ Solution 3 and mix vigorously;

[0198] (7) Incubate at 30℃ for 2 hours, mixing 2-3 times during the process;

[0199] (8) Take 50-150 μL of the incubated bacterial solution, spread it on a defective SD plate (SD-Ura), air dry it, and then incubate it upside down at 30℃ for 48-96 h.

[0200] 4. PCR identification of recombinant bacteria

[0201] (1) Pick a single colony and resuspend it in 10 μL of sterile water. Take 1.5 μL into a PCR tube and add 1.5 μL of yeast lysis buffer (2% Triton X-100, 1% SDS, 1 mM EDTA, 100 mM NaCl, 10 mM Tris-HCl, pH 8.0). Mix well and heat at 99 °C for 30 min.

[0202] (2) Specific identification primers were designed based on different chromosomal loci (see Table 4). PCR amplification was performed using lysis buffer as a template. The reaction system was: TOYOBO KOD One. TM5 μL of PCR Master Mix, 0.2 μL each of primers, 1 μL of genomic DNA template, and distilled water were added to a total volume of 10 μL. The reaction conditions were: 98℃ pre-denaturation for 5 min (1 cycle); 98℃ denaturation for 15 s, 55℃ annealing for 15 s, 68℃ extension at 3 kb / min (30 cycles); 68℃ extension for 5 min (1 cycle).

[0203] (3) Positive strains were inoculated into YPD liquid medium and cultured at 30°C with shaking for 12 hours. After dilution, they were spread onto SC+5FOA solid medium to remove the corresponding specific gRNA plasmids.

[0204] Table 4 Primer sequences

[0205]

[0206]

[0207] II. Construction of CEN.PK2-1D(a) strain

[0208] Using yeast CEN.PK2-1D as the starting strain, the gene expression cassette P was expressed using homologous recombination. TEF1 -Cas9-T CYC1t The strain obtained by integrating into the XI-5 site of the yeast CEN.PK2-1D chromosome was CEN.PK2-1D(a) strain (ShanYang et al., 2020, Applied Microbiology and Biotechnology, 104(7):3037-3047).

[0209] III. Construction of Recombinant Bacterium C20

[0210] The gal80-gRNA plasmid and the complement fragment P were sequentially added. GAL1 -ERG10-T TPI1 -P GAL10 -ERG13-T PGI ypl062w-gRNA plasmid + complement fragment P GAL1 -tHMG1-T ADH1 -P GAL10 -ERG12-T PDC1 XI-3-gRNA plasmid + complement fragment P GAL1 -ERG8-T RPS2 -P GAL10 -ERG19-T TDH1 rox1-gRNA plasmid + complement fragment P GAL1 -IDI1-T CCW12 -P GAL10-BTS1-ERG20-T RPL9A YPRCΔ15-gRNA plasmid + complement fragment P GAL1 -tHMG1-T ADH1 -P GAL10 -EfmvaE-T PDC1 The recombinant bacteria CEN.PK2-1D(a) was integrated into the corresponding site on the chromosome. The correct positive clone was identified by PCR. The corresponding specific gRNA plasmid was removed to obtain the recombinant bacteria C20.

[0211] IV. Construction of recombinant bacteria C20-01 to C20-05

[0212] The XII-1-gRNA plasmid was conjugated with different complement fragments (P... GAL1 -TwCPS3-T CYC1 -P GAL10 -TwKSL2-T ADH1 P GAL2 -PpCPS / KS-T PDC1 P GAL2 -PpCPS / KS-MBP-T PDC1 P GAL2 -t1PpCPS / KS-MBP-T PDC1 P GAL2 -t2PpCPS / KS-MBP-T PDC1 The samples were mixed and integrated into the XII-1 site of chromosome C20 of the recombinant bacteria. The correct positive clones were identified by PCR, and the corresponding specific gRNA plasmids were removed to obtain recombinant bacteria C20-01 to C20-05.

[0213] V. Construction of recombinant bacteria C20-06 to C20-11

[0214] The XII-5-gRNA plasmid was conjugated with different complement fragments (P... GAL1 -AtKO-T CYC1 -P GAL10 -AtCPR2-T ADH1 P GAL1 -AtKO-T CYC1 -P GAL10 -TwCPR3-T ADH1 P GAL1 -AtKO-T CYC1 -P GAL10 -TwCPR5-T ADH1 P GAL1 -TwKO-T CYC1 -P GAL10 -AtCPR2-T ADH1 P GAL1-TwKO-T CYC1 -P GAL10 -TwCPR3-T ADH1 P GAL1 -TwKO-T CYC1 -P GAL10 -TwCPR5-T ADH1 The samples were mixed and integrated into the XII-5 site of chromosome C20-05 of the recombinant bacteria. The correct positive clones were identified by PCR, and the corresponding specific gRNA plasmids were removed to obtain recombinant bacteria C20-06 to C20-11.

[0215] VI. Construction of recombinant bacteria C20-12 to C20-17

[0216] The Int4-gRNA plasmid was combined with the complementation fragment P. GAL1 -GgFPS F112A -T CYC1 -P GAL10 -XdGGPPS-T ADH1 The mixtures were then integrated into the Int4 sites on chromosomes C20-06 to C20-11 of the recombinant bacteria. The correct positive clones were identified by PCR, and the corresponding specific gRNA plasmids were removed to obtain recombinant bacteria C20-12 to C20-17.

[0217] VII. Construction of recombinant bacteria C20-18 to C20-23

[0218] The Int14-gRNA plasmid was combined with the complement fragment (P) GAL1 -AtKO-T CYC1 P GAL1 -AtKO-T CYC1 -P GAL10 -AaCYB5-T ADH1 P GAL1 -AtKO-T CYC1 -P GAL10 -CrCYB5-T ADH1 P GAL1 -AtKO-T CYC1 -P GAL10 -RsCYB5-T A DH1 P GAL1 -AtKO-T CYC1 -P GAL10 -TwCYB5-AT ADH1 The samples were mixed and integrated into the Int14 site of chromosome C20-13 of the recombinant bacteria. The correct positive clones were identified by PCR, and the corresponding specific gRNA plasmids were removed to obtain recombinant bacteria C20-18 to C20-22.

[0219] The Int17-gRNA plasmid was combined with the complementation fragment P. GAL1 -ScZWF1-T CYC1 -P GAL10 -ScGND1-T ADH1 The mixture was integrated into the Int17 site of chromosome C20-20 of the recombinant bacteria. The correct positive clone was identified by PCR, and the corresponding specific gRNA plasmid was removed to obtain the recombinant bacteria C20-23.

[0220] Information on all the recombinant bacteria mentioned above is shown in Table 5.

[0221] Table 5 Information on recombinant bacteria

[0222]

[0223]

[0224] Example 3: Application of recombinant bacteria in the production of KA and 16-OH KOA

[0225] 1. Cultivation of engineered bacteria and extraction of products

[0226] All yeast strains prepared in Example 2 were activated in YPD solid selective medium. Seed culture was prepared in YPD liquid medium (30°C, 250 rpm, 16 h). 1% of the seed culture was inoculated into 50 mL shake flasks containing 10 mL of YPD liquid medium and cultured at 30°C with shaking at 250 rpm for 3 days. 1 mL of fermentation broth was taken, an equal volume of ethyl acetate was added, and the mixture was thoroughly shaken and centrifuged at 10,000 rpm for 5 min. The organic phase was collected for later use.

[0227] 2. Qualitative and quantitative analysis of KA and 16-OH KOA precursor compounds

[0228] Organic phases of the recombinant bacteria (C20-01 to C20-05) prepared in Example 2 were used to detect the fermentation products using gas chromatography-mass spectrometry (GC-MS). The GC-MS system was a Thermo TRACE 1310 / TSQ 8000 gas chromatograph, with an injection volume of 1 μL, splitless mode (25:1), a Thermo Scientific TG-5MS column (30 m × 0.25 mm × 0.25 μm), a helium flow rate of 1.0 mL / min, an injection port temperature of 280 °C, an ion source temperature of 280 °C, a temperature program of 50 °C for 2 min, a ramp-up to 250 °C at 40 °C / min, a ramp-up to 300 °C at 10 °C / min, a hold-up to 10 min, an electron energy of 70 eV, and a sample scan in the range of 50-500 m / z. Qualitative and quantitative analyses were performed using standards of KA and 16-OH KOA, which were purchased from Sigma-Aldrich.

[0229] 3. Derivatization of KA and 16-OH KOA and its qualitative and quantitative analysis

[0230] (1) Derivatization of KA and 16-OH KOA

[0231] Take the organic phase of the recombinant bacteria (C20-06 to C20-23) prepared in Example 2, dry the above organic phase sample with nitrogen in a fume hood, add 100 μL of methanol and 100 μL of (trimethylsilyl) diazomethane, mix well and let stand at room temperature for 10 min to obtain the derivatized substance; dry it with nitrogen in a fume hood, add 100 μL of ethyl acetate to redissolve and perform GC-MS detection.

[0232] (2) GC-MS detection of fermentation products

[0233] Fermentation products were detected using gas chromatography-mass spectrometry (GC-MS). The GC-MS system was a Thermo TRACE1310 / TSQ 8000 gas chromatograph, with an injection volume of 1 μL, splitless mode (25:1). The GC column was a Thermo Scientific TG-5MS (30 m × 0.25 mm × 0.25 μm). The helium flow rate was 1.0 mL / min, the injection port temperature was 280 °C, the ion source temperature was 280 °C, and the temperature program was as follows: 50 °C for 2 min, then ramped up to 250 °C at 40 °C / min, then to 300 °C at 10 °C / min, and held for 10 min. The electron energy was 70 eV, and the sample was scanned in the range of 50–500 m / z. Qualitative and quantitative analysis were performed using KA and 16-OH KOA standards, which were purchased from Sigma-Aldrich.

[0234] The yield of the target product after 72 hours of fermentation by each engineered strain is as follows: Figure 3 and Figure 4 As shown.

[0235] from Figure 3 It can be seen that the fermentation products of recombinant bacteria C20-04 and C20-05 can isolate the precursor of KA, Kau, while the fermentation products of recombinant bacteria C20-02 and C20-05 can isolate the precursor of 16-OH KOA, 16-OH Kau. Among these, C20-05 showed the highest efficiency and can be used as a starting strain for further modification. C20-04 also exhibited relatively high efficiency. Figure 4 It can be seen that KA and 16-OH KOA can be separated from the fermentation products of recombinant bacteria C20-12 to C20-23. Among them, C20-23 has the highest efficiency, and C20-18 to C20-20 also have high efficiency.

[0236] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. A method for constructing a recombinant yeast strain for producing enantiocarboxylic acid and 16α-hydroxyenantiocarboxane-19-carboxylic acid, characterized in that, Includes the following steps: 1) Gene expression cassette P TEF1 -Cas9-T CYC1 The recombinant strain CEN.PK2-1D was obtained by importing it into the starting yeast CEN.PK2-1D; 2) By increasing the expression or activity of the following genes in the recombinant Saccharomyces cerevisiae CEN.PK2-1D(a): acetyl-CoA acyltransferase gene ERG10, HMG-CoA synthase gene ERG13, HMG-CoA reductase N-terminal truncated gene tHMG1, mevalonate kinase gene ERG12, mevalonate phosphate kinase gene ERG8, mevalonate phosphate decarboxylase gene ERG19, IPP isomerase gene IDI1, FPP synthase gene ERG20, GGPP synthase gene BTS1, and acetyl-CoA acyltransferase / HMG-CoA reductase gene EfmvaE, recombinant strain C20 was obtained. 3) In recombinant strain C20, the following enzymes were expressed: enantio-cobamic pyrophosphate synthase TwCPS3, enantio-kaurene synthase TwKSL2, bifunctional kaurene synthase PpCPS / KS, enantio-kaurene oxidases TwKO and AtKO, cytochrome P450 reductases TwCPR3, TwCPR5, and AtCPR2, cytochrome b5 AaCYB5, CrCYB5, RsCYB5, TwCYB5-A, and FPP / GGPP synthase GgFPS. F112A At least one of GGPP synthase XdGGPPS, glucose-6-phosphate dehydrogenase ScZWF1, and 6-phosphate gluconate dehydrogenase ScGND1 was used to obtain a recombinant yeast strain for the production of enantiocarboxylic acid and 16α-hydroxyenantiocarboxane-19-carboxylic acid.

2. The construction method according to claim 1, characterized in that, Step 3) includes: 31) One of the following genes was introduced into recombinant bacteria C20: TwCPS3 and TwKSL2, PpCPS / KS, fusion protein gene PpCPS / KS-MBP, fusion protein gene t1PpCPS / KS-MBP, or fusion protein gene t2PpCPS / KS-MBP to obtain recombinant bacteria 1. 32) One of the AtKO and AtCPR2 genes, AtKO and TwCPR3 genes, AtKO and TwCPR5 genes, TwKO and AtCPR2 genes, TwKO and TwCPR3 genes, and TwKO and TwCPR5 genes was introduced into recombinant bacteria 1 to obtain recombinant bacteria 2. 33) Gene GgFPS F112A XdGGPPS was introduced into recombinant strain 2 to obtain recombinant strain 3, which is a recombinant yeast strain that produces enantio-kauronic acid and 16α-hydroxyenantio-kaurane-19-carboxylic acid.

3. The construction method according to claim 2, characterized in that, Step 3) includes: 34) One of the AtKO gene, AtKO and AaCYB5 gene, AtKO and CrCYB5 gene, AtKO and RsCYB5 gene, and AtKO and TwCYB5-A gene was introduced into recombinant strain 3 to obtain recombinant strain 4. Recombinant strain 4 is a recombinant yeast strain that produces enantio-kaurethane and 16α-hydroxyenantio-kaurethane-19-carboxylic acid.

4. The construction method according to claim 3, characterized in that, Step 3) includes: 35) The ScZWF1 and ScGND1 genes were introduced into recombinant strain 4 to obtain recombinant strain 5. Recombinant strain 5 is a recombinant yeast strain that produces enantio-kauronic acid and 16α-hydroxyenantio-kauronic acid-19-carboxylic acid.

5. The construction method according to any one of claims 1-4, characterized in that, The method for gene introduction involves integrating a gene expression cassette containing the gene into the chromosome of the starting bacterium.

6. The construction method according to claim 5, characterized in that, The gene expression cassette for expressing the protein or fusion protein in vivo includes a promoter, the coding nucleic acid of the protein or fusion protein, and a terminator; the promoter is TEF1, GAL1, GAL10, or GAL2; the terminator is CYC1, RPS2, CCW12, RPL9A, PGI, ADH1, or PDC1; wherein the nucleotide sequence of TEF1 is shown in SEQ ID No. 31; the nucleotide sequence of GAL1 is shown in SEQ ID No. 32; the nucleotide sequence of GAL2 is shown in SEQ ID No. 33; the nucleotide sequence of GAL10 is shown in SEQ ID No. 34; the nucleotide sequence of CYC1 is shown in SEQ ID No. 35; the nucleotide sequence of TPI1 is shown in SEQ ID No. 36; the nucleotide sequence of PGI is shown in SEQ ID No. 37; the nucleotide sequence of ADH1 is shown in SEQ ID No. 38; the nucleotide sequence of PDC1 is shown in SEQ ID No. 39; the nucleotide sequence of RPS2 is shown in SEQ ID No. 40; and the nucleotide sequence of TDH1 is shown in SEQ ID No.

39. The nucleotide sequence of CCW12 is shown in SEQ ID No. 41; the nucleotide sequence of RPL9A is shown in SEQ ID No. 42; and the nucleotide sequence of RPL9A is shown in SEQ ID No.

43.

7. The construction method according to claim 1, characterized in that, In step 2, the nucleotide sequence of the ERG10 gene is shown in SEQ ID No. 1; the nucleotide sequence of the ERG13 gene is shown in SEQ ID No. 2; the nucleotide sequence of the tHMG1 gene is shown in SEQ ID No. 3; the nucleotide sequence of the ERG12 gene is shown in SEQ ID No. 4; the nucleotide sequence of the ERG8 gene is shown in SEQ ID No. 5; the nucleotide sequence of the ERG19 gene is shown in SEQ ID No. 6; the nucleotide sequence of the IDI1 gene is shown in SEQ ID No. 7; the nucleotide sequence of the ERG20 gene is shown in SEQ ID No. 8; the nucleotide sequence of the BTS1 gene is shown in SEQ ID No. 9; and the nucleotide sequence of the EfmvaE gene is shown in SEQ ID No.

10. In step 3), the nucleotide sequence of the enantio-cobamic pyrophosphate synthase gene TwCPS3 is shown in SEQ ID No. 11; the nucleotide sequence of the enantio-kaurene synthase gene TwKSL2 is shown in SEQ ID No. 12; the nucleotide sequence of the bifunctional kaurene synthase gene PpCPS / KS is shown in SEQ ID No. 13; the nucleotide sequence of the enantio-kaurene oxidase gene TwKO is shown in SEQ ID No. 14; the nucleotide sequence of the kaurene oxidase gene AtKO is shown in SEQ ID No. 15; the nucleotide sequence of the cytochrome P450 reductase gene AtCPR2 is shown in SEQ ID No. 16; the nucleotide sequence of the cytochrome P450 reductase gene TwCPR3 is shown in SEQ ID No. 17; the nucleotide sequence of the cytochrome P450 reductase TwCPR5 gene is shown in SEQ ID No. 18; the nucleotide sequence of the cytochrome b5 AaCYB5 gene is shown in SEQ ID No. 19; and the nucleotide sequence of the cytochrome b5 CrCYB5 gene is shown in SEQ ID No.

10. The nucleotide sequence of the cytochrome b5 gene RsCYB5 is shown in SEQ ID No. 20; the nucleotide sequence of the cytochrome b5 TwCYB5-A gene is shown in SEQ ID No. 22; the FPP / GGPP synthase gene GgFPS is shown in SEQ ID No.

20. F112A The nucleotide sequences of the following are shown in SEQ ID No. 23; the nucleotide sequences of the GGPP synthase gene XdGGPPS are shown in SEQ ID No. 24; the nucleotide sequences of the glucose-6-phosphate dehydrogenase gene ScZWF1 are shown in SEQ ID No. 25; and the nucleotide sequences of the 6-phosphate gluconate dehydrogenase gene ScGND1 are shown in SEQ ID No.

26. The fusion protein gene is composed, in sequence, of the bifunctional kaurene synthase gene PpCPS / KS or its truncated variant 1 (t1PpCPS / KS), truncated variant 2 (t2PpCPS / KS), a linker peptide, and the maltose-binding protein gene MBP. The nucleotide sequences of the t1PpCPS / KS gene are shown in SEQ ID No. 27; the nucleotide sequences of the t2PpCPS / KS gene are shown in SEQ ID No. 28; the nucleotide sequences of the linker peptide are shown in SEQ ID No. 29; and the nucleotide sequences of the MBP gene are shown in SEQ ID No.

30.

8. The recombinant bacteria constructed by any one of the methods described in claims 1-7.

9. The use of the recombinant bacteria constructed by any of the methods of claims 1-7 or the recombinant bacteria of claim 8 in the preparation of enantio-kauronic acid and 16α-hydroxyenantio-kauronic acid-19-carboxylic acid.

10. A method for preparing enantio-kauronic acid and 16α-hydroxyenantio-kaurane-19-carboxylic acid, characterized in that, The method includes culturing the recombinant bacteria of claim 8 to obtain a fermentation product; and obtaining enantio-kauronic acid and 16α-hydroxyenantio-kaurane-19-carboxylic acid from the fermentation product.

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