CN111471704B

CN111471704B - Recombinant bacterium for producing rare ginsenoside 20S-O-Glc-DM and application thereof

Info

Publication number: CN111471704B
Application number: CN201910063187.2A
Authority: CN
Inventors: 杨金玲; 朱平; 胡宗风; 顾安頔; 姜逢霖; 巩婷; 陈晶晶
Original assignee: Institute of Materia Medica of CAMS
Current assignee: Institute of Materia Medica of CAMS
Priority date: 2019-01-23
Filing date: 2019-01-23
Publication date: 2024-02-06
Anticipated expiration: 2039-01-23
Also published as: CN111471704A

Abstract

The invention discloses a recombinant bacterium for producing rare ginsenoside 20S-O-Glc-DM and application thereof, and particularly provides a method for producing dammarenediol-II glycoside 20S-O-Glc-DM and a construction method of the recombinant bacterium, the recombinant bacterium obtained by the method, and application of the recombinant bacterium in preparation of dammarenediol-II glycoside 20S-O-Glc-DM.

Description

Recombinant bacterium for producing rare ginsenoside 20S-O-Glc-DM and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a construction method of recombinant bacteria for producing dammarenediol-II glycoside 20S-O-Glc-DM, the recombinant bacteria obtained by the method, and application of the recombinant bacteria in preparation of dammarenediol-II glycoside 20S-O-Glc-DM.

Background

Ginseng radix (Panax ginseng C.A. Meyer) is a traditional rare medicinal material, and has various pharmacological activities such as anticancer, antiaging, antidiabetic, antihypertensive, immunoregulatory and neuroprotection, wherein ginsenoside is the main bioactive component of Ginseng radix. To date, 150 or more natural ginsenosides have been isolated and identified from ginseng plants. The structural and functional diversity of ginsenosides depends on the aglycone structure as well as the type, number and position of the glycosyl ligands. According to the different aglycone skeletons, the dammarane type tetracyclic triterpene saponins and oleanane type pentacyclic triterpene saponins can be classified. Dammarane-type saponins, which are the vast majority of ginsenosides, are further classified into protopanoxadiol (PPD) type and protopanoxatriol (PPT) type ginsenosides. PPD type ginsenoside is synthesized by glycosylation of PPD at C3-OH and/or C20-OH, while PPT type ginsenoside is synthesized by glycosylation of PPT at C6-OH and/or C20-OH. In addition, the difference in the positions and amounts of hydroxyl groups and sugar groups leads to a variety of biological activities of ginsenoside.

It has been reported that the cytotoxic activity of dammarane-type ginsenosides is inversely related to the number of hydroxy groups of its aglycone. However, triterpene saponins using dammarenediol-II (DM) as a substrate have never been isolated from Panax plants. DM as a direct precursor of PPD has fewer hydroxyl groups than PPD and PPT, and only two hydroxyl groups at the C3 and C20 positions, so it is presumed that C3-glycosylated DM and C20-glycosylated DM may have higher cytotoxic activity than PPD-type and PPT-type ginsenosides. In vitro pharmacological activity detection shows that 20S-O-Glc-DM has growth inhibition effect on a plurality of colon cancer cell lines; the in vivo pharmacological evaluation showed that 20S-O-Glc-DM, alone or in combination with 5-FU, significantly inhibited growth of C26 colon carcinoma xenografts, compared to control Rg3 and Compound K.

In recent years, researchers have cloned and identified various UDP-glycosyltransferase (UGT) genes from Panax plants, wherein UGTPg1 from Panax plants can selectively catalyze glycosylation of DM and C20-OH of PPD to generate 20S-O-Glc-DM and CK respectively. The UGT research related to ginsenoside biosynthesis lays a foundation for producing natural or unnatural ginsenoside through metabolic engineering.

The invention clones the genes of encoding dammarenediol-II synthase (DS) and UGTPG1 from ginseng respectively. UGTPG1 is subjected to heterologous expression in escherichia coli BL21 (DE 3), and DM glycoside 20S-O-Glc-DM is obtained through in vitro enzymatic reaction. The biosynthesis pathway of 20S-O-Glc-DM was constructed by introducing codon-optimized DS and UGTPG1 genes into Saccharomyces cerevisiae with hexokinase 2 gene knockdown, using the yeast endogenous terpenoid biosynthesis genes. DS and UGTPg1 genes are integrated into a yeast genome through a CRISPR/Cas9 system, several key enzymes upstream of a 20S-O-Glc-DM biosynthesis pathway are overexpressed, strategies such as competitive branch metabolic pathway downregulation and transcription activator HAC1 overexpression are optimized, and the biosynthesis pathway of recombinant bacteria is optimized so as to improve the yield of 20S-O-Glc-DM. The present study provides an effective method for the production of 20S-O-Glc-DM, which provides a candidate compound for new drug studies.

Disclosure of Invention

The present inventors have found that the yield of DM in recombinant bacteria is significantly increased when the dammarenediol-II synthase (DS) is expressed in fusion with Green Fluorescent Protein (GFP) in the recombinant bacteria producing DM.

The inventors have also found that the ginseng glycosyltransferase UGTPG1 is capable of selectively catalyzing the C20-OH of DM to produce 20S-O-Glc-DM.

Further, the inventors have found that knocking out hexokinase 2, a key enzyme of the glycolytic pathway, can modulate the metabolic flux of the glycolytic pathway, thereby increasing the yield of DM in recombinant bacteria.

In order to obtain a recombinant strain producing 20S-O-Glc-DM and a method of constructing the strain, the present invention provides in the following paragraphs:

[1] a method of constructing a recombinant bacterium, the method comprising the steps of: knocking out hexokinase 2 gene in saccharomyces cerevisiae, and introducing a coding gene expression cassette of fusion protein of DS and GFP and a coding gene expression cassette of ginseng UGTPg1 into the saccharomyces cerevisiae.

[2] The method according to [1], further comprising the steps of: enhancing the activity of 3-hydroxy-3-methylglutaryl-coa reductase in the saccharomyces cerevisiae.

[3] The method of any one of [1] or [2], further comprising one or more of:

enhancing activity of isopentenyl pyrophosphate isomerase IDI1 in saccharomyces cerevisiae;

enhancing the activity of farnesyl pyrophosphate synthase ERG20 in Saccharomyces cerevisiae;

improving the activity of squalene monooxygenase ERG1 in Saccharomyces cerevisiae;

improving the activity of squalene synthase ERG9 in Saccharomyces cerevisiae;

reducing the activity of lanosterol synthase ERG7 in Saccharomyces cerevisiae;

Increasing the level of the chaperone BiP in saccharomyces cerevisiae;

increasing the level of the transcription factor HAC1 in saccharomyces cerevisiae; or alternatively

Increasing the level of disulfide isomerase PDI1 in Saccharomyces cerevisiae.

[4] The method according to any one of [1] to [3], wherein the gene expression cassette encoding a fusion protein of DS and GFP comprises a gene encoding a fusion protein of DS and GFP represented by SEQ ID NO. 1.

[5] The method according to any one of [1] to [4], wherein the UGTPg 1-encoding gene expression cassette comprises a UGTPg 1-encoding gene represented by SEQ ID NO. 2.

[6] The method according to [2], wherein the activity of 3-hydroxy-3-methylglutaryl-CoA reductase in the Saccharomyces cerevisiae is increased by introducing a gene tHMG1 expression cassette encoding 3-hydroxy-3-methylglutaryl-CoA reductase into the Saccharomyces cerevisiae.

[7] The method according to any one of [3] to [6], wherein,

said increasing the activity of isopentenyl pyrophosphate isomerase IDI1 in saccharomyces cerevisiae is performed by introducing into saccharomyces cerevisiae an expression cassette encoding a gene encoding isopentenyl pyrophosphate isomerase IDI 1;

said increasing the activity of farnesyl pyrophosphate synthase ERG20 in Saccharomyces cerevisiae is performed by introducing into Saccharomyces cerevisiae an expression cassette encoding a gene encoding farnesyl pyrophosphate synthase ERG 20;

Said increasing the activity of squalene monooxygenase ERG1 in Saccharomyces cerevisiae is performed by introducing into Saccharomyces cerevisiae a coding gene expression cassette for squalene monooxygenase ERG 1;

said increasing the activity of squalene synthase ERG9 in Saccharomyces cerevisiae is performed by introducing into Saccharomyces cerevisiae a gene expression cassette encoding squalene synthase ERG 9;

said decreasing the activity of lanosterol synthase ERG7 in saccharomyces cerevisiae is performed by introducing an expression cassette for an antisense fragment of lanosterol synthase ERG7 into saccharomyces cerevisiae;

said increasing the level of chaperone BiP in saccharomyces cerevisiae is performed by introducing into saccharomyces cerevisiae an expression cassette encoding the chaperone BiP;

said increasing the level of transcription factor HAC1 in saccharomyces cerevisiae is performed by introducing into saccharomyces cerevisiae an expression cassette encoding the transcription factor HAC 1; or alternatively

The increasing of the level of disulfide isomerase PDI1 in Saccharomyces cerevisiae is performed by introducing a gene expression cassette encoding disulfide isomerase PDI1 into Saccharomyces cerevisiae.

[8] The method according to [7], characterized in that:

the nucleotide sequence for encoding IDI1 is the sequence shown in SEQ ID NO. 4;

the nucleotide sequence for encoding ERG20 is the sequence shown in SEQ ID NO. 5;

The nucleotide sequence for encoding ERG1 is the sequence shown in SEQ ID NO. 6;

the nucleotide sequence for encoding ERG9 is the sequence shown in SEQ ID NO. 7;

the nucleotide sequence of the antisense fragment of ERG7 is the sequence shown in SEQ ID NO. 8;

the nucleotide sequence for encoding BiP is a sequence shown as SEQ ID NO. 8;

the nucleotide sequence for encoding HAC1 is the sequence shown by SEQ ID NO. 10; or alternatively

The nucleotide sequence encoding PDI1 is the sequence shown by SEQ ID NO. 11.

[9] The method of any one of [1] to [8], wherein the integration of the expression cassette into the saccharomyces cerevisiae genome is performed, preferably using CRISPR/Cas 9.

[10] A recombinant bacterium obtained by the method according to any one of [1] to [9 ].

[11] The use of the recombinant bacterium according to [9] for producing 20S-O-Glc-DM.

[12] A method for producing 20S-O-Glc-DM, which comprises fermenting the recombinant bacterium according to [9] to obtain 20S-O-Glc-DM.

Detailed Description

In a first aspect, the present invention provides a method of constructing a recombinant bacterium, the method comprising the steps of: knocking out hexokinase 2 gene in saccharomyces cerevisiae, and introducing a coding gene expression cassette of fusion protein of dammarenediol-II synthase and GFP (hereinafter referred to as DS-GFP) and a coding gene expression cassette of ginseng glycosyltransferase UGTPG1 into the saccharomyces cerevisiae.

In the present invention, the dammarenediol-II synthase may be a ginseng-derived dammarenediol-II synthase. In a preferred embodiment, the ginseng dammarenediol-II synthase gene ds (No. AB265170.1) may be used.

The green fluorescent protein GFP is a fluorescent protein separated from Victoria multi-tube jellyfish, and can emit green fluorescence under the excitation of blue light of 450-490 nm, so that the green fluorescent protein GFP is an ideal reporter molecule. Numerous studies have expressed the target protein fused to GFP, subcellular localization of the target protein by observing green fluorescence, and exploration of its biological function. In the present invention, the GFP-encoding gene may be any polynucleotide capable of encoding GFP.

In the present invention, GFP may be fused to the C-terminus of the dammarenediol-II synthase.

In the present invention, GFP may be directly linked to the dammarenediol-II synthase, or a spacer sequence may also be present between GFP and dammarenediol-II synthase, for example, 2 to 40 amino acids, preferably 5 to 20 amino acids. In a preferred embodiment of the invention, GFP may be fused to the C-terminus of the dammarenediol-II synthase.

In a preferred embodiment, the coding gene for DS-GFP is prepared by the method described in SEQ ID NO. 1 (which is described in Liang et al, ginseng radix dammarenediol-II synthase expression, localization and functional studies in Saccharomyces cerevisiae, pharmaceutical journal, acta Pharmaceutica Sinica 2016, 51 (6): 998-1003, which is incorporated herein by reference in its entirety).

In one embodiment of the present invention, the DS-GFP expression cassette further specifically comprises a promoter TEF1, a gene encoding DS-GFP, and a terminator CYC1.

In a preferred embodiment of the invention, the optimized gene UGTPg1 sequence (SEQ ID NO: 2) is synthesized according to the codon preference of Saccharomyces cerevisiae based on cDNA sequence information of the ginseng glycosyltransferase UGTPg1 (No. KF377585.1).

In one embodiment of the invention, the gene expression cassette encoding the ginseng glycosyltransferase UGTPg1 further specifically comprises the promoters TDH3, UGTPg1 encoding gene, terminator ADH2.

In the present invention, knockdown of hexokinase 2 gene in Saccharomyces cerevisiae is performed by means known in the art. For example, the hexokinase 2 gene HXK2 is knocked out by homologous recombination.

In the present invention, diploid and haploid Saccharomyces cerevisiae mutants deleted for gene HXK2 may be used, and haploid Saccharomyces cerevisiae mutants are preferably used.

In a further embodiment of the invention, the method further comprises the steps of: enhancing the activity of 3-hydroxy-3-methylglutaryl-coa reductase in the saccharomyces cerevisiae.

3-hydroxy-3-methyl-glutaryl CoA reductase (HMGR) is the first key enzyme in the mevalonate metabolic pathway, catalyzing the production of mevalonate from 3-hydroxy-3-methylglutaryl CoA, which is also the first rate limiting step in the ginsenoside biosynthetic pathway. HMGR contains an N-terminal transmembrane domain and a C-terminal catalytic domain, which exert a localization and catalytic effect, respectively. Overexpression of HMGR in cells results in feedback inhibition of the mevalonate metabolic pathway, i.e., downstream products of HMGR catalysis activate HMGR anchored to the endoplasmic reticulum membrane into the degradation pathway, and thus the localization of its transmembrane domain plays an important role in the HMGR degradation pathway. In view of this, by removing the transmembrane domain, truncating the HMGR gene and overexpressing it, the feedback inhibition of the mevalonate metabolic pathway can be effectively reduced, thereby promoting biosynthesis of downstream products. In s.cerevisiae, there are two members of the mevalonate pathway, hmg1p and Hmg2p, respectively, encoded by the genes HMG1 and HMG2, wherein HMG 1-encoded Hmg1p plays a major role. The cDNA sequence of gene tHMG1 encoding the HMGR catalytic domain (No. NM-001182434.1) is the sequence shown by SEQ ID NO. 3.

In the present invention, the overexpression of the HMGR catalytic domain increases the supply of upstream precursor 2, 3-oxidosqualene, and at the same time avoids feedback inhibition caused by downstream product accumulation, and finally, the DM content in the recombinant bacterium is significantly increased.

Thus, in a preferred mode of the present invention, the method of increasing the activity level of 3-hydroxy-3-methyl-glutaryl CoA reductase may be the introduction of the 3-hydroxy-3-methyl-glutaryl CoA reductase encoding gene tHMG1 expression cassette.

In one embodiment of the present invention, the 3-hydroxy-3-methyl-glutaryl CoA reductase encoding gene expression cassette further specifically comprises the promoters PGK1, tHMG1, terminator ADH1.

In the method of the invention, the method further comprises one or more of the following:

improving the activity of squalene synthase ERG9 in Saccharomyces cerevisiae;

reducing the activity of lanosterol synthase ERG7 in Saccharomyces cerevisiae;

increasing the level of the chaperone BiP in saccharomyces cerevisiae;

Increasing the level of disulfide isomerase PDI1 in Saccharomyces cerevisiae.

In a specific embodiment, the activity of isopentenyl pyrophosphate isomerase IDI1 in saccharomyces cerevisiae is increased by introducing into saccharomyces cerevisiae an expression cassette encoding the isopentenyl pyrophosphate isomerase IDI 1;

increasing the activity of farnesyl pyrophosphate synthase ERG20 in Saccharomyces cerevisiae by introducing a gene expression cassette encoding farnesyl pyrophosphate synthase ERG20 into Saccharomyces cerevisiae;

increasing the activity of squalene monooxygenase ERG1 in Saccharomyces cerevisiae by introducing a coding gene expression cassette of squalene monooxygenase ERG1 into Saccharomyces cerevisiae;

increasing the activity of squalene synthase ERG9 in Saccharomyces cerevisiae by introducing a coding gene expression cassette of squalene synthase ERG9 into Saccharomyces cerevisiae;

reducing the activity of lanosterol synthase ERG7 in Saccharomyces cerevisiae by introducing an expression cassette of an antisense fragment of lanosterol synthase ERG7 into Saccharomyces cerevisiae;

increasing the level of chaperone BiP in saccharomyces cerevisiae by introducing a gene expression cassette encoding chaperone BiP into saccharomyces cerevisiae;

increasing the level of transcription factor HAC1 in saccharomyces cerevisiae by introducing a gene expression cassette encoding transcription factor HAC1 into saccharomyces cerevisiae; or alternatively

The level of disulfide isomerase PDI1 in Saccharomyces cerevisiae was increased by introducing a gene expression cassette encoding disulfide isomerase PDI1 into Saccharomyces cerevisiae.

In one embodiment, the gene expression cassette encoding isopentenyl pyrophosphate isomerase IDI1 may comprise a promoter TDH3, a gene encoding isopentenyl pyrophosphate isomerase IDI1, and a terminator TPI1.

In one embodiment, the nucleotide sequence encoding IDI1 is the sequence shown by SEQ ID NO. 4.

In one embodiment, the gene expression cassette encoding farnesyl pyrophosphate synthase ERG20 may comprise a promoter PGK1, a gene encoding farnesyl pyrophosphate synthase ERG20, a terminator ADH1.

In one embodiment, the nucleotide sequence encoding ERG20 is the sequence set forth in SEQ ID NO. 5.

In one embodiment, the squalene monooxygenase ERG1 encoding gene expression cassette may comprise a promoter PGK1, a squalene monooxygenase ERG1 encoding gene, and a terminator ADH1.

In one embodiment, the nucleotide sequence encoding ERG1 is the sequence set forth in SEQ ID NO. 6.

In one embodiment, the squalene synthase ERG9 encoding gene expression cassette may comprise a promoter TEF1, a squalene synthase ERG9 encoding gene, and a terminator TPI1.

In one embodiment, the nucleotide sequence encoding ERG9 is the sequence set forth in SEQ ID NO. 7.

In one embodiment, the gene expression cassette encoding the lanosterol synthase ERG7 antisense fragment may comprise the gene of the promoter TEF1, lanosterol synthase ERG7 antisense fragment, terminator CYC1.

In one embodiment, the nucleotide sequence encoding the ERG7 antisense fragment is the sequence shown by SEQ ID NO. 8.

In one embodiment, the gene expression cassette encoding the chaperone BiP may include a promoter TEF1, a gene encoding the chaperone BiP, a terminator CYC1.

In one embodiment, the nucleotide sequence encoding BiP is the sequence set forth in SEQ ID NO. 9.

In one embodiment, the expression cassette of the coding gene of the transcription factor HAC1 may include a promoter TEF1, a coding gene of the transcription factor HAC1, and a terminator CYC1.

In one embodiment, the nucleotide sequence encoding HAC1 is the sequence set forth in SEQ ID NO. 10.

In one embodiment, the disulfide isomerase PDI1 encoding gene expression cassette may include a promoter TEF1, a disulfide isomerase PDI1 encoding gene, and a terminator CYC1.

In one embodiment, the nucleotide sequence encoding PDI1 is the sequence set forth in SEQ ID NO. 11.

In embodiments of the invention, the above-described expression cassettes may be integrated into the genome of a Saccharomyces cerevisiae cell, respectively. The expression cassettes described above can also be linked to one another and integrated into the genome of the Saccharomyces cerevisiae cells. For example, all expression cassettes can be linked in series and integrated into the genome of a s.cerevisiae cell. Alternatively, the above expression cassette may be constructed as a plurality of expression modules and then integrated into the genome of a s.cerevisiae cell. In the present invention, an expression module refers to two or more expression cassettes operably linked. In the present invention, the expression cassette and/or expression module may be integrated into one or more sites. In the present invention, CRISPR/Cas9 is preferably employed for the integration of expression cassettes and/or expression modules

In a preferred embodiment, the gene expression cassette encoding the fusion protein of dammarenediol-II synthase with GFP, the gene expression cassette encoding the ginseng glycosyltransferase UGTPg1 and the gene expression cassette encoding the 3-hydroxy-3-methylglutaryl-CoA reductase may be integrated into the Saccharomyces cerevisiae genome delta 1 site. The expression cassette encoding the isopentenyl pyrophosphate isomerase IDI1, the expression cassette encoding the farnesyl pyrophosphate synthase ERG20, the expression cassette encoding the squalene monooxygenase ERG1, the expression cassette encoding the squalene synthase ERG9, and the expression cassette of the lanosterol synthase ERG7 antisense fragment may be integrated into the saccharomyces cerevisiae genome delta 4 site. The gene expression cassette encoding the chaperone BiP, the gene expression cassette encoding the transcription factor HAC1, and the gene expression cassette encoding the disulfide isomerase PDI1 may be integrated into the saccharomyces cerevisiae genome rDNA site.

In a preferred embodiment, the gene expression cassette encoding the fusion protein of dammarenediol-II synthase with GFP, the gene expression cassette encoding the ginseng glycosyltransferase UGTPG1 and the gene expression cassette encoding the 3-hydroxy-3-methylglutaryl-CoA reductase may be further constructed as an expression module and integrated into the s.cerevisiae genome delta 1 site. The expression cassette encoding the isopentenyl pyrophosphate isomerase IDI1, the expression cassette encoding the farnesyl pyrophosphate synthase ERG20, the expression cassette encoding the squalene monooxygenase ERG1, the expression cassette encoding the squalene synthase ERG9, and the expression cassette for the lanosterol synthase ERG7 antisense fragment may be further constructed as expression modules and integrated into the saccharomyces cerevisiae genome delta 4 site. The gene expression cassette encoding the chaperone BiP, the gene expression cassette encoding the transcription factor HAC1, and the gene expression cassette encoding the disulfide isomerase PDI1 may be constructed as an expression module and integrated into the saccharomyces cerevisiae genome rDNA site.

In a further preferred mode, the invention constructs a CRISPR/Cas9 system based on a Saccharomyces cerevisiae genome delta 1 locus, and the yeast engineering bacteria are obtained by utilizing double strand break mediated by endonuclease Cas9 in the system and through a homologous recombination mechanism.

The Saccharomyces cerevisiae of the present invention may be any Saccharomyces cerevisiae available in the art. For example, commercially available Saccharomyces cerevisiae INVSc1, saccharomyces cerevisiae BY4742, saccharomyces cerevisiae YPH499, or Saccharomyces cerevisiae W303-1B, etc.

The coding gene expression cassette is integrated into a saccharomyces cerevisiae genome; alternatively, these coding gene expression cassettes are present in the Saccharomyces cerevisiae cells in the form of plasmids.

In some embodiments, the plasmid vector is selected from the group consisting of pESC-HIS, pESC-URA, pESC-TRP, and pESC-TRP (Invitrogen, USA).

The dammarenediol-II synthase, GFP, glycosyltransferase UGTPG1, 3-hydroxy-3-methylglutaryl coenzyme A reductase, isopentenyl pyrophosphate isomerase, farnesyl pyrophosphate synthase, squalene monooxygenase, squalene synthase, lanosterol synthase, chaperone BiP, transcription factor HAC1 or disulfide isomerase PDI1 in the invention can be:

(a) Naturally occurring wild-type enzymes;

(b) A polypeptide which is formed by substitution, deletion or addition of one or more amino acid residues of a polypeptide of a wild-type enzyme or is formed by addition of a signal peptide sequence and has corresponding activity;

(c) A polypeptide comprising the polypeptide sequence of (a) or (b) in its sequence;

(d) A polypeptide having an amino acid sequence which is not less than 85% or not less than 90% (preferably not less than 95%) identical to the amino acid sequence of the wild-type enzyme and has wild-type enzyme activity.

In the present invention, the coding gene used may be a natural polynucleotide sequence (e.g., cDNA sequence, genomic sequence, RNA, etc.) encoding any one of the above (a) - (d), or degenerate variants thereof. As used herein, "degenerate variant" refers in the present invention to a polynucleotide sequence capable of encoding a protein of any one of (a) - (d) above, but which differs from the native polynucleotide sequence. Preferably, codon-optimized DNA sequences are used. Polynucleotides are generally obtained by PCR amplification, recombinant methods or synthetic methods.

The exogenous gene expression cassette can be integrated into the genome for expression in Saccharomyces cerevisiae, or can be expressed in the form of plasmid isolated from the genome.

The Saccharomyces cerevisiae episomal vector may be a commercially available vector, or any vector having the same function. For example, episomal vectors can be pESC-series vectors, including pESC-HIS, pESC-URA, pESC-TRP, and pESC-TRP; pYES2; or pAUR123 (Invitrogen, USA).

The method of integrating the exogenous gene into the yeast genome is mainly homologous recombination. The method of homologous recombination comprises amplifying the sequence upstream and downstream of the integration site as an upstream homology arm or downstream homology arm, constructing a gene expression cassette of interest (comprising a promoter, a gene of interest and a terminator), usually further comprising a selection marker gene in the upstream homology arm, and then ligating the above-mentioned members in the order of the upstream homology arm, the gene expression cassette and the downstream homology arm to form a fragment useful for homologous recombination; the obtained fragment for homologous recombination is introduced into Saccharomyces cerevisiae, and positive transformants are selected according to the selection markers, thereby obtaining integrated recombinant Saccharomyces cerevisiae.

The integration site in the Saccharomyces cerevisiae genome may be selected from the following: a delta site, 1-10 random positions in a plurality of delta genes on the saccharomyces cerevisiae chromosome; rDNA sites, 1-10 random positions in a plurality of ribosomal genes on Saccharomyces cerevisiae chromosome; a HIS3 site, a HIS3 gene position in the histidine biosynthetic pathway on the Saccharomyces cerevisiae chromosome; alternatively, the Trp1 site is the Trp1 gene position in the tryptophan biosynthetic pathway on the Saccharomyces cerevisiae chromosome.

Useful Saccharomyces cerevisiae gene integration selectable markers may be any selectable marker known to those of skill in the art, provided that the selectable markers used when integrating different fragments into the same Saccharomyces cerevisiae strain are different from one another. Common selection markers are auxotrophic selection markers and resistance selection markers. Wherein the auxotrophic selection marker may be selected from LEU, HIS, URA or TRP. The resistance selection marker may be G418 or HYG.

The promoter may be any promoter that can be used in Saccharomyces cerevisiae. For example, the promoter may be selected from the group consisting of: pPGK, pADH1, pTDH3, pTEF2, pPDC1, and pTPI1. The terminator may be any terminator that can be used in Saccharomyces cerevisiae. For example, the terminator may be selected from the group consisting of: PGK1t, ADH1t and FBA1t.

The homologous recombinant fragments or recombinant plasmids are introduced into Saccharomyces cerevisiae using methods known in the art. Among them, various transformation methods known to those skilled in the art, such as an electrotransformation method, a lithium acetate chemical transformation method, and the like, can be used for the method of transforming Saccharomyces cerevisiae.

The level of ergosterol synthase activity in Saccharomyces cerevisiae can be reduced by reducing the expression level of the ergosterol synthase gene erg7 or by reducing the activity of the ergosterol synthase protein.

In the methods of constructing recombinant strains of the invention, any method known to those of skill in the art may be used to reduce the expression level of a target gene (e.g., erg7 gene) or to reduce ergosterol synthase activity (including inactivating the target gene), such methods including, but not limited to: gene knockout, site-directed mutagenesis, or RNA interference (RNAi).

In the embodiment of the present invention involving RNAi, the method for achieving RNAi is not particularly limited, and various RNAi techniques known to those skilled in the art can be employed, for example, transcription or translation of a target gene (e.g., erg7 gene) can be blocked by using small interfering RNA (siRNA), antisense nucleic acid, microRNA (microRNA), or the like, thereby causing a decrease in the expression level of the target gene.

In a second aspect, the invention provides recombinant bacteria produced by the method of the first aspect of the invention.

A third aspect of the invention is the use of a recombinant bacterium according to the second aspect for the production of 20S-O-Glc-DM.

In a fourth aspect, the invention provides a method for producing 20S-O-Glc-DM comprising fermenting a recombinant bacterium as described in the second aspect to produce 20S-O-Glc-DM.

In the present invention, fermentation of recombinant bacteria may be performed according to various methods known in the art.

Beneficial technical effects

According to the invention, the recombinant strain for producing the dammarenediol-II glycoside 20S-O-Glc-DM is obtained by transferring the dammarenediol-II synthase gene and the ginseng glycosyltransferase UGTPG1 gene into the saccharomyces cerevisiae knocked out by hexokinase HXK2 gene. On the basis, the CRISPR/Cas9 technology is adopted to promote the integration of exogenous genes in a saccharomyces cerevisiae genome, the upstream key enzymes of isopentenyl pyrophosphate isomerase, farnesyl pyrophosphate synthase, squalene monooxygenase and squalene synthase are overexpressed, the antisense technology is adopted to down regulate the expression of lanosterol synthase, and the transcription factor HAC1 is overexpressed, so that the yield of 20S-O-Glc-DM in engineering bacteria is further improved. The invention obtains the high-yield engineering bacteria for producing rare ginsenoside 20S-O-Glc-DM for the first time, and lays a foundation for the mass production of the engineering bacteria.

Drawings

FIG. 1 shows a schematic construction of the knockout element LoxP-KanMX-LoxP.

FIG. 2 shows the amplification electrophoresis of the left and right homology arms of the Saccharomyces cerevisiae primary metabolism related gene HXK2 (hexokinase 2) gene. Wherein, 1 HXK2 left homology arm; 2 HXK2 right homology arm

FIG. 3 shows an electrophoretogram of KanMX gene expression cassette amplification.

FIG. 4 shows the result of electrophoresis of the knockout element LoxP-KanMX-LoxP. Wherein, 1 HXK2 gene knockout element

FIG. 5 shows a schematic representation of diagnostic PCR primers verified by gene knockout strains.

FIG. 6 shows the results of the electrophoretic verification of HXK2 knockout strains. Wherein, lane 1 and lane 5 primers: HXK2-1F/KanMX-R; lane 2 and lane 6 primers: kanMX-F/HXK2-2R; lane 3 and lane 7 primers: HXK2-1F/HXK2-2R; lane 4 and lane 8 primers: HXK2-YF/YR. Wherein Y-delta HXK2: knocking out YPH499 of HXK2 gene; WT: YPH499 genome.

FIG. 7 shows the electrophoresis results of integration modules I and II.

FIG. 8 shows the results of electrophoresis of integrated modules III, IV and V.

FIG. 9 shows the results of electrophoresis of integration modules VI, VII and VIII.

Fig. 10 shows a schematic diagram of the construction of each integrated module.

FIG. 11 shows Cas9 expression cassette element P _TEF1 -Cas9-T _CYC1 (1) And fusion element gRNA-P _TEF1 -Cas9-T _CYC1 (2) Is a result of electrophoresis.

FIG. 12 shows a standard curve for standard 20S-O-Glc-DM.

FIG. 13 shows the HPLC detection result of 20S-O-Glc-DM in recombinant bacteria.

FIG. 14 shows the results of LC-MS detection of 20S-O-Glc-DM in recombinant bacteria.

FIG. 15 shows the genotype of recombinant Y2C and the results of high-yield strain selection. FIG. 15A shows the genotype of recombinant Y2C; FIG. 15B shows the yield of 20S-O-Glc-DM of recombinant strains Y2C-1 to Y2C-20.

FIG. 16 shows the genotype of recombinant Y2CS and the results of high-yield strain selection. FIG. 16A shows the genotype of recombinant Y2 CS; FIG. 16B shows the yield of 20S-O-Glc-DM of recombinant strains Y2CS-1 to Y2 CS-20.

FIG. 17 shows genotypes of recombinant Y2CSB, Y2CSH and Y2CSP and high-yielding strain selection results. FIG. 17A shows genotypes of recombinant Y2CSB, Y2CSH and Y2 CSP; FIG. 17B shows the yields of recombinant bacteria Y2CSB-1 to Y2CSB-10, Y2CSH-1 to Y2CSH-10, and 20S-O-Glc-DM of Y2CSP-1 to Y2 CSP-10.

FIG. 18 shows the quantitative determination of recombinant yeast producing 20S-O-Glc-DM.

FIG. 19 shows the results of high-density fermentation yield assays for recombinant yeasts producing 20S-O-Glc-DM.

Detailed Description

The following describes exemplary embodiments of the invention, and it should be understood by those skilled in the art that the following embodiments are not limiting of the specific embodiments of the invention, but are to be construed to include all variations, equivalents, or alternatives falling within the spirit and scope of the invention. Various modifications and other embodiments are within the ability of one of ordinary skill in the art and are contemplated as falling within the scope of the present invention.

Unless otherwise indicated, the experimental methods used hereinafter are conventional methods well known to those skilled in the art, and can be carried out, for example, using standard procedures described in the following works: sambrook et al, molecular cloning guidelines (3 rd edition) (Molecular Cloning: A Laboratory Manual, cold Spring Harbor Laboratory Press, cold Spring Harbor, n.y., USA, 2001); davis et al, basic methods of molecular biology (Basic Methods in Molecular Biology, elsevier Science Publishing, inc., new York, USA, 1995); juan S.Bonifacino et al, guidelines for cell biology laboratory (Current Protocols in Cell Biology, john Wiley and Sons, inc.).

Examples

The invention will be better understood by means of the following examples, which are given solely for illustration of the invention and should not be construed as limiting it.

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

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

Example 1 construction of Yeast Gene knockout element LoxP-KanMX-LoxP

The gene knockout element comprises a part with two ends homologous to the 5 'and 3' non-coding regions of the target gene, and a kanamycin resistance gene (KanMX) is contained in the middle as a reporter gene for screening gene deletion mutant strains. The Kan-resistant gene expression cassette (kanMX) was amplified using the plasmid pUC6 as a template and the primer KAN-F/R. And (3) respectively fusing the left and right homologous arms of the target gene and a Kan resistance gene expression cassette (kanMX) by using an overlap extension PCR (OE-PCR) technology to obtain a target gene knockout element. The LoxP-KanMX-LoxP knockout element was obtained as shown in FIG. 1.

The nucleotide sequence (NC_ 001139.9) of the Saccharomyces cerevisiae primary metabolism related gene HXK2 (hexokinase 2) was searched and obtained according to GenBank Nucleotide (http:// www.ncbi.nlm.nih.gov/nuccore /), primers were designed according to sequence information (Table 1), HXK2-1F/R and 2F/R were used as upstream and downstream primers, saccharomyces cerevisiae INVSc1 genomic DNA was used as a template, and the left and right homology arms (SEQ ID NO:12 and SEQ ID NO:13, respectively) of each target gene were amplified by using high fidelity enzymes (FIG. 2), and the band sizes were about 360-400 bp.

TABLE 1 primer sequences for gene knockout elements

Primers (Table 2) were designed based on the sequence information of the plasmid pUC6, and Kan-resistant expression cassette (kanMX) (SEQ ID NO: 14) (FIG. 3) was amplified using the plasmid as a template and the primers KAN-F and KAN-R in Table 2, with a target band size of 1613bp, and the expressed aminoglycoside phosphotransferase was able to inactivate kanamycin, so that gene knockout bacteria were selected as a resistance marker.

TABLE 2 cloning primer sequences for resistance genes

The PCR amplification system was as follows (50. Mu.L):

the PCR amplification conditions were as follows:

98℃，30s；

98 ℃ for 10s;55-60 ℃ for 30s;72 ℃,1kb/15-30s;30 cycles;

72℃，5min；

4℃，∞；

the PCR products were detected by agarose gel electrophoresis at 1.0%.

The target gene left and right homologous arms and the resistance gene expression cassette are recovered, and the target gene knockout element is obtained by using OE-PCR and primer CF/CR amplification.

OE-PCR reaction System (50 uL):

OE-PCR amplification conditions:

95℃，30s；

95 ℃ for 10s;50-60 ℃ for 30s;72 ℃,1kb/15-30s;10 cycles;

95 ℃ for 10s;55 ℃ for 30s;72 ℃,1kb/15-30s;20 cycles;

72℃，5min；

4℃，∞；

the electrophoresis results showed that the actual size of the fusion fragment was consistent with the theoretical value (FIG. 4).

4 mu L of OE-PCR product is connected with 1 mu L of cloning vector pEASY-Blunt simple vector, and Trans1-T1 competent cells are transformed; screening positive transformant, extracting plasmid, sequencing plasmid to obtain target gene knockout element LoxP-KanMX-LoxP complete sequence, and named pEASY-HXK2.

Example 2 selection of transformed and knocked out strains of Saccharomyces cerevisiae

The yeast gene knockout element LoxP-KanMX-LoxP was transferred into Saccharomyces cerevisiae YPH499 competent cells. Using the LiAC/ssDNA/PEG yeast transfection method, the following transfection mixtures were added sequentially:

with 50. Mu.L ddH ₂ O replaces the target gene knockout element as a control. And (5) taking a proper amount of Saccharomyces cerevisiae YPH499 competent cells, and adding the system to perform a yeast transformation experiment. After transformation, willThe cell sap of the experimental group and the control group were spread on a solid medium plate containing antibiotic G418 (0.3 mg/ml) with a glass rod, respectively, and cultured at 30℃for 2d until the transformants appeared. (Note: after addition of PEG3350, the cell fluid was sufficiently suspended; ssDNA was melted by treatment at 100℃for 10min in advance, and immediately cooled on ice).

The corresponding transformant with good growth vigor on the G418 resistant plate is selected, the genome DNA of the Saccharomyces cerevisiae transformant is extracted as a template, the corresponding diagnosis PCR primer HXK2-1F/HXK2-2R, HXK2-1F/M-A, M-B/HXK2-2R, HXK2-YF/HXK2-YR (table 1 and table 3) is selected, and the positive transformant with the knocked-out gene is selected. The diagnostic PCR primer design is shown in FIG. 5.

TABLE 3 diagnostic PCR primer sequences

Positive transformants were screened for gene knockout by diagnostic PCR, and 5uL of diagnostic PCR samples were taken, respectively, for agarose gel electrophoresis detection, and the results showed that diploid and haploid saccharomyces cerevisiae mutants with gene HXK2 deletion were successfully screened by using a homologous recombination mechanism (fig. 6).

As shown in FIG. 6, the single colony of Saccharomyces cerevisiae YPH499 transformed with HXK2 knockout element and corresponding wild type yeast genome DNA are used as templates, 3 pairs of diagnostic primers HXK2-1F/KanMX-R, kanMX-F/HXK2-2R, HXK-1F/2R are used for carrying out PCR diagnostic tests, and compared with a control group, the stripes which are consistent with the theoretical size of the knockout element appear in the transformants respectively, which indicates that homologous recombination of the knockout element occurs and the target gene HXK2 on the genome is replaced; meanwhile, when the gene self primer HXK2-YF/YR is used for verification, the corresponding band of the target gene does not appear in the conversion group, which indicates that the blurred band with about 1.0Kb appearing in the conversion group is nonspecifically amplified instead of the single knockout of the diploid gene, and the result is synthesized, which indicates that the target gene on the saccharomyces cerevisiae genome is successfully replaced by the knockout element by utilizing a homologous recombination mechanism, thus obtaining the saccharomyces cerevisiae HXK2 gene defect strain which is named as Y-delta HXK2.

The Saccharomyces cerevisiae gene-deficient strains constructed in this example are shown in Table 4.

TABLE 4 Gene-deficient Saccharomyces cerevisiae strains

In order to examine the growth state of Saccharomyces cerevisiae in different media after gene knockout, a time growth curve was determined for Y-DeltaHXK 2. Inoculating the genetically modified Saccharomyces cerevisiae and wild Saccharomyces cerevisiae into 2mL YPD liquid culture medium, and shake culturing at 30deg.C and 220rpm for 14 hr to obtain seed solution. The seed liquid is respectively at the initial OD ₆₀₀ A proportion of 0.4 was inoculated into 30mL of YPD or YPG liquid medium, and 3 groups were arranged in parallel. The bacterial solutions were cultured at 30℃and 220rpm with shaking, and absorbance (OD) at 600nm was measured at the UV spectrometer of the gene-deficient Saccharomyces cerevisiae bacterial solutions obtained in the different culture media at the time points of 4 hours, 8 hours, 12 hours, 16 hours, etc., respectively. Experimental results show that the growth speed of the knockout bacterium Y-delta HXK2 in the two culture media is slightly higher than that of the control bacterium YPH499, and the knockout bacterium Y-delta HXK2 gene does not influence the normal growth of the YPH 499.

Example 3 construction of Gene expression cassettes

Construction of genes DS-GFP, UGTPG1 and tHMG1 expression cassettes

cDNA (No. AB265170.1) sequence information of the dammarenediol-II synthase gene DS derived from ginseng was obtained from GenBank registration information, and an optimized DS gene and DS-GFP sequence (SEQ ID NO: 1) were synthesized based on the codon preference of Saccharomyces cerevisiae to obtain plasmid pESC-HIS-DS-GFP (prepared according to the methods described in the Can-et al, renskagaku-et al, rensdamenediol-II synthase expression, localization and function studies in Saccharomyces cerevisiae, pharmaceutical journal, acta Pharmaceutica Sinica 2016, 51 (6): 998-1003, which was incorporated herein by reference in its entirety).

By plasmidspESC-HIS-DS-GFP is used as a template, and primers DS-TEF1-F and DS-CYC1-R in the table 5 are used for PCR amplification to obtain synDS-GFP gene (3036 bp); the Saccharomyces cerevisiae INVSc1 genomic DNA was used as a template, and overlapping extension primers A-TEF1-delta1-F/TEF1-DS-R and CYC1-DS-F/CYC1-PGK1-R in Table 5 were used, respectively, to amplify and obtain Saccharomyces cerevisiae promoter TEF1 (430bp,SEQ ID NO:15) and terminator CYC1 (189bp,SEQ ID NO:16) sequence fragments. The primers A-TEF1-delta1-F and CYC1-PGK1-R in Table 5 were used for OE-PCR, and fused to give synDS-GFP gene expression cassette elements: p (P) _TEF1 -synDS-GFP-T _CYC1 。

cDNA sequence information of a glycosyltransferase gene UGTPG1 (KF 377585.1) from ginseng is obtained according to GenBank registration information, and an optimized gene UGTPG1 sequence (SEQ ID NO: 2) is synthesized according to the codon preference of saccharomyces cerevisiae to obtain a plasmid pUC57-UGTPG1.

The plasmid pUC57-UGTPg1 is used as a template, and primers UGTPg1-TDH3-F and UGTPg1-ADH2-R in the table 5 are used for amplification to obtain synUGTPg1 gene (1428 bp); the Saccharomyces cerevisiae promoter TDH3 (800bp,SEQ ID NO:17) and terminator ADH2 (566bp,SEQ ID NO:18) sequence fragments are amplified by using Saccharomyces cerevisiae INVSc1 genomic DNA as a template and primers TDH3-ADH1-F/TDH3-UGTPg1-R and ADH2-UGTPg1-F/ADH2-HIS-R in Table 5 respectively. The primers TDH3-ADH1-F and ADH2-HIS-R in Table 5 were used for OE-PCR, and fused to obtain synUGTPg1 gene expression cassette elements: p (P) _TDH3 -synUGTPg1-T _ADH2 。

Primers tHMG1-PGK1-F/tHMG1-ADH1-R were designed based on the cDNA sequence (No. NM-001182434.1) of the GenBank registered Saccharomyces cerevisiae (S.cerevisiae) 3-hydroxy-3-methylglutaryl CoA reductase gene HMG1.

Primers tHMG1-PGK1-F and tHMG1-ADH1-R in Table 5 were used as templates to amplify the tHMG1 gene encoding the HMGR catalytic domain (1634bp,SEQ ID NO:3); the Saccharomyces cerevisiae INVSc1 genomic DNA was used as a template, and primers PGK1-CYC1-F/PGK1-tHMG1-R and ADH1-tHMG1-F/ADH1-R in Table 5 were used for amplification to obtain sequences of Saccharomyces cerevisiae promoter PGK1 (750bp,SEQ ID NO:19) and terminator ADH1 (158bp,SEQ ID NO:20), respectively. OE-PC with primers PGK1-CYC1-F and ADH1-R Using OE-PCRR, fusion to obtain a tHMG1 gene expression cassette element: p (P) _PGK1 _tHMG1-T _ADH1 。

TABLE 5 cloning primer sequences for genes

4 mu L of OE-PCR product is connected with 1 mu L of cloning vector pEASY-Blunt-Zero vector, and Trans1-T1 competent cells are transformed; positive transformants were selected and plasmids were extracted and designated pEASY-DS-GFP, pEASY-UGTPG1 and pEASY-tHMG1, respectively. After sequencing, the sequencing result of the target fragment is consistent with the theoretical sequence.

Genes IDI1, ERG20, ERG1, ERG9 and ERG7 ^- Construction of expression cassettes

Primers IDI1-TDH3-F and IDI1-TPI1-R (Table 6) were designed based on the DNA sequence (No. NC-001148.4) of the Saccharomyces cerevisiae isopentenyl pyrophosphate isomerase IDI1 gene registered in GenBank.

PCR amplification was performed using Saccharomyces cerevisiae YPH499 genomic DNA as a template and primers IDI1-TDH3-F and IDI1-TPI1-R of Table 6 to obtain IDI1 gene (867bp,SEQ ID NO:4); the saccharomyces cerevisiae YPH499 genome DNA is used as a template, and overlapped extension primers TDH3-TY4-1-F/TDH3-IDI1-R and TPI1-IDI1-F/TPI1-PGK1-R are respectively used for amplification to obtain a saccharomyces cerevisiae promoter TDH3 (800bp,SEQ ID NO:17) sequence fragment and a terminator TPI1 (422bp,SEQ ID NO:21) sequence fragment. The primer TDH3-TY4-1-F and the primer TPI1-PGK1-R are used for carrying out OE-PCR, and fusion is carried out to obtain the IDI1 gene expression cassette element: p (P) _TDH3 -IDI1-T _TPI1 。

Primers ERG20-PGK1-F/ERG20-ADH1-R (Table 6) were designed based on the DNA sequence of the GenBank registered Saccharomyces cerevisiae farnesyl pyrophosphate synthase gene ERG20 (No. NC-001142.9).

PCR amplification was performed using Saccharomyces cerevisiae YPH499 genomic DNA as a template and the primers ERG20-PGK1-F and ERG20-ADH1-R of Table 6 to obtain ERG20 gene (1059bp,SEQ ID NO:5); to make wineYeast YPH499 genomic DNA was used as a template, and overlapping extension primers PGK1-TPI1-F/PGK1-ERG20-R and ADH1-ERG20-F/ADH1-TEF1-R in Table 6 were used to amplify the sequences to obtain Saccharomyces cerevisiae promoter PGK1 (750bp,SEQ ID NO:19) and terminator ADH1 (158bp,SEQ ID NO:20). OE-PCR was performed with primers PGK1-TPI1-F and ADH1-TEF1-R using OE-PCR, and fused to obtain ERG20 gene expression cassette elements: p (P) _PGK1 -ERG20-T _ADH1 。

Primers ERG1-PGK1-F/ERG1-ADH1-R (Table 6) were designed based on the DNA sequence of the Saccharomyces cerevisiae squalene monooxygenase gene ERG1 registered in GenBank (No. NC-001139.9).

PCR amplification was performed using Saccharomyces cerevisiae YPH499 genomic DNA as a template and the primers ERG1-PGK1-F/ERG1-ADH1-R of Table 6 to obtain ERG1 gene (1491bp,SEQ ID NO:6); saccharomyces cerevisiae YPH499 genomic DNA is used as a template, and overlapping extension primers PGK1-CYC1-F/PGK1-ERG1-R and ADH1-ERG1-F/ADH1-TEF1-R in Table 6 are respectively used for amplification to obtain Saccharomyces cerevisiae promoter PGK1 (750bp,SEQ ID NO:19) and terminator ADH1 (158bp,SEQ ID NO:20) sequence fragments. OE-PCR was performed with primers PGK1-CYC1-F and ADH1-TEF1-R using OE-PCR, and fused to obtain ERG1 gene expression cassette elements: p (P) _PGK1 -ERG1-T _ADH1 。

Primers ERG9-TEF1-F/ERG9-CYC1-R (Table 6) were designed based on the DNA sequence of the Saccharomyces cerevisiae squalene synthase gene ERG9 registered in GenBank (No. NC-001140.6).

PCR amplification was performed using Saccharomyces cerevisiae YPH499 genomic DNA as a template and the primers ERG9-TEF1-F/ERG9-CYC1-R of Table 6 to obtain ERG9 gene (1335bp,SEQ ID NO:7); saccharomyces cerevisiae promoter TEF1 (430bp,SEQ ID NO:15) and terminator CYC1 (189bp,SEQ ID NO:16) sequence fragments are amplified by using Saccharomyces cerevisiae YPH499 genomic DNA as a template and overlapping extension primers TEF1-ADH1-F/TEF1-ERG9-R and CYC1-ERG9-F/CYC1-PGK1-R in Table 6 respectively. OE-PCR was performed with primers TEF1-ADH1-F and CYC1-PGK1-R using OE-PCR, and fused to obtain ERG9 gene expression cassette elements: p (P) _TEF1 -ERG9-T _CYC1 。

With plasmid pESC-URA-ERG7 ^- For the template (Wang Qinghua et al, antisense RNA technology was used to inhibit the expression of the Saccharomyces cerevisiae lanosterol synthase gene, pharmaceutical report, acta Pharmaceutic Sinica 2015,50 (1): 118-122, which is incorporated herein by reference in its entirety), amplifying an ERG7 antisense gene long fragment with primers ERG7-TEF1-F/ERG7-CYC1-R in Table 6 (2247bp,SEQ ID NO:8); the Saccharomyces cerevisiae promoter TEF1 (430bp,SEQ ID NO:15) and terminator CYC1 (189bp,SEQ ID NO:16) sequence fragments were amplified using Saccharomyces cerevisiae genomic DNA as a template and the primers TEF1-ADH1-F/TEF1-ERG7-R and CYC1-ERG7-F/CYC1-LEU-R listed in Table 6, respectively. OE-PCR was performed with primers TEF1-ADH1-F and CYC1-LEU-R using OE-PCR, and fusion to obtain ERG7 antisense gene expression cassette elements: p (P) _TEF1 -ERG7 ^- -T _CYC1 。

TABLE 6 cloning primer sequences for genes

4 mu L of OE-PCR product is connected with 1 mu L of cloning vector pEASY-Blunt-Zero vector, and Trans1-T1 competent cells are transformed; positive transformants were selected and plasmids were extracted and designated pEASY-IDI1, pEASY-ERG1, pEASY-ERG20, pEASY-ERG9 and pEASY-ERG7, respectively. Sequencing confirmed that the sequence of the fragment of interest was identical to that expected.

Construction of the expression cassettes for the genes BiP, HAC1 and PDI1

Primers BIP-TEF1-F and BIP-CYC1-R (Table 7) were designed based on the DNA sequence of the Saccharomyces cerevisiae chaperone BiP gene registered in GenBank (No. NC-001142.9).

PCR amplification was performed using Saccharomyces cerevisiae YPH499 genomic DNA as a template and the primers BIP-TEF1-F and BIP-CYC1-R in Table 7 to obtain the BiP gene (2049bp,SEQ ID NO:9); saccharomyces cerevisiae genomic DNA is used as a template, and overlapped extension primers GJ-F/TEF1-BIP-R and CYC1-BIP-F/GJ-R in Table 7 are respectively used for amplification to obtain sequences of Saccharomyces cerevisiae promoter TEF1 (430bp,SEQ ID NO:15) and terminator CYC1 (189bp,SEQ ID NO:16). OE-PCR was performed with primers GJ-F and GJ-R using OE-PCR, fusionObtaining a BiP gene expression cassette element: p (P) _TEF1 -BiP-T _CYC1 。

Primers HAC1-TEF1-F and HAC1-CYC1-R (Table 7) were designed based on the DNA sequence of the Saccharomyces cerevisiae transcription factor HAC1 gene registered in GenBank (No. NC-001138.5).

PCR amplification was performed using Saccharomyces cerevisiae YPH499 genomic DNA as a template and the primers HAC1-TEF1-F and HAC1-CYC1-R of Table 7 to obtain the HAC1 gene (717bp,SEQ ID NO:10); saccharomyces cerevisiae YPH499 genomic DNA is used as a template, and overlapping extension primers GJ-F/TEF1-HAC1-R and CYC1-HAC1-F/GJ-R in Table 7 are respectively used for amplification to obtain Saccharomyces cerevisiae promoter TEF1 (430bp,SEQ ID NO:15) and terminator CYC1 (189bp,SEQ ID NO:16) sequence fragments. And (3) carrying out OE-PCR by using OE-PCR and primers GJ-F and GJ-R, and fusing to obtain the BiP gene expression cassette element: p (P) _TEF1 -HAC1-T _CYC1 。

Primers PDI1-TEF1-F and HAC1-CYC1-R (Table 7) were designed based on the DNA sequence (No. NC-001135.5) of the Saccharomyces cerevisiae chaperone (disulfide isomerase) PDI1 gene registered in GenBank.

PCR amplification was performed using Saccharomyces cerevisiae YPH499 genomic DNA as a template and the primers PDI1-TEF1-F and PDI1-CYC1-R in Table 7 to obtain PDI1 gene (1569bp,SEQ ID NO:11); saccharomyces cerevisiae YPH499 genome DNA is used as a template, and overlapped extension primers GJ-F/TEF1-PDI1-R and CYC1-PDI1-F/GJ-R are respectively used for amplification to obtain sequences of Saccharomyces cerevisiae promoter TEF1 (430bp,SEQ ID NO:15) and terminator CYC1 (189bp,SEQ ID NO:16). And (3) carrying out OE-PCR by using OE-PCR and primers GJ-F and GJ-R, and fusing to obtain the PDI1 gene expression cassette element: p (P) _TEF1 -PDI1-T _CYC1 。

TABLE 7 cloning of genes and plasmid construction primer sequences

4 mu L of OE-PCR product is connected with 1 mu L of cloning vector pEASY-Blunt-Zero vector, and Trans1-T1 competent cells are transformed; positive transformants were selected and plasmids were extracted and designated pEASY-BiP, pEASY-HAC1 and pEASY-PDI1, respectively. Sequencing confirmed that the sequence of the fragment of interest was identical to that expected.

EXAMPLE 4 construction of genomic integration modules

Integration module Iδ1-1-P _TEF1 -synDS-GFP-T _CYC1 -P _PGK1 -tHMG1-T _ADH1 Construction of (3)

The Saccharomyces cerevisiae INVSc1 genomic DNA was used as a template, and primers Delta1-2F and Delta2-1R listed IN Table 8 were used, respectively, to amplify a Saccharomyces cerevisiae genome Delta1 (Delta 1) site sequence fragment (410bp,SEQ ID NO:22), which was ligated to pEASY-Blunt-Simple vector, to obtain plasmid pEASY-IN Delta. The plasmid pEASY-IN Delta was used as template and the primers Delta1-2F and Delta1-TEF1-2R listed IN Table 8 were used to amplify the fragment of the genomic integration site Delta 1-1 and the primers Delta1-2F and CYC1-PGK1-R IN Table 8 were used to amplify the fragment of Delta 1-1 and P by OE-PCR _TEF1 -synDSGFP-T _CYC1 Fusion is carried out to obtain the element delta 1-1-P _TEF1 -synDS-GFP-T _CYC1 。

Reference to OE-PCR reaction System and conditions, as element δ1-1-P _TEF1 -synDS-GFP-T _CYC1 And P _PGK1 -tHMG1-T _ADH1 As templates, a second round of OE-PCR was performed with primers Delta1-2F and ADH1-TDH3-R as set forth in Table 8, and fused to give an integration module I.delta.1-1-P _TEF1 -synDS-GFP-T _CYC1 -P _PGK1 -tHMG1-T _ADH1 . 4 mu L of the second round OE-PCR product is connected with 1 mu L of cloning vector pEASY-Blunt-Zero vector, and Trans1-T1 competent cells are transformed; positive transformants were selected, plasmids were extracted and sequenced to verify that the recombinant plasmid was designated pEASY-3. Gel electrophoresis and DNA sequencing showed that the amplified fragments were identical to the theoretical sequences (FIG. 7).

Integration module II overlap-P _TDH3 -synUGTPg1-T _ADH2 Construction of HIS-delta 1-2

The 455bp tHMGR gene expression cassette 3' fragment amplified using the plasmid pEASY-3 as a template, the primers B-400F and ADH1-TDH3-R as set forth in Table 8 was used for OE-PCR with reference to the OE-PCR reaction system and conditions, and element P _TDH3 -synUGTPg1-T _ADH2 And the fragment obtained by the amplification was used as a template, and OE-PCR was performed with primers B400-F and ADH2-HIS-R as shown in Table 8, and fused to obtain element overlay-P _TDH3 -synUGTPg1-T _ADH2 。

The plasmid pESC-HIS was used as a template, and the primers HIS-ADH2-F and HIS-Delta2-R listed in Table 8 were used to amplify the resistance marker gene HIS expression cassette sequence (1169bp,SEQ ID NO:23); the genomic integration site δ1-2 fragment was amplified using plasmid pEASY-IN Delta as template and the primers Delta2-HIS-1F and Delta2-1R listed IN Table 8. Referring to the OE-PCR reaction system and conditions, the δ1-2 fragment was fused with the gene HIS expression cassette sequence using primers HIS-ADH2-F and Delta2-1R in Table 8 to obtain HIS- δ1-2.

With reference to OE-PCR reaction System and conditions, the elements overlap-P _TDH3 -synUGTPg1-T _ADH2 And HIS-Delta 1-2 as templates, and performing a second round of OE-PCR with primers B400-F and Delta2-1R listed in Table 8, and fusing to obtain an integration module II overlapping-P _TDH3 -synUGTPg1-T _ADH2 HIS-delta 1-2. 4 mu L of the second round OE-PCR product is connected with 1 mu L of cloning vector pEASY-Blunt-Zero vector, and Trans1-T1 competent cells are transformed; positive transformants were selected, plasmids were extracted and sequenced to verify, and the recombinant plasmid was designated pEASY-2. Gel electrophoresis and DNA sequencing showed that the amplified fragments were identical to the theoretical sequences (FIG. 7).

Integration module III delta 4-1-P _TDH3 -IDI1-T _TPI1 -P _PGK1 -ERG20-T _ADH1 Construction of (3)

The Saccharomyces cerevisiae YPH499 genomic DNA was used as a template, and primers TY4-F1 and TY4-R2 listed in Table 8 were used to amplify a Saccharomyces cerevisiae genome Delta4 (Delta 4) site sequence fragment (371bp,SEQ ID NO:24), which was ligated to pEASY-Blunt-Simple vector to obtain plasmid pEASY-TY4. The plasmid pEASY-TY4 was used as a template, and primers TY4-F1 and TY4-1-TDH3-R listed in Table 8 were used to amplify the fragment of the genomic integration site δ4-1, and the primers TY4-F1 and TPI1-PGK1-R of Table 8 were used to amplify the fragment of δ4-1 and P by OE-PCR _TDH3 -IDI1-T _TPI1 Fusion is carried out to obtain the element delta 4-1-P _TDH3 -IDI1-T _TPI1 。

Reference to OE-PCR reaction System and conditions, as element delta 4-1-P _TDH3 -IDI1-T _TPI1 And P _PGK1 -ERG20-T _ADH1 As templates, a second round of OE-PCR was performed with primers TY4-F1 and ADH1-TEF1-R as listed in Table 8, and fused to obtain integration module IIIδ4-1-P _TDH3 -IDI1-T _TPI1 -P _PGK1 -ERG20-T _ADH1 . 4 mu L of the second round OE-PCR product is connected with 1 mu L of cloning vector pEASY-Blunt-Zero vector, and Trans1-T1 competent cells are transformed; positive transformants were selected, plasmids were extracted and sequenced to verify, and the recombinant plasmid was designated pEASY-S28. Gel electrophoresis and DNA sequencing showed that the amplified fragments were identical to the theoretical sequences (FIG. 8).

Integrated module VP _PGK1 -ERG1-T _ADH1 -P _TEF1 -ERG7 ^- -T _CYC1 Construction of LEU-delta 4-2

The plasmid pESC-LEU was used as a template, and the primers LEU-CYC1-F and LEU-TY4-2-R listed in Table 8 were used to amplify the expression cassette sequence of the resistance marker gene LEU2 (2178bp,SEQ ID NO:25); the plasmid pEASY-TY4 was used as a template and the primers TY4-2-LEU-F and TY4-2R listed in Table 8 were used to amplify the fragment of the genomic integration site δ4-2. The delta 4-2 fragment and the gene LEU2 expression cassette sequence are fused by using a primer LEU-CYC1-F/TY4-2R according to an OE-PCR reaction system and conditions to obtain LEU 2-delta 4-2.

Reference to OE-PCR reaction System and conditions, element P _PGK1 -ERG1-T _ADH1 ，P _TEF1 -ERG7 ^- -T _CYC1 And LEU 2-delta 4-2 as template, and performing a second round of OE-PCR with primers PGK1-CYC1-F and TY4-2R listed in Table 8, and fusing to obtain an integrated module VP _PGK1 -ERG1-T _ADH1 -P _TEF1 -ERG7 ^- -T _CYC1 LEU 2-delta 4-2. 4 mu L of the second round OE-PCR product is connected with 1 mu L of cloning vector pEASY-Blunt-Zero vector, and Trans1-T1 competent cells are transformed; positive transformants were selected, plasmids were extracted and sequenced for verification, and the recombinant plasmid was designated pEASY-S1319. Gel electrophoresis and DNA sequencing showed that the amplified fragments were identical to the theoretical sequences (FIG. 8).

Integration module IVoverlap-P _TEF1 -ERG9-T _CYC1 Construction of an overlay

The fragment at the 3' end of the 515bp ERG20 gene expression cassette was amplified using plasmid pEASY-S28 as a template and the primers S28-400F/TEF1-ERG9-R as set forth in Table 8 as a first fragment for OE-PCR; amplified with the plasmid pEASY-S1319 as a template and the primers PGK1-CYC1-F and S1319-400R as set forth in Table 8Fragments 5' to 548bp of the ERG1 gene expression cassette as a second fragment for OE-PCR; reference to OE-PCR reaction System and conditions, element P _TEF1 -ERG9-T _CYC1 And the two fragments obtained by the amplification are used as templates, and primers S28-400F and S1319-400R listed in Table 8 are used for OE-PCR, and are fused to obtain an integration module IVoverlap-P _TEF1 -ERG9-T _CYC1 -overlap. 4 mu L of the second round OE-PCR product is connected with 1 mu L of cloning vector pEASY-Blunt-Zero vector, and Trans1-T1 competent cells are transformed; positive transformants were selected, plasmids were extracted and sequenced for verification, and the recombinant plasmid was designated as pEASY-S813. Gel electrophoresis and DNA sequencing showed that the amplified fragments were identical to the theoretical sequences (FIG. 8).

Construction of recombinant vector prDNA-TRP

The primers rDNA1-MQWD-F and rDNA2-MQWD-R listed in Table 8 were used as templates to amplify rDNA locus sequences (1264bp,SEQ ID NO:26) of Saccharomyces cerevisiae genome, which were ligated to pEASY-Blunt-Simple vector to obtain plasmid pEASY-rDNA. The plasmid pEASY-rDNA is used as a template, and primers GJ-RDNA1-2U-F and rDNA1-MQWD-R listed in Table 8 are used for amplification to obtain a genome integration site rDNA-1 fragment; amplifying rDNA2-TRP-F and GJ-RDNA2-PUC-R with primers listed in Table 8 to obtain a genomic integration site rDNA-2 fragment; the plasmid pESC-TRP was used as a template, and the primers TRP-MQWD-F and TRP-rDNA2-R listed in Table 8 were used for amplification to obtain a resistance marker gene TRP expression cassette sequence (1365bp,SEQ ID NO:27); plasmid backbone sequences were amplified using plasmid pESC-TRP as a template and the primers PUC-GJ-RDNA2-F and 2U-GJ-RDNA1-R as listed in Table 8. Seamless connection is carried out on the four fragments by referring to an eFuse reaction system and conditions; transforming the ligation product into Trans1-T1 competent cells; positive transformants were screened, plasmids were extracted and sequenced to verify that the recombinant plasmids were designated as forming plasmid prDNA-TRP.

TABLE 8 genomic integration module primer sequences

Integration module VIrDNA1-P _TEF1 -BiP-T _CYC1 Construction of rDNA2

Plasmid prDNA-TRP and pEASY-BiP (obtained in example 3) were subjected to double cleavage treatment using restriction enzymes Sal I and Xho I; cutting glue by using a glue recovery kit to recover the target gene fragment and the plasmid vector after enzyme cutting; p was ligated by T4 DNA ligase _TEF1 -BiP-T _CYC1 Ligating to the prDNA-TRP to obtain a recombinant prDNA-TRP-BiP; plasmid prDNA-TRP-BiP was treated with restriction enzymes BamH I and Sac I to obtain integration module VIrDNA1-P _TEF1 -BiP-T _CYC1 rDNA2. Gel electrophoresis and DNA sequencing showed that the amplified fragments were identical to the theoretical sequences (FIG. 9).

Integration module VII rDNA1-P _TEF1 -HAC1-T _CYC1 Construction of rDNA2

Plasmid prDNA-TRP and pEASY-HAC1 (obtained in example 3) were subjected to double cleavage treatment using restriction enzymes Sal I and Xho I; cutting glue by using a glue recovery kit to recover the target gene fragment and plasmid vector after enzyme cutting; p was ligated by T4 DNA ligase _TEF1 -HAC1-T _CYC1 Ligating to the prDNA-TRP to obtain a recombinant prDNA-TRP-HAC1; plasmid prDNA-TRP-HAC1 was treated with restriction enzymes BamH I and Sac I to obtain integration module VII rDNA1-P _TEF1 -HAC1-T _CYC1 rDNA2. Gel electrophoresis and DNA sequencing showed that the amplified fragments were identical to the theoretical sequences (FIG. 9).

Integration module VIII rDNA1-P _TEF1 -PDI1-T _CYC1 Construction of rDNA2

Plasmid prDNA-TRP and pEASY-PDI1 (obtained in example 3) were subjected to double cleavage treatment using restriction enzymes Sal I and Xho I; cutting glue by using a glue recovery kit to recover the target gene fragment and the plasmid vector after enzyme cutting; p was ligated by T4 DNA ligase _TEF1 -PDI1-T _CYC1 Ligating to the prDNA-TRP to obtain a recombinant prDNA-TRP-PDI1; plasmid prDNA-TRP-PDI1 was treated with restriction enzymes BamHI and SacI to obtain integrated module VIIIrDNA1-P _TEF1 -PDI1-T _CYC1 rDNA2. Gel electrophoresis and DNA sequencing showed that the amplified fragments were identical to the theoretical sequences (FIG. 9).

The construction schematic of each integration module is shown in fig. 10.

Wherein, the double enzyme digestion system and enzyme digestion conditions are as follows:

double cleavage System (100. Mu.L):

cleavage was carried out overnight at 37 ℃.

Wherein the connection system and connection conditions used are as follows:

ligation system (20 μl):

e.coli Trans1-T1 competent cells were transformed immediately by ligation for 30min at room temperature; positive transformants were screened and plasmids were extracted.

The eFuse connection system and connection conditions used are as follows:

ligation system (15 μl):

Construction of Cas9 expression plasmids

A CRISPR-Cas9 expression system based on the saccharomyces cerevisiae genome delta 1 site was constructed using a codon humanized Cas9 protein derived from streptococcus pyogenes (Streptococcus pyogenes).

PCR was performed using plasmid FM-1 (Zhang et al, 2016,Fungal Genet Biol,86:47-57) as a template and the primers Cas9-TEF1-F/Cas9-ADH2-R set forth in Table 9,amplifying to obtain a codon humanized Cas9 gene sequence (4272bp,SEQ ID NO:28) from streptococcus pyogenes; saccharomyces cerevisiae genome YPH499 was used as a template, and primers TEF1-SUP4t-MSC-F/TEF1-Cas9-R, ADH2-Cas9-F/ADH2-pESC-R listed in Table 9 were used as primers, respectively, to amplify and obtain sequences of Saccharomyces cerevisiae promoter TEF1p (430bp,SEQ ID NO:15) and terminator ADH2t (566bp,SEQ ID NO:29). OE-PCR was performed with primers TEF1-SUP4t-MSC-F and ADH2-pESC-R listed in Table 9, and fused to obtain Cas9 gene expression cassette elements: p (P) _TEF1 -Cas9-T _CYC1 . Gel electrophoresis and DNA sequencing showed that the amplified fragments were identical to the theoretical sequences (FIG. 11).

A delta 1 site-specific gRNA expression cassette sequence fragment (458bp,SEQ ID NO:30) was synthesized, which contained the RNA polymerase III nucleolar microRNA (snorRNA) promoter SNR52p and the yeast tRNA gene terminator SUP4t, and was ligated to plasmid pUC57, designated pUC57-sgRNA. The delta 1 site-specific gRNA expression cassette sequence fragments were amplified using the plasmid pUC57-sgRNA as template and the primers SNR52p-MSC-pESC-F/SUP4t-MSC-TEF1-R as listed in Table 9, respectively. The plasmid backbone fragment was obtained by amplification using pESC-URA as a template and the primers pESC-ADH2-F/pESC-SNR52P-MCS-R as shown in Table 9.

Reference to OE-PCR reaction System and conditions, element P _TEF1 -Cas9-T _CYC1 And the gRNA expression cassette sequence as a template, and were subjected to a second round of OE-PCR with primers SNR52P-MSC-pESC-F and ADH2-pESC-R as set forth in Table 9, to obtain DNA element gRNA-P _TEF1 -Cas9-T _CYC1 . Gel electrophoresis and DNA sequencing showed that the amplified fragments were identical to the theoretical sequences (FIG. 11).

The DNA element gRNA-P was subjected to eFuse reaction system and conditions _TEF1 -Cas9-T _CYC1 Seamless connection is carried out with the plasmid skeleton segment; transforming the ligation product into Trans1-T1 competent cells; positive transformants were screened, plasmids were extracted and sequenced to verify that the recombinant plasmid was designated p-Cas 9-delta.

TABLE 9 construction of CRISPR-Cas9 expression plasmid primer tables

Table 10 constructed recombinant plasmid

Example 5 Saccharomyces cerevisiae transformation and recombinant screening

The integration module Idelta 1-1-P is transformed by LiAc/SS Carrier DNA/PEG _TEF1 -synDS-GFP-T _CYC1 -P _PGK1 -tHMG1-T _ADH1 And integration module II overlap-P _TDH3 -synUGTPg-T _ADH2 -HIS-delta 1-2 transformed Saccharomyces cerevisiae mutant Y-delta HXK2 to construct a recombinant yeast Y2 producing 20S-O-Glc-DM; at the same time, the integrated module Idelta 1-1-P _TEF1 -synDS-GFP-T _CYC1 -P _PGK1 -tHMG1-T _ADH1 Integration module II overlay-P _TDH3 -synUGTPg1-T _ADH2 The HIS-delta 1-2 and Cas9 expression plasmid p-Cas 9-delta are used for transforming saccharomyces cerevisiae mutant strain Y-delta HXK2 to construct yeast recombinant strain Y2C. Positive transformants were selected on SD auxotroph medium. Transformants are picked from SD plates respectively, genomic DNA is extracted as a template, corresponding specific primers are selected for PCR amplification, and correct introduction of the gene module is verified.

Will integrate module III delta 4-1-P _TDH3 -IDI1-T _TPI1 -P _PGK1 -ERG20-T _ADH1 Integration module IVoverlap-P _TEF1 -ERG9-T _CYC1 -overlay and integration module VP _PGK1 -ERG1-T _ADH1 -P _TEF1 -ERG7 ^- -T _CYC1 And (3) converting the Saccharomyces cerevisiae recombinant strain Y2C by LEU-delta 4-2 to construct a yeast recombinant strain Y2CS. Positive transformants were selected on SD auxotroph medium. Transformants were picked from SD plates, respectively, and genomic DNA was extracted as a templateAnd (3) selecting corresponding specific primers for PCR amplification, and verifying the correct introduction of the gene module.

The integration modules VIrDNA1-P are respectively _TEF1 -BiP-T _CYC1 rDNA2, integration module VII rDNA1-P _TEF1 -HAC1-T _CYC1 rDNA2, integration module VIII rDNA1-P _TEF1 -PDI1-T _CYC1 And (3) converting rDNA2 into saccharomyces cerevisiae recombinant bacteria Y2CS12, and constructing yeast recombinant bacteria Y2CSB, Y2CSH and Y2CSP. Positive transformants were randomly selected on SD auxotroph medium. Transformants are picked from SD plates respectively, genomic DNA is extracted as a template, corresponding specific primers are selected for PCR amplification, and correct introduction of the gene module is verified.

Example 6 Standard Curve of Standard 20S-O-Glc-DM

Precisely weighing 5.0mg of 20S-O-Glc-DM, dissolving with methanol to prepare 1.0mg/mL mother liquor, respectively preparing five standard solutions of 1.0mg/mL, 0.5mg/mL, 0.25mg/mL, 0.125mg/mL and 0.0625mg/mL, and taking 10 mu L of sample under chromatographic conditions (HPLC condition detection condition: cosmosil C18 reverse phase column, 4.6X106 mm, flow rate of 1mL/min, ultraviolet detection wavelength of 203nm, mobile phase condition: 0min,58% ACN, 30min,58% ACN) for 3 times, taking the average value of peak areas as ordinate, and sample concentration as abscissa, and taking a standard curve.

A standard curve was drawn with the mean of peak areas as ordinate and the sample concentration as abscissa (fig. 12). The linear regression equation for 20S-O-Glc-DM in the range of 0.0625-1.0 mg/mL is y= 4901.6x-51.057, R ² ＝0.9994。

Example 7 screening of recombinant bacteria producing 20S-O-Glc-DM and optimized recombinant bacteria

And extracting recombinant bacterium fermentation products, and carrying out HPLC and LC-MS detection. HPLC results showed that compounds consistent with UV absorption and Rt of the 20S-O-Glc-DM standard appeared in both recombinant and culture medium extracts (FIG. 13). LC-MS results showed that the fragment ion peak of this compound was consistent with the 20S-O-Glc-DM standard (fig. 14).

HPLC condition detection conditions: cosmosil C18 reverse phase column, 4.6X106 mm, flow rate of 1mL/min, ultraviolet detection wavelength of 203nm, and sample injection of 10. Mu.L. The mobile phase conditions were: 0min,58% ACN;30min,58% ACN.

Randomly picking 20 positive transformants of the yeast recombinant bacteria Y2 and Y2C respectively, and culturing for 12h at 30 ℃ and 200rpm in 10mL YPD culture medium; determination of OD ₆₀₀ (10-20), a proper amount of culture solution is taken and transferred to 50mL YPD medium to make the final OD ₆₀₀ 0.2. Culturing recombinant bacteria Y2 and Y2C 3d at 30 ℃ and 220rpm, and respectively centrifuging and collecting the bacteria.

Taking 1.0g of recombinant fungus dry fungus, adding 100mL of 70% ethanol, refluxing at 70 ℃ for 1h, naturally cooling, filtering to remove fungus residues, evaporating the extracting solution under reduced pressure, dissolving with 100mL of water, extracting with 100mL of water saturated n-butanol for 3 times respectively, and standing for 1h each time. The extracts were combined and evaporated to dryness, and the resulting mixture was filtered through a 0.22 μm filter, and then 10. Mu.L of the mixture was introduced, followed by HPLC detection, whereby the content of 20S-O-Glc-DM in the recombinant cells was determined according to a standard curve. The result shows that only two strains of 20S-O-Glc-DM are produced in 20 transformants of recombinant strain Y2; in the yeast recombinant strain mediated by CRISPR/Cas9 technology, the yield of 20S-O-Glc-DM is obviously improved, wherein the yield of the No. 14 recombinant strain is highest. The genotype of recombinant Y2C and the screening result are shown in FIG. 15.

And respectively randomly picking 20 positive transformants of the yeast recombinant strain Y2CS, and carrying out recombinant strain fermentation and product extraction detection. And detecting by HPLC, and determining the content of 20S-O-Glc-DM in the recombinant bacteria according to a standard curve. The results show that the yield of 20S-O-Glc-DM in the recombinant strain is further increased by overexpressing a key enzyme upstream of the 20S-O-Glc-DM biosynthetic pathway, wherein the yield of the recombinant strain of yeast No. 12 is the highest. The genotype of recombinant Y2CS and the screening result are shown in FIG. 16.

10 positive transformants of the yeast recombinant strain Y2CSB, Y2CSH and Y2CSP are randomly selected respectively, and recombinant strain fermentation and product extraction detection are carried out. And (3) detecting by HPLC, and determining the content of 20S-O-Glc-DM in each recombinant strain according to a standard curve. The results show that the yield of the transcription factor HAC1, 20S-O-Glc-DM is significantly increased by overexpressing the transcription factor in the yeast recombinant strain Y2CS, wherein the yield of the yeast recombinant strain No. 10 20S-O-Glc-DM is the highest. The genotypes of the recombinant bacteria Y2CSB, Y2CSH and Y2CSP and the screening results are shown in FIG. 17.

Example 8 quantitative detection of 20S-O-Glc-DM recombinant bacteria

Culturing the recombinant yeast strains Y2, Y2C, Y CS and Y2CSH produced by activation of YPD solid plates at 30 ℃ and 220rpm for 24 hours; single colonies were picked up and inoculated into 10mLYPD liquid medium at 30℃and 220rpm, cultured for 12 hours, and then transferred to 100mL YPD medium to give a final OD ₆₀₀ 0.2. Culturing yeast recombinant at 30deg.C and 220rpm, wherein 5mL of feed medium is added to the culture medium at 48h, 72h and 96h respectively, culturing is continued until 6d, respectively centrifuging to collect thallus and fermentation liquid, and cooling to dry.

1.0g of dry fungus is weighed, 100mL of 70% ethanol is added, reflux is carried out at 70 ℃ for 1h, natural cooling is carried out, bacterial residues are removed by suction filtration, the extracting solution is evaporated to dryness under reduced pressure, 100mL of water is used for dissolving, 100mL of water saturated n-butanol is used for extracting for 3 times respectively, and each time is kept stand for 1h. The extracts were combined and evaporated to dryness, and the resulting mixture was filtered through a 0.22 μm filter, and then 10. Mu.L of the mixture was introduced, followed by HPLC detection, whereby the content of 20S-O-Glc-DM in the recombinant cells was determined according to a standard curve.

100mL of the supernatant of the fermentation broth after centrifugation is taken, and is extracted with 100mL of water saturated n-butanol for 3 times respectively, and the mixture is kept stand for 1h each time. The extracts were combined and evaporated to dryness, and the resulting mixture was filtered through a 0.22 μm filter and then subjected to HPLC detection in an amount of 10. Mu.L, followed by measuring the 20S-O-Glc-DM content of the fermentation broth according to a standard curve.

The total yield of the yeast recombinant strain Y2 of 20S-O-Glc-DM is 18.6mg/L by HPLC quantitative analysis; the CRISPR/Cas9 technology is adopted, the total 20S-O-Glc-DM yield of the yeast recombinant strain Y2C obtained by mediating the integration of exogenous genes into yeast genome is 265.1mg/L, and compared with the yield of Y2, the yield of the yeast recombinant strain Y2C is improved by 14.3 times. The total yield of 20S-O-Glc-DM of the obtained yeast recombinant Y2CS is 491.9mg/L by over-expressing the key enzyme upstream of the biosynthesis pathway of 20S-O-Glc-DM, and is improved by 1.86 times compared with the yield of Y2C. The total 20S-O-Glc-DM yield of the yeast recombinant Y2CSH obtained by overexpressing the transcriptional activator HAC1 in the yeast recombinant was 752.8mg/L, which was 1.53-fold higher than the Y2CS yield (FIG. 18).

Example 9 high Density fermentation of 20S-O-Glc-DM engineering bacteria Using exponential fed-batch

Activating engineering bacteria Y2CSH on SD auxotroph solid culture medium, picking single colony, inoculating into 100mL YPD liquid culture medium, and culturing at 30deg.C and 220 rpm. The seed solution was introduced into a 3L fermenter (Shanghai Baoxing biological equipments Co., ltd.) containing 1L of YPD fermentation medium at an inoculum size of 10%. The fermentation temperature is 30 ℃, the ventilation amount is 3L/min, the Dissolved Oxygen (DO) value is 30%, and the stirring speed is 300-900 rpm. The pH was maintained at 5.5.+ -. 0.2 by 5M ammonia. After 20h of fermentation, an exponential feed was started, wherein the feed medium contained 578g/L glucose, 9g/L KH ₂ PO ₄ ，5.12g/L MgSO ₄ ·7H ₂ O，3.5g/L K ₂ SO ₄ ，0.28g/L Na ₂ SO ₄ 2.1g/L adenine, 2.5g/L uracil, 5g/L lysine, 10mL/L microelement solution (15g EDTA,10.2g ZnSO) ₄ ·7H ₂ O，0.5g MnCl ₂ ·4H ₂ O，0.5g CuSO ₄ ，0.86g CoCl ₂ ·6H ₂ O，0.56g Na ₂ MoO ₄ ·2H ₂ O，3.84g CaCl ₂ ·2H ₂ O and 5.12g FeSO ₄ ·7H ₂ O to 1L distilled water, filter sterilized, stored at 4 ℃) and 12mL/L vitamin solution (0.05 g biotin, 1g ubiquitin calcium, 1g niacin, 25g inositol, 1g thiamine hydrochloride, 1g pyridoxine hydrochloride, 0.2g aminobenzoic acid to 1L distilled water, filter sterilized, stored at 4 ℃). The glucose concentration is controlled below 1.0g/L, and the ethanol concentration is not higher than 5.0g/L.

Samples were taken every 24 hours and biomass and product content were determined. Biomass continues to increase before 96h, followed by plateau, fermentation broth OD at 168h ₆₀₀ Reaching a maximum 1125. The yield of 20S-O-Glc-DM increased with cell growth, reaching a maximum of 5.6g/L at 144h (FIG. 19).

It will be apparent to those skilled in the art that various modifications and variations can be made in the methods and recombinant bacteria of the present invention without departing from the spirit or scope of the invention. Accordingly, it is intended that the present invention cover the modifications and variations of this invention provided they come within the scope of the appended claims and their equivalents.

Sequence listing

<110> institute of medicine at the national academy of medical science

<120> recombinant bacterium for producing rare ginsenoside 20S-O-Glc-DM and application thereof

<160> 30

<170> SIPOSequenceListing 1.0

<210> 1

<211> 3036

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 1

atgtggaagc taaaggtagc tcaaggaaat gatccatatt tgtatagcac taacaacttt 60

gttggcagac aatattggga gtttcagccc gatgctggta ctccagaaga gagggaagag 120

gttgaaaaag cacgcaagga ttatgtaaac aacaagaagc ttcatggtat tcatccatgt 180

agtgatatgc tgatgcgcag gcagcttatt aaagaaagtg gaatcgatct cctaagcata 240

ccgccgttga gattagatga aaacgaacaa gtgaactacg atgcagttac aaccgctgtg 300

aagaaagctc ttcgattgaa ccgggcaatt caagcacacg atggtcactg gccagctgaa 360

aatgcaggct ctttacttta tacacctccc cttatcattg ccctatatat cagcggaacg 420

attgacacta ttctgacaaa acaacacaag aaggaactga ttcgcttcgt ttacaaccat 480

caaaatgagg atggtggatg gggatcctat attgaggggc acagcacgat gattgggtca 540

gtacttagct acgtgatgtt acgtttgcta ggagaaggat tagctgaatc tgatgatgga 600

aatggtgcag ttgagagagg ccggaagtgg atacttgatc atggaggtgc agccggcata 660

ccctcttggg gaaagactta tctagcggtg cttggagtat atgagtggga agggtgtaac 720

ccacttccac cagaattttg gctttttcca tctagttttc cttttcatcc agcaaaaatg 780

tggatctact gccggtgcac ttacatgcca atgtcgtatt tgtatgggaa gagatatcat 840

ggaccaataa ccgatcttgt tttatctttg aggcaagaaa tttacaacat tccttatgag 900

cagataaagt ggaatcaaca gcgccataac tgttgcaagg aggatctcta ctaccctcat 960

acccttgtac aagacctggt ttgggatggt cttcactact ttagtgaacc attcctcaaa 1020

cgttggccct tcaacaaact gcgaaaaaga ggtctaaaaa gagttgttga actaatgcgc 1080

tatggtgcca ccgagaccag attcataacc acaggaaatg gggaaaaagc tttacaaata 1140

atgagttggt gggcagaaga tcccaatggt gatgagttta agcatcatct tgctagaatc 1200

ccagattttt tgtggattgc tgaggatgga atgacagtac agagttttgg tagtcaacta 1260

tgggactgta ttcttgctac tcaagcaatt atcgccacca atatggttga agaatacgga 1320

gattctctta agaaggcgca tttcttcatc aaagaatcgc agataaaaga aaatccaaga 1380

ggagacttcc taaaaatgtg tcgacagttt accaaaggtg cgtggacttt ctctgatcaa 1440

gatcatggtt gcgttgtctc ggactgcaca gctgaagcgc taaagtgcct actgttactt 1500

tcacaaatgc cacaggatat tgtcggagaa aaacctgagg ttgagcgatt atatgaggct 1560

gtgaatgttc ttctctattt gcagagtcgt gtaagtggtg gtttcgcagt ttgggagcct 1620

ccagttccaa aaccatattt ggagatgttg aatccttcag aaatttttgc agacattgtt 1680

gttgagagag agcacattga atgcactgca tctgtaatca aaggtctgat ggcatttaaa 1740

tgcttgcatc ctgggcatcg tcagaaagag atagaggatt ctgtggcgaa agccatccgt 1800

tatcttgaaa gaaaccaaat gcctgatggt tcatggtatg gcttttgggg aatttgtttc 1860

ctctatggga cattttttac cctatctggg tttgcttctg ctgggaggac ttatgacaac 1920

agtgaagcag ttcgtaaggg tgttaaattt ttcctttcaa cacaaaatga agaaggtggt 1980

tggggggaga gtcttgaatc atgcccaagc gaaaaattta caccactcaa gggaaacaga 2040

acaaatctag tacaaacatc atgggctatg ctaggtctta tgtttggtgg acaggccgag 2100

agagatccga cacctctgca tagagcagca aagttgttga tcaatgcgca aatggataat 2160

ggagatttcc ctcaacagga aattactgga gtatactgta aaaatagtat gttacattat 2220

gcggagtaca gaaatatatt tcctctttgg gcactcggag aatatcggaa acgtgtttgg 2280

ttgcctaagc accagcagct caaaattgcg gccgcgatgg tgagcaaggg cgaggagctg 2340

ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc 2400

agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc 2460

tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc 2520

gtgcagtgct tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc 2580

atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag 2640

acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc 2700

atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc 2760

cacaacgtct atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc 2820

cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc 2880

atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccgccctg 2940

agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc 3000

gggatcactc tcggcatgga cgagctgtac aagtaa 3036

<210> 2

<211> 1428

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 2

atgaagtccg aattaatttt cttgcctgca ccagctattg gacacttagt cggaatggtc 60

gaaatggcca agttgttcat tagtagacac gagaacttgt cagtaacagt gttgattgca 120

aagttctata tggataccgg agtggataat tacaataagt cattattaac caatcccact 180

cctagattga caattgtgaa cttgccagaa accgatccac agaattatat gttgaaaccc 240

agacacgcta tctttccttc tgtaatcgaa actcagaaga ctcatgtaag agacatcatt 300

tctggaatga ctcaaagtga aagtacaaga gttgtaggat tattggcaga tttgttgttc 360

attaatataa tggatattgc aaacgagttt aacgttccca cctatgtgta ttctccagct 420

ggtgccggtc atttgggttt ggcatttcat ttgcaaacat tgaatgacaa gaagcaagat 480

gttacagagt ttaggaatag tgacaccgaa ttgttggtgc caagtttcgc taacccagtg 540

cctgccgaag tgttaccatc tatgtatgtt gataaagaag gtggttatga ttacttattc 600

tctttgttta gaaggtgtag agaatccaag gctatcatca tcaatacctt tgaggagtta 660

gaaccctacg ctataaactc tttgagaatg gactctatga tccctccaat atacccagtc 720

ggtccaatat tgaatttgaa cggagatggt cagaactctg atgaagcagc cgtaatattg 780

ggatggttgg atgatcagcc accttcctca gttgtgttct tgtgctttgg ttcttatggt 840

acatttcagg agaaccaagt caaagaaatt gcaatgggct tagagaggtc cggacataga 900

ttcttgtggt cattaagacc atctattcct aagggcgaaa ccaaattaca gttgaaatat 960

tccaacttag aagagatatt gccagttggt ttcttagaca ggacctcctg cgttggaaag 1020

gtgattggtt gggcacctca agtagccgta ttgggtcatg aagctgtagg tggcttctta 1080

tcccattgcg gttggaactc caccttggag tcagtatggt gcggagtacc tgtcgccact 1140

tggcccatgt acggtgaaca acaattgaat gcatttgaaa tggtgaaaga attaggaatc 1200

gctgtcgaaa tagaagtgga ttataagaat gactacttca atatgaagaa tgatttcatt 1260

gtcagagccg aagaaattga aactaagatt aagaagttaa tgatggatga gaataattct 1320

gagattagaa agaaagttaa ggaaatgaag gagaaatcaa gagcagctat gtccgagaat 1380

ggttcatcct ataatagttt agccaaatta tttgaggaga ttatgtaa 1428

<210> 3

<211> 1634

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 3

atggaccaat tggtgaaaac tgaagtcacc aagaagtctt ttactgctcc tgtacaaaag 60

gcttctacac cagttttaac caataaaaca gtcatttctg gatcgaaagt caaaagttta 120

tcatctgcgc aatcgagctc atcaggacct tcatcatcta gtgaggaaga tgattcccgc 180

gatattgaaa gcttggataa gaaaatacgt cctttagaag aattagaagc attattaagt 240

agtggaaata caaaacaatt gaagaacaaa gaggtcgctg ccttggttat tcacggtaag 300

ttacctttgt acgctttgga gaaaaaatta ggtgatacta cgagagcggt tgcggtacgt 360

aggaaggctc tttcaatttt ggcagaagct cctgtattag catctgatcg tttaccatat 420

aaaaattatg actacgaccg cgtatttggc gcttgttgtg aaaatgttat aggttacatg 480

cctttgcccg ttggtgttat aggccccttg gttatcgatg gtacatctta tcatatacca 540

atggcaacta cagagggttg tttggtagct tctgccatgc gtggctgtaa ggcaatcaat 600

gctggcggtg gtgcaacaac tgttttaact aaggatggta tgacaagagg cccagtagtc 660

cgtttcccaa ctttgaaaag atctggtgcc tgtaagatat ggttagactc agaagaggga 720

caaaacgcaa ttaaaaaagc ttttaactct acatcaagat ttgcacgtct gcaacatatt 780

caaacttgtc tagcaggaga tttactcttc atgagattta gaacaactac tggtgacgca 840

atgggtatga atatgatttc taaaggtgtc gaatactcat taaagcaaat ggtagaagag 900

tatggctggg aagatatgga ggttgtctcc gtttctggta actactgtac cgacaaaaaa 960

ccagctgcca tcaactggat cgaaggtcgt ggtaagagtg tcgtcgcaga agctactatt 1020

cctggtgatg ttgtcagaaa agtgttaaaa agtgatgttt ccgcattggt tgagttgaac 1080

attgctaaga atttggttgg atctgcaatg gctgggtctg ttggtggatt taacgcacat 1140

gcagctaatt tagtgacagc tgttttcttg gcattaggac aagatcctgc acaaaatgtt 1200

gaaagttcca actgtataac attgatgaaa gaagtggacg gtgatttgag aatttccgta 1260

tccatgccat ccatcgaagt aggtaccatc ggtggtggta ctgttctaga accacaaggt 1320

gccatgttgg acttattagg tgtaagaggc ccgcatgcta ccgctcctgg taccaacgca 1380

cgtcaattag caagaatagt tgcctgtgcc gtcttggcag gtgaattatc cttatgtgct 1440

gccctagcag ccggccattt ggttcaaagt catatgaccc acaacaggaa acctgctgaa 1500

ccaacaaaac ctaacaattt ggacgccact gatataaatc gtttgaaaga tgggtccgtc 1560

acctgcatta aatcctaaac ttagtcatac gtcattggta ttctcttgaa aaagaagcac 1620

aacagcacca tgtg 1634

<210> 4

<211> 867

<212> DNA

<213> Saccharomyces cerevisiae (Saccharomyces cerevisiae)

<400> 4

atgactgccg acaacaatag tatgccccat ggtgcagtat ctagttacgc caaattagtg 60

caaaaccaaa cacctgaaga cattttggaa gagtttcctg aaattattcc attacaacaa 120

agacctaata cccgatctag tgagacgtca aatgacgaaa gcggagaaac atgtttttct 180

ggtcatgatg aggagcaaat taagttaatg aatgaaaatt gtattgtttt ggattgggac 240

gataatgcta ttggtgccgg taccaagaaa gtttgtcatt taatggaaaa tattgaaaag 300

ggtttactac atcgtgcatt ctccgtcttt attttcaatg aacaaggtga attactttta 360

caacaaagag ccactgaaaa aataactttc cctgatcttt ggactaacac atgctgctct 420

catccactat gtattgatga cgaattaggt ttgaagggta agctagacga taagattaag 480

ggcgctatta ctgcggcggt gagaaaacta gatcatgaat taggtattcc agaagatgaa 540

actaagacaa ggggtaagtt tcacttttta aacagaatcc attacatggc accaagcaat 600

gaaccatggg gtgaacatga aattgattac atcctatttt ataagatcaa cgctaaagaa 660

aacttgactg tcaacccaaa cgtcaatgaa gttagagact tcaaatgggt ttcaccaaat 720

gatttgaaaa ctatgtttgc tgacccaagt tacaagttta cgccttggtt taagattatt 780

tgcgagaatt acttattcaa ctggtgggag caattagatg acctttctga agtggaaaat 840

gacaggcaaa ttcatagaat gctataa 867

<210> 5

<211> 1059

<212> DNA

<213> Saccharomyces cerevisiae (Saccharomyces cerevisiae)

<400> 5

atggcttcag aaaaagaaat taggagagag agattcttga acgttttccc taaattagta 60

gaggaattga acgcatcgct tttggcttac ggtatgccta aggaagcatg tgactggtat 120

gcccactcat tgaactacaa cactccaggc ggtaagctaa atagaggttt gtccgttgtg 180

gacacgtatg ctattctctc caacaagacc gttgaacaat tggggcaaga agaatacgaa 240

aaggttgcca ttctaggttg gtgcattgag ttgttgcagg cttacttctt ggtcgccgat 300

gatatgatgg acaagtccat taccagaaga ggccaaccat gttggtacaa ggttcctgaa 360

gttggggaaa ttgccatcaa tgacgcattc atgttagagg ctgctatcta caagcttttg 420

aaatctcact tcagaaacga aaaatactac atagatatca ccgaattgtt ccatgaggtc 480

accttccaaa ccgaattggg ccaattgatg gacttaatca ctgcacctga agacaaagtc 540

gacttgagta agttctccct aaagaagcac tccttcatag ttactttcaa gactgcttac 600

tattctttct acttgcctgt cgcattggcc atgtacgttg ccggtatcac ggatgaaaag 660

gatttgaaac aagccagaga tgtcttgatt ccattgggtg aatacttcca aattcaagat 720

gactacttag actgcttcgg taccccagaa cagatcggta agatcggtac agatatccaa 780

gataacaaat gttcttgggt aatcaacaag gcattggaac ttgcttccgc agaacaaaga 840

aagactttag acgaaaatta cggtaagaag gactcagtcg cagaagccaa atgcaaaaag 900

attttcaatg acttgaaaat tgaacagcta taccacgaat atgaagagtc tattgccaag 960

gatttgaagg ccaaaatttc tcaggtcgat gagtctcgtg gcttcaaagc tgatgtctta 1020

actgcgttct tgaacaaagt ttacaagaga agcaaatag 1059

<210> 6

<211> 1491

<212> DNA

<213> Saccharomyces cerevisiae (Saccharomyces cerevisiae)

<400> 6

atgtctgctg ttaacgttgc acctgaattg attaatgccg acaacacaat tacctacgat 60

gcgattgtca tcggtgctgg tgttatcggt ccatgtgttg ctactggtct agcaagaaag 120

ggtaagaaag ttcttatcgt agaacgtgac tgggctatgc ctgatagaat tgttggtgaa 180

ttgatgcaac caggtggtgt tagagcattg agaagtctgg gtatgattca atctatcaac 240

aacatcgaag catatcctgt taccggttat accgtctttt tcaacggcga acaagttgat 300

attccatacc cttacaaggc cgatatccct aaagttgaaa aattgaagga cttggtcaaa 360

gatggtaatg acaaggtctt ggaagacagc actattcaca tcaaggatta cgaagatgat 420

gaaagagaaa ggggtgttgc ttttgttcat ggtagattct tgaacaactt gagaaacatt 480

actgctcaag agccaaatgt tactagagtg caaggtaact gtattgagat attgaaggat 540

gaaaagaatg aggttgttgg tgccaaggtt gacattgatg gccgtggcaa ggtggaattc 600

aaagcccact tgacatttat ctgtgacggt atcttttcac gtttcagaaa ggaattgcac 660

ccagaccatg ttccaactgt cggttcttcg tttgtcggta tgtctttgtt caatgctaag 720

aatcctgctc ctatgcacgg tcacgttatt cttggtagtg atcatatgcc aatcttggtt 780

taccaaatca gtccagaaga aacaagaatc ctttgtgctt acaactctcc aaaggtccca 840

gctgatatca agagttggat gattaaggat gtccaacctt tcattccaaa gagtctacgt 900

ccttcatttg atgaagccgt cagccaaggt aaatttagag ctatgccaaa ctcctacttg 960

ccagctagac aaaacgacgt cactggtatg tgtgttatcg gtgacgctct aaatatgaga 1020

catccattga ctggtggtgg tatgactgtc ggtttgcatg atgttgtctt gttgattaag 1080

aaaataggtg acctagactt cagcgaccgt gaaaaggttt tggatgaatt actagactac 1140

catttcgaaa gaaagagtta cgattccgtt attaacgttt tgtcagtggc tttgtattct 1200

ttgttcgctg ctgacagcga taacttgaag gcattacaaa aaggttgttt caaatatttc 1260

caaagaggtg gcgattgtgt caacaaaccc gttgaatttc tgtctggtgt cttgccaaag 1320

cctttgcaat tgaccagggt tttcttcgct gtcgcttttt acaccattta cttgaacatg 1380

gaagaacgtg gtttcttggg attaccaatg gctttattgg aaggtattat gattttgatc 1440

acagctatta gagtattcac cccatttttg tttggtgagt tgattggtta a 1491

<210> 7

<211> 1335

<212> DNA

<213> Saccharomyces cerevisiae (Saccharomyces cerevisiae)

<400> 7

atgggaaagc tattacaatt ggcattgcat ccggtcgaga tgaaggcagc tttgaagctg 60

aagttttgca gaacaccgct attctccatc tatgatcagt ccacgtctcc atatctcttg 120

cactgtttcg aactgttgaa cttgacctcc agatcgtttg ctgctgtgat cagagagctg 180

catccagaat tgagaaactg tgttactctc ttttatttga ttttaagggc tttggatacc 240

atcgaagacg atatgtccat cgaacacgat ttgaaaattg acttgttgcg tcacttccac 300

gagaaattgt tgttaactaa atggagtttc gacggaaatg cccccgatgt gaaggacaga 360

gccgttttga cagatttcga atcgattctt attgaattcc acaaattgaa accagaatat 420

caagaagtca tcaaggagat caccgagaaa atgggtaatg gtatggccga ctacatctta 480

gatgaaaatt acaacttgaa tgggttgcaa accgtccacg actacgacgt gtactgtcac 540

tacgtagctg gtttggtcgg tgatggtttg acccgtttga ttgtcattgc caagtttgcc 600

aacgaatctt tgtattctaa tgagcaattg tatgaaagca tgggtctttt cctacaaaaa 660

accaacatca tcagagatta caatgaagat ttggtcgatg gtagatcctt ctggcccaag 720

gaaatctggt cacaatacgc tcctcagttg aaggacttca tgaaacctga aaacgaacaa 780

ctggggttgg actgtataaa ccacctcgtc ttaaacgcat tgagtcatgt tatcgatgtg 840

ttgacttatt tggccggtat ccacgagcaa tccactttcc aattttgtgc cattccccaa 900

gttatggcca ttgcaacctt ggctttggta ttcaacaacc gtgaagtgct acatggcaat 960

gtaaagattc gtaagggtac tacctgctat ttaattttga aatcaaggac tttgcgtggc 1020

tgtgtcgaga tttttgacta ttacttacgt gatatcaaat ctaaattggc tgtgcaagat 1080

ccaaatttct taaaattgaa cattcaaatc tccaagatcg aacagtttat ggaagaaatg 1140

taccaggata aattacctcc taacgtgaag ccaaatgaaa ctccaatttt cttgaaagtt 1200

aaagaaagat ccagatacga tgatgaattg gttccaaccc aacaagaaga agagtacaag 1260

ttcaatatgg ttttatctat catcttgtcc gttcttcttg ggttttatta tatatacact 1320

ttacacagag cgtga 1335

<210> 8

<211> 2247

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 8

tccaaaaaaa agtaagaatt tttgaaaatt cgaattcaac cctcactaaa gggcggccgc 60

cgtttgcggc ttgctgaggg gttaaatatt cccagctttc tcggcctagc tcatcagttc 120

tcagtctcca aagacgtgga tctgtctttg gtagaccgat tgtgtcagaa taaaattctg 180

tcatctgttt tgtactttct ttgtgggcga cgattattgg taaggttatt gggcacagca 240

gtacattact ggagaggctt gtgctcaatg gctttattaa aagctgaggg catgtattat 300

attttttttt ttttgagaaa atgtatatct gtaaataacc gatataaaaa cttttcacct 360

aaacgattat aaacgaaaag tttccgcaga tatcaaatct agaaaagatg ctattgaaag 420

tactatatat gattgcaaat taaaataggg gtggagagaa agctagaaat tatcatggtt 480

cccattttgc ttattcttca ctattgagaa tttaatagta atgttaggga gtcatgcatt 540

aggattcaga aatgtacaat gagatggaca aaaactaagc atgaaaaatg aggaagccga 600

accataacaa gccatatgtc tctgctaatc atgcaacttt accccattag tagcatactt 660

gaaattttaa cacaatatcc ggaaacatcg actttatcga ggaacactaa tacaactctt 720

aactaaacca agctacacca aattaaacgc cagcgcctat aacacggtct agaaatggca 780

taatcaaaat gaatcgaaag gctaagggaa cttgaagtta taaaatgacc cttcagaacc 840

aggtttcatt taaaccgtta ggttgtaaca gtaatcacga atagcagcag attttcttgc 900

actacataat agaggtatac aaaacgcata ccgttaaaac aaaccaactg tgcaccatac 960

aacagcaatc ccagtaattt gccgttatat atgtgattct ttcacctttc cttgcattac 1020

tgttataccc gctctactgt attttttttc attcgatagc aggtacagtg acatctaatg 1080

aaagatgaat gggtatttag ataattctta aaattctgtc ataataaaag ctataatatt 1140

ttaggtgtat ataatataga agacattctc ctcaaggggt attatctccc ttttatcttt 1200

acatactgtc gttcattatc ctattatatt atcaaacctt gcatttcagc tttcattggg 1260

tttggtgact tatctcaatc ttgacgccaa taaacactca aatgagttat ctttttaaca 1320

tcagaatttc aacaaatagt aagcggattt agctcagttg ggagagcgcc agactgaaga 1380

aaaaacttcg gtcaagttat ctggaggtcc tgtgttcgat ccacagaatt cgcatttttg 1440

taaacacaca ctattttttt tttttaaaaa atatttgtat ttcctaacat gacagggcac 1500

atagctgaat agatcaaaag ctcagcaagg aaaaaaaaaa aaaaaaagtt aatttattga 1560

tgaatgaaag atatataaca ttttcgaaat taacaagatt taatctgttt ccaagaattt 1620

gcatcataat cattgtctga ttctctgggt ctcttcaccg gagcgttggc gggagttccg 1680

acgcgcaatt tcgctttcat taaagtgttc gtttcccata gcaaaaactc gtcatatgga 1740

atggccagct cataattcag cttacccaga caccatcttt ccattagaga tagatccttt 1800

gcatggagcc cacttataat ttgccaattt ttcatcgagt aagtgttgtc gttcaaaaat 1860

ttgtgcgata gaataagaca gcagagaaaa attctgcggg aacagtgcga gaattcaggc 1920

aatgaggact catcccggac acctctggat tggtgaatct tttgaaagta gaaagtggct 1980

aacacagcgt tttctttact gcacttcgat ctcttaagca cttcatttag aaattttaaa 2040

atgttgtggg tagaattatt gattttgcca tttgaaagag gccgactgat ctcggacaag 2100

aaattcgata tgataattgc tagatttgtt ttctttctgc tgtaatttgt ctgatgatga 2160

gtaccgttgt gctctaaggg cggagtctga tacggatttc tgccagtgga agactcgagc 2220

tcttaattaa caattcttcg ccagagg 2247

<210> 9

<211> 2049

<212> DNA

<213> Saccharomyces cerevisiae (Saccharomyces cerevisiae)

<400> 9

atgtttttca acagactaag cgctggcaag ctgctggtac cactctccgt ggtcctgtac 60

gcccttttcg tggtaatatt acctttacag aattctttcc actcctccaa tgttttagtt 120

agaggtgccg atgatgtaga aaactacgga actgttatcg gtattgactt aggtactact 180

tattcctgtg ttgctgtgat gaaaaatggt aagactgaaa ttcttgctaa tgagcaaggt 240

aacagaatca ccccatctta cgtggcattc accgatgatg aaagattgat tggtgatgct 300

gcaaagaacc aagttgctgc caatcctcaa aacaccatct tcgacattaa gagattgatc 360

ggtttgaaat ataacgacag atctgttcag aaggatatca agcacttgcc atttaatgtg 420

gttaataaag atgggaagcc cgctgtagaa gtaagtgtca aaggagaaaa gaaggttttt 480

actccagaag aaatttctgg tatgatcttg ggtaagatga aacaaattgc cgaagattat 540

ttaggcacta aggttaccca tgctgtcgtt actgttcctg cttatttcaa tgacgcgcaa 600

agacaagcca ccaaggatgc tggtaccatc gctggtttga acgttttgag aattgttaat 660

gaaccaaccg cagccgccat tgcctacggt ttggataaat ctgataagga acatcaaatt 720

attgtttatg atttgggtgg tggtactttc gatgtctctc tattgtctat tgaaaacggt 780

gttttcgaag tccaagccac ttctggtgat actcatttag gtggtgaaga ttttgactat 840

aagatcgttc gtcaattgat aaaagctttc aagaagaagc atggtattga tgtgtctgac 900

aacaacaagg ccctagctaa attgaagaga gaagctgaaa aggctaaacg tgccttgtcc 960

agccaaatgt ccacccgtat tgaaattgac tccttcgttg atggtatcga cttaagtgaa 1020

accttgacca gagctaagtt tgaggaatta aacctagatc tattcaagaa gaccttgaag 1080

cctgtcgaga aggttttgca agattctggt ttggaaaaga aggatgttga tgatatcgtt 1140

ttggttggtg gttctactag aattccaaag gtccaacaat tgttagaatc atactttgat 1200

ggtaagaagg cctccaaggg tattaaccca gatgaagctg ttgcatacgg tgcagccgtt 1260

caagctggtg tcttatccgg tgaagaaggt gtcgaagata ttgttttatt ggatgtcaac 1320

gctttgactc ttggtattga aaccactggt ggtgtcatga ctccattaat taagagaaat 1380

actgctattc ctacaaagaa atcccaaatt ttctctactg ccgttgacaa ccaaccaacc 1440

gttatgatca aggtatacga gggtgaaaga gccatgtcta aggacaacaa tctattaggt 1500

aagtttgaat taaccggcat tccaccagca ccaagaggtg tacctcaaat tgaagtcaca 1560

tttgcacttg acgctaatgg tattctgaag gtgtctgcca cagataaggg aactggtaaa 1620

tccgaatcta tcaccatcac taacgataaa ggtagattaa cccaagaaga gattgataga 1680

atggttgaag aggctgaaaa attcgcttct gaagacgctt ctatcaaggc caaggttgaa 1740

tctagaaaca aattagaaaa ctacgctcac tctttgaaaa accaagttaa tggtgaccta 1800

ggtgaaaaat tggaagaaga agacaaggaa accttattag atgctgctaa cgatgtttta 1860

gaatggttag atgataactt tgaaaccgcc attgctgaag actttgatga aaagttcgaa 1920

tctttgtcca aggtcgctta tccaattact tctaagttgt acggaggtgc tgatggttct 1980

ggtgccgctg attatgacga cgaagatgaa gatgacgatg gtgattattt cgaacacgac 2040

gaattgtag 2049

<210> 10

<211> 717

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 10

atggaaatga ctgattttga actaactagt aattcgcaat cgaacttggc tatccctacc 60

aacttcaagt cgactctgcc tccaaggaaa agagccaaga caaaagagga aaaggaacag 120

cgaaggatcg agcgtatttt gagaaacaga agagctgctc accagagcag agagaaaaaa 180

agactacatc tgcagtatct cgagagaaaa tgttctcttt tggaaaattt actgaacagc 240

gtcaaccttg aaaaactggc tgaccacgaa gacgcgttga cttgcagcca cgacgctttt 300

gttgcttctc ttgacgagta cagggatttc cagagcacga ggggcgcttc actggacacc 360

agggccagtt cgcactcgtc gtctgatacg ttcacacctt cacctctgaa ctgtacaatg 420

gagcctgcga ctttgtcgcc caagagtatg cgcgattccg cgtcggacca agagacttca 480

tgggagctgc agatgtttaa gacggaaaat gtaccagagt cgacgacgct acctgccgta 540

gacaacaaca atttgtttga tgcggtggcc tcgccgttgg cagacccact ctgcgacgat 600

atagcgggaa acagtctacc ctttgacaat tcaattgatc ttgacaattg gcgtaatcca 660

gaagcgcagt caggtttgaa ttcatttgaa ttgaatgatt tcttcatcac ttcatga 717

<210> 11

<211> 1569

<212> DNA

<213> Saccharomyces cerevisiae (Saccharomyces cerevisiae)

<400> 11

atgaagtttt ctgctggtgc cgtcctgtca tggtcctccc tgctgctcgc ctcctctgtt 60

ttcgcccaac aagaggctgt ggcccctgaa gactccgctg tcgttaagtt ggccaccgac 120

tctttcaatg agtacattca gtcgcacgac ttggtgcttg cggagttttt tgctccatgg 180

tgtggccact gtaagaacat ggctcctgaa tacgttaaag ccgccgagac tttagttgag 240

aaaaacatta ccttggccca gatcgactgt actgaaaacc aggatctgtg tatggaacac 300

aacattccag ggttcccaag cttgaagatt ttcaaaaaca gcgatgttaa caactcgatc 360

gattacgagg gacctagaac tgccgaggcc attgtccaat tcatgatcaa gcaaagccaa 420

ccggctgtcg ccgttgttgc tgatctacca gcttaccttg ctaacgagac ttttgtcact 480

ccagttatcg tccaatccgg taagattgac gccgacttca acgccacctt ttactccatg 540

gccaacaaac acttcaacga ctacgatttt gtctccgctg aaaacgcaga cgatgatttc 600

aagctttcta tttacttgcc ctccgccatg gacgagcctg tagtatacaa tggtaagaaa 660

gccgatatcg ctgacgctga tgtttttgaa aaatggttgc aagtggaagc cttgccctac 720

tttggtgaaa tcgacggttc cgttttcgcc caatacgtcg aaagcggttt gcctttgggt 780

tacttgttct acaatgacga ggaagaattg gaagaataca aacctctctt taccgagttg 840

gccaaaaaga acagaggtct aatgaacttt gttagcatcg atgccagaaa attcggcaga 900

cacgccggca acttgaacat gaaggaacaa ttccctctat ttgccatcca cgacatgact 960

gaagacttga agtacggttt gcctcaactc tctgaagagg cgtttgacga attgagcgac 1020

aagatcgtgt tggaatctaa ggctattgaa tctttggtta aggacttctt gaaaggtgat 1080

gcctccccaa tcgtgaagtc ccaagagatc ttcgagaacc aagattcctc tgtcttccaa 1140

ttggtcggta agaaccatga cgaaatcgtc aacgacccaa agaaggacgt tcttgttttg 1200

tactatgccc catggtgtgg tcactgtaag agattggccc caacttacca agaactagct 1260

gatacctacg ccaacgccac atccgacgtt ttgattgcta aactagacca cactgaaaac 1320

gatgtcagag gcgtcgtaat tgaaggttac ccaacaatcg tcttataccc aggtggtaag 1380

aagtccgaat ctgttgtgta ccaaggttca agatccttgg actctttatt cgacttcatc 1440

aaggaaaacg gtcacttcga cgtcgacggt aaggccttgt acgaagaagc ccaggaaaaa 1500

gctgctgagg aagccgatgc tgacgctgaa ttggctgacg aagaagatgc cattcacgat 1560

gaattgtaa 1569

<210> 12

<211> 390

<212> DNA

<213> Saccharomyces cerevisiae (Saccharomyces cerevisiae)

<400> 12

ccaaaaaaac cacaagccag aaagggttcc atggccgatg tgccaaagga attgatgcaa 60

caaattgaga attttgaaaa aattttcact gttccaactg aaactttaca agccgttacc 120

aagcacttca tttccgaatt ggaaaagggt ttgtccaaga agggtggtaa cattccaatg 180

attccaggtt gggttatgga tttcccaact ggtaaggaat ccggtgattt cttggccatt 240

gatttgggtg gtaccaactt gagagttgtc ttagtcaagt tgggcggtga ccgtaccttt 300

gacaccactc aatctaagta cagattacca gatgctatga gaactactca aaatccagac 360

gaattgtggg aatttattgc cgactctttg 390

<210> 13

<211> 364

<212> DNA

<213> Saccharomyces cerevisiae (Saccharomyces cerevisiae)

<400> 13

cccagccaga atcgaggaag atccattcga gaacctagaa gataccgatg acttgttcca 60

aaatgagttc ggtatcaaca ctactgttca agaacgtaaa ttgatcagac gtttatctga 120

attgattggt gctagagctg ctagattgtc cgtttgtggt attgctgcta tctgtcaaaa 180

gagaggttac aagaccggtc acatcgctgc agacggttcc gtttacaaca gatacccagg 240

tttcaaagaa aaggctgcca atgctttgaa ggacatttac ggctggactc aaacctcact 300

agacgactac ccaatcaaga ttgttcctgc tgaagatggt tccggtgctg gtgccgctgt 360

tatt 364

<210> 14

<211> 1613

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 14

gcaggtcgac aacccttaat ataacttcgt ataatgtatg ctatacgaag ttattaggtc 60

tagagatctg tttagcttgc ctcgtccccg ccgggtcacc cggccagcga catggaggcc 120

cagaataccc tccttgacag tcttgacgtg cgcagctcag gggcatgatg tgactgtcgc 180

ccgtacattt agcccataca tccccatgta taatcatttg catccataca ttttgatggc 240

cgcacggcgc gaagcaaaaa ttacggctcc tcgctgcaga cctgcgagca gggaaacgct 300

cccctcacag acgcgttgaa ttgtccccac gccgcgcccc tgtagagaaa tataaaaggt 360

taggatttgc cactgaggtt cttctttcat atacttcctt ttaaaatctt gctaggatac 420

agttctcaca tcacatccga acataaacaa ccatgggtaa ggaaaagact cacgtttcga 480

ggccgcgatt aaattccaac atggatgctg atttatatgg gtataaatgg gctcgcgata 540

atgtcgggca atcaggtgcg acaatctatc gattgtatgg gaagcccgat gcgccagagt 600

tgtttctgaa acatggcaaa ggtagcgttg ccaatgatgt tacagatgag atggtcagac 660

taaactggct gacggaattt atgcctcttc cgaccatcaa gcattttatc cgtactcctg 720

atgatgcatg gttactcacc actgcgatcc ccggcaaaac agcattccag gtattagaag 780

aatatcctga ttcaggtgaa aatattgttg atgcgctggc agtgttcctg cgccggttgc 840

attcgattcc tgtttgtaat tgtcctttta acagcgatcg cgtatttcgt ctcgctcagg 900

cgcaatcacg aatgaataac ggtttggttg atgcgagtga ttttgatgac gagcgtaatg 960

gctggcctgt tgaacaagtc tggaaagaaa tgcataagct tttgccattc tcaccggatt 1020

cagtcgtcac tcatggtgat ttctcacttg ataaccttat ttttgacgag gggaaattaa 1080

taggttgtat tgatgttgga cgagtcggaa tcgcagaccg ataccaggat cttgccatcc 1140

tatggaactg cctcggtgag ttttctcctt cattacagaa acggcttttt caaaaatatg 1200

gtattgataa tcctgatatg aataaattgc agtttcattt gatgctcgat gagtttttct 1260

aatcagtact gacaataaaa agattcttgt tttcaagaac ttgtcatttg tatagttttt 1320

ttatattgta gttgttctat tttaatcaaa tgttagcgtg atttatattt tttttcgcct 1380

cgacatcatc tgcccagatg cgaagttaag tgcgcagaaa gtaatatcat gcgtcaatcg 1440

tatgtgaatg ctggtcgcta tactgctgtc gattcgatac taacgccgcc atccagtgtc 1500

gaaaacgagc tctcgagaac ccttaatata acttcgtata atgtatgcta tacgaagtta 1560

ttaggtgata tcagatccac tagtggccta tgccccagcc agaatcgagg aag 1613

<210> 15

<211> 430

<212> DNA

<213> Saccharomyces cerevisiae (Saccharomyces cerevisiae)

<400> 15

agtgatcccc cacacaccat agcttcaaaa tgtttctact ccttttttac tcttccagat 60

tttctcggac tccgcgcatc gccgtaccac ttcaaaacac ccaagcacag catactaaat 120

ttcccctctt tcttcctcta gggtgtcgtt aattacccgt actaaaggtt tggaaaagaa 180

aaaagagacc gcctcgtttc tttttcttcg tcgaaaaagg caataaaaat ttttatcacg 240

tttctttttc ttgaaaattt ttttttttga tttttttctc tttcgatgac ctcccattga 300

tatttaagtt aataaacggt cttcaatttc tcaagtttca gtttcatttt tcttgttcta 360

ttacaacttt ttttacttct tgctcattag aaagaaagca tagcaatcta atctaagttt 420

taattacaaa 430

<210> 16

<211> 189

<212> DNA

<213> Saccharomyces cerevisiae (Saccharomyces cerevisiae)

<400> 16

atccgctcta accgaaaagg aaggagttag acaacctgaa gtctaggtcc ctatttattt 60

tttttaatag ttatgttagt attaagaacg ttatttatat ttcaaatttt tctttttttt 120

ctgtacaaac gcgtgtacgc atgtaacatt atactgaaaa ccttgcttga gaaggttttg 180

ggacgctcg 189

<210> 17

<211> 800

<212> DNA

<213> Saccharomyces cerevisiae (Saccharomyces cerevisiae)

<400> 17

atactagcgt tgaatgttag cgtcaacaac aagaagttta atgacgcgga ggccaaggca 60

aaaagattcc ttgattacgt aagggagtta gaatcatttt gaataaaaaa cacgcttttt 120

cagttcgagt ttatcattat caatactgcc atttcaaaga atacgtaaat aattaatagt 180

agtgattttc ctaactttat ttagtcaaaa aattagcctt ttaattctgc tgtaacccgt 240

acatgcccaa aatagggggc gggttacaca gaatatataa catcgtaggt gtctgggtga 300

acagtttatt cctggcatcc actaaatata atggagcccg ctttttaagc tggcatccag 360

aaaaaaaaag aatcccagca ccaaaatatt gttttcttca ccaaccatca gttcataggt 420

ccattctctt agcgcaacta cagagaacag gggcacaaac aggcaaaaaa cgggcacaac 480

ctcaatggag tgatgcaacc tgcctggagt aaatgatgac acaaggcaat tgacccacgc 540

atgtatctat ctcattttct tacaccttct attaccttct gctctctctg atttggaaaa 600

agctgaaaaa aaaggttgaa accagttccc tgaaattatt cccctacttg actaataagt 660

atataaagac ggtaggtatt gattgtaatt ctgtaaatct atttcttaaa cttcttaaat 720

tctactttta tagttagtct tttttttagt tttaaaacac caagaactta gtttcgaata 780

aacacacata aacaaacaaa 800

<210> 18

<211> 566

<212> DNA

<213> Saccharomyces cerevisiae (Saccharomyces cerevisiae)

<400> 18

gcggatctct tatgtcttta cgatttatag ttttcattat caagtatgcc tatattagta 60

tatagcatct ttagatgaca gtgttcgaag tttcacgaat aaaagataat attctacttt 120

ttgctcccac cgcgtttgct agcacgagtg aacaccatcc ctcgcctgtg agttgtaccc 180

attcctctaa actgtagaca tggtagcttc agcagtgttc gttatgtacg gcatcctcca 240

acaaacagtc ggttatagtt tgtcctgctc ctctgaatcg tctccctcga tatttctcat 300

tttccttcgc atgccagcat tgaaatgatc gaagttcaat gatgaaacgg taattcttct 360

gtcatttact catctcatct catcaagtta tataattcta tacggatgta atttttcact 420

tttcgtcttg acgtccaccc tataatttca attattgaac cctcacaaat gatgcactgc 480

aatgtacaca ccctcatata gtttctcagg gcttgatcag ggttccgtag atgggaattt 540

gagaagtata agggagataa cggtaa 566

<210> 19

<211> 750

<212> DNA

<213> Saccharomyces cerevisiae (Saccharomyces cerevisiae)

<400> 19

acgcacagat attataacat ctgcataata ggcatttgca agaattactc gtgagtaagg 60

aaagagtgag gaactatcgc atacctgcat ttaaagatgc cgatttgggc gcgaatcctt 120

tattttggct tcaccctcat actattatca gggccagaaa aaggaagtgt ttccctcctt 180

cttgaattga tgttaccctc ataaagcacg tggcctctta tcgagaaaga aattaccgtc 240

gctcgtgatt tgtttgcaaa aagaacaaaa ctgaaaaaac ccagacacgc tcgacttcct 300

gtcttcctat tgattgcagc ttccaatttc gtcacacaac aaggtcctag cgacggctca 360

caggttttgt aacaagcaat cgaaggttct ggaatggcgg gaaagggttt agtaccacat 420

gctatgatgc ccactgtgat ctccagagca aagttcgttc gatcgtactg ttactctctc 480

tctttcaaac agaattgtcc gaatcgtgtg acaacaacag cctgttctca cacactcttt 540

tcttctaacc aagggggtgg tttagtttag tagaacctcg tgaaacttac atttacatat 600

atataaactt gcataaattg gtcaatgcaa gaaatacata tttggtcttt tctaattcgt 660

agtttttcaa gttcttagat gctttctttt tctctttttt acagatcatc aaggaagtaa 720

ttatctactt tttacaacaa atataaaaca 750

<210> 20

<211> 158

<212> DNA

<213> Saccharomyces cerevisiae (Saccharomyces cerevisiae)

<400> 20

agttataaaa aaaataagtg tatacaaatt ttaaagtgac tcttaggttt taaaacgaaa 60

attcttattc ttgagtaact ctttcctgta ggtcaggttg ctttctcagg tatagcatga 120

ggtcgctctt attgaccaca cctctaccgg catgccga 158

<210> 21

<211> 422

<212> DNA

<213> Saccharomyces cerevisiae (Saccharomyces cerevisiae)

<400> 21

gattaatata attatataaa aatattatct tcttttcttt atatctagtg ttatgtaaaa 60

taaattgatg actacggaaa gcttttttat attgtttctt tttcattctg agccacttaa 120

atttcgtgaa tgttcttgta agggacggta gatttacaag tgatacaaca aaaagcaagg 180

cgctttttct aataaaaaga agaaaagcat ttaacaattg aacacctcta tatcaacgaa 240

gaatattact ttgtctctaa atccttgtaa aatgtgtacg atctctatat gggttactca 300

taagtgtacc gaagactgca ttgaaagttt atgttttttc actggaggcg tcattttcgc 360

gttgagaaga tgttcttatc caaatttcaa ctgttatata gacgcacaga tattataaca 420

tc 422

<210> 22

<211> 410

<212> DNA

<213> Saccharomyces cerevisiae (Saccharomyces cerevisiae)

<400> 22

ggaagctgaa acgtctaacg gatcttgatt tgtgtggact tccttagaag taaccgaagc 60

acaggcgcta ccatgagaaa tgggtgaatg ttgagataat tgttgggatt ccattgttga 120

taaaggctat aatattaggt atacagaata tactagaagt tctcctcgag gatataggaa 180

tcctcaaaat ggaatctata tttctacata ctaatattac gattattcct cattccgttt 240

tatatgttta tattcattga tcctattaca ttatcaatcc ttgcgtttca gcttccacta 300

atttagatga ctatttctca tcatttgcgt catcttctaa caccgtatat gataatatac 360

tagtaatgta aatactagtt agtagatgat agttgatttc tattccaaca 410

<210> 23

<211> 1169

<212> DNA

<213> Saccharomyces cerevisiae (Saccharomyces cerevisiae)

<400> 23

tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60

cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120

ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180

accataaatt cccgttttaa gagcttggtg agcgctagga gtcactgcca ggtatcgttt 240

gaacacggca ttagtcaggg aagtcataac acagtccttt cccgcaattt tctttttcta 300

ttactcttgg cctcctctag tacactctat atttttttat gcctcggtaa tgattttcat 360

tttttttttt cccctagcgg atgactcttt ttttttctta gcgattggca ttatcacata 420

atgaattata cattatataa agtaatgtga tttcttcgaa gaatatacta aaaaatgagc 480

aggcaagata aacgaaggca aagatgacag agcagaaagc cctagtaaag cgtattacaa 540

atgaaaccaa gattcagatt gcgatctctt taaagggtgg tcccctagcg atagagcact 600

cgatcttccc agaaaaagag gcagaagcag tagcagaaca ggccacacaa tcgcaagtga 660

ttaacgtcca cacaggtata gggtttctgg accatatgat acatgctctg gccaagcatt 720

ccggctggtc gctaatcgtt gagtgcattg gtgacttaca catagacgac catcacacca 780

ctgaagactg cgggattgct ctcggtcaag cttttaaaga ggccctactg gcgcgtggag 840

taaaaaggtt tggatcagga tttgcgcctt tggatgaggc actttccaga gcggtggtag 900

atctttcgaa caggccgtac gcagttgtcg aacttggttt gcaaagggag aaagtaggag 960

atctctcttg cgagatgatc ccgcattttc ttgaaagctt tgcagaggct agcagaatta 1020

ccctccacgt tgattgtctg cgaggcaaga atgatcatca ccgtagtgag agtgcgttca 1080

aggctcttgc ggttgccata agagaagcca cctcgcccaa tggtaccaac gatgttccct 1140

ccaccaaagg tgttcttatg tagtgacac 1169

<210> 24

<211> 371

<212> DNA

<213> Saccharomyces cerevisiae (Saccharomyces cerevisiae)

<400> 24

tgttggaacg agagtaatta atagtgacat gagttgctat ggtaacaatc taatgcttac 60

atcgtatatt aatgtacaac tcgtatacgt ttaagtgtga ttgcgcctat tgcagaagga 120

atgttaaacg agaagctcag acaatactga agctgtgtta aagacctatt agttgaacat 180

gttatggtag gtacatatat gaggaatatg agtcgtcaca tcaatgtata gtaactaccg 240

gaatcactat tatattggtc atgattaata tgaccaatcg gcgtgtgttt tatatacctc 300

tcttatttag tataagaaga tcagtactca cttcttcatt aatactaatt tttaacctct 360

aattatcaac a 371

<210> 25

<211> 2178

<212> DNA

<213> Saccharomyces cerevisiae (Saccharomyces cerevisiae)

<400> 25

ctccatcaaa tggtcaggtc attgagtgtt ttttatttgt tgtatttttt tttttttaga 60

gaaaatcctc caatattaaa attaggaata gtagtttcat gattttctgt tacacctaac 120

tttttgtgtg gtgccctcct ccttgtcaat attaatgtta aagtgcaatt ctttttcctt 180

atcacgttga gccattagta tcaatttgct tacctgtatt cctttacatc ctcctttttc 240

tccttcttga taaatgtatg tagattgcgt atatagtttc gtctacccta tgaacatatt 300

ccattttgta atttcgtgtc gtttctatta tgaatttcat ttataaagtt tatgtacaaa 360

tatcataaaa aaagagaatc tttttaagca aggattttct taacttcttc ggcgacagca 420

tcaccgactt cggtggtact gttggaacca cctaaatcac cagttctgat acctgcatcc 480

aaaacctttt taactgcatc ttcaatggcc ttaccttctt caggcaagtt caatgacaat 540

ttcaacatca ttgcagcaga caagatagtg gcgatagggt tgaccttatt ctttggcaaa 600

tctggagcag aaccgtggca tggttcgtac aaaccaaatg cggtgttctt gtctggcaaa 660

gaggccaagg acgcagatgg caacaaaccc aaggaacctg ggataacgga ggcttcatcg 720

gagatgatat caccaaacat gttgctggtg attataatac catttaggtg ggttgggttc 780

ttaactagga tcatggcggc agaatcaatc aattgatgtt gaaccttcaa tgtagggaat 840

tcgttcttga tggtttcctc cacagttttt ctccataatc ttgaagaggc caaaacatta 900

gctttatcca aggaccaaat aggcaatggt ggctcatgtt gtagggccat gaaagcggcc 960

attcttgtga ttctttgcac ttctggaacg gtgtattgtt cactatccca agcgacacca 1020

tcaccatcgt cttcctttct cttaccaaag taaatacctc ccactaattc tctgacaaca 1080

acgaagtcag tacctttagc aaattgtggc ttgattggag ataagtctaa aagagagtcg 1140

gatgcaaagt tacatggtct taagttggcg tacaattgaa gttctttacg gatttttagt 1200

aaaccttgtt caggtctaac actaccggta ccccatttag gaccacccac agcacctaac 1260

aaaacggcat cagccttctt ggaggcttcc agcgcctcat ctggaagtgg aacacctgta 1320

gcatcgatag cagcaccacc aattaaatga ttttcgaaat cgaacttgac attggaacga 1380

acatcagaaa tagctttaag aaccttaatg gcttcggctg tgatttcttg accaacgtgg 1440

tcacctggca aaacgacgat cttcttaggg gcagacatta gaatggtata tccttgaaat 1500

atatatatat atattgctga aatgtaaaag gtaagaaaag ttagaaagta agacgattgc 1560

taaccaccta ttggaaaaaa caataggtcc ttaaataata ttgtcaactt caagtattgt 1620

gatgcaagca tttagtcatg aacgcttctc tattctatat gaaaagccgg ttcgcggcct 1680

ctcacctttc ctttttctcc caatttttca gttgaaaaag gtatatgcgt caggcgacct 1740

ctgaaattaa caaaaaattt ccagtcatcg aatttgattc tgtgcgatag cgcccctgtg 1800

tgttctcgtt atgttgagga aaaaaataat ggttgctaag agattcgaac tcttgcatct 1860

tacgatacct gagtattccc acagttaact gcggtcaaga tatttcttga atcaggcgcc 1920

ttagaccgct cggccaaaca accaattact tgttgagaaa tagagtataa ttatcctata 1980

aatataacgt ttttgaacac acatgaacaa ggaagtacag gacaattgat tttgaagaga 2040

atgtggattt tgatgtaatt gttgggattc catttttaat aaggcaataa tattaggtat 2100

gtagatatac tagaagttct cctcgaccgg tcgatatgcg gtgtgaaata ccgcacagat 2160

gcgtaaggag aaaatacc 2178

<210> 26

<211> 1264

<212> DNA

<213> Saccharomyces cerevisiae (Saccharomyces cerevisiae)

<400> 26

atgagagtag caaacgtaag tctaaaggtt gttttatagt agttaggatg tagaaaatgt 60

attccgatag gccattttac atttggaggg acggttgaaa gtggacagag gaaaaggtgc 120

ggaaatggct gattttgatt gtttatgttt tgtgtgatga ttttacattt ttgcatagta 180

ttaggtagtc agatgaaaga tgaatagaca taggagtaag aaaacataga atagttaccg 240

ttattggtag gagtgtggtg gggtggtata gtccgcattg ggatgttact ttcctgttat 300

ggcatggatt tccctttagg gtctctgaag cgtatttccg tcaccgaaaa aggcagaaaa 360

agggaaactg aagggaggat agtagtaaag tttgaatggt ggtagtgtaa tgtatgatat 420

ccgttggttt tggtttcggt tgtgaaaagt tttttggtat gatattttgc aagtagcata 480

tatttcttgt gtgagaaagg tatattttgt atgttttgta tgttcccgcg cgtttccgta 540

ttttccgctt ccgcttccgc agtaaaaaat agtgaggaac tgggttaccc ggggcacctg 600

tcactttgga aaaaaaatat acgctaagat ttttggagaa tagcttaaat tgaagttttt 660

ctcggcgaga aatacgtagt taaggcagag cgacagagag ggcaaaagaa aataaaagta 720

agattttagt ttgtaatggg agggggggtt tagtcatgga gtacaagtgt gaggaaaagt 780

agttgggagg tacttcatgc gaaagcagtt gaagacaagt tcgaaaagag tttggaaacg 840

aattcgagta ggcttgtcgt tcgttatgtt tttgtaaatg gcctcgtcaa acggtggaga 900

gagtcgctag gtgatcgtca gatctgccta gtctctatac agcgtgttta attgacatgg 960

gttgatgcgt attgagagat acaatttggg aagaaattcc cagagtgtgt ttcttttgcg 1020

tttaacctga acagtctcat cgtgggcatc ttgcgattcc attggtgagc agcgaaggat 1080

ttggtggatt actagctaat agcaatctat ttcaaagaat tcaaacttgg gggaatgcct 1140

tgttgaatag ccggtcgcaa gactgtgatt cttcaagtgt aacctcctct caaatcagcg 1200

atatcaaacg taccattccg tgaaacaccg gggtatctgt ttggtggaac ctgattagag 1260

gaaa 1264

<210> 27

<211> 1365

<212> DNA

<213> Saccharomyces cerevisiae (Saccharomyces cerevisiae)

<400> 27

cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac 60

agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt 120

tggcgggtgt cggggctggc ttaactatgc ggcatcagag cagattgtac tgagagtgca 180

ccataaacga cattactata tatataatat aggaagcatt taatagacag catcgtaata 240

tatgtgtact ttgcagttat gacgccagat ggcagtagtg gaagatattc tttattgaaa 300

aatagcttgt caccttacgt acaatcttga tccggagctt ttcttttttt gccgattaag 360

aattaattcg gtcgaaaaaa gaaaaggaga gggccaagag ggagggcatt ggtgactatt 420

gagcacgtga gtatacgtga ttaagcacac aaaggcagct tggagtatgt ctgttattaa 480

tttcacaggt agttctggtc cattggtgaa agtttgcggc ttgcagagca cagaggccgc 540

agaatgtgct ctagattccg atgctgactt gctgggtatt atatgtgtgc ccaatagaaa 600

gagaacaatt gacccggtta ttgcaaggaa aatttcaagt cttgtaaaag catataaaaa 660

tagttcaggc actccgaaat acttggttgg cgtgtttcgt aatcaaccta aggaggatgt 720

tttggctctg gtcaatgatt acggcattga tatcgtccaa ctgcatggag atgagtcgtg 780

gcaagaatac caagagttcc tcggtttgcc agttattaaa agactcgtat ttccaaaaga 840

ctgcaacata ctactcagtg cagcttcaca gaaacctcat tcgtttattc ccttgtttga 900

ttcagaagca ggtgggacag gtgaactttt ggattggaac tcgatttctg actgggttgg 960

aaggcaagag agccccgaaa gcttacattt tatgttagct ggtggactga cgccagaaaa 1020

tgttggtgat gcgcttagat taaatggcgt tattggtgtt gatgtaagcg gaggtgtgga 1080

gacaaatggt gtaaaagact ctaacaaaat agcaaatttc gtcaaaaatg ctaagaaata 1140

ggttattact gagtagtatt tatttaagta ttgtttgtgc acttgcctat gcggtgtgaa 1200

ataccgcaca attgaaagga gaaaataccg catcaggaaa ttgtaaacgt taatattttg 1260

ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc 1320

ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttg 1365

<210> 28

<211> 4272

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 28

atggactata aggaccacga cggagactac aaggatcatg atattgatta caaagacgat 60

gacgataaga tggccccaaa gaagaagcgg aaggtcggta tccacggagt cccagcagcc 120

gacaagaagt acagcatcgg cctggacatc ggcaccaact ctgtgggctg ggccgtgatc 180

accgacgagt acaaggtgcc cagcaagaaa ttcaaggtgc tgggcaacac cgaccggcac 240

agcatcaaga agaacctgat cggagccctg ctgttcgaca gcggcgaaac agccgaggcc 300

acccggctga agagaaccgc cagaagaaga tacaccagac ggaagaaccg gatctgctat 360

ctgcaagaga tcttcagcaa cgagatggcc aaggtggacg acagcttctt ccacagactg 420

gaagagtcct tcctggtgga agaggataag aagcacgagc ggcaccccat cttcggcaac 480

atcgtggacg aggtggccta ccacgagaag taccccacca tctaccacct gagaaagaaa 540

ctggtggaca gcaccgacaa ggccgacctg cggctgatct atctggccct ggcccacatg 600

atcaagttcc ggggccactt cctgatcgag ggcgacctga accccgacaa cagcgacgtg 660

gacaagctgt tcatccagct ggtgcagacc tacaaccagc tgttcgagga aaaccccatc 720

aacgccagcg gcgtggacgc caaggccatc ctgtctgcca gactgagcaa gagcagacgg 780

ctggaaaatc tgatcgccca gctgcccggc gagaagaaga atggcctgtt cggaaacctg 840

attgccctga gcctgggcct gacccccaac ttcaagagca acttcgacct ggccgaggat 900

gccaaactgc agctgagcaa ggacacctac gacgacgacc tggacaacct gctggcccag 960

atcggcgacc agtacgccga cctgtttctg gccgccaaga acctgtccga cgccatcctg 1020

ctgagcgaca tcctgagagt gaacaccgag atcaccaagg cccccctgag cgcctctatg 1080

atcaagagat acgacgagca ccaccaggac ctgaccctgc tgaaagctct cgtgcggcag 1140

cagctgcctg agaagtacaa agagattttc ttcgaccaga gcaagaacgg ctacgccggc 1200

tacattgacg gcggagccag ccaggaagag ttctacaagt tcatcaagcc catcctggaa 1260

aagatggacg gcaccgagga actgctcgtg aagctgaaca gagaggacct gctgcggaag 1320

cagcggacct tcgacaacgg cagcatcccc caccagatcc acctgggaga gctgcacgcc 1380

attctgcggc ggcaggaaga tttttaccca ttcctgaagg acaaccggga aaagatcgag 1440

aagatcctga ccttccgcat cccctactac gtgggccctc tggccagggg aaacagcaga 1500

ttcgcctgga tgaccagaaa gagcgaggaa accatcaccc cctggaactt cgaggaagtg 1560

gtggacaagg gcgcttccgc ccagagcttc atcgagcgga tgaccaactt cgataagaac 1620

ctgcccaacg agaaggtgct gcccaagcac agcctgctgt acgagtactt caccgtgtat 1680

aacgagctga ccaaagtgaa atacgtgacc gagggaatga gaaagcccgc cttcctgagc 1740

ggcgagcaga aaaaggccat cgtggacctg ctgttcaaga ccaaccggaa agtgaccgtg 1800

aagcagctga aagaggacta cttcaagaaa atcgagtgct tcgactccgt ggaaatctcc 1860

ggcgtggaag atcggttcaa cgcctccctg ggcacatacc acgatctgct gaaaattatc 1920

aaggacaagg acttcctgga caatgaggaa aacgaggaca ttctggaaga tatcgtgctg 1980

accctgacac tgtttgagga cagagagatg atcgaggaac ggctgaaaac ctatgcccac 2040

ctgttcgacg acaaagtgat gaagcagctg aagcggcgga gatacaccgg ctggggcagg 2100

ctgagccgga agctgatcaa cggcatccgg gacaagcagt ccggcaagac aatcctggat 2160

ttcctgaagt ccgacggctt cgccaacaga aacttcatgc agctgatcca cgacgacagc 2220

ctgaccttta aagaggacat ccagaaagcc caggtgtccg gccagggcga tagcctgcac 2280

gagcacattg ccaatctggc cggcagcccc gccattaaga agggcatcct gcagacagtg 2340

aaggtggtgg acgagctcgt gaaagtgatg ggccggcaca agcccgagaa catcgtgatc 2400

gaaatggcca gagagaacca gaccacccag aagggacaga agaacagccg cgagagaatg 2460

aagcggatcg aagagggcat caaagagctg ggcagccaga tcctgaaaga acaccccgtg 2520

gaaaacaccc agctgcagaa cgagaagctg tacctgtact acctgcagaa tgggcgggat 2580

atgtacgtgg accaggaact ggacatcaac cggctgtccg actacgatgt ggaccatatc 2640

gtgcctcaga gctttctgaa ggacgactcc atcgacaaca aggtgctgac cagaagcgac 2700

aagaaccggg gcaagagcga caacgtgccc tccgaagagg tcgtgaagaa gatgaagaac 2760

tactggcggc agctgctgaa cgccaagctg attacccaga gaaagttcga caatctgacc 2820

aaggccgaga gaggcggcct gagcgaactg gataaggccg gcttcatcaa gagacagctg 2880

gtggaaaccc ggcagatcac aaagcacgtg gcacagatcc tggactcccg gatgaacact 2940

aagtacgacg agaatgacaa gctgatccgg gaagtgaaag tgatcaccct gaagtccaag 3000

ctggtgtccg atttccggaa ggatttccag ttttacaaag tgcgcgagat caacaactac 3060

caccacgccc acgacgccta cctgaacgcc gtcgtgggaa ccgccctgat caaaaagtac 3120

cctaagctgg aaagcgagtt cgtgtacggc gactacaagg tgtacgacgt gcggaagatg 3180

atcgccaaga gcgagcagga aatcggcaag gctaccgcca agtacttctt ctacagcaac 3240

atcatgaact ttttcaagac cgagattacc ctggccaacg gcgagatccg gaagcggcct 3300

ctgatcgaga caaacggcga aaccggggag atcgtgtggg ataagggccg ggattttgcc 3360

accgtgcgga aagtgctgag catgccccaa gtgaatatcg tgaaaaagac cgaggtgcag 3420

acaggcggct tcagcaaaga gtctatcctg cccaagagga acagcgataa gctgatcgcc 3480

agaaagaagg actgggaccc taagaagtac ggcggcttcg acagccccac cgtggcctat 3540

tctgtgctgg tggtggccaa agtggaaaag ggcaagtcca agaaactgaa gagtgtgaaa 3600

gagctgctgg ggatcaccat catggaaaga agcagcttcg agaagaatcc catcgacttt 3660

ctggaagcca agggctacaa agaagtgaaa aaggacctga tcatcaagct gcctaagtac 3720

tccctgttcg agctggaaaa cggccggaag agaatgctgg cctctgccgg cgaactgcag 3780

aagggaaacg aactggccct gccctccaaa tatgtgaact tcctgtacct ggccagccac 3840

tatgagaagc tgaagggctc ccccgaggat aatgagcaga aacagctgtt tgtggaacag 3900

cacaagcact acctggacga gatcatcgag cagatcagcg agttctccaa gagagtgatc 3960

ctggccgacg ctaatctgga caaagtgctg tccgcctaca acaagcaccg ggataagccc 4020

atcagagagc aggccgagaa tatcatccac ctgtttaccc tgaccaatct gggagcccct 4080

gccgccttca agtactttga caccaccatc gaccggaaga ggtacaccag caccaaagag 4140

gtgctggacg ccaccctgat ccaccagagc atcaccggcc tgtacgagac acggatcgac 4200

ctgtctcagc tgggaggcga caaaaggccg gcggccacga aaaaggccgg ccaggcaaaa 4260

aagaaaaagt aa 4272

<210> 29

<211> 566

<212> DNA

<213> Saccharomyces cerevisiae (Saccharomyces cerevisiae)

<400> 29

gcggatctct tatgtcttta cgatttatag ttttcattat caagtatgcc tatattagta 60

tatagcatct ttagatgaca gtgttcgaag tttcacgaat aaaagataat attctacttt 120

ttgctcccac cgcgtttgct agcacgagtg aacaccatcc ctcgcctgtg agttgtaccc 180

attcctctaa actgtagaca tggtagcttc agcagtgttc gttatgtacg gcatcctcca 240

acaaacagtc ggttatagtt tgtcctgctc ctctgaatcg tctccctcga tatttctcat 300

tttccttcgc atgccagcat tgaaatgatc gaagttcaat gatgaaacgg taattcttct 360

gtcatttact catctcatct catcaagtta tataattcta tacggatgta atttttcact 420

tttcgtcttg acgtccaccc tataatttca attattgaac cctcacaaat gatgcactgc 480

aatgtacaca ccctcatata gtttctcagg gcttgatcag ggttccgtag atgggaattt 540

gagaagtata agggagataa cggtaa 566

<210> 30

<211> 458

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 30

cctcttcgct attacgccag ctgggatccg cggccgctct ttgaaaagat aatgtatgat 60

tatgctttca ctcatattta tacagaaact tgatgttttc tttcgagtat atacaaggtg 120

attacatgta cgtttgaagt acaactctag attttgtagt gccctcttgg gctagcggta 180

aaggtgcgca ttttttcaca ccctacaatg ttctgttcaa aagattttgg tcaaacgctg 240

tagaagtgaa agttggtgcg catgtttcgg cgttcgaaac ttctccgcag tgaaagataa 300

atgatctata ctagaagttc tcctcgtttt agagctagaa atagcaagtt aaaataaggc 360

tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg gtgctttttt tgttttttat 420

gtctgtcgac aagcttagtg atcccccaca caccatag 458

Claims

1. A method of constructing a recombinant bacterium, the method comprising the steps of: knocking out hexokinase 2 gene in saccharomyces cerevisiae, introducing a gene expression cassette of fusion protein of dammarenediol-II synthase and GFP and a gene expression cassette of ginseng glycosyltransferase UGTPg1 into the saccharomyces cerevisiae, wherein the gene of the dammarenediol-II synthase is a ginseng dammarenediol-II synthase gene ds shown in No. AB265170.1, the gene of the ginseng glycosyltransferase UGTPg1 is a ginseng glycosyltransferase UGTPg1 shown in No. KF377585.1, and fusing GFP at the C end of the dammarenediol-II synthase.

2. The method according to claim 1, wherein the expression cassette of the gene encoding the fusion protein of dammarenediol-II synthase with GFP comprises the gene encoding the fusion protein of dammarenediol-II synthase with GFP shown in SEQ ID NO. 1.

3. The method according to claim 1, wherein the gene expression cassette encoding the ginseng glycosyltransferase UGTPg1 comprises the gene encoding the ginseng glycosyltransferase UGTPg1 shown by SEQ ID No. 2.

4. The method according to claim 1, further comprising the step of: enhancing the activity of 3-hydroxy-3-methylglutaryl-coa reductase in the saccharomyces cerevisiae.

5. The method according to claim 4, wherein the activity of 3-hydroxy-3-methylglutaryl-CoA reductase in said Saccharomyces cerevisiae is increased by introducing a gene expression cassette encoding 3-hydroxy-3-methylglutaryl-CoA reductase into said Saccharomyces cerevisiae.

6. The method according to claim 5, wherein the 3-hydroxy-3-methylglutaryl-CoA reductase encoding gene expression cassette comprises the gene tHMG1 encoding the 3-hydroxy-3-methylglutaryl-CoA reductase catalytic domain shown by SEQ ID NO. 3.

7. The method of claim 1, further comprising one or more of:

Increasing the activity of isopentenyl pyrophosphate isomerase IDI1 in said saccharomyces cerevisiae;

increasing the activity of farnesyl pyrophosphate synthase ERG20 in the saccharomyces cerevisiae;

increasing the activity of squalene monooxygenase, ERG1, in the saccharomyces cerevisiae;

increasing the activity of squalene synthase ERG9 in the Saccharomyces cerevisiae;

decreasing the activity of lanosterol synthase ERG7 in the saccharomyces cerevisiae;

increasing the level of the chaperone BiP in the saccharomyces cerevisiae;

increasing the level of the transcription factor HAC1 in said saccharomyces cerevisiae; or alternatively

Increasing the level of disulfide isomerase PDI1 in the Saccharomyces cerevisiae.

8. The method according to claim 7, wherein,

increasing the activity of isopentenyl pyrophosphate isomerase IDI1 in the saccharomyces cerevisiae by introducing into the saccharomyces cerevisiae an expression cassette encoding the isopentenyl pyrophosphate isomerase IDI 1;

increasing the activity of farnesyl pyrophosphate synthase ERG20 in the Saccharomyces cerevisiae by introducing into the Saccharomyces cerevisiae an expression cassette encoding the gene encoding farnesyl pyrophosphate synthase ERG 20;

increasing the activity of squalene monooxygenase ERG1 in the Saccharomyces cerevisiae by introducing a coding gene expression cassette of squalene monooxygenase ERG1 into the Saccharomyces cerevisiae;

Increasing the activity of squalene synthase ERG9 in the Saccharomyces cerevisiae by introducing into the Saccharomyces cerevisiae a gene expression cassette encoding squalene synthase ERG 9;

reducing the activity of lanosterol synthase ERG7 in the saccharomyces cerevisiae by introducing into the saccharomyces cerevisiae an expression cassette for an antisense fragment of lanosterol synthase ERG 7;

increasing the level of chaperone BiP in the saccharomyces cerevisiae by introducing into the saccharomyces cerevisiae an expression cassette of the encoding gene of chaperone BiP;

increasing the level of transcription factor HAC1 in the saccharomyces cerevisiae by introducing into the saccharomyces cerevisiae an expression cassette encoding the transcription factor HAC 1; or alternatively

The level of disulfide isomerase PDI1 in the Saccharomyces cerevisiae is increased by introducing into the Saccharomyces cerevisiae an expression cassette encoding a disulfide isomerase PDI 1.

9. The method according to claim 8, wherein,

the nucleotide sequence for encoding BiP is a sequence shown as SEQ ID NO. 9;

The nucleotide sequence encoding PDI1 is the sequence shown by SEQ ID NO. 11.

10. The method of any one of claims 1-9, wherein integrating the expression cassette into the saccharomyces cerevisiae genome is performed using CRISPR/Cas9 technology.

11. Recombinant bacterium produced by the method of any one of claims 1-10.

12. Use of the recombinant bacterium of claim 11 for the production of 20S-O-Glc-DM.

13. A method of producing 20S-O-Glc-DM comprising fermenting the recombinant bacterium of claim 11 to produce 20S-O-Glc-DM.