Expression constructs, cells and methods of obtaining cells

Abstract

To provide an expression construct capable of improving resistance to a specific target substance, a cell transformed by the expression construct, and a method for obtaining a cell by the expression construct.SOLUTION: A first promoter having constitutive activity and functioning in a cell, a first nucleotide sequence encoding a CRISPR enzyme operably linked to the first promoter, a second promoter functioning in the cell, a second nucleotide sequence encoding a guide RNA operably linked to the second promoter, a third promoter for expression and functioning in the cell, a third nucleotide sequence encoding a protein for introducing a point mutation into the genome of the cell operably linked to the third promoter, a drug resistance gene, and a replication origin, wherein the guide RNA is capable of complementarily binding to at least a part of a multicopy sequence present in the genome.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to an expression construct, a cell and a method for obtaining the cell. [Background technology]

[0002] Research is being conducted into techniques that alter the metabolic capacity and / or resistance of cells by inserting both point mutations and structural mutations into the genome of cells. Chemical substances such as ethyl methanesulfonate and ultraviolet light have often been used to introduce mutations into the yeast Saccharomyces cerevisiae. However, these methods have a trade-off between the rate of mutation introduction and cell viability, making it difficult to introduce mutations efficiently.

[0003] As an alternative method, attempts have been made to transform cells by introducing a plasmid to insert both a point mutation and a structural mutation into the genome. For example, in Non-Patent Document 1, a plasmid that causes a point mutation and a plasmid that causes a structural mutation are simultaneously introduced into the genome of Saccharomyces cerevisiae, a yeast capable of producing 2,3-butanediol. The yeast into which the mutations have been introduced is conferred 2,3-butanediol resistance and shows suppressed growth inhibition. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Mizobata A et al., J. Biosci. Bioeng., vol.131, No. 3,pp. 283-289, 2021 Summary of the Invention [Problem to be solved by the invention]

[0005] The method of Non-Patent Document 1 improves resistance to a specific target substance by inducing point mutations and structural mutations in the genome, but there is room for further improvement. An object of the present invention is to provide an expression construct that can improve resistance to a specific target substance, cells transformed with the expression construct, and a method for obtaining cells using the expression construct. [Means for solving the problem]

[0006] The present invention includes the following aspects. [1] An expression construct comprising: a first promoter having constitutive activity that functions in a cell; a first nucleotide sequence operably linked to the first promoter and encoding a CRISPR enzyme; a second promoter that functions in the cell; a second nucleotide sequence operably linked to the second promoter and encoding a guide RNA; a third promoter for expression that functions in the cell; a third nucleotide sequence operably linked to the third promoter and encoding a protein that introduces a point mutation into the genome of the cell; a drug resistance gene; and a replication origin, wherein the guide RNA is capable of complementarily binding to at least a portion of a multicopy sequence present in the genome. [2] The expression construct according to [1], wherein the cell is any one type of cell selected from yeast, filamentous fungi, Corynebacterium bacteria, Escherichia coli, and Bacillus subtilis. [3] The expression construct according to [1] or [2], wherein the CRISPR enzyme is Cas9. [4] A cell transformed with the expression construct according to any one of [1] to [3]. [5] The cell according to [4], which has resistance to a target substance due to the transformation. [6] The cell described in [5], wherein the target substance is at least one selected from 2,3-butanediol, isobutyl alcohol, and lactic acid. [7] A method for obtaining a cell having resistance to a target substance, comprising introducing into a cell an expression construct according to any one of [1] to [3], causing a point mutation in the genome of the cell, causing a structural mutation in the multicopy sequence, and conferring resistance to the target substance to the cell by the point mutation and the structural mutation. [8] The method for obtaining cells according to [7], wherein the target substance is at least one selected from 2,3-butanediol, isobutyl alcohol, and lactic acid. [9] A method for obtaining the cells described in [7] or [8], further comprising re-introducing the expression construct into the cells to which resistance to the target substance has been conferred, thereby conferring resistance to a target substance other than the target substance. [Effects of the Invention]

[0007] According to the above aspect, it is possible to provide an expression construct that can improve resistance to a specific target substance, a cell transformed with the expression construct, and a method for obtaining a cell using the expression construct. [Brief explanation of the drawings]

[0008] [Figure 1] FIG. 1 is a schematic diagram illustrating an expression construct according to one embodiment. [Figure 2] 1 is a graph showing the ΔOD600 of the YPH499 / pEWPMSM_Co mutant strain and the YPH499 / pEWPMSM mutant strain. [Figure 3] 1 is a graph showing the change in OD600 of YPD medium over time when each of the YPH499 / pEWPMSM_Co mutant strains and YPH499 was cultured in 2,3-butanediol-free YPD medium. [Figure 4] 1 is a graph showing the change in OD600 over time when each of the YPH499 / pEWPMSM_Co mutant strains and YPH499 was cultured in a YPD medium containing 100 g / L of 2,3-butanediol (2,3-BDO). [Figure 5]1 is a graph showing the change in OD600 over time when each of the YPH499 / pEWPMSM_Co mutant strains and YPH499 was cultured in a YPD medium containing 150 g / L of 2,3-butanediol (2,3-BDO). [Figure 6] 1 is a graph showing the relative OD600 values ​​of the YPH499 / pEWPMSM_Co mutant strain to the OD600 of YPH499 after 72 hours of culture. [Figure 7] 1 is a graph showing the relative OD600 values ​​of the YPH499 / pEWPMSM_Co mutant strain to the OD600 value of YPH499 after 72 hours of culture in a YPD medium containing 15 g / L of isobutyl alcohol. [Figure 8] 1 is a graph showing the relative OD600 values ​​of the YPH499 / Co58 mutant strain to the OD600 value of YPH499 after 72 hours of culture in a YPD medium containing 15 g / L of isobutyl alcohol. [Figure 9] 1 is a graph showing the relative OD600 values ​​of the YPH499 / pEWPMSM_Co2 mutant strain to the OD600 of YPH499 after 72 hours of culture. [Figure 10] 1 is a graph showing the relative OD600 values ​​of the SH-4 / pEWPMSM_Co2 mutant strain to the OD600 value of SH-4 after 72 hours of culture. [Figure 11] 1 is a graph showing the relative OD600 values ​​of the YPH499 / Co58 mutant strain to the OD600 value of YPH499 after 72 hours of culture in YPD medium containing 150 g / L of 2,3-butanediol (2,3-BDO). DETAILED DESCRIPTION OF THE INVENTION

[0009] <Definition> "Nucleic acid" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) or a polymer thereof in either single- or double-stranded form. "Nucleotide sequence" refers to a polymer of DNA or RNA in either single- or double-stranded form.

[0010] The term "gene" refers to a segment of DNA involved in producing or encoding a polypeptide chain. It may include regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons).

[0011] A "promoter" is defined as an array of nucleic acid control sequences that direct transcription of a nucleic acid.

[0012] The "CRISPR / Cas" system refers to a broad class of bacterial systems for defense against exogenous nucleic acids. CRISPR / Cas systems are classified into Class 1 and 2, and further subdivided into Types I to VI.

[0013] When a numerical range is stated, for example, as "1 to 10," the numerical range is defined to include the lower limit of 1 and the upper limit of 10.

[0014] <Expression construct> The expression construct of this embodiment comprises a first promoter having constitutive activity that functions in a cell, a first nucleotide sequence operably linked to the first promoter and encoding a CRISPR enzyme, a second promoter that functions in the cell, a second nucleotide sequence operably linked to the second promoter and encoding a guide RNA, a third promoter for expression that functions in the cell, a third nucleotide sequence operably linked to the third promoter and encoding a protein that introduces a point mutation into the genome of the cell, a drug resistance gene, and a replication origin, wherein the guide RNA is capable of complementarily binding to at least a portion of a multicopy sequence present in the genome.

[0015] 1 is a schematic diagram showing an expression construct according to this embodiment. Expression construct 1 includes a first promoter 11, a first nucleotide sequence 12, a second promoter 13, a second nucleotide sequence 14, a third promoter 15, a third nucleotide sequence 16, a drug resistance gene 17, and an origin of replication 18.

[0016] The first promoter 11 has constitutive activity that functions in cells into which the expression construct 1 is introduced. In other words, the first promoter 11 is a constitutive expression promoter.

[0017] The first nucleotide sequence 12 is operably linked to the first promoter 11. The first nucleotide sequence 12 encodes a CRISPR enzyme. The CRISPR enzyme is an enzyme with RNA-guided DNA endonuclease activity. The nuclease domain of the CRISPR enzyme is activated by recognizing a PAM (Protospacer adjacent motif) sequence. The CRISPR enzyme introduces a structural mutation into the genome of a cell into which the expression construct 1 is introduced. A method for introducing a structural mutation will be described below.

[0018] Examples of the CRISPR enzyme encoded by the first nucleotide sequence 12 include Cas9 and Cas12a, and Cas9 is preferred.

[0019] The second promoter 13 is a promoter specific to the transcription of the second nucleotide sequence 14 encoding the guide RNA. The second promoter 13 may be a constitutive promoter.

[0020] The second nucleotide sequence 14 is operably linked to the second promoter 13. The second nucleotide sequence 14 encodes a guide RNA. The guide RNA includes a guide sequence and a protein-binding sequence. The guide sequence includes a sequence complementary to a target nucleic acid in the genome of a cell into which the expression construct 1 is introduced. In this embodiment, the target nucleic acid is at least a portion of a multicopy sequence present in the genome. A multicopy sequence is a sequence present in the introns of various cells, and tens to hundreds of identical sequences exist in the genome. An example of a multicopy sequence in yeast is a delta sequence. The guide sequence may be a sequence complementary to the entire sequence of a multicopy sequence, or a sequence complementary to a portion of the sequence of a multicopy sequence. Preferably, one guide sequence has, for example, 19 to 27 bases. Of the total number of bases in the guide sequence, the number of bases that complementarily bind to the target multicopy sequence is, for example, preferably 95 to 100%.

[0021] The protein binding sequence is a sequence to which the CRISPR enzyme associates, and is appropriately selected depending on the organism and type of CRISPR enzyme encoded by the first nucleotide sequence 12.

[0022] The third promoter 15 is a promoter specific to the transcription of the third nucleotide sequence 16 that encodes a protein that introduces a point mutation into the genome of a cell into which the expression construct 1 is introduced. The third promoter 15 may be a constitutive promoter.

[0023] The third nucleotide sequence 16 is operably linked to the third promoter 15. The third nucleotide sequence 16 encodes a protein that introduces a point mutation into the genome of the cell. Methods for introducing point mutations are described below.

[0024] The drug resistance gene 17 is contained in the expression construct 1 for selection purposes when the expression construct 1 is mass-produced using a host such as Escherichia coli. As the drug resistance gene 17, a common drug resistance gene can be used, and examples thereof include an ampicillin resistance gene, a chloramphenicol resistance gene, a hygromycin resistance gene, a kanamycin resistance gene, a tetracycline resistance gene, and a zeocin resistance gene.

[0025] The replication origin 18 is appropriately selected depending on the cell type into which the expression construct 1 is to be introduced. The expression construct 1 may have a host replication origin 19 when mass-produced using a host such as E. coli.

[0026] In this embodiment, the expression construct has a first promoter operably linked to a first nucleotide sequence encoding a CRISPR enzyme for introducing structural mutations, which has constitutive activity. This allows cell culture in the presence of glucose, which is thought to result in the expression of a large amount of CRISPR enzyme under conditions of better cellular metabolism than in the case of a typical galactose-inducible promoter. As a result, the CRISPR enzyme cleaves multiple multicopy sequences in the genome, resulting in numerous structural mutations, and it is thought that cells into which the expression construct has been introduced can acquire high resistance to specific target substances.

[0027] <Method for obtaining cells resistant to a target substance>

[0028] The method for obtaining cells having resistance to a target substance of this embodiment includes introducing the above-mentioned expression construct, causing point mutations in the genome of the cells, causing structural mutations in the multicopy sequence, and conferring resistance to the target substance to the cells through the point mutations and structural mutations. Details are described below.

[0029] The expression construct is introduced into cells for which resistance to a target substance is to be improved. The expression construct can be introduced into cells by a conventional method, such as the lithium acetate method.

[0030] The cells are not particularly limited as long as they are of a cell type having a multicopy sequence in the genome. Examples include Saccharomyces cerevisiae, a yeast used for alcohol production, etc., Pichia pastoris, a yeast used for protein production, etc., Candida utilis, a yeast used in the food industry, Aspergillus oryzae, a filamentous fungus used in the food industry, Corynebacterium glutamicum, a Corynebacterium bacterium used for amino acid production, etc., Escherichia coli, an Escherichia coli used for various purposes, and Bacillus subtilis, a Bacillus subtilis used for various purposes. When the cells are yeast, the yeast may be haploid or diploid.

[0031] When the expression construct is introduced into a cell, the first to third nucleotide sequences are transcribed or translated within the cell, and the CRISPR enzyme, guide RNA, and protein that introduces a point mutation into the genome are expressed.

[0032] The CRISPR enzyme and guide RNA cleave the multicopy sequence, which is the target nucleic acid, through the CRISPR / Cas system, introducing a structural mutation into the genome. As described above, the guide RNA includes a guide sequence and a protein-binding sequence. The guide sequence binds to at least a portion of the multicopy sequence. The protein-binding sequence also recruits the CRISPR enzyme to the multicopy sequence, which is the target nucleic acid. This allows the guide RNA, CRISPR enzyme, and multicopy sequence to associate with each other. The CRISPR enzyme cleaves the multicopy sequence near the PAM sequence.

[0033] As mentioned above, because multicopy sequences exist in introns, their cleavage does not alter the function or specific activity of proteins expressed by cells. However, cleavage of multicopy sequences can alter the expression levels of multiple proteins or cause significant changes in phenotype.

[0034] Examples of proteins that introduce point mutations into the genome include proofreading-deficient polymerases. Proofreading-deficient polymerases introduce point mutations randomly into the genome, so when point mutations are introduced into exons, protein function and activity may change. However, phenotypic changes are minimal.

[0035] The target substance can be appropriately selected depending on the target cells, and examples thereof include alcohols, carboxylic acids, etc. When the cells are Saccharomyces cerevisiae, examples of the target substance include alcohols such as 2,3-butanediol and isobutyl alcohol, and carboxylic acids such as lactic acid.

[0036] A method for selecting cells that have been conferred resistance to a target substance includes culturing cells in a medium containing the target substance to obtain cells that can grow, and more preferably, performing subculture while gradually increasing the concentration of the target substance contained in the medium to obtain cells that can grow.

[0037] An expression construct may be further introduced into cells that have been conferred resistance to a target substance (referred to as the first target substance) selected by the above-mentioned method. This simultaneously introduces point mutations and structural mutations into the cells. By selecting these mutant cells using a second target substance different from the first target substance, cells that have been conferred resistance to the second target substance can be obtained. Cells that have been conferred resistance to the second target substance may maintain their resistance to the first target substance. In other words, cells obtained by this method may be conferred resistance to two or more of the target substances.

[0038] In this embodiment, the method for obtaining cells resistant to a target substance allows for simultaneous introduction of point mutations and structural mutations into the cell genome. Furthermore, in the above-described expression construct, the promoter operably linked to the nucleotide sequence encoding the CRISPR enzyme involved in the introduction of the structural mutation has constitutive activity. This allows for CRISPR enzyme expression even when cells are cultured in the presence of glucose, which is thought to result in greater expression of CRISPR enzymes under conditions of better cellular metabolism than in the case of a typical galactose-inducible promoter. As a result, more structural mutations are generated, and cells into which the expression construct has been introduced are thought to acquire high resistance to a specific target substance.

[0039] <Cells transformed with the expression construct> The cells in this embodiment are cells transformed with the above-mentioned expression construct. Examples of the cells include those described above.

[0040] The cells may be resistant to a target substance by transformation. Examples of the cells include cells having a multicopy sequence in their genome as described in <Method for Obtaining Cells Resistant to a Target Substance>. Examples of the target substance include the target substances as described in <Method for Obtaining Cells Resistant to a Target Substance>. Cells resistant to a target substance can be obtained by the method described in <Method for Obtaining Cells Resistant to a Target Substance>.

[0041] In the cells of this embodiment, point mutations and structural mutations are simultaneously introduced into the genome by the above-described expression construct. Furthermore, in the above-described expression construct, the promoter operably linked to the nucleotide sequence encoding the CRISPR enzyme involved in the introduction of the structural mutation has constitutive activity. This allows the CRISPR enzyme to be expressed even when cells are cultured in the presence of glucose, which is thought to result in a greater amount of CRISPR enzyme being expressed under conditions of better cellular metabolism than in the case of a typical galactose-inducible promoter. As a result, many structural mutations occur, and it is thought that cells transformed with the expression construct can acquire high resistance to specific target substances. [Example]

[0042] The present invention will be specifically described below with reference to examples, but the present invention is not limited to the descriptions of the following examples.

[0043] (Construction of pEWPMSM_Co plasmid) PCR was performed using the pEWPMSM plasmid described in Yamada et al., November 2023, Research Square, 10.21203 / rs.3.rs-3623691 / v1 as a template and Cas-pTEF(F)_Ass (gcaatCTAATCTAAGTTTTAATTACAAAAAAAAACCtctagATGGCTGATAAGCCAGGTC, SEQ ID NO: 1) and pRPR-pTEF(R)_Ass (AGCTATGGTGTGTGGTtctagaCTCGAGGATCTGCCAATTG, SEQ ID NO: 2) as primers. This resulted in a nucleic acid fragment containing the entire sequence except for the GAL1 promoter (the promoter for galactose-inducible expression). In other words, the amplified nucleic acid fragment contained the first to third nucleotide sequences, the second and third promoters, the drug resistance gene, and the replication origin. In pEWPMSM, a nucleotide sequence encoding a CRISPR enzyme was operably linked to the promoter for galactose-inducible expression.

[0044] PCR was performed using Saccharomyces cerevisiae YPH499 genomic DNA as a template and pTEF-pRPR(F)_Ass (CAGATCCTCGAGtctagaACCACACACCATAGCTTCAAAATG, SEQ ID NO: 3) and pTEF-Cas(R)_Ass (GGACCTGGCTTATCAGCCATctagaGGTTTTTTTTTGTAATTAAAACTTAGATTAG, SEQ ID NO: 4) as primers to obtain a nucleic acid fragment containing the sequence of the TEF1 promoter (i.e., the first promoter).

[0045] The two nucleic acid fragments obtained by PCR described above were ligated to construct pEWPMSM_Co, in which a nucleotide sequence encoding the CRISPR enzyme is operably linked to a promoter with constitutive activity.

[0046] (Creation of mutant strains by simultaneous introduction of point mutations and structural mutations) pEWPMSM or pEWPMSM_Co was introduced into S. cerevisiae YPH499 (obtained from the National Institute of Technology and Evaluation Biotechnology Center) by the lithium acetate method to generate YPH499 / pEWPMSM cells and YPH499 / pEWPMSM_Co cells.

[0047] <Comparative Example 1> YPH499 / pEWPMSM cells were cultured in a test tube containing 5 mL of SG medium (containing 6.7 g / L of yeast nitrogen base without amino acids, 20 g / L of galactose, 10 g / L of raffinose, and appropriate amino acids and nucleic acids) containing 50 g / L of 2,3-butanediol, at an OD 600 The bacteria were inoculated so that the OD was 0.3, and cultured at 30°C with shaking at 150 rpm. 600 Measure the OD 600 From the test tubes where the OD exceeded 1.0, the OD was increased by 10 g / L in fresh SG medium. 600 The bacteria were inoculated so that the OD was 0.3, and cultured with shaking under the above conditions. 600 The 2,3-butanediol concentration was increased repeatedly until the σ did not exceed 1.0.

[0048] Among the YPH499 / pEWPMSM cells grown in SG medium containing 180 g / L of 2,3-butanediol, 20 strains were isolated as YPH499 / pEWPMSM mutant strains.

[0049] Example 1 The same procedures as in Comparative Example 1 were carried out, except that YPH499 / pEWPMSM_Co cells were cultured in SD medium (containing 6.7 g / L of yeast nitrogen base without amino acids, 20 g / L of glucose, and appropriate amino acids and nucleic acids) containing 50 g / L of 2,3-butanediol instead of SG medium. The 2,3-butanediol concentration was also increased in 10 g / L increments, as in Comparative Example 1, and the experiment was carried out.

[0050] Among the YPH499 / pEWPMSM_Co cells grown in SD medium containing 210 g / L of 2,3-butanediol, 20 strains were isolated as YPH499 / pEWPMSM_Co mutant strains.

[0051] (Screening of 2,3-butanediol-resistant mutants) Twenty mutant strains each obtained in Example 1 and Comparative Example 1 were cultured in a deep well plate containing 1.2 mL of SD medium at 30° C. and 1500 rpm with shaking for 48 hours (preculture).

[0052] 0.06 mL of the preculture solution was inoculated into 1.14 mL of SD medium containing 150 g / L of 2,3-butanediol, and cultured in a deep-well plate at 30°C with shaking at 1500 rpm for 48 hours. 600 Difference (ΔOD 600 The activity (sometimes referred to as "activity") was measured using a plate reader.

[0053] FIG. 2 shows the ΔOD values ​​of the YPH499 / pEWPMSM_Co mutant strain of Example 1 and the YPH499 / pEWPMSM mutant strain of Comparative Example 1. 600 10 is a graph showing the ΔOD . It was shown that the YPH499 / pEWPMSM_Co mutant strain was conferred higher 2,3-butanediol resistance than the YPH499 / pEWPMSM mutant strain. Note that the parent strain YPH499, which was not transformed with either the pEWPMSM_Co or pEWPMSM plasmid, had a ΔOD 600 was 0.

[0054] (Growth ability evaluation of 2,3-butanediol-resistant mutants) <Example 2> The YPH499 / pEWPMSM_Co mutant strains (Co2, Co34, Co36, Co40, Co53, and Co58 strains) obtained in the screening of 2,3-butanediol-resistant mutants were cultured in test tubes containing 5 mL of SD medium at 30°C, 150 rpm, and with shaking for 48 hours (preculture).

[0055] The preculture was grown to OD 600 The bacteria were inoculated into 100 mL (500 mL Erlenmeyer flask) of YPD medium (containing 10 g / L yeast extract, 20 g / L peptone, and 20 g / L glucose) containing a predetermined concentration of 2,3-butanediol so that the β-glucan content was 0.05, and cultured with shaking at 30°C, 150 rpm, and 72 hours.

[0056] <Comparative Example 2> Cultivation was carried out in the same manner as in Example 2, except that the YPH499 / pEWPMSM_Co mutant strain was replaced with the parent strain YPH499, which was not transformed with either the pEWPMSM_Co or pEWPMSM plasmid.

[0057] 3 to 5 show the OD of YPD medium when each YPH499 / pEWPMSM_Co mutant strain and YPH499 were cultured in YPD medium containing 0 g / L, 100 g / L, and 150 g / L of 2,3-butanediol (2,3-BDO), respectively. 600 Figure 6 shows the OD of YPH499 after 72 hours of culture in YPD medium containing 100 g / L or 150 g / L of 2,3-butanediol (2,3-BDO). 600 OD of the YPH499 / pEWPMSM_Co mutant strain 6003 to 6 show the relative values ​​of α,β- and β-actin levels. Figures 3 to 6 demonstrate that the YPH499 / pEWPMSM_Co mutant strain exhibits higher growth ability than YPH499 when cultured for 72 hours in a medium containing 2,3-butanediol. The YPH499 / pEWPMSM_Co mutant strain exhibited the highest growth ability, which was 8.5-fold higher than YPH499 in YPD medium containing 100 g / L of 2,3-butanediol and 34.8-fold higher than YPH499 in YPD medium containing 150 g / L of 2,3-butanediol. This indicates that the YPH499 / pEWPMSM_Co mutant strain has high tolerance to 2,3-butanediol.

[0058] (Screening of isobutyl alcohol-resistant mutants and evaluation of their growth ability) Example 3 YPH499 / pEWPMSM_Co cells were cultured in a test tube containing 5 mL of SG medium (containing 6.7 g / L of yeast nitrogen base without amino acids, 20 g / L of galactose, 10 g / L of raffinose, and appropriate amino acids and nucleic acids) containing 5.0 g / L of isobutyl alcohol until the OD 600 The bacteria were inoculated so that the OD was 0.3, and cultured at 30°C with shaking at 150 rpm. 600 Measure the OD 600 From the test tubes where the OD exceeded 1.0, the OD was increased by 1.0 g / L in fresh SG medium. 600 The bacteria were inoculated so that the OD was 0.3, and cultured with shaking under the above conditions. 600 The isobutyl alcohol concentration was increased repeatedly until the value did not exceed 1.0.

[0059] Five strains (IB_35, IB_40, IB_43, IB_44, and IB_80) of YPH499 / pEWPMSM_Co cells grown in SD medium containing 16 g / L isobutyl alcohol were isolated as YPH499 / pEWPMSM_Co mutants.

[0060] The IB_35, IB_40, IB_43, IB_44, and IB_80 strains were each cultured in a test tube containing 5 mL of SD medium at 30°C, 150 rpm, and shaking for 48 hours (preculture).

[0061] The preculture was grown to OD 600 The bacteria were inoculated into 100 mL (500 mL Erlenmeyer flask) of YPD medium (containing 10 g / L yeast extract, 20 g / L peptone, and 20 g / L glucose) containing 15 g / L isobutyl alcohol so that the β-glucan content was 0.05, and cultured at 30°C, 150 rpm, and with shaking for 72 hours.

[0062] <Comparative Example 3> Cultivation was carried out in the same manner as in Example 3, except that the YPH499 / pEWPMSM_Co mutant strain was replaced with the parent strain YPH499, which was not transformed with either the pEWPMSM_Co or pEWPMSM plasmid.

[0063] FIG. 7 shows the OD of YPH499 (Comparative Example 3) after 72 hours of culture in YPD medium containing 15 g / L of isobutyl alcohol. 600 OD of the YPH499 / pEWPMSM_Co mutant strain (Example 3) 600 7 is a graph showing the relative values ​​of β- and β-actin levels. Figure 7 shows that when cultured for 72 hours in a medium containing 15 g / L of isobutyl alcohol, the YPH499 / pEWPMSM_Co mutant strain exhibited higher growth ability than YPH499. The YPH499 / pEWPMSM_Co mutant strain IB_43 exhibited the highest growth ability, which was 3.57-fold higher than that of YPH499. This indicates that the YPH499 / pEWPMSM_Co mutant strain has high tolerance to isobutyl alcohol.

[0064] (Creation of isobutyl alcohol-resistant mutants using YPH499 / pEWPMSM_Co mutants) Example 4 pEWPMSM_Co was introduced into the Co58 strain, which is the YPH499 / pEWPMSM_Co mutant obtained in Example 1, by the lithium acetate method to prepare YPH499 / Co58 mutant cells.

[0065] (Screening of isobutyl alcohol-resistant mutants and evaluation of their growth ability) The YPH499 / Co58 mutant cells were screened using the same procedure as in Example 3, except that the isobutyl alcohol concentration was increased to 20 g / L. Five strains of the YPH499 / Co58 mutant cells (strains IB_17, IB_39, IB_47, IB_49, and IB_55) were isolated as YPH499 / pEWPMSM_Co mutant strains.

[0066] The proliferation ability of the IB_17, IB_39, IB_47, IB_49, and IB_55 strains was evaluated using the same procedure as described in Example 3.

[0067] FIG. 8 shows the OD of YPH499 (Comparative Example 3) after 72 hours of culture in YPD medium containing 15 g / L of isobutyl alcohol. 600 OD of the YPH499 / Co58 mutant strain (Example 4) 600 8 shows the relative values ​​of β- and β-actin levels. Figure 8 shows that the YPH499 / pEWPMSM_Co mutant strain exhibited higher growth ability than YPH499 when cultured for 72 hours in a medium containing 15 g / L of isobutyl alcohol. The YPH499 / Co58 mutant strain IB_39, which exhibited the highest growth ability, exhibited 118-fold higher growth ability than YPH499. This indicates that introducing additional mutations into the YPH499 / pEWPMSM_Co mutant strain, which is resistant to the heterologous alcohol 2,3-butanediol, can yield a mutant strain with dramatically increased tolerance to isobutyl alcohol. This suggests that introducing pEWPMSM_Co into the YPH499 / pEWPMSM_Co mutant strain and introducing additional mutations can significantly increase its tolerance to other organic solvents.

[0068] (Screening of lactate-resistant mutants and evaluation of their growth ability) <Example 5> In this example, the pEWPMSM_Co2 plasmid was used, in which the selection marker of the pEWPMSM_Co plasmid was changed to URA3, and YPH499 / pEWPMSM_Co2 cells were obtained by the method described in (Creation of mutant strains by simultaneous introduction of point mutations and structural mutations).

[0069] YPH499 / pEWPMSM_Co2 cells were cultured in a test tube containing 5 mL of SG medium (containing 6.7 g / L yeast nitrogen base without amino acids, 20 g / L galactose, 10 g / L raffinose, and appropriate amino acids and nucleic acids) containing 10 g / L lactic acid, at OD 600 The bacteria were inoculated so that the OD was 0.3, and cultured at 30°C with shaking at 150 rpm. 600 Measure the OD 600 From the test tubes where the OD exceeded 1.0, the OD was increased by 2.0 g / L in fresh SG medium. 600 The bacteria were inoculated so that the OD was 0.3, and cultured with shaking under the above conditions. 600 The lactic acid concentration was increased repeatedly until the β-glucan concentration did not exceed 1.0.

[0070] Among the YPH499 / pEWPMSM_Co2 cells grown in SD medium containing 16 g / L of lactic acid, the YPH499 / LA_86 strain was isolated as a YPH499 / pEWPMSM_Co2 mutant.

[0071] The YPH499 / LA_86 strain was cultured in a test tube containing 5 mL of SD medium at 30°C, 150 rpm, and with shaking for 48 hours (preculture).

[0072] The preculture was grown to OD 600 The bacteria were inoculated into 100 mL (500 mL Erlenmeyer flask) of YPD medium (containing 10 g / L yeast extract, 20 g / L peptone, and 20 g / L glucose) containing 10 g / L, 14 g / L, or 18 g / L of lactic acid so that the β-glucan content was 0.05, and cultured with shaking at 30°C, 150 rpm, and 72 hours.

[0073] <Comparative Example 4> Cultivation was carried out in the same manner as in Example 5, except that the YPH499 / pEWPMSM_Co2 mutant strain (YPH499 / LA_86 strain) was replaced with the parent strain YPH499 that was not transformed with either the pEWPMSM_Co plasmid or the pEWPMSM plasmid.

[0074] FIG. 9 shows the OD of YPH499 (Comparative Example 4) after 72 hours of culture in YPD medium containing 10 g / L, 14 g / L, or 18 g / L of lactic acid. 600 OD of YPH499 / LA_86 strain (Example 5) 600 9 is a graph showing the relative values ​​of lactic acid tolerance and lactic acid tolerance. Figure 9 shows that the YPH499 / LA_86 strain exhibited higher growth ability than YPH499 when cultured for 72 hours in a medium containing 10 g / L, 14 g / L, or 18 g / L of lactic acid. When the YPH499 / LA_86 strain was cultured in a YPD medium containing 14 g / L of lactic acid, it was found to grow 3.77-fold more than YPH499. This indicates that the YPH499 / pEWPMSM_Co2 mutant (YPH499 / LA_86 strain) has high resistance to lactic acid.

[0075] (Screening of lactic acid-tolerant mutants using practical yeast and evaluation of their growth ability) Example 6 The pEWPMSM_Co2 plasmid was introduced into the industrial yeast SH-4 (diploid, shochu yeast, obtained from the National Research Institute of Brewing) by the lithium acetate method to generate SH-4 / pEWPMSM_Co2 mutant cells.

[0076] The SH-4 / pEWPMSM_Co2 mutant cells were screened using the same procedure as in Example 5 for screening mutant strains, except that the lactic acid concentration was increased to 20 g / L, and the SH-4 / LA_42 strain was isolated as the SH-4 / pEWPMSM_Co2 mutant strain.

[0077] The SH-4 / LA_42 strain was subjected to growth ability evaluation using the same procedure as described in Example 5.

[0078] <Comparative Example 5> Cultivation was carried out in the same manner as in Example 6, except that the SH-4 / pEWPMSM_Co2 mutant strain (SH-4 / LA_42 strain) was replaced with the parent strain, practical yeast SH-4, which was not transformed with either the pEWPMSM_Co2 plasmid or the pEWPMSM plasmid.

[0079] FIG. 10 shows the OD of SH-4 (Comparative Example 5) after 72 hours of culture in YPD medium containing 10 g / L, 14 g / L, or 18 g / L of lactic acid. 600 OD of SH-4 / LA_42 strain (Example 6) 600 10 is a graph showing the relative values ​​of lactic acid tolerance and lactic acid tolerance. Figure 10 shows that when cultured for 72 hours in a medium containing 14 g / L or more of lactic acid, the SH-4 / LA_42 strain exhibited higher growth ability than SH-4. When the SH-4 / LA_42 strain was cultured in a YPD medium containing 14 g / L of lactic acid, it was found to grow 40.1 times more than SH-4. In other words, the SH-4 / pEWPMSM_Co2 mutant strain (SH-4 / LA_42 strain) was shown to have high resistance to lactic acid.

[0080] These results demonstrate that the use of the expression construct pEWPMSM_Co2 of the present invention can enhance the resistance of cells to organic acids, and that mutations can be introduced into diploid industrial yeast, which is generally considered difficult to introduce mutations into.

[0081] (Evaluation of 2,3-butanediol resistance in the YPH499 / Co58 mutant strain IB_39) Example 7 The YPH499 / Co58 mutant IB_39 strain obtained in Example 4 is an isobutyl alcohol-resistant mutant, whereas the parent Co58 strain is a 2,3-butanediol-resistant mutant. An experiment was conducted to confirm whether the YPH499 / Co58 mutant IB_39 strain maintained 2,3-butanediol resistance.

[0082] The IB_39 strain was cultured in a test tube containing 5 mL of SD medium at 30°C, 150 rpm, and with shaking for 48 hours (preculture).

[0083] The preculture was grown to OD 600 The bacteria were inoculated into 100 mL (500 mL Erlenmeyer flask) of YPD medium (containing 10 g / L yeast extract, 20 g / L peptone, and 20 g / L glucose) containing 150 g / L of 2,3-butanediol so that the β-glucan content was 0.05, and cultured with shaking at 30°C, 150 rpm, and 72 hours.

[0084] FIG. 11 shows the OD of YPH499 (Comparative Example 3) after 72 hours of culture. 600 OD of IB_39 strain (Example 7) 600 11 is a graph showing the relative values ​​of β-actin and β-actin. Figure 11 shows that when cultured for 72 hours in a medium containing 2,3-butanediol, the IB_39 strain exhibited higher growth ability than YPH499. Specifically, the IB_39 strain exhibited 29.1-fold higher growth ability than YPH499 in YPD medium containing 150 g / L of 2,3-butanediol. This indicates that the IB_39 strain, a YPH499 / Co58 mutant cell, not only possesses isobutyl alcohol tolerance but also maintains high tolerance to 2,3-butanediol. [Explanation of symbols]

[0085] 1...expression construct 1, 11...first promoter, 12...first nucleotide sequence, 13...second promoter 1, 14...second nucleotide sequence, 15...third promoter, 16...third nucleotide sequence, 17...drug resistance gene, 18...replication origin

Claims

1. a first promoter that functions in cells and has constitutive activity; a first nucleotide sequence encoding a CRISPR enzyme operably linked to the first promoter; a second promoter that functions in the cell; a second nucleotide sequence encoding a guide RNA operably linked to the second promoter; a third promoter for expression that functions in the cell; a third nucleotide sequence operably linked to the third promoter, the third nucleotide sequence encoding a protein that introduces a point mutation into the genome of the cell; Drug resistance genes, an origin of replication, An expression construct, wherein the guide RNA is capable of binding complementarily to at least a portion of a multicopy sequence present in the genome.

2. 2. The expression construct according to claim 1, wherein the cell is any one selected from the group consisting of yeast, filamentous fungi, Corynebacterium bacteria, Escherichia coli, and Bacillus subtilis.

3. 2. The expression construct of claim 1, wherein the CRISPR enzyme is Cas9.

4. A cell transformed with the expression construct according to any one of claims 1 to 3.

5. The cell according to claim 4 , which is resistant to a target substance due to the transformation.

6. The cell according to claim 5, wherein the target substance is at least one selected from 2,3-butanediol, isobutyl alcohol, and lactic acid.

7. Introducing the expression construct according to any one of claims 1 to 3 into a cell, creating a point mutation in the genome of the cell; generating structural mutations in the multicopy sequence; and conferring resistance to a target substance to the cell by the point mutation and the structural mutation. A method for obtaining cells that are resistant to a target substance.

8. The method for obtaining cells according to claim 7, wherein the target substance is at least one selected from the group consisting of 2,3-butanediol, isobutyl alcohol, and lactic acid.

9. The method for obtaining the cell described in claim 7 further comprises re-introducing the expression construct into the cell to which resistance to the target substance has been conferred, thereby conferring resistance to a target substance other than the target substance.

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