WO2022231037A1 - Nouveau variant et procédé de production d'imp faisant appel à celui-ci - Google Patents
Nouveau variant et procédé de production d'imp faisant appel à celui-ci Download PDFInfo
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- WO2022231037A1 WO2022231037A1 PCT/KR2021/005459 KR2021005459W WO2022231037A1 WO 2022231037 A1 WO2022231037 A1 WO 2022231037A1 KR 2021005459 W KR2021005459 W KR 2021005459W WO 2022231037 A1 WO2022231037 A1 WO 2022231037A1
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Classifications
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- C—CHEMISTRY; METALLURGY
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
- C12N15/77—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Corynebacterium; for Brevibacterium
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- C12N9/0004—Oxidoreductases (1.)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N9/10—Transferases (2.)
- C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
- C12P19/28—N-glycosides
- C12P19/30—Nucleotides
- C12P19/32—Nucleotides having a condensed ring system containing a six-membered ring having two N-atoms in the same ring, e.g. purine nucleotides, nicotineamide-adenine dinucleotide
Definitions
- the present application relates to a novel variant, a Corynebacterium stasis strain comprising the variant, and an IMP production method using the strain.
- the present inventors completed the present application by developing a novel variant that increases IMP production, a Corynebacterium stationis strain including the variant, and an IMP production method using the strain.
- An object of the present application is (i) one or more variants of the present application (ii) one or more polynucleotides encoding the variant, or (iii) a combination thereof, and having IMP (5'-inosine monophosphate) producing ability , Corynebacterium station is to provide a strain ( Corynebacterium stationis ).
- Another object of the present application is to include (i) one or more variants of the present application (ii) one or more polynucleotides encoding the variant, or (iii) a combination thereof, and having IMP-producing ability, Corynebacter It is to provide a method for producing an IMP, comprising the step of culturing the strain of Leeum stationionis in a medium.
- One aspect of the present application is (i) any one or more variants selected from the group consisting of the following (a) to (o), (ii) one or more polynucleotides encoding the variant, or (iii) a combination thereof It provides a Corynebacterium stationionis strain comprising a.
- a 1,4-alpha-glucan-branching enzyme variant consisting of the amino acid sequence set forth in SEQ ID NO: 17, wherein aspartic acid, an amino acid corresponding to the 6th position of SEQ ID NO: 19, is substituted with asparagine;
- glucosamine-6-phosphate deaminase variant consisting of the amino acid sequence shown in SEQ ID NO: 25 in which alanine, an amino acid corresponding to position 157 of SEQ ID NO: 27, is substituted with valine;
- a peptide methionine sulfoxide reductase variant consisting of the amino acid sequence shown in SEQ ID NO: 33 in which alanine, an amino acid corresponding to the 6th position of SEQ ID NO: 35, is substituted with valine;
- (k) a formate-dependent phosphoribosylglycinamide formyltransferase variant consisting of the amino acid sequence set forth in SEQ ID NO: 41 in which valine, an amino acid corresponding to position 255 of SEQ ID NO: 43, is substituted with isoleucine;
- one or more is 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, or 15 or more, but is not limited thereto.
- the strain of the present application is (i) 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more selected from the group consisting of (a) to (o), 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, or 15 or more variants, (ii) 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more encoding the variant or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, or 15 or more polynucleotides, or (iii) a combination thereof, but is not limited thereto.
- the variant and the variant of (ii) above may be selected from the group consisting of (a) to (o), but may be the same or different, but is not limited thereto.
- the strain of the present application may include (a) a polynucleotide encoding the variant; and (b) a strain comprising a combination of variants, but is not limited thereto.
- variants of the present application are SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 21, SEQ ID NO: 25, SEQ ID NO: 29, SEQ ID NO: 33, SEQ ID NO: 37, SEQ ID NO: 41, SEQ ID NO: 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, or 15 or more, or may consist essentially of the amino acid sequence.
- variant of the present application may include any one or more amino acid sequences selected from the group consisting of the following (aa) to (oo).
- amino acid sequence set forth in SEQ ID NO: 1 the amino acid corresponding to position 526 based on the amino acid sequence of SEQ ID NO: 3 is isoleucine, and at least 70%, 75%, 80% of the amino acid sequence set forth in SEQ ID NO: 1 , an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% homology or identity;
- the amino acid corresponding to position 209 based on the amino acid sequence of SEQ ID NO: 7 in the amino acid sequence set forth in SEQ ID NO: 5 is methionine, and at least 70%, 75%, 80% of the amino acid sequence set forth in SEQ ID NO: 5 , an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% homology or identity;
- amino acid corresponding to position 130 based on the amino acid sequence of SEQ ID NO: 11 in the amino acid sequence set forth in SEQ ID NO: 9 is aspartic acid, and at least 70%, 75%, 80% with the amino acid sequence set forth in SEQ ID NO: 9 an amino acid sequence having at least %, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% homology or identity;
- the amino acid corresponding to position 232 based on the amino acid sequence of SEQ ID NO: 15 is phenylalanine, and at least 70%, 75%, 80% of the amino acid sequence set forth in SEQ ID NO: 13 , an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% homology or identity;
- the amino acid corresponding to position 6 based on the amino acid sequence of SEQ ID NO: 19 is asparagine, and at least 70%, 75%, 80% of the amino acid sequence set forth in SEQ ID NO: 17 , an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% homology or identity;
- amino acid corresponding to position 294 based on the amino acid sequence of SEQ ID NO: 23 in the amino acid sequence set forth in SEQ ID NO: 21 is asparagine, and at least 70%, 75%, 80% of the amino acid sequence set forth in SEQ ID NO: 21 , an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% homology or identity;
- (gg) the amino acid corresponding to position 157 based on the amino acid sequence of SEQ ID NO: 27 in the amino acid sequence set forth in SEQ ID NO: 25 is valine, and at least 70%, 75%, 80% of the amino acid sequence set forth in SEQ ID NO: 25 , an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% homology or identity;
- amino acid corresponding to position 80 based on the amino acid sequence of SEQ ID NO: 31 is threonine, and at least 70%, 75%, 80% of the amino acid sequence set forth in SEQ ID NO: 29 , an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% homology or identity;
- the amino acid corresponding to position 6 based on the amino acid sequence of SEQ ID NO: 35 is valine, and at least 70%, 75%, 80% of the amino acid sequence set forth in SEQ ID NO: 33 , an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% homology or identity;
- amino acid corresponding to position 425 based on the amino acid sequence of SEQ ID NO: 39 is asparagine, and at least 70%, 75%, 80% from the amino acid sequence set forth in SEQ ID NO: 37 , an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% homology or identity;
- the amino acid corresponding to position 255 based on the amino acid sequence of SEQ ID NO: 43 in the amino acid sequence set forth in SEQ ID NO: 41 is isoleucine, and at least 70%, 75%, 80% of the amino acid sequence set forth in SEQ ID NO: 41 , an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% homology or identity;
- the amino acid corresponding to position 351 based on the amino acid sequence of SEQ ID NO: 47 in the amino acid sequence set forth in SEQ ID NO: 45 is valine, and at least 70%, 75%, 80% of the amino acid sequence set forth in SEQ ID NO: 45 , an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% homology or identity;
- the amino acid corresponding to position 172 based on the amino acid sequence of SEQ ID NO: 51 in the amino acid sequence set forth in SEQ ID NO: 49 is aspartic acid, and at least 70%, 75%, 80% with the amino acid sequence set forth in SEQ ID NO: 49 an amino acid sequence having at least %, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% homology or identity;
- amino acid corresponding to position 122 based on the amino acid sequence of SEQ ID NO: 55 in the amino acid sequence set forth in SEQ ID NO: 53 is threonine, and at least 70%, 75%, 80% of the amino acid sequence set forth in SEQ ID NO: 53 , an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% homology or identity;
- the amino acid corresponding to position 108 based on the amino acid sequence of SEQ ID NO: 59 in the amino acid sequence set forth in SEQ ID NO: 57 is serine, and at least 70%, 75%, 80% of the amino acid sequence set forth in SEQ ID NO: 57 , an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% homology or identity;
- variants having an amino acid sequence in which some sequences are deleted, modified, substituted, conservatively substituted or added are also included within the scope of the present application. is self-evident
- sequence additions or deletions naturally occurring mutations, silent mutations or conservation within the N-terminus, C-terminal and/or within the amino acid sequence that do not alter the function of the variants of the present application It is a case of having an enemy substitution.
- conservative substitution means substituting an amino acid for another amino acid having similar structural and/or chemical properties. Such amino acid substitutions may generally occur based on similarity in the polarity, charge, solubility, hydrophobicity, hydrophilicity and/or amphipathic nature of the residues. Typically, conservative substitutions may have little or no effect on the activity of the protein or polypeptide.
- variant means that one or more amino acids are conservatively substituted and/or modified so that they differ from the amino acid sequence before the mutation of the variant, but have functions or properties. refers to a polypeptide that is maintained. Such variants can generally be identified by modifying one or more amino acids in the amino acid sequence of the polypeptide and evaluating the properties of the modified polypeptide. That is, the ability of the variant may be increased, unchanged, or decreased compared to the polypeptide before the mutation. In addition, some variants may include variants in which one or more portions, such as an N-terminal leader sequence or a transmembrane domain, have been removed.
- variants may include variants in which a portion is removed from the N- and/or C-terminus of the mature protein.
- variant may be used interchangeably with terms such as mutant, modified, mutant polypeptide, mutated protein, mutant and mutant (in English, modified, modified polypeptide, modified protein, mutant, mutein, divergent, etc.) and, as long as it is a term used in a mutated sense, it is not limited thereto.
- the variant includes a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 in which threonine, an amino acid corresponding to position 526 of the amino acid sequence of SEQ ID NO: 3, is substituted with isoleucine; a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 5 in which valine, an amino acid corresponding to position 209 of the amino acid sequence of SEQ ID NO: 7, is substituted with methionine; a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 9 in which glycine, an amino acid corresponding to position 130 of the amino acid sequence of SEQ ID NO: 11, is substituted with aspartic acid; a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 13 in which serine, an amino acid corresponding to position 232 of the amino acid sequence of SEQ ID NO: 15, is substituted with phenylalanine; a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 in which th
- variants may include deletions or additions of amino acids that have minimal effect on the properties and secondary structure of the polypeptide.
- a signal (or leader) sequence involved in protein translocation may be conjugated to the N-terminus of the mutant, either co-translationally or post-translationally.
- the variants may also be conjugated with other sequences or linkers for identification, purification, or synthesis.
- the term 'homology' or 'identity' refers to the degree of similarity between two given amino acid sequences or nucleotide sequences and may be expressed as a percentage.
- the terms homology and identity can often be used interchangeably.
- Sequence homology or identity of a conserved polynucleotide or polypeptide is determined by standard alignment algorithms, with default gap penalties established by the program used may be used. Substantially homologous or identical sequences are generally capable of hybridizing with all or part of a sequence under moderate or high stringent conditions. It is apparent that hybridization also includes hybridization with polynucleotides containing common codons or codons taking codon degeneracy into account in the polynucleotide.
- a GAP program can be defined as the total number of symbols in the shorter of the two sequences divided by the number of similarly aligned symbols (ie, nucleotides or amino acids).
- Default parameters for the GAP program are: (1) a binary comparison matrix (containing values of 1 for identity and 0 for non-identity) and Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation , pp. 353-358 (1979), Gribskov et al (1986) Nucl. Acids Res. 14: weighted comparison matrix of 6745 (or EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix); (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap (or a gap open penalty of 10, a gap extension penalty of 0.5); and (3) no penalty for end gaps.
- the variants of the present application are 3D-(3,5/4)-trihydroxycyclohexane-1,2-dione acylhydrolase, anaerobic coproporphyrinogen III oxidase, D -alanine-D-alanine ligase, formamidopyrimidine-DNA glycosylase, 1,4-alpha-glucan-branching enzyme, formimidoylglutamase, glucosamine-6-phosphate deaminase, selenide, Water dikinase, peptide methionine sulfoxide reductase, phytoene desaturase, formate-dependent phosphoribosylglycinamide formyltransferase, bifunctional phosphoribosylaminoimidazolecarboxamide formyltransferase/IMP cyclohydrola ase, 5-(carboxyamino)imidazolibonucleotide synthe
- one or more variants or a combination thereof of the present application may have an activity to increase the IMP production capacity compared to the wild-type polypeptide.
- the term "3D-(3,5/4)-trihydroxycyclohexane-1,2-dione acylhydrolase (acetolactate synthase)" is a polypeptide having an activity of catalyzing hydrolysis.
- 3D-(3,5/4)-trihydroxycyclohexane-1,2-dione acylhydrolase of the present application (3D-(3,5/4)-trihydroxycyclohexane-1,2- dione acylhydrolase) can be used in combination with IolD.
- sequence of the 3D-(3,5/4)-trihydroxycyclohexane-1,2-dione acylhydrolase can be obtained from GenBank of NCBI, a known database. Specifically, it may be a polypeptide having 3D-(3,5/4)-trihydroxycyclohexane-1,2-dione acylhydrolase activity encoded by iolD , but is not limited thereto.
- anaerobic coproporphyrinogen III oxidase is an oxygen-independent, decarboxylation of coproporphyrinogen III to produce protoporphyrinogen IV. It is a polypeptide having a catalytic activity.
- the anaerobic coproporphyrinogen III oxidase of the present application is coproporphyrinogen-III oxidase, coproporphyrinogen-III oxidase or HemN.
- anaerobic coproporphyrinogen III oxidase can obtain its sequence from NCBI's GenBank, a known database.
- anaerobic coproporphyrinogen III encoded by hemN It may be a polypeptide having oxidase activity, but is not limited thereto.
- D-alanine-D-alanine ligase (D-alanine--D-alanine ligase) is derived from two D-alanine Alanyl-D-alanine) is a polypeptide having an activity to catalyze production.
- D-alanine-D-alanine ligase of the present application may be used in combination with D-alanine-D-alanine ligase or Ddl.
- the sequence of the D-alanine-D-alanine ligase can be obtained from GenBank of NCBI, a known database. Specifically, it may be a polypeptide having D-alanine-D-alanine ligase activity encoded by ddl , but is not limited thereto.
- the term "formamidopyrimidine-DNA glycosylase” is involved in base excision repair, and catalyzes recognition and removal of oxidized purines in damaged DNA. It is a polypeptide having the activity of Specifically, the formamidopyrimidine-DNA glycosylase of the present application is DNA glycosylase, DNA-formamidopyrimidine glycosylase, DNA-formamidopyrimidine glycosidase (formamidopyrimidine-DNA glycosidase), Alternatively, it may be used in combination with MutM.
- the formamidopyrimidine-DNA glycosylase sequence can be obtained from GenBank of NCBI, a known database. Specifically, it may be a polypeptide having a formamidopyrimidine-DNA glycosylase activity encoded by mutM , but is not limited thereto.
- 1,4-alpha-glucan-branching enzyme refers to an alpha-1,6-glycosidic bond having an activity of catalyzing the branching of glycogen. is a polypeptide.
- the 1,4-alpha-glucan-branching enzyme of the present application may be used in combination as glycogen-branching enzyme, branching enzyme or GlgB.
- the sequence of the 1,4-alpha-glucan-branching enzyme can be obtained from GenBank of NCBI, a known database. Specifically, it may be a polypeptide having a 1,4-alpha-glucan-branching enzyme activity encoded by glgB , but is not limited thereto.
- the term “formimidoylglutamase” refers to N-formimidoyl-L-glutamate and water as substrates L-glutamate and It is a polypeptide that produces formamide.
- the formimidoyl glutamase of the present application is formiminoglutamase (Formiminoglutamase), N-formiminoglutamate hydrolase (N-formiminoglutamate hydrolase), N-formimino-L- glutamate formimino Hydrolase (N-formimino-L-glutamate formiminohydrolase), N-formimidoyl-L-glutamate formimidoyl hydrolase (N-formimidoyl-L-glutamate formimidoylhydrolase) or HutG may be used in combination.
- the sequence of the formimidoyl glutamase can be obtained from GenBank of NCBI, which is a known database. Specifically, it may be a polypeptide having a formimidoylglutamase activity encoded by hutG , but is not limited thereto.
- glucosamine-6-phosphate deaminase refers to fructose 6-phosphate and NH 3 using glucosamine-6-phosphate and water as substrates. producing polypeptides.
- the glucosamine-6-phosphate deaminase of the present application is 2-amino-2-deoxy-D-glucose-6-phosphate aminohydrolase (2-amino-2-deoxy-D-glucose-6-phosphate aminohydrolase) (Ketol isomerizing), Glucosaminephosphate isomerase, Glucosamine-6-phosphate isomerase, Phosphoglucosaminisomerase, Glucosamine phosphate deaminase Glucosamine phosphate deaminase), aminodeoxyglucosephosphate isomerase, phosphoglucosamine isomerase, or NagB may be used in combination.
- the sequence of the glucosamine-6-phosphate deaminase can be obtained from GenBank of NCBI, a known database. Specifically, it may be a polypeptide having glucosamine-6-phosphate deaminase activity encoded by nagB , but is not limited thereto.
- the term "selenide, water dikinase (Selenide, water dikinase)” is adenosine triphosphate (Adenosine triphosphate, ATP), selenide (Selenide) and water as a substrate adenosine monophosphate (Adenosine monophosphate, AMP), It is a polypeptide that produces selenophosphate and phosphate.
- selenide and water dikinase of the present application may be used in combination with ATP: selenide, water phosphotransferase (ATP: Selenide, water phosphotransferase), selenophosphate synthetase or SelD.
- the selenide and water dikinase sequences can be obtained from NCBI's GenBank, a known database. Specifically, it may be a polypeptide having selenide and water dikinase activity encoded by selD , but is not limited thereto.
- peptide methionine sulfoxide reductase is a polypeptide that reduces methionine sulfoxide to methionine in a protein.
- the peptide methionine sulfoxide reductase of the present application may be used in combination with methionine sulfoxide reductase or MsrA.
- the sequence of the peptide methionine sulfoxide reductase can be obtained from GenBank of NCBI, a known database. Specifically, it may be a polypeptide having a peptide methionine sulfoxide reductase activity encoded by msrA , but is not limited thereto.
- phytoene desaturase refers to a colorless 15-cis-phytoene in a biochemical pathway called a poly-trans pathway to a bright red color. It is a polypeptide that converts to lycopene.
- the phytoene desaturase of the present application may be used in combination with phytoene desaturase or CrtI.
- the sequence of the phytoene desaturase can be obtained from GenBank of NCBI, which is a known database. Specifically, it may be a polypeptide having a phytoene desaturase activity encoded by crtI , but is not limited thereto.
- formate-dependent phosphoribosylglycinamide formyltransferase refers to 10-formyltetrahydrofolate + N1-(5-phospho-D-ribosyl)glycinamide ⁇ tetrahydrofolate + N2-formyl-N1 -(5-phospho-D-ribosyl)glycinamide has the activity of catalyzing the chemical reaction.
- formate-dependent phosphoribosylglycinamide formyltransferase of the present application may be used interchangeably with PurT.
- the formate-dependent phosphoribosylglycinamide formyltransferase may have its sequence obtained from NCBI's GenBank, a known database. Specifically, it may be a polypeptide having a formate-dependent phosphoribosylglycinamide formyltransferase activity encoded by purT , but is not limited thereto.
- bifunctional phosphoribosylaminoimidazolecarboxamide formyltransferase/IMP cyclohydrolase refers to 10-formyltetrahydrofolate + 5-amino-1-(5 Phosphoribosylaminoimidazolecarboxamide formyltransferase, which catalyzes the chemical reaction of -phospho-D-ribosyl)imidazole-4-carboxamide ⁇ tetrahydrofolate + 5-formamido-1-(5-phospho-D-ribosyl)imidazole-4-carboxamide (EC:2.1.2.3) and IMP + H 2 O ⁇ 5-formamido-1-(5-phospho-D-ribosyl)imidazole-4-carboxamide catalyzing the chemical reaction of IMP cyclohydrolase (EC:3.5.4.10) containing two domains of activity.
- the bifunctional phosphoribosylaminoimidazolecarboxamide formyltransferase/IMP cyclohydrolase of the present application may be used in combination with PurH.
- the sequence of the bifunctional phosphoribosylaminoimidazolecarboxamide formyltransferase/IMP cyclohydrolase can be obtained from GenBank of NCBI, a known database. Specifically, it may be a polypeptide having bifunctional phosphoribosylaminoimidazolecarboxamide formyltransferase/IMP cyclohydrolase activity encoded by purH , but is not limited thereto.
- 5- (carboxyamino) imidazole ribonucleotide synthase is ATP + 5-amino-1- (5-phospho-D-ribosyl) imidazole + HCO 3 - ⁇
- 5-(carboxyamino)imidazole ribonucleotide synthase 5-(carboxyamino)imidazole ribonucleotide synthase) of the present application may be used interchangeably with PurK.
- the sequence of the 5-(carboxyamino)imidazolibonucleotide synthetase can be obtained from GenBank of NCBI, which is a known database. Specifically, it may be a polypeptide having 5-(carboxyamino)imidazolibonucleotide synthetase activity encoded by purK , but is not limited thereto.
- adenine phosphoribosyltransferase catalyzes the salvage reaction of AMP formation, which is less energy-consuming than de novo synthesis.
- adenine phosphoribosyltransferase of the present application may be used in combination with Apt.
- the sequence of the adenine phosphoribosyl group transferase can be obtained from GenBank of NCBI, a known database. Specifically, it may be a polypeptide having an adenine phosphoribosyl group transferase activity encoded by apt , but is not limited thereto.
- bifunctional pyr operon transcriptional regulator/uracil phosphoribosyltransferase PyrR refers to a pyrimidine nucleotide (pyr) operon in a way that depends on uridine. Controls the transcriptional attenuation of Specifically, the bifunctional pyr operon transcriptional regulator/uracil phosphoribosyltransferase PyrR (bifunctional pyr operon transcriptional regulator/uracil phosphoribosyltransferase PyrR) of the present application may be used interchangeably with PyrR.
- the bifunctional pyr operon transcriptional regulator/uracil phosphoribosyl transfer enzyme PyrR can be sequenced from GenBank of NCBI, a known database. Specifically, it may be a polypeptide having a bifunctional pyr operon transcriptional regulator/uracil phosphoribosyl transfer enzyme PyrR activity encoded by pyrR , but is not limited thereto.
- corresponding to refers to an amino acid residue at a listed position in a polypeptide, or to an amino acid residue that is similar, identical or homologous to a listed residue in a polypeptide. Identifying an amino acid at a corresponding position may be determining a specific amino acid in a sequence that refers to a specific sequence.
- corresponding region generally refers to a similar or corresponding position in a related protein or reference protein.
- any amino acid sequence is aligned with SEQ ID NO: 3, and based on this, each amino acid residue of the amino acid sequence can be numbered with reference to the numerical position of the amino acid residue corresponding to the amino acid residue of SEQ ID NO: 3 .
- a sequence alignment algorithm such as that described in this application can identify the position of an amino acid, or a position at which modifications, such as substitutions, insertions, or deletions, occur compared to a query sequence (also referred to as a "reference sequence").
- Such alignments include, for example, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453), the Needleman program in the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al. , 2000), Trends Genet. 16: 276-277), etc., but is not limited thereto, and a sequence alignment program known in the art, a pairwise sequence comparison algorithm, etc. may be appropriately used.
- polynucleotide refers to a DNA or RNA strand of a certain length or longer as a polymer of nucleotides in which nucleotide monomers are linked in a long chain by covalent bonds, and more specifically, encoding the variant. polynucleotide fragments.
- Polynucleotides encoding the variants of the present application are SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 21, SEQ ID NO: 25, SEQ ID NO: 29, SEQ ID NO: 33, SEQ ID NO: 37, SEQ ID NO: 41, SEQ ID NO: 45, SEQ ID NO: 49, SEQ ID NO: 53, and may include one or more base sequences encoding any one or more of the amino acid sequences set forth in SEQ ID NO: 57.
- the polynucleotide of the present application is SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 22, SEQ ID NO: 26, SEQ ID NO: 30, SEQ ID NO: 34, SEQ ID NO: 38, SEQ ID NO: 42, SEQ ID NO: 46, SEQ ID NO: 50, SEQ ID NO: 54, and SEQ ID NO: 58 may have, comprise, consist, or consist essentially of any one or more sequences.
- the polynucleotides of the present application are various in the coding region within the range that does not change the amino acid sequence of the variants of the present application. Deformation can be made.
- polynucleotide of the present application is SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 22, SEQ ID NO: 26, SEQ ID NO: 30, SEQ ID NO: 34, SEQ ID NO: 38, SEQ ID NO: SEQ ID NO: 42, SEQ ID NO: 46, SEQ ID NO: 50, SEQ ID NO: 54, and at least 70%, at least 75%, at least 80%, at least 85%, at least 90% homology or identity to any one or more sequences of SEQ ID NO: 58; having or comprising a nucleotide sequence that is 95% or more, 96% or more, 97% or more, 98% or more, and less than 100%, or SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 18, sequence SEQ ID NO: 22, SEQ ID NO: 26, SEQ ID NO: 30, SEQ ID NO: 34, SEQ ID NO:
- the codon encoding the amino acid corresponding to the 526th position of SEQ ID NO: 1 is one of the codons encoding isoleucine;
- the codon encoding the amino acid corresponding to position 209 of SEQ ID NO: 5 is one of the codons encoding methionine;
- the codon encoding the amino acid corresponding to position 130 of SEQ ID NO: 9 is one of the codons encoding aspartic acid;
- the codon encoding the amino acid corresponding to position 232 of SEQ ID NO: 13 is one of the codons encoding phenylalanine;
- the codon encoding the amino acid corresponding to the 6th position of SEQ ID NO: 17 is one of the codons encoding asparagine;
- the codon encoding the amino acid corresponding to position 294 of SEQ ID NO: 21 is one of the codons encoding asparagine;
- polynucleotide of the present application may be included without limitation as long as it can hybridize under stringent conditions with a probe that can be prepared from a known gene sequence, for example, a sequence complementary to all or part of the polynucleotide sequence of the present application.
- stringent condition means a condition that enables specific hybridization between polynucleotides. These conditions are described in J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989; F.M. Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, 9.50-9.51, 11.7-11.8).
- polynucleotides with high homology or identity 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or a condition in which polynucleotides having 99% or more homology or identity hybridize with each other and polynucleotides with lower homology or identity do not hybridize, or a washing condition of conventional Southern hybridization at 60°C, 1 ⁇ SSC, 0.1% SDS, specifically 60° C., 0.1 ⁇ SSC, 0.1% SDS, more specifically 68° C., 0.1 ⁇ SSC, 0.1% SDS at a salt concentration and temperature equivalent to one wash, specifically two to three times conditions can be enumerated.
- Hybridization requires that two nucleic acids have complementary sequences, although mismatch between bases is possible depending on the stringency of hybridization.
- complementary is used to describe the relationship between nucleotide bases capable of hybridizing to each other.
- adenine is complementary to thymine
- cytosine is complementary to guanine.
- the polynucleotides of the present application may also include substantially similar nucleic acid sequences as well as isolated nucleic acid fragments complementary to the overall sequence.
- a polynucleotide having homology or identity to the polynucleotide of the present application can be detected using the hybridization conditions including a hybridization step at a Tm value of 55°C and using the above-described conditions.
- the Tm value may be 60 °C, 63 °C, or 65 °C, but is not limited thereto and may be appropriately adjusted by those skilled in the art according to the purpose.
- the appropriate stringency for hybridizing the polynucleotides depends on the length of the polynucleotides and the degree of complementarity, and the parameters are well known in the art (eg, J. Sambrook et al., supra).
- the vector of the present application may include a DNA preparation comprising the nucleotide sequence of a polynucleotide encoding the target polypeptide operably linked to a suitable expression control region (or expression control sequence) so that the target polypeptide can be expressed in a suitable host.
- the expression control region may include a promoter capable of initiating transcription, an optional operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence regulating the termination of transcription and translation.
- the vector After transformation into an appropriate host cell, the vector can replicate or function independently of the host genome, and can be integrated into the genome itself.
- the vector used in the present application is not particularly limited, and any vector known in the art may be used.
- Examples of commonly used vectors include plasmids, cosmids, viruses and bacteriophages in a natural or recombinant state.
- pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A may be used as phage vectors or cosmid vectors, and pDZ-based, pBR-based, and pUC-based plasmid vectors may be used.
- pBluescript II-based pGEM-based, pTZ-based, pCL-based, pET-based and the like
- pDZ pDC, pDCM2, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vectors and the like
- pC1BAC vectors and the like can be used.
- a polynucleotide encoding a target polypeptide may be inserted into a chromosome through a vector for intracellular chromosome insertion.
- the insertion of the polynucleotide into the chromosome may be performed by any method known in the art, for example, homologous recombination, but is not limited thereto.
- It may further include a selection marker (selection marker) for confirming whether the chromosome is inserted.
- the selection marker is used to select cells transformed with the vector, that is, to determine whether a target nucleic acid molecule is inserted, and selectable phenotypes such as drug resistance, auxotrophy, resistance to cytotoxic agents, or surface polypeptide expression. Markers to be given can be used. In an environment treated with a selective agent, only the cells expressing the selectable marker survive or exhibit other expression traits, so that the transformed cells can be selected.
- the term "transformation” refers to introducing a vector including a polynucleotide encoding a target polypeptide into a host cell or microorganism so that the polypeptide encoded by the polynucleotide can be expressed in the host cell.
- the transformed polynucleotide may include all of them regardless of whether they are inserted into the chromosome of the host cell or located outside the chromosome, as long as they can be expressed in the host cell.
- the polynucleotide includes DNA and/or RNA encoding a target polypeptide.
- the polynucleotide may be introduced in any form as long as it can be introduced and expressed into a host cell.
- the polynucleotide may be introduced into a host cell in the form of an expression cassette, which is a gene construct including all elements necessary for self-expression.
- the expression cassette may include a promoter operably linked to the polynucleotide, a transcription termination signal, a ribosome binding site, and a translation termination signal.
- the expression cassette may be in the form of an expression vector capable of self-replication.
- the polynucleotide may be introduced into a host cell in its own form and operably linked to a sequence required for expression in the host cell, but is not limited thereto.
- operably linked means that a promoter sequence that initiates and mediates transcription of a polynucleotide encoding the target variant of the present application and the polynucleotide sequence are functionally linked.
- polypeptides 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, or 15 of the present application
- One or more variant polypeptides 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more encoding the polypeptide 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, or 15 or more polynucleotides, including polynucleotides of the present application or more, 13 or more, 14 or more, or 15 or more vectors, or combinations thereof.
- strain or microorganism
- strain includes both wild-type microorganisms and microorganisms in which genetic modification has occurred naturally or artificially.
- a specific mechanism is weakened or enhanced as a microorganism, and may be a microorganism including genetic modification for the production of a desired polypeptide, protein or product.
- the strain of the present application includes a strain comprising any one or more of one or more variants of the present application, one or more polynucleotides of the present application, and one or more vectors comprising the polynucleotides of the present application; strains modified to express one or more variants of the present application or one or more polynucleotides of the present application; a strain expressing one or more variants of the present application, or one or more polynucleotides of the present application (eg, a recombinant strain); Or it may be a strain (eg, a recombinant strain) having one or more variant activities of the present application, but is not limited thereto.
- the strain of the present application may be a strain having IMP-producing ability.
- the strain of the present application is naturally 3D-(3,5/4)-trihydroxycyclohexane-1,2-dione acylhydrolase, anaerobic coproporphyrinogen III oxidase, D-alanine-D -alanine ligase, formamidopyrimidine-DNA glycosylase, 1,4-alpha-glucan-branching enzyme, formimidoylglutamase, glucosamine-6-phosphate deaminase, selenide, water dikinase, Peptide methionine sulfoxide reductase, phytoene desaturase, formate-dependent phosphoribosylglycinamide formyltransferase, bifunctional phosphoribosylaminoimidazolecarboxamide formyltransferase/IMP cyclohydrolase, 5- (carboxyamino) imidazolibonucleotide synthetase, adenine
- the strain of the present application is a cell or microorganism that is transformed with one or more polynucleotides encoding one or more variants of the present application or one or more vectors comprising the same, and expresses one or more variants of the present application
- the strain of the present application may include all microorganisms capable of producing IMP, including one or more variants of the present application.
- the strain of the present application may be a recombinant strain having increased IMP-producing ability by introducing a polynucleotide encoding one or more variants of the present application into a natural wild-type microorganism or a microorganism producing IMP.
- the recombinant strain having an increased IMP-producing ability may be a microorganism having an increased IMP-producing ability compared to a natural wild-type microorganism or an unmodified microorganism (ie, a microorganism that does not express the mutant), but is not limited thereto.
- the strain with increased IMP-producing ability of the present application is SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 23, SEQ ID NO: 27, SEQ ID NO: 31, SEQ ID NO: 35, sequence No.
- SEQ ID NO: 43 SEQ ID NO: 47, SEQ ID NO: 51, SEQ ID NO: 55, or a microorganism having an increased IMP-producing ability compared to Corynebacterium stasis comprising a polypeptide of SEQ ID NO: 59 or a polynucleotide encoding the same may be, but is not limited thereto.
- the non-modified microorganism which is the target strain for comparing whether the increase in the IMP production ability is increased, may be the CJI2332 strain (KCCM12277P, KR 10-1950141 B1), but is not limited thereto.
- the recombinant strain with increased production capacity is about 1% or more, specifically about 1% or more, about 10% or more, about 15% or more, or about 20 compared to the IMP production capacity of the parent strain or unmodified microorganism before mutation.
- % or more the upper limit is not particularly limited, for example, it may be about 200% or less, about 150% or less, about 100% or less, or about 50% or less. It is not limited thereto, as long as it has an increase in the + value compared to the productivity.
- the recombinant strain with increased production capacity has an IMP production capacity of about 1.01 times or more, about 1.05 times or more, about 1.10 times or more, 1.15 times or more, or about 1.2 times or more (The upper limit is not particularly limited, for example, it may be about 20 times or less) may be increased, but is not limited thereto.
- the term “about” is a range including all of ⁇ 0.5, ⁇ 0.4, ⁇ 0.3, ⁇ 0.2, ⁇ 0.1, etc. not limited
- the term "unmodified microorganism” does not exclude a strain containing a mutation that can occur naturally in a microorganism, it is a wild-type strain or a natural-type strain itself, or a genetic variation caused by natural or artificial factors. It may mean the strain before being changed.
- the unmodified microorganism may refer to a strain into which one or more variants described herein have not been introduced or have been introduced.
- the "unmodified microorganism” may be used interchangeably with "strain before modification", “microbe before modification”, “unmodified strain”, “unmodified strain”, “unmodified microorganism” or "reference microorganism”.
- the microorganism of the present application is Corynebacterium glutamicum ( Corynebacterium glutamicum ), Corynebacterium crudilactis ), Corynebacterium deserti ( Corynebacterium deserti ), Cory Nebacterium efficiens ( Corynebacterium efficiens ), Corynebacterium callunae ), Corynebacterium stationis , Corynebacterium stationis ), Corynebacterium singulare ( Corynebacterium singulare ), Corynebacterium halo Tolerans ( Corynebacterium halotolerans ), Corynebacterium striatum ( Corynebacterium striatum ), Corynebacterium ammoniagenes ( Corynebacterium ammoniagenes ), Corynebacterium pollutisoli ( Corynebacterium pollutisoli ), Corynebacterium imitans imitans imitans imitans imit
- the term “attenuation” of polypeptide activity is a concept that includes both reduced or no activity compared to intrinsic activity.
- the attenuation may be used interchangeably with terms such as inactivation, deficiency, down-regulation, decrease, reduce, attenuation, and the like.
- the attenuation is when the activity of the polypeptide itself is reduced or eliminated compared to the activity of the polypeptide possessed by the original microorganism due to mutation of the polynucleotide encoding the polypeptide, etc.
- the overall polypeptide activity level and/or concentration (expression amount) in the cell is lower than that of the native strain due to (translation) inhibition, etc., when the expression of the polynucleotide is not made at all, and/or when the expression of the polynucleotide is Even if there is no activity of the polypeptide, it may also be included.
- the "intrinsic activity” refers to the activity of a specific polypeptide originally possessed by the parent strain, wild-type or unmodified microorganism before transformation when the trait is changed due to genetic mutation caused by natural or artificial factors. This may be used interchangeably with “activity before modification”. "Inactivation, deficiency, reduction, downregulation, reduction, attenuation” of the activity of the polypeptide compared to the intrinsic activity means that the activity of the specific polypeptide originally possessed by the parent strain or unmodified microorganism before transformation is lowered.
- Attenuation of the activity of such a polypeptide may be performed by any method known in the art, but is not limited thereto, and may be achieved by application of various methods well known in the art (eg, Nakashima N et al., Bacterial cellular engineering by genome editing and gene silencing. Int J Mol Sci. 2014;15(2):2773-2793, Sambrook et al. Molecular Cloning 2012, etc.).
- the attenuation of the polypeptide activity of the present application is
- an antisense oligonucleotide eg, antisense RNA
- an antisense oligonucleotide that complementarily binds to the transcript of said gene encoding the polypeptide
- deletion of a part or all of the gene encoding the polypeptide may be the removal of the entire polynucleotide encoding the endogenous target polypeptide in the chromosome, replacement with a polynucleotide in which some nucleotides are deleted, or replacement with a marker gene.
- the expression control region includes, but is not limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence regulating the termination of transcription and translation.
- the base sequence modification encoding the start codon or 5'-UTR region of the gene transcript encoding the polypeptide is, for example, a base encoding another start codon having a lower expression rate of the polypeptide compared to the intrinsic start codon It may be substituted with a sequence, but is not limited thereto.
- the modification of the amino acid sequence or polynucleotide sequence of 3) and 4) above deletes, inserts, non-conservative or conservative substitution of the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide so as to weaken the activity of the polypeptide.
- a combination thereof may result in a mutation in sequence, or replacement with an amino acid sequence or polynucleotide sequence improved to have weaker activity or an amino acid sequence or polynucleotide sequence improved to have no activity, but is not limited thereto.
- the expression of a gene may be inhibited or attenuated, but is not limited thereto.
- antisense oligonucleotide eg, antisense RNA
- antisense RNA an antisense oligonucleotide that complementarily binds to the transcript of the gene encoding the polypeptide
- Weintraub, H. et al. Antisense-RNA as a molecular tool. for genetic analysis, Reviews - Trends in Genetics, Vol. 1(1) 1986].
- RTE reverse transcription engineering
- the term "enhancement" of a polypeptide activity means that the activity of the polypeptide is increased compared to the intrinsic activity.
- the reinforcement may be used interchangeably with terms such as activation, up-regulation, overexpression, and increase.
- activation, enhancement, up-regulation, overexpression, and increase may include all of those exhibiting an activity that was not originally possessed, or exhibiting an improved activity compared to an intrinsic activity or an activity prior to modification.
- intrinsic activity refers to the activity of a specific polypeptide originally possessed by the parent strain or unmodified microorganism before transformation when the trait is changed due to genetic mutation caused by natural or artificial factors.
- Enhance, "up-regulation”, “overexpression” or “increase” in the activity of a polypeptide compared to its intrinsic activity means the activity of a specific polypeptide originally possessed by the parent strain or unmodified microorganism before transformation. And / or concentration (expression amount) means improved compared to.
- the enrichment can be achieved by introducing an exogenous polypeptide, or by enhancing the activity and/or concentration (expression amount) of the endogenous polypeptide. Whether or not the activity of the polypeptide is enhanced can be confirmed from the increase in the level of activity, expression level, or the amount of product excreted from the polypeptide.
- the enhancement of the activity of the polypeptide can be applied by various methods well known in the art, and is not limited as long as it can enhance the activity of the target polypeptide compared to the microorganism before modification. Specifically, it may be one using genetic engineering and/or protein engineering well known to those skilled in the art, which is a routine method of molecular biology, but is not limited thereto (eg, Sitnicka et al. Functional Analysis of Genes. Advances in Cell). Biology 2010, Vol. 2. 1-16, Sambrook et al. Molecular Cloning 2012, etc.).
- modification of the polynucleotide sequence encoding the polypeptide to enhance the polypeptide activity eg, modification of the polynucleotide sequence of the polypeptide gene to encode a polypeptide modified to enhance the activity of the polypeptide
- the increase in the intracellular copy number of the polynucleotide encoding the polypeptide is achieved by introduction into the host cell of a vector to which the polynucleotide encoding the polypeptide is operably linked, which can replicate and function independently of the host it may be Alternatively, the polynucleotide encoding the polypeptide may be achieved by introducing one copy or two or more copies into a chromosome in a host cell.
- the introduction into the chromosome may be performed by introducing a vector capable of inserting the polynucleotide into the chromosome in the host cell into the host cell, but is not limited thereto.
- the vector is the same as described above.
- Replacing the gene expression control region (or expression control sequence) on the chromosome encoding the polypeptide with a sequence with strong activity is, for example, deletion, insertion, non-conservative or Conservative substitution or a combination thereof may result in a mutation in the sequence, or replacement with a sequence having a stronger activity.
- the expression control region is not particularly limited thereto, but may include a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence controlling the termination of transcription and translation.
- the original promoter may be replaced with a strong promoter, but is not limited thereto.
- Examples of known strong promoters include CJ1 to CJ7 promoters (US 7662943 B2), lac promoter, trp promoter, trc promoter, tac promoter, lambda phage PR promoter, PL promoter, tet promoter, gapA promoter, SPL7 promoter, SPL13 (sm3) promoter (US Patent US 10584338 B2), O2 promoter (US Patent US 10273491 B2), tkt promoter, yccA promoter, etc., but is not limited thereto.
- Modification of the nucleotide sequence encoding the start codon or 5'-UTR region of the gene transcript encoding the polypeptide is, for example, a nucleotide sequence encoding another start codon having a higher expression rate of the polypeptide compared to the intrinsic start codon. It may be a substitution, but is not limited thereto.
- the modification of the amino acid sequence or polynucleotide sequence of 4) and 5) above may include deletion, insertion, non-conservative or conservative substitution of the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide to enhance the activity of the polypeptide;
- a combination thereof may result in sequence mutation, or replacement with an amino acid sequence or polynucleotide sequence improved to have stronger activity or an amino acid sequence or polynucleotide sequence improved to increase activity, but is not limited thereto.
- the replacement may be specifically performed by inserting a polynucleotide into a chromosome by homologous recombination, but is not limited thereto.
- the vector used may further include a selection marker for confirming whether or not the chromosome is inserted.
- the selection marker is the same as described above.
- the introduction of the foreign polynucleotide exhibiting the activity of the polypeptide may be the introduction of the foreign polynucleotide encoding the polypeptide exhibiting the same/similar activity as the polypeptide into a host cell.
- the foreign polynucleotide is not limited in its origin or sequence as long as it exhibits the same/similar activity as the polypeptide.
- the method used for the introduction can be performed by appropriately selecting a known transformation method by those skilled in the art, and the introduced polynucleotide is expressed in a host cell to generate a polypeptide and increase its activity.
- Codon optimization of the polynucleotide encoding the polypeptide is codon-optimized so that the transcription or translation of the endogenous polynucleotide is increased in the host cell, or the transcription and translation of the foreign polynucleotide is optimized in the host cell. It may be that its codons are optimized so that the
- Selecting an exposed site by analyzing the tertiary structure of the polypeptide and modifying or chemically modifying it is, for example, by comparing the sequence information of the polypeptide to be analyzed with a database in which sequence information of known proteins is stored to determine the degree of sequence similarity. Accordingly, it may be to determine a template protein candidate, check the structure based on this, and select an exposed site to be modified or chemically modified and modified or modified.
- Such enhancement of polypeptide activity is to increase the activity or concentration of the corresponding polypeptide based on the activity or concentration of the polypeptide expressed in the wild-type or pre-modified microbial strain, or increase the amount of product produced from the polypeptide.
- the present invention is not limited thereto.
- Modification of part or all of the polynucleotide in the microorganism of the present application is (a) homologous recombination using a vector for chromosome insertion in the microorganism or genome correction using engineered nuclease (e.g., CRISPR-Cas9) and/or (b) It may be induced by light and/or chemical treatments such as, but not limited to, ultraviolet and radiation.
- the method for modifying part or all of the gene may include a method by DNA recombination technology.
- nucleotide sequence or a vector containing a nucleotide sequence homologous to a target gene into the microorganism to cause homologous recombination, deletion of a part or all of a gene may be made.
- the injected nucleotide sequence or vector may include a dominant selection marker, but is not limited thereto.
- Corynebacterium stasis strain comprising (i) one or more variants of the present application, (ii) one or more polynucleotides encoding the variant, or (iii) a combination thereof. It provides a method for producing IMP, comprising the step of culturing in a medium.
- the method for producing IMP of the present application may include culturing a Corynebacterium stationionis strain comprising the mutant of the present application or the polynucleotide of the present application or the vector of the present application in a medium.
- the term "cultivation” means growing the Corynebacterium stasis strain of the present application under moderately controlled environmental conditions.
- the culture process of the present application may be made according to a suitable medium and culture conditions known in the art. Such a culturing process can be easily adjusted and used by those skilled in the art according to the selected strain.
- the culture may be a batch, continuous, and/or fed-batch, but is not limited thereto.
- the term "medium” refers to a material in which nutrients required for culturing the Corynebacterium stasis strain of the present application are mixed as a main component, and nutrients including water essential for survival and development and growth factors.
- any medium and other culture conditions used for culturing the Corynebacterium stasis strain of the present application may be used without particular limitation as long as it is a medium used for culturing conventional microorganisms, but the Corynebacterium of the present application Rium stasis strains can be cultured while controlling temperature, pH, etc. under aerobic conditions in a conventional medium containing an appropriate carbon source, nitrogen source, phosphorus, inorganic compound, amino acid and/or vitamin.
- the culture medium for the Corynebacterium sp. strain can be found in the literature ["Manual of Methods for General Bacteriology” by the American Society for Bacteriology (Washington D.C., USA, 1981)].
- the carbon source includes carbohydrates such as glucose, saccharose, lactose, fructose, sucrose, maltose, and the like; sugar alcohols such as mannitol and sorbitol; organic acids such as pyruvic acid, lactic acid, citric acid and the like; amino acids such as glutamic acid, methionine, lysine, and the like may be included.
- natural organic nutrient sources such as starch hydrolyzate, molasses, blackstrap molasses, rice winter, cassava, sugar cane offal and corn steep liquor can be used, specifically glucose and sterilized pre-treated molasses (i.e., converted to reducing sugar). molasses) may be used, and other appropriate amounts of carbon sources may be variously used without limitation. These carbon sources may be used alone or in combination of two or more, but is not limited thereto.
- nitrogen source examples include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, anmonium carbonate, and ammonium nitrate; Amino acids such as glutamic acid, methionine, glutamine, and organic nitrogen sources such as peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolyzate, fish or degradation products thereof, defatted soybean cake or degradation products thereof, etc. can be used These nitrogen sources may be used alone or in combination of two or more, but is not limited thereto.
- inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, anmonium carbonate, and ammonium nitrate
- Amino acids such as glutamic acid, methionine, glutamine
- organic nitrogen sources such as peptone, NZ-amine, meat extract, yeast extract
- the phosphorus may include potassium first potassium phosphate, second potassium phosphate, or a sodium-containing salt corresponding thereto.
- potassium first potassium phosphate potassium phosphate
- second potassium phosphate or a sodium-containing salt corresponding thereto.
- sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, etc. may be used, and in addition, amino acids, vitamins and/or suitable precursors may be included. These components or precursors may be added to the medium either batchwise or continuously. However, the present invention is not limited thereto.
- the pH of the medium may be adjusted.
- an antifoaming agent such as fatty acid polyglycol ester may be used to suppress bubble formation.
- oxygen or oxygen-containing gas may be injected into the medium, or nitrogen, hydrogen or carbon dioxide gas may be injected without or without gas to maintain anaerobic and microaerobic conditions, it is not
- the culture temperature may be maintained at 20 to 45 °C, specifically, 25 to 40 °C, and may be cultured for about 10 to 160 hours, but is not limited thereto.
- the IMP produced by the culture of the present application may be secreted into the medium or may remain in the cell.
- the IMP production method of the present application includes the steps of preparing the Corynebacterium stasis strain of the present application, preparing a medium for culturing the strain, or a combination thereof (regardless of the order, in any order) , for example, prior to the culturing step, may further include.
- the IMP production method of the present application may further include recovering the IMP from the culture medium (cultured medium) or the Corynebacterium stationionis strain of the present application.
- the recovering step may be further included after the culturing step.
- the recovery may be to collect the desired IMP using a suitable method known in the art according to the culture method of the microorganism of the present application, for example, a batch, continuous or fed-batch culture method.
- a suitable method known in the art according to the culture method of the microorganism of the present application, for example, a batch, continuous or fed-batch culture method.
- centrifugation, filtration, treatment with a crystallized protein precipitating agent (salting out method) extraction, ultrasonic disruption, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity
- Various chromatography such as island chromatography, HPLC, or a combination thereof may be used, and a desired IMP may be recovered from a medium or a microorganism using a suitable method known in the art.
- the IMP production method of the present application may additionally include a purification step.
- the purification may be performed using a suitable method known in the art.
- the recovery step and the purification step are performed continuously or discontinuously, regardless of the order, or performed simultaneously or integrated into one step may be, but is not limited thereto.
- variants, polynucleotides, vectors, strains, and the like are as described in the other aspects above.
- Corynebacterium comprising one or more variants of the present application, one or more polynucleotides encoding the variants, one or more vectors including the polynucleotides, or one or more polynucleotides of the present application stationary strains; the culture medium; Or to provide a composition for producing IMP comprising a combination of two or more of them.
- composition of the present application may further include any suitable excipients commonly used in compositions for IMP production, and these excipients may be, for example, preservatives, wetting agents, dispersing agents, suspending agents, buffering agents, stabilizing agents or isotonic agents, etc.
- excipients commonly used in compositions for IMP production
- these excipients may be, for example, preservatives, wetting agents, dispersing agents, suspending agents, buffering agents, stabilizing agents or isotonic agents, etc.
- the present invention is not limited thereto.
- composition of the present application variants, polynucleotides, vectors, strains, media and IMPs are the same as those described in the other aspects above.
- Example 1 Evaluation of IMP production ability of microorganisms expressing 3D-(3,5/4)-trihydroxycyclohexane-1,2-dione acylhydrolase mutant
- Example 1-1 3D-(3,5/4)-trihydroxycyclohexane-1,2-dione acylhydrolase in microorganisms Vector construction for mutant expression
- 3D-(3,5/4)-trihydroxycyclohexane-1,2-dione acylhydrolase mutant (T526I; sequence) in which the threonine at position 526 of the amino acid sequence (SEQ ID NO: 3) is substituted with isoleucine; No. 1) using the plasmid pDCM2 (Korea Publication No. 10-2020-0136813) for the insertion and replacement of genes in the Corynebacterium chromosome to determine the effect of No. 1) on IMP production It was prepared as follows.
- PCR was performed using a primer pair of sequences of SEQ ID NOs: 61 and 62 and a pair of primers of sequences of SEQ ID NOs: 63 and 64, respectively.
- overlapping PCR was performed again using the primer pair of SEQ ID NO: 61 and SEQ ID NO: 64 to obtain a fragment.
- PCR was repeated 30 times at 94°C for 30 seconds, at 55°C for 30 seconds, and at 72°C for 1 minute and 30 seconds, and then at 72°C for 5 minutes.
- the pDCM2 vector was treated with smal and the PCR product obtained above was fusion cloned. Fusion cloning was performed using the In-Fusion® HD cloning kit (Clontech). The resulting plasmid was named pDCM2-iolD(T526I).
- Example 1-2 3D-(3,5/4)-trihydroxycyclohexane-1,2-dione acylhydrolase Evaluation of IMP-producing ability of microorganisms expressing variants
- Example 1-1 The vector prepared in Example 1-1 was transformed into the Corynebacterium stationionis CJI2332 strain (KCCM12277P, KR 10-1950141 B1).
- the strain into which the mutant was introduced was selected using SEQ ID NOs: 65 and 66.
- the selected strain was named CJI2332_iolD_T526I.
- IMP production capacity was analyzed by evaluating the flask fermentation titer of each strain and control parent strain prepared in Example 1-2-1.
- each strain was inoculated into a test tube with a diameter of 18 mm containing 2 ml of the seed medium, and cultured with shaking at 30 ° C. for 24 hours was used as a seed culture medium.
- 2 ml of the seed culture solution was inoculated into a 250ml corner-baffle flask containing 29ml of production medium (24ml of main medium + 5ml of separate sterile medium) and cultured at 30°C for 72 hours at 170rpm.
- the production capacity of IMP was measured by HPLC. IMP concentration and concentration increase rate in the culture medium for each strain tested are shown in Table 1 below.
- the CJ2332_iolD_T526I strain exhibited a 12.1% increase in IMP production capacity compared to the control group.
- the CJI2332_iolD_T526I was named CN01-2641, and was deposited at the Korea Microorganism Conservation Center, an institution under the Budapest Treaty, as of November 30, 2020. Therefore, it was given an accession number KCCM12836P.
- Example 2 Evaluation of IMP-producing ability of microorganisms expressing anaerobic coproporphyrinogen III oxidase variants
- Example 2-1 Anaerobic coproporphyrinogen III oxidase in microorganisms Vector construction for mutant expression
- a vector for constructing an expression strain thereof was produced as follows using the plasmid pDCM2 (Korea Publication No. 10-2020-0136813) for the insertion and replacement of genes in the Corynebacterium chromosome.
- PCR was performed using a primer pair of sequences of SEQ ID NOs: 67 and 68 and a pair of primers of SEQ ID NOs: 69 and 70 using gDNA (genomic DNA) of wild-type Corynebacterium stasis ATCC6872 as a template, respectively.
- gDNA genomic DNA
- overlapping PCR was performed again using the primer pair of SEQ ID NO: 67 and SEQ ID NO: 70 to obtain a fragment.
- PCR was repeated 30 times at 94°C for 30 seconds, at 55°C for 30 seconds, and at 72°C for 1 minute and 30 seconds, and then at 72°C for 5 minutes.
- the pDCM2 vector was treated with smal and the PCR product obtained above was fusion cloned. Fusion cloning was performed using the In-Fusion® HD cloning kit (Clontech). The resulting plasmid was named pDCM2-hemN (V209M).
- Example 2-2 Anaerobic coproporphyrinogen III oxidase Evaluation of IMP-producing ability of microorganisms expressing variants
- Example 2-1 The vector prepared in Example 2-1 was transformed into the Corynebacterium stationionis CJI2332 strain (KCCM12277P, KR 10-1950141 B1).
- the strain into which the mutant was introduced was selected using SEQ ID NOs: 71 and 72.
- the selected strain was named CJI2332_hemN_V209M.
- IMP production capacity was analyzed by evaluating the flask fermentation titer of each strain and the control parent strain prepared in Example 2-2-1.
- each strain was inoculated into a test tube with a diameter of 18 mm containing 2 ml of the seed medium, and cultured with shaking at 30 ° C. for 24 hours was used as a seed culture medium.
- 2 ml of the seed culture solution was inoculated into a 250ml corner-baffle flask containing 29ml of production medium (24ml of main medium + 5ml of separate sterile medium) and cultured at 30°C for 72 hours at 170rpm.
- the production capacity of IMP was measured by HPLC. IMP concentration and concentration increase rate in the culture medium for each strain tested are shown in Table 2 below.
- the CJI2332_hemN_V209M strain exhibited an IMP-producing ability increased by 20.7% compared to the control.
- the CJI2332_hemN_V209M was named CN01-2642, and it was deposited with the Korea Microorganism Conservation Center, an institution under the Budapest Treaty, on November 30, 2020, and was given an accession number KCCM12837P.
- Example 3 D-alanine--D-alanine ligase Evaluation of IMP-producing ability of microorganisms expressing variants
- Example 3-1 D-alanine--D-alanine ligase in microorganisms Vector construction for mutant expression
- D-alanine-D-alanine ligase The mutant (G130D; SEQ ID NO: 9) in which the glycine at position 130 of the amino acid sequence (SEQ ID NO: 11) is substituted with aspartic acid (G130D; SEQ ID NO: 9) for the production of an expression strain thereof to determine the effect on IMP production
- the vector was constructed as follows using the plasmid pDCM2 (Korea Publication No. 10-2020-0136813) for the insertion and replacement of genes in the Corynebacterium chromosome.
- PCR was performed using a primer pair of sequences of SEQ ID NOs: 73 and 74 and a pair of primers of sequences of SEQ ID NOs: 75 and 76, respectively.
- overlapping PCR was performed again using the primer pair of SEQ ID NO: 73 and SEQ ID NO: 76 to obtain a fragment.
- PCR was repeated 30 times at 94°C for 30 seconds, at 55°C for 30 seconds, and at 72°C for 1 minute and 30 seconds, and then at 72°C for 5 minutes.
- the pDCM2 vector was treated with smal and the PCR product obtained above was fusion cloned. Fusion cloning was performed using the In-Fusion® HD cloning kit (Clontech). The resulting plasmid was named pDCM2-ddl (G130D).
- Example 3-2 D-alanine--D-alanine ligase Evaluation of IMP-producing ability of microorganisms expressing variants
- Example 3-1 The vector prepared in Example 3-1 was transformed into Corynebacterium stasis CJI2332 strain (KCCM12277P, KR 10-1950141 B1).
- the strain into which the mutant was introduced was selected using SEQ ID NOs: 77 and 78.
- the selected strain was named CJI2332_ddl_G130D.
- IMP production capacity was analyzed by evaluating the flask fermentation titer of each strain and control parent strain prepared in Example 3-2-1.
- each strain was inoculated into a test tube with a diameter of 18 mm containing 2 ml of the seed medium, and cultured with shaking at 30 ° C. for 24 hours was used as a seed culture medium.
- 2 ml of the seed culture solution was inoculated into a 250ml corner-baffle flask containing 29ml of production medium (24ml of main medium + 5ml of separate sterile medium) and cultured at 30°C for 72 hours at 170rpm.
- the production capacity of IMP was measured by HPLC. IMP concentration and concentration increase rate in the culture medium for each strain tested are shown in Table 3 below.
- the CJI2332_ddl_G130D was named CN01-2644, and it was deposited with the Korea Microorganism Conservation Center, an institution under the Budapest Treaty, on November 30, 2020, and was given an accession number KCCM12839P.
- Example 4 Evaluation of IMP-producing ability of microorganisms expressing formamidopyrimidine-DNA glycosylase mutants
- Example 4-1 Formamidopyrimidine-DNA glycosylase in microorganisms Vector construction for mutant expression
- Formamidopyrimidine-DNA glycosylase amino acid sequence (SEQ ID NO: 15) of the 232 position serine substituted with phenylalanine (S232F; SEQ ID NO: 13) to determine the effect on IMP production for the production of its expression strain
- the vector was constructed as follows using the plasmid pDCM2 (Korea Publication No. 10-2020-0136813) for the insertion and replacement of genes in the Corynebacterium chromosome.
- PCR was performed using a pair of primers of SEQ ID NOs: 79 and 80 and a pair of primers of SEQ ID NOs: 81 and 82 using gDNA (genomic DNA) of wild-type Corynebacterium stasis ATCC6872 as a template, respectively.
- gDNA genomic DNA
- overlapping PCR was performed again using the primer pair of SEQ ID NO: 79 and SEQ ID NO: 82 to obtain a fragment.
- PCR was repeated 30 times at 94°C for 30 seconds, at 55°C for 30 seconds, and at 72°C for 1 minute and 30 seconds, and then at 72°C for 5 minutes.
- the pDCM2 vector was treated with smal and the PCR product obtained above was fusion cloned. Fusion cloning was performed using the In-Fusion® HD cloning kit (Clontech). The resulting plasmid was named pDCM2-mutM (S232F).
- Example 4-2 formamidopyrimidine-DNA glycosylase Evaluation of IMP-producing ability of microorganisms expressing variants
- the vector prepared in Example 4-1 was transformed into the Corynebacterium stationionis CJI2332 strain (KCCM12277P, KR 10-1950141 B1).
- the strain into which the mutant was introduced was selected using SEQ ID NOs: 83 and 84.
- the selected strain was named CJI2332_mutM_S232F.
- IMP production ability was analyzed by evaluating the flask fermentation titer of each strain and control parent strain prepared in Example 4-2-1.
- each strain was inoculated into a test tube with a diameter of 18 mm containing 2 ml of the seed medium, and cultured with shaking at 30 ° C. for 24 hours was used as a seed culture medium.
- 2 ml of the seed culture solution was inoculated into a 250ml corner-baffle flask containing 29ml of production medium (24ml of main medium + 5ml of separate sterile medium) and cultured at 30°C for 72 hours at 170rpm.
- the production capacity of IMP was measured by HPLC. IMP concentration and concentration increase rate in the culture medium for each strain tested are shown in Table 4 below.
- ⁇ Seed medium (pH 7.5)> glucose 1%, peptone 1%, broth 1%, yeast extract 1%, sodium chloride 0.25%, adenine 100mg/l, guanine 100mg/l (based on 1 liter of distilled water)
- the CJI2332_mutM_S232F strain exhibited a 32.2% increased IMP-producing ability compared to the control.
- the CJI2332_mutM_S232F was named CN01-2643, and it was deposited with the Korea Microorganism Conservation Center, an institution under the Budapest Treaty, on November 30, 2020, and was given an accession number KCCM12838P.
- Example 5 IMP-producing ability of microorganisms expressing 1,4-alpha-glucan-branching enzyme variants
- Example 5-1 1,4-alpha-glucan-branching enzyme in microorganisms Vector construction for mutant expression
- 1,4-alpha-glucan-branching enzyme amino acid sequence (SEQ ID NO: 19) in the 6th position of the aspartic acid substituted with asparagine (D6N; SEQ ID NO: 17) to determine the effect on IMP production for the production of the expression strain
- the vector was constructed as follows using the plasmid pDCM2 (Korea Publication No. 10-2020-0136813) for the insertion and replacement of genes in the Corynebacterium chromosome.
- PCR was performed using a pair of primers of SEQ ID NOs: 85 and 86 and a pair of primers of SEQ ID NOs: 87 and 88 using gDNA (genomic DNA) of wild-type Corynebacterium stasis ATCC6872 as a template, respectively.
- gDNA genomic DNA
- overlapping PCR was performed again using the primer pair of SEQ ID NO: 85 and SEQ ID NO: 88 to obtain a fragment.
- PCR was repeated 30 times at 94°C for 30 seconds, at 55°C for 30 seconds, and at 72°C for 1 minute and 30 seconds, and then at 72°C for 5 minutes.
- the pDCM2 vector was treated with smal and the PCR product obtained above was fusion cloned. Fusion cloning was performed using the In-Fusion® HD cloning kit (Clontech). The resulting plasmid was named pDCM2-glgB(D6N).
- Example 5-2 1,4-alpha-glucan-branching enzyme Evaluation of IMP-producing ability of microorganisms expressing variants
- Example 5-1 The vector prepared in Example 5-1 was transformed into a Corynebacterium stationionis CJI2332 strain (KCCM12277P, KR 10-1950141 B1).
- the strain into which the mutant was introduced was selected using SEQ ID NOs: 89 and 90.
- the selected strain was named CJI2332_glgB_D6N.
- IMP production capacity was analyzed by evaluating the flask fermentation titer of each strain and control parent strain prepared in Example 5-2-1.
- each strain was inoculated into a test tube with a diameter of 18 mm containing 2 ml of the seed medium, and cultured with shaking at 30 ° C. for 24 hours was used as a seed culture medium.
- 2 ml of the seed culture solution was inoculated into a 250ml corner-baffle flask containing 29ml of production medium (24ml of main medium + 5ml of separate sterile medium) and cultured at 30°C for 72 hours at 170rpm.
- the production capacity of IMP was measured by HPLC. IMP concentration and concentration increase rate in the culture medium for each strain tested are shown in Table 5 below.
- the CJI2332_glgB_D6N strain exhibited an IMP-producing ability that was increased by 6.9% compared to the control.
- the CJI2332_glgB_D6N was named CN01-2645, and was deposited with the Korea Microorganism Conservation Center, a trustee under the Budapest Treaty, on November 30, 2020, and was given an accession number KCCM12840P.
- Example 6 Evaluation of IMP-producing ability of microorganisms expressing a formimidoyl glutamase mutant
- Example 6-1 Formimidoyl glutamase in microorganisms (Formimidoylglutamase) Vector construction for mutant expression
- Formimidoyl glutamase amino acid sequence (SEQ ID NO: 23) in the 294th position of the aspartic acid substituted with asparagine (D294N; SEQ ID NO: 21) IMP (5'-inosine monophosphate) to determine the effect on the production of its expression strain
- a vector for construction was prepared as follows using the plasmid pDCM2 (Korea Publication No. 10-2020-0136813) for the insertion and replacement of genes in the Corynebacterium chromosome.
- Wild-type Corynebacterium stationis ( Corynebacterium stationis ) Using gDNA (genomic DNA) of ATCC6872 as a template, primer pairs of sequences of SEQ ID NOs: 91 and 92 and primer pairs of sequences of SEQ ID NOs: 93 and 94 PCR was performed using a pair of primers, respectively did. Using the mixture of the two fragments obtained above as a template, overlapping PCR was performed again using a pair of primers of SEQ ID NO: 91 and SEQ ID NO: 94 to obtain a fragment.
- PCR was repeated 30 times at 94°C for 30 seconds, at 55°C for 30 seconds, and at 72°C for 1 minute and 30 seconds, and then at 72°C for 5 minutes.
- the pDCM2 vector was treated with smal and the PCR product obtained above was fusion cloned. Fusion cloning was performed using the In-Fusion® HD cloning kit (Clontech). The resulting plasmid was named pDCM2-hutG (D294N).
- Example 6-2 IMP-producing ability evaluation of microorganisms expressing the formimidoyl glutamase mutant
- Example 6-1 The vector prepared in Example 6-1 was transformed into the Corynebacterium stationionis CJI2332 strain (KCCM12277P, KR 10-1950141 B1).
- the strain into which the mutant was introduced was selected using SEQ ID NOs: 95 and 96.
- the selected strain was named CJI2332_hutG_D294N.
- IMP production ability was analyzed by evaluating the flask fermentation titer of each strain and control parent strain prepared in Example 6-2-1.
- each strain was inoculated into a test tube with a diameter of 18 mm containing 2 ml of the seed medium, and cultured with shaking at 30 ° C. for 24 hours was used as a seed culture medium.
- 2 ml of the seed culture solution was inoculated into a 250ml corner-baffle flask containing 29ml of production medium (24ml of main medium + 5ml of separate sterile medium) and cultured at 30°C for 72 hours at 170rpm.
- the production capacity of IMP was measured by HPLC. IMP concentration and concentration increase rate in the culture medium for each strain tested are shown in Table 6 below.
- the CJI2332_hutG_D294N was named CN01-2646, and it was deposited with the Korea Microorganism Conservation Center, an institution under the Budapest Treaty, on November 30, 2020, and was given an accession number KCCM12841P.
- Example 7 Evaluation of IMP-producing ability of microorganisms expressing glucosamine-6-phosphate deaminase variants
- Example 7-1 Glucosamine-6-phosphate deaminase in microorganisms Vector construction for mutant expression
- Wild-type Corynebacterium stationis ( Corynebacterium stationis ) Using gDNA (genomic DNA) of ATCC6872 as a template, a primer pair of sequences of SEQ ID NOs: 97 and 98 and a primer pair of sequences of SEQ ID NOs: 99 and 100 Perform PCR, respectively did. Using the mixture of the two fragments obtained above as a template, overlapping PCR was performed again using a primer pair of SEQ ID NO: 97 and SEQ ID NO: 100 to obtain a fragment.
- PCR was repeated 30 times at 94°C for 30 seconds, at 55°C for 30 seconds, and at 72°C for 1 minute and 30 seconds, and then at 72°C for 5 minutes.
- the pDCM2 vector was treated with smal and the PCR product obtained above was fusion cloned. Fusion cloning was performed using the In-Fusion® HD cloning kit (Clontech). The resulting plasmid was named pDCM2-nagB (A157V).
- Example 7-2 Evaluation of IMP-producing ability of microorganisms expressing glucosamine-6-phosphate deaminase variants
- Example 7-1 The vector prepared in Example 7-1 was transformed into Corynebacterium stasis CJI2332 strain (KCCM12277P, KR 10-1950141 B1).
- the strain into which the mutant was introduced was selected using SEQ ID NOs: 101 and 102.
- the selected strain was named CJI2332_nagB_A157V.
- IMP production ability was analyzed by evaluating the flask fermentation titer of each strain and control parent strain prepared in Example 7-2-1.
- each strain was inoculated into a test tube with a diameter of 18 mm containing 2 ml of the seed medium, and cultured with shaking at 30 ° C. for 24 hours was used as a seed culture medium.
- 2 ml of the seed culture solution was inoculated into a 250ml corner-baffle flask containing 29ml of production medium (24ml of main medium + 5ml of separate sterile medium) and cultured at 30°C for 72 hours at 170rpm.
- the production capacity of IMP was measured by HPLC. IMP concentration and concentration increase rate in the culture medium for each strain tested are shown in Table 7 below.
- the CJI2332_nagB_A157V was named CN01-2648, and it was deposited with the Korea Microorganism Conservation Center, an institution under the Budapest Treaty, on November 30, 2020, and was given an accession number KCCM12843P.
- Example 8 Evaluation of IMP-producing ability of microorganisms expressing selenide and water dikinase oxidase mutants
- Example 8-1 Selenide, water dikinase in microorganisms (Selenide, water dikinase) Vector construction for mutant expression
- Wild-type Corynebacterium stationis ( Corynebacterium stationis ) Using gDNA (genomic DNA) of ATCC6872 as a template, a primer pair of sequences of SEQ ID NOs: 103 and 104 and a primer pair of sequences of SEQ ID NOs: 105 and 106 Perform PCR, respectively did. Using the mixture of the two fragments obtained above as a template, overlapping PCR was performed again using the primer pair of SEQ ID NO: 103 and SEQ ID NO: 106 to obtain a fragment.
- PCR was repeated 30 times at 94°C for 30 seconds, at 55°C for 30 seconds, and at 72°C for 1 minute and 30 seconds, and then at 72°C for 5 minutes.
- the pDCM2 vector was treated with smal and the PCR product obtained above was fusion cloned. Fusion cloning was performed using the In-Fusion® HD cloning kit (Clontech). The resulting plasmid was named pDCM2-selD(A80T).
- Example 8-2 Selenide, IMP-producing ability evaluation of microorganisms expressing water dikinase mutants
- Example 8-1 The vector prepared in Example 8-1 was transformed into a Corynebacterium stationionis CJI2332 strain (KCCM12277P, KR 10-1950141 B1).
- the strain into which the mutant was introduced was selected using SEQ ID NOs: 107 and 108.
- the selected strain was designated as CJI2332_selD_A80T.
- IMP production ability was analyzed by evaluating the flask fermentation titer of each strain and control parent strain prepared in Example 8-2-1.
- each strain was inoculated into a test tube with a diameter of 18 mm containing 2 ml of the seed medium, and cultured with shaking at 30 ° C. for 24 hours was used as a seed culture medium.
- 2 ml of the seed culture solution was inoculated into a 250ml corner-baffle flask containing 29ml of production medium (24ml of main medium + 5ml of separate sterile medium) and cultured at 30°C for 72 hours at 170rpm.
- the production capacity of IMP was measured by HPLC. IMP concentration and concentration increase rate in the culture medium for each strain tested are shown in Table 8 below.
- the CJI2332_selD_A80T strain exhibited a 32.2% increased IMP-producing ability compared to the control.
- the CJI2332_selD_A80T was named CN01-2647, and was deposited with the Korea Microorganism Conservation Center, a trustee under the Budapest Treaty, on November 30, 2020, and was given an accession number KCCM12842P.
- Example 9 Evaluation of IMP-producing ability of microorganisms expressing peptide methionine sulfoxide reductase mutants
- Example 9-1 Peptide methionine sulfoxide reductase in microorganisms Vector construction for mutant expression
- a vector for construction was prepared as follows using the plasmid pDCM2 (Korea Publication No. 10-2020-0136813) for the insertion and replacement of genes in the Corynebacterium chromosome.
- Wild-type Corynebacterium stationis ( Corynebacterium stationis ) Using gDNA (genomic DNA) of ATCC6872 as a template, primer pairs of sequences of SEQ ID NOs: 109 and 110 and primer pairs of sequences of SEQ ID NOs: 111 and 112 PCR was performed using a pair of primers, respectively did. Using the mixture of the two fragments obtained above as a template, overlapping PCR was performed again using the primer pair of SEQ ID NO: 109 and SEQ ID NO: 112 to obtain a fragment.
- PCR was repeated 30 times at 94°C for 30 seconds, at 55°C for 30 seconds, and at 72°C for 1 minute and 30 seconds, and then at 72°C for 5 minutes.
- the pDCM2 vector was treated with smal and the PCR product obtained above was fusion cloned. Fusion cloning was performed using the In-Fusion® HD cloning kit (Clontech). The resulting plasmid was named pDCM2-msrA(A6V).
- Example 9-2 Evaluation of IMP production ability of microorganisms expressing peptide methionine sulfoxide reductase mutant
- Example 9-1 The vector prepared in Example 9-1 was transformed into the Corynebacterium stationionis CJI2332 strain (KCCM12277P, KR 10-1950141 B1).
- the strain into which the mutant was introduced was selected using SEQ ID NOs: 113 and 114.
- the selected strain was named CJI2332_msrA_A6V.
- IMP production capacity was analyzed by evaluating the flask fermentation titer of each strain and control parent strain prepared in Example 3-2-1.
- each strain was inoculated into a test tube with a diameter of 18 mm containing 2 ml of the seed medium, and cultured with shaking at 30 ° C. for 24 hours was used as a seed culture medium.
- 2 ml of the seed culture solution was inoculated into a 250ml corner-baffle flask containing 29ml of production medium (24ml of main medium + 5ml of separate sterile medium) and cultured at 30°C for 72 hours at 170rpm.
- the production capacity of IMP was measured by HPLC. IMP concentration and concentration increase rate in the culture medium for each strain tested are shown in Table 9 below.
- the CJI2332_msrA_A6V was named CN01-2650, and it was deposited with the Korea Microorganism Conservation Center, a trustee institution under the Budapest Treaty, on November 30, 2020, and was given an accession number KCCM12845P.
- Example 10 Evaluation of IMP production ability of microorganisms expressing phytoene desaturase variants
- Example 10-1 Phytoene desaturase in microorganisms Vector construction for mutant expression
- Wild-type Corynebacterium stationis ( Corynebacterium stationis ) Using gDNA (genomic DNA) of ATCC6872 as a template, primer pairs of sequences of SEQ ID NOs: 115 and 116 and primer pairs of sequences of SEQ ID NOs: 117 and 118 Perform PCR, respectively did. Using the mixture of the two fragments obtained above as a template, overlapping PCR was performed again using the primer pair of SEQ ID NO: 115 and SEQ ID NO: 118 to obtain a fragment. After denaturation at 94°C for 5 minutes, PCR was repeated 30 times at 94°C for 30 seconds, at 55°C for 30 seconds, and at 72°C for 1 minute and 30 seconds, and then at 72°C for 5 minutes.
- the pDCM2 vector was treated with smal and the PCR product obtained above was fusion cloned. Fusion cloning was performed using the In-Fusion® HD cloning kit (Clontech). The resulting plasmid was named pDCM2-crtI (D425N).
- Example 10-2 Evaluation of IMP production ability of microorganisms expressing phytoene desaturase variants
- Example 10-1 The vector prepared in Example 10-1 was transformed into the Corynebacterium stationionis CJI2332 strain (KCCM12277P, KR 10-1950141 B1).
- the strain into which the mutant was introduced was selected using SEQ ID NOs: 119 and 120.
- the selected strain was named CJI2332_crtI_D425N.
- IMP production ability was analyzed by evaluating the flask fermentation titer of each strain and control parent strain prepared in Example 10-2-1.
- each strain was inoculated into a test tube with a diameter of 18 mm containing 2 ml of the seed medium, and cultured with shaking at 30 ° C. for 24 hours was used as a seed culture medium.
- 2 ml of the seed culture solution was inoculated into a 250ml corner-baffle flask containing 29ml of production medium (24ml of main medium + 5ml of separate sterile medium) and cultured at 30°C for 72 hours at 170rpm.
- the production capacity of IMP was measured by HPLC. IMP concentration and concentration increase rate in the culture medium for each strain tested are shown in Table 10 below.
- the CJI2332_crtI_D425N strain exhibited an IMP-producing ability increased by 20.7% compared to the control.
- the CJI2332_crtI_D425N was named CN01-2649, and was deposited with the Korea Microorganism Conservation Center, a trustee institution under the Budapest Treaty, on November 30, 2020, and was given an accession number KCCM12844P.
- Example 11 Evaluation of IMP-producing ability of microorganisms expressing formate-dependent phosphoribosylglycinamide formyltransferase variants
- Example 11-1 Construction of a vector for the expression of formate-dependent phosphoribosylglycinamide formyltransferase mutants in microorganisms
- a vector was prepared as follows using the plasmid pDCM2 (Korea Publication No. 10-2020-0136813) for the insertion and replacement of genes in the Corynebacterium chromosome.
- PCR was performed using the primer pair of SEQ ID NOs: 121 and 122 and the primer pair of SEQ ID NOs: 123 and 124 using gDNA (genomic DNA) of wild-type Corynebacterium stasis ATCC6872 as a template, respectively.
- gDNA genomic DNA
- overlapping PCR was performed again using the primer pair of SEQ ID NO: 121 and SEQ ID NO: 124 to obtain a fragment.
- PCR was repeated 30 times at 94°C for 30 seconds, at 55°C for 30 seconds, and at 72°C for 1 minute and 30 seconds, and then at 72°C for 5 minutes.
- the pDCM2 vector was treated with SmaI and the PCR product obtained above was fusion cloned. Fusion cloning was performed using the In-Fusion® HD cloning kit (Clontech). The resulting plasmid was named pDCM2-purT(V255I).
- Example 11-2 Evaluation of IMP-producing ability of microorganisms expressing formate-dependent phosphoribosylglycinamide formyltransferase mutants
- Example 11-1 The vector prepared in Example 11-1 was transformed into Corynebacterium stasis CJI2332 strain (KCCM12277P, KR 10-1950141 B1).
- the strain into which the mutant was introduced was selected using SEQ ID NOs: 125 and 126.
- the selected strain was named CJI2332_purT_V255I.
- IMP production ability was analyzed by evaluating the flask fermentation titer of the strain prepared in Example 11-2-1 and the control parent strain.
- each strain was inoculated into a test tube with a diameter of 18 mm containing 2 ml of the seed medium, and cultured with shaking at 30 ° C. for 24 hours was used as a seed culture medium.
- 2 ml of the seed culture solution was inoculated into a 250ml corner-baffle flask containing 29ml of production medium (24ml of main medium + 5ml of separate sterile medium) and cultured at 30°C for 72 hours at 170rpm.
- the production capacity of IMP was measured by HPLC. IMP concentration and concentration increase rate in the culture medium for each strain tested are shown in Table 11 below.
- the CJI2332_purT_V255I strain exhibited an IMP-producing ability increased by 25.3% compared to the control.
- Example 12 Evaluation of IMP production ability of microorganisms expressing bifunctional phosphoribosylaminoimidazolecarboxamide formyltransferase/IMP cyclohydrolase variants
- Example 12-1 Bifunctional phosphoribosylaminoimidazolecarboxamide formyltransferase/IMP cyclohydrolase mutant expression in microorganisms Vector construction
- the vector for the production of the expression strain was prepared as follows using the plasmid pDCM2 (Korea Publication No. 10-2020-0136813) for the insertion and replacement of genes in the Corynebacterium chromosome.
- PCR was performed using the primer pair of SEQ ID NOs: 127 and 128 and the primer pair of SEQ ID NOs: 129 and 130 using gDNA (genomic DNA) of wild-type Corynebacterium stasis ATCC6872 as a template, respectively.
- gDNA genomic DNA
- overlapping PCR was performed again using the primer pair of SEQ ID NO: 127 and SEQ ID NO: 130 to obtain a fragment.
- PCR was repeated 30 times at 94°C for 30 seconds, at 55°C for 30 seconds, and at 72°C for 1 minute and 30 seconds, and then at 72°C for 5 minutes.
- the pDCM2 vector was treated with SmaI and the PCR product obtained above was fusion cloned. Fusion cloning was performed using the In-Fusion® HD cloning kit (Clontech). The resulting plasmid was named pDCM2-purH (A351V).
- Example 12-2 Evaluation of IMP production ability of microorganisms expressing bifunctional phosphoribosylaminoimidazole carboxamide formyltransferase/IMP cyclohydrolase mutant
- Example 12-1 The vector prepared in Example 12-1 was transformed into the Corynebacterium stationionis CJI2332 strain (KCCM12277P, KR 10-1950141 B1).
- the strain into which the variant was introduced was selected using SEQ ID NOs: 131 and 132.
- the selected strain was named CJI2332_purH_A351V.
- IMP production ability was analyzed by evaluating the flask fermentation titer of the strain prepared in Example 12-2-1 and the control parent strain.
- each strain was inoculated into a test tube with a diameter of 18 mm containing 2 ml of the seed medium, and cultured with shaking at 30 ° C. for 24 hours was used as a seed culture medium.
- 2 ml of the seed culture solution was inoculated into a 250ml corner-baffle flask containing 29ml of production medium (24ml of main medium + 5ml of separate sterile medium) and cultured at 30°C for 72 hours at 170rpm.
- the production capacity of IMP was measured by HPLC. IMP concentration and concentration increase rate in the culture medium for each strain tested are shown in Table 12 below.
- the CJI2332_purH_A351V strain exhibited a 20.2% increased IMP-producing ability compared to the control.
- Example 13 5- (carboxyamino) imidazolibonucleotide synthase Evaluation of IMP-producing ability of microorganisms expressing variants
- Example 13-1 5- (carboxyamino) imidazolibonucleotide synthase in microorganism Vector construction for mutant expression
- PCR was performed using the primer pair of SEQ ID NOs: 133 and 134 and the primer pair of SEQ ID NOs: 135 and 136 using gDNA (genomic DNA) of wild-type Corynebacterium stasis ATCC6872 as a template, respectively.
- gDNA genomic DNA
- overlapping PCR was performed again using the primer pair of SEQ ID NO: 133 and SEQ ID NO: 136 to obtain a fragment.
- PCR was repeated 30 times at 94°C for 30 seconds, at 55°C for 30 seconds, and at 72°C for 1 minute and 30 seconds, and then at 72°C for 5 minutes.
- the pDCM2 vector was treated with SmaI and the PCR product obtained above was fusion cloned. Fusion cloning was performed using the In-Fusion® HD cloning kit (Clontech). The resulting plasmid was named pDCM2-purK (G172D).
- Example 13-2 Evaluation of IMP-producing ability of microorganisms expressing 5-(carboxyamino)imidazolibonucleotide synthetase mutants
- Example 13-1 The vector prepared in Example 13-1 was transformed into the Corynebacterium stationionis CJI2332 strain (KCCM12277P, KR 10-1950141 B1).
- the strain into which the mutant was introduced was selected using SEQ ID NOs: 137 and 138.
- the selected strain was named CJI2332_purK_G172D.
- IMP production ability was analyzed by evaluating the flask fermentation titer of the strain prepared in Example 13-2-1 and the control parent strain.
- each strain was inoculated into a test tube with a diameter of 18 mm containing 2 ml of the seed medium, and cultured with shaking at 30 ° C. for 24 hours was used as a seed culture medium.
- 2 ml of the seed culture solution was inoculated into a 250ml corner-baffle flask containing 29ml of production medium (24ml of main medium + 5ml of separate sterile medium) and cultured at 30°C for 72 hours at 170rpm.
- the production capacity of IMP was measured by HPLC. IMP concentration and concentration increase rate in the culture medium for each strain tested are shown in Table 13 below.
- the CJI2332_purK_G172D strain exhibited an IMP-producing ability increased by 23.7% compared to the control.
- Example 14 Evaluation of IMP production ability of microorganisms expressing adenine phosphoribosyl group transferase mutants
- Example 14-1 Construction of a vector for expression of adenine phosphoribosyl group transferase mutant in microorganisms
- PCR was performed using the primer pair of SEQ ID NOs: 139 and 140 and the primer pair of SEQ ID NOs: 141 and 142 using gDNA (genomic DNA) of wild-type Corynebacterium stasis ATCC6872 as a template, respectively.
- gDNA genomic DNA
- overlapping PCR was performed again using a primer pair of SEQ ID NO: 139 and SEQ ID NO: 142 to obtain a fragment.
- PCR was repeated 30 times at 94°C for 30 seconds, at 55°C for 30 seconds, and at 72°C for 1 minute and 30 seconds, and then at 72°C for 5 minutes.
- the pDCM2 vector was treated with SmaI and the PCR product obtained above was fusion cloned. Fusion cloning was performed using the In-Fusion® HD cloning kit (Clontech). The resulting plasmid was named pDCM2-apt(A122T).
- Example 14-2 Evaluation of IMP production ability of microorganisms expressing adenine phosphoribosyl group transferase mutant
- Example 14-1 The vector prepared in Example 14-1 was transformed into the Corynebacterium stationionis CJI2332 strain (KCCM12277P, KR 10-1950141 B1).
- the strain into which the mutant was introduced was selected using SEQ ID NOs: 143 and 144.
- the selected strain was named CJI2332_apt_A122T.
- IMP production ability was analyzed by evaluating the flask fermentation titer of the strain prepared in Example 14-2-1 and the control parent strain.
- each strain was inoculated into a test tube with a diameter of 18 mm containing 2 ml of the seed medium, and cultured with shaking at 30 ° C. for 24 hours was used as a seed culture medium.
- 2 ml of the seed culture solution was inoculated into a 250ml corner-baffle flask containing 29ml of production medium (24ml of main medium + 5ml of separate sterile medium) and cultured at 30°C for 72 hours at 170rpm.
- the production capacity of IMP was measured by HPLC. IMP concentration and concentration increase rate in the culture medium for each strain tested are shown in Table 14 below.
- the CJI2332_apt_A122T strain exhibited a 24.9% increased IMP-producing ability compared to the control.
- Example 15 Bifunctional pyr operon transcriptional regulator / uracil phosphoribosyl transfer enzyme IMP production capacity evaluation of microorganisms expressing the PyrR mutant
- Example 15-1 Bifunctional pyr operon transcriptional regulator / uracil phosphoribosyl transfer enzyme PyrR mutant expression in microorganisms Vector construction
- a vector for the production of the expression strain was prepared as follows using the plasmid pDCM2 (Korea Publication No. 10-2020-0136813) for the insertion and replacement of genes in the Corynebacterium chromosome.
- PCR was performed using a pair of primers of SEQ ID NOs: 145 and 146 and a pair of primers of SEQ ID NOs: 147 and 148 using gDNA (genomic DNA) of wild-type Corynebacterium stasis ATCC6872 as a template, respectively.
- gDNA genomic DNA
- overlapping PCR was performed again using a primer pair of SEQ ID NO: 145 and SEQ ID NO: 148 to obtain a fragment.
- PCR was repeated 30 times at 94°C for 30 seconds, at 55°C for 30 seconds, and at 72°C for 1 minute and 30 seconds, and then at 72°C for 5 minutes.
- the pDCM2 vector was treated with SmaI and the PCR product obtained above was fusion cloned. Fusion cloning was performed using the In-Fusion® HD cloning kit (Clontech). The resulting plasmid was named pDCM2-pyrR (P108S).
- Example 15-2 Bifunctional pyr operon transcriptional regulator / uracil phosphoribosyl transfer enzyme IMP production capacity evaluation of microorganisms expressing the PyrR mutant
- Example 15-1 The vector prepared in Example 15-1 was transformed into Corynebacterium stasis CJI2332 strain (KCCM12277P, KR 10-1950141 B1).
- the strain into which the mutant was introduced was selected using SEQ ID NOs: 149 and 150.
- the selected strain was designated as CJI2332_pyrR_P108S.
- IMP production ability was analyzed by evaluating the flask fermentation titer of the strain prepared in Example 15-2-1 and the control parent strain.
- each strain was inoculated into a test tube with a diameter of 18 mm containing 2 ml of the seed medium, and cultured with shaking at 30 ° C. for 24 hours was used as a seed culture medium.
- 2 ml of the seed culture solution was inoculated into a 250ml corner-baffle flask containing 29ml of production medium (24ml of main medium + 5ml of separate sterile medium) and cultured at 30°C for 72 hours at 170rpm.
- the production capacity of IMP was measured by HPLC. IMP concentration and concentration increase rate in the culture medium for each strain tested are shown in Table 15 below.
- the CJI2332_pyrR_P108S strain exhibited an IMP-producing ability increased by 27.9% compared to the control.
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Abstract
La présente invention concerne un nouveau variant, une souche de Corynebacterium stationis comprenant ledit variant, et un procédé de production d'IMP faisant appel à ladite souche.
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