Yeast capable of high-level production of l-methionine

Abstract

[Problem] To provide, for example, a mutant Met13 protein suitable for high-level production of methionine. [Solution] The problem is solved by a mutant methylenetetrahydrofolate reductase protein (Met13 protein) having a Ser443Phe substitution, the mutant Met13 protein comprising any one of the following amino acid sequences (a) to (c): (a) an amino acid sequence that is set forth in SEQ ID NO: 1 or that has a Ser443Phe substitution in an amino acid sequence encoded by a MET13 gene of a yeast belonging to the genus Saccharomyces; (b) an amino acid sequence that is derived from the amino acid sequence of (a) by deletion, substitution, or addition of one or several amino acids and has the function of a Met13 protein; and (c) an amino acid sequence that has 90% or more sequence identity with the amino acid sequence of (a) and has the function of a Met13 protein.

Description

L-methionine-producing yeast

[0001] This invention relates to a yeast that produces high levels of L-methionine (hereinafter referred to as "methionine").

[0002] Mammals cannot biosynthesize essential amino acids through endogenous pathways and therefore must obtain all nine essential amino acids from food. Recent research has demonstrated that essential amino acids play a crucial role in maintaining brain homeostasis and are associated with the progression of Alzheimer's disease, which is characterized by mild cognitive impairment. Therefore, essential amino acids are attracting significant attention as a way to slow neurodegenerative processes in the elderly, contributing to extended healthy lifespans and improved quality of life. However, due to age-related loss of appetite and fluctuations in the essential amino acid content of food, it is difficult for the elderly to obtain sufficient essential amino acids from their daily diet. The yeast Saccharomyces cerevisiae is generally considered safe and is widely used in the production of nutritional supplements. Therefore, if yeast containing accumulated essential amino acids can be provided, the elderly can easily obtain appropriate amounts of essential amino acids.

[0003] Among the essential amino acids, methionine (Met) is widely used as a pharmaceutical and feed additive. Methionine is involved in carbon metabolism as a precursor to S-adenosylmethionine (SAM), which is a methyl group donor for various intracellular metabolites. As shown in Figure 1(a), methionine biosynthesis consists of two parts: the formation of homocysteine ​​from aspartic acid (Asp) through five enzymatic steps, and the addition of a methyl group to homocysteine. In the first half of the methionine biosynthesis pathway, the first four reactions are shared with the threonine biosynthesis pathway, and it branches off from homocysteine ​​to cysteine ​​(Cys) biosynthesis. In the second half, two different pathways, the methionine cycle and the folate cycle, are involved.

[0004] As with other amino acid biosynthesis, methionine biosynthesis in S. cerevisiae is strictly regulated, including allosteric inhibition of methylenetetrahydrofolate reductase (MTHFR) activity by SAM. MTHFR uses flavin adenine dinucleotide (FAD) as its coenzyme and catalyzes the reduction of methylenetetrahydrofolate (MTHF) by NAD(P)H. Allosteric regulation of MTHFR activity by SAM is important for controlling the transfer of methyl groups from the folate cycle to the methionine cycle (one-carbon metabolism).

[0005] MTHFR dysfunction can cause human diseases such as cancer and mental illness. Therefore, elucidating the functional and structural characteristics of MTHFR can contribute to the microbial production of monocarbon metabolites such as purine nucleotides, amino acids, and glutathione, as well as to promoting human health. In the genomic DNA of S. cerevisiae, two genes encoding MTHFR, MET12 and MET13, have been identified. Phenotypic analysis has shown that under standard laboratory conditions, the Met13 protein functions as the primary MTHFR in S. cerevisiae. Furthermore, enzymatic analysis revealed that the Met13 protein catalyzes NADPH-dependent reduction of MTHF using the cofactor FAD, and, similar to mammalian MTHFR, is sensitive to SAM-mediated inhibition of enzymatic activity.

[0006] As shown in Figure 1(b), according to the amino acid sequence, the Met13 protein consists of an N-terminal catalytic domain and a C-terminal regulatory domain, which is a typical domain configuration for eukaryotic MTHFRs. Crystal structure analysis of human MTHFR (hMTHFR) and Chaetomium thermophilum MTHFR (cMTHFR) confirmed that the allosteric inhibitor SAM is recognized by amino acid residues in the regulatory domain. Furthermore, these amino acid residues responsible for SAM recognition are highly conserved at positions 440-449 of the amino acid sequence in various Met13 proteins. Previous studies on Met13 proteins have shown that introducing alanine substitutions to the amino acid residues corresponding to SAM-binding residues in hMTHFR reduces sensitivity to allosteric inhibition by SAM through amino acid substitutions of Pro354Ala, Arg357Ala, and Glu422Ala (Bhatia et al., 2020: Non-Patent Literature 1). Furthermore, in cMTHFR, dynamic structural changes in the catalytic domain and linker region were induced by SAM. While the similarity in domain structure and conservation of amino acid residues suggest that the allosteric regulation of Met13 protein by SAM is similar to that of cMTHFR, the detailed mechanism of SAM-mediated regulation of Met13 protein has not been fully elucidated. Research has also been conducted on yeast that produces high levels of Met (Patent Documents 1 and 2).

[0007] Japanese Patent Publication No. 2023-071865, Japanese Patent Publication No. 2020-526205

[0008] Bhatia, M.et al. (2020) ‘Allosteric inhibition of MTHFR prevents futile SAM cycling andmaintains nucleotide pools in one-carbon metabolism’, Journal of BiologicalChemistry, 295(47), pp. 16037-16057. Available at:https: / / doi.org / 10.1074 / jbc.RA120.015129.Cherest, H. et al. (1973) ‘Effects of regulatory mutations uponmethionine biosynthesis in Saccharomyces cerevisiae: loci eth2 eth3 eth10’,Journal of Bacteriology, 115(3), pp. 1084-1093. Available at:https: / / doi.org / 10.1128 / jb.115.3.1084-1093.1973.Georgieva, N. and Alexieva, Z. (2005) Selection and characterizationof L-ethionine resistant mutants of Trichosporon cutaneum, Zeitschrift furNaturforschung - Section C Journal of Biosciences. Available at:https: / / doi.org / 10.1515 / znc-2005-9-1001.Froese, D.S. et al. (2018) ‘Structural basis for the regulation ofhuman 5,10-methylenetetrahydrofolate reductase by phosphorylation andS-adenosylmethionine inhibition’, Nature Communications, 9(1), pp. 1-13.Available at: https: / / doi.org / 10.1038 / s41467-018-04735-2.Yamada, K., Mendoza, J. and Koutmos, M. (2024) Structural basis ofS-adenosylmethionine-dependent allosteric transition from active to inactive states in methylenetetrahydrofolate reductase, Nature Communications. Availableat: https: / / doi.org / 10.1038 / s41467-024-49327-5.

[0009] This research was conducted in view of the above-mentioned issues, and its purpose is to provide a mutant Met13 protein capable of high methionine production. In this specification, the Met13 protein, which is MTHFR as a protein, will be written with only the first letter capitalized as "Met13" or "Met13 protein". The MTHFR gene will be written with all letters capitalized as "MET13" or "MET13 gene". Furthermore, the Met13 having the Ser443Phe substitution of the present invention will be written as "Ser443Phe mutant Met13", "Ser443Phe mutant Met13 protein", "mutant Met13", or "mutant Met13 protein". The wild-type MTHFR without the Ser443Phe substitution will be written as "WT-Met13", "WT-Met13 protein", "wild-type Met13", or "wild-type Met13 protein".

[0010] The inventors isolated a Met-accumulating mutant from a diploid experimental strain of Saccharomyces cerevisiae using a conventional mutagenesis method and identified an amino acid substitution of serine (Ser) to phenylalanine (Phe) at position 443 of the Met13 protein. Enzymatic analysis of the Ser443Phe mutant Met13 protein revealed that substituting Ser with Phe at position 443 eliminated sensitivity to inhibition by SAM even in the presence of 2 mM SAM. Furthermore, the Ser443Phe substitution showed an increase in the specific activity of MTHFR. In addition, when the Ser443Phe mutant Met13 protein was expressed in Saccharomyces cerevisiae cells, it was confirmed that the intracellular Met content increased compared to cells expressing wild-type Met13 protein, thus completing the present invention. Thus, the amino acid sequence according to the present invention is a mutant methylenetetrahydrofolate reductase protein (Met13 protein) having a Ser443Phe substitution, which has one of the amino acid sequences described in (a) to (c) below: (a) the amino acid sequence of Sequence ID No. 1, or an amino acid sequence having a Ser443Phe substitution in the amino acid sequence encoded by the MET13 gene of yeast belonging to the genus Saccharomyces; (b) an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence of (a) above, and which has the function of the Met13 protein; (c) an amino acid sequence having 90% or more sequence identity with the amino acid sequence of (a) above, and which has the function of the Met13 protein.

[0011] Sequence ID 1 is (the underlined "F" is 443Phe).

[0012] With respect to (b), there is no particular upper limit on the number of amino acid residues that may be substituted, deleted, inserted, and / or added, as long as the protein has the function of Met13. The upper limit may be, for example, 7, 6, 5, 4, 3, 2, or 1. With respect to (c), the amino acid sequence identity is 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100%. Sequence identity can be checked using homology searches such as FASTA, BLAST, and PSI-BLAST at homology search sites using the Internet, such as the DNA Data Bank of Japan (DDBJ). It can also be checked using BLAST at the National Center for Biotechnology Information (NCBI). "Having the function of a Met13 protein" means that it uses FAD as a cofactor and exhibits activity to catalyze the reduction reaction of MTHF by NAD(P)H. Yeast is a general term for eukaryotic, single-celled microorganisms that are non-motile, possess cell walls, lack photosynthetic ability, and break down external organic matter to absorb nutrients. It includes, for example, the genus Saccharomyces, a type of budding yeast used in food products. Yeast can perform alcoholic fermentation, breaking down sugar into alcohol and carbon dioxide. In this invention, examples include baker's yeast, sake yeast, wine yeast, beer yeast, soy sauce yeast, and bioethanol yeast. Furthermore, both haploid and diploid yeasts can be used.

[0013] Furthermore, the nucleic acid relating to the other invention is a nucleic acid encoding a mutant methylenetetrahydrofolate reductase protein (Met13 protein) having a Ser443Phe substitution having one of the following amino acid sequences (a) to (c): (a) the amino acid sequence of Sequence ID No. 1, or an amino acid sequence having a Ser443Phe substitution in the amino acid sequence encoded by the MET13 gene of yeast belonging to the genus Saccharomyces; (b) an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence of (a) above, and which has the function of the Met13 protein; (c) an amino acid sequence having 90% or more sequence identity with the amino acid sequence of (b) above, and which has the function of the Met13 protein. The genetic code that codes for amino acids has 64 types as triplets (sets of 3), and there are 20 types of amino acids, so multiple triplets correspond to the same amino acid. For this reason, the nucleic acid sequence that identifies the Met13 protein having a Ser443Phe substitution is not limited to one type. According to the present invention, any nucleic acid encoding a Met13 protein having a Ser443Phe substitution can provide yeast that produces high levels of methionine.

[0014]

[0015] Furthermore, a plasmid according to another invention comprises the above-mentioned nucleic acid and is capable of expressing a protein having the function of the Met13 protein. Furthermore, a high-producing L-methionine yeast according to another invention is equipped with the above-mentioned mutant Met13 protein in a form capable of expressing it. Being capable of expressing the protein means, for example, incorporating nucleic acid encoding the mutant Met13 protein into a plasmid for expressing the protein (including at least a promoter and a ribosome binding sequence; it may also include a terminator, antibiotic resistance gene, origin of replication, Rop (a protein that limits the copy number), etc.) into yeast, incorporating nucleic acid encoding the mutant Met13 protein together with the promoter into the yeast chromosome, or converting wild-type MET13 in yeast to mutant MET13 by homologous recombination.

[0016] The above yeast produces methionine at a higher rate than yeast possessing only wild-type MET13, and can therefore be used directly for methionine production. Furthermore, methionine can be efficiently obtained by ingesting the yeast itself. Therefore, we can offer food and beverages containing this yeast. These can be used in beverages (milk-containing drinks, coffee, tea, juice, processed milk, sports drinks, etc.), bakeries (bread, pizza, pies, etc.), Western-style confectionery (cookies, crackers, biscuits, cakes, sponge cake, etc.), noodles, pasta, snacks, sweets (candy, caramel, gum, chocolate, etc.), frozen desserts (ice cream, sherbet, etc.), dairy products (cream, cheese, mousse, powdered milk, condensed milk, milk beverages, etc.), and Western-style fresh confectionery (jelly, pudding, mousse, yogurt, buttercream, etc.). This includes starch cream, Japanese sweets (gyuhi, uiro, mochi, ohagi, dorayaki, etc.), processed fruits and vegetables (jam, marmalade, syrup-preserved foods, candied fruits, etc.), pastes (flour paste, fruit paste, peanut paste, etc.), seasonings (soy sauce, sauces, dips, noodle soup base, dashi stock, soup stock, etc.), frozen and refrigerated foods (ham, sausage, bacon, hamburgers, meatballs, croquettes, dumplings, pilaf, rice balls, etc.), and processed seafood (chikuwa, kamaboko, etc.). Food and beverages are not limited to human consumption but also include those for pets (dogs, cats, etc.). Food and beverages also include healthcare products such as supplements.

[0017] According to the present invention, it is possible to provide yeast that produces high levels of methionine using a mutant Met13 protein.

[0018] This diagram illustrates the function of MTHFR. (a) A schematic diagram showing the steps of methionine biosynthesis in yeast. Met and Cys are biosynthesized from Asp via homocysteine, a common intermediate. In Met biosynthesis, a methyl group derived from the folate cycle binds to homocysteine ​​to produce Met. In the Met cycle, Met is converted to SAM. SAM is the major methyl group donor in cells. MTHFR catalyzes the NADPH-dependent reduction reaction of MTHF with FAD as a cofactor, and its activity is allosterically inhibited by SAM. In the diagram, "SHMT" means serine hydroxyl methyltransferase. (b) The catalytic domain, linker, and regulatory domain of the Met13 protein are shown in Saccharomyces cerevisiae, the thermophilic fungus (Chaetomium thermophilum), and humans (Homo Sapiens). Below that, the amino acids at positions 440-449 of Saccharomyces cerevisiae, thermophilic fungi, and humans are shown. The individual amino acid sequences are shown in SEQ ID NOs: 7 to 9.

[0019] This figure shows the results of isolating yeast mutants with intracellular Met accumulation. (a) A photograph showing the results of examining the sensitivity to ETH by spotting yeast cells into SD+Alt medium (w / o ETH) or medium containing 0.05 mg / mL ETH (+0.05 mg / mL ETH) after culturing them to the initial logarithmic growth phase. (b) A graph showing the results of measuring the intracellular amino acid content after culturing X2180-WT, ETH-80, and ETH-129 strains in SD+Am medium for 48 hours. Data are shown as the mean ± standard deviation of three independent experiments. Statistical significance was evaluated by one-way ANOVA and Tukey's test (multi-group comparison). In the figure, "*" indicates p < 0.05 (X2180-WT vs. ETH-80), and "**" indicates p < 0.05 (X2180-WT vs. ETH-129). This graph shows the results of investigating the effect of Ser443Phe substitution on the inhibition of Met13 protein by SAM. The relative MTHFR activity of WT-Met13 protein (WT) and Ser443Phe mutant Met protein (S443F) was measured by varying the SAM concentration. Enzyme activity in the absence of SAM was defined as 100% (WT-Met13 protein was 44 U / mg, and S443F mutant Met13 protein was 127 U / mg). Data are shown as the mean ± standard deviation of three independent experiments.

[0020] This figure shows the results of a structural comparison between the WT-Met13 protein and the Ser443Phe mutant Met13 protein. (a) Figure showing the local structure around the putative SAM binding pocket of the WT-Met13 protein, (b) Figure showing the local structure around the putative SAM binding pocket of the Ser443Phe mutant Met13 protein. The model structures of WT-Met13 and Ser443Phe mutant Met13 proteins were constructed using cMTHFR bound to SAM as a template (PDB ID: 8UY2). The predicted hydrogen bond between the amino acid residue at position 443 and SAM is shown by a dashed line. (c) Figure showing the local structure around the residue at position 443 in the WT-Met13 protein, (d) Figure showing the local structure around the residue at position 443 in the Ser443Phe mutant Met13 protein. The model structure was constructed using cMTHFR without SAM as a template (PDB ID: 8UY1). MET13 S443F This graph shows the results of a study investigating the effect of gene expression on intracellular amino acid content. Yeast cells were cultured in SD+Am medium, and intracellular amino acid content was measured. Data are presented as the mean ± standard deviation of three independent experiments. Statistical significance was evaluated by one-way ANOVA and Tukey's test (multi-group comparison). In the figure, "*" indicates p < 0.05 (EV vs. Met13). S443F ), ``**'' is p < 0.05 (Met13 WT vs Met13 S443F This indicates "not detected". "nd" means "not detected".

[0021] Next, embodiments of the present invention will be described with reference to the figures and tables. The technical scope of the present invention is not limited by these embodiments, and it can be implemented in various forms without changing the gist of the invention. <Test Method> 1. Diploid laboratory yeast strain X2180 (wild type (WT), MATa / α) and haploid laboratory yeast strain X2180 / ura3Δ (ura3::KanMX4) of yeast strain and culture medium were used as the parent strain for mutagenesis and the host strain for MET13 gene expression, respectively. For yeast culture, nutrient medium YPD (10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose) and synthetic dextrose minimal medium SD+Am (1.7 g / L amino acids and a nitrogen source base for yeast culture that does not contain ammonium sulfate (manufactured by DIFCO Laboratories), 20 g / L glucose and 5 g / L ammonium sulfate) were used. To isolate ethionine (ETH)-resistant strains, strain X2180 was cultured in SD+Alt medium (SD medium supplemented with 5 g / L allantoin as a nitrogen source instead of ammonium sulfate). E. coli strains DH5α (F-λ-Φ80lacZΔM15 Δ(lacZYA argF)U169 deoR recA1 endA1 hsdR17(rk-mk+)supE44 thi-1 gyrA96) and BL21(DE3) (F- ompT hsdS(rB- mB-)gal dcmλ(DE3)(λ(DE3):lacI,lacUV5-T7 gene1 ind1 sam7 nin5) were used for plasmid construction and recombinant Met13 protein expression, respectively. LB medium (5 g / L yeast extract, 10 g / L tryptone, 5 g / L NaCl) or TB medium (12 g / L yeast extract, 24 g / L tryptone, 5.02 g / L glycerol, 170 mM KH2PO4, 720 mM) containing appropriate antibiotics were used. Cultures were performed in K2HPO4. Unless otherwise specified, all chemicals were purchased from Wako Pure Chemical Industries, Nacalai Tesque, and Sigma-Aldrich.

[0022] 2. Construction of a plasmid for MET13 gene expression. Open reading frame (ORF) encoding the wild-type Met13 protein (MET13 WT)(SEQ ID NO: 3: 5’-GGG GAC AAG TTT GTA CAA AAA AGC AGG CTT AAT GAA GAT CAC AGAAAA ATT-3’ and SEQ ID NO: 4: 5’-GGG GAC CAC TTT GTA CAA GAA AGC TGG GTG TTA TAG GCT TAG TAGGAT GG-3’) was amplified from the genomic DNA of X2180 by PCR using these primers. The DNA fragment amplified by PCR was cloned into pDONR221 using BP Clonase II to obtain pDONR_MET13 WT . The point mutation corresponding to the Ser443Phe substitution was introduced into pDONR_Met13 WT using two primers (SEQ ID NO: 5: 5’-TCA TCA CTA TAA ACT tTC AAC CTC AAG TCA A -3’ and SEQ ID NO: 6: 5’-TTG ACTTGA GGT TGA aAG TTT ATA GTG ATG A-3’. In the SEQ ID NOs, the lowercase t and a indicate the positions where the Ser443Phe substitution was introduced.) to generate pDONR_MET13 S443F . To construct an expression plasmid for the recombinant Met13 protein with an N-terminal histidine tag, the MET13 ORF was transferred into the pET53-dest expression vector (Thermo Scientific) using LR Clonase II (Thermo Scientific) to obtain pET_MET13 WT and pET_MET13 S443F . To construct a plasmid expressing the MET13 gene under the constitutive glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter in yeast cells, the MET13 ORF was transferred into the pAG415_GPD expression vector using the same procedure as above to obtain pAG415_MET13WT and pAG415_MET13 S443F .

[0023] 3. Isolation of ETH-resistant mutant strains: Mutations were introduced by treating strain X2180 with 5.5% ethyl methanesulfonic acid (EMS) in phosphate buffer (pH 7.0) at 30°C for 60 minutes. EMS-treated cells were washed twice with 10% (w / v) sodium thiosulfate and spread onto SD+Alt medium containing 0.05 mg / mL ETH. After incubation at 30°C for 3 days, the resulting colonies were collected and their amino acid productivity was evaluated. The viability of yeast cells after EMS treatment was approximately 35%. 4. Spotting test: Strains derived from X2180 were cultured in SD medium or SD+Am medium at 30°C for 2 days, and then inoculated into the same medium. Yeast cells in the exponential growth phase were collected and washed twice with sterile water. Serially diluted yeast cells were spotted onto SD+Alt plates in or without ETH. The plates were incubated at 30°C for 3 days.

[0024] 5. Quantification of intracellular amino acid content: Yeast cells were cultured in SD+Am medium at 30°C for 2 days, and OD was measured. 600 When the concentration reached 0.1, the cells were inoculated into the same medium. After culturing with shaking at 30°C for 48 hours, the yeast cells were collected by centrifugation and washed twice. The obtained yeast pellet was resuspended in sterile water, and the suspension was prepared using OD. 600 The solution was adjusted to equal 200. Next, amino acids were extracted from 0.2 mL of the cell suspension by treating it at 100°C for 20 minutes. After centrifugation at 15,000 × g for 5 minutes, the amino acid content in each supernatant was quantified using an amino acid analyzer (JLC-500 / V2: JEOL Corporation) equipped with ion exchange chromatography and a post-column ninhydrin derivatization device. The content of each amino acid is shown as "μmol / g dry cell weight (DCW)".

[0025] 6. Whole Genome Analysis: Cells X2180, ETH-80, and ETH-129 were cultured in YPD medium at 30°C for 1 day with shaking. Cells were then harvested, and genomic DNA was extracted using the Gen-Toru-kun (for yeast) High Recovery Kit (Takara Bio). Libraries for sequencing analysis were prepared using the NEB Next Ultra DNA Library Prep Kit (New England & Biolabs). 150 bp paired-end short reads were generated using IlluminaNovaSeq 6000 (Illumina). The yeast S288C genome sequence (https: / / www.ncbi.nlm.nih.gov / assembly / GCF_000146045.2 / ) was used as the reference sequence. The sequencing process was outsourced to a commercial DNA sequencing service (Relixa).

[0026] 7. Expression and purification of recombinant Met13 protein pET_MET13 WT and pET_MET13 S443F E. coli BL21 (DE3) containing [the specified substance] was cultured in TB medium containing ampicillin and OD at 37°C. 600 After growing to 1.5, the cells were cooled on ice for 5 minutes, and IPTG (isopropyl β-thiogalactopyranoside) was added to a final concentration of 0.2 mM. After culturing at 18°C ​​for 20 hours, the cells were harvested by centrifugation and suspended in buffer A (50 mM potassium phosphate (pH 7.2), 500 mM NaCl, and 10% (w / v) glycerol). The cell suspension was homogenized under cooling, and the insoluble fraction was removed by centrifugation. The supernatant was filtered through a 0.45 μm filter and then coupled to a nickel affinity column (Ni Sepharose 6 Fast flow (GE Healthcare)). The column was washed with buffer A containing 40 mM imidazole, and the recombinant protein was eluted with buffer A supplemented with 500 mM imidazole.

[0027] 8. Measurement of MTHFR Activity of Recombinant Met13 Protein MTHFR activity was measured using menadione as an electron acceptor with minor modifications to the previously reported method. The reaction mixture (final volume: 0.2 mL) contained 100 mM potassium phosphate (pH 7.2), 0.25 mM menadione, 0.2 mM NADPH, 0.025 mM FAD, and purified wild-type and mutant Met13 proteins (0.42 μg of wild-type and 0.15 μg of Ser443Phe mutant protein). The reaction mixture except for NADPH was pre-equilibrated at 30°C for 3 minutes, and the reaction was initiated by adding NADPH. Met13-dependent NADPH oxidation was monitored at 340 nm using a SpectraMax Mini spectrophotometer (Molecular Devices) and maintained at 30°C. To examine the sensitivity of Met13 protein to inhibition by SAM, SAM was added to the reaction mixture at concentrations ranging from 0.025 mM to 2 mM. The reaction rate was calculated using the extinction coefficient of NADH of 6,220 M -1 ·cm -1 . One activity unit was defined as the amount of enzyme required to oxidize 1 μmol of NADPH per minute. The kinetic parameters of each enzyme were calculated using non-linear regression analysis with GraphPad Prism version 9 (GraphPad Software).

[0028] 9. Structural Analysis of Met13 Protein A homology model of yeast-derived MTHFR (Met13, [UniProtKB accession No. P53128]) was constructed using the SWISS-MODEL online tool. For thermophilic fungal MTHFR (sequence identity with Met13 was 53%, similarity was 70%), the model coupled with SAM [PDB ID No. 8UY2] (Figure 4(a)) and the model not coupled with SAM [PDB ID No. 8UY1] (Figure 4(b)) were used as templates. The amino acid substitution from serine 443 to phenylalanine was calculated using PyMOL software (PyMOL Molecular Graphics System version 2.5). Structural models of WT and Ser443Phe mutant Met13 proteins were plotted using Pymol software (http: / / www.pymol.org). 10. Statistical analysis data are presented as mean ± standard deviation (SD). One-way ANOVA was used for statistical significance analysis, and Tukey's test was used for multi-group comparisons. Prism 9 was used for statistical analysis. A p-value of less than 5% (p < 0.05) was considered statistically significant.

[0029] <Test Results> 1. Isolation of Yeast Mutants with Met Accumulation First, ETH-resistant mutants were isolated using the diploid experimental yeast X2180 as the parent strain. Because ETH is structurally similar to Met, if it is mistakenly incorporated as a component into a protein, it causes protein dysfunction and acts as an inhibitor of Met biosynthesis, thereby causing cell death. The toxicity of ETH can be reversed when Met accumulates in the cell. Therefore, it is expected that ETH-resistant mutants will produce a large amount of Met in the cell (Cherest et al., 1973: Non-Patent Literature 2, Georgieva and Alexieva, 2005: Non-Patent Literature 3). When yeast cells of the parent strain X2180 were randomly mutated by EMS treatment, approximately 150 ETH-resistant mutants were obtained. Of these ETH-resistant mutants, strains ETH-80 and ETH-129 produced 14 times and 10 times more Met, respectively, compared to the parent strain (X2180-WT) (Figure 2(a)). Intracellular Cys was increased in both mutant strains compared to X2180-WT, but Asp was decreased. Intracellular Gly in ETH-80 and ETH-129 strains was 2.1 times higher compared to X2180-WT (Figure 2(b)). In the folate cycle, serine hydroxymethyltransferase (SHMT) forms MTHF from THF and Ser to produce glycine (Figure 1(a)). These results suggest that mutations in ETH-80 and ETH129 strains enhance the flow of the folate cycle, leading to increased availability of MTHF with glycine accumulation, and thereby increased Met production in yeast cells.

[0030] 2. Characteristics of MET13 gene mutations in ETH-80 and ETH-129 strains To identify the mutations responsible for Met overproduction, whole-genome sequence analysis of ETH-80 and ETH-129 strains was performed. Compared with the parental strain X2180, about 200 and about 300 amino acid substitution mutations were detected in the genomes of ETH-80 and ETH-129 strains, respectively. Among the mutated genes, a heterozygous mutation encoding the Ser445Phe mutation with cytosine and thymine mixed at nucleotide position 1328 of the MET13 gene in the genomes of ETH-80 and ETH-129 strains was observed. The Ser residue corresponding to Ser443 of the Met13 protein is located in the SAM-binding pocket of the crystal structures of hMTHFR and cMTHFR (Figure 1(b)). Therefore, a hypothesis was obtained that the inhibition of MTHFR activity via SAM is released by the Ser445Phe substitution of the Met13 protein, increasing the production of Met in yeast cells.

[0031] 3. Effect of Ser443Phe substitution on MTHFR activity To confirm the above hypothesis, using Escherichia coli cells, the Met13 protein (WT) and the Ser443Phe mutant Met13 recombinant protein were expressed and purified. Menadione was used as an artificial electron acceptor, and the NADPH-dependent reduction activity of the Ser443Phe mutant Met13 protein was measured. The specific activity of Ser443Phe mutant Met13 (127 U / mg) was approximately 2.9 times that of the inactivity of WT-Met13 (44 U / mg). Next, the sensitivity to SAM inhibition was evaluated. In the presence of SAM, the NADPH-dependent reduction activity of WT-Met13 was clearly inhibited, and when 2 mM SAM was added, the activity decreased to 25% compared with the case without SAM. On the other hand, the Ser443Phe mutant Met13 was unresponsive to SAM even in the presence of 2 mM SAM (Figure 3).

[0032] Studies on hMTHFR and cMTHFR have shown that the Ser residue corresponding to Ser443 in the Met13 protein is located near the inhibitor SAM (Froese et al., 2018: Non-Patent Literature 4; Yamada, Mendoza and Koutmos, 2024: Non-Patent Literature 5), suggesting that the Ser443Phe substitution affects the binding of SAM to the Met13 protein. To verify this, model structures of WT-Met13 protein and Ser443Phe mutant Met13 protein were constructed using cMTHFR bound to SAM as a template. In the model structure of WT-Met13 protein, the hydroxyl group of the side chain of Ser443 may interact with N3 of the adenine moiety of SAM (Figure 4(a)). On the other hand, the phenol side chain of Phe443 in the Ser443Phe mutant Met13 protein was predicted to cause steric hindrance with SAM, resulting in inhibition of SAM binding to Met13 and insensitivity to SAM inhibition (Figure 4(b)).

[0033] Furthermore, by comparing the model structures of the WT-Met13 protein constructed using cMTHFR without SAM with those of the Ser443Phe mutant Met13 protein, we investigated the effect of the Ser-to-Phe substitution at position 443 on catalytic activity (Figure 4(c), Figure 4(d)). As a result, it was found that the large phenol group of Phe443 acts as steric hindrance between Ile423, Ile426, and Leu430, altering the local structure of the Ser443Phe mutant Met13 protein. Therefore, it was considered that the change in local structure caused by the Ser-to-Phe substitution at position 443 increases the affinity for the substrate and / or cofactor, resulting in a higher specific activity of the Ser443Phe mutant Met13 protein compared to the WT-Met13 protein. Further biochemical and structural analysis is needed to elucidate the detailed mechanisms of the change in sensitivity to SAM inhibition and the change in catalytic properties of the Ser443Phe mutant Met13 protein.

[0034] 4. Effects of Ser443Phe substitution on intracellular Met production. The increased specific activity and decreased sensitivity to SAM inhibition of the Ser443Phe mutant Met13 protein indicate that expression of the mutant protein leads to high levels of Met production in yeast cells. Therefore, using monomer laboratory yeast, WT-Met13 protein and Ser443Phe mutant Met13 protein were expressed under constitutive GAPDH promoter conditions, and intracellular amino acid content was measured (Figure 5). Yeast cells expressing WT-Met13 protein (Met13 WT The intracellular Met content of the (strain) was similar to that of cells containing an empty vector (EV strain). Met13 WT In the strain, despite increased Met13 protein levels compared to the EV strain due to gene expression under a constitutive GAPDH promoter, the MTHFR activity of WT-Met13 protein was inhibited by intracellular SAM. On the other hand, yeast cells expressing the Ser443Phe mutant Met13 protein (Met13 S443F (Stocks) are EV stocks and Met13 WT Compared to the previous strain, the intracellular Met content increased 4.0 times. Also, Met13 S443F In this strain, the Ser443Phe mutant Met13 protein is expressed, resulting in EV strains possessing WT-Met13 protein and Met13 WT Compared to stocks, Gly increased 3.0 times and 2.1 times, respectively.

[0035] These results indicate that the Ser443Phe mutant Met13 protein is insensitive to inhibition by SAM, leading to increased availability of MTHF and enhanced folate cycle flow along with Gly production by SHMT. As a result, yeast cells expressing the Ser443Phe mutant Met13 protein (ETH-80 strain, ETH-129 strain in Figure 2(b) and Met13 in Figure 5) S443F Cells possessing the WT-Met13 protein (X2180-WT, EV strain and Met13) WT Compared to the previous strain, production of Met and Gly increased. Similarly, Met13 S443FThe Cys content of the stock is EV stock and Met13 WT Although it increased compared to the stock, the Asp content remained virtually unchanged (Figure 5). Also, EV stock and Met13 WT Met13 compared to stocks S443F The increase in Met content of the strain (4.0x) was lower than the increase in ETH-80 and ETH129 strains compared to X2180-WT strain (14x and 10x, respectively). MET13 S443F When the gene is expressed under a constitutive GAPDH promoter, Met13 S443F The amount of Met13 protein in the strain was increased compared to ETH-80 and ETH-129 strains. There are reports that yeast cells expressing a mutant protein with deregulated Met13 showed impaired growth compared to yeast cells containing WT-Met13 protein when cultured in minimal media with different sulfur sources. Therefore, the amount of Met13 protein in ETH-80 and ETH-129 strains was increased. S443F The phenotypic differences observed between strains may be due to the toxicity of the Ser443Phe mutant Met13 protein.

[0036] 5. Conclusion In this study, we isolated yeast mutant strains ETH-80 and ETH-129 that accumulate Met in cells using known mutagenesis methods, and identified a common MET13 mutant gene (MET13 Ser443Phe We identified the mutant Met13 protein. By substituting Ser443Phe, inhibition by SAM was released, improving Met production in yeast cells. Thus, this contributes to the development of yeast strains that can accumulate Met, and by supplementing essential amino acids that are lacking in daily diets, it may be possible to extend the healthy lifespan of the elderly and improve their quality of life. In this way, according to this embodiment, we were able to provide yeast that produces high levels of methionine using the mutant Met13 protein.

Claims

1. A mutant methylenetetrahydrofolate reductase protein (Met13 protein) having a Ser443Phe substitution in any of the amino acid sequences described in (a) to (c) below: (a) the amino acid sequence of Sequence ID No. 1, or an amino acid sequence having a Ser443Phe substitution in the amino acid sequence encoded by the MET13 gene of yeast belonging to the genus Saccharomyces; (b) an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence of (a) above, and which has the function of the Met13 protein; (c) an amino acid sequence having 90% or more sequence identity with the amino acid sequence of (a) above, and which has the function of the Met13 protein.

2. A nucleic acid encoding a mutant methylenetetrahydrofolate reductase protein (Met13 protein) having a Ser443Phe substitution in any of the following amino acid sequences (a) to (c): (a) the amino acid sequence of Sequence ID No. 1, or an amino acid sequence having a Ser443Phe substitution in the amino acid sequence encoded by the MET13 gene of yeast belonging to the genus Saccharomyces; (b) an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence of (a) above, and which has the function of the Met13 protein; (c) an amino acid sequence having 90% or more sequence identity with the amino acid sequence of (b) above, and which has the function of the Met13 protein.

3. The nucleic acid according to claim 2, wherein the nucleic acid is the one described in Sequence ID No.

2.

4. A plasmid comprising the nucleic acid described in claim 2, capable of expressing a protein having the function of the Met13 protein.

5. L-methionine-producing yeast comprising the mutant Met13 protein described in claim 1 in a form capable of expressing it.

6. Food or beverage containing the yeast described in claim 5.

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