JP2015195771A - Protein having leucine acid production activity and use thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a protein having the activity of producing leucine acid from MOA, a transformant in which leucine acid production ability is enhanced, and use thereof.SOLUTION: This invention relates to a transformant that is transformed by the DNA encoding a protein having the activity of producing leucine acid from 4-methyl-2-oxopentanoate and is enhanced in leucine acid production ability.

Description

  The present specification relates to a protein having leucine acid production activity and use thereof.

  Ethyl leucine is known as a ginjo aroma component that is important for enhancing the aroma of sake. It has been reported that ethyl leucineate is produced by conversion of leucine in steamed rice, which is a raw material for sake, into leucine acid (α-hydroxy-isocaproic acid) by the koji mold and further yeast action. Ginjo incense greatly affects the quality of sake.

  Regarding the enhancement of ethyl leucineate, it is disclosed that strains for brewing sake belonging to the genus Aspergillus were screened to obtain strains producing 20 ppm or more of leucine acid (Patent Document 1). Moreover, ethyl leucineate high productivity Saccharomyces yeast is disclosed (patent document 2).

JP-A 63-52875 Japanese Patent Laid-Open No. 7-143871

  However, until now, the production route to leucine acid has not been clarified, and there has been no knowledge of the enzyme involved.

  The present specification provides a protein having leucine acid production activity and use thereof.

  The present inventors inferred the production pathway of leucine in Aspergillus oryzae (A. oryzae) as follows. That is, 4-methyl-2-oxopentanoic acid (MOA) is produced from leucine by an aminotransferase derived from an unknown gonococcus, and further MOA is reduced by an unknown MOA reductase. Inferred to be converted to It was also inferred that the leucine acid thus produced was then converted to ethyl leucine by the action of a lipase derived from yeast.

  In addition, it has been found that enzymes belonging to the 2-keto acid dehydrogenase family derived from lactic acid bacteria have MOA reductase activity. Therefore, the present inventors focused on 2-keto acid dehydrogenase derived from lactic acid bacteria, screened for proteins belonging to the 2-keto acid dehydrogenase family, and identified a dehydrogenase that produces leucine acid from MOA. According to the disclosure of the present specification, the following means are provided based on these findings.

(1) A transformant transformed with a DNA encoding the protein according to any one of the following (a) to (e) and having enhanced leucine acid-producing ability.
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 2 (b) having an amino acid sequence in which one or more amino acids in SEQ ID NO: 2 are substituted, deleted, inserted and / or added; A protein having an activity of producing leucine acid from methyl-2-oxopentanoic acid (c) having an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 2, A protein having an activity of producing leucine acid from oxopentanoic acid (d) a protein encoded by DNA consisting of the base sequence represented by SEQ ID NO: 1 (e) a base sequence represented by SEQ ID NO: 1 and 80% or more A tamper having an amino acid sequence encoded by an identical nucleotide sequence and having an activity of producing leucine acid from 4-methyl-2-oxopentanoic acid (2) The protein according to any one of the above (a) to (e) is further aligned with the amino acid sequence represented by SEQ ID NO: 2 in position 86 in the amino acid sequence represented by SEQ ID NO: 2. The site corresponding to is glycine (G), and the site corresponding to positions 159 to 164 is GXGXXG (each X is an arbitrary amino acid independently selected from natural amino acids), The site corresponding to position 242 is arginine (R), the site corresponding to position 271 is glutamic acid (E), and the site corresponding to position 289 is histidine (H). Transformant.
(3) The transformant according to (1) or (2), wherein the transformant is a eukaryotic microorganism.
(4) The transformant according to (3), wherein the eukaryotic microorganism is a koji mold.
(5) The transformant according to (4), wherein the koji mold is Aspergillus oryzae.
(6) The transformant according to (1) or (2), wherein the transformant is a prokaryotic microorganism.
(7) A recombinant vector for enhancing the ability to produce leucine acid, which retains a DNA encoding the protein according to any one of (a) to (e) below.
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 2 (b) having an amino acid sequence in which one or more amino acids in SEQ ID NO: 2 are substituted, deleted, inserted and / or added; A protein having an activity of producing leucine acid from methyl-2-oxopentanoic acid (c) having an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO: 2, A protein having an activity of producing leucine acid from oxopentanoic acid (d) a protein encoded by a DNA consisting of the base sequence represented by SEQ ID NO: 1 (e) a base sequence represented by SEQ ID NO: 1 and 90% or more A tamper having an amino acid sequence encoded by an identical nucleotide sequence and having an activity of producing leucine acid from 4-methyl-2-oxopentanoic acid Quality (8) below (a) ~ introducing a DNA encoding the protein of the host to any one of (e) comprises, a method of producing transformant leucine acid-producing ability is enhanced.
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 2 (b) having an amino acid sequence in which one or more amino acids in SEQ ID NO: 2 are substituted, deleted, inserted and / or added; A protein having an activity of producing leucine acid from methyl-2-oxopentanoic acid (c) having an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO: 2, A protein having an activity of producing leucine acid from oxopentanoic acid (d) a protein encoded by a DNA consisting of the base sequence represented by SEQ ID NO: 1 (e) a base sequence represented by SEQ ID NO: 1 and 90% or more A tamper having an amino acid sequence encoded by an identical nucleotide sequence and having an activity of producing leucine acid from 4-methyl-2-oxopentanoic acid The transformant according to any one of the quality (9) (1) to (6), comprising the step of culturing under conditions where the 4-methyl-2-oxo-pentanoic acid may be present, the method of producing leucine acid.
(10) A step of culturing the transformant according to any one of (1) to (6) under conditions where 4-methyl-2-oxopentanoic acid may be present to produce leucine acid,
Converting the leucine acid to ethyl leucine;
A process for producing ethyl leucine.
(11) A method for producing a koji-containing material, comprising a step of culturing koji mold that is the transformant according to any one of (1) to (6).
(12) A method for producing alcohol, comprising a step of culturing koji mold and yeast that are transformants according to any one of (1) to (6).
(13) A screening method for a leucine high productivity mutant, wherein the test mutant is a protein represented by any one of the following (a) to (e) or a polynucleotide encoding the protein: Screening for leucine high productivity mutants,
A method comprising:
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 2 (b) having an amino acid sequence in which one or more amino acids in SEQ ID NO: 2 are substituted, deleted, inserted and / or added; A protein having an activity of producing leucine acid from methyl-2-oxopentanoic acid (c) having an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO: 2, A protein having an activity of producing leucine acid from oxopentanoic acid (d) a protein encoded by a DNA consisting of the base sequence represented by SEQ ID NO: 1 (e) a base sequence represented by SEQ ID NO: 1 and 90% or more A tamper having an amino acid sequence encoded by an identical nucleotide sequence and having an activity of producing leucine acid from 4-methyl-2-oxopentanoic acid (14) A screening method for leucine acid-producing enzyme, comprising the step of evaluating the activity of producing leucine acid from 4-methyl-2-oxopentanoic acid of a test protein having 2-keto acid dehydrogenase activity, Screening method.
(15) A method for producing leucine acid,
A step of producing leucine acid from 4-methyl-2-oxopentanoic acid using the protein represented by any of the following (a) to (e):
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 2 (b) having an amino acid sequence in which one or more amino acids in SEQ ID NO: 2 are substituted, deleted, inserted and / or added; A protein having an activity of producing leucine acid from methyl-2-oxopentanoic acid (c) having an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO: 2, A protein having an activity of producing leucine acid from oxopentanoic acid (d) a protein encoded by a DNA consisting of the base sequence represented by SEQ ID NO: 1 (e) a base sequence represented by SEQ ID NO: 1 and 90% or more A tamper having an amino acid sequence encoded by an identical nucleotide sequence and having an activity of producing leucine acid from 4-methyl-2-oxopentanoic acid (16) A method for producing ethyl leucine acid, wherein leucine acid is produced from 4-methyl-2-oxopentanoic acid using a protein represented by any of the following (a) to (e): A step of esterifying the leucine acid obtained in the step into ethyl leucineate,
A production method comprising:
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 2 (b) having an amino acid sequence in which one or more amino acids in SEQ ID NO: 2 are substituted, deleted, inserted and / or added; A protein having an activity of producing leucine acid from methyl-2-oxopentanoic acid (c) having an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO: 2, A protein having an activity of producing leucine acid from oxopentanoic acid (d) a protein encoded by a DNA consisting of the base sequence represented by SEQ ID NO: 1 (e) a base sequence represented by SEQ ID NO: 1 and 90% or more A tamper having an amino acid sequence encoded by an identical nucleotide sequence and having an activity of producing leucine acid from 4-methyl-2-oxopentanoic acid Quality

2 shows a molecular phylogenetic tree of the 2-keto acid dehydrogenase family. The result of SDS-PAGE about 2-HADH4 is shown. The calculation results of Michaelis-Menten constant: K _m value, maximum reaction rate: V _max value, and rotation speed: k _cat value for each substrate of 2-HADH4 are shown. The GC chromatogram of the reaction product of MOA by 2-HADH4 and the mass fragment pattern of the reaction product of MOA by 2-HADH4 are shown. The result of the electrophoresis of the wild type strain and the transformed strain transformed with the 2-HADH4 gene is shown. The comparison of the expression level of 2-HADH4 in the mRNA level of the wild strain and the transformed strain transformed with the 2-HADH4 gene is shown. The comparison of the leucine acid production amount of the wild type strain and the transformed strain transformed with the 2-HADH4 gene is shown. The result of the expression analysis of 2-HADH4 when leucine, MOA, or leucine acid is added to a culture medium is shown.

  The present specification relates to a protein having an activity of producing leucine acid from MOA (hereinafter also simply referred to as leucine acid production activity) and use thereof. According to the protein having leucine acid-producing activity disclosed herein (hereinafter also simply referred to as this protein), leucine and ethyl leucine, which are the raw materials for ginjoka leucine acid, are efficiently obtained by genetic engineering techniques. Can be produced.

  For example, a transformant with enhanced leucine acid-producing ability can be obtained by using DNA encoding this protein. By using such a transformant or the present protein, leucine acid useful as a raw material for the ginjo aroma component can be produced. In addition, by using the transformant or the produced leucine acid itself, ethyl leucineate as a ginjo scent component can be produced efficiently. In particular, by using a transformant, which is a koji mold, in a sake production process, it becomes possible to produce sake with enhanced Ginjoka leucine acid.

  Leucic acid is also a metabolite of leucine in vivo and has been reported to suppress muscle fatigue and atrophy after exercise (Am J Physiol Endocrinol Metab 305: E416-E428, 2013). It is also useful.

  Hereinafter, regarding the disclosure of the present specification, the present protein, DNA encoding the present protein, transformants and production methods thereof, recombinant vectors, and the like will be sequentially described.

(This protein)
This protein has an activity of producing leucine acid from MOA (hereinafter also referred to as leucine acid production activity). The MOA production activity of this protein means the activity of catalyzing the following reaction.
MOA + NADPH → Leucic acid + NADP ⁺

  One embodiment of this protein is a protein consisting of the amino acid sequence represented by SEQ ID NO: 2. The protein consisting of the amino acid sequence represented by SEQ ID NO: 2 is 2-hydroxyacid dehydrogenase (2-HADH) derived from A. oryzae. As a result of extensive searches for the 2-HADA family, GRHPR (glycol) It is selected from proteins belonging to oxylate reductase / hydroxypyruvate reductase (GRHPR (glyoxylate reductase / hydroxyl pyruvate reductase)).

  The protein comprising the amino acid sequence represented by SEQ ID NO: 2 is particularly useful as the present protein because it is derived from A. oryzae used in various fermented foods including Japanese sake.

  As another aspect of this protein, the amino acid sequence represented by SEQ ID NO: 2 has an amino acid sequence in which one or more amino acids are substituted, deleted, inserted and / or added, and has leucine acid production activity. The protein which has. Any one of amino acid mutations relative to the amino acid sequence represented by SEQ ID NO: 2, ie, substitution, deletion, insertion and addition may be used, or two or more may be combined. Further, the total number of these mutations is not particularly limited, but is preferably 1 or more and 30 or less, more preferably 1 or more and 20 or less, and further preferably 1 or more and 15 or less, more More preferably, it is about 1 or more and 10 or less. Most preferably, it is 1 or more and 5 or less.

  As an example of amino acid substitution, conservative substitution is preferable, and specific examples include substitution within the following groups. (Glycine, alanine) (valine, isoleucine, leucine) (aspartic acid, glutamic acid) (asparagine, glutamine) (serine, threonine) (lysine, arginine) (phenylalanine, tyrosine).

Leucic acid production activity can be evaluated as follows, for example. By adding MOA as a substrate and NADPH as a coenzyme to the protein of interest and measuring leucine acid and / or NADP ⁺ produced by the reduction reaction, or by measuring the decrease in NADPH, leucine acid Productive activity can be measured. Preferably, the decrease amount of NADPH is measured by a visible light absorption method. Measuring the amount of NADPH decrease and measuring enzyme activity is a technique well known to those skilled in the art.

  The phrase “having leucine acid production activity” is sufficient if it has leucine acid production activity, and the degree thereof is not limited. Preferably, it is 50% or more of the leucine acid production activity of the protein consisting of the amino acid sequence represented by SEQ ID NO: 2, more preferably 60% or more, still more preferably 70% or more, and still more preferably 80%. % Or more, still more preferably 90% or more, more preferably 95% or more, and most preferably 100% or more.

  Still another embodiment of the present protein includes a protein having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 2 and having leucine acid production activity. More preferably, the identity is 85% or more, further 90% or more, still more preferably 95% or more, still more preferably 96% or more, still more preferably 97% or more, and much more. Preferably it is 98% or more, More preferably, it is 99% or more.

  As used herein, identity or similarity is a relationship between two or more proteins or two or more polynucleotides determined by comparing sequences, as is known in the art. “Identity” in the art refers to a protein or polynucleotide sequence as determined by alignment between protein or polynucleotide sequences, or in some cases by alignment between a series of such sequences. Means the degree of sequence invariance between. Similarity is also the correlation between protein or polynucleotide sequences, as determined by alignment between protein or polynucleotide sequences, or in some cases by alignment between a series of partial sequences. Means the degree of More specifically, it is determined by sequence identity and conservation (substitutions that maintain specific amino acids in the sequence or physicochemical properties in the sequence). The similarity is referred to as “Similarity” in the sequence homology search result of BLAST described later. The method for determining identity and similarity is preferably a method designed to align the longest between the sequences to be compared. Methods for determining identity and similarity are provided as programs available to the public. For example, BLAST (Basic Local Alignment Search Tool) program by Altschul et al. (Eg Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ., J. Mol. Biol., 215: p403-410 (1990), Altschyl SF, Madden TL, Schaffer AA, Zhang J, Miller W, Lipman DJ., Nucleic Acids Res. 25: p3389-3402 (1997)). The conditions for using software such as BLAST are not particularly limited, but it is preferable to use default values.

  In the present protein, when aligned with the amino acid sequence represented by SEQ ID NO: 2, the site corresponding to position 86 in the amino acid sequence represented by SEQ ID NO: 2 is glycine (G), and positions 159 to The site corresponding to position 164 is GXGXXXG (each X is an arbitrary amino acid independently selected from natural amino acids), the site corresponding to position 242 is arginine (R), and 271 The site corresponding to the position is preferably glutamic acid (E), and the site corresponding to position 289 is preferably histidine (H). Sites corresponding to positions 159 to 164 are known as NAD binding motifs and are motifs that are widely conserved in 2-HADH. In addition, amino acids at other specific sites are amino acids that are widely conserved in 2-HADH as substrate binding sites and the like. By having these amino acids and motifs, it can have activity as 2-HADH. The alignment can be performed as described above.

  This protein is also a protein encoded by DNA consisting of the base sequence represented by SEQ ID NO: 1. The base sequence represented by SEQ ID NO: 1 encodes 2-HADH derived from A. oryzae. Furthermore, as another aspect of the present protein, a protein having an amino acid sequence encoded by a base sequence having 80% or more identity with the base sequence represented by SEQ ID NO: 1 and having leucine acid production activity can be mentioned. . More preferably, the identity is 85% or more, further 90% or more, still more preferably 95% or more, still more preferably 96% or more, still more preferably 97% or more, and much more. Preferably it is 98% or more, More preferably, it is 99% or more.

  A protein having an amino acid sequence having a certain identity or higher with the amino acid sequence represented by SEQ ID NO: 2 or an amino acid sequence encoded by a base sequence having a certain identity or more with the base sequence represented by SEQ ID NO: 1 As the protein, for example, a protein derived from Aspergillus flavus (accession number: XP_001826015), a protein derived from Aspergillus clavatus (accession number XP_001268820), a protein derived from Aspergillus fumigattus (accession number: XP_752810) (accession number) : XP_001210726), proteins derived from Aspergillus nidulans (accession number: XP_658379).

  This protein can be synthesized chemically or genetically engineered based on, for example, amino acid sequence information or base sequence information described later. A person skilled in the art can obtain a protein based on the sequence information.

  In addition, DNAs encoding the proteins of the above various aspects (hereinafter referred to as the present DNA) include, for example, DNA extracted from Aspergillus or the like using primers designed based on the base sequence represented by SEQ ID NO: 1, A nucleic acid fragment can be obtained by performing PCR amplification using a nucleic acid derived from a cDNA library or a genomic DNA library as a template. Moreover, it can obtain as a nucleic acid fragment by performing hybridization using a nucleic acid derived from the above-mentioned library or the like as a template and a DNA fragment which is a part of the present DNA as a probe. Alternatively, the present DNA may be synthesized as a nucleic acid fragment by various nucleic acid sequence synthesis methods known in the art such as chemical synthesis methods.

  In addition, the present DNA is obtained by, for example, converting a DNA encoding the amino acid sequence represented by SEQ ID NO: 2 (for example, comprising the base sequence represented by SEQ ID NO: 1) into a conventional mutagenesis method or site-specific mutation. It can be obtained by modification by a molecular evolution method using a method, error-prone PCR, or the like. As such a technique, a known technique such as the Kunkel method or the Gapped duplex method or a method similar thereto can be mentioned. For example, a mutation introduction kit using site-directed mutagenesis (for example, Mutant-K (manufactured by TAKARA) ) Or Mutant-G (manufactured by TAKARA)) or the like, or a TAKARA LA PCR in vitro Mutagenesis series kit.

  In addition, those skilled in the art should refer to Molecular Cloning (Sambrook, J. et al., Molecular Cloning: a Laboratory Manual 2nd ed., Cold Spring Harbor Laboratory Press, 10 Skyline Drive Plainview, NY (1989)). Thus, for example, the present DNA can be obtained based on a known sequence such as SEQ ID NO: 1 or 2.

  Note that the amino acid sequence represented by SEQ ID NO: 2 or the base sequence encoding the amino acid sequence having a certain relationship with the amino acid sequence as described above does not change the amino acid sequence of the protein in accordance with the degeneracy of the genetic code. At least one base of a base sequence encoding a predetermined amino acid sequence can be substituted with another kind of base. Therefore, this DNA also includes DNA encoding a base sequence converted by substitution based on the degeneracy of the genetic code.

(Transformant)
One aspect of the transformant disclosed herein (hereinafter referred to as the present transformant) is a cell transformed with a DNA encoding this protein. This transformant is useful not only as a production source of this protein, but also as a leucine acid producer in the presence of leucine or MOA. Furthermore, it is also useful as a production material for ethyl leucineate.

(Host)
The host of this transformant should just be a host cell which can express this protein, and the kind is not limited. The transformant may be, for example, a eukaryotic microorganism or a prokaryotic microorganism, but is preferably a eukaryotic microorganism. Examples of prokaryotic microorganisms include bacteria such as Escherichia coli, actinomycetes such as Streptomyces, and cyanobacteria such as Microcystis aeruginosa.

  Examples of eukaryotic microorganisms include koji molds, yeasts, fungi, and algae. Yeast and bacilli are preferred. Aspergillus oryzae, Aspergillus aculeatus, Aspergillus niger, Aspergillus nidulans, Aspergillus soya, Aspergillus kawai, Aspergillus kawai Examples thereof include Aspergillus awamori and Aspergillus saitoi.

  As the eukaryotic microorganism, A. oryzae is preferable, particularly a koji mold commonly used for fermentation such as sake. From these hosts, the present DNA can be obtained, and industrially more useful koji molds can be obtained by introducing the present DNA into the eukaryotic microorganism so as to enhance expression. In addition, gonococci such as A. oryzae have the ability to produce MOA from leucine, so this transformant, which is a gonococcus, produces leucine acid by culturing it using rice-derived materials. Can do. In addition, since this protein is derived from A. oryzae, it is self-cloning when introduced into A. oryzae, which is significant.

  Examples of yeast include yeasts of the genus Saccharomyces cerevisiae such as Saccharomyces cerevisiae, yeasts of the genus Schizosaccharomyces pombe such as Schizosaccharomyces pombe, and genus Candida such as Candida shehatae. Yeast, yeast of the genus Pichia such as Pichia stipitis, yeast of the genus Hansenula, yeast of the genus Trichosporon, yeast of the genus Brettanomyces, yeast of the genus Pachysolen, Yamadajima ( Examples include yeasts of the genus Yamadazyma, yeasts of the genus Kluyveromyces such as Kluyveromyces marxianus and Kluveromyces lactis. Of these, Saccharomyces yeasts are preferred from the viewpoints of industrial availability and the like, and from the viewpoint of being widely used for fermentation of sake and the like. Of these, Saccharomyces cerevisiae is preferable. Yeasts such as S. cerevisiae can convert leucine acid to leuco acid ethyl leucine, which is a ginjo fragrance component. Therefore, by imparting the ability to produce leucine acid to yeast, at least the yeast can synthesize leucine acid from MOA and, by extension, ethyl leucine acid.

  The present transformant is not particularly limited as long as it retains the present DNA so that it can be expressed. The present DNA may be retained outside the host genome, but is preferably retained on the host genome. The present DNA may be provided so that it can be inducibly expressed, or may be provided so that it can be constitutively expressed. However, from the viewpoint of enhancing the production of this protein and the ability to produce leucine acid, preferably it can be expressed constitutively Is provided.

  The transformant preferably has enhanced leucine production ability. The fact that the leucine production ability is enhanced includes both the case where the leucine production ability is newly imparted and the case where the originally provided leucine production ability is enhanced. For example, in transformants such as A. oryzae, for example, 10 times or more, more preferably 30 times or more, still more preferably 50 times or more, more preferably 70 times or more, and more preferably, compared to the wild type. It has a leucine producing ability of 90 times or more, more preferably 100 times or more.

  The ability of this transformant to produce leucine acid can be evaluated based on, for example, the amount of leucine acid produced in addition to the transcription product (mRNA) and translation product (protein) of the present DNA by the transformant.

(Recombinant vector)
The recombinant vector disclosed herein (hereinafter referred to as the present vector) can retain the DNA encoding the protein. The present recombinant vector is also an expression vector for enhancing the ability to produce leucine acid by expressing the present DNA. Recombinant vectors and their construction methods are well known to those skilled in the art and are disclosed in Molecular Cloning 3rd Edition, Current Protocols in Molecular Biology and the like. In addition, the form of a vector can take various forms according to a usage form. For example, it can take the form of a DNA fragment, and can also take the form of a plasmid.

  This vector can be provided with various necessary elements depending on the host and known necessary elements for enabling expression of the present DNA in addition to various elements depending on the vector medium. That is, in addition to various regulatory regions such as promoters and terminators, homologous recombination regions and the like can be provided when introduced into the host genome. In addition, a marker gene can be provided to select recombinants.

  The promoter used in the vector is not particularly limited, and can be appropriately selected from known promoters depending on the host and the expression form. For example, when the host is Neisseria gonorrhoeae, the tef1 promoter, enolase promoter, ADH1 promoter, phosphoglycerate kinase (PGK) promoter, α-amylase promoter, glucoamylase promoter, cellulase promoter, cellobiohydrase promoter, acetamidase promoter, etc. Or the like can be used. In order to enhance leucine production ability, a promoter capable of constitutively expressing a gene under control is preferably used.

(Production method of this transformant)
The production method of the present transformant can include a step of introducing the present DNA into a host. Specifically, this vector is introduced into a host and transformed. In the method for producing the transformant, various aspects of the vector and the host described above can be applied. Methods for producing transformants themselves are well known to those skilled in the art, and those skilled in the art can implement this step by appropriately referring to the above-mentioned documents and the like according to the type of host. Typically, various conventionally known methods such as transformation method, transfection method, conjugation method, protoplast method, electroporation method, lipofection method, lithium acetate method and the like can be used.

(Leucic acid production method)
The method for producing leucine acid disclosed in the present specification can include a step of culturing the transformant under conditions where MOA can exist as a substrate. According to this production method, the production amount of leucine acid can be increased by the ability of this transformant to produce leucine acid. Leucic acid is useful in itself and can be used as a raw material for ethyl leucineate.

  The culture process can be appropriately set according to the type of host of the transformant. When this transformant is koji mold, koji mold is considered to be able to produce leucine from the protein in the medium as a raw material and further produce leucine acid via MOA. Accordingly, leucine acid can be produced by culturing appropriately using a raw material containing protein such as rice.

  When the transformant is other than koji molds such as yeast, it can be co-cultured with a microorganism capable of producing MOA from leucine in the same manner as koji molds or koji molds. Further, the transformant medium may contain MOA as a substrate for this protein, or may contain a culture supernatant of Neisseria gonorrhoeae or similar microorganisms.

  Whether the transformant is a koji mold or not, for efficient production of leucine acid, if necessary, a fermentation raw material containing a protein (polypeptide) containing leucine as an amino acid, Preferably, the medium contains leucine and / or MOA.

  The culture step can be performed under appropriate conditions in consideration of the host. The culture can be stationary culture, shaking culture, aeration-agitation culture, or the like. The aeration conditions can be appropriately selected from anaerobic conditions, microaerobic conditions, aerobic conditions, and the like. For example, if the host is an aerobic bacterium such as Neisseria gonorrhoeae or membrane-producing yeast, aerobic conditions can be selected. The culture temperature is not particularly limited, and can be in the range of 25 ° C. to 55 ° C. For example, if the host is a koji mold, yeast or the like, it can be selected from 20 ° C. to 40 ° C., more preferably from 25 ° C. to 35 ° C. Moreover, although culture | cultivation time is also set as needed, it can be set as about 6 hours or more and 150 hours or less. For example, if the host is a koji mold, the culture time can be 24 hours or more and 120 hours or less. The pH can be adjusted using an inorganic or organic acid, an alkaline solution, or the like. For example, if the host is gonococcus, yeast or the like, it can be 4.0 or more and 8.0 or less. During the culture, antibiotics such as ampicillin and tetracycline can be added to the medium as necessary. In addition, after completion | finish of a culture | cultivation process, you may implement the process which removes microorganisms from a culture solution, collect | recovers a leucine-acid containing fraction, and also concentrates this.

  The production method of leucine acid disclosed in the present specification may include a step of producing leucine acid from MOA under conditions where MOA can exist as a substrate, using the present protein, not as a culture step. Good. You may perform this process simultaneously with the process of culture | cultivating this transformant demonstrated previously and producing a leucine acid. That is, leucine acid may be produced from MOA using this protein in a medium while producing leucine acid from MOA in the present transformant.

  In this production process, the present protein obtained by any method can be allowed to act on MOA as a substrate. This protein may be extracted from wild-type A. oryzae or obtained from this transformant. MOA as a substrate is preferably added as MOA. In this production process, it is preferable to produce leucine acid under the optimum conditions of this protein. As such conditions, for example, the pH can be set to 4.0 or more and 8.0 or less, for example. About temperature, it can be 20 degreeC or more and 40 degrees C or less, for example, More preferably, it can be 25 degreeC or more and 35 degrees C or less. About salt concentration, it can be 0.6 mass% or more and 0.9 mass% or less.

(Method for producing ethyl leucineate)
The production method of ethyl leucine disclosed in the present specification can comprise a step of producing leucine acid from MOA and a step of converting leucine acid into ethyl leucineate. According to this production method, ethyl leucine can be obtained by efficiently converting leucine with this transformant. Ethyl leucine is useful as a ginjo aroma component for sake.

  As described above, the leucine acid production process may be a process of culturing the present transformant under conditions where MOA can exist as a substrate, and MOA exists as a substrate regardless of the microorganism. It may be a step of allowing the present protein to act under possible conditions, or a combination thereof. In addition, even when production of ethyl leucine is intended, it is preferable to include leucine and / or MOA in the medium as necessary for efficient production of ethyl leucine.

  The step of converting leucine acid to ethyl leumate is a step of culturing a microorganism such as yeast that expresses esterase or lipase that can catalyze the conversion reaction in the presence of leucine acid obtained in the leucine acid production step. Alternatively, it may be a step in which these enzymes are allowed to act without depending on microorganisms, or a combination thereof. In addition, esterases and lipases expressed by microorganisms may act on leucine acid in the microbial cell, or may act on leucine acid outside the microbial cell.

  As such lipases, lipases derived from yeast such as S. cerevisiae are known. The esterase and lipase that catalyze the reaction can be easily found by screening or the like. Examples thereof include acarboxyl esterase, reel esterase, triacylglycerol lipase, phospholipase A2, lysophospholipase, acetyl esterase, acetylcholinesterase, cholinesterase and the like. Moreover, it does not specifically limit as reaction conditions, The conditions normally applied to these enzymes are employable.

  The leucine acid production step and the step of converting to ethyl leumate can be performed separately, but from the viewpoint of efficiency, it is preferable to perform them simultaneously, that is, as one step. In that case, either or both of the leucine production step by the transformant and the leucine production step by the protein as the leucine production step, and the conversion step by the lipase-expressing microorganism such as yeast, the lipase, etc. Either or both of the conversion steps can be appropriately combined. When performing the leucine acid production step and the conversion step to ethyl leucine in one step, the culture conditions, enzyme reaction conditions, and the like are appropriately set.

(Method for producing straw-containing material)
The method for producing a koji-containing material disclosed in the present specification can include a step of culturing the present transformant that is a koji mold. According to this production method, it is possible to obtain a koji-containing material with enhanced productivity of ethyl leucine such as seed koji or koji.

  As the koji mold, those already described can be mentioned. Further, it is appropriately selected from yellow koji mold, white koji mold, and black koji mold depending on the application. As a raw material used in the step of culturing the present transformant, which is a koji mold, a material used for producing known koji or seed koji can be appropriately selected. For example, rice, wheat, soybeans, etc. that have been appropriately heat-treated can be mentioned. A method for producing a koji-containing material is conventionally known, and those skilled in the art can obtain a koji-containing material such as seed koji or koji by appropriately applying a known production process to the transformant.

  The culturing step may be a step of culturing yeast, lactic acid bacteria, and the like simultaneously. In addition, leucine or MOA can also be included in a culture raw material as needed.

  According to the disclosure of the present specification, brewed foods such as miso, soy sauce, sake (sake), mirin, etc., and koji seasonings can be further produced using such koji-containing materials. In addition, cosmetics and their raw materials can be produced. In particular, such a koji-containing material is useful as a koji or seed koji for sake because it is suitable for the production of ethyl leucineate, a ginjo aroma of sake. Examples of sake include various types of sake made mainly from rice, as well as distilled spirits such as shochu and awamori. Of these, sake is preferred.

(Alcohol production method)
The method for producing alcohol disclosed in the present specification can include a step of culturing the present transformant, which is a koji mold, and yeast, and subjecting the yeast to alcohol fermentation. According to this production method, it is possible to produce an alcohol having a high content of ethyl leucine, which is a ginjo aroma component. The alcohol production method is applied to so-called various types of sake, and is preferably sake made mainly from rice.

  The culture process in this production method may include a process corresponding to the type of alcohol in addition to the culture process, using the koji-containing material. For example, a method for producing sake can include the following steps. Prior to the culturing step, a rice milling process, a rice steaming process, a manufacturing process of the koji-containing material already described, and the like can be provided. Moreover, after the culture | cultivation process, the process of squeezing the solid substance (moromi) produced by culture | cultivation and solid-separating, and the process of pasteurizing may be provided. In the culture step of the production method, leucine or MOA may be included in the raw material.

  Alcohol containing ethyl leucine obtained by this production method itself constitutes a liquor suitable for drinking as an alcohol, and is also used as a raw material for ginjo aroma to add ginjo aroma to other brewed foods such as sake. You can also

  In addition, leucine acid can be obtained by culturing the transformant under conditions where MOA can be present. However, alcohol other than sake can be produced by co-culturing the transformant and yeast capable of alcohol fermentation. can do.

(Screening method of leucine high productivity mutant)
The screening method for a leucine high productivity mutant disclosed in the present specification comprises a step of screening a leucine high productivity mutant for the test mutant using the present protein or a polynucleotide encoding the present protein as an index. Can do.

  The DNA introduced into the test mutant is preferably a DNA encoding a protein having 2-keto acid dehydrogenase activity. The protein having 2-keto acid dehydrogenase activity is a protein having glyoxylate reductase / hydroxypyruvate reductase (GRHPR (glyoxylate reductase / hydroxyl pyruvate reductase)) activity, D-lactate dehydrogenase (D-LDH (lactate dehydrogenase)) activity A protein having formate dehydrogenase (FDH (formate dehydrogenase)) activity, a protein having D-malate dehydrogenase (D-MDH (D-malate dehydrogenase)) activity, D-3-phosphoglycerate dehydrogenase (3- It includes proteins having PGDH (D-3-phosphoglycerate dehydrogenase) activity. More preferably, the DNA introduced into the test mutant is a DNA encoding a protein having GRHPR activity. This protein is a protein having GRHPR activity. The DNA introduced into the test mutant is a DNA encoding a protein having GRHPR activity, and has a certain identity (for example, 60% or more, 70% or more) with the amino acid sequence represented by SEQ ID NO: 2. 80% or more, 90% or more, 95% or more, 97% or more, 98% or more, and the like. Further, as described above, a protein having a specific amino acid residue or motif at a predetermined position is preferable. In addition, the present protein derived from Aspergillus is preferably used as the test protein.

  In the screening step, when the present protein is used as an index, for example, it can also be carried out by measuring the leucine acid-producing ability produced by the test mutant by the method disclosed in Examples and the like. In the screening step, when the polynucleotide encoding the present protein is used as an index, for example, an RNA sample containing RNA is prepared from the test mutant, and for this sample, for example, quantitative real-time PCR is performed on the mRNA. Or cDNA can be obtained and evaluated by expression level.

(Leucic acid producing enzyme screening method)
The screening method for a leucine acid-producing enzyme disclosed in the present specification can include a step of evaluating the leucine acid-producing activity of a test protein.

  The test protein is preferably the protein described in the above mutant screening method. What is necessary is just to implement also about the process of evaluating leucine acid production activity as already demonstrated.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated concretely, these do not limit this invention.

(Preparation of transformants expressing gonococcal MOA reductase (MOAR) candidates)
(1) Search for candidate genes It has been found that enzymes belonging to the 2-keto acid dehydrogenase family derived from lactic acid bacteria have MOAR activity. Therefore, a blast search was performed on the genome database of Aspergillus oryzae using a gene highly homologous to the 2-keto acid dehydrogenase gene (accession number: M26929) of lactic acid bacteria having MOAR activity. Based on the results, three types of MOAR candidate genes shown in Table 1 were selected.

(2) Creation of molecular phylogenetic tree Using the amino acid sequences of the three types of MOAR candidate genes (2-HADH1, 4, 6), search for homologous proteins from other organisms, and use the amino acid sequences. A molecular phylogenetic tree was created. FIG. 1 shows the created molecular phylogenetic tree. As shown in FIG. 1, 2-HADH1 belongs to a family of proteins having D-LDH activity, 2-HADH4 belongs to a family of proteins having GRHPR activity, and 2-HADH6 has D-MDH activity. It has been found that it belongs to the family of proteins it has. In FIG. 1, SERA indicates a gene encoding a protein having 3-PGDH activity.

(3) Construction of plasmid Aspergillus oryzae KBN8243 was inoculated into 50 ml of liquid M medium (2% Malt Extract, 0.1% polypeptone, 2% glucose) prepared in a Sakaguchi flask, followed by shaking culture at 28 ° C. for 20 hours. .

  After filtering the culture solution with suction, the cells were frozen using liquid nitrogen, crushed with a mortar and pestle, and the cells were collected in a 1.5 ml microcentrifuge tube.

  RNA was extracted from the bacterial cells with RNeasy Plant Mini Kit (QIAGEN), and cDNA was reverse transcribed with PrimeScript (registered trademark) 1st cDNA Synthesis Kit (TaKaRa) to obtain cDNA. Using the synthesized cDNA as a template, each gene fragment (2-HADH1, 4, 6) was amplified by PCR. The primers used are shown in Table 2, the composition of the PCR reaction solution is shown in Table 3, and the PCR reaction conditions are shown in Table 4. After electrophoresis of the PCR product by agarose gel electrophoresis, the target gene fragment was excised, and DNA was extracted from the agarose gel and purified by UltraClean ™ 15 DNA Purification Kit (MO BIO).

  PCR products were cloned using Mighty TA-cloning Kit (TaKaRa). Ligation was performed at 16 ° C. for 2 hours according to the attached protocol, 2 μl of the ligation solution was added to 50 μl of competent cells (E. coli DH10B for cloning), and transformation was performed on ice for 10 minutes, 42 ° C. for 40 seconds, and 2 minutes on ice . Table 5 shows the composition of the restriction enzyme treatment sample solution, and Table 6 shows the composition of the ligation sample solution. After adding 500 μl of SOC medium, shaking culture was performed at 37 ° C. for 30 minutes. All subsequent transformations were performed under these conditions. Thereafter, the mixture was applied to an LB plate containing 50 μg / ml ampicillin, 0.2% X-Gal, and 0.1% IPTG, followed by stationary culture at 37 ° C. overnight.

  Colony PCR was performed from E. coli colonies grown overnight using GoTaq ™ DNA Polymerase (Promega). Colonies in which the target gene fragment was confirmed by agarose gel electrophoresis were inoculated into 5 ml of liquid LB medium prepared in a test tube, and cultured with shaking at 37 ° C. for 12 hours. Thereafter, the plasmid was purified by an alkali extraction method. It was confirmed whether the plasmid was correctly constructed by agarose gel electrophoresis by performing DNA sequencing.

  Next, the purified plasmid is treated with a restriction enzyme, the target gene fragment is confirmed by agarose gel electrophoresis, cut out from the agarose gel, and then DNA is extracted using the UltraClean ™ 15 DNA Purification Kit (MO BIO). Was purified. A plasmid vector for protein expression was treated in the same manner.

  Ligation was performed at 16 ° C. for 2 hours using T4 DNA Ligase (TaKaRa), and the gene fragment was inserted into a plasmid vector. E. coli DH10B for cloning was transformed. Thereafter, the mixture was applied to an LB plate containing 50 μg / ml ampicillin and statically cultured at 37 ° C. overnight. Colony PCR was performed from the grown E. coli colonies. When amplification of the target gene fragment was confirmed by agarose gel electrophoresis, Escherichia coli was inoculated into 5 ml of liquid LB medium prepared in a test tube, cultured at 37 ° C. for 12 hours, and the plasmid was purified by alkali extraction. . E.coli BL21-CodonPlus, which is E. coli for protein expression, was transformed with a plasmid that was subjected to PCR (using GoTaq ™ DNA Polymerase) and restriction enzyme treatment and confirmed that the plasmid was correctly constructed by agarose gel electrophoresis.

(1) Expression of MOAR candidate The transformant prepared in Example 1 was applied to an LB plate containing 50 μg / ml ampicillin, allowed to stand overnight at 37 ° C., and then recombined enzyme using the grown Escherichia coli. Was expressed.

  The prepared Escherichia coli was inoculated into 5 ml of liquid LB medium and cultured with shaking at 37 ° C. for 12 hours. 2 ml of the bacterial solution was inoculated into 200 ml of a liquid LB medium prepared in a 500 ml Erlenmeyer flask and cultured with shaking at 37 ° C. for 2 to 3 hours (preculture). Using a spectrophotometer, the absorbance of the culture solution at 600 nm was measured. When the absorbance reached 0.5 to 0.7, IPTG was added to a final concentration of 0.1 mM and shaken at 20 ° C. for 8 hours. Cultured (main culture). After 8 hours, the culture solution was transferred to one 50 ml plastic centrifuge tube and centrifuged at 8000 rpm, 4 ° C. for 5 minutes to remove the supernatant. Samples contained in 50 ml plastic centrifuge tubes were stored in a freezer (−80 ° C.). As in the case of adding IPTG, before adding IPTG, a part of the culture solution is taken out and incubated at 95 ° C. for 5 minutes to obtain a sample for SDS-PAGE. Used to perform SDS-PAGE. After completion of the electrophoresis, CBB staining was performed.

  Take out the preserved 50 ml centrifuge tube, add 15 ml of 50 mM phosphate buffer (pH 7.2, containing 0.15 M NaCl, hereinafter referred to as phosphate buffer) and a small amount of lysozyme, and pipette the cells. Was dissolved and crushed using an ultrasonic homogenizer.

  The disrupted solution and protein sample were incubated at 95 ° C. for 5 minutes to form a sample for SDS-PAGE, and SDS-PAGE was performed using a 12% polyacrylamide gel. After completion of the electrophoresis, CBB staining was performed. FIG. 2 shows the results of SDS-PAGE for 2-HADH4.

  As shown in FIG. 2, in (3) the soluble fraction after the main culture and (6) the purified fraction, a clear band appeared at a position of about 35 kDa, indicating that 2-HADH4 is expressed. confirmed. In (6), it was confirmed that 2-HADH4 was purified because almost no bands other than the band appeared. From the results of SDS-PAGE, the expression of 2-HADH1 and 6 was also confirmed (not shown).

  From the results of SDS-PAGE, 2-HADH1, 4, 6 in which expression of the recombinant enzyme was confirmed was purified with 2 ', 5'-ADP Sepharose 4B. After equilibrating the column by dropping 30 ml or more of phosphate buffer 1 onto 2 ', 5'-ADP Sepharose 4B, the sample was dropped. After washing the column, 2-HADH was purified by dropwise addition of 8 ml of phosphate buffer containing 2 mM NADPH. Each of the non-adsorbed fraction that was not adsorbed to 2 ′, 5′-ADP Sepharose 4B and the purified fraction after purification was incubated at 95 ° C. for 5 minutes to obtain a sample for SDS-PAGE. SDS-PAGE was performed using a% polyacrylamide gel.

  Next, 25 ml of 20 mM phosphate buffer (pH 7.2) was dropped onto the desalting column PD-10 (GE Healthcare) to equilibrate the column, and then 2.5 ml of the purified sample solution was dropped. Subsequently, 3 ml of a phosphate buffer not containing NaCl was added dropwise for elution.

  To a 1.5 ml microcentrifuge tube, 490 μl of Quick Start Bradford 1 × Dye Reagent (Bio-Rad) and 10 μl of the desalted sample were added and mixed. 150 μl was added to the measurement plate, the absorbance value at a wavelength of 595 nm was measured with a plate reader, and the protein concentration was calculated.

(2) MOAR activity measurement A 20 mM phosphate buffer (pH 7.2) was used as a buffer for activity measurement. Add a phosphate buffer (prepared to a total volume of 500 μl), purified 2-HADH1,4,6, and 100 mM NADPH (final concentration 125 μM) to a glass cuvette (1 ml) for absorbance measurement, and stir. It was. Thereto was added 100 mM MOA (final concentration 2 mM) as a substrate, pipetting was performed, and the absorbance value at a wavelength of 340 nm was measured for 5 minutes using a visible spectrophotometer. The specific activity and the enzyme using the absorbance value, _{K m values, V max} values _were calculated _{k cat} values. The same experiment was conducted with phenylpyruvic acid, pyruvic acid, glyoxylic acid, β-hydroxypyruvic acid, and 3-methyloxovaleric acid instead of MOA.

Regarding the results of measuring the MOAR activity of 2-HADH4, the absorbance value at a wavelength of 340 nm decreased after the reaction after the reaction. From this, it was confirmed that NADPH decreased, that is, that leucine acid was produced from MOA. Figure 3, _{K m} values for each substrate 2-HADH4 based on absorbance _{values, V max} value _indicates the calculation results of the _{k cat} values. As shown in FIG. 3, it was found that 2-HADH4 also has an activity of promoting a reduction reaction using not only MOA but also phenylpyruvic acid, pyruvic acid and glyoxylic acid as substrates. It was also found that 2-HADH4 does not use β-hydroxypyruvic acid or 3-methyloxovaleric acid as a substrate. As for 2-HADH1 and 6, since the absorbance value at 340 nm hardly decreased before and after the reaction, it was found that there was no MOAR activity (not shown).

  In order to clarify the reaction product of MOA by 2-HADH4, GC-MS analysis was performed. Specifically, after the above reaction, the reaction was stopped by adding HCL to the reaction product. Then, the reaction product was extracted with ethyl acetate, trimethylsilylated, and then subjected to GC-MS analysis. FIG. 4 shows a GC chromatogram and a mass-mass fragment pattern of the reaction product.

  As shown in FIG. 4, it was found that the addition of 2-HADH4 decreased the substrate MOA, increased the reaction product, and the reaction product was leucine acid. That is, it was found that 2-HADH4 functions as MOAR and reduces the substrate MOA to leucine acid. Hereinafter, 2-HADH4 having MOAR activity is referred to as MOAR1.

(Preparation of koji mold with high leucine acid production)
(1) Amplification of MOAR1 gene In order to prepare a leucine acid high-producing gonococci with high expression of MOAR1 gene, the strains and plasmids shown in Table 7 (Yoshino-Yasuda S. et al. Food Sci Technol, Res. 18, 59- 65, 2012) and primers were used.

  The plasmid pTAaphA has a site where pyrG, tefI promoter and acid phosphatase sequence are linked (Yoshino-Yasuda S. et al. Food Sci Technol, Res. 18, 59-65, 2012). From here, the DNA sequence in which pyrG and the tefI promoter were linked was amplified by PCR using fupyrGN and tefPrv as primers. In addition, amplification was performed using tefmoa368 and moa368downSphI using the ORF and terminator part of the novel MOAR1 gene derived from Aspergillus oryzae as primers.

(2) Preparation of culture media
As the medium for E. coli, LB medium (Tryptone 1.0 g, Yeast extract 0.5 g, NaCl 0.5 g / 100 ml) was used. Ampicillin was added to a final concentration of 50 μg / ml. In the case of a solid medium, agar was added so as to be 1.5%. When performing blue-white selection, 0.4% X-gal (5-bromo-4-chloro-3-indolyl-beta-D-galactoside), 0.1% IPTG (Isopropyl β-D-1-thiogalactopyranoside) Was added.

As a medium for A. oryzae, Czapek-Dox medium (pH 6.0 with 1N HCl, NaNO ₃ 0.3 g, KH ₂ PO ₄ 0.1 g, MgSO ₄ · 7H ₂ O 0.05 g, KCl 0.05 g, FeSO4 · 7H ₂ O 0.01 g, Sucrose 3.0 g, Agar 1.5 g / 100 ml).

As the Czapek-Dox selective medium for protoplast regeneration, the following lower layer medium and upper layer medium were used. In the lower layer medium, it was added to Czapek-Dox medium so that the final concentrations were 0.2 M sucrose and 1.5% agar, respectively. Furthermore, Trace elements solution (FeSO4 ・ 7H ₂ O 1.0g, ZnSO ₄・ 7H ₂ O 8.8g, CuSO ₄・ 5H ₂ O 0.4 g, Na ₂ B ₄ O ₇・ 10H ₂ O 0.1 g, (NH ₄ ) ₆ Mo ₇ O ₂₄ · 4H ₂ O 0.05 g / 1L) was added to a final concentration of 0.1%. In the upper layer medium, it was added to Czapek-Dox medium so that the final concentrations were 1 M sucrose and 0.5% agar, respectively. Trace elements solution was added to a final concentration of 0.1%.

  As a complete medium, a medium (pH 6.5 with 1N KOH, Malt extract 2.0 g, Bacto pepton 0.1 g, Glucose 2.0 g / 100 ml) was used.

Protoplast solution (10 mM-Na phosphate 0.8 M-NaCl buffer (pH 6.0) 30 ml, YatalaseTM (TaKaRa) 90 mg, Lysing enzymes l2265 (SIGMA) 9.0 g, BSA 0.3 g / 30 ml) Prepared on the day of conversion and sterilized with filters (Millex® Syringe Filter Units, Non-Sterile).

  Solution 1 (0.8 M NaCl, 10 mM CaCl2, 10 mM Tris-HCl (pH 7.5)) was sterilized by autoclaving. Solution 2 (40% (w / v) PEG4000, 50 mM CaCl2, 50 mM Tris-HCl (pH 7.5)) was sterilized with a filter. As a spore suspension solution, 0.1% Tween80, 0.9% NaCl solution was used.

  As additives, Pyrimidine (uridine, uracil) (Uridine 1.2 g, Uracil 1.12 g / 1L) and 5-FOA (pH 6.5 with 1N KOH, 5-FOA 0.1 g / 100 ml) were used. When 5-FOA (5-Fluoroorotic Acid) was used, it was dissolved in 1/4 volume of sterile water of the target medium and sterilized by filter. This solution was mixed with the target medium prepared in 3/4 volume only in the amount of sterilized water to obtain a 5-FOA-added medium.

(3) Construction of plasmid E. coli was transformed using electroporation.

  For electrophoresis of DNA, 0.8% agarose gel and TAE buffer were used, and electrophoresis was performed at 100V. After completion of the electrophoresis, DNA was visualized by staining with 10 mg / ml ethidium bromide solution for 20 minutes and irradiating with ultraviolet rays in the dark. When extracting the target DNA from the gel, UltraClean 15 DNA Purification Kit was used. The method followed the attached instructions.

  After preparing the PCR reaction solution of Table 8, the annealing temperature was set to 52, 56, 60, 64, and 68 ° C., and PCR was performed under the reaction conditions shown in Table 8. The extension time X was set according to the length of the target sequence (1 kb = 1 min).

  The ligated DNA was ligated to the vector. Using pGEM®-T Easy Vector System, the method followed the attached instructions. Several colonies were taken with a sterilized toothpick, and the plasmid was extracted after culturing at 30 ° C. for 12 hours in LB medium.

  A small amount of cells were removed from the transformed E. coli colony with a sterilized toothpick and inoculated into 5 ml of LB liquid medium containing ampicillin. After shaking culture for 12 to 16 hours, 1.5 ml of the bacterial solution was transferred to a microcentrifuge tube. Centrifugation was carried out at 13000 rpm for 3 minutes, and the supernatant was removed to collect bacteria. Plasmids were extracted using QIAprep Spin Miniprep Kit. The method followed the attached instructions.

(4) Transformation of A. oryzae KBN8243 A. oryzae KBN8243 is cultured at 30 ° C. for 4-7 days in a Czapek-Dox flat medium satisfying auxotrophy, and 10 ml of a spore spore suspension preparation is added thereto. The spores were scraped off using a large rod. This suspension was collected with a pipette to obtain a spore suspension.

Suspended spores were inoculated into 50 ml of complete medium and aerobically shaken at 30 ° C. for 20 hours. After incubation, the cells were collected by centrifugation at 3000 rpm for 5 minutes, and the supernatant was removed by decantation. Then, 30 ml of a protoplast solution was added and gently shaken at 30 ° C. for 3 hours. This solution was filtered through sterilized Miracloth, and the filtrate was centrifuged at 3000 rpm for 5 minutes to precipitate protoplasts. After removing the supernatant, 10 ml of Solution 1 was added and suspended, and centrifuged again to discard the supernatant, thereby washing the protoplasts. Protoplasts were suspended in solution 1 to 2 × 10 ⁸ / ml, and 1/4 of the solution 2 was added and mixed. 20 μl or less of various plasmid DNA solutions were added to 0.2 ml of protoplast suspension to gently suspend, and allowed to stand in ice for 30 minutes. Further, 1 ml of the solution 2 was added and the mixture was allowed to stand at room temperature for 15 minutes. The total amount of this solution was mixed with 5 ml of the upper layer medium kept at 50 ° C., and spread evenly on the lower layer medium that had been solidified beforehand in the petri dish. When the medium solidified, it was cultured at 30 ° C. for 4-7 days.

  After culturing the transformant, a small amount of spores were scraped and suspended in 50 μl of buffer (100 mM Tris-HCl pH 9.5, 1 M KCl, 10 mM EDTA). The solution was incubated at 95 ° C. for 10 minutes and then stirred vigorously with a Vortex mixer. After centrifugation, 1 μl of the supernatant was added to the PCR reaction solution shown in Table 9, and PCR was carried out under the reaction conditions shown in Table 9. The size of the band was confirmed after electrophoresis.

  FIG. 5 shows the result of electrophoresis of DNA extracted from the transformant. When PCR was performed using primers tef 150 check Fw and Moa 350 check Rv, it was expected that a band was detected at a position of 1000 bp in the genomic DNA of the MOAR1 high expression strain. In the transformed strain, a band was detected at a position of 1000 bp, indicating that the plasmid for high expression of MOAR1 was integrated into the genomic DNA.

(5) Comparison of the expression level of MOAR1 at the mRNA level by real-time PCR
After filtering the culture solution of A.oryzae KBN8243 with suction, the cells recovered with liquid nitrogen were frozen and crushed using a mortar and pestle. RNA was extracted from the bacterial cells with RNeasy Plant Mini Kit (QIAGEN), and cDNA was reverse transcribed with PrimeScript (registered trademark) 1st cDNA Synthesis Kit (TaKaRa) to obtain cDNA. Real-time PCR was performed using GeneAce SYBR (registered trademark) qPCR Mix plus ROX Tube.

In a PCR tube, 1 μl of Oligo dT primer solution, 1 μl of dNTP mixture and 8 μl of extracted RNA solution were added and incubated at 65 ° C. for 5 minutes. After cooling on ice, reverse transcription reaction was performed by adding 4 μl of 5 × Prime Script II buffer, 0.5 μl of RNase Inhibitor, 1 μl of PrimeScript II RTase, 4.5 μl of RNase Free dH ₂ O, and 10 μl of the solution containing RNA incubated for 5 minutes at 65 ° C. went. The reaction cycle was performed as shown in Table 10. This was used as a cDNA solution.

  SYBR Premix Ex Taq II (12.5 μl), primer AO0368 417Fw solution 1.0 μl, primer AO0368 607Rv solution 1.0 μl, sterile water 8.5 μl, cDNA solution 2 μl were mixed to make a total volume of 25 μl and subjected to real-time PCR. FIG. 6 shows a comparison of the expression level of MOAR1 at the mRNA level between the wild strain and the transformed strain.

  As shown in FIG. 6, the transformed strain had a 9-fold higher expression level of MOAR1 than the wild-type strain. From this, it was found that the transformant was a MOAR1 high expression strain.

(6) Quantification of intracellular leucine acid by LC-MS / MS A 500 strain Czapek liquid medium was inoculated with a wild strain (KBN8243) and a MOAR1 high expression strain, and cultured with shaking at 30 ° C. for 3 days. After suction filtration of the culture solution, 3.0 g of bacterial cells were recovered. After freeze-fracturing with liquid nitrogen, intracellular metabolites were extracted with a 50% methanol solution. This was freeze-dried and a sample redissolved in 300 μl of 50% ethanol solution was analyzed by LC-MS / MS. FIG. 7 shows a comparison between the wild strain, the transformed strain, and the leucine acid production amount.

  As shown in FIG. 7, the leucine acid production amount of the wild strain was 3.0 pmol / g wet weight, and the transformed strain was 290 pmol / g wet weight. From this, it was found that the transformed strain produced 97 times more leucine acid than the wild strain.

(7) Expression analysis of MOAR1 when adding substrate by real-time PCR Wild strains were inoculated into 5 ml of Czapek liquid medium alone and 2 mM leucine, 2 mM MOA, and 2 mM leucine acid, respectively, and 28 ° C., 100 rpm And cultured with shaking for 3 days. As described above, the culture medium is suction filtered, and the cells are disrupted. Then, RNA is extracted using the RNeasy Plant Mini Kit (QIAGEN), and then converted into cDNA using the PrimeScript (registered trademark) 1st cDNA Synthesis Kit (TaKaRa). Reverse transcribed. Real-time PCR was performed with GeneAce SYBR® qPCR Mix plus ROX using AO0368 417Fw and AO0368 607RV primers. FIG. 9 shows the results of MOAR1 expression analysis upon substrate addition.

  As shown in FIG. 9, when leucine and MOA were added, the MOAR1 expression level was higher than when no leucine was added, but when leucine acid was added, the expression level was lower than when no leucine was added. MOAR1 was found to be induced by leucine and MOA and suppressed by leucine acid. From the above, in order to obtain leucine acid, addition of leucine or MOA is effective. On the other hand, feedback inhibition by the product (leucine acid) is caused, so conversion to ethyl leucine is effective. I understood it.

Sequence number 3-18: Primer

Claims (16)

A transformant which is transformed with DNA encoding the protein according to any one of the following (a) to (e) and has enhanced leucine acid-producing ability.
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 2 (b) having an amino acid sequence in which one or more amino acids in SEQ ID NO: 2 are substituted, deleted, inserted and / or added; A protein having an activity of producing leucine acid from methyl-2-oxopentanoic acid (c) having an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 2, A protein having an activity of producing leucine acid from oxopentanoic acid (d) a protein encoded by a DNA consisting of a base sequence represented by SEQ ID NO: 1 (e) a base sequence represented by SEQ ID NO: 1 and 80% or more of A tamper having an amino acid sequence encoded by a base sequence having identity and having an activity of producing leucine acid from 4-methyl-2-oxopentanoic acid Quality   When the protein according to any one of (a) to (e) is further aligned with the amino acid sequence represented by SEQ ID NO: 2, the site corresponding to position 86 in the amino acid sequence represented by SEQ ID NO: 2 Is glycine (G), and the site corresponding to positions 159 to 164 is GXGXXG (each X is an arbitrary amino acid independently selected from natural amino acids), and position 242 The transformant according to claim 1, wherein the corresponding site is arginine (R), the site corresponding to position 271 is glutamic acid (E), and the site corresponding to position 289 is histidine (H). .   The transformant according to claim 1 or 2, wherein the transformant is a eukaryotic microorganism.   The transformant according to claim 3, wherein the eukaryotic microorganism is a koji mold.   The transformant according to claim 4, wherein the koji mold is Aspergillus oryzae.   The transformant according to claim 1 or 2, wherein the transformant is a prokaryotic microorganism. A recombinant vector for enhancing the ability to produce leucine acid, which retains a DNA encoding the protein according to any one of (a) to (e) below.
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 2 (b) having an amino acid sequence in which one or more amino acids in SEQ ID NO: 2 are substituted, deleted, inserted and / or added; A protein having an activity of producing leucine acid from methyl-2-oxopentanoic acid (c) having an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO: 2, A protein having an activity of producing leucine acid from oxopentanoic acid (d) a protein encoded by a DNA consisting of the base sequence represented by SEQ ID NO: 1 (e) a base sequence represented by SEQ ID NO: 1 and 90% or more A tamper having an amino acid sequence encoded by a base sequence having identity and having an activity of producing leucine acid from 4-methyl-2-oxopentanoic acid Quality A method for producing a transformant having enhanced leucine acid production ability, comprising a step of introducing a DNA encoding the protein according to any one of (a) to (e) below into a host.
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 2 (b) having an amino acid sequence in which one or more amino acids in SEQ ID NO: 2 are substituted, deleted, inserted and / or added; A protein having an activity of producing leucine acid from methyl-2-oxopentanoic acid (c) having an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO: 2, A protein having an activity of producing leucine acid from oxopentanoic acid (d) a protein encoded by a DNA consisting of the base sequence represented by SEQ ID NO: 1 (e) a base sequence represented by SEQ ID NO: 1 and 90% or more A tamper having an amino acid sequence encoded by a base sequence having identity and having an activity of producing leucine acid from 4-methyl-2-oxopentanoic acid Quality   A method for producing leucine acid, comprising a step of culturing the transformant according to any one of claims 1 to 6 under conditions in which 4-methyl-2-oxopentanoic acid may be present. Culturing the transformant according to any one of claims 1 to 6 under a condition in which 4-methyl-2-oxopentanoic acid may be present to produce leucine acid;
Converting the leucine acid to ethyl leucine;
A process for producing ethyl leucine.   A method for producing a koji-containing material, comprising the step of culturing koji mold which is the transformant according to any one of claims 1 to 6.   A method for producing alcohol, comprising a step of culturing koji mold and yeast that are transformants according to any one of claims 1 to 6. A screening method for leucine high productivity mutants, comprising:
Screening a mutant with high productivity of leucine using the protein represented by any one of the following (a) to (e) or a polynucleotide encoding the protein as an index,
A method comprising:
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 2 (b) having an amino acid sequence in which one or more amino acids in SEQ ID NO: 2 are substituted, deleted, inserted and / or added; A protein having an activity of producing leucine acid from methyl-2-oxopentanoic acid (c) having an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO: 2, A protein having an activity of producing leucine acid from oxopentanoic acid (d) a protein encoded by a DNA consisting of the base sequence represented by SEQ ID NO: 1 (e) a base sequence represented by SEQ ID NO: 1 and 90% or more A tamper having an amino acid sequence encoded by a base sequence having identity and having an activity of producing leucine acid from 4-methyl-2-oxopentanoic acid Quality A screening method for leucine acid-producing enzyme,
Evaluating the activity of producing leucine acid from 4-methyl-2-oxopentanoic acid of a test protein having 2-keto acid dehydrogenase activity;
A screening method comprising: A method for producing leucine acid, comprising:
A step of producing leucine acid from 4-methyl-2-oxopentanoic acid using the protein represented by any of the following (a) to (e):
A production method comprising:
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 2 (b) having an amino acid sequence in which one or more amino acids in SEQ ID NO: 2 are substituted, deleted, inserted and / or added; A protein having an activity of producing leucine acid from methyl-2-oxopentanoic acid (c) having an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO: 2, A protein having an activity of producing leucine acid from oxopentanoic acid (d) a protein encoded by a DNA consisting of the base sequence represented by SEQ ID NO: 1 (e) a base sequence represented by SEQ ID NO: 1 and 90% or more A tamper having an amino acid sequence encoded by a base sequence having identity and having an activity of producing leucine acid from 4-methyl-2-oxopentanoic acid Quality A method for producing ethyl leucine,
A step of producing leucine acid from 4-methyl-2-oxopentanoic acid using the protein represented by any of the following (a) to (e):
Esterifying the leucine acid obtained in the above step to ethyl leucineate;
A production method comprising:
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 2 (b) having an amino acid sequence in which one or more amino acids in SEQ ID NO: 2 are substituted, deleted, inserted and / or added; A protein having an activity of producing leucine acid from methyl-2-oxopentanoic acid (c) having an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO: 2, A protein having an activity of producing leucine acid from oxopentanoic acid (d) a protein encoded by a DNA consisting of the base sequence represented by SEQ ID NO: 1 (e) a base sequence represented by SEQ ID NO: 1 and 90% or more A tamper having an amino acid sequence encoded by a base sequence having identity and having an activity of producing leucine acid from 4-methyl-2-oxopentanoic acid Quality