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WO2021080277A1 - Nouvelle enzyme pour la production de d-thréonine, et procédé de production stéréospécifique de d-thréonine l'utilisant - Google Patents

Nouvelle enzyme pour la production de d-thréonine, et procédé de production stéréospécifique de d-thréonine l'utilisant Download PDF

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Publication number
WO2021080277A1
WO2021080277A1 PCT/KR2020/014292 KR2020014292W WO2021080277A1 WO 2021080277 A1 WO2021080277 A1 WO 2021080277A1 KR 2020014292 W KR2020014292 W KR 2020014292W WO 2021080277 A1 WO2021080277 A1 WO 2021080277A1
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threonine
seq
amino acid
enzyme
producing
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Korean (ko)
Inventor
이승구
염수진
김하성
박성현
이대희
나유진
이혜원
권길광
안정웅
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Korea Research Institute of Bioscience and Biotechnology KRIBB
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Korea Research Institute of Bioscience and Biotechnology KRIBB
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y401/00—Carbon-carbon lyases (4.1)
    • C12Y401/02—Aldehyde-lyases (4.1.2)
    • C12Y401/02042—D-Threonine aldolase (4.1.2.42)

Definitions

  • the present invention relates to a novel D-threonine producing enzyme.
  • amino acids in nature have alpha carbons that exhibit optical activity, and are divided into L-amino acids and D-amino acids according to their stereospecificity.
  • Most of the proteins in nature are composed of L-amino acids, but exceptionally, the components of microbial peptidoglycans, peptide-based antibiotics, and physiologically active substances of higher plants are composed of D-amino acids. have.
  • D-threonine is an intermediate or precursor for synthesizing physiologically active substances such as neurotransmitters, vaccines, synthetic sweeteners, antibiotics, and hormones. It is widely used, and thus, methods for producing D-threonine have been developed.
  • Methods of producing D-threonine can be largely divided into chemical synthesis methods and biological synthesis methods using enzymes or microorganisms.
  • threonine production by chemical synthesis since the product is obtained as a racemic mixture of D-threonine and L-threonine, it is difficult to undergo another complicated optical purification process to obtain pure D-threonine. have.
  • the chemical synthesis method as described above also has a problem that serious environmental pollution is caused due to by-products generated in the process.
  • D-threonine can be produced stereospecifically without environmental pollution, while not only having high activity and production, but also dramatically reducing the ratio of impurities such as D-allothreonine to produce pure D-threonine.
  • Research and development of a novel D-threonine-producing enzyme is still in need.
  • the present invention is capable of stereospecific production of only D-threonine, not a mixture of L-threonine and D-threonine, while overcoming the conventional problem that D-allotreonine is produced together, thereby producing D-threonine with high purity.
  • An object of the present invention is to provide a novel enzyme with characteristics and a method for producing D-threonine using the same.
  • an aspect of the present invention is the 147th arginine, 179th glycine of D-threonine aldolase derived from Filomicrobium marinum. And it provides a D-threonine-producing enzyme in which at least one amino acid selected from the group consisting of 312th serine is substituted with alanine.
  • another aspect of the present invention provides a gene encoding the D-threonine producing enzyme.
  • another aspect of the present invention provides a recombinant expression vector containing the gene and a transformant in which the expression vector is introduced into a host cell.
  • another aspect of the present invention comprises the steps of culturing the transformant; And separating the D-threonine-producing enzyme from the culture of the transformant.
  • another aspect of the present invention is a composition for producing D-threonine comprising the D-threonine-producing enzyme as an active ingredient, and the D-threonine-producing enzyme reacted with glycine and acetaldehyde. It provides a method for producing D-threonine in an in vitro containing;
  • another aspect of the present invention provides a method for producing D-threonine in vivo, comprising culturing the transformant in the presence of glycine and acetaldehyde. do.
  • the D-threonine-producing enzyme of the present invention converts glycine and acetaldehyde into D-threonine as substrates, reducing the ratio and production amount of D-allothreonine that can be produced together, thereby producing D-threonine with high purity. There is an effect.
  • 1 is a molecular mass characteristic of a protein having an amino acid sequence of SEQ ID NO: 1, overexpressed in a transformant, purified and isolated, by SDS-PGAE (A) and gel-filtration chromatography (B). This is the result of analysis.
  • FIG. 2 is a graph showing the degree of conversion of D-threonine by a protein having the amino acid sequence of SEQ ID NO: 1.
  • FIG. 3 is a graph confirming the type (A) of metal cations and the concentration (B) of metal cations affecting the enzyme activity of the protein having the amino acid sequence of SEQ ID NO: 1.
  • Figure 5 is a graph confirming the effect of pH on the enzyme activity of the protein having the amino acid sequence of SEQ ID NO: 1, MOPS (pH 6.5 to 7.5), HEPES (pH 7.5 to 8.0), EPPS (pH 8.0 to 8.5) as a buffer solution. ) And CHES (pH 8.5 to 10) buffer solutions, respectively.
  • PBP pyridoxal-5-phosphate
  • FIG. 7 is a photograph showing the X-ray crystal structure (A) of the protein enzyme having the amino acid sequence of SEQ ID NO: 1 and the residue (B) predicted to be involved in the diastereomer ratio change at the active site.
  • WT 9 is a wild-type protein (WT) having the amino acid sequence of SEQ ID NO: 1, the relative specific activity and diastereomers (D-threonine and D-allotreonine) of each protein in which one amino acid residue of SEQ ID NO: 1 is replaced with another. This is a graph confirming the rate change.
  • Figure 10 is a wild-type protein having the amino acid sequence of SEQ ID NO: 1 (Wild-type), SEQ ID NOs: 3, 4, 5 substituted with alanine, and each protein having a sequence substituted with an amino acid other than alanine was confirmed by SDS-PAGE. It is the result.
  • Figure 11 is a wild-type protein having the amino acid sequence of SEQ ID NO: 1 (Wild-type), SEQ ID NOs: 3, 4, 5 substituted with alanine, and relative specific activity and portions of each protein having a sequence substituted with an amino acid other than alanine. This is a graph confirming the change in the ratio of stereoisomers (D-threonine and D-allotreonine).
  • FIG. 12 is a result of confirming by SDS-PAGE a wild-type protein having the amino acid sequence of SEQ ID NO: 1 (Wild-type) and each protein having the amino acid sequence of SEQ ID NOs: 9 to 11 into which multiple alanine substitutions have been introduced.
  • A is SEQ ID NO: 1 (WT)
  • B is SEQ ID NO: 4 (G179A)
  • C is SEQ ID NO: 10 (G179A + S312A)
  • A is SEQ ID NO: 1 (WT)
  • B is SEQ ID NO: 4 (G179A)
  • C is the ratio of acetaldehyde as a substrate to the proteins having the amino acid sequence of SEQ ID NO: 10 (G179A + S312A) is D-threonine This is a graph confirming the effect on production.
  • FIG. 16A is SEQ ID NO: 1 (WT)
  • B is SEQ ID NO: 4 (G179A)
  • C is SEQ ID NO: 10 (G179A + S312A) by reacting proteins having the amino acid sequence of the amino acid sequence under optimal substrate conditions, D-threonine and D- This is a graph comparing the production of allotreonine.
  • the "D-threonine-producing enzyme” of the present invention means an enzyme having the activity of producing D-threonine from glycine and acetaldehyde as shown in the following scheme.
  • One aspect of the present invention is the 147th arginine, 179th glycine and 312th serine of D-threonine aldolase derived from Filomicrobium marinum. It provides a D-threonine-producing enzyme in which at least one amino acid selected from the group consisting of alanine is substituted.
  • the D-threonine-producing enzyme is that the 147th arginine of the D-threonine aldolase is substituted with alanine, the 179th glycine is substituted with alanine, the 312th serine is substituted with alanine, or The 179th glycine and the 312th serine may be substituted with alanine, respectively.
  • another aspect of the present invention provides a gene of a D-threonine-producing enzyme encoding the D-threonine-producing enzyme.
  • the D-threonine-producing enzyme may catalyze a reaction for producing D-threonine using glycine and acetaldehyde as substrates.
  • the D-threonine aldolase derived from Filomicrobium marinum may include the amino acid sequence of SEQ ID NO: 1, and the D-threonine aldolase is within a range that does not affect the function of the protein.
  • Amino acid residue deletion, insertion, substitution, or a combination thereof may be a variant of an amino acid having a different sequence, or fragments.
  • Amino acid exchange at the protein and peptide level that does not totally alter the activity of the D-threonine aldolase is known in the art. In some cases, it may be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, and farnesylation.
  • the D-threonine aldolase includes a protein having an amino acid sequence substantially identical to that of a protein comprising the amino acid sequence of SEQ ID NO: 1, and a variant thereof or an active fragment thereof.
  • the amino acid sequence of SEQ ID NO: 1 may be encoded by the nucleotide sequence of SEQ ID NO: 2.
  • the D-threonine producing enzyme of the present invention may include any one amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11,
  • the D-threonine-producing enzyme may be mutants or fragments of amino acids having different sequences by deletion, insertion, substitution, or a combination of amino acid residues within a range that does not affect the function of the protein. Amino acid exchange at the protein and peptide level that does not totally alter the activity of the D-threonine producing enzyme of the present invention is known in the art.
  • the present invention has an amino acid sequence that is substantially identical to a protein comprising any one amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11. Proteins and variants or active fragments thereof.
  • the term'X#Y' is clearly recognized in the art, and'#' represents a substituted position with respect to the amino acid number of the protein, and'X' is a wild-type protein. Represents an amino acid found at that position in the amino acid sequence of, and'Y' represents a substituted amino acid at that position.
  • the amino acid sequence of SEQ ID NO: 3 is that the 312th serine of the amino acid sequence of SEQ ID NO: 1 is substituted with alanine (S312A).
  • the amino acid sequence of SEQ ID NO: 4 is that glycine at the 179th of the amino acid sequence of SEQ ID NO: 1 is substituted with alanine (G179A).
  • the amino acid sequence of SEQ ID NO: 5 is one in which arginine at the 147th of the amino acid sequence of SEQ ID NO: 1 is substituted with alanine (R147A).
  • amino acid sequence of SEQ ID NO: 9 arginine at the 147th of the amino acid sequence of SEQ ID NO: 1 is substituted with alanine, and glycine at the 179th is substituted with alanine (R147A, G179A).
  • the amino acid sequence of SEQ ID NO: 10 is that glycine at the 179th of the amino acid sequence of SEQ ID NO: 1 is substituted with alanine and serine at the 312th is substituted with alanine (G179A, S312A).
  • the amino acid sequence of SEQ ID NO: 11 is that the 147th arginine of the amino acid sequence of SEQ ID NO: 1 is substituted with alanine, the 179th glycine is substituted with alanine, and the 312th serine is substituted with alanine (R147A, G179A, S312A). .
  • the D-threonine producing enzyme of the present invention can produce D-threonine and D-allotreonine, which is a diastereoisomer thereof.
  • the D-threonine producing enzyme of the present invention can produce D-threonine so that the proportion of D-threonine in the reaction product is 85% or more, and specifically 87.5% or more, 90% or more, 92% or more, 95% or more, It can be produced to be 97% or more, 97.5% or more, 98% or more, 98.5% or more, or 99% or more.
  • the D-threonine-producing enzyme can produce more D-threonine than D-allothreonine so that the diastereomeric excess (de) calculated by the following formula is 75% or more, and specifically de Is 77% or more, 79% or more, 80% or more, 82% or more, 85% or more, 87% or more, 90% or more, 92% or more, or 93% or more.
  • the gene encoding the D-threonine producing enzyme of the present invention is composed of any one nucleotide sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14 Can be.
  • the gene encoding the D-threonine-producing enzyme of the present invention and its mutant or active fragment thereof is variously modified in the coding region within a range that does not change the amino acid sequence of the enzyme and its mutant or active fragment thereof expressed from the coding region. This can be achieved, and various mutations can be made within a range that does not affect the expression of the gene even in portions other than the coding region, and such mutant genes are also included in the scope of the present invention.
  • the present invention consists of a nucleotide sequence substantially identical to a gene consisting of any one nucleotide sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14 It includes a gene and a fragment of the gene.
  • Genes consisting of the substantially identical nucleotide sequence means those having sequence homology of 80% or more, preferably 90% or more, and most preferably 95% or more, but are not limited thereto, and sequence homology of 80% or more. And, if the encoded protein has the same enzymatic activity, it is included in the present invention.
  • nucleic acid bases may be mutated by substitution, deletion, insertion, or a combination thereof. It is also included in the scope of the present invention.
  • nucleotide sequences may be single-stranded or double-stranded, and may be DNA molecules or RNA molecules.
  • Another aspect of the present invention provides a recombinant expression vector containing the gene of the D-threonine producing enzyme of the present invention and a transformant into which the recombinant expression vector has been introduced.
  • the recombinant expression vector of the present invention includes a gene encoding the D-threonine-producing enzyme, and thus can be usefully used as a vector capable of producing the D-threonine-producing enzyme.
  • the description of the D-threonine-producing enzyme is the same as described in the'new D-threonine-producing enzyme and its genes'.
  • the expression vector includes, but is not limited to, a plasmid vector, a cozmid vector, a bacteriophage vector, and a viral vector.
  • the recombinant expression vector contains an expression control sequence such as a promoter, a terminator, or an enhancer, or a sequence for secretion, depending on the type of host cell for which the D-threonine producing enzyme of the present invention is to be produced. It can be combined according to the purpose as appropriate.
  • the expression vector may further include a selection marker for selecting a host cell into which the vector has been introduced, and may include an origin of replication in the case of a replicable expression vector.
  • the recombinant expression vector may include a sequence for facilitating purification of the expressed protein, and specifically, the gene encoding the tag for separation and purification so as to be operable to the gene encoding the D-threonine producing enzyme of the present invention is Can be connected.
  • the tags for separation and purification may be used alone, such as GST, poly-Arg, FLAG, histidine-tag, c-myc, or two or more of them may be sequentially connected.
  • the gene encoding the D-threonine-producing enzyme can be cloned through the restriction enzyme cleavage site, and when the gene encoding the protein-cleaving enzyme recognition site is used in the vector, the gene and the frame of the D-threonine-producing enzyme are different. It is connected to fit (in frame), when the enzyme is obtained and then digested with a protein cleavage enzyme, the original form of D-threonine-producing enzyme can be prepared.
  • the recombinant expression vector may be prepared by inserting a gene encoding a D-threonine-producing enzyme into a plasmid vector pET28a(+), and in addition to pET28a(+) used in the preparation of the cloning vector, various vectors for prokaryotic cells or Since vectors for eukaryotic cells (pPIC and pPICZ, etc.) are known, various expression vectors other than the above vectors can be used depending on the purpose of expression.
  • the recombinant expression vector containing the gene encoding the D-threonine producing enzyme of the present invention is introduced.
  • the description of the D-threonine producing enzyme and its gene is the same as described in the'new D-threonine producing enzyme and its gene'.
  • a transformant can be prepared by transforming the recombinant expression vector according to the present invention into any suitable host cell selected from the group consisting of bacteria, yeast, E. coli, fungi, plant cells, and animal cells depending on the purpose of expression.
  • the host cell may be E. coli ( E. coli BL21(DE3), DH5 ⁇ , etc.) or yeast cells ( Saccharomyces genus, Pichia genus, etc.).
  • E. coli E. coli BL21(DE3), DH5 ⁇ , etc.
  • yeast cells Saccharomyces genus, Pichia genus, etc.
  • a known technique that is, a heat shock method, an electric shock method, or the like can be used.
  • the D-threonine-producing enzyme Since the protein expressed from the transformant is the D-threonine-producing enzyme of the present invention, the D-threonine-producing enzyme can be easily mass-produced by mass-culturing the transformant to express the gene.
  • Another aspect of the present invention provides a method for preparing a D-threonine-producing enzyme using a transformant into which a recombinant expression vector containing a gene encoding a D-threonine-producing enzyme of the present invention has been introduced.
  • the method for producing a D-threonine-producing enzyme of the present invention comprises the steps of culturing a transformant into which a recombinant expression vector containing a gene encoding the D-threonine-producing enzyme of the present invention has been introduced; And separating the D-threonine-producing enzyme from the culture of the transformant.
  • the method for preparing the D-threonine-producing enzyme may further include inducing expression of a gene encoding the D-threonine-producing enzyme in the cultured transformant.
  • a gene encoding a tag for separation and purification may be additionally linked to the N-terminus of the gene encoding the D-threonine-producing enzyme, thereby making it possible to obtain a D-threonine-producing enzyme.
  • a protein cleavage enzyme cleavage site may be additionally linked to the N-terminus, and thus, purification of a recombinant D-threonine-producing enzyme may be possible.
  • the cultivation of the transformant may be performed according to a known method, and conditions such as cultivation temperature, cultivation time, and pH of the medium may be appropriately adjusted.
  • the culture method may include batch culture, continuous culture, and fed-batch culture. The culture medium used must adequately meet the requirements of the specific strain.
  • Separation of the D-threonine-producing enzyme from the culture of the transformant may be performed through a method commonly performed in the art, such as centrifugation and filtration.
  • the D-threonine-producing enzyme isolated by the above method can be purified in a conventional manner, such as salting out (eg, ammonium sulfate precipitation, sodium phosphate precipitation), solvent precipitation (protein fraction precipitation using acetone, ethanol, etc.) , Dialysis, gel filtration, ion exchange, chromatography such as reverse phase column chromatography, ultrafiltration, and the like may be used alone or in combination to purify the enzyme of the present invention.
  • Another aspect of the present invention provides a composition for producing D-threonine for producing D-threonine with high purity.
  • the composition for producing D-threonine includes the enzyme for producing D-threonine of the present invention as an active ingredient.
  • the description of the D-threonine-producing enzyme is the same as described in the'new D-threonine-producing enzyme and its gene', and the D-threonine-producing enzyme has an activity capable of producing D-threonine with high purity, A composition containing the enzyme can be used for efficient production of D-threonine.
  • the composition for producing D-threonine may further include glycine and acetaldehyde, which are substrates for D-threonine producing enzymes.
  • the glycine and acetaldehyde may be included in a sufficient amount according to the amount of desired D-threonine, and the glycine and acetaldehyde are in a ratio of 1:1 to 10:1, such as a ratio of 2:1 to 8:1, 3 It may be included in a ratio of :1 to 5:1, or in a ratio of 4:1 to 4.5:1.
  • the composition may further contain a metal cation and/or pyridoxal-5-phosphate together with the D-threonine-producing enzyme.
  • the metal cation may be a divalent metal cation, and the divalent metal cation may be a manganese cation or a magnesium cation.
  • Another aspect of the present invention provides a method for producing D-threonine in vitro.
  • the method for producing D-threonine in vitro includes the step of reacting the D-threonine producing enzyme of the present invention with glycine and acetaldehyde.
  • the description of the D-threonine-producing enzyme is the same as described in the'new D-threonine-producing enzyme and its genes'.
  • the glycine and acetaldehyde may be injected from the outside, and the glycine and acetaldehyde must be injected in a sufficient amount according to the amount of the desired D-threonine.
  • the glycine and acetaldehyde may be reacted with the enzyme in a ratio of 1:1 to 10:1, for example, in a ratio of 2:1 to 8:1, a ratio of 3:1 to 5:1, or 4:1 to It can be reacted in a ratio of 4.5:1.
  • the injection of glycine and acetaldehyde may be performed continuously.
  • D-threonine may be continuously produced by the D-threonine producing enzyme.
  • the step of reacting the D-threonine-producing enzyme with glycine and acetaldehyde includes metal cation and/or pyridoxal-5-phosphate with the D-threonine-producing enzyme to enhance the activity of the D-threonine-producing enzyme. It may be to react by including more.
  • the metal cation may be a divalent metal cation, and the divalent metal cation may be a manganese cation or a magnesium cation.
  • the reacting step may be performed at a temperature of 20° C. to 55° C., for example, 22° C. to 45° C., or 25° C. to 40° C., but is not limited thereto.
  • the reaction may be carried out at a pH of 7 to 10, such as a pH of 8 to 9.5, or a pH of 8.5 to 9, but is not limited thereto.
  • the method for producing D-threonine in vitro may further include recovering D-threonine from the reaction product of the D-threonine producing enzyme, glycine and acetaldehyde.
  • Another aspect of the present invention provides a method for producing D-threonine in vivo.
  • the method for producing D-threonine in vivo includes culturing the transformant of the present invention in the presence of glycine and acetaldehyde.
  • the description of the transformant is the same as described in the'expression vector of the D-threonine-producing enzyme, the transformant, and a method for producing a D-threonine-producing enzyme using the same.'
  • the glycine and acetaldehyde In the step of culturing the transformant in the presence of glycine and acetaldehyde, the glycine and acetaldehyde must be present in a sufficient amount according to the amount of the desired D-threonine, and exist in a ratio of 1:1 to 10:1. And, for example, in a ratio of 2:1 to 8:1, in a ratio of 3:1 to 5:1, or in a ratio of 4:1 to 4.5:1.
  • the glycine and acetaldehyde may be artificially supplied from the outside.
  • glycine and acetaldehyde may be added and supplied together in a medium used for culturing the transformant, or an environment in which glycine and acetaldehyde are produced may be provided.
  • the environment in which glycine and acetaldehyde are produced may be provided by transforming genes encoding at least one enzyme involved in the production of glycine and acetalde
  • the cultivation of the transformant as described above may be performed according to an appropriate medium and culture conditions known in the art. Those of ordinary skill in the art can easily adjust and use the medium and culture conditions according to the type of host cell of the selected transformant.
  • the culture method may include a batch type, continuous type, fed-batch type, or a combination culture thereof.
  • the medium may contain various carbon sources, nitrogen sources, and trace element components.
  • the carbon source is, for example, glucose, sucrose, lactose, fructose, maltose, starch, carbohydrates such as cellulose, soybean oil, sunflower oil, castor oil, fats such as coconut oil, palmitic acid, stearic acid, fatty acids such as linoleic acid, Alcohols such as glycerol and ethanol, organic acids such as acetic acid, or combinations thereof.
  • the cultivation may be performed using glucose as a carbon source.
  • the nitrogen source is an organic nitrogen source and urea such as peptone, yeast extract, broth, malt extract, corn steep liquor (CSL), and soybean meal, an inorganic nitrogen source such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate, or Combinations of these may be included.
  • the medium may include, for example, potassium dihydrogen phosphate and dipotassium hydrogen phosphate as a source of phosphorus, and a corresponding metal salt such as sodium-containing salt, magnesium sulfate, and iron sulfate.
  • amino acids, vitamins, and suitable precursors may be included in the medium.
  • the medium or individual components may be added to the culture medium in a batch or continuous manner.
  • the generation of air bubbles can be suppressed by using an antifoaming agent such as fatty acid polyglycol ester during culture.
  • a metal cation and/or pyridoxal-5-phosphate may be further included in the medium to enhance the activity of the D-threonine-producing enzyme of the present invention prepared by culturing by the transformant.
  • the metal cation may be a divalent metal cation, and the divalent metal cation may be a manganese cation or a magnesium cation.
  • the cultivation of the transformant as described above may be carried out at 20 °C to 50 °C, for example, it may be carried out at 25 °C to 45 °C, or 30 °C to 40 °C.
  • the cultivation of the transformant is performed in a temperature range of less than 20° C. or more than 50° C., a sufficient amount of an intermediate product is not produced, and as a result, the production amount of D-threonine, which is a final product, may not be sufficient.
  • cultivation of the transformant as described above may be performed at pH 5 to pH 10, for example, pH 6 to pH 9, or pH 6.5 to pH 8, but is not limited thereto.
  • the pH conditions for culturing the transformants as described above can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid and sulfuric acid to the culture medium of the transformant.
  • the culture pH condition of the transformant is out of the above range, the growth of the transformant is inhibited, so that the expression of the D-threonine-producing enzyme decreases, and thus the production of D-threonine may be reduced.
  • the method for producing D-threonine in vivo may further include inducing expression of a gene encoding the D-threonine-producing enzyme in the cultured transformant.
  • the method for producing D-threonine in vivo may further include recovering D-threonine from the culture of the transformant.
  • the gene of the nucleotide sequence of SEQ ID NO: 2 was synthesized by requesting Bioneer, and a primer was designed for amplifying the D-threonine sequence using the synthesized gene as a template.
  • the nucleotide sequence of SEQ ID NO: 2 was amplified by performing PCR using a pair of forward primers (5'-tggtgccgcgcggcagccatatgcgcgcaccagcacggct-3') and reverse primers (5'-ggtggtggtggtggtgctcgagctagaacacacacaacctcgcgc-3').
  • the PCR product containing the nucleotide sequence of SEQ ID NO: 2 amplified as described above using Gibson assembly (New England Biolabs, USA) was used for vector amplification forward sequence (5'-gcgcgaggttgtgtgttctagctcgagcaccaccaccaccacc-3') and reverse sequence (5' -agccgtgctggtgcgcgcatatggctgccgcgcggcacca-3') was inserted into the multiple cloning site of the amplified plasmid vector pET28a(+) (Novagen, USA).
  • Example [1-1] The transformants stored frozen in Example [1-1] were pre-inoculated in 3 ml of LK solid medium (LB medium + 2 ⁇ g/ml kanamycin) and cultured at 37° C. for 10 hours or longer.
  • LK solid medium LB medium + 2 ⁇ g/ml kanamycin
  • One colony appearing in the solid medium was inoculated into a test tube containing 3 ml of LK solid medium through colony separation, and seed culture was performed for 12 hours in a shaking incubator at 37°C.
  • 2 ml of the seed cultured culture solution was added to a 1,000 ml flask containing 200 ml of LK medium to perform main culture, and the final concentration when the absorbance at 600 nm was 0.6 was 0.1 mM.
  • the culture medium of the transformant in which the overexpression of the protein having the amino acid sequence of SEQ ID NO: 1 is induced was dispensed into 50 ml of a conical tube (SPL Life Sciences Co., Ltd., Korea), and To the pellet from which the supernatant was separated by centrifugation at 3,000 rpm for 20 minutes at 4°C, 10 ml of Profinia 1X Lysis buffer (Bio-Rad Laboratories, USA) was added, and sonication (sonication) By disrupting the cells by the method, a cell lysate of the transformant was obtained.
  • the cell lysate obtained as described above was centrifuged again at 14,000 rpm for 20 minutes at 4° C. to obtain a supernatant, and high-speed protein liquid chromatography equipped with an IMAC Kit ® His tag adsorption column (Bio-Rad Laboratories, USA)
  • a protein having an overexpressed amino acid sequence of SEQ ID NO: 1 was isolated from the supernatant obtained as described above using Fast Protein Liquid Chromatography (Bio-Rad Laboratories, USA).
  • the thus-separated protein was mixed with a 4X protein dye mixed with 100 ⁇ l of 2-mercaptoethanol and 900 ⁇ l of Laemmli Sample Buffer (#1610747, Bio-Rad Laboratories, USA) at a ratio of 3:1. , Put in boiling water and heated for 10 minutes.
  • the protein is denatured and has a linear amino acid sequence, so it can be separated according to its molecular weight through SDS-PAGE. It was confirmed that the protein having the amino acid sequence of SEQ ID NO: 1 of 40.88 kDa to which His-Tag is attached was overexpressed by the above-described series of processes, and purified and separated from the cell lysate of the transformant (Fig. 1A). .
  • the protein having the amino acid sequence of SEQ ID NO: 1 purified and isolated through Example 1 has an activity as a'D-threonine producing enzyme' as shown in the following scheme, and the activity was confirmed by reacting glycine and acetaldehyde. .
  • Example [1-2] 0.01 mg/ml of the protein having the amino acid sequence of SEQ ID NO: 1 purified and isolated in Example [1-2] was mixed with 100 mM glycine and 100 mM acetaldehyde, and 100 ⁇ M pyridoxal-5. -Added to 50 mM CHES buffer (pH 9.0) containing phosphoric acid (pyridoxal-5-phosphate, PLP) and 1 mM MnCl 2 , reacted at 37° C. for 10 minutes, and then at 100° C. for 10 minutes. After the reaction was terminated by boiling water, the concentration of D-threonine was measured in the buffer solution at which the reaction was completed.
  • phosphoric acid pyridoxal-5-phosphate
  • the generated D-threonine was analyzed through HPLC after OPA/NAC pretreatment (OPA/NAC derivatization).
  • OPA o-phthalaldelhyde
  • NAC N-acetylcysteine
  • Example 2 since the protein having the amino acid sequence of SEQ ID NO: 1 acts as a D-threonine-producing enzyme, conditions affecting its activity were identified, and the protein having the amino acid sequence of SEQ ID NO: 1 Optimal activity conditions were derived.
  • Example [1-2] Mg/ml, 100 mM glycine, 100 mM acetaldehyde, and 100 ⁇ M pyridoxal-5-phosphate in 50 mM CHES buffer (pH 9.0) containing 1 mM EDTA or metal cations (Mn 2+ , Mg 2+ ). , Co 2+ , Zn 2+ ) and reacted at 35° C. for 10 minutes, followed by bathing at 100° C. for 10 minutes to terminate the reaction. And in the same manner as in Example 2, the concentrations of D-threonine and D-allothreonine were measured in the buffer solution in which the reaction was terminated as described above, and their relative values were compared.
  • the activity of the D-threonine-producing enzyme was improved when manganese cation and magnesium cation were added among various types of metal cations, and among them, when manganese cation was used. It was confirmed that the activity of the D-threonine-producing enzyme was the most excellent (Fig. 3A).
  • Example [1-2] In order to confirm the effect of the concentration of metal cation on the enzyme activity of the protein having the amino acid sequence of SEQ ID NO: 1, 0.01 mg of the protein having the amino acid sequence of SEQ ID NO: 1 purified and isolated in Example [1-2] /Ml, 100 mM glycine, 100 mM acetaldehyde, and 100 ⁇ M pyridoxal-5-phosphate in 50 mM CHES buffer solution (pH 9.0) with manganese cations 0 mM, 0.1 mM, 0.25 mM, 0.5 mM, 1 Mm, 2.5 mM, and 5 mM were added respectively and reacted at 35° C. for 10 minutes, followed by bathing at 100° C. for 10 minutes to terminate the reaction. And in the same manner as in Example 2, the concentrations of D-threonine and D-allothreonine were measured in the buffer solution in which the reaction was terminated as described above, and their relative values were compared.
  • the protein having the amino acid sequence of SEQ ID NO: 1 exhibits the activity of the D-threonine-producing enzyme only in an environment in which a manganese cation is present (FIG. 3B).
  • Example [1-2] 100 mM of glycine and 100 mM of acetaldehyde were added to 50 mM CHES buffer (pH 9.0) containing 100 ⁇ M of pyridoxal-5-phosphoric acid and 1 mM of Mn 2+, and then at 25°C to 55°C. The reaction was performed at a temperature for 10 minutes, and a bath was performed at 100° C. for 10 minutes to terminate the reaction. And, in the same manner as in Example 2, the concentrations of D-threonine and D-allotreonine were measured in the buffer solution in which the reaction was terminated as described above, and their relative values were compared.
  • the enzymatic reaction was performed at a temperature of 35° C. for 10 minutes, and then bathed at 100° C. for 10 minutes to terminate the reaction. And in the same manner as in Example 2, the concentrations of D-threonine and D-allothreonine were measured in the buffer solution in which the reaction was terminated as described above, and their relative values were compared.
  • Example [1-2] 0 ⁇ M, 0.1 ⁇ M of pyridoxal-5-phosphate in 50 mM CHES buffer solution (pH 9.0) containing 0.01 mg/ml of protein having the sequence, 100 mM glycine, 100 mM acetaldehyde, and 1 mM Mn 2+, 0.5 ⁇ M, 2.5 ⁇ M, 5 ⁇ M, 25 ⁇ M, 50 ⁇ M, 100 ⁇ M, and 200 ⁇ M were added respectively to react at 35° C.
  • Example 3 As confirmed in Example 3, as it was confirmed that the protein having the amino acid sequence of SEQ ID NO: 1 has high activity as a D-threonine-producing enzyme, the enzyme was improved for the purpose of specifically synthesizing D-threonine. I did.
  • residues predicted to be involved in diastereomer changes in the active site in the crystal structure of the protein enzyme having the amino acid sequence of SEQ ID NO: 1 were selected, and the ratio of the activity to the diastereomer by substituting the residues with alanine Changes were analyzed.
  • residues that improve stereospecificity for D-threonine were derived, and it was confirmed that the stereospecificity for D-threonine was improved to 99% or more.
  • the protein structure having the amino acid sequence of SEQ ID NO: 1 was obtained based on the previously reported D-threonine aldolase X-ray crystal structure derived from Alcaligenes xylosoxidans (FIG. 7), and was determined. At the active site of the structure, amino acid residues that interact with substrates (glycine and acetaldehyde), pyridoxal-5-phosphate and manganese ions at a distance of 4 angstroms were selected.
  • each primer was designed based on the nucleotide sequence of SEQ ID NO: 2 and synthesized by requesting Macrogen.
  • the DNA containing the nucleotide sequence of SEQ ID NO: 2 is used as a template, and each PCR product amplified by performing PCR using each of the primer pairs is ligated using Phusion, Q5, and KOD polymerases.
  • Each of the ends of the nucleotide sequence was ligated by the kination method, and through sequencing (Macrogen, Inc.), it was confirmed that the corresponding residues were accurately substituted with nucleotide sequences encoding amino acids such as alanine.
  • the corresponding nucleotide sequences were transformed into E. coli C2566 strain (Novagen, USA), respectively, and stored frozen by adding a 20% glycerin solution.
  • Example [4-1] the transformants frozen in Example [4-1] were pre-inoculated in 3 ml of LK solid medium (LB medium + kanamycin 25 ⁇ g/ml) and at 37°C. Incubated for more than 10 hours.
  • One colony appearing in the solid medium was inoculated into a test tube containing 3 ml of LK solid medium in the process of separating a single colony, and seed culture was performed for 12 hours in a shaking incubator at 37°C.
  • 2 ml of the seed cultured culture solution was added to a 1,000 ml flask containing 200 ml of LK medium to perform main culture, and the final concentration when the absorbance at 600 nm was 0.6 was 0.1 mM.
  • IPTG isopropyl-1-thio- ⁇ -D-galactopyranoside
  • the stirring speed was adjusted to maintain 200 rpm and the culture temperature at 37°C, and after the addition of IPTG, the stirring speed was adjusted to 150 rpm and the culture temperature to 18°C, followed by incubation for 20 hours.
  • the culture medium of the transformant in which the overexpression of each of the single substituted proteins was induced was dispensed into a 50 ml conical tube (SPL Life Sciences Co., Ltd., Korea), and then at 4° C. at 3,000 rpm. By centrifuging for 20 minutes to separate the supernatant, 10 ml of Profinia 1X Lysis buffer (Bio-Rad Laboratories, USA) was added, and the cells were disrupted by sonication. , To obtain a cell lysate of the transformant (cell lysate).
  • the cell lysate obtained as described above was centrifuged again at 14,000 rpm for 20 minutes at 4° C. to obtain a supernatant, and high-speed protein liquid chromatography equipped with an IMAC Kit ® His tag adsorption column (Bio-Rad Laboratories, USA) The single substituted proteins overexpressed were isolated from the supernatant using Fast Protein Liquid Chromatography (Bio-Rad Laboratories, USA).
  • Example [4-1] The activity of each of the single substituted proteins obtained in Example [4-1] and the diastereomer ratio change of the reaction product catalyzed by them were confirmed.
  • 0.01 mg/ml of the proteins obtained in Example [4-1] were mixed with 100 mM glycine and 100 mM acetaldehyde, and 100 ⁇ M of pyridoxal-5-phosphate (pyridoxal-5).
  • -phosphate, PLP) and 1 mM MnCl 2 were added to 50 mM CHES buffer (pH 9.0), and reacted at 37° C. for 10 minutes. After the reaction was terminated by bathing at 100° C. for 10 minutes, the concentration of D-threonine was measured in the buffer solution at which the reaction was completed.
  • the amount of D-threonine produced was analyzed through HPLC after OPA/NAC pretreatment (OPA/NAC derivatization).
  • OPA o-phthalaldelhyde
  • NAC N-acetylcysteine
  • the mobile phase A and the mobile phase B were maintained at a ratio of 3:7 for 0 to 3 minutes, and the mobile phase A and mobile phase B at a ratio of 7:3 for 3 to 10 minutes.
  • commercially available D-threonine Sigma-aldrich, USA
  • D-allotreonine TOKYO CHEMICAL INDUSTRY CO., LTD., Japan
  • R147A, G179A, and S312A having the amino acid sequence of SEQ ID NO: 5, 4, and 3 have lower specific activity compared to the wild-type having the amino acid sequence of SEQ ID NO: 1, but the diastereomer excess (de) is wild-type 55.4 Compared to %, it was confirmed that it was significantly improved to 79.5%, 93.2%, and 79.5%, respectively. Therefore, it was confirmed that R147, G179, and S312 are residues involved in the diastereomer ratio change.
  • the DNA containing the nucleotide sequence of SEQ ID NO: 2 is used as a template, and each PCR product amplified by performing PCR using each of the primer pairs is ligated using Phusion, Q5, and KOD polymerases. Each end of the nucleotide sequence was ligated by the kination method, and through sequencing (Macrogen, Inc.), it was confirmed that the corresponding residues were correctly substituted with nucleotide sequences encoding amino acids of lysine, glutamine, valine, leucine, and threonine. . The corresponding nucleotide sequences were transformed into E. coli C2566 strain (Novagen, USA), respectively, and stored frozen by adding a 20% glycerin solution.
  • PCR products amplified through PCR as described above were linked to the ends of the nucleotide sequence by ligation/kination method using Phusion, Q5, and KOD polymerases, respectively, and through nucleotide sequencing (Macrogen Co., Ltd.), the corresponding It was confirmed that the residues were correctly substituted with nucleotide sequences encoding multiple alanines.
  • the corresponding nucleotide sequences were transformed into E. coli C2566 strain (Novagen, USA), respectively, and stored frozen by adding a 20% glycerin solution.
  • the protein was prepared in the same manner as in Examples [4-2] and [4-3]. Expression and purification were confirmed (FIG. 12), and enzyme reactions and analysis were performed and compared with specific activity. As a result, it was confirmed that when both amino acid residues G179 and S312 were substituted with alanine, the diastereomer excess rate (d.e.) increased to 99.4%, thereby improving the diastereomer specificity (FIG. 13). Therefore, when both G179 and S312 amino acids were substituted with alanine, it was confirmed that D-threonine-specific production activity was improved.
  • Example [4-3] 0.01 mg/ml of wild-type, R147A, G179A, and S312A enzymes each having the purified amino acid sequence of SEQ ID NOs: 1, 5, 4, 3 were added to 50 mM CHES (pH 9.0. ), 0.1 mM pyridoxal-5-phosphate, 1 mM manganese ion, and reacted under the conditions of 35° C., and 0, 1, 2.5, 5, 10, 25, 50, and 100 mM of D-threonine or D-allo When reacted with threonine for 10 minutes, the degree of decomposition thereof was measured by HPLC.
  • Example [4-2] 50 mM CHES buffer solution (pH 9.0) containing 100 ⁇ M of -5-phosphate and 1 mM Mn 2+ was reacted at 35° C. for 15, 30, 60, 90, 120, 150, and 180 minutes. And in order to terminate the reaction, the reaction solution was bathed at 100° C. for 10 minutes, and through HPLC analysis via OPA/NAC derivatization, which is the same method as in Example [4-3], D-threonine and D- The concentration of allothreonine was measured and its relative values were compared.
  • D-threonine was specifically produced in the enzyme G179A and the enzyme G179A+S312A having the amino acid sequences of SEQ ID NOs: 4 and 10 compared to the wild type having the amino acid sequence of SEQ ID NO: 1. It was confirmed that the enzyme G179A + S312A having the amino acid sequence of produced 46 mM D-threonine at a ratio of 95% diastereomeric excess in 60 minutes (FIG. 16).
  • the enzymes having the amino acid sequences of SEQ ID NOs: 4 and 10 of the present invention each produced 16.4 mM and 18.7 mM D-threonine in 10 minutes, respectively, 95% and 98% diastereomers. It can be specifically synthesized with an excess of isomers.
  • the enzyme having the amino acid sequence of SEQ ID NO: 10 (G179A+S312A) is capable of synthesizing D-threonine with the same level of purity as commercially available D-threonine. -It has the best specificity for threonine.

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Abstract

La présente invention concerne une nouvelle enzyme pour la production de D-thréonine. Lorsque des réactions sont effectuées à l'aide de l'enzyme de la présente invention, le ration en diastéréoisomère D-allo-thréonine, pouvant être produit conjointement avec de la D-thréonine, est abaissé et une D-thréonine de pureté supérieure peut être obtenue.
PCT/KR2020/014292 2019-10-23 2020-10-20 Nouvelle enzyme pour la production de d-thréonine, et procédé de production stéréospécifique de d-thréonine l'utilisant Ceased WO2021080277A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
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CN115109769A (zh) * 2022-05-20 2022-09-27 华东理工大学 一种l-苏氨酸醛缩酶突变体以及在l-丝氨酸合成中的应用

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DATABASE Nucleotide GenBank; ANONYMOUS: "Candidatus Filomicrobium marinum strain Y genome assembly, chromosome: 1", XP055805807, retrieved from NCBI *
DATABASE Protein GenBank; ANONYMOUS: "MULTISPECIES: DSD1 family PLP-dependent enzyme [Filomicrobium]", XP055805805, retrieved from NCBI *
HENRIQUES ANA C., DE MARCO PAOLO: "Complete Genome Sequences of Two Strains of " Candidatus Filomicrobium marinum," a Methanesulfonate-Degrading Species", GENOME ANNOUNCEMENTS, vol. 3, no. 3, 25 June 2015 (2015-06-25), XP055805802, DOI: 10.1128/genomeA.00160-15 *
QIJIA CHEN, CHEN XI, CUI YUNFENG, REN JIE, LU WEI, FENG JINHUI, WU QIAQING, ZHU DUNMING: "A new D-threonine aldolase as a promising biocatalyst for highly stereoselective preparation of chiral aromatic β-hydroxy-α-amino acids", CATALYSIS SCIENCE & TECHNOLOGY, ROYAL SOCIETY OF CHEMISTRY, UK, vol. 7, no. 24, 1 January 2017 (2017-01-01), UK, pages 5964 - 5973, XP055695514, ISSN: 2044-4753, DOI: 10.1039/C7CY01774J *
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115109769A (zh) * 2022-05-20 2022-09-27 华东理工大学 一种l-苏氨酸醛缩酶突变体以及在l-丝氨酸合成中的应用
CN115109769B (zh) * 2022-05-20 2023-07-14 华东理工大学 一种l-苏氨酸醛缩酶突变体以及在l-丝氨酸合成中的应用

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