KR20200081339A - 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 an expression vector for optimizing expression of a mutated transcriptional regulatory factor Upc2p and a sterol precursor overproducing method using the same. More specifically, the method is to produce a recombined enzyme, in which expression of mutated transcriptional regulatory factor (Upc2p) with decreased affinity to ergosterol is optimized to reduce feedback adjustment, such that the entire biosynthesis path of the ergosterol is amplified, thereby enhancing biosynthesis performance of a sterol precursor, such as epoxy squalene and squalene, and a ginsenoside precursor, such as dammarenediol. Accordingly, a mutated Upc2p expression cassette, a replacement cassette using a CRISPR system, and recombined enzyme strains using the cassettes of the present invention can be used for producing massive biosynthesis recombined enzymes for ginsenoside or ursodeoxycholic acid (UDCA) precursor materials through additional metabolic engineering.

Description

Expression vectors for optimal expression of mutated Upc2p and method for overproducing sterol precursors using the same}

The present invention is to produce a recombinant yeast strain having an increased ergosterol and related precursor biosynthesis activity by producing a variant transcription factor Upc2 protein and optimizing expression, and specifically, an amino acid region important for binding to ergosterol is substituted. By optimizing the expression of the UPC2 mutant gene encoding the Upc2 mutant protein through regulation of copy number and promoter activity and alteration of metabolic flow to the expression host, a recombinant yeast strain that highly expresses sterol precursors including epoxy squalene is produced. In addition, the UPC2 gene is replaced with a variant on the chromosome using a CRISPR system to encode the Upc2 mutant protein substituted with an amino acid site important for binding to ergosterol using a CRISPR system to produce a recombinant yeast strain.

Saccharomyces of traditional yeast Saccharomyces cerevisiae ) is a strain used in fermentation of beer and bread for a long time, and is a GRAS (Generally Recognized as Safe) microorganism that is guaranteed for human safety. In particular, yeast has a gene transcription and translation system that is very similar to that of higher organisms, and also has a protein secretory, so it can produce activated proteins through post-translational modification, making it a more useful host for mass production of pharmaceutical proteins derived from higher organisms than E. coli. It is used as a system. In addition, it is a host system that is useful for industrial production of food and recombinant pharmaceutical proteins because the scale-up is simple due to easy cultivation of the strain and purification of the extracellular secreted protein.

Meanwhile, yeast is attracting attention as a host capable of expressing by introducing various secondary metabolism biosynthetic pathways. Secondary metabolites produced by various living organisms are important sources of high value-added chemical compounds and in many cases have important medicinal properties. In particular, metabolites of plants prevent infections such as bacteria, viruses, and fungi through antioxidant or antibiotic functions, and are also useful as a functional substance that is beneficial to human health. . Yeast, whose secondary metabolic biosynthetic pathway is limited by itself, does not interfere with or compete with foreign metabolic pathways introduced through genetic engineering. Above all, various ohmic analysis systems are well-established, so that yeast hosts can be analyzed through transcriptome and metabolite analysis. It has the advantage of being able to secure overall information about the physiological state. In addition, a detailed model of metabolic processes has been developed, and in silico yeast is built to predict the behavior of a modified metabolic network, which makes it easier to design and manufacture artificial cells utilizing it. Furthermore, as a single-cell eukaryotic microorganism, yeast is a host suitable for expression of foreign enzymes such as cytochrome P450, which must be expressed in organelles such as endoplasmic reticulum and mitochondria, and is also a prokaryotic microbial host that has the ability to modify proteins after protein translation necessary for enzyme activity derived from plants and animals It is an advantage compared to

Sterol forms part of the cell membrane that regulates fluidity and function, and plays an important role in the physiology of eukaryotic organisms. In yeast, ergosterol is the main sterol, a cell membrane component, and a commercially important metabolite as a precursor to vitamin D2. In addition, its intermediate metabolite, isoprenoid, is widely used commercially as antioxidant (carotenoids), flavors (terpens), nutraceuticals (ubiquinone) and anti-cancer compounds (taxol). Another intermediate metabolite, squalene, is the most well-known health-promoting food, and is usefully used as a skin moisturizer and cosmetic ingredient along with the next level of metabolites, lanosterol. In addition to the industrial fields using intermediate metabolites, the metabolic engineering field in which more useful substances are produced in yeast has been recently spotlighted by changing the process of synthesizing ergosterol to genetic engineering. Next step of squalene, an intermediate metabolite, epoxysqualene, can be converted to dammarenediol by dammarenediol II synthase from Panax ginseng, and two types of cytochrome P450 and several sugar transfers It is made of various ginsenosides by enzymes (glucosyltransferase). In addition, by blocking metabolic pathways from zymosterol, an intermediate metabolite, to ergosterol and introducing and expressing new genes from the outside, anti-inflammatory steroid hormones used in the treatment of arthritis, skin diseases, adrenal insufficiency, etc. Recombinant yeast for the production of pharmaceutical precursors such as cholesterol, which is a precursor of phosphorus hydrocortisone or ursodeoxycholic acid (UDCA), which is a treatment for liver disease, can be produced (FIG. 1).

On the other hand, Upc2p, a transcriptional regulator that induces the expression of several genes involved in the ergosterol biosynthesis process of yeast, recognizes and binds ergosterol, the main component of the cell membrane, and remains inactivated in the cytoplasm and binds when ergosterol is insufficient This non-Upc2p can enter the nucleus and bind to the promoter of genes involved in ergosterol synthesis in a dimeric form. Through this binding, the expression of genes involved in sterol synthesis is induced, and as a result, it plays a role in promoting the ergosterol synthesis process.

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

An object of the present invention is saccharide as MY access celebrity bicyclic Ke (Saccharomyces cerevisiae ) derived Upc2p ligand binding domain site, a recombinant expression vector comprising a mutated UPC2 mutation gene, a Saccharomyces cerevisiae recombinant strain transformed with the recombinant expression vector, and a method for enhancing total cholesterol production using the recombinant strain And it is to provide a method for enhancing the production of epoxy squalene and dammarenediol using the recombinant strain.

Another object of the present invention includes a UPC2 mutation gene insertion cassette, the UPC2 mutation gene insertion cassette, which includes the UPC2 promoter gene, the UPC2 mutation gene represented by SEQ ID NO: 1, the UPC2 termination gene, the URA3 marker gene, and the UPC2 termination gene in sequence. It is to provide a recombinant vector, a recombinant strain of Saccharomyces cerevisiae transformed with the recombinant vector, and a method for enhancing total cholesterol production using the recombinant strain.

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

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 from Saccharomyces cerevisiae is mutated.

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

In addition, the present invention comprises the steps of transforming the Saccharomyces cerevisiae strain having the UPC2 gene deleted with the recombinant expression vector; And culturing the transformed strain.

In addition, the present invention comprises the steps of transforming the Saccharomyces cerevisiae strain producing epoxy squalene and dammarenediol with the recombinant expression vector; And it provides a method for enhancing the production of epoxy squalene and dammarenediol comprising culturing the transformed strain.

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.

In addition, the present invention is a CRISPR-Cas9 recombination vector comprising a single guide RNA (sgRNA) targeting the Cas9 gene and the UPC2 gene; And a Saccharomyces cerevisiae recombinant strain transformed with donor DNA for UPC2 mutation gene introduction.

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

The present invention relates to a variant transcription control factor Upc2p expression optimization vector and a method for overproduction of a sterol precursor using the same, in detail, to produce a cassette expressing the C-terminal amino acid substitution variant UPC2 _G888D gene expression level through the UPC2 promoter itself. And by reducing the gene copy number to 1 to 2 copies, the expression of ergosterol metabolic pathway genes was enhanced to induce production of ginsenosides and cholesterol precursors including epoxy squalene and zymosterol. Specifically, the C-terminal amino acid substitution variant UPC2 _G888D (= UPC2-1 ) is used using the donor DNA in the process of inducing and double- stranding the DNA in the intrinsic UPC2 gene using the CRISPR-CAS9 expression vector. As much as possible. Through this, the expression of ergosterol biosynthetic metabolic pathway genes was enhanced to induce production of ginsenoside precursors containing epoxy squalene. UPC2 _G888D encoding an ergosterol binding site substitution Upc2p mutant protein Recombinant yeast with optimized expression of the mutant gene has been significantly improved in the production efficiency of sterol precursors including epoxy squalene, thereby producing large amounts of cholesterol, a precursor of ginsenoside precursors, ginsenosides, which are excellent in immunity increase effects, or UDCA, a liver disease treatment agent. Developed as a host, it can be used as a useful industrial host that increases the stability of products and economics of price.

Figure 1 shows a schematic diagram of the production of useful metabolites using the ergosterol biosynthetic pathway in yeast.
Figure 2 shows a strategy for producing recombinant yeast with an ergosterol biosynthetic pathway in which feedback regulation is reduced through the development of the transcription factor Upc2p mutant.
Figure 3 shows a schematic diagram of the structure (A) and modified amino acid sequence (B) of various variant Upc2p produced by C- or N-terminal domain deletion or ligand binding domain substitution, and various combinations of expression vectors (C). will be. The mutation G888D (glycine -> asphaltic acid) position of the amino acid present at the C-terminus of the yeast S. cerevisiae transcription regulator Upc2p is shown in bold, and the removed both terminal N- and C- domains are underlined.
Figure 4 is a comparative analysis and analysis of the antifungal agent fluconazole (fluconazole) resistance of recombinant yeast strains according to the condition of expressing various variant Upc2p as TEF1 promoter (A) or its own UPC2 promoter (B), CEN or 2μ vector (C) This is the result of comparative analysis (D) of the change in the total sterol production by introducing the mutated Upc2p whose effect has been confirmed by spectrophotometry.
FIG. 5 is a comparison of the production efficiency of epoxy squalene and dammarenediol through HPLC after transforming the recombinant yeast strains producing epoxy squalene and dammarenediol with YCpH-Tp-UPC2-1 and YCpH-np-UPC2-1, respectively. One result. Recombinant yeast cell extract cultured with medium without methionine added (A), and recombinant yeast cell extract cultured with medium with added methionine (B). MET3 by adding methionine By suppressing the ERG7 gene whose expression is regulated by a promoter, the accumulation rate of epoxy squalene and dammarenediol was increased.
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 It is a schematic diagram of an expression cassette diagram (A) that can be replaced with a gene, and a production strategy (B) of Upc2-1 mutants using the same .
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 After the gene pop-out, it is the result of the analysis of the spectrophotometer for reconfirming the polymerase reaction for confirming the mutant strain and the overall sterol biosynthesis efficiency.
Figure 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). Genetic sequence GGT in which mutation occurs is capitalized, the DNA sequence recognized by sgRNA is underlined, and the PAM site that defines the double-stranded cleavage of the Cas9 enzyme is inclined.
Figure 9 is a Cas9 enzyme used for mutation of the Upc2 protein and UPC2 in the genome of this enzyme It is a schematic diagram (A) of the vector YCpH-SpCas9-sgUPC2 expressing sgRNA that leads to the gene position and a schematic diagram (B) showing the process of mutation in the intrinsic UPC2 gene in the genome when the stomach vector is expressed in yeast.
10 is a polymerase chain reaction of the C-terminal portion of the UPC2 gene for verification and the spectrophotometer analysis results (A) for comparison of sterol production per cell of CEN.PK candidate strains transformed with YCpH-SpCas9-sgUPC2 vector The sequencing result (B) and the spectrophotometer analysis result (C) and the polymerase chain reaction of the C-terminal part for re-comparison of sterol production per cell after removing the YCpH-SpCas9-sgUPC2 vector through continuous culture in YPD medium and This is the sequencing result (D). EV is a strain in which a blank vector having a HIS3 marker is put in a wild-type CEN.PK strain, and #1 to 6 are YCpH-SpCas9-sgUPC2 vector transformation 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 analysis results and the polymerase chain reaction and sequencing result (C) of the UPC2 gene C-terminal part of some of the candidate strains of the two strains. EV is a strain in which a wild type BY4742 strain and an EmD/tHle7 strain have empty vectors with HIS3 markers, and #1 to 5 are YCpH-SpCas9-sgUPC2 vector transformation candidate strains.
FIG. 12 shows YCpH - npUPC2, a vector having a UPC2 promoter and a UPC2 -1 ORF with a BY4742 mutant strain (Cas9), a wild type with an empty vector (WT+EV), and a centromere replication mechanism replaced with a UPC2 -1 mutant gene. Wild type with -1 (WT+YCpH-npUPC2-1), BY-△ upc2 deletion with an empty vector (upc2+EV) and BY-△ upc2 deletion with an YCpH-npUPC2-1 vector (upc2+npUPC2-1) ) This is the result of spectrophotometer analysis (B) for comparing the production of sterol per cell of the strains used for the above experiment and the spating experiment (A), which observes the resistance under the condition of adding Fluconazole, a substance that inhibits the sterol biosynthesis for the strains.
Figure 13 is the SC-H selection medium (A) for comparing the growth rate and final growth of the BY4742 mutant strains used in the experiment of Figure 12 and the EmD/ of the empty vector and CRISPR-CAS9 used in the experiment of Figure 11 tHle7 strain, Centromere replication mechanism, and the vector having the UPC2 promoter and the UPC2-1 ORF YCpH-npUPC2-1 containing the EmD/tHle7 strain to compare the growth level of the HIS3 marker vector SC- It is the result of OD measurement in the L-selection medium (B) and SC-LUTH selection medium (C) in which the vector is not removed.

Accordingly, the present inventors produced a recombinant yeast having an ergosterol biosynthetic pathway activated through protein engineering and expression optimization of an ergosterol biosynthesis-related electron regulator (Upc2p), thereby completing the present invention (FIG. 2 ).

Amino acid substitution variant of ergosterol attachment domain developed in the present invention UPC2-1 (Upc2p _G888D ) The expression optimization cassette induces an increase in production of several ergosterol biosynthetic precursors, including epoxy squalene and zymosterol, and thus can be usefully used for mass production of cholesterol, a precursor of UDCA, in addition to the ginsenoside precursor.

In addition, the present inventors introduced the CRISPR-CAS9 method to produce a variant transcription factor UPC2 gene that is not inhibited by ergosterol activity, thereby replacing the wild-type UPC2 gene on the chromosome with a variant, thereby synthesizing ergosterol biosynthesis in total. Recombinant yeast with an activated pathway was developed. The YCpH-SpCas9-sgUPC2 vector and donor DNA sequence information produced in the present invention is efficiently embedded UPC2 It can cause mutations in the gene and causes only a single-base polymorphism on the genome. When exporting a vector, antibiotic resistance and auxotrophic markers are not left in the yeast, making it suitable for GRAS evaluation.

The present invention is Saccharomyces Saccharomyces cerevisiae ) provides a recombinant expression vector comprising a UPC2 mutant gene with a ligand binding domain region of Upc2p derived.

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

The NCBI accession no. of the UPC2 gene is 851799, and the NCBI accession no. of the Upc2p protein may be LBMA01000004.1, but is not limited thereto.

Preferably, the recombinant expression vector may include a UPC2 promoter gene, a UPC2 mutant gene represented by SEQ ID NO: 1, a GAL7 termination gene, and a HIS3 marker gene in order, but is not limited thereto.

In the present invention, "vector" refers to a self-replicating DNA molecule used to carry a clone gene (or other piece of clone DNA).

In the present invention, “expression vector” refers to a recombinant DNA molecule comprising a desired coding sequence and an appropriate nucleic acid sequence essential for expressing a coding sequence operably linked in a particular host organism. The expression vector may preferably include one or more selectable markers. The marker is a nucleic acid sequence having a property that can be selected by a chemical method, and all genes capable of distinguishing transformed cells from non-transformed cells are included. Examples include, but are not limited to, antibiotic resistance genes such as Ampicillin, Kanamycin, Geneticin (G418), Bleomycin, Hygromycin, Chloramphenicol, etc. It does not become, and can be appropriately selected by those skilled in the art.

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

Preferably, the recombinant strain is UPC2 A gene-deficient Saccharomyces cerevisiae strain may be transformed with the recombinant expression vector, more preferably, the recombinant strain is Saccharomyces cerevisiae deposited with KCTC 13401BP BY-upc2Δ/ npUPC2-1 may be a strain, but is not limited thereto. On the other hand, the Saccharomyces cerevisiae BY-upc2Δ/npUPC2-1 strain deposited with the KCTC 13401BP is a strain with enhanced total cholesterol production.

Preferably, the recombinant strain may be a Saccharomyces cerevisiae strain producing epoxy squalene and dammarenediol transformed with the recombinant expression vector. More preferably, the Saccharomyces cerevisiae strain producing the epoxy squalene and dammarenediol may be the Saccharomyces cerevisiae EmD/tH1e7 strain deposited as KCTC 13399BP, and the recombinant strain is KCTC 13400BP Saccharomyces cerevisiae deposited as may be a strain EmD / tHle7 / npUPC2-1, but is not limited thereto. On the other hand, the Saccharomyces cerevisiae EmD/tHle7/npUPC2-1 strain deposited with the KCTC 13400BP is a strain with enhanced production of epoxy squalene and dammarenediol.

In addition, the present invention comprises the steps of transforming the Saccharomyces cerevisiae strain having the UPC2 gene deleted with the recombinant expression vector; And culturing the transformed strain. Preferably, the transformed strain may be Saccharomyces cerevisiae BY-upc2Δ/npUPC2-1 strain deposited as KCTC 13401BP, but is not limited thereto.

In addition, the present invention comprises the steps of transforming the Saccharomyces cerevisiae strain producing epoxy squalene and dammarenediol with the recombinant expression vector; And it provides a method for enhancing the production of epoxy squalene and dammarenediol comprising culturing the transformed strain.

Preferably, the Saccharomyces cerevisiae strain producing the epoxy squalene and dammarenediol may be the Saccharomyces cerevisiae EmD/tH1e7 strain deposited as KCTC 13399BP, and the transformed strain is KCTC Saccharomyces cerevisiae deposited as 13400BP may be an EmD/tHle7/npUPC2-1 strain, but is not limited thereto.

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

In addition, the present invention UPC2 promoter gene, UPC2 mutation gene represented by SEQ ID NO: 1, UPC2 termination gene, URA3 Provided is a UPC2 variant gene insertion cassette comprising a marker gene and a UPC2 termination gene in sequence.

In addition, the present invention provides a recombinant vector comprising the UPC2 mutant 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. The -1 strain may be an endogenous UPC2 gene substituted with the UPC2 mutant gene represented by SEQ ID NO: 1, but is not limited thereto.

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

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

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

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

In addition, the present invention is a CRISPR-Cas9 recombination vector comprising a single guide RNA (sgRNA) targeting the Cas9 gene and the UPC2 gene; And a Saccharomyces cerevisiae recombinant strain transformed with donor DNA for UPC2 mutation gene introduction.

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

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

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

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

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

Hereinafter, the present invention will be described in more detail through examples. These examples are only intended to illustrate the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

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

It was intended to increase the overall sterol biosynthetic activity including ergosterol by alleviating the feedback regulation of the Upc2p transcription regulator, which plays a key role in regulating the ergosterol biosynthetic pathway activity. YCpH-Tp-UPC2, a wild-type Upc2p expression vector, is polymerized by UPC2-fw and UPC2-bw primers to synthesize a wild-type UPC2 gene, and then the YCpH-Tp vector and UPC2 gene are cut by SpeI/XhoI . Next, it was produced by pasting. In order to synthesize the gene encoding the ercosterol binding site mutation Upc2p, N-, respectively, using UPC2-fw, UPC2-bw, UPC2-1-F2-fw, and UPC2-1-F1-bw among the primers shown in Table 1 After obtaining and securing the C-fragment, a fusion polymerase chain reaction was performed to produce a single base mutation in the 888th glycine codon (GGT), thereby producing a UPC2-1 gene substituted with aspartic acid codon (GAT). Specifically, UPC2-fw and UPC2-1-F1-bw were used to synthesize the 2,673 bp N-PCR fragment from the start codon ATG of the UPC2 gene to the mutation introduction site, and 86 bp included from the mutation position to the termination codon. UPC2-1-F2-fw and UPC2-bw were used to synthesize C-PCR fragments. The obtained UPC2-1 gene was cut with YCpH-Tp-UPC2 vector and SpeI/XhoI to insert YCpH-Tp-UPC2-1 vector instead of wild type UPC2 . In addition, in order to manufacture Upc2p mutants that deleted the N-domain part, fusion polymerase chaining using UPC2-fw, UPC2-NTT-F1-bw, UPC2-NTT-F2-fw, UPC2-NTT-F2-bw primers A UPC2 _NΔ fragment with 100-390 amino acid sequence portion removed was synthesized using a 300 bp N-fragment starting from the start codon ATG and a C-fragment having a length of 300 bp starting from amino acid codon 390. The obtained UPC2 _NΔ mutant gene fragment and YCpH-Tp-UPC2 vector were digested with SpeI/SauI restriction enzymes to obtain wild type UPC2. Gene cloned In the YCpH-Tp-UPC2 it contains the 390 amino acid sequence from the start codon of the SpeI UPC2 / SauI A vector YCpH-Tp expressing a gene encoding UPC2 _NΔ (N-terminal 100-390 amnio acids truncation), in which the linker portion connecting the DNA binding and ligand binding domains is removed through the process of replacing with the UPC2 _NΔ fragment. -UPC2 _NΔ was produced. 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 UPC2 _CΔ (C-terminal 792) in which the ligand binding domain was removed by proceeding to replace the UPC2 C-fragment containing the 792-913 amino acid sequence with the wild type UPC2 by linking with the truncated YCpH-Tp-UCP2 vector -913 amino acids truncation) expression vector YCpH-Tp-UPC2 _CΔ It was produced. 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 _NΔ expressing the gene was constructed (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 Table 1, Table 2 and Table 3, respectively.

gene primer Sequence purpose HIS3 ScHIS3-FW 5'-catg ccatgg taattctcgttttaagagcttg-3' Screening
Gene amplification
ScHIS3-BW-ApaI 5'-gcgc gggccc ctcgagttcaagagaaaaaaaaag-3' UPC2 UPC2-fw 5'-gcgc tctagactagt atgagcgaagtcggtatac-3' Mutant
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 5'-gcgc gtcgac gcagaatattcgaaatagattccc-3' 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' Gene replacement and pop-out confirmation Upc2-1-int-confirm-bw(ura3) 5'-cagtcaagatatccacatgtg-3'

¹ Restriction enzymes are underlined.

^{2 The} sequence in which glycine #888 was changed to asphaltic acid is shown 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-upc2Δ MATα;ura3Δ0 ; leu2Δ0 ; his3Δ1 ; lys2Δ0 ; upc2::kanMX4 Open Biosystems BY4742/EV BY4742 harboring YCpU-Tp or YCpH-Tp The present invention BY-upc2Δ/EV BY-upc2Δharboring YCpU-Tp or YCpH-Tp The present invention BY/Tp-UPC2 BY4742 harboring YCpH-Tp-UPC2 The present invention BY/Tp-UPC2-1 BY4742 harboring YCpH-Tp-UPC2-1 The present invention BY/Tp-UPC2 _CΔ BY4742 harboring YCpH-Tp-UPC2 _CΔ The present invention BY/Tp-UPC2 _NΔ BY4742 harboring YCpH-Tp-UPC2 _NΔ The present invention BY/Tp-UPC2-1 _NΔ BY4742 harboring YCpU-Tp-UPC2-1 _NΔ The present invention BY/npUPC2 BY4742 harboring YCpH-np-UPC2 The present invention BY/npUPC2-1 BY4742 harboring YCpH-np-UPC2-1 The present invention BY-upc2Δ/Tp-UPC2 BY-upc2Δharboring YCpH-Tp-UPC2 The present invention BY-upc2Δ/Tp-UPC2-1 BY-upc2Δharboring YCpH-Tp-UPC2-1 The present invention BY-upc2Δ/npUPC2 BY-upc2Δharboring YCpH-np-UPC2 The present invention BY-upc2Δ/npUPC2-1 BY-upc2Δharboring YCpH-np-UPC2-1 The present invention BY-upc2Δ/npUPC2 _CΔ BY-upc2Δharboring YCpH-np-UPC2 _CΔ The present invention BY-upc2Δ/npUPC2 _NΔ BY-upc2Δharboring YCpH-np-UPC2 _NΔ The present invention BY-upc2Δ/npUPC2-1 _NΔ BY-upc2Δharboring YCpH-np-UPC2-1 _NΔ The present invention BY/YE-np-UPC2-1 BY4742 harboring YEpH-np-UPC2-1 The present invention BY-upc2Δ/YE-npUPC2-1 BY-upc2Δharboring YEpH-np-UPC2-1 The present invention E _m D/tH1e7/Tp-UPC2-1 Em/D/tH1e7 harboring YCpH-Tp-UPC2-1 The present invention E _m D/tH1e7/npUPC2-1 Em/D/tH1e7 harboring YCpH-np-UPC2-1 The present invention CEN.PK-upc2-1 CEN-PK2-1C replaced with UPC2-1 mutant allele The present invention

Plasmid Description Reference pRE316 p ScGAL1/10-URA3-CEN vector Cheon et al, (2010) pTcUR190 ScURA3 containing vector without yeast replication origin The present invention YCpU-Tp CEN-based pRE316 derivative containing the TEF1 promoter, the GAL7 terminatoer and the URA3 marker The present invention YCpH-Tp YCpU-Tp derivative containing the HIS3 marker The present invention YCpH-Tp-UPC2 YCpH-Tp containing the wild type UPC2 fused to the TEF1 promoter The present invention YCpH-Tp-UPC2-1 YCpH-Tp containing a single amino acid variant UPC2 _G888D fused to the TEF1 promoter The present invention YCpH-Tp-UPC2 _CΔ YCpH-Tp containing a C-terminal truncated UPC2 fused to the TEF1 promoter The present invention YCpH-Tp-UPC2 _NΔ YCpH-Tp containing a N-terminal truncated UPC2 fused to the TEF1 promoter The present invention YCpH-Tp-UPC2-1 _NΔ YCpH-Tp containing the double variant UPC2-1 _NΔ fused to the TEF1 promoter The present invention YCpH-np-UPC2 YCpH derivative expressing the wild type UPC2 under the control of its own promoter The present invention YCpH-np-UPC2-1 YCpH derivative expressing UPC2 -1 under the control of its own promoter The present invention YCpH-np-UPC2 _CΔ YCpH derivative expressing a C-terminal truncated UPC2 under the control of ts own promoter The present invention YCpH-np-UPC2 _NΔ YCpH derivative expressing a N-terminal truncated UPC2 under the control of its own promoter The present invention YCpH-np-UPC2-1 _NΔ YCpH derivative expressing a double variant UPC2-1 _NΔ under the control of its own promoter The present invention YEpH-np-UPC2 2μ -based vector expressing UPC2 under the control of its own promoter and carrying HIS3 marker The present invention YEpH-np-UPC2-1 2μ -based vector expressing UPC2 -1 under the control of its own promoter and carrying HIS3 marker The present invention
NP-UPC2-1-integration UPC2 promoter, UPC2 -1, UPC2 terminator and URA3 marker The present invention

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

The prepared YCpH-Tp-UPC2 vectors were introduced into the wild-type Saccharomyces cerevisiae BY4742 strain and the UPC2 gene crushed BY-upc2 Δ mutant to select HIS+ transformants growing in SC-HIS medium. As a result of measuring the resistance of these recombinant yeast strains to fluconazole, an antimicrobial agent that inhibits ergosterol biosynthesis, wild-type Upc2p overexpression in wild-type strains rather caused a decrease in fluconazole resistance, and the over-expression of mutant Upc2p proteins slightly increased. Showed fluconazole resistance. On the other hand, in the upc2 △defective mutant strain, fluconazole resistance was restored to the level of the wild type strain only when the wild type Upc2p was overexpressed, and the level of recovery of the variant upc2p overexpression was lowered (FIG. 4A). This suggests that the transcriptional activity of the produced Upc2p mutant proteins is significantly lower than that of the wild type.

The present inventors have recognized that it is important to optimize the expression level of the protein Upc2p always pUPC2-FW-HpaI-BamHI to be expressed by its own promoter UPC2 TEF1 promoter in place of maintaining a high level of expression, Seq-ScUPC2-3B A polymerase chain reaction was performed on the Saccharomyces cerevisiae genome using primers to amplify the UPC2 N-fragment (658 bp) with the UPC2 promoter (p UPC2 , 480 bp). The N-fragment with the secured 1,138 bp UPC2 promoter was cut with BamHI and EcoRI restriction enzymes and YCpU-Tp-UPC2, YCpU-Tp-UPC2-1, YCpH-Tp-UPC2 _CΔ UPC2 C-fragment, UPC2 -1 fragment, UPC2 _CΔ obtained by cutting vectors with EcoRI and XhoI restriction enzymes Fragment BamHI / XhoI YCpH-np-UPC2, YCpH-np-UPC2-1, and YCpH-np-UPC2 _CΔ were prepared by performing a three-piece ligation with pRE316 vector digested with restriction enzymes (FIG. 3C). The YCpH-np-UPC2 _NΔ vector was used to create the YCpH-pT-UPC2 _NΔ vector. UPC2-fw, UPC2-NTT-F1-bw, UPC2-NTT-F2-fw, UPC2-NTT-F2-bw primer set using fusion polymerase chain reaction using pUPC2-FW-BamHI-HpaI primer instead of UPC2-fw Proceed with 1,070 bp gene fragment containing UPC2 promoter and N-segment and C-fragment having a length of 300 bp starting from amino acid codon 390, and then cutting with BamHI and Eco81I restriction enzymes, followed by YCpH-np-UPC2 vector It was completed by replacing the BamHI/Eco81I UPC2 fragment of. After transforming the upc2 △defective mutant with these YCpH-np-UPC2 vectors, the comparative analysis of fluconazole resistance showed the highest pruconazole resistance to the strains in which UPc2-1p mutant protein was expressed at an appropriate level from the UPC2 promoter itself. (Figure 4B). The importance of this expression level was reconfirmed as a result of significantly reduced fluconazole resistance in both wild-type strains and mutant strains when expressed with a high copy number of 2 μ-based even when expressed from the UPC2 promoter itself (FIG. 4C ). As a result of comparing and analyzing the total cholesterol productivity of the recombinant yeast strain expressing the produced Upc2-1p mutant as its own promoter by spectrometry, upc2 compared to the wild-type strain and upc2-Δdefective strain compared to the wild-type strain. When 1p was expressed, it was confirmed that the sterol biosynthesis efficiency was further increased (FIG. 4D ).

Example 3: Expression optimized Upc2 Epoxy squalene by introduction to -1p and Dammarenediol Increased 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 After each transformation, it was analyzed by HPLC. SC (except for YNB+amino acid mixture, leucine, uracil, tryptophan, histidine) medium and inoculated with strains 1 to 2 colony, followed by pre-culture, and then in the SC medium with or without 10 mM methionine to make initial inoculation OD600=0.5. Cultured. After incubation for 24 hours (28°C, 220 rpm), the yeast cells OD ₆₀₀ =100 were recovered in a 2 ml tube, suspended in 1 ml of a solution of KOH in 13% dissolved in 70% ethanol, and reacted in an incubator at 85°C for 1 hour. To crush the yeast cells. Thereafter, 800 ul of heptane was added to each sample, mixed with vortexing for 30 seconds, and centrifuged (12,000 rpm, 1 min, 25°C) to recover the heptane supernatant (600 ul) and dried. HPLC analysis was performed with the sample dissolved by adding 200 ul of acetone to the dried sample. For HPLC analysis, Cosmosil C18-PAQ (4.6 mm*250 mm) was used as the column, the flow rate was 1 ml/min, the analysis 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 ( In the recombinant yeast strain (E _m D/tHle7/npUPC2-1) introduced with YCpH-np-UPC2-1), it was confirmed that the amount of biosynthesis of dammarenediol in addition to epoxy squalene increased significantly (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 noticeable increase was observed (FIG. 5A ), ERG7 due to methionine attachment. In the culture conditions where the expression was suppressed, the overall production of epoxy squalene, dammarenediol and squalene produced in the E _m D/tH1e7 recombinant strain was greatly increased, and ergosterol biosynthesis was not significantly increased after Upc2-1p introduction, but with epoxy squalene and It was observed that the productivity of dammarenediol increased significantly (FIG. 5B).

Example 4: Expression optimized UPC2 -One Generating and utilizing cassettes for gene replacement

Upc2-1p is upc2 When expressed in the defective Saccharomyces cerevisiae, it has the highest resistance to the antifungal agent fluconazole (Figure 4C) and produces the highest amount of sterol (Figure 4D), ginsenoside, cholesterol precursor and 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. To this end, the YCpH-np-UPC2-1 vector and the pTcUR190 vector were digested with BamHI/NdeI restriction enzyme and then UPC2 obtained from the YCpH -np-UPC2-1 vector. The promoter -UPC2-1 ORF (1-889 amino acid) fragment was put into the pTcUR190 vector, and NP-UPC2-int. I created the step1 vector. Subsequently, the UPC2tL-FW and UPC2tL-BW-NdeI primers shown in Table 1 were used to polymerize fragments containing the ORF of the 889-912 amino acid region and the UPC2 native terminator of 424bp in the wild-type Saccharomyces cerevisiae strain. Obtained by chain reaction. NP-UPC2-int produced earlier. step1 vector and UPC2 ORF (889-912 amino acid)- The UPC2 terminator fragment was treated with NdeI restriction enzyme, and then the fragment was NP-UPC2-int. After introducing it into the step1 vector, check the directionality to NP-UPC2-int. I created a step2 vector. UPC2tR-FW-SalI and UPC2tR-BW-XhoI primers in Table 1 were used to prepare 885 bp long UPC2 terminator fragments to remove the URA3 gene using homologous recombination in wild-type Saccharomyces cerevisiae strains once more. . This fragment and NP-UPC2-int. After processing the SalI/XhoI restriction enzyme in the step2 vector, a fragment was inserted into the vector to prepare a final NP-UPC2-1 integration vector (FIG. 6A).

The produced NP-UPC2-1 vector is SpeI / XhoI Wild type UPC2 after linearization by digestion with restriction enzymes 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 ( Figure 6B). Primers Upc2-1-int-confrim-fw(2) were used to convert transformants whose UPC2-1 gene cassette was inserted into the endogenous UPC2 region precisely after obtaining transformants with the URA3 selection label in the medium from which Uracil was removed. -1)/ was screened through a polymerase chain reaction using Upc2-1-int-confirm-bw (ura3) (FIG. 7A, left panel). In addition, the maintenance of the ability to increase the production of cholesterol precursors of URA3 transformants was also analyzed by measurement with a spectrophotometer to select transformant #3 showing a pattern consistent with the results of polymerase chain reaction analysis (FIG. 7A, right panel), and amplification. the PCR fragment was sequenced to verify that the finally substituted by UPC2 -1 gene (Figure 7A, the panel below). Two UPC2 contained in the UPC2 -1 gene insertion cassette Transformation #3 in a medium supplemented with 5-Fluoroorotic Acid and Uracil in order to homologous recombination between the terminator sequences and pop-out the inserted URA3 gene to restore the original form of UPC2 terminator. Were cultured. Upc2-1-int-confrim-fw(2-1), Upc2-1-int-confirm-bw() used for transformant screening that the URA3 selection marker was removed accurately through homologous recombination between two UPC2 terminator sequences. 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 cassette, and the UPC2-1 variant obtained (CEN.PK-upc2-1) retained a higher level of sterol synthesis ability than the wild type. It was confirmed by measuring and analyzing it with a spectrophotometer. As a result, transformant #3-2 has this strain URA3. After the pop-out process, it was confirmed that sterol synthesis was maintained at a higher level than that of the wild type (FIG. 7B, right panel).

Example 5: UPC2 -One Genetic mutation replacement Chrisper - CAS 9 base YCpH - SpCas9 -sgUPC2 vector production

UPC2 him do as Saccharomyces expressing the wild-type gene UPC2 Mai Seth Serenity curd present in the genome to have a gene mutation UPC2-1 The Cas9 system was used to more efficiently change the DNA sequence of a gene. Using the Coex 413-Cas9-UCC1 gRNA vector provided by Seoul National University as a template, the 181bp polymerase chain reaction product obtained using UPC2-gRNA-L-FW and UPC2-gRNA-L-BW primers shown in Table 4 and UPC2- Two 291 bp polymerase chain reaction products obtained using gRNA-R-FW and UPC2-gRNA-R-BW primers were used as a template once again using UPC2-gRNA-L-FW and UPC2-gRNA-R-BW primers. The spacerRNA portion that has a base sequence complementary to the ORF portion of the URA3 gene is complementary to the C-terminal portion (2,641-2,660bp) of the UPC2 gene. A fragment of 452 bp made to have a sequence was produced. XbaI / SalI to Coex 413-Cas9-UCC1 gRNA vector Restriction enzyme treatment to URA3 After removing the spacerRNA portion having a sequence complementary to the ORF of the gene, the synthesized polymerase chain reaction fragment is treated with XbaI/SalI restriction enzyme and linked through ligation to UPC2. A YCpH-SpCas9-sgUPC2 vector causing double-strand break at the C-terminus of the gene was constructed (FIG. 9A).

Cas9 UPC2 The site of sgRNA that leads to the gene location and the portion of the PAM sequencing that can occur to determine the DNA cleavage site are described in Figure 8A. By cas9 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 As a template as DNA, it must be transformed together in yeast to be used for genome recovery. To this end, the UPC2-1 donor-DNA-FW and UPC2-1 donor-DNA-BW primers shown in Table 1 were used to form a 78 bp gene sequence corresponding to the UPC2-1 C-terminus using a polymerase chain reaction without a template. DNA fragments were obtained (Figure 8B).

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

gene primer Sequence purpose UPC2 UPC2-gRNA-L-FW 5'-gcgc tctaga ttttgtagtgccctct-3' SpacerRNA replacement fragment amplification in UPC2 recognition sgRNA 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 mutation gene replacement donor DNA amplification UPC2-1 donor-DNA-BW 5'-gtaatccgccaccgaggaaatctagcatcatatgcataTcaccacctcca-3'

¹ Restriction enzyme recognition sites are underlined.

^{2 The} DNA sequence in which 888 amino acid glycine was changed to asphaltic acid is capitalized.

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 The present invention

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

Example 6: Chrisper In the genome through system introduction UPC2 gene Variant Acquisition and analysis

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 in SC-HIS selection medium. A small amount of vector and 3 μg donor DNA was used for transformation. In order to obtain a strain having a mutation on the genome, sterol quantitative analysis was performed as a secondary selection process following the use of selective media. 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, the culture solution was removed, and then dissolved in 1 ml of an ethanol solution containing 6.25% KOH and 8.75% H ₂ O and reacted at 85° C. for 1 hour. When the reaction is over, add 0.8 ml of heptane, mix well, and transfer the heptane supernatant collected in the upper layer to a new tube by separating the layers. After mixing the obtained heptane supernatant with 100% ethanol 1:5, the value is measured at a wavelength of 240-310 nm using the scanning function of the spectrophotometer. CEN.PK2-1C yeast containing an empty vector expressing HIS3 was used as a reference strain, and a solution of a mixture of pure heptane and ethanol was used as a control for quantitative analysis of sterol. As a result of the analysis, 5 of the 6 candidate strains showed higher sterol synthesis level than the strain containing the empty vector (FIG. 10A). GDNA of 6 candidate strains was obtained, and the UPC2 gene C-terminal part was used with Upc2-1-int-confirm-fw(2-1) and Upc2-1-confirm-bw(2-1) primers shown in Table 1. As a result of sequencing analysis secured through the polymerase chain reaction, the intended GGT to GAT mutation occurred in all six through the CRISPR system, and additionally 2,655 nt in candidate strain 5, which showed low sterol synthesis level It was confirmed that a single base deletion occurred in (FIG. 10B).

When the Cas9 enzyme causes a double-strand break on the genome by the PAM sequence, the donor DNA corresponding to the sequence of the UPC2-1 gene is recovered as a template. At this time, the PAM sequence of the wild type UPC2 gene, NGG, is changed to an NGA sequence in the UPC2-1 mutant gene, and is designed to prevent double strand breaks from occurring in the genome where the Cas9 enzyme once mutated (FIG. 9A). However, even with a low probability, an additional double-strand break occurs, and UPC2 Since non-Homologous-End-Joining can cause non-specific mutations inside the gene, the transformants are continuously cultivated in YPD medium to confirm that the vector is missing, and then the sterol quantification and sequencing analysis is performed again. After exiting, it was confirmed that there was no change in sterol biosynthesis function or gene sequence (FIGS. 10C and 10D ).

Example 7: Chrisper Of other strains using the system Saccharomyces In Serbia UPC2 -One Gene replacement

There are several types of strains in Saccharomyces cerevisiae and little by little differences in the DNA sequence of the genome. Therefore, transformation was carried out to check whether the chrisper system produced in this patent is applied equally to several strains or to a strain in which genetic manipulation other than wild type has occurred. Oxidosqualene and squalene are over-produced and accumulated because they have mutations and foreign genes in some of the genes commonly used in laboratories, BY4742 and CEN.PK2-1C, and sterol synthesis derived from CEN.PK2-1C. It was transformed into an EmD/tHle7 (Table 2) strain capable of producing dammarenediol, a precursor of ginsenoside. As a result, it was easy to obtain transformable candidate strains in both strains, and these candidate strains were also subjected to quantitative analysis and sequencing of sterols. As a result, 9 out of 10 candidate strains except for 1 were designed as UPC2-1 mutant genes. It was confirmed to over-produce sterol by replacing with (Fig. 11A, Fig. 11B and Fig. 11C). For the other 1 strain, it was confirmed that a single base defect occurred at a different site from the previous CEN.PK2-1C strain.

Example 8: Upc2-1 Test for resistance to sterol biosynthesis inhibitors of gene replacement strains

Fluconazole is used as an antifungal agent by inhibiting the sterol biosynthesis of cells by inhibiting the function of Erg11p present in yeast ergosterol synthesis. By using the fact that growth inhibition is weakened in yeast with increased sterol synthesis ability, application of the CRISPR system obtained in the above experiment to SC-H medium containing fluconazole at a concentration of 0, 8, 15, 20 μg/ml BY4742 candidate strain and Example 2 As a result of performing a spatting experiment cultivated with the strains produced in (FIG. 12A), the Cas9 strain replaced with the Upc2-1 gene showed similar growth to the BY4742 wild type strain containing YCpH-npUPC2-1. In the case of a strain containing the YCpH-npUPC2-1 vector in the BY-upc2Δ mutant, it exhibited a higher resistance to fluconazole, and the strain containing the blank vector in the wild type BY4742 strain had weak resistance, and the strain containing the empty vector in the BY-upc2Δ mutant strain Resistance was very low. As in the experiment of Example 3, using the strains used in the spating experiment, a quantitative analysis of sterol was performed (FIG. 12B), and the YCpH-npUPC2-1 vector was added to the BY-upc2Δ mutant, which was the most resistant to the sterol biosynthesis inhibitor. It showed the highest level of sterol biosynthesis in the strain. In addition, the strains that put the vector into the BY4742 wild type and the BY-upc2Δ mutant showed the lowest level of sterol biosynthesis at a similar level, and the WT+YCpH-npUPC2-1 strain and Chris expressing the wild type UPC2 gene and UPC2-1 gene together. The results of the similar sterol levels of the UPC2-1 mutant replacement strain ( Cas9 ) obtained through the fur system were confirmed. In addition, while conducting a spating experiment, in the case of strains (upc2+YCpH-npUPC2-1) having a YCpH-npUPC2-1 vector in a wild-type, BY-upc2Δ mutant, an empty vector was added to the reference condition SC-H medium. It was confirmed that the growth level lower than the strains.

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

For the UPC2-1 mutant replacement strain ( Cas9 ) produced by applying the CRISPR system to the wild type strain, the wild type (WT+2-1) and upc2Δ mutant (upc2+2-1) strains with the YCpH-npUPC2-1 vector Growth inhibition was not observed in the SC-H medium. To confirm this, a growth curve and a final growth degree in SC-H medium were confirmed (FIG. 13A). As a result, as in the sputtering experiment, the strains having the YCpH-npUPC2-1 vector (upc2+YCpH-npUPC2-1) in the wild type, upc2Δ mutant strains are about 10 higher than the reference strains containing the growth vector in SC-H medium. It appeared as low as -30%. On the other hand, it was confirmed that the UPC2-1 mutant gene replacement strain showed a similar or higher growth degree than the reference strains containing the empty vector.

As a result of comparing the growth degree of Dhammarenediol in the production strain EmD/tHle7, the strain containing the empty vector (Em+EV) has the highest growth in SC-L medium, which is a condition where YCpH-npUPC2-1 vector can escape. As shown in Fig. 5, the strain having the YCpH-npUPC2-1 vector (Em+2-1) was grown next, and the UPC2-1 mutant gene replacement strain (Em+Cas) ) Showed the lowest growth (FIG. 13B ). However, there was no significant difference within the OD value of 1. As a result of growth in SC-LUTH medium, which is a condition in which the vector does not escape, the strain having the empty vector still showed the highest growth, and the UPC2-1 mutant gene replacement strain 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.

Since the specific parts of the present invention have been described above in detail, it is clear that for those skilled in the art, these specific techniques are only preferred embodiments, and the scope of the present invention is not limited thereto. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Korea Research Institute of Bioscience and Biotechnology KCTC13399BP 20171120 Korea Research Institute of Bioscience and Biotechnology KCTC13400BP 20171120 Korea Research Institute of Bioscience and Biotechnology KCTC13401BP 20171120 Korea Research Institute of Bioscience and Biotechnology 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-2020-0092 <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 caacagcagc 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 gacgaataca gtggaggtgg 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 Arg 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 Glu 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 Gln 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 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 Met 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 Gly Ala Met Arg Ile Met 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 His 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 (5)

CRISPR-Cas9 recombination vector comprising a single guide RNA (sgRNA) targeting the Cas9 gene and UPC2 gene; And Saccharomyces cerevisiae recombinant strain transformed with donor DNA for introduction of the UPC2 mutant gene represented by SEQ ID NO: 8. The recombinant strain of claim 1, wherein the sgRNA targeting the UPC2 gene is represented by SEQ ID NO: 7. The recombinant strain of claim 1, wherein the CRISPR-Cas9 recombination vector is a YCpH-SpCas9-sgUPC2 recombination vector having a cleavage map of FIG. 9(A). According to claim 1, wherein the recombinant strain is a recombinant strain characterized in that the endogenous (endogenous) UPC2 gene in the chromosome is replaced with the UPC2 mutation gene represented by SEQ ID NO: 1. A method for enhancing sterol production, comprising culturing the recombinant strain according to any one of claims 1 to 4.

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