CN114807211B

CN114807211B - Recombinant saccharomyces cerevisiae for producing ginsenoside CK by metabolizing glycerol and construction method

Info

Publication number: CN114807211B
Application number: CN202210519519.5A
Authority: CN
Inventors: 卢文玉; 张传波; 田锦平; 刘瑞霞
Original assignee: Tianjin University
Current assignee: Tianjin University
Priority date: 2022-05-13
Filing date: 2022-05-13
Publication date: 2023-06-27
Anticipated expiration: 2042-05-13
Also published as: CN114807211A

Abstract

The invention discloses a recombinant saccharomyces cerevisiae for producing ginsenoside CK by metabolizing glycerol and a construction method thereof, wherein the construction method comprises the following steps: (1) Introducing a glycosyltransferase UGT1 gene expression cassette, a PGM2 gene expression cassette of glucose phosphomutase and a UDP glucose pyrophosphorylase UGP1 gene expression cassette into Saccharomyces cerevisiae capable of synthesizing protopanaxadiol to obtain recombinant bacteria 1; (2) Introducing a glycerol transporter CjFPS1 gene expression cassette, a glycerol dehydrogenase OpGDH gene expression cassette and a dihydroxyacetone kinase DAK1 gene expression cassette into recombinant bacterium 1 to obtain recombinant bacterium 2; (3) Introducing a 3-hydroxy-3-methylglutaryl-CoA reductase HMGr gene expression cassette into the recombinant bacterium 2 to obtain a recombinant bacterium 3; experiments prove that the yields of the ginsenoside CK of the recombinant bacteria 1, 2 and 3 are 109.84mg/L,152.12mg/L and 330.94mg/L in sequence.

Description

Recombinant saccharomyces cerevisiae for producing ginsenoside CK by metabolizing glycerol and construction method

Technical Field

The invention relates to the technical field of biology, in particular to recombinant saccharomyces cerevisiae for producing ginsenoside CK by metabolizing glycerol, and a construction method and application thereof.

Background

Ginsenoside CK (CK for short) belongs to protopanoxadiol type saponin, and has biological properties of anticancer, antiallergic, antiaging, and antidiabetic. CK is extremely low in ginseng, and the ginsenoside Rb1, rd, rg2 and the like are mainly obtained by hydrolyzing the ginsenoside through mild acid hydrolysis alkali treatment, microbial transformation and enzymatic transformation, so that the single cost is still high. In recent years, research on synthetic biology has been advanced to enable microorganisms to synthesize animal and plant-derived compounds. Although researchers constructed strains capable of producing ginsenoside CK, the yield still remained to be improved.

In the prior art, the ginsenoside is produced by taking glucose as a substrate, and the glycerol has higher reduction degree compared with the glucose, is a main product in the petrochemical industry and has the characteristic of low cost, so that the synthesis of the ginsenoside CK by taking the glycerol as the substrate has potential in theory. However, no studies have been made to produce terpenoid natural products, particularly triterpenoid saponins, by modifying glycerol metabolism.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a recombinant saccharomyces cerevisiae for producing ginsenoside CK by metabolizing glycerol.

The second object of the invention is to provide a construction method of recombinant saccharomyces cerevisiae for producing ginsenoside CK by metabolizing glycerol.

The third object of the invention is to provide an application of recombinant saccharomyces cerevisiae for producing ginsenoside CK by metabolism of glycerol in the production of ginsenoside CK by fermentation.

The fourth object of the present invention is to provide another recombinant Saccharomyces cerevisiae for metabolizing glycerol to produce ginsenoside CK.

The fifth object of the invention is to provide another construction method of recombinant saccharomyces cerevisiae for producing ginsenoside CK by metabolizing glycerol.

The sixth object of the invention is to provide an application of another recombinant saccharomyces cerevisiae for producing ginsenoside CK by metabolism of glycerol in the production of ginsenoside CK by fermentation.

The technical scheme of the invention is summarized as follows:

the construction method of the recombinant saccharomyces cerevisiae for producing the ginsenoside CK by metabolizing glycerol comprises the following steps:

(1) Introducing a glycosyltransferase UGT1 gene expression cassette, a PGM2 gene expression cassette of glucose phosphomutase and a UDP glucose pyrophosphorylase UGP1 gene expression cassette into Saccharomyces cerevisiae capable of synthesizing protopanaxadiol to obtain recombinant bacteria 1;

the nucleotide sequence of the glycosyltransferase UGT1 gene is shown in SEQ ID NO. 1;

the nucleotide sequence of the glucose phosphomutase PGM2 gene is shown in SEQ ID NO. 2;

the nucleotide sequence of the UDP-glucose pyrophosphorylase UGP1 gene is shown in SEQ ID NO. 3;

(2) Introducing a glycerol transporter CjFPS1 gene expression cassette, a glycerol dehydrogenase OpGDH gene expression cassette and a dihydroxyacetone kinase DAK1 gene expression cassette into recombinant bacterium 1 to obtain recombinant bacterium 2;

the nucleotide sequence of the glycerol transporter 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.

Recombinant saccharomyces cerevisiae for producing ginsenoside CK by metabolizing glycerol constructed by the method.

The application of the recombinant saccharomyces cerevisiae for producing the ginsenoside CK by metabolizing glycerol to producing the ginsenoside CK is provided.

Another construction method of recombinant saccharomyces cerevisiae for producing ginsenoside CK by metabolizing glycerol comprises the following steps:

(3) Introducing a 3-hydroxy-3-methylglutaryl-CoA reductase HMGr gene expression cassette into the recombinant bacterium 2 to obtain a recombinant bacterium 3;

the nucleotide sequence of the HMGr gene of the 3-hydroxy-3-methylglutaryl-CoA reductase is shown in SEQ ID NO. 7.

The invention has the advantages that:

the invention successfully constructs the recombinant saccharomyces cerevisiae for producing the ginsenoside CK by metabolizing glycerol. Experiments prove that the recombinant saccharomyces cerevisiae for producing the ginsenoside CK by metabolizing glycerol is fermented to obtain the ginsenoside CK, and the yields of the ginsenoside CK of the recombinant bacteria 1, 2 and 3 are 109.84mg/L,152.12mg/L and 330.94mg/L in sequence.

Drawings

FIG. 1A is a diagram showing the yield of ginsenoside CK synthesized by fermentation of recombinant Saccharomyces cerevisiae, which metabolizes glycerol to ginsenoside CK. Wherein the recombinant bacterium 1 is fermented by YPD liquid culture medium, and the recombinant bacterium 2 and the recombinant bacterium 3 are fermented by YPG liquid culture medium.

Detailed Description

The invention is further illustrated by the following examples.

Saccharomyces cerevisiae capable of synthesizing protopanoxadiol is used as an initial strain of the invention, and the initial strain selected by the invention is from Chinese patent, patent number 201410735927.X, and the invention name is: a fusion protein capable of improving the conversion efficiency of dammarenediol, a construction method and application thereof, namely a yeast strain W3a for synthesizing protopanaxadiol obtained in the embodiment 3, is called as 'Saccharomyces cerevisiae W3 a' for short in the invention, but the invention is not limited.

Other recombinant bacteria including the coding sequence of dammarenediol synthase DS, protopanaxadiol synthase PPDS, and cytochrome P450 enzyme reductase AtCPR1 may also be used in the present invention.

The experimental methods used in the examples below are conventional methods unless otherwise specified.

Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

Saccharomyces cerevisiae ATCC208352, saccharomyces cerevisiae ATCC4040002, time purchased, 2016.6, website: https:// www.atcc.org /)

EXAMPLE 1 construction method of recombinant Saccharomyces cerevisiae 1 (recombinant bacterium 1)

And introducing a glycosyltransferase UGT1 gene expression cassette, a glucose phosphomutase PGM2 gene expression cassette and a UDP glucose pyrophosphorylase UGP1 gene expression cassette into Saccharomyces cerevisiae W3a to obtain recombinant bacteria 1.

The glycosyltransferase UGT1 is derived from Ginseng (Ginseng), and the nucleotide sequence of the glycosyltransferase UGT1 gene is shown in SEQ ID NO.1 after the codon optimization of saccharomyces cerevisiae of WU Jin Kairui bioengineering limited company; the nucleotide sequence of the glucose phosphomutase PGM2 gene is derived from Saccharomyces cerevisiae and is shown in SEQ ID NO. 2; the nucleotide sequence of the UDP-glucose pyrophosphorylase UGP1 gene is derived from Saccharomyces cerevisiae and is shown in SEQ ID NO. 3.

(1) Expression cassette construction

As shown in Table 1, the HOL fragment was amplified by PCR using the Saccharomyces cerevisiae ATCC208352 genome as a template and HOL-F (SEQ ID NO. 8) and TEF1-HOL-R (SEQ ID NO. 9); PCR amplification of P using Saccharomyces cerevisiae ATCC208352 genome as template and HOL-TEF1-F (SEQ ID NO. 10) and PGM2-TEF1-R (SEQ ID NO. 11) _TEF1 Fragments; PCR amplification of PGM2-T using Saccharomyces cerevisiae ATCC208352 genome as template and TEF1-PGM2-F (SEQ ID NO. 12) and TDH3-PGM2T-R (SEQ ID NO. 13) _PGM2 Fragments; PCR amplification of P using the Saccharomyces cerevisiae ATCC208352 genome as a template with PGM2t-TDH3-F (SEQ ID NO. 14) and UGP1-TDH3-R (SEQ ID NO. 15) _TDH3 Fragments; PCR amplification of UGP1-T using Saccharomyces cerevisiae ATCC208352 genome as template and TDH3-UGP1-F (SEQ ID NO. 16) and PGK1-UGP1T-R (SEQ ID NO. 17) _UGP1 Fragments; PCR amplification of P using Saccharomyces cerevisiae ATCC208352 genome as template and UGP1t-PGK1-F (SEQ ID NO. 18) and UGT1-PGK1-R (SEQ ID NO. 19) _PGK1 Fragments; PCR amplification of UGT1 fragment using PGK1-UGT1-F (SEQ ID NO. 20) and ADH1-UGT1-R (SEQ ID NO. 21) with the nucleic acid sequence of UGT1 gene (SEQ ID NO. 1) as a template; PCR amplification of T using Saccharomyces cerevisiae ATCC208352 genome as template and UGT1-ADH1-F (SEQ ID NO. 22) and ADE-ADH1-R (SEQ ID NO. 23) _ADH1 Fragments; ADE fragments were PCR amplified using the Saccharomyces cerevisiae ATCC4040002 genome as a template and ADH1-ADE-F (SEQ ID NO. 24) and HOR-ADE-R (SEQ ID NO. 25); the HOR fragment was PCR amplified using the Saccharomyces cerevisiae ATCC208352 genome as a template and ADE-HOR-F (SEQ ID NO. 26) and HOR-R (SEQ ID NO. 27). Amplifying the obtained HOL, P _TEF1 With PGM2-T _PGM2 Fragment fusion to obtain HOL-P _TEF1 -PGM2-T _PGM2 A gene expression cassette; p obtained by amplification _TDH3 With UGP1-T _UGP1 Fragment fusion to obtain P _TDH3 -UGP1-T _UGP1 A gene expression cassette; p obtained by amplification _PGK1 ，UGT1，T _ADH1 ADE and HOR fragments are fused to obtain P _PGK1 -UGT1-T _ADH1 ADE-HOR gene expression cassette.

(2) Saccharomyces cerevisiae transformation

Inoculating single colony of Saccharomyces cerevisiae W3a into 3ml YPD liquid culture medium, and culturing in shaking table at 30deg.C and 220rpm for 12 hr; transferring the cultured yeast seed liquid into a new 3ml YPD liquid culture medium, and culturing for 5 hours at the temperature of 30 ℃ and the rotating speed of 220 rpm; taking 1ml of bacterial liquid in a 1.5ml centrifuge tube which is sterilized in advance, centrifuging for 3min by a 4000rpm centrifuge, discarding the supernatant, and collecting bacterial cells; 1ml of sterile water was washed once, centrifuged at 4000rpm for 3min, and the supernatant was discarded to collect the cells. Then 1ml of 100mM LiAc aqueous solution was added to the cells and mixed well, and the mixture was allowed to stand at room temperature for 5 minutes. The cells after the completion of the standing were centrifuged at 4000rpm for 3min, and the LiAc liquid was removed by a pipette. Salmon sperm DNA (Solarbio, 10 mg/ml) was boiled in boiling water for 5min and then rapidly placed on prepared ice for cooling.

Sequentially adding 120 μl PEG3350 (50 g PEG3350/100ml water), 18 μl 1.0M LiAc aqueous solution, 5 μl salmon sperm DNA cooled by boiling with boiled water for 5min, 200ng HOL-P into a bacterial precipitation centrifuge tube _TEF1 -PGM2-T _PGM2 Fragments, 200ng P _TDH3 -UGP1-T _UGP1 Fragments, 200ng P _PGK1 -UGT1-T _ADH1 ADE-HOR fragment, final volume was made up to 180. Mu.l with sterile water. Then gently blow with a pipette for 1min to mix evenly; placing the evenly mixed centrifuge tube in a water bath kettle with the temperature of 42 ℃ for heat shock for 30min, centrifuging for 3min by a 4000rpm centrifuge, and removing the supernatant; continuously adding clean 1ml YPD liquid culture medium, and carrying out resuscitating culture for 2h at 30 ℃ by a shaking table at 220 rpm; centrifuging the resuscitated centrifuge tube at 4000rpm for 3min, discarding the upper culture medium, and washing twice with 1ml of sterile water; finally, 200 mu l of sterile water is added into a centrifuge tube containing thalli, the mixture is uniformly mixed, the mixture is coated on an SC culture medium flat plate lacking adenine, the mixture is placed in a constant temperature incubator at 30 ℃ for 2 days, and after single colony grows out, recombinant bacterium 1 is obtained by screening.

TABLE 1 recombinant bacterium 1 construction primer and template

EXAMPLE 2 construction method of recombinant Saccharomyces cerevisiae 2 (recombinant 2)

And (3) introducing a glycerol transporter CjFPS1 gene expression cassette, a glycerol dehydrogenase OpGDH gene expression cassette and a Dihydroxyacetone (DHA) kinase DAK1 gene expression cassette into the recombinant bacterium 1 to obtain recombinant bacterium 2.

The glycerol transporter CjFPS1 is derived from Cyberlindnera jadinii, and after the codon optimization of saccharomyces cerevisiae of Wohan Jin Kairui bioengineering Co., ltd., the nucleic acid sequence of the obtained glycerol transporter CjFPS1 gene is shown by SEQ ID NO. 4; the glycerol dehydrogenase OpGDH is derived from Ogataeapara polymorpha, and the nucleotide sequence of the obtained glycerol dehydrogenase OpGDH gene is shown in SEQ ID NO.5 after the codon optimization of Saccharomyces cerevisiae of WU Jin Kairui bioengineering limited company; the Dihydroxyacetone (DHA) kinase DAK1 is derived from Saccharomyces cerevisiae, and the nucleic acid sequence is shown in SEQ ID NO. 6; .

(1) Gene expression cassette construction

As shown in Table 2, the GUT1L fragment was PCR amplified using the Saccharomyces cerevisiae ATCC208352 genome as a template and GUT1L-F (SEQ ID NO. 28) and TDH3-GUT1L-R (SEQ ID NO. 29); PCR amplification of P using Saccharomyces cerevisiae ATCC208352 genome as template and GUT1L-TDH3-F (SEQ ID NO. 30) and CjFPS1-TDH3-R (SEQ ID NO. 31) _TDH3 Fragments; PCR amplification of the CjFPS1 fragment using the nucleic acid sequence of the CjFPS1 gene (SEQ ID NO. 4) as a template and TDH3-CjFPS1-F (SEQ ID NO. 32) and CYC1-CjFPS1-R (SEQ ID NO. 33); PCR amplification of T using Saccharomyces cerevisiae ATCC208352 genome as template and CjFPS1-CYC1-F (SEQ ID NO. 34) and PGK1-CYC1-R (SEQ ID NO. 35) _CYC1 Fragments; PCR amplification of P using Saccharomyces cerevisiae ATCC208352 genome as template and CYC1-PGK1-F (SEQ ID NO. 36) and OpGDH-PGK1-R (SEQ ID NO. 37) _PGK1 Fragments; PCR amplifying the OpGDH fragment using the nucleic acid sequence of the OpGDH gene (SEQ ID NO. 5) as a template and PGK1-OpGDH-F (SEQ ID NO. 38) and ADH3-OpGDH-R (SEQ ID NO. 39); PCR amplification of T using Saccharomyces cerevisiae ATCC208352 genome as template and OpGDH-ADH3-F (SEQ ID NO. 40) and TEF1-ADH3-R (SEQ ID NO. 41) _ADH3 Fragments; PCR amplification of P with ADH3-TEF1-F (SEQ ID NO. 42) and DAK1-TEF1-R (SEQ ID NO. 43) Using Saccharomyces cerevisiae ATCC208352 genome as template _TEF1 Fragments; saccharomyces cerevisiaeATCC208352 genome as template, and PCR amplification of DAK1-T Using TEF1-DAK1-F (SEQ ID NO. 44) and hyhMX6-DAK1T-R (SEQ ID NO. 45) _DAK1 Fragments; the hyhMX6 fragment was PCR amplified using the plasmid pFA6 a-link-yECitrarine-Hygro (purchased from Addgene, http:// www.addgene.org /) as template and DAK1t-hyhMX6-F (SEQ ID NO. 46) and GUT1R-hyhMX6-R (SEQ ID NO. 47); the GUT1R fragment was PCR amplified using Saccharomyces cerevisiae ATCC208352 genome as a template and hyhMX6-GUT1R-F (SEQ ID NO. 48) and GUT1R-R (SEQ ID NO. 49). Amplifying the obtained GUT1L, P _TDH3 ，CjFPS1，T _cyc1 Fragment fusion to obtain GUT1L-P _TDH3 -CjFPS1-T _cyc1 A gene expression cassette; p obtained by amplification _PGK1 ，OpGDH，T _ADH3 Fragment fusion to obtain P _PGK1 -OpGDH-T _ADH3 A gene expression cassette; p obtained by amplification _TEF1 ，DAK1-T _DAK1 Fusion of hyhMX6 with GUT1R fragment to obtain P _TEF1 -DAK1-T _DAK1 -hyhMX6-GUT1R gene expression cassette.

(2) Saccharomyces cerevisiae transformation

200ng of GUT1L-P obtained in step (1) was subjected to the transformation method according to step (2) of example 1 _TDH3 -CjFPS1-T _cyc1 、200ng P _PGK1 -OpGDH-T _ADH3 、200ng P _TEF1 The DAK1-DAK1t-hyhMX6-GUT1R gene expression cassette is transformed into recombinant bacterium 1, and the recombinant bacterium is coated on a YPD plate containing 500mg/L hygromycin for screening to obtain recombinant bacterium 2.

TABLE 2 recombinant bacterium 2 construction primer and template

EXAMPLE 3 construction method of recombinant Saccharomyces cerevisiae 3 (recombinant bacterium 3)

Introducing a 3-hydroxy-3-methylglutaryl-CoA reductase HMGr gene expression cassette into the recombinant bacterium 2 to obtain a recombinant bacterium 3;

the 3-hydroxy-3-methylglutaryl-CoA reductase HMGr is derived from Silicibacter pomeroyi, and the nucleic acid sequence of the obtained 3-hydroxy-3-methylglutaryl-CoA reductase HMGr gene is shown in SEQ ID NO.7 after the codon optimization of Saccharomyces cerevisiae of Wohan Jin Kairui bioengineering Co.

(1) Expression cassette construction

As shown in Table 3, the MET17L fragment was PCR amplified using the Saccharomyces cerevisiae ATCC208352 genome as a template and MET17L-F (SEQ ID NO. 50) and Bleo-MET17L-R (SEQ ID NO. 51); the Bleo fragment was PCR amplified using the pSH65 plasmid (purchased from Wuhan vast Biotechnology Co., ltd.) as a template and MET17L-Bleo-F (SEQ ID NO. 52) and TDH3-Bleo-R (SEQ ID NO. 53); PCR amplification of P using Saccharomyces cerevisiae ATCC208352 genome as template and Bleo-TDH3-F (SEQ ID NO. 54) and HMGr-TDH3-R (SEQ ID NO. 55) _TDH3 Fragments; PCR amplification of HMGr fragment using the nucleic acid sequence of the 3-hydroxy-3-methylglutaryl-CoA reductase HMGr gene (SEQ ID NO. 7) as template and TDH3-HMGr-F (SEQ ID NO. 56) and ADH1-HMGr-R (SEQ ID NO. 57); PCR amplification of T using Saccharomyces cerevisiae ATCC208352 genome as template, HMGr-ADH1-F (SEQ ID NO. 58) and MET17R-ADH1-R (SEQ ID NO. 59) _ADH1 Fragments; the MET17R fragment was PCR amplified using the Saccharomyces cerevisiae ATCC208352 genome as a template and ADH1-MET17R-F (SEQ ID NO. 60) and MET17R-R (SEQ ID NO. 61). MET17L, bleo, P obtained by amplification _TDH3 ，HMGr，T _ADH1 Fusion of the MET17R fragment to obtain MET17L-Bleo-P _TDH3 -HMGr-T _ADH1 -MET17R gene expression cassette.

(2) Saccharomyces cerevisiae transformation

200ng of MET17L-Bleo-P obtained in step (1) of this example was subjected to the transformation method of step (2) of example 1 _TDH3 -HMGr-T _ADH1 The MET17R gene expression cassette is transformed into recombinant bacterium 2, and the recombinant bacterium 3 is obtained by screening with YPD plates added with 300mg/L bleomycin.

TABLE 3 recombinant bacterium 3 construction primer and template

EXAMPLE 4 fermentation Process of recombinant bacteria and extraction and determination of metabolites

(1) Fermentation process and metabolite extraction of recombinant bacteria

And (3) carrying out microbial fermentation by using each constructed recombinant bacterium, and detecting the yield of ginsenoside CK in the metabolite.

Recombinant strain 1 was inoculated into 2mL of YPD liquid medium at 30℃and 200rpm overnight for cultivation. And (3) inoculating a proper amount of seed liquid into a shake flask containing 50mL of YPD liquid culture medium, so that the initial OD of the fermentation liquid is 0.1, and fermenting for 4 days.

Recombinant 2 single colonies were inoculated into 2mL of YPD liquid medium at 30℃and 200rpm overnight for cultivation. And (3) inoculating a proper amount of seed liquid into a shake flask containing 50mL of YPG liquid culture medium, enabling the initial OD of the fermentation liquid to be 0.1, and fermenting for 4 days.

Recombinant 3 single colonies were inoculated into 2mL YPD liquid medium at 30℃and 200rpm overnight for cultivation. And (3) inoculating a proper amount of seed liquid into a shake flask containing 50mL of YPG liquid culture medium, enabling the initial OD of the fermentation liquid to be 0.1, and fermenting for 4 days.

After the fermentation, 400. Mu.L of n-butanol and quartz sand corresponding to the volume of the cells were added to 1mL of the fermentation broth, respectively, followed by shaking by vortexing for 30 minutes and centrifuging at 12000rpm for 10 minutes, and the upper organic phase was collected and filtered with a 0.22 μm filter membrane for HPLC-MS analysis.

(2) HPLC-MS determination of metabolites

Chromatographic conditions: HPLC analysis was performed using a Hypersil C18 column (4.6mm.times.250 mm,5 μm; elite Analytical Instruments Co., ltd., dai.C. China) with a detection wavelength of 203nm and a column temperature of 50℃and a mobile phase of 80% acetonitrile. A standard curve of the concentration of ginsenoside CK is prepared for quantitatively analyzing the yield of ginsenoside CK in the strain metabolite.

Mass spectrometry conditions: signal source type, ESI; ion polarity, positive; all spectra were obtained in the m/z range of 50/1200; a dry gas flow of 6.0L/min; the drying temperature is 180 ℃; the atomizer pressure was 0.8bar; probe voltage +4.5kV

(3) Measurement results

The yields of the ginsenoside CK in the recombinant bacteria 1, 2 and 3 are 109.84mg/L,152.12mg/L and 330.94mg/L in sequence. .

(4) The medium used in the examples

YPD liquid medium: the final concentration of glucose is 25g/L, the final concentration of yeast extract powder is 10g/L, and the final concentration of peptone is 20g/L, and the yeast extract powder is prepared by distilled water. YPD solid medium was prepared by adding 20g/L agar powder to YPD liquid medium.

YPG liquid medium: the final concentration of glycerin is 25.6g/L, the final concentration of yeast extract powder is 10g/L, and the final concentration of peptone is 20g/L, and the yeast extract powder is prepared by distilled water.

SC medium: the final concentration of glucose is 20g/L, the final concentration of nitrogen source (YNB) of the non-amino yeast is 6.7g/L, the final concentration of the amino acid mixture is 0.2g/L, the agar powder of 20g/L is prepared by distilled water, and the deletion of the amino acid mixture refers to the removal of the corresponding components in the amino acid mixture.

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; serine, 2.0g; isoleucine, 2.0g; inositol, 2.0g; 2.0g of glutamine; 0.2g of para aminobenzoic acid; adenine, 0.5g; leucine 10g; methionine, 2g; tryptophan, 2g; histidine, 2g; uracil, 2g.

Sequence listing

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atacagaatg aattctgggc aaagtacggc cgtactttct tcactcgtta tgattttgaa 1320

aaagttgaaa cagaaaaagc taacaagatt gtcgatcaat tgagagcata tgttaccaaa 1380

tcgggtgttg ttaattccgc cttcccagcc gatgagtctc ttaaggtcac cgattgtggt 1440

gatttttcat acacagattt ggacggttct gtttctgacc atcaaggttt atatgtcaag 1500

ctttccaatg gtgcaagatt cgttctaaga ttgtcaggta caggttcttc aggtgctacc 1560

attagattgt acattgaaaa atactgcgat gataaatcac aataccaaaa gacagctgaa 1620

gaatacttga agccggtgaa cagtttattc ctggcatcaa ttattaactc ggtcatcaag 1680

ttcttgaact ttaaacaagt tttaggaact gaagaaccaa cggttcgtac ttaaaacgaa 1740

tgatttacta atggcttaat gattttcacc tttttcaatg aatattaacg gtaaagaaga 1800

aaatttcaat tttttgaaca catactttat atacttaata gatccatatt tcgacatatt 1860

agcaaacgat tgcataggtt tctgagtctt tttttttttt ttttcataag gaggagaata 1920

ttttggttaa tcgcagtatc ttcttcataa gtgctgtttc taattatatc taattcacga 1980

atttttccca aattagcgta tccccgaatt cagattacct accccgagtt ttttattata 2040

tttccctcga gaaatctgta aaatggccgt catccttaga tttataaata aaatgataaa 2100

attcagccaa agtgctccta aaccagaatt gttcaactgg gt 2142

<210> 3

<211> 1881

<212> DNA

<213> Saccharomyces cerevisiae (Saccharomyces cerevisiae)

<400> 3

atgtccacta agaagcacac caaaacacat tccacttatg cattcgagag caacacaaac 60

agcgttgctg cctcacaaat gagaaacgcc ttaaacaagt tggcggactc tagtaaactt 120

gacgatgctg ctcgcgctaa gtttgagaac gaactggatt cgtttttcac gcttttcagg 180

agatatttgg tagagaagtc ttctagaacc accttggaat gggacaagat caagtctccc 240

aacccggatg aagtggttaa gtatgaaatt atttctcagc agcccgagaa tgtctcaaac 300

ctttccaaat tggctgtttt gaagttgaac ggtgggctgg gtacctccat gggctgcgtt 360

ggccctaaat ctgttattga agtgagagag ggaaacacct ttttggattt gtctgttcgt 420

caaattgaat acttgaacag acagtacgat agcgacgtgc cattgttatt gatgaattct 480

ttcaacactg acaaggatac ggaacacttg attaagaagt attccgctaa cagaatcaga 540

atcagatctt tcaatcaatc caggttccca agagtctaca aggattcttt attgcctgtc 600

cccaccgaat acgattctcc actggatgct tggtatccac caggtcacgg tgatttgttt 660

gaatctttac acgtatctgg tgaactggat gccttaattg cccaaggaag agaaatatta 720

tttgtttcta acggtgacaa cttgggtgct accgtcgact taaaaatttt aaaccacatg 780

atcgagactg gtgccgaata tataatggaa ttgactgata agaccagagc cgatgttaaa 840

ggtggtactt tgatttctta cgatggtcaa gtccgtttat tggaagtcgc ccaagttcca 900

aaagaacaca ttgacgaatt caaaaatatc agaaagttta ccaacttcaa cacgaataac 960

ttatggatca atctgaaagc agtaaagagg ttgatcgaat cgagcaattt ggagatggaa 1020

atcattccaa accaaaaaac tataacaaga gacggtcatg aaattaatgt cttacaatta 1080

gaaaccgctt gtggtgctgc tatcaggcat tttgatggtg ctcacggtgt tgtcgttcca 1140

agatcaagat tcttgcctgt caagacctgt tccgatttgt tgctggttaa atcagatcta 1200

ttccgtctgg aacacggttc tttgaagtta gacccatccc gttttggtcc aaacccatta 1260

atcaagttgg gctcgcattt caaaaaggtt tctggtttta acgcaagaat ccctcacatc 1320

ccaaaaatcg tcgagctaga tcatttgacc atcactggta acgtcttttt aggtaaagat 1380

gtcactttga ggggtactgt catcatcgtt tgctccgacg gtcataaaat cgatattcca 1440

aacggctcca tattggaaaa tgttgtcgtt actggtaatt tgcaaatctt ggaacattga 1500

aggtgcggct tgcaatccct tttttacttt caattctccg ttaggttatt tattctacta 1560

cacgcagttt ttttttttca atgggttata tatctctaat atatacacat ttatatgttt 1620

agacttatac agaagcaaaa aaaaatgcaa aagaagctta gtgttcataa ttcttcactc 1680

acgtcatgtt atatatttgt taagcgtagc ttcagtatat atagagattt tcctttggcg 1740

gggtaagggt ctgtgaactt tagggaaata gaataaacgt aagttaaagg tgacaagctc 1800

ggatcgtcat ctttcaatta tcaccccaaa agatattgta aaaaagcaag ttcaatctcg 1860

tttgttacat ttgtcccgta t 1881

<210> 4

<211> 1035

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 4

atgactggtg aacttttggc ctctggtgag ggttgcagtt ctgacatcgt cttgaccaac 60

tctaccgccg ccccttcttc tggtttggaa aggagggcca acatcaccga ccatatcagt 120

tgcgagcact tcaccgcctt gcagagattt agatacggat ttagagagta cttcgccgag 180

ttcatcggaa ccatgatcct tgtcatgttc ggagacggtg tcgtcgctca atacaccttg 240

tctaagggat ctgccggtaa ttacaccacc atcgccttct cttgggctac tgccgtcttc 300

cttggatact gctgctctgc tggtatctct ggtgcccact tgaatccagc tgttaccttg 360

tctgccgcca ctttcagaca gttcccatgg aggaaggtct tgggttacat gttcgcccaa 420

ggtttgggtg gttacatcgg agcccttatc gtctacggta cttacatcca atctatcaac 480

aactactctg gtgaaggtca aaggatcgcc gttggagaca agagtaccgg aggtatcttc 540

tgcaccttcc cacagccata cttgaacacc aagggacaag ttaccagtga acttgtcacc 600

accgctttgc ttcagttcgg tatcttttct atgaccgatc cacataacgc cccacttggt 660

aacttcttcc catttggttt gtggattttg atctatggac ttggaaccag tttcggttac 720

cagaccggat acgccatcaa cttcgccaga gactttaccc caaggttggc tgctttgacc 780

gtcggttatg gtaccgagat gttcaccgcc tactaccact acttctgggt ccctatgatc 840

atccctttca tcggagcctt gcttggtgcc ttcatttacg acttcttcat ttaccaaggt 900

ttggactctc ctttgaacca gccaaagttc ggatacgaca ttagaaaaaa aaaaatccaa 960

gaattcgaat ttaaattgga aaattataaa cttgacttca atccagaagc ctctcacgga 1020

aggcttgatg cttaa 1035

<210> 5

<211> 1143

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 5

atgaaaggac ttttgtatta tggtaccaac gacattaggt actctgagac cgtcccagag 60

ccagaaatca agaaccctaa cgacgtcaaa attaaggtca gttactgcgg tatttgcggt 120

accgacttga aggagttcac ctacagtggt ggtccagttt tcttccctaa gcaaggtacc 180

aaagataaaa tcagtggata tgagttgcca ctttgccccg gtcacgagtt ctctggtacc 240

gttgttgagg tcggttctgg tgtcacctct gttaaacccg gtgatagagt tgctgtcgaa 300

gctacctctc actgctctga tagaagtagg tacaaggaca ccgttgccca agatttgggt 360

ttgtgcatgg cttgtcaaag tggttctcct aactgctgcg cctctttgag tttctgcggt 420

ttgggtggag cttctggtgg tttcgccgag tatgtcgttt acggtgagga ccacatggtc 480

aagttgccag acagtatccc agatgacatc ggtgccttgg tcgagccaat cagtgttgct 540

tggcatgctg tcgagagggc tagatttcag cccggtcaaa ccgccttggt tttgggaggt 600

ggtcctatcg gtttggctac catccttgcc cttcaaggtc accatgctgg taagatcgtc 660

tgcagtgaac cagctttgat cagaaggcaa ttcgccaagg agttgggtgc tgaagtcttc 720

gaccctagta cttgtgatga cgctaatgcc gtcttgaagg ctatggtccc agaaaacgaa 780

ggattccacg ccgccttcga ttgttctgga gtccctcaga ccttcaccac cagtatcgtt 840

gccactggtc catctggtat cgccgttaac gttgccgttt ggggagacca cccaatcggt 900

ttcatgccaa tgtctttgac ctaccaagag aagtacgcca ccggtagtat gtgctatacc 960

gtcaaggact tccaagaagt tgtcaaggcc ttggaggacg gtcttatctc tttggataag 1020

gctagaaaga tgatcaccgg taaggttcac ttgaaggatg gtgtcgagaa gggttttaaa 1080

caacttattg aacacaaaga aaataatgtt aaaattttgg ttaccccaaa cgaagttagt 1140

taa 1143

<210> 6

<211> 2025

<212> DNA

<213> Saccharomyces cerevisiae (Saccharomyces cerevisiae)

<400> 6

atgtccgcta aatcgtttga agtcacagat ccagtcaatt caagtctcaa agggtttgcc 60

cttgctaacc cctccattac gctggtccct gaagaaaaaa ttctcttcag aaagaccgat 120

tccgacaaga tcgcattaat ttctggtggt ggtagtggac atgaacctac acacgccggt 180

ttcattggta agggtatgtt gagtggcgcc gtggttggcg aaatttttgc atccccttca 240

acaaaacaga ttttaaatgc aatccgttta gtcaatgaaa atgcgtctgg cgttttattg 300

attgtgaaga actacacagg tgatgttttg cattttggtc tgtccgctga gagagcaaga 360

gccttgggta ttaactgccg cgttgctgtc ataggtgatg atgttgcagt tggcagagaa 420

aagggtggta tggttggtag aagagcattg gcaggtaccg ttttggttca taagattgta 480

ggtgccttcg cagaagaata ttctagtaag tatggcttag acggtacagc taaagtggct 540

aaaattatca acgacaattt ggtgaccatt ggatcttctt tagaccattg taaagttcct 600

ggcaggaaat tcgaaagtga attaaacgaa aaacaaatgg aattgggtat gggtattcat 660

aacgaacctg gtgtgaaagt tttagaccct attccttcta ccgaagactt gatctccaag 720

tatatgctac caaaactatt ggatccaaac gataaggata gagcttttgt aaagtttgat 780

gaagatgatg aagttgtctt gttagttaac aatctcggcg gtgtttctaa ttttgttatt 840

agttctatca cttccaaaac tacggatttc ttaaaggaaa attacaacat aaccccggtt 900

caaacaattg ctggcacatt gatgacctcc ttcaatggta atgggttcag tatcacatta 960

ctaaacgcca ctaaggctac aaaggctttg caatctgatt ttgaggagat caaatcagta 1020

ctagacttgt tgaacgcatt tacgaacgca ccgggctggc caattgcaga ttttgaaaag 1080

acttctgccc catctgttaa cgatgacttg ttacataatg aagtaacagc aaaggccgtc 1140

ggtacctatg actttgacaa gtttgctgag tggatgaaga gtggtgctga acaagttatc 1200

aagagcgaac cgcacattac ggaactagac aatcaagttg gtgatggtga ttgtggttac 1260

actttagtgg caggagttaa aggcatcacc gaaaaccttg acaagctgtc gaaggactca 1320

ttatctcagg cggttgccca aatttcagat ttcattgaag gctcaatggg aggtacttct 1380

ggtggtttat attctattct tttgtcgggt ttttcacacg gattaattca ggtttgtaaa 1440

tcaaaggatg aacccgtcac taaggaaatt gtggctaagt cactcggaat tgcattggat 1500

actttataca aatatacaaa ggcaaggaag ggatcatcca ccatgattga tgctttagaa 1560

ccattcgtta aagaatttac tgcatctaag gatttcaata aggcggtaaa agctgcagag 1620

gaaggtgcta aatccactgc tacattcgag gccaaatttg gcagagcttc gtatgtcggc 1680

gattcatctc aagtagaaga tcctggtgca gtaggcctat gtgagttttt gaagggggtt 1740

caaagcgcct tgtaagtact tggctcacga atacatatca agatacttat gatatatata 1800

tatagaaaaa gcttactttt cttggagtta ttgttattat catcgcgaag aacgattgta 1860

taacccggtt caacgcgaaa cgaatcgtta aactggtgaa atgttaacgc gagtgtcaga 1920

gatatacata gtatgagagt agctagatgt tgaatcggtg gtaagaacaa gaaggaaata 1980

ccgttaacaa gtgaaggaac aatctagtat tgttgaacaa gaatt 2025

<210> 7

<211> 1302

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 7

atgactggta agaccggtca tattgatggt ttgaactcca gaatcgaaaa gatgagagat 60

ttggatccag ctcaaagatt ggttagagtt gctgaagctg ctggtttgga accagaagct 120

atttctgctt tggctggtaa tggtgctttg ccattgtctt tggctaatgg tatgatcgaa 180

aacgtcatcg gtaagttcga attgccattg ggtgttgcta ctaatttcac tgttaacggt 240

agagactact tgattccaat ggctgttgaa gaaccatctg ttgttgctgc tgcttcttat 300

atggctagaa ttgctagaga aaacggtggt tttactgctc atggtactgc tccattgatg 360

agagcacaaa ttcaagttgt tggtttgggt gatccagaag gtgctagaca aagattattg 420

gctcataagg ctgcttttat ggaagctgca gatgctgttg atccagtttt ggttggttta 480

ggtggtggtt gtagagatat cgaagttcac gtttttagag atactccagt tggtgccatg 540

gttgtcttgc atttgatagt tgatgttaga gatgctatgg gtgctaacac tgttaatacc 600

atggctgaaa gattggctcc agaagttgaa agaattgctg gtggtactgt tagattgagg 660

atcttgtcta atttggccga tttgagatta gttagagcca gagttgaatt ggctcctgaa 720

actttgacta ctcaaggtta tgatggtgct gatgttgcta gaggtatggt tgaagcttgt 780

gctttagcta tcgttgatcc atatagagct gctactcata acaagggtat tatgaacggt 840

atcgatccag ttgttgttgc cactggtaat gattggagag ctattgaagc tggtgcacat 900

gcttatgctg ctagaactgg tcattatact tcattgacca gatgggaatt agccaacgat 960

ggtagattgg ttggtactat tgaattgcct ttggccttgg gtttagtagg tggtgctaca 1020

aaaactcatc caactgctag agctgcattg gctttgatgc aagttgaaac tgctactgaa 1080

ttggcacaag ttactgctgc tgtaggtttg gctcaaaaca tggctgctat tagagctttg 1140

gctactgaag gtattcaaag gggtcacatg actttacatg ctagaaacat tgctattatg 1200

gctggtgcta ctggtgcaga tattgataga gttactagag ttattgtcga agccggtgat 1260

gtttctgttg caagagctaa acaagttttg gagaacacct aa 1302

<210> 8

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 8

cttgatggta ggataataat aattc 25

<210> 9

<211> 36

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 9

gtgtggggga tcactgtaca taaattcagg gatttg 36

<210> 10

<211> 38

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 10

cctgaattta tgtacagtga tcccccacac accatagc 38

<210> 11

<211> 47

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 11

caatttgaaa tgacattttg taattaaaac ttagattaga ttgctat 47

<210> 12

<211> 37

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 12

tttaattaca aaatgtcatt tcaaattgaa acggttc 37

<210> 13

<211> 34

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 13

attcaacgct agtataccca gttgaacaat tctg 34

<210> 14

<211> 38

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 14

attgttcaac tgggtatact agcgttgaat gttagcgt 38

<210> 15

<211> 35

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 15

cttcttagtg gacattttgt ttgtttatgt gtgtt 35

<210> 16

<211> 32

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 16

taaacaaaca aaatgtccac taagaagcac ac 32

<210> 17

<211> 37

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 17

ataatatctg tgcgtatacg ggacaaatgt aacaaac 37

<210> 18

<211> 35

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 18

acatttgtcc cgtatacgca cagatattat aacat 35

<210> 19

<211> 47

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 19

caattctgac ttcattgttt tatatttgtt gtaaaaagta gataatt 47

<210> 20

<211> 43

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 20

aacaaatata aaacaatgaa gtcagaattg attttcttgc cag 43

<210> 21

<211> 46

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 21

cttatttttt ttataacttt acataatttc ttcgaataat ttagcc 46

<210> 22

<211> 36

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 22

cgaagaaatt atgtaaagtt ataaaaaaaa taagtg 36

<210> 23

<211> 32

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 23

tgttctagaa tccattcggc atgccggtag ag 32

<210> 24

<211> 43

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 24

ctaccggcat gccgaatgga ttctagaaca gttggtatat tag 43

<210> 25

<211> 34

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 25

gtacccatct gagtcgatct tatgtatgaa attc 34

<210> 26

<211> 36

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 26

ttcatacata agatcgactc agatgggtac gttgtg 36

<210> 27

<211> 28

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 27

agcatctagc acatactcga tttttacc 28

<210> 28

<211> 22

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 28

atgtttccct ctctcttccg ac 22

<210> 29

<211> 40

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 29

taacattcaa cgctagtatc aataccgtag ttaacaattg 40

<210> 30

<211> 36

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 30

gttaactacg gtattgatac tagcgttgaa tgttag 36

<210> 31

<211> 31

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 31

gttcaccagt cattttgttt gtttatgtgt g 31

<210> 32

<211> 32

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 32

ataaacaaac aaaatgactg gtgaactttt gg 32

<210> 33

<211> 31

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 33

ctaattacat gattaagcat caagccttcc g 31

<210> 34

<211> 36

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 34

aggcttgatg cttaatcatg taattagtta tgtcac 36

<210> 35

<211> 34

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 35

ataatatctg tgcgtgcaaa ttaaagcctt cgag 34

<210> 36

<211> 33

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 36

aaggctttaa tttgcacgca cagatattat aac 33

<210> 37

<211> 38

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 37

aagtcctttc attgttttat atttgttgta aaaagtag 38

<210> 38

<211> 39

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 38

aaatataaaa caatgaaagg acttttgtat tatggtacc 39

<210> 39

<211> 29

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 39

gtaacacgct attaactaac ttcgtttgg 29

<210> 40

<211> 29

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 40

gaagttagtt aatagcgtgt tacgcaccc 29

<210> 41

<211> 28

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 41

gtgtggggga tcactgatgt tttagttc 28

<210> 42

<211> 28

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 42

tcgaactaaa acatcagtga tcccccac 28

<210> 43

<211> 33

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 43

cgatttagcg gacattttgt aattaaaact tag 33

<210> 44

<211> 34

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 44

agttttaatt acaaaatgtc cgctaaatcg tttg 34

<210> 45

<211> 34

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 45

ctgggcctcc atgtcaattc ttgttcaaca atac 34

<210> 46

<211> 35

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 46

tgttgaacaa gaattgacat ggaggcccag aatac 35

<210> 47

<211> 37

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 47

ataggccact aaatgcagta tagcgaccag cattcac 37

<210> 48

<211> 37

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 48

ctggtcgcta tactgcattt agtggcctat tcgctcc 37

<210> 49

<211> 28

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 49

gttgttattg gaagttttct agaacctg 28

<210> 50

<211> 21

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 50

gccatcctca tgaaaactgt g 21

<210> 51

<211> 40

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 51

aggagtagaa acattgacct tttcgaattc ttctggattg 40

<210> 52

<211> 40

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 52

gaagaattcg aaaaggtcaa tgtttctact ccttttttac 40

<210> 53

<211> 35

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 53

cattcaacgc tagtatgcaa attaaagcct tcgag 35

<210> 54

<211> 40

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 54

gaaggcttta atttgcatac tagcgttgaa tgttagcgtc 40

<210> 55

<211> 40

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 55

ggtcttacca gtcattttgt ttgtttatgt gtgtttattc 40

<210> 56

<211> 43

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 56

cacacataaa caaacaaaat gactggtaag accggtcata ttg 43

<210> 57

<211> 33

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 57

tcataagaaa ttcgcttagg tgttctccaa aac 33

<210> 58

<211> 36

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 58

ttggagaaca cctaagcgaa tttcttatga tttatg 36

<210> 59

<211> 38

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 59

cttggtaaca ccagatgaga tcatgataca taaaagcg 38

<210> 60

<211> 46

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 60

cttttatgta tcatgatctc atctggtgtt accaaggact taattc 46

<210> 61

<211> 27

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 61

tcaacgatgg aattccaaca gctttac 27

Claims

1. The construction method of the recombinant saccharomyces cerevisiae for producing the ginsenoside CK by metabolizing glycerol is characterized by comprising the following steps:

2. The construction method of the recombinant saccharomyces cerevisiae for producing the ginsenoside CK by metabolizing glycerol is characterized by comprising the following steps:

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

3. Recombinant saccharomyces cerevisiae constructed by the method of claim 1 or 2 for metabolizing glycerol to produce ginsenoside CK.

4. Use of the recombinant saccharomyces cerevisiae for producing ginsenoside CK by metabolizing glycerol according to claim 3 for producing ginsenoside CK.