Recombinant saccharomyces cerevisiae for producing protopanoxadiol by metabolizing glycerol and construction method thereof

Abstract

The invention discloses recombinant saccharomyces cerevisiae for producing protopanoxadiol by metabolizing glycerol and a construction method thereof, and the construction method comprises the following steps: introducing a dammarendiol synthase DS gene expression cassette, a PPD synthase and reductase fusion protein PPDS-ATR1 gene expression cassette and a squalene oxidase ERG1 gene expression cassette into saccharomyces cerevisiae to obtain a recombinant bacterium 1; introducing a glycerol transport protein CjFPS1 gene expression cassette, a glycerol dehydrogenase OpGDH gene expression cassette, a dihydroxy acetone kinase DAK1 gene expression cassette and a 3-hydroxy-3-methylglutaryl coenzyme A reductase HMGR gene expression cassette into the recombinant bacterium 1 to obtain a recombinant bacterium 2; introducing citric acid lyase AnACLa and AnACLb gene expression cassettes and citric acid transporter protein YHM2 and CTP1 gene expression cassettes into the recombinant bacterium 2 to obtain a recombinant bacterium 3; experiments prove that the yield of the recombinant bacteria 2, 3PPD is 124 mg / L and 220 mg / L.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biotechnology, and in particular to a recombinant Saccharomyces cerevisiae for producing protopanaxadiol (PPD) by metabolizing glycerol, a construction method and application thereof. BACKGROUND

[0002] Protopanaxadiol (PPD) belongs to triterpenoids, has strong anticancer activity, can effectively kill tumor cells, has no toxic and side effects on the human body, and can also enhance the body's immunity. When used in combination with other anticancer drugs, the anticancer efficacy can be effectively enhanced. PPD has a very low content in ginseng. According to reports, it is mainly obtained by hydrolyzing ginsenosides Rb1, Rd and Rg2 and the like ginsenosides through mild alkaline hydrolysis treatment, microbial transformation and enzymatic transformation, but the cost is still high. In recent years, the research on synthetic biology has been continuously developed, so that microorganisms can synthesize compounds of animal and plant origin. Although researchers have constructed strains capable of producing protopanaxadiol (PPD), the yield still needs to be improved.

[0003] Most of the existing researches produce ginsenosides by using glucose as a substrate. Glycerol has a higher degree of reduction than glucose, and glycerol is the main product of the petroleum chemical industry and has the characteristics of low cost. However, there is no report on the production of protopanaxadiol by modifying glycerol metabolism. SUMMARY

[0004] The present application aims to overcome the shortcomings of the prior art and provide a recombinant Saccharomyces cerevisiae for producing protopanaxadiol by metabolizing glycerol.

[0005] A second object of the present application is to provide a construction method of a recombinant Saccharomyces cerevisiae for producing protopanaxadiol by metabolizing glycerol.

[0006] A third object of the present application is to provide an application of a recombinant Saccharomyces cerevisiae for producing protopanaxadiol by metabolizing glycerol.

[0007] A fourth object of the present application is to provide another recombinant Saccharomyces cerevisiae for producing protopanaxadiol by metabolizing glycerol.

[0008] A fifth object of the present application is to provide another construction method of a recombinant Saccharomyces cerevisiae for producing protopanaxadiol by metabolizing glycerol.

[0009] A sixth object of the present application is to provide an application of another recombinant Saccharomyces cerevisiae for producing protopanaxadiol by metabolizing glycerol.

[0010] The technical solutions of the present application are summarized as follows:

[0011] A construction method of a recombinant Saccharomyces cerevisiae for producing protopanaxadiol by metabolizing glycerol, comprising the following steps:

[0012] (1) The dammarene diol synthase DS gene expression cassette, the PPD synthase and its reductase fusion protein PPDS-ATR1 gene expression cassette and the squalene oxidase ERG1 gene expression cassette were introduced into Saccharomyces cerevisiae BY4741 to obtain recombinant strain 1.

[0013] The nucleotide sequence of the dammarene diol synthase DS gene is shown in SEQ ID NO.1;

[0014] The nucleotide sequence of the PPD synthase and its reductase fusion protein PPDS-ATR1 gene is shown in SEQ ID NO.2;

[0015] The nucleotide sequence of the squalene oxidase ERG1 gene is shown in SEQ ID NO.3;

[0016] (2) The glycerol transport protein CjFPS1 gene expression cassette, glycerol dehydrogenase OpGDH gene expression cassette, dihydroxyacetone kinase DAK1 gene expression cassette and 3-hydroxy-3-methylglutaryl-CoA reductase HMGR gene expression cassette were introduced into recombinant strain 1 to obtain recombinant strain 2, that is, the recombinant Saccharomyces cerevisiae that metabolizes glycerol to produce protopanaxadiol (PPD).

[0017] The nucleotide sequence of the glycerol transport protein CjFPS1 gene is shown in SEQ ID NO.4;

[0018] The nucleotide sequence of the glycerol dehydrogenase OpGDH gene is shown in SEQ ID NO.5;

[0019] The nucleotide sequence of the dihydroxyacetone kinase DAK1 gene is shown in SEQ ID NO. 6;

[0020] The nucleotide sequence of the 3-hydroxy-3-methylglutaryl-CoA reductase HMGR gene is shown in SEQ ID NO.7.

[0021] Another method for constructing a recombinant Saccharomyces cerevisiae strain that metabolizes glycerol to produce protopanaxadiol includes the following steps:

[0022] (1) The dammarene diol synthase DS gene expression cassette, the PPD synthase and its reductase fusion protein PPDS-ATR1 gene expression cassette and the squalene oxidase ERG1 gene expression cassette were introduced into Saccharomyces cerevisiae BY4741 to obtain recombinant strain 1.

[0023] The nucleotide sequence of the dammarene diol synthase DS gene is shown in SEQ ID NO.1;

[0024] The nucleotide sequence of the PPD synthase and its reductase fusion protein PPDS-ATR1 gene is shown in SEQ ID NO.2;

[0025] The nucleotide sequence of the squalene oxidase ERG1 gene is shown in SEQ ID NO.3;

[0026] (2) The glycerol transport protein CjFPS1 gene expression cassette, glycerol dehydrogenase OpGDH gene expression cassette, dihydroxyacetone kinase DAK1 gene expression cassette and 3-hydroxy-3-methylglutaryl-CoA reductase HMGR gene expression cassette were introduced into recombinant strain 1 to obtain recombinant strain 2.

[0027] The nucleotide sequence of the glycerol transport protein CjFPS1 gene is shown in SEQ ID NO.4;

[0028] The nucleotide sequence of the glycerol dehydrogenase OpGDH gene is shown in SEQ ID NO.5;

[0029] The nucleotide sequence of the dihydroxyacetone kinase DAK1 gene is shown in SEQ ID NO. 6;

[0030] The nucleotide sequence of the 3-hydroxy-3-methylglutaryl-CoA reductase HMGR gene is shown in SEQ ID NO.7.

[0031] (3) Introduce the citrate lyase AnACLa gene expression cassette and the citrate lyase AnACLb gene expression cassette, the citrate transporter YHM2 gene expression cassette and the citrate transporter CTP1 gene expression cassette into recombinant strain 2 to obtain recombinant strain 3, which is another recombinant Saccharomyces cerevisiae that metabolizes glycerol to produce protopanaxadiol.

[0032] The nucleotide sequence of the citrate lyase AnACLa gene is shown in SEQ ID NO. 8;

[0033] The nucleotide sequence of the citrate lyase AnACLb gene is shown in SEQ ID NO. 9;

[0034] The nucleotide sequence of the citrate transporter YHM2 gene is shown in SEQ ID NO.10;

[0035] The nucleotide sequence of the citrate transporter CTP1 gene is shown in SEQ ID NO.11.

[0036] The above method was used to construct a recombinant Saccharomyces cerevisiae strain for metabolizing glycerol to produce protopanaxadiol.

[0037] The above-mentioned recombinant Saccharomyces cerevisiae is used to produce protopanaxadiol by metabolizing glycerol.

[0038] Advantages of this invention:

[0039] This invention successfully constructed a recombinant Saccharomyces cerevisiae strain that metabolizes glycerol to produce protopanaxadiol (PPD). Experiments showed that the recombinant Saccharomyces cerevisiae strain of this invention produced protopanaxadiol (PPD) through fermentation. The protopanaxadiol (PPD) yields of recombinant strains 2 and 3 were 124 mg / L and 220 mg / L, respectively, which were higher than the yield of the control strain (recombinant strain 1, 15.6 mg / L). Attached Figure Description

[0040] Figure 1 The yield of protopanaxadiol (PPD) produced by fermentation of recombinant Saccharomyces cerevisiae for the production of protopanaxadiol (PPD) from glycerol metabolism. Detailed Implementation

[0041] The present invention will be further described below through specific embodiments.

[0042] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0043] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0044] The *Saccharomyces cerevisiae* BY4741 (ATCC: ATCC4040002) used in this invention (purchased in June 2016, website: https: / / www.atcc.org / ) is the starting strain. Hereinafter referred to as *Saccharomyces cerevisiae* BY4741.

[0045] Example 1: The method for constructing recombinant bacteria 1 includes the following steps:

[0046] Recombinant strain 1 was obtained by introducing dammarene diol synthase DS gene expression cassette, PPD synthase (PPDS) and its reductase (ATR1) fusion protein PPDS-ATR1 gene expression cassette and squalene oxidase ERG1 gene expression cassette into Saccharomyces cerevisiae BY4741.

[0047] The dammarene diol synthase DS is derived from ginseng (Panax ginseng). After codon optimization by Saccharomyces cerevisiae from Suzhou Genewise Co., Ltd., the nucleotide sequence of the dammarene diol synthase DS gene is shown in SEQ ID NO.1.

[0048] The PPD synthase PPDS and its reductase ATR1 are derived from ginseng. After codon optimization by Suzhou Genewiz Company, the nucleotide sequence of the fusion protein PPDS-ATR1 gene of PPD synthase PPDS and its reductase ATR1 is shown in SEQ ID NO.2. The nucleotide sequence of the squalene oxidase ERG1 gene is derived from Saccharomyces cerevisiae BY4741, as shown in SEQ ID NO.3.

[0049] (1) Expression box construction

[0050] Using the primer pairs and templates in Table 1, HOU and P were amplified by PCR. PGK1 DS, T CYC1 P TEF1 PPDS-ATR1, T ADH2 P TDH3 ERG1, T ADH3 MET and HOD fragments.

[0051] The amplified HOU, P PGK1 DS and T CYC1 Fragment fusion to obtain HOU-P PGK1 -DS-T CYC1 Gene expression cassette (dammarene diol synthase DS gene expression cassette, also known as fragment 1);

[0052] The amplified P TEF1 PPDS-ATR1, T ADH2 Fragment fusion to obtain P TEF1 -PPDS-ATR1-T ADH2 Gene expression cassette (PPDS-ATR1 gene expression cassette, also known as fragment 2);

[0053] The amplified P TDH3 ERG1, T ADH3 The MET and HOD fragments are fused to obtain P. TDH3 -ERG1-T ADH3 -MET-HOD gene expression cassette (squalene oxidase ERG1 gene expression cassette, also known as fragment 3);

[0054] (2) Conversion of brewing yeast

[0055] A single colony of *Saccharomyces cerevisiae* BY4741 was inoculated into 3 ml of YPD liquid medium and cultured for 12 h at 30 °C and 220 rpm in a shaker. The cultured yeast seed culture was then transferred to a fresh 3 ml of YPD liquid medium and cultured for 5 h at 30 °C and 220 rpm. 1 ml of the bacterial culture was transferred to a pre-sterilized 1.5 ml centrifuge tube and centrifuged at 4000 rpm for 3 min. The supernatant was discarded, and the bacterial cells were collected. The cells were washed once with 1 ml of sterile water and centrifuged at 4000 rpm for 3 min. The supernatant was discarded, and the bacterial cells were collected. Then, 1 ml of 100 mM LiAc aqueous solution was added to the bacterial cells and mixed well. The mixture was allowed to stand at room temperature for 5 min. After standing, the cells were centrifuged at 4000 rpm for 3 min, and the LiAc liquid was removed using a pipette.

[0056] Boil salmon DNA (Solarbio, 10 mg / ml) in boiling water for 5 minutes, then quickly place it on prepared ice to cool.

[0057] Add 120 μl of PEG3350 (50 g PEG3350 / 100 ml water), 18 μl of 1.0 M LiAc aqueous solution, 5 μl of salmon sperm DNA (boiled in boiling water for 5 min and cooled), 200 ng of fragment 1, 200 ng of fragment 2, and 200 ng of fragment 3 to the bacterial pellet centrifuge tube in sequence, and finally make up the volume to 180 μl with sterile water. Then, gently pipette the mixture for 1 minute to mix it thoroughly. Place the mixed centrifuge tube in a 42°C water bath for 30 minutes, then centrifuge at 4000 rpm for 3 minutes and remove the supernatant. Add 1 ml of clean YPD liquid medium and incubate at 30°C and 220 rpm for 2 hours. Centrifuge the incubated centrifuge tube at 4000 rpm for 3 minutes, discard the upper culture medium, and wash twice with 1 ml of sterile water. Finally, add 200 μl of sterile water to the centrifuge tube containing the bacteria and mix well. Spread the mixture on methionine-deficient SC medium plates and incubate at 30°C for 2 days. Once single colonies have grown, screen to obtain recombinant bacteria 1.

[0058] Table 1 Primers and templates for constructing recombinant strain 1

[0059]

[0060] Example 2: The method for constructing recombinant bacteria 2 includes the following steps:

[0061] The glycerol transport protein CjFPS1 gene expression cassette, the glycerol dehydrogenase OpGDH gene expression cassette, the dihydroxyacetone (DHA) kinase DAK1 gene expression cassette, and the 3-hydroxy-3-methylglutaryl-CoA reductase HMGR gene expression cassette were introduced into recombinant strain 1 to obtain recombinant strain 2, which is a recombinant Saccharomyces cerevisiae that metabolizes glycerol to produce protopanaxadiol (PPD).

[0062] The glycerol transport protein CjFPS1 was derived from Cyberlindnera jadinii. After codon optimization by Saccharomyces cerevisiae from Wuhan Jinkairui Biotechnology Co., Ltd., the nucleotide sequence of the glycerol transport protein CjFPS1 gene was obtained as shown in SEQ ID NO.4.

[0063] The glycerol dehydrogenase OpGDH was derived from Ogataeapara polymorpha. After codon optimization by Saccharomyces cerevisiae from Wuhan Jinkairui Biotechnology Co., Ltd., the nucleotide sequence of the glycerol dehydrogenase OpGDH gene was obtained as shown in SEQ ID NO.5.

[0064] The dihydroxyacetone (DHA) kinase DAK1 is derived from Saccharomyces cerevisiae BY4741, and the nucleotide sequence of the DAK1 gene is shown in SEQ ID NO.6;

[0065] The 3-hydroxy-3-methylglutaryl-CoA reductase HMGR was derived from *Silicibacter pomeroyi*. After codon optimization by Wuhan Jinkairui Biotechnology Co., Ltd., the nucleotide sequence of the 3-hydroxy-3-methylglutaryl-CoA reductase HMGR gene was obtained, as shown in SEQ ID NO.7.

[0066] (1) Gene expression cassette construction

[0067] Using the primer pairs and templates in Table 2, GUT1U and P were amplified by PCR. TEF2 DAK1, P TDH3 、CjFPS1、T CYC1 P PGK1 OpGDH, T ADH3 P TDH3 HMGR, T ADH1 His, GUT1D fragments;

[0068] GUT1U, P TEF2 Fusing DAK1 fragments yields GUT1U-P. TEF2 -DAK1 gene expression cassette (DAK1 gene expression cassette, also known as fragment 4);

[0069] P TDH3 、CjFPS1、T CYC1 Fragment fusion yields P TDH3 -CjFPS1-T CYC1 Gene expression cassette (glycerol transport protein CjFPS1 gene expression cassette, also known as fragment 5);

[0070] P PGK1 OpGDH, T ADH3 Fragment fusion yields P PGK1 -OpGDH-T ADH3 Gene expression cassette (glycerol dehydrogenase OpGDH gene expression cassette, also known as fragment 6);

[0071] P TDH3 HMGR, TADH1 Fusing the His and GUT1D fragments yields P. TDH3 -HMGR-T ADH1 -His-GUT1D gene expression cassette (3-hydroxy-3-methylglutaryl-CoA reductase HMGR gene expression cassette, also known as fragment 7).

[0072] (2) Conversion of brewing yeast

[0073] Following the transformation method in step (2) of Example 1, the 200ng fragments 4, 200ng fragments 5, 200ng fragments 6 and 200ng fragments 7 obtained in step (1) were transformed into recombinant bacteria 1. The recombinant bacteria were plated on histidine-deficient SC medium plates for screening to obtain recombinant bacteria 2.

[0074] Table 2 Primers and templates for constructing recombinant bacteria 2

[0075]

[0076]

[0077] Example 3: The method for constructing recombinant bacteria 3 includes the following steps:

[0078] The citrate lyase AnACLa gene expression cassette and the citrate lyase AnACLb gene expression cassette, the citrate transporter YHM2 gene expression cassette and the citrate transporter CTP1 gene expression cassette were introduced into recombinant strain 2 to obtain recombinant strain 3, which is the second type of recombinant Saccharomyces cerevisiae that metabolizes glycerol to produce protopanaxadiol.

[0079] The citrate lyases AnACLa and AnACLb are derived from *Aspergillus nidulans*. After codon optimization by Beijing Qingke Biotechnology Co., Ltd., the nucleotide sequence of the citrate lyase AnACLa gene is shown in SEQ ID NO. 8, and the nucleotide sequence of the citrate lyase AnACLb gene is shown in SEQ ID NO. 9. The citrate transporter YHM2 is derived from *Saccharomyces cerevisiae* BY4741, and its nucleotide sequence is shown in SEQ ID NO. 10. The citrate transporter CTP1 is derived from *Saccharomyces cerevisiae* BY4741, and its nucleotide sequence is shown in SEQ ID NO. 11.

[0080] (1) Expression box construction

[0081] Using the primer pairs and templates in Table 2, PYC1U and P were amplified by PCR. ENO1 YHM2, T CPS1 P SPG1 CTP1, T ERG20 PTMA10 、AnACLa、T PRM9 P TAR1 AnACLb, T SPG5 Leu, PYC1D fragments;

[0082] PYC1U, P ENO1 YHM2, T CPS1 Fusion yields PYC1U-P ENO1 -YHM2-T CPS1 Gene expression cassette (citrate transporter YHM2 gene expression cassette, also known as fragment 8),

[0083] P SPG1 CTP1, T ERG20 Fusion, resulting in P SPG1 -CTP1-T ERG20 Gene expression cassette (CTP1 citrate transporter gene expression cassette, also known as fragment 9),

[0084] P TMA10 、AnACLa、T PRM9 Fusion, resulting in P TMA10 -AnACLa-T PRM9 Gene expression cassette (citrate lyase AnACLa gene expression cassette, also known as fragment 10),

[0085] P TAR1 AnACLb, T SPG5 By fusing Leu and PYC1D, we obtain P. TAR1 -AnACLb-T SPG5 -Leu-PYC1D gene expression cassette (citrate lyase AnACLb gene expression cassette, also known as fragment 11).

[0086] (2) Conversion of brewing yeast

[0087] Following the transformation method in step (2) of Example 1, the 200ng fragments 8, 200ng fragments 9, 200ng fragments 10 and 200ng fragments 11 obtained in step (1) of this Example were transformed into recombinant bacteria 2. The recombinant bacteria were plated on leucine-deficient SC medium plates for screening to obtain recombinant bacteria 3.

[0088] Table 3 Primers and templates for constructing recombinant bacteria 3

[0089]

[0090]

[0091] Example 4: Fermentation process and extraction and determination of metabolites of recombinant bacteria

[0092] (1) Fermentation process and metabolite extraction of recombinant bacteria

[0093] Microbial fermentation was carried out using the constructed recombinant bacteria, and the yield of protopanaxadiol (PPD) in the metabolites was detected.

[0094] A single colony of recombinant strain 1 was inoculated into 3 mL of YPD liquid medium and cultured overnight at 30°C and 220 rpm. An appropriate amount of seed culture was inoculated into a shake flask containing 30 mL of YPD liquid medium, and the initial OD of the fermentation broth was set to 0.1. Fermentation was carried out for 4 days.

[0095] A single colony of recombinant strain 2 was inoculated into 3 mL of YPD liquid medium and cultured overnight at 30°C and 220 rpm. An appropriate amount of seed culture was inoculated into a shake flask containing 30 mL of YPG liquid medium, and the initial OD of the fermentation broth was set to 0.1. Fermentation was carried out for 4 days.

[0096] Three single colonies of the recombinant bacteria were inoculated into 3 mL of YPD liquid medium and cultured overnight at 30°C and 220 rpm. An appropriate amount of seed culture was inoculated into a shake flask containing 30 mL of YPG liquid medium to make the initial OD of the fermentation broth 0.1, and fermentation was carried out for 4 days.

[0097] After fermentation, 500 μL of n-butanol and an amount of quartz sand equal to the volume of the bacterial cells were added to 1 mL of fermentation broth. The mixture was vortexed for 30 minutes, centrifuged at 12,000 rpm for 10 minutes, and the upper organic phase was collected. After filtration through a 0.22 μm filter membrane, the mixture was used for HPLC-MS analysis.

[0098] (2) HPLC-MS determination of metabolites

[0099] Chromatographic conditions: HPLC analysis was performed using a Hypersil C18 column (4.6 mm × 250 mm, 5 μm; Elite Analytical Instruments Co., Ltd., Dalian, China). The detection wavelength was 203 nm, the column temperature was 50 °C, and the mobile phase was methanol:acetonitrile = 4:6. A standard curve for protopanaxadiol (PPD) concentration was prepared for quantitative analysis of the yield of protopanaxadiol (PPD) in the strain's metabolites.

[0100] Mass spectrometry conditions: signal source type, ESI; ion polarity, positive; all spectra were obtained in the 50 / 1200 m / z range; dry gas flow rate, 6.0 L / min; drying temperature, 180 °C; nebulizer pressure, 0.8 bar; probe voltage, +4.5 kV.

[0101] (3) Measurement results

[0102] The PPD yields of ginsenoside diol in recombinant strains 1, 2, and 3 were 15.6 mg / L, 124 mg / L, and 220 mg / L, respectively.

[0103] (4) Culture medium used in the examples

[0104] YPD liquid medium: final glucose concentration 25 g / L, final yeast extract concentration 10 g / L, final peptone concentration 20 g / L, prepared with distilled water. YPD solid medium is YPD liquid medium with 20 g / L agar powder added.

[0105] YPG liquid medium: final concentration of glycerol 25 g / L, final concentration of yeast extract 10 g / L, final concentration of peptone 20 g / L, prepared with distilled water.

[0106] SC medium: final glucose concentration of 20 g / L, final concentration of amino-free yeast nitrogen source (YNB) of 6.7 g / L, final concentration of amino acid mixture of 0.2 g / L, 20 g / L agar powder, prepared with distilled water. The missing amino acid mixture refers to the amino acid mixture with the corresponding components removed.

[0107] Amino acid mixture: glycine, 2.0g; alanine, 2.0g; methionine, 2.0g; lysine, 2.0g; arginine, 2.0g; serine, 2.0g; asparagine, 2.0g; aspartic acid, 2.0g; phenylalanine, 2.0g; cysteine, 2.0g; proline, 2.0g; tyrosine, 2.0g; glutamic acid, 2.0g; valine, 2.0g; threonine, 2.0g; isoleucine, 2.0g; inositol, 2.0g; glutamine, 2.0g; para-aminobenzoic acid, 0.2g; adenine, 0.5g; leucine, 10.0g; tryptophan, 2.0g; histidine, 2.0g; uracil, 2.0g.

Claims

1. A method for constructing a recombinant Saccharomyces cerevisiae strain that metabolizes glycerol to produce protopanaxadiol, characterized in that: Includes the following steps: (1) The dammarene diol synthase DS gene expression cassette, the PPD synthase and its reductase fusion protein PPDS-ATR1 gene expression cassette and the squalene oxidase ERG1 gene expression cassette were introduced into Saccharomyces cerevisiae BY4741 to obtain recombinant strain 1. The nucleotide sequence of the dammarene diol synthase DS gene is shown in SEQ ID NO.1; The nucleotide sequence of the PPD synthase and its reductase fusion protein PPDS-ATR1 gene is shown in SEQ ID NO.2; The nucleotide sequence of the squalene oxidase ERG1 gene is shown in SEQ ID NO.3; (2) The glycerol transport protein CjFPS1 gene expression cassette, glycerol dehydrogenase OpGDH gene expression cassette, dihydroxyacetone kinase DAK1 gene expression cassette and 3-hydroxy-3-methylglutaryl-CoA reductase HMGR gene expression cassette were introduced into recombinant strain 1 to obtain recombinant strain 2, that is, the recombinant Saccharomyces cerevisiae that metabolizes glycerol to produce protopanaxadiol. The nucleotide sequence of the glycerol transport protein CjFPS1 gene is shown in SEQ ID NO.4; The nucleotide sequence of the glycerol dehydrogenase OpGDH gene is shown in SEQ ID NO.5; The nucleotide sequence of the dihydroxyacetone kinase DAK1 gene is shown in SEQ ID NO. 6; The nucleotide sequence of the 3-hydroxy-3-methylglutaryl-CoA reductase HMGR gene is shown in SEQ ID NO.

7.

2. Another method for constructing a recombinant Saccharomyces cerevisiae strain that metabolizes glycerol to produce protopanaxadiol, characterized by: Includes the following steps: (1) The dammarene diol synthase DS gene expression cassette, the PPD synthase and its reductase fusion protein PPDS-ATR1 gene expression cassette and the squalene oxidase ERG1 gene expression cassette were introduced into Saccharomyces cerevisiae BY4741 to obtain recombinant strain 1. The nucleotide sequence of the dammarene diol synthase DS gene is shown in SEQ ID NO.1; The nucleotide sequence of the PPD synthase and its reductase fusion protein PPDS-ATR1 gene is shown in SEQ ID NO.2; The nucleotide sequence of the squalene oxidase ERG1 gene is shown in SEQ ID NO.3; (2) The glycerol transport protein CjFPS1 gene expression cassette, glycerol dehydrogenase OpGDH gene expression cassette, dihydroxyacetone kinase DAK1 gene expression cassette and 3-hydroxy-3-methylglutaryl-CoA reductase HMGR gene expression cassette were introduced into recombinant strain 1 to obtain recombinant strain 2. The nucleotide sequence of the glycerol transport protein CjFPS1 gene is shown in SEQ ID NO.4; The nucleotide sequence of the glycerol dehydrogenase OpGDH gene is shown in SEQ ID NO.5; The nucleotide sequence of the dihydroxyacetone kinase DAK1 gene is shown in SEQ ID NO. 6; The nucleotide sequence of the 3-hydroxy-3-methylglutaryl-CoA reductase HMGR gene is shown in SEQ ID NO.

7. (3) Introduce the citrate lyase AnACLa gene expression cassette and the citrate lyase AnACLb gene expression cassette, the citrate transporter YHM2 gene expression cassette and the citrate transporter CTP1 gene expression cassette into recombinant strain 2 to obtain recombinant strain 3, which is another recombinant Saccharomyces cerevisiae that metabolizes glycerol to produce protopanaxadiol. The nucleotide sequence of the citrate lyase AnACLa gene is shown in SEQ ID NO. 8; The nucleotide sequence of the citrate lyase AnACLb gene is shown in SEQ ID NO. 9; The nucleotide sequence of the citrate transporter YHM2 gene is shown in SEQ ID NO.10; The nucleotide sequence of the citrate transporter CTP1 gene is shown in SEQ ID NO.

11.

3. The recombinant Saccharomyces cerevisiae for producing protopanaxadiol by the method of claim 1 or 2.

4. The application of the recombinant Saccharomyces cerevisiae for producing protopanaxadiol according to claim 3.

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