Yeast that highly produces threonine due to mutant aspartate kinase HOM3

Abstract

To provide a method for producing threonine by using yeast that highly produces threonine due to mutant aspartate kinase Hom3.SOLUTION: Provided is a method for producing threonine by using yeast expressing mutant aspartate kinase Hom3 with Ala462Thr substitution of (a) to (c): (a) a specific amino acid sequence or an amino acid sequence which is encoded by the HOM3 gene of yeast belonging to the genus Saccaromyces, and has Ala462Thr substitution; (b) an amino acid sequence in which one or more amino acids are deleted, substituted, or added to the amino acid sequence of (a), and which has a function of Hom3; and (c) an amino acid sequence which has 90% or more sequence identity with the amino acid sequence of (a), the amino acid sequences of positions 452 to 461 and 463 to 472 of (a) matching the specific sequence and having the function of Hom3.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] The present invention relates to a yeast that produces a high level of threonine by using a mutant aspartate kinase Hom3. [Background technology]

[0002] Amino acids are fundamental molecules for living organisms as building blocks for protein synthesis. Mammals cannot synthesize nine of the 20 amino acids that make up proteins (essential amino acids), making these essential amino acids crucial for their nutrition. Recent studies have shown that decreased blood levels of essential amino acids are associated with the onset of dementia in individuals with mild cognitive impairment, suggesting that essential amino acids play an important role in maintaining brain homeostasis. Therefore, increasing daily intake of essential amino acids may slow the process of neurodegeneration. However, due to age-related appetite loss and fluctuations in the content of essential amino acids in food, it is difficult for elderly people to efficiently obtain essential amino acids from their daily diet. Yeast is a generally recognized safe organism and is widely used in the production of nutritional supplements. Therefore, yeast, which contains many essential amino acids, may help elderly people obtain adequate amounts of essential amino acids, thereby contributing to extending healthy lifespan and improving quality of life.

[0003] Of the nine essential amino acids, threonine is industrially produced using microorganisms and is widely used in food, feed, cosmetics, and pharmaceuticals. In yeast, threonine is biosynthesized from aspartate via homoserine through five enzymatic reactions. As shown in Figure 1(A), the threonine biosynthetic pathway branches from homoserine to methionine, and threonine is further converted to isoleucine. Aspartate kinase (AK), encoded by the HOM3 gene, catalyzes the first reaction in threonine biosynthesis, producing aspartate-4-phosphate by phosphorylating aspartate with ATP. The enzymatic activity of Hom3 is feedback inhibited by the end product, threonine, making this reaction the rate-limiting step in threonine biosynthesis in budding yeast.

[0004] As shown in Figure 1(B), the amino acid sequence map of Hom3 indicates that it consists of an N-terminal catalytic domain and two C-terminal regulatory domains (ACT-1 and ACT-2). The C-terminal regulatory domain exhibits the characteristic secondary structure of aspartate kinase, chorismate mutase, and TyrA (ACT) domains, which are widely involved in the allosteric regulation of amino acid metabolic enzymes. Studies on AKs in various microorganisms have revealed that the interaction of the two ACT domains forms a binding site for inhibitory molecules. Studies on yeast mutants that accumulate threonine have identified amino acid substitutions in the HOM3 gene that contribute to threonine overproduction, such as Glu279Ala and Glu282Asp substitutions in the catalytic domain, Ser399Phe substitution in the ACT-1 domain, and Gly452Asp substitution in the ACT-2 domain. Enzyme analysis revealed that these amino acid substitutions reduce sensitivity to feedback inhibition by threonine, leading to increased threonine production. However, analyses using Glu279Ala and Gly452Asp substitutions showed that these amino acid substitutions also affected the kinetic constants of the enzyme, resulting in a decrease in the catalytic activity of Hom3. This suggests that if there are amino acid substitutions that do not affect catalytic activity and are not subject to feedback inhibition by threonine, they would be advantageous for increasing threonine productivity in yeast (Non-Patent Documents 1, 2, 3). [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Sato H, TakadoY, Toyoda S, Tsukamoto-Yasui M, Minatohara K, Takuwa H, Urushihata T, TakahashiM, Shimojo M, Ono M, Maeda J, Orihara A, Sahara N, Aoki I, Karakawa S, IsokawaM, Kawasaki N, Kawasaki M, Ueno S, Kanda M, Nishimura M, Suzuki K, Mitsui A,Nagao K, Kitamura A, Higuchi M.: Neurodegenerative processes accelerated by protein malnutrition and decelerated by essential amino acids in a tauopathy mouse model, Science Advances, 7, eabd5046, 2021 [Non-patent document 2] BareichDC, Wright GD.: Functionally important acids amino acids in Saccharomyces cerevisiaeaspartate kinase, Biochemical and Biophysical Research Communications, 311, 597-603,2003 [Non-patent document 3] Marina P, Martinez-Costa OH, Calderon IL, Aragon JJ.: Characterization of the aspartatekinase from Saccharomyces cerevisiae and of its interaction with threonine, Biochemicaland Biophysical Research Communications, 321, 584-591, 2004 Summary of the Invention [Problem to be solved by the invention]

[0006] This study was conducted in light of the above-mentioned problems, and its purpose is to provide a yeast that produces high levels of threonine using mutant aspartate kinase Hom3. In this specification, aspartate kinase Hom3 is abbreviated as Hom3, with only the first letter capitalized. The Hom3 gene is abbreviated as HOM3. Furthermore, the aspartate kinase Hom3 of the present invention having the Ala462Thr substitution is abbreviated as mutant Hom3. Wild-type aspartate kinase Hom3 is abbreviated as wild-type Hom3. [Means for solving the problem]

[0007] The present inventors isolated a threonine-accumulating mutant from diploid laboratory yeast using conventional mutagenesis techniques and identified a novel amino acid substitution, Ala462Thr, in Hom3. This Ala462Thr mutant Hom3 was found to be immune to feedback inhibition by threonine, even in the presence of 50 mM Thr. The Ala462Thr amino acid substitution did not affect the catalytic activity of Hom3. Furthermore, expression of the Ala462Thr mutant Hom3 in yeast significantly increased the intracellular threonine content compared with yeast cells expressing wild-type Hom3. Thus, the method for producing threonine according to the present invention includes a method for producing threonine using a yeast in which a mutant aspartate kinase Hom3 having an Ala462Thr substitution and having the amino acid sequences (a) to (c) below has been expressed: (a) the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having an Ala462Thr substitution in the amino acid sequence of the HOM3 gene of yeast belonging to the genus Saccaromyces; (b) an amino acid sequence in which one or several amino acids are deleted, substituted, or added to the amino acid sequence of (a) above, and which has the function of Hom3; (c) An amino acid sequence that has 90% or more (preferably 95% or more) sequence identity with (a) above, and in which the amino acid sequences of positions 452-461 and 463-472 of (a) above match those of SEQ ID NO: 1, and that has the function of Hom3. SEQ ID NO: 1 is MPMDFQPTSSHSNWVVQKFGGTSVGKFPVQIVDDIVKHYSKPDGPNNNVAVVCSARSSYTKAEGTTSRLLKCCDLASQESEFQDIIEVIRQDHIDNADRFILNPALQAKLVDDTNKELELVKKYLNASKVLGEVSSRTVDLVMSCGEKLSCLFMTALCNDRGCKAKYVDLSHIVPSDFSASALDNSFYTFLVQALKEKLAPFVSAKERIVPVFTGFFGLVPT GLLNGVGRGYTDLCAALIAVAVNADELQVWKEVDGIFTADPRKVPEARLLDSVTPEEASELTYYGSEVIHPFTMEQVIRAKIPIRIKNVQNPLGNGTIIYPDNVAKKGESTPPH PPENLSSSFYEKRKRGATAITTKNDIFVINIHSNKKTLSHGFLAQIFTILDKYKLVVDLISTSEVHVSMALPIPDADSLKSLRQAEEKLRILGSVDITKKLSIVSLVGKHMKQYI GIAGTMFTTLTEEGINIEMIS QGANEINISCVINESDSIKALQCIHAKLLSERTNTSNQFEHAIDERLEQLKRLGI. Regarding (b), the upper limit of the number of amino acid residues to be substituted, deleted, inserted, and / or added is not particularly limited as long as the Hom3 function is maintained, and may be, for example, 7, 6, 5, 4, 3, 2, or 1. Regarding (c), the amino acid sequence identity is 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%. Sequence identity can be determined using homology searches such as FASTA, BLAST, and PSI-BLAST at online homology search sites, such as the DNA Data Bank of Japan (DDBJ). It can also be determined using BLAST at the National Center for Biotechnology Information (NCBI). "Having Hom3 function" refers to catalyzing the initial reaction of threonine biosynthesis, i.e., exhibiting activity to catalyze the phosphorylation of aspartate using ATP. Generally, Saccharomyces yeasts are known to biosynthesize threonine via a reaction catalyzed by Hom3. Therefore, it is believed that Hom3 functions in yeasts capable of threonine biosynthesis.

[0008] Yeast is a general term for fungi that are eukaryotic, unicellular microorganisms that are non-motile, have cell walls, lack photosynthetic ability, and decompose external organic matter and absorb it as nutrients. This term includes, for example, the genus Saccharomyces, a type of budding yeast that has been used in foods. Yeast is capable of alcoholic fermentation, which decomposes sugars into alcohol and carbon dioxide. In the present invention, examples of yeast include baker's yeast, sake yeast, wine yeast, brewer's yeast, soy sauce yeast, and bioethanol yeast. Both haploid and diploid yeasts can be used as yeast. Another aspect of the present invention provides a food or drink containing a yeast that has expressed aspartate kinase Hom3 with an Ala462Thr substitution and has the amino acid sequences (a) to (c) below: (a) the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence encoded by the HOM3 gene of a yeast belonging to the genus Saccaromyces, which has an Ala462Thr substitution; (b) an amino acid sequence in (a) above, in which one or several amino acids are deleted, substituted or added, and which has the function of Hom3; (c) An amino acid sequence that has 90% or more (preferably 95% or more) sequence identity with (a) above, and in which the amino acid sequences of positions 452-461 and 463-472 of (a) above match those of SEQ ID NO: 1, and that has the function of Hom3. Examples of food and beverage products include beverages (dairy-containing beverages, coffee, tea, juice, processed milk, sports drinks, etc.), bakery products (bread, pizza, pie, etc.), Western sweets (cookies, crackers, biscuits, cake, castella, etc.), noodles, pasta, snacks, confectionery products (candy, caramel, gum, chocolate, etc.), frozen desserts (ice cream, sorbet, etc.), dairy products (cream, cheese, mousse, powdered milk, condensed milk, dairy drinks, etc.), Western sweets (jelly, pudding, mousse, yogurt, buttercream, etc.), , custard cream, etc.), Japanese sweets (gyuhi, uriwara, mochi, ohagi, dorayaki, etc.), processed fruit and vegetable foods (jam, marmalade, preserved fruit in syrup, candied fruit, etc.), pastes (flower paste, fruit paste, peanut paste, etc.), seasonings (soy sauce, sauce, noodle soup, dashi stock, soup stock, etc.), frozen and refrigerated foods (ham, sausage, bacon, hamburger steak, meatballs, croquettes, gyoza, pilaf, rice balls, etc.), processed seafood foods (chikuwa, kamaboko, etc.), etc. Food and beverages are not limited to those for humans but also include those for pets (e.g., dogs, cats, etc.). Food and beverages also include health care products such as supplements.

[0009] Another aspect of the present invention provides a nucleic acid encoding aspartate kinase Hom3 having an Ala462Thr substitution and having the amino acid sequences (a) to (c) below: (a) a nucleic acid encoding the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence encoded by the HOM3 gene of a yeast belonging to the genus Saccaromyces, which has an Ala462Thr substitution; (b) a nucleic acid encoding an amino acid sequence in (a) above in which one or several amino acids have been deleted, substituted, or added, and which has the function of Hom3; (c) A nucleic acid that has a sequence identity of 90% or more (preferably 95% or more) with (a) above, and in which the amino acid sequences of positions 452-461 and 463-472 of (a) above match those of SEQ ID NO: 1, and encodes an amino acid sequence that has the function of Hom3. The genetic code encoding amino acids consists of 64 triplets (groups of three), and since there are 20 types of amino acids, multiple triplets correspond to the same amino acid. Therefore, the nucleic acid sequence specifying aspartate kinase Hom3 having an Ala462Thr substitution is not limited to one type. According to the present invention, a yeast capable of high threonine production can be provided by using a nucleic acid encoding aspartate kinase Hom3 having an Ala462Thr substitution.

[0010]

[0011] Another aspect of the present invention provides a yeast comprising a nucleic acid encoding the above-described nucleic acid in an expressible state, and which produces high levels of threonine using the mutant aspartate kinase Hom3. Examples of methods for producing the mutant Hom3 in an expressible state include incorporating the nucleic acid encoding the mutant Hom3 into an expression plasmid (which contains at least a promoter and a ribosome-binding sequence, and may also contain a terminator, an antibiotic resistance gene, an origin of replication, and Rop (a protein that limits copy number)) and then incorporating the resulting plasmid into yeast; incorporating the nucleic acid encoding the mutant Hom3 together with a promoter into a yeast chromosome; and converting wild-type Hom3 in yeast to the mutant Hom3 by homologous recombination. This yeast is highly productive of threonine, and can be used directly for the production of threonine. Furthermore, consuming the yeast itself allows for efficient intake of threonine. [Effects of the Invention]

[0012] According to the present invention, a yeast capable of producing a high amount of threonine by using a mutant aspartate kinase Hom3 can be provided. [Brief explanation of the drawings]

[0013] [Figure 1] (A) Diagram showing the threonine biosynthetic pathway, and (B) domain structure of the Hom3 protein. [Figure 2] Graphs showing the results of comparing the intracellular (A) threonine (Thr) content and (B) aspartic acid (Asp), isoleucine (Ile), and methionine (Met) contents when the parent strain (WT) and the mutant strain (HNV-5) were cultured. In the figure, "*" indicates a significant difference at a significance level of 5% (p<0.05). [Figure 3] 1 is a graph showing the results of an investigation into whether the activities of wild-type Hom3 (WT) and mutant Hom3 (A462T) are affected depending on the threonine (Thr) concentration. [Figure 4]Graphs showing the results of comparing the intracellular (A) threonine (Thr) content and (B) aspartic acid (Asp), isoleucine (Ile), and methionine (Met) contents of wild-type Hom3 (WT) and HOM3 gene-deficient haploid laboratory yeast strains (hom3Δ cells) containing the Ala462Thr mutant Hom3 (A462T). In the figure, "*" indicates a significant difference at a 5% level (p<0.05). [Figure 5] (A) Ribbon diagram of the homodimer structure of Hom3 when an inhibitor threonine (Inhibitor Thr) binds. (B) Schematic diagram showing the relationship between Ala462 and Gly452 in wild-type Hom3 (Ala462). (C) Schematic diagram showing the relationship between Thr462 and Gly452 in A462T mutant Hom3 (Thr462). DETAILED DESCRIPTION OF THE INVENTION

[0014] Next, embodiments of the present invention will be described with reference to the drawings. The technical scope of the present invention is not limited to these embodiments, and various embodiments can be implemented without departing from the spirit of the invention. <Test Method> 1. Strains and media The diploid laboratory yeast strain X2180 (wild-type (WT), MATa / α) and the haploid laboratory yeast strain BY4741 / hom3Δ (hom3Δ, MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 hom3::KanMX4) (Horizon Discovery) were used as parent strains for mutant breeding and host strains for HOM3 gene expression, respectively. YPD medium (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 acid and ammonium sulfate-free nitrogen base for yeast culture (Difco Laboratories)), 20 g / L glucose, and 5 g / L ammonium sulfate) were used for yeast culture. To isolate hydroxynorvaline (HNV)-resistant mutants, strain X2180 was cultured in SD+Alt medium (SD medium supplemented with 5 g / L allantoin instead of ammonium sulfate as the nitrogen source).

[0015] 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 Hom3 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 NaCl) containing the appropriate antibiotics was used. Unless otherwise stated, all chemicals were purchased from Wako Pure Chemical Industries, Ltd., Nacalai Tesque, and Sigma-Aldrich.

[0016] 2. Construction of an expression plasmid for the HOM3 gene The open reading frame (ORF) encoding wild-type Hom3 (HOM3 WT) and ORF encoding the Ala462Thr mutant Hom3 (HOM3 A462T ) was amplified from the genomic DNA of a yeast mutant strain (HNV-5 strain) that accumulates threonine by PCR using primers (5'-GGGAC AAG TTT GTA CAA AAA AGC AGG CTT AAT GCC AAT GGA TTT CCA ACC-3' (SEQ ID NO: 3) and 5'-AGG GGA CCA CTT TGT ACA AGA AAG CTG GGT GTT AAA TTC CAA GTC TTT TCA-3' (SEQ ID NO: 4)). The PCR-amplified DNA fragment was cloned into pDONR221 using BP clonase II to construct pDONR_HOM3. WT and pDONR_HOM3 A462T To construct an expression plasmid for the recombinant Hom3 protein, the ORF of the HOM3 gene was transferred to the pET53-dest expression vector (Thermo Scientific) using LR clonase II (Thermo Scientific), resulting in pET_HOM3. WT and pET_HOM3 A462T To construct a plasmid expressing the HOM3 gene under the constitutive glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter in yeast cells, the ORF of the HOM3 gene was transferred to the pAG415_GPD expression vector using the same procedure as above, resulting in pAG415_HOM3. WT and pAG415_HOM3 A462T Plasmid pRS415-CgHIS3MET15, which contains the LEU2, HIS3, and MET15 genes, was used to complement the auxotrophy of BY4741 / hom3Δ cells.

[0017] 3. Isolation of HNV-resistant mutants Mutations were introduced into the X2180 strain by treating it with 5.5% ethyl methanesulfonate (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 plated onto SD+Alt medium containing 5 mg / mL HNV. After 2 days of incubation at 30°C, the resulting colonies were harvested and evaluated for amino acid productivity. The survival rate of yeast cells after EMS treatment was approximately 40%.

[0018] 4. Quantification of Intracellular Amino Acid Content Yeast cells were cultured in SD+Am medium at 30°C for 2 days, then harvested by centrifugation and washed twice. The resulting yeast pellet was resuspended in sterile water and treated at 100°C for 20 minutes to extract the amino acids in the yeast cells. After removing the insoluble fraction by centrifugation, the amino acid content in the supernatant was quantified using an amino acid analyzer (JLC-500 / V2: JEOL). The content of each amino acid was expressed as μmol / g dry cell weight (DCW).

[0019] 5. Expression and Purification of Recombinant Hom3 Protein pET_HOM3 and pET_HOM3 A462T E. coli BL21(DE3) containing the Hom3 gene was cultured in TB medium containing ampicillin and grown at 37°C to an OD600 of 1.5. The culture was then cooled on ice for 5 min, and IPTG (isopropyl β-thiogalactopyranoside) was added to a final concentration of 0.2 mM. After 20 h of incubation at 18°C, the cells were harvested by centrifugation and suspended in Buffer A (50 mM HEPES-NaOH (pH 7.5), 150 mM NaCl, and 10% (w / v) glycerol). The cell suspension was sonicated under cooling and then centrifuged to remove the insoluble fraction. The supernatant was filtered through a 0.45 μm filter and then bound to a nickel affinity column (Ni Sepharose 6 Fast flow, GE Healthcare). The column was washed with Buffer A containing 100 mM imidazole, and the recombinant Hom3 protein was eluted with Buffer A supplemented with 500 mM imidazole.

[0020] 6. Measurement of AK Activity of Recombinant Hom3 Protein The AK activity of each protein was measured by quantifying the ADP produced by the Hom3-mediated oxidation of pyruvate kinase (PK) and lactate dehydrogenase (LDH). Specifically, activity was measured in a reaction solution (final volume 1 mL) containing 100 mM HEPES-NaOH (pH 7.5), 40 mM MgSO4, 10 mM KCl, 1 mM phosphoenolpyruvate, 0.25 mM NADH, 15 U PK / LDH, 0.5 μg purified Hom3, and various concentrations of aspartate and ATP. First, the reaction solution was prepared without aspartate and equilibrated at 30°C for 3 min. The reaction was then initiated by the addition of aspartate. The reaction temperature was maintained at 30°C, and the change in absorbance at 340 nm associated with the Hom3-dependent oxidation of NADH was measured using a DU-800 spectrophotometer (Beckman Coulter). The ATP concentration was fixed at 5 mM, and the aspartate concentration was varied in the range of 1.0 mM to 30 mM. The aspartate concentration was fixed at 20 mM, and the ATP concentration was varied in the range of 0.25 mM to 10 mM. To examine the sensitivity of Hom3 to feedback inhibition, the aspartate and ATP concentrations were fixed at 20 mM and 5 mM, respectively, and threonine was added to the reaction solution in the range of 1.0 mM to 30 mM. The initial reaction rate was determined by the molar extinction coefficient of NADH, 6,220 M -1 ·cm -1 The activity was calculated as follows: The activity unit was defined as the amount of enzyme required to produce 1 μmol of ADP per minute. The reaction kinetic parameters of each enzyme were calculated using nonlinear regression analysis with GraphPad Prism version 9 (GraphPad Software).

[0021] 7. Structural analysis of Hom3 protein A homology model of Saccharomyces cerevisiae aspartate kinase (Hom3, [UniProtKB accession no. P10869], homodimer) was downloaded from the SWISS-MODEL database. The template protein used for model construction was M. jannaschii aspartate kinase ([PDB ID no. 3C1N], 32% sequence identity to Hom3). The alanine-to-threonine 462 amino acid substitution was calculated using PyMOL software (PyMOL Molecular Graphics System version 2.5). The structural models of wild-type and Ala462Thr mutant Hom3 were drawn using Pymol software (http: / / www.pymol.org).

[0022] <Test Results> 1. Isolation of yeast mutants that accumulate threonine intracellularly First, we isolated yeast mutants resistant to HNV, a toxic analog of threonine. HNV, a structural analog of threonine, inhibits AK activity in the same way as threonine and is incorporated into proteins, altering the enzyme's properties and inhibiting yeast growth. Most of the toxicity of HNV can be reversed by accumulating intracellular threonine. Therefore, HNV-resistant yeast mutants are thought to produce large amounts of threonine intracellularly. Approximately 80 HNV-resistant mutants were obtained from diploid laboratory yeast X2180 mutated by EMS. Among the mutants, HNV-5 produced 4.4-fold more threonine (16.9 μmol / g-DCW) than the parent strain (3.8 μmol / g-DCW) (Figure 2(A)). The intracellular methionine (Met) content of the HNV-5 strain increased threefold compared with that of the parent strain, but the aspartic acid (Asp) and isoleucine (Ile) contents of the HNV-5 strain were almost equivalent to those of the parent strain (Fig. 2(B)).

[0023] 2. Identification of HOM3 gene mutations in HNV-5 strains Next, we performed whole-genome sequencing of the HNV-5 strain to identify the mutation responsible for the improved threonine productivity, which was found to have a heterozygous mutation in the HOM3 gene, resulting in an amino acid substitution of Ala462Thr at nucleotide 1384, changing guanine to adenine. 3. Effect of Ala462Thr substitution on AK activity of Hom3 To evaluate the effect of the Ala462Thr substitution on the AK activity of Hom3, wild-type Hom3 (WT) and the Ala462Thr mutant Hom3 (A462T) were expressed in E. coli and the recombinant Hom3 proteins were purified. First, the reaction kinetic parameters of the recombinant Hom3 were determined in the absence of threonine. The results are shown in Table 1.

[0024] [Table 1]

[0025] The kinetic constants (Km, kcat, and kcat / Km ratio) between wild-type and Ala462Thr mutant Hom3 were not significantly different, indicating that the Ala462Thr substitution does not affect the catalytic activity of Hom3, unlike known amino acid substitutions (Glu278Ala, Gly452Asp) that relieve threonine inhibition. Next, we evaluated the sensitivity of both proteins to feedback inhibition by threonine. The AK activity of wild-type Hom3 was inhibited by threonine in a concentration-dependent manner, with a half maximal inhibitory concentration (IC50) of 5.4 ± 0.12 mM. In contrast, the Ala462Thr mutant Hom3 was completely insensitive to threonine inhibition, even in the presence of 50 mM Thr (Figure 3).

[0026] 4. Effect of Ala462Thr substitution on amino acid productivity Next, we analyzed the effect of the Ala462Thr mutant Hom3 on yeast amino acid productivity. Wild-type and Ala462Thr mutant Hom3 were expressed under the constitutive GAPDH promoter in a HOM3-deficient haploid laboratory yeast strain (hom3Δ cells). When yeast cells were cultured in SD+Am minimal medium, the intracellular threonine content of hom3Δ cells expressing the Ala462Thr mutant Hom3 was 2.8-fold higher (113 μmol / g-DCW) than that of hom3Δ cells expressing wild-type Hom3 (39.9 μmol / g-DCW) (Figure 4(A)). Furthermore, the Ala462Thr mutant Hom3 cells showed a 0.6-fold decrease in Asp and a 1.8-fold increase in Ile, but no change in Met, compared with wild-type Hom3 cells (Figure 4(B)).

[0027] In this study, we isolated a mutant strain (HNV-5 strain) that accumulates threonine intracellularly by inducing mutations in yeast. We then isolated a mutation (HOM3) with an amino acid substitution of Ala462Thr introduced into the HOM3 gene of this strain. Ala462Thr The amino acid substitution of Ala462Thr abolished the sensitivity to feedback inhibition by threonine (Fig. 3), resulting in increased threonine production in yeast cells expressing this mutant Hom3 (Figs. 2 and 4).

[0028] The Ala462Thr substitution did not affect the kinetic constants of Hom3, unlike known amino acid substitutions that contribute to reduced sensitivity to threonine feedback inhibition (Table 1). Structural analyses of AK from various organisms have shown that inhibitor binding alters the enzyme's conformation, thereby reducing its phosphorylation activity.

[0029] The 462nd amino acid residue is highly conserved as alanine in yeast AK, and the amino acid residue corresponding to Ala462 is thought to be important for allosteric regulation depending on threonine. In the homodimer structure model of Hom3, Ala462 was located far from the threonine-binding site in the same monomer and did not interact with the threonine-binding site of the adjacent monomer (Figs. 5(A) and 5(B)). On the other hand, when Ala462 was replaced with Thr462, it was predicted that the hydroxyl group of the side chain of Thr462 would form a hydrogen bond with the amino group of the main chain of Gly452 located at the threonine-binding site in the adjacent monomer described above (Fig. 5(C)).

[0030] As one possibility, it was thought that such an interaction prevents the structural change induced by the binding of threonine, which is an inhibitor, and reduces the sensitivity to feedback inhibition by threonine. Also, Gly452 in Hom3 corresponds to the glycine residue that interacts with the inhibitor Thr in AK derived from M. jannaschii (PDB ID: 3C1N). Therefore, as another possibility, it is thought that the formation of the interaction between Thr462 and Gly452* in the Ala462Thr mutant changes the local structure of the inhibitor Thr-binding site and reduces the binding affinity for threonine. Thus, functional analysis of the Ala462Thr mutant of Hom3 found in the threonine-overproducing yeast strain HNV-5 revealed that it reduces the sensitivity to feedback inhibition by threonine and causes threonine overproduction in yeast cells. Since there was no change in the catalytic ability of Hom3 in this mutant, the threonine productivity using yeast can be improved.

[0031] <Wild-type Hom3 possessed by yeast of the Saccharomyces genus> Comparison of the wild-type Hom3 amino acid sequences for 45 yeast species belonging to the genus Saccharomyces revealed a high identity rate of 95% or more, as shown in Table 2. The first sequence accession number in the table, NP_010972.1, coincides with the amino acid sequence of Hom3 from the parent strain of this embodiment. For example, SEQ ID NOs: 5 and 6 show the amino acid sequence and nucleotide sequence of brewer's yeast Hom3 (QID845911.1).

[0032] [Table 2]

[0033] The amino acid sequence is SEQ ID NO: 5 (MDFHTTSSHSNWVVQKFGGTSVGKFPIQIVDDIVKYYSNPDGTNNDVAVVCSARSSYTKAEGTTSRLLKCCDLASQESEFQDIIEVIRQDHIDNADRFILNPALQTRLVDDTNKELELVKKYLNASKVLGEVSSRTVDLVMSCGEKLSCLFITALCNDRGCKAKYVDLTHIVPSDFSASALDNSFYTFLVQALKEKLVPFVSAKERIVPVFTGFFGL VPTGLLNGVGRGYTDLCAALIAVALNADELQVWKEVDGIFSADPRKVPEARLLDSVTPEEASELTYYGSEVIHPFTMEQVIRAKIPIRIKNVQNPLGNGTIIYPDNIAKKGESTPP HPPENLSSSFYEKRKRGATAITTKNDIFVINIHSNKKTLSHGFLAQIFTILDKFKLVVDLISTSEVHVSMALPIPDADSLKSLRQAEEKLKILGSVDVTKKLSIVSLVGKHMKQYI GIAGTMFTTLAEEGINIEMI SQGANEINISCVINETDSIKALQCIHAKLLGGRTSTPSQFEHAVDERLEQLKRLGI).

[0034]

[0035] In the Saccharomyces yeasts shown in Table 2, the amino acid sequence of wild-type Hom3 centered on the amino acid at position 462 matched the amino acid sequence of SEQ ID NO: 1 for 10 amino acids before and after (a total of 21 amino acids, including position 462) (excluding K at position 464 in XP_018222719.1 out of 45 Hom3s). (Furthermore, a further 20 amino acids before and after (a total of 41 amino acids, including position 462) matched the amino acid sequence of SEQ ID NO: 1 (excluding I at position 443 in AJP38320.1 and KHIL at positions 479-482 in CAI4420337.1). From this, it was considered that mutant Hom3 (especially mutant Hom3 from the genus Saccharomyces) in which 462 is Thr and the 20 amino acids at positions 452-461 and 463-472 match those of SEQ ID NO: 1 is highly likely to be able to produce high amounts of threonine, as in the above examples. These findings will contribute to the development of yeast strains that accumulate intracellular threonine, which is thought to extend healthy lifespan and improve the quality of life of elderly people by supplementing essential amino acids that are lacking in the daily diet. As described above, according to this embodiment, a yeast capable of highly producing threonine by using the mutant aspartate kinase Hom3 could be provided.

Claims

1. A method for producing threonine using a yeast expressing a mutant aspartate kinase Hom3 having an Ala462Thr substitution and having the amino acid sequence (a) to (c) below: (a) the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence encoded by the HOM3 gene of a yeast belonging to the genus Saccaromyces, which has an Ala462Thr substitution; (b) an amino acid sequence in which one or several amino acids are deleted, substituted, or added to the amino acid sequence of (a) above, and which has the function of Hom3; (c) An amino acid sequence that has 90% or more sequence identity with the amino acid sequence of (a) above, and the amino acid sequences of positions 452-461 and 463-472 of (a) above match SEQ ID NO: 1, and that has the function of Hom3.

2. A food or drink containing a yeast expressing a mutant aspartate kinase Hom3 having an Ala462Thr substitution and having the amino acid sequence (a) to (c) below: (a) the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence encoded by the HOM3 gene of a yeast belonging to the genus Saccaromyces, which has an Ala462Thr substitution; (b) an amino acid sequence in (a) above, in which one or several amino acids are deleted, substituted or added, and which has the function of Hom3; (c) An amino acid sequence that has 90% or more sequence identity with (a) above, and in which the amino acid sequences of positions 452-461 and 463-472 of (a) above match SEQ ID NO: 1, and that has the function of Hom3.

3. A nucleic acid encoding a mutant aspartate kinase Hom3 having an Ala462Thr substitution and having the amino acid sequence of any one of (a) to (c) below: (a) a nucleic acid encoding the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence encoded by the HOM3 gene of a yeast belonging to the genus Saccaromyces, which has an Ala462Thr substitution; (b) a nucleic acid encoding an amino acid sequence in (a) above in which one or several amino acids have been deleted, substituted, or added, and which has the function of Hom3; (c) A nucleic acid that has a sequence identity of 90% or more with (a) above, and in which the amino acid sequences of positions 452-461 and 463-472 of (a) above coincide with SEQ ID NO: 1, and encodes an amino acid sequence that has the function of Hom3.

4. A nucleic acid having a sequence set forth in SEQ ID NO:

2.

5. A yeast comprising a nucleic acid encoding the nucleic acid according to claim 3 or 4 in an expressible state, which produces a high level of threonine by the mutant aspartate kinase Hom3.

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