KR20190080790A - Expression vectors for optimal expression of mutated Upc2p and method for overproducing sterol precursors using the same - Google Patents

Abstract

The present invention relates to a vector for optimizing expression of Upc2p, which is a mutated transcriptional regulatory factor, and a sterol precursor overproduction method using the same. More specifically, expression of the mutated transcriptional regulatory factor (Upc2p) with reduced affinity to ergosterol is optimized to smoothen feedback adjustment such that an entire ergosterol biosynthesis path is amplified, thereby producing a recombinant yeast with increased biosynthesis performance for a sterol precursor, such as epoxy squalene and squalene, and a ginsenoside precursor such as dammarenediol. Accordingly, recombinant yeast strains produced by using a mutated UPC2 expression cassette and a substitution cassette using a CRISPR system can be used for production of recombinant yeast for massively biosynthesizing ginsenoside or ursodeoxycholic acid (UDCA) precursor materials through additional metabolic engineering. According to the present invention, a recombined expression vector includes a UPC2 mutated gene with a mutated ligand coupling domain portion of Upc2p derived from Saccharomyces cerevisiae.

Description

TECHNICAL FIELD The present invention relates to an expression vector for Upc2p expression and a sterol precursor and a method for producing the same,

The present invention relates to a recombinant yeast strain which is produced by modifying Upc2 protein and optimizing the expression of the mutant transcription regulator, and more particularly, to a recombinant yeast strain in which the amino acid site important for binding to ergosterol is substituted Upc2 variations to the copy number of the expression UPC2 mutant gene encoding a protein and a promoter active control, to optimize the metabolic flux through the switching to the expression host making a recombinant yeast strain expressing the sterol and the epoxy precursor including squalene. In addition, the recombinant yeast strain is produced by replacing the UPC2 gene with a mutation on the chromosome using the Krisper system so as to encode a Upc2 mutation protein substituted with an amino acid site that is important for binding with ergosterol using a crystal system.

Traditional yeast Saccharomyces cerevisiae ) is a strain used in fermentation of beer and bread for a long period of time. It is a safe microorganism of Generally Recognized as Safe (GRAS). In particular, yeast has a protein transcription system, which is very similar to that of higher organisms, and has a protein secretion organs so that it can produce activated proteins through post-translational modification. Therefore, yeast is a more useful host for mass production of medicinal proteins derived from higher organisms System. In addition, it is a host system useful for the industrial production of food and recombinant pharmaceutical proteins because of easy scale-up of the strain and easy purification of extracellular secretory proteins.

On the other hand, yeast is attracting attention as a host capable of introducing and expressing various secondary metabolite biosynthetic pathways. Secondary metabolites produced by various organisms are important sources of high-value chemical compounds and often have important medicinal properties. Especially, the metabolites of plants inhibit bacterial, viral, and fungal infections through antioxidant or antibiotic functions, and are also useful as therapeutic agents for human health. Therefore, there is a high demand for mass production technology utilizing microorganisms . Yeast, which has its own limited biosynthetic biosynthetic pathway, does not interfere with or compete with the exogenous metabolic pathways introduced through genetic engineering, and various omix analysis systems are well established. There is an advantage that the total information on the physiological state can be obtained. In addition, a detailed model of the metabolism has been developed and in silico yeast has been constructed to predict the behavior of the modified metabolic network, which makes it easier to design and manufacture artificial cells using the yeast. Furthermore, as a single-cell eukaryotic microorganism, yeast is a host suitable for expressing a foreign enzyme such as cytochrome P450, which becomes active only when it is expressed in an organelle such as an endoplasmic reticulum and mitochondria. It has a post-translational modification necessary for enzyme activity in plants and animals, .

Sterol plays an important role in the physiology of eukaryotes, forming part of the cell membrane that regulates fluidity and function. In yeast, ergosterol is a major sterol, a membrane component and a commercially important metabolite as a precursor to vitamin D2. In addition, its intermediate metabolite, isoprenoid, is widely used commercially as carotenoids, terpens, ubiquinone, and anti-cancer compounds (taxol). Another intermediate metabolite, squalene, is the most well-known health promotion food, and is being used as a skin moisturizer and cosmetic ingredient along with its next-generation metabolism, lanosterol. In addition to the industrial field using the intermediate metabolite as such, recently, the field of metabolic engineering that produces more useful substances in yeast has been spotlighted by changing the synthesis process of ergosterol into genetic engineering technology. Epoxysqualene, an intermediate metabolite of squalene, can be converted to dhamrenediol Ⅱ synthase by ginseng (Panax ginseng), and two cytochrome P450s and several kinds of glycosides It is made of various ginsenosides by enzymes (glucosyltransferase). In addition, by blocking the metabolic pathway from zymosterol, which is an intermediate metabolite, to ergosterol, and introducing a new gene from the outside to express it, it can be used as an anti-inflammatory steroid hormone used in the treatment of arthritis, skin diseases, adrenal insufficiency, A recombinant yeast producing drug substance precursor such as hydrocortisone or cholesterol which is a precursor of ursodeoxycholic acid (UDCA) as a therapeutic agent for liver disease can be produced (Fig. 1).

On the other hand, Upc2p, which is a transcription regulator that induces the expression of various genes involved in the biosynthesis of ergosterol in yeast, is in a state of being inactivated in the cytoplasm in the state of recognizing and binding to ergosterol, which is a main component of the cell membrane. Uptake of Upc2p into the nucleus allows it to bind dimer to the promoter of genes involved in ergosterol synthesis. This combination induces the expression of the genes involved in the synthesis of sterols and, as a result, promotes the synthesis of ergosterol.

Korean Registered Patent No. 10-1655696 (Registered on September 1, 2016)

An object of the present invention is saccharide as MY access celebrity bicyclic Ke (Saccharomyces a recombinant expression vector containing a UPC2 mutation gene in which the ligand binding domain region of Upc2p derived from S. cerevisiae is mutated, a recombinant Saccharomyces cerevisiae strain transformed with the recombinant expression vector, a method of promoting total cholesterol production using the recombinant strain And a method for promoting production of epoxysqualene and dhamrenediol by using the recombinant strain.

Another object of the present invention is to provide a UPC2 mutant gene insertion cassette comprising the UPC2 promoter gene, the UPC2 mutation gene, the UPC2 termination gene, the URA3 marker gene and the UPC2 termination gene in sequence, the UPC2 mutation insertion cassette , A recombinant Saccharomyces cerevisiae strain transformed with the recombinant vector, and a method for promoting total cholesterol production using the recombinant strain.

Another object of the present invention is to provide a CRISPR-Cas9 recombinant vector comprising a single guide RNA (sgRNA) targeting Cas9 gene and UPC2 gene; And a recombinant Saccharomyces cerevisiae strain transformed with Donor DNA for introducing the UPC2 mutation gene and a method for promoting sterol production using the recombinant strain.

In order to achieve the above object, the present invention provides a recombinant expression vector comprising a UPC2 mutation gene in which the ligand binding domain region of Upc2p derived from Saccharomyces cerevisiae is mutated.

In addition, the present invention provides a recombinant Saccharomyces cerevisiae strain transformed with the recombinant expression vector.

Also, the present invention provides a method for producing a recombinant vector comprising the steps of: transforming a Saccharomyces cerevisiae strain lacking the UPC2 gene with the recombinant expression vector; And culturing the transformed strain. The present invention also provides a method for promoting total cholesterol production.

The present invention also provides a method for producing a recombinant vector comprising the steps of: transforming a Saccharomyces cerevisiae strain producing the epoxysqualene and dhamalene diol with the recombinant expression vector; And culturing the transformed strain. The present invention also provides a method for promoting the production of epoxysqualene and fmalenediol.

The present invention also provides a UPC2 mutant gene insertion cassette containing a mutant gene UPC2, UPC2 terminator gene, URA3 marker gene and the gene end UPC2 represented by UPC2 promoter gene, SEQ ID NO: 1 sequence.

In addition, the present invention provides a recombinant vector comprising a UPC2 mutant gene insertion cassette.

In addition, the present invention provides a recombinant strain of Saccharomyces cerevisiae transformed with the recombinant vector.

In addition, the present invention provides a method for enhancing total cholesterol production comprising culturing the recombinant strain.

The present invention also relates to a CRISPR-Cas9 recombinant vector comprising a single guide RNA (sgRNA) targeting Cas9 gene and UPC2 gene; And a recombinant Saccharomyces cerevisiae strain transformed with donor DNA for introducing a UPC2 mutation gene.

In addition, the present invention provides a method of promoting sterol production comprising culturing the recombinant strain.

The present invention relates to a mutant transcription regulatory factor Upc2p expression optimization vector and a sterol precursor using the same, and more particularly, to a method for producing a cassette expressing the C-terminal amino acid substitution mutant UPC2 _G888D gene expression level through the UPC2 own promoter And increased the expression of ergosterol metabolism pathway genes by controlling the gene copy number to 1 to 2 copies low, thereby inducing the production of ginsenoside and cholesterol precursors including epoxysqualene and zymostosterol. Specifically, a double-stranded DNA fragment was deleted from the internal UPC2 gene using a Cris-Cass9 expression vector, and mutated to a C-terminal amino acid substitution mutant UPC2 _G888D (= UPC2-1 ) using donor DNA . This led to increased production of ginsenoside precursors including epoxysqualene by enhancing the expression of ergosterol biosynthetic pathway genes. UPC2 _G888D coding for the Upg2p mutant protein with the ergosterol binding site substitution The recombinant yeast optimized for the expression of the mutant gene can be produced by mass production of cholesterol, a precursor of the dysmenoride precursor, dysmenoride precursor, or UDCA, which is an agent for treating liver diseases, by remarkably improving the production efficiency of sterol precursors including epoxysqualene It can be used as a useful industrial host which is developed as a host and increases the stability of the product and the cost efficiency.

Figure 1 shows a schematic diagram of production of useful metabolites of ergosterol biosynthesis pathway in yeast.
FIG. 2 shows a strategy for producing a recombinant yeast having an ergosterol biosynthetic pathway in which feedback control through development of a transcription regulatory factor Upc2p mutation is relaxed.
Figure 3 is a schematic representation of the structure (A) and the modified amino acid sequence (B) of various variant Upc2p and various combinations of expression vectors (C) prepared through C- or N-terminal domain deletion or ligand binding domain substitutions will be. The position of the amino acid G888D (glycine-> aspartic acid) at the C-terminus of the yeast S. cerevisiae transcriptional regulator Upc2p is indicated in bold, and the terminal N- and C- domains at both ends are underlined.
4 is a comparative analysis of fluconazole resistance of the recombinant yeast strains according to the conditions of expressing various mutant Upc2p with the TEF1 promoter (A) or its own UPC2 promoter (B), CEN or 2μ vector (C) (D) the change in total sterol production by the introduction of the mutant type Upc2p, which was confirmed to be effective, by a spectrometry measurement.
FIG. 5 is a graph comparing the production efficiencies of epoxysqualene and dharmalene diol by HPLC after transforming the recombinant yeast strains producing epoxysqualene and dendrimerodiol into YCpH-Tp-UPC2-1 and YCpH-np-UPC2-1, respectively This is a result. Recombinant yeast cell extract (A) cultured in medium without methionine, recombinant yeast cell extract (B) cultured in medium supplemented with methionine. MET3 Inhibition of the ERG7 gene whose expression is regulated by the promoter increased the accumulation rate of epoxysqualene and dhamalene dienol .
6 is wild-type on the upc2 △ deficient Saccharomyces My process three Levy jiae chromosome of a host in which the, UPC2-1 mutant gene so as to have the effect of expressing Upc2-1p the strain has a wild-type gene UPC2 UPC2 (A), and a schematic diagram (B) of the Upc2-1 mutant production strategy using the cassette.
Figure 7 is a spectrophotometric analysis (A) for the polymerase reaction results and the total sterol biosynthesis compared to the CEN-based PK2-1C Upc2 -1 mutant confirmed obtained using the expression cassette produced in Figure 6, URA3 (B) is a spectrophotometric analysis result for confirming the mutagenicity of the gene after pop-out and confirming the efficiency of biosynthesis of total sterol.
8 is UPC2 A donor DNA sequence for a double-stranded cleavage and mutation occurring region (A) and 888 differ second amino acid variants UPC2 -1 gene replacement by site, Kas 9 enzyme sgRNA is recognized by the DNA sequence of the gene (B). The GGT gene sequence in which the mutation occurs is in capital letters, the DNA sequence recognized by the sgRNA is underlined, and the PAM region that allows double-strand breaks of the CAS 9 enzyme to occur is tilted.
9 is a graph showing the relationship between the Cas 9 enzyme used for the Upc2 protein mutation and the UPC2 (A) of the vector YCpH-SpCas9-sgUPC2 expressing the sgRNA that leads to the gene locus, and (B) a schematic diagram (B) showing the process in which the mutation occurs in the genomic UPC2 gene in the genome when the viral vector is expressed in the yeast.
FIG. 10 shows spectrophotometric analysis results (A) for the comparison of sterol production per cell of CEN.PK candidate strains transformed with the YCpH-SpCas9-sgUPC2 vector and the polymerase chain reaction of the C-terminal portion of the UPC2 gene for verification Sequencing results (B) and spectrophotometric analysis (C) for the comparison of sterol production per cell after removal of the YCpH-SpCas9-sgUPC2 vector through continuous culture in YPD medium resulted in the polymerase chain reaction (C) and C- Sequencing result (D). EV is a strain in which a wild-type CEN.PK strain has an empty vector having a HIS3 marker, and # 1 to 6 are YCpH-SpCas9-sgUPC2 vector transgenic candidate strains.
The Figure 11 uses the YCpH SpCas9--vector sgUPC2 replaced by UPC2 -1 mutant gene BY4742 strain, Cas9 (A) and Dhamma Lene diol-producing strain of the spectrophotometer for comparing per cell production of sterol EmD / tHle7 (B) The results of the analysis and the sequencing results of the polymerase chain reaction (C) of the C-terminal part of the UPC2 gene in some of the candidate strains of both strains. EV is a strain in which the wild-type BY4742 strain and the EmD / tHle7 strain have the HIS3 marker, respectively, and # 1 to # 5 are the YCpH-SpCas9-sgUPC2 vector transformation candidate strains.
12 is replaced by the UPC2 -1 mutant gene mutant strain BY4742 (Cas9), wild-type, with the empty vector (WT + EV), centromere while having the replication mechanism of the vector with the UPC2 promoter and UPC2 -1 ORF YCpH-npUPC2 (Upc2 + npUPC2-1) with a wild-type (WT + YCpH-npUPC2-1) containing -1, a BY-? Upc2 deletion (upc2 + EV) with a vacant vector, and a BY-? Upc2 deletion with a YCpH- (A) for observing the resistance under the addition of Fluconazole, which is a substance inhibiting the sterol biosynthesis against the strains, and (B) a spectrophotometric analysis result (B) for the comparison of the peroxide production per cell of the strains used in the above experiment.
FIG. 13 is a graph showing changes in the growth rate and final growth rate of the strain BY4742 used in the experiment of FIG. 12, the SC-H selection medium A and the EmD / tHle7 strain, centromere while having the replication mechanism and EmD UPC2 promoter containing vectors YCpH-npUPC2-1 with UPC2-1 ORF / tHle7 bacteria week SC- vector that could fall with the HIS3 marker as compared to a growth level L selection medium (B) and the SC-LUTH selection medium (C) in which the vector is not excluded.

Accordingly, the present inventors completed the present invention by preparing a recombinant yeast in which an ergosterol biosynthetic pathway was activated through protein engineering and optimization of expression of ergosterol biosynthesis-related electron regulatory factor (Upc2p) (FIG. 2).

The amino acid substitution mutation type UPC2-1 (Upc2p _G888D ) of the ergosterol adhering domain, which was developed in the present invention, Expression Optimization Cassettes can be used to develop cholesterol mass production host, which is a precursor of UDCA, in addition to ginsenoside precursors, since it induces production of various ergosterol biosynthetic precursors including epoxysqualene, zymostosterol and the like.

In addition, the present inventors introduced the CYPER -CAS 9 method to prepare a mutant transcription regulatory factor UPC2 gene that does not inhibit the activity by ergosterol, and mutated the wild-type UPC2 gene on the chromosome to mutant forms of ergosterol biosynthesis Pathway activated recombinant yeast was developed. The YCpH-SpCas9-sgUPC2 vector and the donor DNA sequence information prepared in the present invention can be efficiently inserted into the internal UPC2 It is possible to cause mutations in the gene, causing only a single nucleotide polymorphism in the genome, and exporting the vector is suitable for GRAS evaluation because antibiotic resistance and nutritional requirement markers do not remain in the yeast.

The present invention relates to the use of Saccharomyces cerevisiae a recombinant expression vector comprising a UPC2 mutant gene in which the ligand binding domain region of Upc2p derived from S. cerevisiae is mutated.

Preferably, the UPC2 mutant gene can be represented by SEQ ID NO: 1, but is not limited thereto. The UPC2 mutation gene represented by SEQ ID NO: 1 encodes an Upc2p mutant protein represented by the amino acid sequence of SEQ ID NO: 2. The 888th amino acid of SEQ ID NO: 2 was substituted with the aspartic acid (D) codon (GAT) for the glycine (G) codon (GGT) of the wild type Upc2p protein. UPC2 _G888D described in the specification of the present invention means the UPC2 mutation gene represented by SEQ ID NO: 1 described above.

The NCBI accession number of the UPC2 gene is 851799, and the NCBI accession number of the Upc2p protein may be LBMA01000004.1, but is not limited thereto.

Preferably, the recombinant expression vector comprises, but is not limited to, a UPC2 promoter gene, a UPC2 mutation gene represented by SEQ ID NO: 1, a GAL7 termination gene, and a HIS3 marker gene.

In the present invention, "vector" means a DNA molecule that is replicated by itself, which is used to carry the clone gene (or another fragment of the clone DNA).

In the present invention, an "expression vector" means a recombinant DNA molecule comprising a desired coding sequence and a suitable nucleic acid sequence necessary for expressing a coding sequence operably linked in a particular host organism. The expression vector may preferably comprise one or more selectable markers. The marker is typically a nucleic acid sequence having a property that can be selected by a chemical method, and includes all genes capable of distinguishing a transformed cell from a non-transformed cell. Examples include antibiotic resistance genes such as Ampicillin, Kanamycin, Geneticin (G418), Bleomycin, Hygromycin, and Chloramphenicol. And is suitably selectable by a person skilled in the art.

In addition, the present invention provides a recombinant Saccharomyces cerevisiae strain transformed with the recombinant expression vector.

Preferably, the recombinant strain is UPC2 The recombinant expression vector may be obtained by transforming Saccharomyces cerevisiae strain deficient in the gene with the recombinant expression vector. More preferably, the recombinant strain is Saccharomyces cerevisiae BY-upc2ΔΔ deposited with KCTC 13401BP, npUPC2-1 strain, but is not limited thereto. On the other hand, the strain Saccharomyces cerevisiae BY-upc2Δ / npUPC2-1 deposited with KCTC 13401BP is a strain in which total cholesterol production is enhanced.

Preferably, the recombinant strain may be obtained by transforming Saccharomyces cerevisiae strain producing epoxysqualene and dhamarenendiol with the recombinant expression vector. More preferably, the Saccharomyces cerevisiae strain producing the epoxysqualene and dhamrenediol can be Saccharomyces cerevisiae EmD / tH1e7 deposited with KCTC 13399BP, and the recombinant strain is KCTC 13400BP But are not limited to, the Saccharomyces cerevisiae EmD / tHle7 / npUPC2-1 strain deposited with the National Institutes of Health. On the other hand, the Saccharomyces cerevisiae EmD / tHle7 / npUPC2-1 strain deposited with KCTC 13400BP is a strain in which production of epoxysqualene and dhamrenediol is promoted.

Also, the present invention provides a method for producing a recombinant vector comprising the steps of: transforming a Saccharomyces cerevisiae strain lacking the UPC2 gene with the recombinant expression vector; And culturing the transformed strain. The present invention also provides a method for promoting total cholesterol production. Preferably, the transformed strain may be Saccharomyces cerevisiae BY-upc2Δ / npUPC2-1 strain deposited with KCTC 13401BP, but is not limited thereto.

The present invention also provides a method for producing a recombinant vector comprising the steps of: transforming a Saccharomyces cerevisiae strain producing the epoxysqualene and dhamalene diol with the recombinant expression vector; And culturing the transformed strain. The present invention also provides a method for promoting the production of epoxysqualene and fmalenediol.

Preferably, the Saccharomyces cerevisiae strain producing the epoxysqualene and the dhamalene diol may be a Saccharomyces cerevisiae EmD / tH1e7 strain deposited with KCTC 13399BP, and the transformed strain may be KCTC But not limited to, the Saccharomyces cerevisiae EmD / tHle7 / npUPC2-1 strain deposited at 130000BP.

The Saccharomyces cerevisiae EmD / tH1e7 strain deposited with KCTC 13399BP used in the present invention was transformed on the rDNA of squalene and Saccharomyces cerevisiae transformed with the expression vector of dhamalene diol synthase (DDS) The epoxysqualene and dhamalene diol in which the ERG1 mutation gene represented by SEQ ID NO: 3 is inserted, and the recombinant Saccharomyces cerevisiae strain.

The present invention also relates to a UPC2 promoter gene, a UPC2 mutation gene represented by SEQ ID NO: 1, a UPC2 termination gene, URA3 UPC2 containing the marker gene and the terminator gene sequence variations UPC2 provides a gene cassette inserted.

In addition, the present invention provides a recombinant vector comprising the UPC2 mutation gene insertion cassette.

In addition, the present invention provides a recombinant strain of Saccharomyces cerevisiae transformed with the recombinant vector. Preferably, the recombinant strain may be Saccharomyces cerevisiae CEN.PK-upc2-1 strain deposited with KCTC 13404BP, Saccharomyces cerevisiae CEN.PK-upc2 deposited with KCTC 13404BP 1 may be, but not limited to, an endogenous UPC2 gene substituted with a UPC2 mutation gene represented by SEQ ID NO: 1.

The Saccharomyces cerevisiae wild type strain used above is described as Enten and Kotter, Methods in Microbiology (2007) 36: 629-666 as CEN-PK2-1C strain.

In addition, the present invention provides a method for enhancing total cholesterol production comprising culturing the recombinant strain.

The URA3 marker gene and the HIS3 marker gene used in the present invention are used as an auxotrophic selection marker, and the URA3 marker (NCBI accession no. NM_001178836.3) or HIS3 marker gene (GENE ID: 854377) may be used, but are not limited thereto.

The UPC2 promoter gene used in the present invention is represented by SEQ ID NO: 4, the GAL7 termination gene is represented by SEQ ID NO: 5, and the UPC2 termination gene is represented by SEQ ID NO: 6.

The present invention also relates to a CRISPR-Cas9 recombinant vector comprising a single guide RNA (sgRNA) targeting Cas9 gene and UPC2 gene; And a recombinant Saccharomyces cerevisiae strain transformed with donor DNA for introducing a UPC2 mutation gene.

Preferably, the sgRNA targeting the UPC2 gene may be represented by SEQ ID NO: 7, and the donor DNA for introducing the UPC2 mutation gene may be represented by SEQ ID NO: 8, but the present invention is not limited thereto.

Preferably, the CRISPR-Cas9 recombinant vector may be a YCpH-SpCas9-sgUPC2 recombinant vector having a cleavage map of Fig. 9 (A), but is not limited thereto.

Preferably, the recombinant strain may be substituted with a UPC2 mutation gene represented by SEQ ID NO: 1 in a chromosome endogenous UPC2 gene.

In addition, the present invention provides a method of promoting sterol production comprising culturing the recombinant strain.

The CRISPR-Cas9 system used in the present invention is emerging as a genetic engineering tool that can be applied to bacteria and eukaryotes. Cas9 has an endonuclease function that cleaves the target DNA determined by sgRNA. A double-strand break (DSB) by Cas9 occurs at a specific sequence that is the anterior sequence of the protospacer adjacent motif (PAM) designated by the sgRNA. When transformed with the guide RNA and Cas9 expression vector together with the donor DNA having the homologous sequence, it is possible to perform gene correction with high efficiency by the homologous repair (HR) mechanism.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example  1: Various Transcription regulator Upc2p  Production of mutation parts and expression vectors

We tried to increase the total sterol biosynthesis activity including ergosterol by alleviating the feedback regulation of Upc2p transcription factor with a key role in regulating the activity of ergosterol biosynthesis pathway. YCpH-Tp-UPC2, which is a wild-type Upc2p expression vector, synthesizes a wild-type UPC2 gene by polymerase chain reaction with UPC2-fw and UPC2-bw primers and then cuts YCpH-Tp vector and UPC2 gene with SpeI / XhoI Respectively. Mutation of erucosterol binding site In order to synthesize the gene encoding Upc2p, the nucleotide sequences of the N-, C-terminus, and the UPC2-1-F1-bw of UPC2-fw, UPC2-bw, UPC2-1-F2- C-fragment was obtained and secured, followed by fused polymerase chain reaction to produce a UPC2-1 gene substituted with aspartic acid codon (GAT) by generating a single base mutation at 888th glycine codon (GGT). Specifically, UPC2-fw and UPC2-1-F1-bw were used to synthesize an 2,673 bp N-PCR fragment from the initiation codon ATG of the UPC2 gene to the mutation site, and 86 bp UPC2-1-F2-fw and UPC2-bw were used to synthesize the C-PCR fragment. The obtained UPC2-1 gene was digested with YCpH-Tp-UPC2 vector and SpeI / XhoI , and inserted into wild-type UPC2 to construct a YCpH-Tp-UPC2-1 vector. In order to construct Upc2p mutation parts lacking the N-domain part, fusion polymerase chain reaction using UPC2-fw, UPC2-NTT-F1-bw, UPC2-NTT-F2-fw and UPC2-NTT- A UPC2 _N? Fragment was prepared by removing the 100-390 amino acid sequence portion from the 300 bp N-fragment starting from the initiation codon ATG and the C-fragment starting from the 390 amino acid codon and having a length of 300 bp. The obtained UPC2 _NΔ mutant gene fragment and YCpH-Tp-UPC2 vector were digested with SpeI / SauI restriction enzyme to obtain wild type UPC2 When the gene is cloned In the YCpH-Tp-UPC2 contains the 390 amino acid sequence from the start codon of the SpeI UPC2 / SauI UPC2 _NΔ intercept and change the process proceeds to a linker (linker) portion that connects the DNA binding, ligand binding domain removed _{UPC2 NΔ (N-terminal 100-390 amnio} acids truncation) the vector YCpH Tp-expressing the gene encoding -UPC2 _{N ?} . In addition to UPC2-fw, UPC2-CTT- bw proceed polymerase chain reaction with a primer to synthesize a gene UPC2 _CΔ UPC2 C- short part (aa 792-913) is removed and cut into SauI / XhoI restriction enzymes SauI / XhoI The cleaved YCpH-Tp-UCP2 vector was ligated to the UPC2 C-fragment containing the 792-913 amino acid sequence to replace the wild-type UPC2 to obtain a UPC2 _CΔ (C-terminal 792 -913 amino acids truncation) Expression vector YCpH-Tp-UPC2 _C? Respectively. Also YCpH-Tp-UPC2-1 vector UPC2 to cut the sections with _NΔ SpeI / SauI restriction enzyme fragment and replacing the N- UPC2 _NΔ UPC2 of the wild-type gene having a UPC2-1 UPC2-1 mutations at the same time to C- fragment _NΔ A vector YCpH-Tp-UPC2-1 _N ? Expressing the gene was prepared (Fig. 3).

The primer list used in the present invention, the yeast strain used in the present invention, and the plasmid used in the present invention are shown in Tables 1, 2 and 3, respectively.

gene primer Base sequence Usage HIS3 ScHIS3-FW 5'-catg ccatgg taattctcgttttaagagcttg-3 ' For selective marking
Gene amplification
ScHIS3-BW-ApaI 5'-gcgc gggccc ctcgagttcaagagaaaaaaaaag-3 ' UPC2 UPC2-fw Gt ; Variant
Gene amplification UPC2-bw 5'-gcgc ctcgagcccggg ctataacgaaaaatcagagaaatttg-3 ' UPC2-1-Fu2-fw 5'-gaatacagtggaggtggt gat a-3 ' UPC2-1-Fu1-bw 5'-ctagcatcatatgcat atc acc-3 ' UPC2-NTT-F1-bw 5'-ctttctcgtgacatacttc-3 ' UPC2-NTT-F2-fw 5'-gaagtatgtcacgagaaagttacaagctgatgcattatct-3 ' UPC2-NTT-F2-bw 5'-tataggcgccattgtcga-3 ' UPC2-CTT-bw 5'-gcgc ctcgag aatgacatcgcctaaatc-3 ' pUPC2-fw 5'-gcgc ggatcc gcgc gttaac gcgcttactgctaagcc-3 ' pUPC2-FW-BamHI-HpaI 5'-gcgc ggatcc gcgc gttaac gcgcttactgctaagcc-3 ' Seq-ScUPC2-3B 5'-tccagaaggcaatggcggt-3 ' UPC2tL-FW 5'-gttgacgaatacagtggagg-3 ' UPC2tR-FW-SalI Gt ; UPC2tL-BW-NdeI 5'-gcgc catatg cttatcgacccagtgccag-3 ' UPC2tR-BW-XhoI 5'-gcgc ctcgag cctgaatgtcattaccatgg-3 ' Upc2-1-int-confirm-fw (2-1) 5'-gcattgctagacaagacattc-3 ' Check gene replacement and pop-out Upc2-1-int-confirm-bw (ura3) 5'-cagtcaagatatccacatgtg-3 '

¹ Restriction enzymes are marked with an underline.

^{2 The} sequence in which the 888th glycine was replaced with an aspartic acid was indicated in bold.

Strain Genotype Reference CEN-PK2-1C MATa ura3-52 trp1-289 leu2-3,112 his3-1 MAL2-8C SUC2 Entian and Kotter (2007) E _m D / tH1e7 CEN-tH1e7 with an integrated copy of the 403 bp p TEF1 -ERG1 _K311R cassette and harboring YEpL-PgDDS KCTC 13399BP BY4742 MATa His3D1 Leu2D0 Lys2D0 ura3D0 Open Biosystems BY-upc2? MATα; ura3Δ0 ; leu2Δ0 ; his3Δ1 ; lys2Δ0 ; upc2 :: kanMX4 Open Biosystems BY4742 / EV BY4742 harboring YCpU-Tp or YCpH-Tp Invention BY-upc2Δ / EV BY-upc2Δharboring YCpU-Tp or YCpH-Tp Invention BY / Tp-UPC2 BY4742 harboring YCpH-Tp-UPC2 Invention BY / Tp-UPC2-1 BY4742 harboring YCpH-Tp-UPC2-1 Invention BY / Tp-UPC2 _C? BY4742 harboring YCpH-Tp-UPC2 _C? Invention BY / Tp-UPC2 _N? BY4742 harboring YCpH-Tp-UPC2 _N? Invention BY / Tp-UPC2-1 _N? BY4742 harboring YCpU-Tp-UPC2-1 _N? Invention BY / npUPC2 BY4742 harboring YCpH-np-UPC2 Invention BY / npUPC2-1 BY4742 harboring YCpH-np-UPC2-1 Invention BY-upc2? / Tp-UPC2 BY-upC2Δharboring YCpH-Tp-UPC2 Invention BY-upc2? / Tp-UPC2-1 BY-upC2Aharboring YCpH-Tp-UPC2-1 Invention BY-upc2? / NpUPC2 BY-upC2Δharboring YCpH-np-UPC2 Invention BY-upc2? / NpUPC2-1 BY-upC2? Harboring YCpH-np-UPC2-1 Invention BY-upc2? / NpUPC2 _C? BY-upc2? Harboring YCpH-np-UPC2 _C? Invention BY-upc2? / NpUPC2 _N? BY-upc2? Harboring YCpH-np-UPC2 _N? Invention BY-upc2? / NpUPC2-1 _N? BY-upc2? Harboring YCpH-np-UPC2-1 _N? Invention BY / YE-np-UPC2-1 BY4742 harboring YEpH-np-UPC2-1 Invention BY-upc2? / YE-npUPC2-1 BY-upC2Δharboring YEpH-np-UPC2-1 Invention E _m D / tH1e7 / Tp-UPC2-1 Em / D / tH1e7 harboring YCpH-Tp-UPC2-1 Invention E _m D / tH1e7 / npUPC2-1 Em / D / tH1e7 harboring YCpH-np-UPC2-1 Invention CEN.PK-upC2-1 CEN-PK2-1C replaced with UPC2-1 mutant allele Invention

Plasmid Description Reference pRE316 p ScGAL1 / 10-URA3-CEN vector Cheon et al, (2010) pTcUR190 Score3 containing vector without yeast replication origin Invention YCpU-Tp CEN-based pRE316 derivative containing the TEF1 promoter, the GAL7 terminator and the URA3 marker Invention YCpH-Tp YCpU-Tp derivative containing the HIS3 marker Invention YCpH-Tp-UPC2 YCpH-Tp containing the wild type UPC2 fused to the TEF1 promoter Invention YCpH-Tp-UPC2-1 YCpH-Tp containing a single amino acid variant UPC2 _G888D fused to the TEF1 promoter Invention YCpH-Tp-UPC2 _C? YCpH-Tp containing a C-terminal truncated UPC2 fused to the TEF1 promoter Invention YCpH-Tp-UPC2 _N? YCpH-Tp containing a N-terminal truncated UPC2 fused to the TEF1 promoter Invention YCpH-Tp-UPC2-1 _N? YCpH-Tp containing the double variant UPC2-1 _N? fused to the TEF1 promoter Invention YCpH-np-UPC2 YCpH derivative expressing the wild type UPC2 under the control of its own promoter Invention YCpH-np-UPC2-1 YCpH derivative expressing UPC2 -1 under the control of its own promoter Invention YCpH-np-UPC2 _C? YCpH derivative expressing a C-terminal truncated UPC2 under the control of ts own promoter Invention YCpH-np-UPC2 _N? YCpH derivative expressing a N-terminal truncated UPC2 under the control of its own promoter Invention YCpH-np-UPC2-1 _N? YCpH derivative expressing a double variant UPC2-1 _N? under the control of its own promoter Invention YEpH-np-UPC2 2μ -based vector expressing UPC2 under the control of its own promoter and carrying HIS3 marker Invention YEpH-np-UPC2-1 2-based vector expressing UPC2 -1 under the control of its own promoter and carrying HIS3 marker Invention
NP-UPC2-1-integration UPC2 promoter, UPC2-1 , UPC2 terminator and URA3 marker Invention

Example  2 : Upc2p  Increased productivity of ergosterol by optimizing expression of mutated parts

The prepared YCpH-Tp-UPC2 vectors were introduced into wild-type Saccharomyces cerevisiae BY4742 strain and BY- upc2Δ mutant strain in which the UPC2 gene was disrupted to select HIS + transformants grown in the SC-HIS medium. The resistance of these recombinant yeast strains to fluconazole, an antimicrobial agent that inhibits the ergosterol biosynthesis, was measured. As a result, wild type Upc2p overexpression resulted in a decrease in fluconazole resistance and overexpression of the mutant Upc2p protein was slightly increased Resistant fluconazole. On the other hand, in the upc2 △ deletion mutant, only the wild type Upc2p overexpression regained fluconazole resistance to the wild type strain level and the mutant Upc2p overexpression regained its recovery level (FIG. 4A). This suggests that the transcription activity of the prepared Upc2p mutant proteins is considerably lower than that of the wild type.

Therefore, the present inventors have recognized that it is important to optimize the expression level of Upc2p protein and use pUPC2-FW-BamHI-HpaI, Seq-ScUPC2-3B to be expressed by the UPC2 self-promoter instead of the TEF1 promoter which always maintains high expression level The primer was used to amplify the UPC2 N-fragment (658 bp) carrying the UPC2 promoter ( pUPC2 , 480 bp) by performing the polymerase chain reaction on Saccharomyces cerevisiae genome. The thus obtained N-fragment having the 1,138 bp UPC2 promoter was digested with BamHI and EcoRI restriction enzymes and ligated to YCpU-Tp-UPC2, YCpU-Tp-UPC2-1, YCpH- _Tp- Secured by cutting the vector with EcoRI and XhoI restriction enzymes UPC2 C- fragments, fragments UPC2 -1, UPC2 _CΔ The fragment was digested with BamHI / XhoI YCpH-np-UPC2, YCpH-np-UPC2-1, and YCpH-np-UPC2 _CΔ were prepared by performing three-piece ligation with pRE316 vector digested with restriction enzymes (FIG. The YCpH-np-UPC2 _NΔ vector was used to construct the YCpH-pT-UPC2 _NΔ vector FW-BamHI-HpaI primer instead of UPC2-fw in the UPC2-fw, UPC2-NTT-F1-bw, UPC2-NTT-F2-fw and UPC2- A 1,070 bp gene fragment containing the UPC2 promoter and N-segment was ligated with a C-fragment having a length of 300 bp starting from the 390 amino acid codon, followed by digestion with BamHI and Eco81I restriction enzymes, and the YCpH-np-UPC2 vector Of the BamHI / Eco81I UPC2 fragment. From these YCpH-np-UPC2 vectors results UPC2 own promoter compared to fluconazole resistant analysis was upc2 △ transforming a mutant strain deficient in the showed the highest variation Upc2-1p fruit to Kona sol resistant to a strain in which the protein is expressed at moderate levels (Fig. 4B). The importance of this level of expression was reaffirmed as a result of significantly reduced fluconazole resistance in both wild-type and mutant strains when expressed in a high copy number based on 2μ-even when expressed from the UPC2 own promoter (FIG. 4C). The total cholesterol productivity of the recombinant yeast strains expressing the produced Upc2-1p variant component as a self-promoter was measured by a spectrophotometer. As a result, as compared with the wild-type strain, upc2Δ deletion strain was upc2- 1p, the efficiency of sterol biosynthesis was further increased (FIG. 4D).

Example  3: Expression Optimized Upc2 Epoxy squalene by introduction into -1p and Fenadenosine  Increase productivity

After confirming the completion of the Executive Producer of promoting cholesterol production effect with Upc2-1p optimize expression, the E _m D / tH1e7 recombinant strain producing epoxy squalene and Dhamma Lene diol YCpH-Tp-UPC2-1 and YCpH-np-UPC2-1 Each was transformed and then analyzed by HPLC. The cells were preincubated with 1 ~ 2 colony strains in SC (YNB + amino acid mixture, except leucine, uracil, tryptophan, histidine) medium and then incubated in SC medium with or without 10 mM methionine Lt; / RTI > After culturing for 24 hours (28 ° C, 220 rpm), yeast cells OD ₆₀₀ = 100 were collected in a 2 ml tube, suspended in 1 ml of a solution of KOH in 13% of 70% ethanol and incubated in an incubator at 85 ° C for 1 hour And the yeast cells were disrupted. Then, 800 μl of heptane was added to each sample, and the mixture was vortexed for 30 seconds. After centrifugation (12,000 rpm, 1 min, 25 ° C), heptane supernatant (600 μl) was recovered and dried. HPLC analysis was carried out on the dried sample by adding 200 ul of acetone to the dissolved sample. For the HPLC analysis, Cosmosil C18-PAQ (4.6 mm * 250 mm) was used, the flow rate was 1 ml / min, the analytical solvent was 100% acetonitrile and the analysis time was 30 min. As a result, the recombinant yeast strain Upc2-1p expression vector is introduced by the TEF1 promoter for _{(E m D / tHle7 / Tp} -UPC2-1) , whereas there is no much increased effect, Upc2-1p expression by UPC2 promoter vector ( (E _m D / tHle7 / npUPC2-1) into which YCpH-np-UPC2-1 was introduced (FIG. 5). When cultured in SC medium that is not the D E _m / tH1e7 recombinant strain of 10 mM methionine ERG7 that expression is controlled by the introduction, the attachment Upc2-1p as well as epoxy squalene and ergosterol biosynthesis Dhamma Lene diol biosynthesis by MET3 promoter Fig. A significant increase was observed (Figure 5A), due to methionine attachment, ERG7 The expression is suppressed in the culture conditions, the overall yield of the epoxy squalane, squalene Dhamma Lene diol and produced in E _m D / tH1e7 recombinant strain is greatly increased, and the state after the introduction Upc2-1p ergosterol biosynthesis is not significantly increase the epoxy squalene and A significant increase in the productivity of dendrimer dendiol was observed (Fig. 5B).

Example  4: Expression Optimized UPC2 -One  Production and utilization of gene replacement cassettes

Upc2-1p is upc2 (Fig. 4C) and the highest amount of sterol (Fig. 4D), the expression of ginsenosides, cholesterol precursors and cholesterol precursors while eliminating the endogenous UPC2 Esaka to access My Celebi jiae with endogenous (endogenous) UPC2 genes for higher sterol production strains produced for the production at the same time to prepare a vector that can be replaced by inserting a gene UPC2-1. For this, the YCpH-np-UPC2-1 vector and the pTcUR190 vector were digested with BamHI / NdeI restriction enzyme, and the UPC2 obtained from the YCpH -np-UPC2-1 vector The promoter- UPC2-1 ORF (1-889 amino acid) fragment was inserted into the pTcUR190 vector to obtain NP-UPC2-int. I made the step1 vector. The UPC2tL-FW and UPC2tL-BW-NdeI primers shown in Table 1 were used to amplify the fragment containing the ORF of 889-912 amino acid region and the UPC2 specific terminator of 424 bp in the wild-type Saccharomyces cerevisiae strain, Chain reaction. The NP-UPC2-int. Step 1 vector and UPC2 ORF (889-912 amino acid) - The UPC2 terminator fragment was treated with NdeI restriction enzyme and the fragment was designated NP-UPC2-int. step1 vector and check the directionality of NP-UPC2-int. I made a step2 vector. A UPC2 terminator fragment of 885 bp in length was prepared to remove the URA3 gene using homologous recombination in the wild-type Saccharomyces cerevisiae strain using the UPC2tR-FW-SalI and UPC2tR-BW-XhoI primers shown in Table 1 . This fragment and NP-UPC2-int. The step 2 vector was treated with SalI / XhoI restriction enzyme, and a fragment was inserted into the vector to prepare a final NP-UPC2-1 integration vector (FIG. 6A).

The prepared NP-UPC2-1 vector was amplified by SpeI / XhoI After digestion with restriction enzymes and linearization, wild-type UPC2 While eliminating the endogenous UPC2 by double homologous recombination with transgenic UPC2 by promoter and terminator part of my process Celebi jiae CEN-PK2-1C Saccharomyces strains into which genes have been inserted to replace UPC2 -1 gene is induced ( 6B). After transformants having URA3 selection markers were first obtained in the Uracil-free medium, the transformants in which the UPC2-1 gene cassette was inserted and replaced into the endogenous UPC2 site were amplified with primers Upc2-1-int-confrim-fw (2 -1) / Upc2-1-int-confirm-bw (ura3) (Fig. 7A, left panel). In addition, the ability of the URA3 transformants to increase or decrease the amount of cholesterol precursor production was also measured by a spectrophotometer, and the transformant # 3 exhibiting the same pattern as that of the PCR was selected (Fig. 7A, right panel) the PCR fragment was sequenced to verify that the finally substituted by UPC2 -1 gene (Figure 7A, the panel below). Two UPC2 included in UPC2 -1 gene cassette inserted Transformants # 3 and Uracil were added in a medium supplemented with 5-Fluoroorotic Acid and Uracil to restore the UPC2 terminator in its original form by popping out the inserted URA3 gene causing homologous recombination between the terminator sequences Were cultured. Upc2-1-int-confrim-fw ( 2-1), Upc2-1-int-confirm-bw previously used in two UPC2 transformant screening the URA3 selection marker is suspended through the homologous recombination between the transfected terminator sequence removed ( ura3) using a primer proceeds Polymerase chain reaction analysis by a URA3 pop-out can be obtained of the UPC2 -1 mutant (Fig. 7B, left panel). Finally, the Saccharomyces cerevisiae wild-type CEN.PK2-1C strain was transformed with the above cassette to maintain a higher level of sterol aggregation performance than the wild type in the UPC2-1 mutant (CEN.PK-upc2-1) Were measured and analyzed using a spectrophotometer. As a result, the transformant # 3-2 was transformed into URA3 After the pop-out process, it was confirmed that the sterol synthesis was maintained at a higher level than the wild type (Fig. 7B, right panel).

Example  5: UPC2 -One  Replacement of gene mutation Crisper - CAS 9  base YCpH - SpCas9 -sgUPC2 vector production

UPC2 a kid with UPC2 Saccharomyces expressing the wild-type gene present in the genome Mai Seth Serenity busy to have a gene mutation UPC2-1 A CAS 9 system was used to more efficiently alter the DNA sequence of the gene. The 181 bp PCR product obtained using UPC2-gRNA-L-FW and UPC2-gRNA-L-BW primers shown in Table 4 and the UPC2-gRNA-L-BW primer shown in Table 4 using the Coex 413- Two 291-bp PCR products obtained by using gRNA-R-FW and UPC2-gRNA-R-BW primers were used as templates again using UPC2-gRNA-L-FW and UPC2-gRNA- The part of the spacer RNA, which had a complementary base sequence to the ORF part of the URA3 gene, was substituted with the C-terminal part (2,641-2,660 bp) of the UPC2 gene, A 452 bp fragment was prepared. Coex 413-Cas9-UCC1 gRNA vector was loaded with XbaI / SalI After treatment with restriction enzymes, URA3 After eliminating the portion of the spacer RNA having a complementary sequence to the ORF of the gene, the synthesized polymerase chain reaction fragment was treated with XbaI / SalI restriction enzyme and ligated to obtain UPC2 A YCpH-SpCas9-sgUPC2 vector causing double-strand break at the C-terminus of the gene was prepared (Fig. 9A).

Cas9 to UPC2 The possible sites for the sgRNA binding and the DNA cleavage site leading to the gene location are shown in Figure 8A. By CAS 9 enzyme When you double-stranded cleavage occurs in the genome so that it can be recovered in the existing state of the wild-type gene or UPC2 UPC2-1 genes instead of causing non-specific variation in the process of recovering them into the donor DNA fragments corresponding to genes intentionally UPC2 -1 DNA must be transformed into yeast to be used for genome recovery as a template. For this purpose, two primers, UPC2-1 donor-DNA-FW and UPC2-1 donor-DNA-BW, shown in Table 1 were used to amplify the 78bp DNA fragments were obtained (Fig. 8B).

The list of primers used in this example, the plasmid used in the present invention and the yeast strain used in the present invention are shown in Tables 4, 5 and 6, respectively.

gene primer Base sequence Usage UPC2 UPC2-gRNA-L-FW 5'-gcgc tctaga ttttgtagtgccctct-3 '  Spacer RNA replacement fragment in sgRNA for UPC2 recognition UPC2-gRNA-L-BW 5'-ccacctccactgtattcgtcgatcatttatctttcactgcgg-3 ' UPC2-gRNA-R-FW 5'-gacgaatacagtggaggtgggttttagagctagaaatagcaa-3 ' UPC2-gRNA-R-BW 5'-gcgc gtcgac gaataatgtaatcgtg-3 ' UPC2-1 donor-DNA-FW 5'-tgctgcctcaagatgttgacgaatacagtggaggtggtgAtatgcatatg-3 ' UPC2 -1 mutant donor DNA amplification for gene replacement UPC2-1 donor-DNA-BW 5'-gtaatccgccaccgaggaaatctagcatcatatgcataTcaccacctcca-3 '

¹ restriction enzyme recognition sites are indicated by underlined letters.

^{2 The} DNA sequence that replaced the 888 amino acid glycine with aspartic acid was shown in capital letters.

Plasmid Description Reference Coex 413-Cas9-UCC1 gRNA Yeast CEN - HIS3 vector containing Cas9 and UCC1 targeting sgRNA SNU, Hahn J.-S. (unpublished) YCpH-SpCas9-sgUPC2 YCpH-SpCas9-sgURA3 derivative containing sgRNA targeting UPC2 gene Invention

Strain Genotype Reference Cas9 BY4742 replaced with UPC2 -1 mutant allele Invention EmD / tHe7-Cas EmD / tHe7 replaced with UPC2 -1 mutant allele Invention

Example  6: Crisper  Genome phase through system introduction UPC2 gene Mutant  Securing and analyzing

The prepared YCpH-SpCas9-sgUPC2 vector and UPC2-1 donor DNA were introduced into the wild-type Saccharomyces cerevisiae CEN.PK2-1C strain to select HIS ⁺ transformants growing on the SC-HIS selection medium. Small amounts of vector and 3 μg of donor DNA were used for transformation. Sterol quantitative analysis was performed by using a selective medium followed by a secondary screening procedure to obtain strains with mutations in the genome. Six of the obtained transformants were used as candidates and cultured in SC-HIS liquid medium for 24 hours to obtain yeast of OD ₆₀₀ 10. After transferring the yeast to a 2 ml microtube, remove the culture, dissolve in 1 ml of ethanol solution containing 6.25% KOH and 8.75% H ₂ O and react at 85 ℃ for 1 hour. After completion of the reaction, 0.8 ml of heptane is added and mixed well. Then, the layer is separated and the heptane supernatant collected in the upper layer is transferred to a new tube. The heptane supernatant is mixed with 100% ethanol at a ratio of 1: 5 and the value is measured at a wavelength of 240-310 nm using a spectrophotometer scanning function. CEN.PK2-1C yeast containing HIS3-expressing vector was used as a reference strain, and a solution of pure heptane and ethanol was used for sterol quantitative analysis control. As a result of the analysis, five strains out of the six candidate strains showed a higher level of sterol synthesis than strains containing a co-vector (FIG. 10A). GDNAs of 6 candidate strains were obtained and the C-terminal portion of UPC2 gene was amplified using Upc2-1-int-confirm-fw (2-1) and Upc2-1-confirm-bw (2-1) primers shown in Table 1 Sequence analysis was carried out by polymerase chain reaction. As a result, GGT mutated from GGT to GAT in all six cases, and the 5th candidate strain with low level of sterol synthesis showed an additional 2,655 nt Lt; / RTI > (Fig. 10B).

When the CA 9 enzyme undergoes double strand breaks on the genome by the PAM sequence, the donor DNA corresponding to the sequence of the UPC2-1 gene is recovered as a template. In this case, NGG, which is a PAM sequence of the wild-type UPC2 gene, is designed so that the UPC2-1 mutation gene is replaced with the NGA sequence and the mutation of the CAS 9 enzyme is once mutated so that double strand breakage is no longer caused (Fig. 9A). However, even with a low probability, additional double strand breaks may occur, resulting in UPC2 Since nonspecific mutations including non-homologous-end-joining may occur in the gene, the transformants are continuously cultured in YPD medium to confirm that the vector is missing, and then the sperm determination and sequencing analysis are performed again. It was confirmed that there was no change in sterol biosynthesis function or gene sequence (Fig. 10C and Fig. 10D).

Example  7: Crisper  System using different strains Sakaromayses  From Serebiisha UPC2 -One Gene replacement

Saccharomyces cerevisiae has a variety of strains and slightly different genomic DNA sequences. Therefore, transformation was carried out in order to confirm whether the Crisper system produced in this patent is applied to the same strain or to a strain which has genetic manipulation other than wild type. In addition to CEN.PK2-1C, BY4742, which is frequently used in laboratories and CEN.PK2-1C, has a mutation and a foreign gene in some of the genes involved in sterol synthesis, resulting in the production and accumulation of oxydosqualene and squalene And was transformed into EmD / tHle7 (Table 2) strain capable of producing dhamaranediol, a precursor of ginsenoside. As a result, the candidate strains were easily obtained from both strains. Sterol analysis and sequencing of these candidate strains showed that UPC2-1 mutant gene, which was designed from 9 out of 10 candidate strains, (Fig. 11A, Fig. 11B and Fig. 11C). In the case of the remaining one strain, it was confirmed that a single base defect occurred in another site than the previous CEN.PK2-1C strain.

Example 8: Upc2-1 Sterol biosynthesis inhibitor resistance test of gene replacement strain

Fluconazole is used as an antifungal agent by inhibiting the biosynthesis of sterols in cells by inhibiting the function of Erg11p present in yeast ergosterol synthesis. In order to confirm that the yeast strain with the increased ability to synthesize sterol is weakened by inhibition of growth inhibition, SC-H medium containing fluconazole at concentrations of 0, 8, 15, and 20 μg / (Fig. 12A). The Cas9 strain, which was replaced with Upc2-1 gene, showed similar growth to the BY4742 wild-type strain containing YCpH-npUPC2-1. In the BY-upc2Δ mutant, YCpH-npUPC2-1 vector showed higher resistance to fluconazole, while the wild-type BY4742 strain had weak resistance and the vector containing BY-upc2Δ mutant had weak resistance. The resistance was very low. Sterols were also quantitatively analyzed (FIG. 12B) by using the strains used for the experiment in Example 3, and the YCpH-npUPC2-1 vector was inserted into the BY-upc2Δ mutation, which was the most resistant to the most sterol biosynthesis inhibitor Showed the highest level of sterol biosynthesis in the strain. In addition, the BY4742 wild-type and BY-upc2Δ mutant strains showed the lowest levels of sterol biosynthesis at similar levels, and the WT + YCpH-npUPC2-1 strain expressing the wild-type UPC2 gene and the UPC2-1 gene Sterol levels of the UPC2-1 mutant strain ( Cas9 ) obtained from the per system were similar. In addition, in the case of the wild-type, strain YCpH-npUPC2-1 having the YCpH-npUPC2-1 vector (upc2 + YCpH-npUPC2-1) in the wild-type BY-upc2Δ mutation, The growth rate was lower than that of the strains.

Example 9: UPC2-1 gene Confirmation of the growth inhibition of the replacement strain

In the case of the wild type strain Chris flops manufactured by applying the system UPC2-1 variations replaced strain (Cas9), YCpH-npUPC2-1 wild type (WT + 2-1) and upc2Δ mutation with a vector (upc2 + 2-1) strain Showed no inhibition of growth in SC-H medium. To confirm this, the growth curve and the final growth level in SC-H medium were confirmed (Fig. 13A). As a result, the strain (upc2 + YCpH-npUPC2-1) having a YCpH-npUPC2-1 vector in wild-type and upc2Δ mutant strains was found to have a growth rate of about 10 -30%. On the other hand, it was confirmed that the UPC2-1 mutant gene showed a similar or higher growth rate than the reference strain containing the empty vector.

As a result of comparing the degree of growth in EmD / tHle7, which is a production strain of dendrimer-rene diol, in the SC-L medium, in which the YCpH-npUPC2-1 vector can escape, the strain (Em + EV) (Em + 2-1) having the YCpH-npUPC2-1 vector showed the following degree of growth, and the strain of the UPC2-1 mutant gene (Em + Cas ) Showed the lowest growth (Fig. 13B). However, there was no significant difference within the OD value of 1. Growth in the SC-LUTH medium, a condition in which the vector did not escape, showed that the strain with the empty vector still showed the highest growth, and that the UPC2-1 mutant gene had the next sequence, the YCpH-npUPC2-1 vector The strain showed the lowest growth (Fig. 13C). With this result, the high, but are sterol biosynthesis per cell strain having a YCpH-npUPC2-1 vector appears the growth inhibition, UPC2 -1 gene mutation that replaced strain is not an inhibitory effect on the growth, while there is found little effect sterol biosynthesis It can be seen that there are advantages.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Korea Biotechnology Research Institute KCTC13399BP 20171120 Korea Biotechnology Research Institute KCTC13400BP 20171120 Korea Biotechnology Research Institute KCTC13401BP 20171120 Korea Biotechnology Research Institute KCTC13404BP 20171124

<110> Chung-Ang University Industry-Academy Cooperation Foundation          Intelligent Synthetic Biology Center <120> Expression vectors for optimal expression of mutated Upc2p and          method for overproducing sterol precursors using the same <130> ADP-2018-0372 <150> KR 10-2017-0182422 <151> 2017-12-28 <160> 8 <170> Kopatentin 2.0 <210> 1 <211> 2739 <212> DNA <213> Saccharomyces cerevisiae <400> 1 atgagcgaag tcggtataca gaatcacaag aaagcggtga caaaacccag aagaagagaa 60 aaagtcatcg agctaattga agtggacggc aaaaaggtga gtacgacttc aaccggtaaa 120 cgtaaattcc ataacaaatc aaagaatggg tgcgataact gtaaaagaag aagagttaag 180 tgtgatgaag ggaagccagc ctgtaggaag tgcacaaata tgaagttgga atgtcagtat 240 acaccaatcc atttaaggaa aggtagagga gcaacagtag tgaagtatgt cacgagaaag 300 gcagacggta gcgtggagtc tgattcatcg gtagatttac ctcctacgat caagaaggag 360 cagacaccgt tcaatgatat ccaatcagcg gtaaaagctt caggctcatc caatgattcc 420 tttccatcaa gcgcctctac aactaagagt gagagcgagg aaaagtcatc ggcccctata 480 gaggacaaaa acaatatgac tcctctaagt atgggcctcc agggtaccat caataagaaa 540 gatatgatga ataacttttt ctctcaaaat ggcactattg gttttggttc tcctgaaaga 600 ttgaattcag gtatcgatgg cttactatta ccgccattgc cttctggaaa tatgggtgcg 660 ttccaacttc agcaacagca gcaagtgcag cagcaatctc aaccacagac ccaagcgcag 720 caagcaagtg gaactccaaa cgagagatat ggttcattcg atcttgcggg tagtcctgca 780 ttgcaatcca cgggaatgag cttatcaaat agtctaagcg ggatgttact atgtaacagg 840 attccttccg gccaaaacta cactcaacaa caattacaat atcaattaca ccagcagctg 900 caattgcaac agcatcagca agttcagctg cagcagtatc aacaattacg tcaggaacaa 960 caccaacaag ttcagcaaca acaacaggaa caactccagc aataccaaca acattttttg 1020 caacaacagc aacaagtact gcttcagcaa gagcaacaac ctaacgatga ggaaggtggc 1080 gttcaggaag aaaacagcaa aaaggtaaag gaagggcctt tacaatcaca aacaagcgaa 1140 actactttaa acagcgatgc tgctacatta caagctgatg cattatctca gttaagtaag 1200 atggggctaa gcctaaagtc gttaagtacc tttccaacag ctggtattgg tggtgtttcc 1260 tatgactttc aggaactgtt aggtattaag tttccaataa ataacggcaa ttcaagagct 1320 actaaggcca gcaacgcaga ggaagctttg gccaatatgc aagagcatca tgaacgtgca 1380 gctgcttctg taaaggagaa tgatggtcag ctctctgata cgaagagtcc agcgccatcg 1440 aataacgccc aagggggaag tgctagtatt atggaacctc aggcggctga tgcggtttcg 1500 acaatggcgc ctatatcaat gattgaaaga aacatgaaca gaaacagcaa catttctcca 1560 tcaacgccct ctgcagtgtt gaatgatagg caagagatgc aagattctat aagttctcta 1620 ggaaatctga caaaagcagc cttggagaac aacgaaccaa cgataagttt acaaacatca 1680 cagacagaga atgaagacga tgcatcgcgg caagacatga cctcaaaaat taataacgaa 1740 gctgaccgaa gttctgtttc tgctggtacc agtaacatcg ctaagctttt agatctttct 1800 accaaaggca atctgaacct gatagacatg aaactgtttc atcattattg cacaaaggtc 1860 tggcctacga ttacagcggc caaagtttct gggcctgaaa tatggaggga ctacataccg 1920 gagttagcat ttgactatcc atttttaatg cacgctttgt tggcattcag tgccacccat 1980 ctttcgagga ctgaaactgg actggagcaa tacgtttcat ctcaccgcct agacgctctg 2040 agattattaa gagaagctgt tttagaaata tctgagaata acaccgatgc gctagttgcc 2100 agcgccctga tactaatcat ggactcgtta gcaaatgcta gtggtaacgg cactgtagga 2160 aaccaaagtt tgaatagcat gtcaccaagc gcttggatct ttcatgtcaa aggtgctgca 2220 acaattttaa ccgctgtgtg gcctttgagt gaaagatcta aatttcataa cattatatct 2280 gttgatctta gcgatttagg cgatgtcatt aaccctgatg ttggaacaat tactgaattg 2340 gtatgttttg atgaaagtat tgccgatttg tatcctgtcg gcttagattc gccatatttg 2400 ataacactag cttatttaga taaattgcac cgtgaaaaaa accagggtga ttttattctg 2460 cgggtattta catttccagc attgctagac aagacattcc tggcattact gatgacaggt 2520 gatttaggtg caatgagaat tatgagatca tattataaac tacttcgagg atttgccaca 2580 gaggtcaagg ataaagtctg gtttctcgaa ggagtcacgc aggtgctgcc tcaagatgtt 2640 ggggatgtgg tgatatgcat atgatgctag atttcctcgg tggcggatta 2700 ccatcgatga caacaacaaa tttctctgat ttttcgtta 2739 <210> 2 <211> 913 <212> PRT <213> Saccharomyces cerevisiae <400> 2 Met Ser Glu Val Gly Ile Gln Asn His Lys Lys Ala Val Thr Lys Pro   1 5 10 15 Arg Arg Arg Glu Lys Val Ile Glu Leu Ile Glu Val Asp Gly Lys Lys              20 25 30 Val Ser Thr Thr Ser Thr Gly Lys Arg Lys Phe His Asn Lys Ser Lys          35 40 45 Asn Gly Cys Asp Asn Cys Lys Arg Arg Arg Val Lys Cys Asp Glu Gly      50 55 60 Lys Pro Ala Cys Arg Lys Cys Thr Asn Met Lys Leu Glu Cys Gln Tyr  65 70 75 80 Thr Pro Ile His Leu Arg Lys Gly Arg Gly Ala Thr Val Val Lys Tyr                  85 90 95 Val Thr Arg Lys Ala Asp Gly Ser Val Glu Ser Asp Ser Ser Val Asp             100 105 110 Leu Pro Pro Thr Ile Lys Lys Glu Gln Thr Pro Phe Asn Asp Ile Gln         115 120 125 Ser Ala Val Lys Ala Ser Gly Ser Ser Asn Asp Ser Phe Pro Ser Ser     130 135 140 Ala Ser Thr Thr Lys Ser Glu Ser Glu Glu Lys Ser Ser Ala Pro Ile 145 150 155 160 Glu Asp Lys Asn Asn Met Thr Pro Leu Ser Met Gly Leu Gln Gly Thr                 165 170 175 Ile Asn Lys Lys Asp Met Met Asn Asn Phe Phe Ser Gln Asn Gly Thr             180 185 190 Ile Gly Phe Gly Ser Pro Glu Leu Asn Ser Gly Ile Asp Gly Leu         195 200 205 Leu Leu Pro Pro Leu Pro Ser Gly Asn Met Gly Ala Phe Gln Leu Gln     210 215 220 Gln Gln Gln Gln Val Gln Gln Gln Ser Gln Pro Gln Thr Gln Ala Gln 225 230 235 240 Gln Ala Ser Gly Thr Pro Asn Glu Arg Tyr Gly Ser Phe Asp Leu Ala                 245 250 255 Gly Ser Pro Ala Leu Gln Ser Thr Gly Met Ser Leu Ser Asn Ser Leu             260 265 270 Ser Gly Met Leu Leu Cys Asn Arg Ile Pro Ser Gly Gln Asn Tyr Thr         275 280 285 Gln Gln Gln Leu Gln Tyr Gln Leu His Gln Gln Leu Gln Leu Gln Gln     290 295 300 His Gln Gln Val Gln Leu Gln Gln Tyr Gln Gln Leu Arg Gln Glu Gln 305 310 315 320 His Gln Gln Val Gln Gln Gln Gln Gln Gllu Gln Leu Gln Gln Tyr Gln                 325 330 335 Gln His Phe Leu Gln Gln Gln Gln Gln Val Leu Leu Gln Gln Glu Gln             340 345 350 Gln Pro Asn Asp Glu Glu Gly Gly Val Glu Glu Glu Asn Ser Lys Lys         355 360 365 Val Lys Glu Gly Pro Leu Gln Ser Gln Thr Ser Glu Thr Thr Leu Asn     370 375 380 Ser Asp Ala Ala Thr Leu Gln Ala Asp Ala Leu Ser Gln Leu Ser Lys 385 390 395 400 Met Gly Leu Ser Leu Lys Ser Leu Ser Thr Phe Pro Thr Ala Gly Ile                 405 410 415 Gly Gly Val Ser Tyr Asp Phe Gln Glu Leu Leu Gly Ile Lys Phe Pro             420 425 430 Ile Asn Asn Gly Asn Ser Arg Ala Thr Lys Ala Ser Asn Ala Glu Glu         435 440 445 Ala Leu Ala Asn Met Gln Glu His His Glu Arg Ala Ala Ala Ser Val     450 455 460 Lys Glu Asn Asp Gly Gln Leu Ser Asp Thr Lys Ser Pro Ala Pro Ser 465 470 475 480 Asn Asn Ala Gln Gly Gly Ser Ala Ser Ile Met Glu Pro Gln Ala Ala                 485 490 495 Asp Ala Val Ser Thr Met Ala Pro Ile Ser Met Ile Glu Arg Asn Met             500 505 510 Asn Arg Asn Ser Asn Ile Ser Pro Ser Thr Pro Ser Ala Val Leu Asn         515 520 525 Asp Arg Gln Glu Met Gln Asp Ser Ile Ser Ser Leu Gly Asn Leu Thr     530 535 540 Lys Ala Ala Leu Glu Asn Asn Glu Pro Thr Ile Ser Leu Gln Thr Ser 545 550 555 560 Gln Thr Glu Asn Glu Asp Asp Ala Ser Arg Gln Asp Met Thr Ser Lys                 565 570 575 Ile Asn Asn Glu Ala Asp Arg Ser Ser Val Ser Ala Gly Thr Ser Asn             580 585 590 Ile Ala Lys Leu Leu Asp Leu Ser Thr Lys Gly Asn Leu Asn Leu Ile         595 600 605 Asp Met Lys Leu Phe His His Tyr Cys Thr Lys Val Trp Pro Thr Ile     610 615 620 Thr Ala Lys Val Ser Gly Pro Glu Ile Trp Arg Asp Tyr Ile Pro 625 630 635 640 Glu Leu Ala Phe Asp Tyr Pro Phe Leu Met His Ala Leu Leu Ala Phe                 645 650 655 Ser Ala Thr His Leu Ser Arg Thr Glu Thr Gly Leu Glu Gln Tyr Val             660 665 670 Ser Ser His Arg Leu Asp Ala Leu Arg Leu Leu Arg Glu Ala Val Leu         675 680 685 Glu Ile Ser Glu Asn Asn Thr Asp Ala Leu Val Ala Ser Ala Leu Ile     690 695 700 Leu Ile Met Asp Ser Leu Ala Asn Ala Ser Gly Asn Gly Thr Val Gly 705 710 715 720 Asn Gln Ser Leu Asn Ser Ser Ser Pro Ser Ala Trp Ile Phe His Val                 725 730 735 Lys Gly Ala Ala Thr Ile Leu Thr Ala Val Trp Pro Leu Ser Glu Arg             740 745 750 Ser Lys Phe His Asn Ile Ile Ser Val Asp Leu Ser Asp Leu Gly Asp         755 760 765 Val Ile Asn Pro Asp Val Gly Thr Ile Thr Glu Leu Val Cys Phe Asp     770 775 780 Glu Ser Ile Ala Asp Leu Tyr Pro Val Gly Leu Asp Ser Pro Tyr Leu 785 790 795 800 Ile Thr Leu Ala Tyr Leu Asp Lys Leu His Arg Glu Lys Asn Gln Gly                 805 810 815 Asp Phe Ile Leu Arg Val Phe Thr Phe Pro Ala Leu Leu Asp Lys Thr             820 825 830 Phe Leu Ala Leu Leu Met Thr Gly Asp Leu         835 840 845 Arg Ser Tyr Tyr Lys Leu Leu Arg Gly Phe Ala Thr Glu Val Lys Asp     850 855 860 Lys Val Trp Phe Leu Glu Gly Val Thr Gln Val Leu Pro Gln Asp Val 865 870 875 880 Asp Glu Tyr Ser Gly Gly Gly Asp Met Met Met Leu Asp Phe Leu                 885 890 895 Gly Gly Gly Leu Pro Ser Met Thr Thr Thr Asn Phe Ser Asp Phe Ser             900 905 910 Leu     <210> 3 <211> 1488 <212> DNA <213> Saccharomyces cerevisiae <400> 3 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 agatttagag 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 tgattggt 1488 <210> 4 <211> 490 <212> DNA <213> Saccharomyces cerevisiae <400> 4 gcgcgttaac gcgcttactg ctaagcctgt ttctaaacat gatccccagg gtgaatatct 60 cccatagtgg ctgtgttatt ctgtctaaat gtggcagcta aataaggttt tttccaagct 120 atttttttct ctaaacgagc gcaccttagc gggatcgcgt caaattttcg cccaacgaat 180 attattctat tgtcttcagt gaatgtgctt gtcgaaatct atatatgcgc cgccctggca 240 ggatcagcaa atacaagtta cttggaaagt cgcggcatag aaagtagaca cttcttttgt 300 caaagattga tattgctttc agggtgcgat agtgctgact gttcctctaa acagcgattt 360 tttacataca ttaacttttt gaagttgtag gaactatctt ttctcattta tccaacacta 420 aaacaaaatt gagctttcca gaaatagtga atcaaaaaaa gttaagtaca aatatttaca 480 gttcagcagt 490 <210> 5 <211> 434 <212> DNA <213> Saccharomyces cerevisiae <400> 5 ttgaacggag tgacaatata tatatatata tatttaataa tgacatcatt atctgtaaat 60 ctgattctta atgctattct agttatgtaa gagtggtcct ttccataaaa aaaaaaaaaa 120 agaaaaaaga attttaggaa tacaatgcag cttgtaagta aaatctggaa tattcatatc 180 gccacaactt cttatgctta taaaagcact aatgcctgaa tttatgttga aaatatgtgt 240 cacaaataaa gaaactgtga catctgacac atttccactt tattgacaag aatagaattt 300 ctttaagttt cccctctaga ttatttattt tcaaatttta ggctctgttg aagtttatta 360 cgtagaaatt cctacgatag ttattagtcc taattggatg ttgcagcaag gctcattgtc 420 ggtgtcgtta tcga 434 <210> 6 <211> 885 <212> DNA <213> Saccharomyces cerevisiae <400> 6 tgatagaaaa tttaaaagtg cagaatattc gaaatagatt cccaaaaaaa aaaaaaggga 60 tattgtgtta cccagttcac aatggaattt tctagaagaa aaaacaaaga agcctcgcaa 120 caaaccaaga tggattcaaa atgcctactt accgcattgt atgtgggaga atgaacatat 180 ctgtaattgt attatataaa agtagttagt tttttttttt tttttttttt tttttttttt 240 ttatgtgtaa tgcaaataca aaaagaactg caggcatttt gaaagttcac tcgtatagga 300 aactttgata gagaggcaat tccttacttt cgttattcct ttcagtctta ttcttaatcg 360 tttatagtag caacaatata tcaatatggt cgtgaataac ccaaataact ggcactgggt 420 cgataagaac tgcatcggat gggccaagga gtatttcaaa cagaaactgg ttggagtgga 480 ggccggttcg gtgaaggata aaaagtatgc caaaatcaag tctgtttcgt ccattgaagg 540 tgattgtgaa gttaatcagc gtaaggggaa ggttatatct ttgtttgatt tgaaaatcac 600 ggtgttaata gagggacacg tggactctaa ggacggatcg gcattgccat tcgagggtag 660 cattaacgtt cctgaagttg ccttcgatag cgaggcctca agctatcaat ttgacatatc 720 tatatttaag gagactagcg aattgagtga agccaagcca ctaattagat ccgagttatt 780 gcccaagttg agacaaattt tccaacaatt tggcaaggat ttgctggcca cccatggtaa 840 tgacattcag gtgcccgaat ctcaggtgaa atctaactac acaag 885 <210> 7 <211> 20 <212> RNA <213> Artificial Sequence <220> <223> sgRNA <400> 7 gacgaataca gtggaggtgg 20 <210> 8 <211> 78 <212> DNA <213> Artificial Sequence <220> <223> Donor DNA <400> 8 tgctgcctca agatgttgac gaatacagtg gaggtggtga tatgcatatg atgctagatt 60 tcctcggtgg cggattac 78

Claims (28)

Saccharomyces &lt; RTI ID = 0.0 &gt; A recombinant expression vector comprising a UPC2 mutation gene in which the ligand binding domain region of Upc2p derived from S. cerevisiae is mutated. 2. The recombinant expression vector according to claim 1, wherein the UPC2 mutation gene is represented by SEQ ID NO: 2. The recombinant expression vector according to claim 1, wherein the recombinant expression vector comprises a UPC2 promoter gene, a UPC2 mutation gene represented by SEQ ID NO: 1, a GAL7 termination gene, and a HIS3 marker gene. A recombinant Saccharomyces cerevisiae strain transformed with the recombinant expression vector according to any one of claims 1 to 3. 5. The recombinant strain according to claim 4, wherein the recombinant strain is obtained by transforming Saccharomyces cerevisiae strain lacking UPC2 gene with the recombinant expression vector. The recombinant strain according to claim 5, wherein the recombinant strain is Saccharomyces cerevisiae BY-upc2Δ / npUPC2-1 strain deposited with KCTC 13401BP. The recombinant strain according to claim 6, wherein the strain Saccharomyces cerevisiae BY-upc2Δ / npUPC2-1 deposited with KCTC 13401BP is a strain in which total cholesterol production is promoted. Transforming a Saccharomyces cerevisiae strain lacking the UPC2 gene with a recombinant expression vector according to any one of claims 1 to 3; And
And culturing the transformed strain. The method according to claim 8, wherein the transformed strain is Saccharomyces cerevisiae BY-upc2Δ / npUPC2-1 strain deposited with KCTC 13401BP. 5. The recombinant strain according to claim 4, wherein the recombinant strain is obtained by transforming Saccharomyces cerevisiae strain producing epoxysqualene and dhamrenediol with the recombinant expression vector. [Claim 11] The recombinant microorganism of claim 10, wherein the Saccharomyces cerevisiae strain producing the epoxy squalene and the fmalene diol is a Saccharomyces cerevisiae E _m D / tH1e7 strain deposited with KCTC 13399BP . [Claim 11] The recombinant strain according to claim 10, wherein the recombinant strain is Saccharomyces cerevisiae E _m D / tHle7 / npUPC2-1 strain deposited with KCTC 13400BP. 13. The recombinant strain according to claim 12, wherein the Saccharomyces cerevisiae E _m D / tHle7 / npUPC2-1 strain deposited with KCTC 13400BP is a strain in which production of epoxysqualene and dhamrenediol is promoted. Transforming a Saccharomyces cerevisiae strain producing an epoxysqualene and a dhamalene diol with a recombinant expression vector according to any one of claims 1 to 3; And
And culturing said transformed strain. &Lt; RTI ID = 0.0 &gt; 8. &lt; / RTI &gt; 15. The pharmaceutical composition according to claim 14, wherein the saccharomyces cerevisiae strain producing the epoxy squalene and the dhamrenediol is a Saccharomyces cerevisiae E _m D / tH1e7 strain deposited with KCTC 13399BP. And promoting the production of dendritic radendiol. 15. The method according to claim 14, wherein the transformed strain is Saccharomyces cerevisiae E _m D / tHle7 / npUPC2-1 strain deposited with KCTC 13400BP. UPC2 promoter gene, UPC2 containing the mutant gene UPC2, UPC2 gene terminator, URA3 marker gene and the terminator gene UPC2 sequence shown in SEQ ID NO: 1 Mutation gene insertion cassette. 17. A recombinant vector comprising the UPC2 mutant gene insertion cassette according to claim 17. 18. A recombinant strain of Saccharomyces cerevisiae transformed with the recombinant vector according to claim 18. The recombinant strain according to claim 19, wherein the recombinant strain is Saccharomyces cerevisiae CEN.PK-upc2-1 strain deposited with KCTC 13404BP. 20. The method according to claim 20, wherein the Saccharomyces cerevisiae CEN.PK-upc2-1 strain deposited with KCTC 13404BP is characterized in that an endogenous UPC2 gene is substituted with a UPC2 mutation gene represented by SEQ ID NO: 1 Lt; / RTI &gt; 22. A method for promoting total cholesterol production comprising culturing a recombinant strain according to any one of claims 19 to 21. A CRISPR-Cas9 recombinant vector containing a single guide RNA (sgRNA) targeting Cas9 gene and UPC2 gene; And a recombinant Saccharomyces cerevisiae strain transformed with Donor DNA for introducing a UPC2 mutation gene. The recombinant strain according to claim 23, wherein the sgRNA targeting the UPC2 gene is represented by SEQ ID NO: 7. The recombinant strain according to claim 23, wherein the donor DNA for introducing the UPC2 mutation gene is represented by SEQ ID NO: 8. The recombinant strain according to claim 23, wherein the CRISPR-Cas9 recombinant vector is a YCpH-SpCas9-sgUPC2 recombinant vector having a cleavage map of FIG. 9 (A). 24. The recombinant strain according to claim 23, wherein the recombinant strain is an endogenous UPC2 gene in the chromosome, which is substituted with the UPC2 mutation gene represented by SEQ. ID. 28. A method for promoting sterol production comprising culturing a recombinant strain according to any one of claims 23 to 27.

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