Novel ribose-phospho-bisphosphate kinase variant and method for producing L-amino acids using it

Abstract

This application relates to: a novel ribophosphate diphosphate kinase variant polypeptide; a polynucleotide encoding the variant polypeptide; a microorganism comprising the variant polypeptide or the polynucleotide encoding the variant polypeptide; a method for producing L-amino acids, comprising the step of culturing the microorganism in a culture medium; and the use of the polypeptide, the polynucleotide, and the microorganism for the production of L-amino acids.

Description

Technical Field

[0001] This disclosure relates to a novel variant polypeptide of ribophosphate diphosphate kinase; a polynucleotide encoding the variant polypeptide; a microorganism containing the variant polypeptide or encoding the polynucleotide of the variant polypeptide; and a method for producing L-amino acids, the method comprising the step of culturing the microorganism in a culture medium. Background Technology

[0002] Processes for producing target substances (e.g., amino acids) in microorganisms have been investigated in various ways as environmentally friendly and safe production methods. Among these, ongoing research has focused on producing large quantities of target substances from Corynebacterium sp. microorganisms. Corynebacterium sp., particularly Corynebacterium glutamicum, is a widely used Gram-positive microorganism for the production of L-amino acids and other useful substances.

[0003] L-amino acids are the basic structural units of proteins and are used as important materials in pharmaceuticals, food additives, animal feed, nutritional supplements, pesticides, and fungicides. Various studies have been conducted to develop efficient production microorganisms and technologies for fermentation processes in order to produce L-amino acids and other useful substances. For example, target-specific methods are primarily used, such as increasing the expression of genes encoding enzymes involved in L-lysine biosynthesis or removing genes unnecessary for biosynthesis (US8048650 B2).

[0004] L-Tryptophan is an essential amino acid and is widely used in feed additives, pharmaceutical raw materials such as infusions, and health food ingredients. L-Tryptophan can be produced through chemical synthesis, enzymatic reactions, and fermentation, but currently, direct fermentation using microorganisms is the primary method. Previous research, including intracellular quantitative analysis, has shown that among 20 amino acids, tryptophan biosynthesis requires the highest level of energy (Proc. Natl. Acad. Sci. USA, (2002) V99, pp3695-3700). Therefore, research is needed to effectively increase L-Tryptophan production.

[0005] L-histidine is one of the 20 standard amino acids and is classified as an essential amino acid for children's growth. L-histidine participates in important physiological processes such as antioxidation and immune regulation, and is used in the medical industry as a raw material for the treatment of gastric ulcers, circulatory system therapy, and amino acid infusion. Because histidine is particularly abundant in hemoglobin, it is mainly produced through protein hydrolysis extraction using blood meal as a raw material; however, this method has drawbacks such as low efficiency and environmental pollution. Microbial fermentation for the production of L-histidine is possible, but large-scale industrialization has not yet been achieved. Therefore, further research is needed to effectively increase the production of L-histidine.

[0006] Meanwhile, phosphoribosyl pyrophosphate (PRPP) is one of the precursors required for the biosynthesis of tryptophan and histidine, and is essential for increasing tryptophan or histidine production. Previous studies have reported that when ribophosphate diphosphate kinase is overexpressed, the supply of phosphoribosyl pyrophosphate increases, leading to increased histidine biosynthesis (EP 1529839 A1). Summary of the Invention

[0007] Technical issues

[0008] The objective of this disclosure is to provide a variant polypeptide of ribophosphate diphosphate kinase, wherein the amino acid at position 239 of the amino acid sequence corresponding to SEQ ID NO: 1 is replaced by another amino acid; a polynucleotide encoding the variant polypeptide; a microorganism comprising the variant polypeptide or the polynucleotide encoding the variant polypeptide; a method for producing L-amino acids, the method comprising the step of culturing the microorganism in a culture medium; and the use of the polypeptide, polynucleotide, or microorganism in the production of L-amino acids.

[0009] Technical solution

[0010] One aspect of this disclosure provides a variant polypeptide of ribophosphate diphosphate kinase, wherein the amino acid at position 239 of the amino acid sequence corresponding to SEQ ID NO: 1 is replaced by another amino acid.

[0011] In one specific embodiment, regarding the variant polypeptide, the amino acid at position 239 of the amino acid sequence corresponding to SEQ ID NO: 1 may be replaced by valine, proline, asparagine, methionine, glycine, or tyrosine.

[0012] In another specific embodiment, the variant polypeptide may consist of any amino acid sequence selected from SEQ ID NO: 15 to 20.

[0013] Another aspect of this disclosure provides a polynucleotide encoding a variant polypeptide of ribose-phosphobisphosphonate kinase.

[0014] Another aspect of this disclosure provides microorganisms comprising a variant polypeptide of ribose-phosphodiesterase or a polynucleotide encoding said variant polypeptide.

[0015] In one specific embodiment, the microorganism may have increased L-amino acid production capacity compared to unmodified microorganisms.

[0016] Regarding the microorganism according to any one of the foregoing specific embodiments, the microorganism may be a Corynebacterium genus microorganism.

[0017] Regarding the microorganism according to any one of the foregoing specific embodiments, the Corynebacterium genus microorganism may be Corynebacterium glutamicum.

[0018] Regarding the microorganism according to any one of the foregoing specific embodiments, the L-amino acid may be any one or more selected from the group consisting of L-tryptophan and L-histidine.

[0019] Another aspect of this disclosure provides a method for producing L-amino acids, the method comprising the step of culturing the microorganism in a culture medium.

[0020] In one specific implementation, the method may further include the step of recovering the target substance from cultured microorganisms, cultures of said microorganisms, fermentation products of said microorganisms, or culture media.

[0021] Another aspect of this disclosure provides a composition for producing L-amino acids, the composition comprising: a variant polypeptide of ribophosphate diphosphate kinase; a polynucleotide encoding the variant polypeptide; a microorganism comprising the variant polypeptide or the polynucleotide encoding the variant polypeptide; a culture of the microorganism; and a combination of two or more thereof.

[0022] Another aspect of this disclosure provides a variant polypeptide of ribophosphate diphosphate kinase, a polynucleotide encoding the variant polypeptide, a microorganism containing the variant polypeptide or the polynucleotide encoding the variant polypeptide, and the use of a culture of the microorganism for the production of L-amino acids.

[0023] Beneficial effects

[0024] When microorganisms containing variant peptides of the ribophosphate diphosphate kinase disclosed herein are cultured, it is possible to produce L-amino acids in high yields compared to unmodified microorganisms. Detailed Implementation

[0025] This disclosure will be described in detail below. Furthermore, each description and embodiment disclosed herein can be applied to other descriptions and embodiments. That is, all combinations of the various elements disclosed herein fall within the scope of this disclosure. Moreover, the scope of this disclosure is not limited to the specific embodiments described below. In addition, numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to further clarify the level and scope of the subject matter to which this disclosure pertains.

[0026] definition

[0027] As used in the specification and appended claims of this disclosure, the singular forms (“a,” “an,” and “the”) include plural objects unless the context otherwise requires. Unless the context otherwise requires, singular terms shall include plurals, and plural terms shall include singulars. As used in the specification and appended claims of this disclosure, unless otherwise required, the use of “or” may be used to include “and / or”.

[0028] As used herein, the term "approximately" may appear before a specific numerical value. The term "approximately" as used herein includes not only the exact number listed after the term, but also a range that is close to or near that number. Considering the context in which the number appears, it can be determined whether any number is close to or near the specific number that appears. For example, the term "approximately" may refer to a range of -10% to +10% of a value. As another example, the term "approximately" may refer to a range of -5% to +5% of a given numerical value, but is not limited to these.

[0029] As used herein, descriptions such as the terms “first, second, third…”, “i), ii), iii), or “or (a), (b), (c), (d)” can be used to distinguish individual components. When these terms are used to refer to steps of a method, use, or determination, they do not imply a sequence or order of execution; for example, there may be no time interval between these steps, or they may be performed simultaneously, or they may be performed sequentially, in reverse, or randomly, with intervals of seconds, minutes, hours, days, or months.

[0030] As used herein, the term "composed of" means that the total percentage of the specific features, steps, components, or other elements listed in the term is 100%. The features, steps, components, or other elements listed in the term "composed of" may be necessary or mandatory. For example, any other features, steps, components, or other elements, or non-essential features, steps, components, or other elements, may be excluded in addition to those listed in the term "composed of".

[0031] As used herein, the term "substantially composed of" can mean that the one or more undetailed features, steps, components or other elements claimed herein may be present without being substantially affected by the presence of one or more undetailed features, steps, components or other elements.

[0032] As used herein, the term "comprising" means the presence of the features, steps, components, or other elements listed after the term, and does not exclude the presence of one or more additional features, steps, components, or other elements. In this document, the features, steps, components, or other elements listed after the term "comprising" may be necessary or mandatory. However, in some embodiments, the term may also include any other or non-essential features, steps, components, or other elements.

[0033] Proteins, polypeptides, variant polypeptides

[0034] As used herein, the terms "protein" or "peptide" refer to a polymer or oligomer of consecutive amino acid residues. In this disclosure, the terms "peptide," "protein," and "peptide" are used interchangeably.

[0035] As used herein, the term "mature polypeptide or mature protein" refers to a polypeptide or protein that does not have a signal sequence or propeptide sequence. A mature polypeptide or mature protein can be the functional form of a polypeptide or protein. A mature polypeptide or mature protein can be the polypeptide in its final form after translation and / or post-translational modifications. Examples of post-translational modifications include, but are not limited to, N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, leader sequence removal, etc.

[0036] As used herein, the term "wild-type" refers to a naturally occurring state without artificial modification. When the term "wild-type" is used to refer to a polypeptide, it refers to a naturally occurring polypeptide that does not have artificial mutations (substitutions, insertions, deletions, etc.) at one or more amino acid positions. Similarly, when the term "wild-type" is used to refer to a polynucleotide, it refers to a naturally occurring polynucleotide that does not have artificial modifications (substitutions, insertions, deletions, etc.) at one or more nucleotide positions. However, polynucleotides encoding wild-type polypeptides are not limited to naturally occurring polynucleotides and include sequences encoding any wild-type polypeptide.

[0037] As used herein, the parental sequence or backbone refers to the reference sequence in which modifications are introduced to produce the variant polypeptide. That is, the parental sequence can serve as the starting sequence, in which modifications such as substitution, addition, and / or deletion can be introduced. The parental sequence can be naturally occurring or wild-type, or a variant of the naturally occurring or wild-type sequence with one or more substitutions, insertions, or deletions, or it can be a synthetically produced sequence.

[0038] As used herein, the term "reference sequence" refers to a sequence used as a reference for specifying the N position in a particular amino acid sequence. For example, when a particular amino acid sequence and a reference sequence are aligned using sequence alignments known in the art, and based thereon, when each amino acid residue in the particular amino acid sequence is numbered with reference to the numerical position of an amino acid residue in the reference sequence, the position of the amino acid corresponding to the N position of the reference sequence can be determined in the particular amino acid sequence.

[0039] As used herein, the term "N-position" in an amino acid sequence can refer to any amino acid position other than the N-position itself. Specifically, the N-position can include amino acid positions corresponding to any amino acid residue in a mature polypeptide, disclosed in a particular amino acid sequence. A particular amino acid sequence can be the amino acid sequence of a reference sequence.

[0040] As used herein, the term "corresponding to" refers to an amino acid residue at a position listed in the polypeptide, or an amino acid residue that is similar to, identical to, or homologous to a residue listed in the polypeptide. Identifying an amino acid at a corresponding position can be done by determining a specific amino acid in a sequence, which refers to a specific sequence. As used herein, the term "corresponding region" generally refers to a similar or corresponding position in the amino acid sequence of a related protein or a reference protein.

[0041] In this disclosure, the amino acid sequence of SEQ ID NO: 1 can be used as a reference sequence to determine the position of amino acids in any amino acid sequence.

[0042] In other words, the amino acid sequence of SEQ ID NO: 1 disclosed herein can be used to identify the corresponding amino acid residues in any polypeptide. Unless otherwise stated in this disclosure, the residues of a particular amino acid sequence are based on the amino acid sequence number of SEQ ID NO: 1.

[0043] For example, any amino acid sequence can be aligned with the amino acid sequence of SEQ ID NO: 1, and based on this alignment, each amino acid residue in the arbitrary amino acid sequence can be numbered with reference to the numerical position of the amino acid residue corresponding to the amino acid residue in SEQ ID NO: 1. For example, the sequence alignment algorithm described herein can identify the position of amino acids compared to the query sequence (also known as the "reference sequence"), or the location of modifications such as substitutions, insertions, or deletions.

[0044] In such alignments, for example, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453), the Needle program of the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000), Trends Genet. 16: 276-277), but not limited thereto, may be used, as well as sequence alignment programs, pairwise sequence comparison algorithms, etc., known in the art as appropriate.

[0045] In addition, the corresponding amino acid residues in another polypeptide can be identified by multiple sequence alignment. Examples of known multiple sequence alignment programs in this field include MUSCLE (multiple sequence comparison by log-expectation; version 3.5 or later; Edgar, 2004, Nucleic Acids Research 32: 1792-1797), MAFFT (version 6.857 or later; Katoh and Kuma, 2002, Nucleic Acids Research 30: 3059-3066; Katoh et al., 2005, Nucleic Acids Research 33: 511-518; Katoh and Toh, 2007, Bioinformatics 23: 372-374; Katoh et al., 2009, Methods in Molecular Biology 537: 39-64; Katoh and Toh, 2010, Bioinformatics 26: ). The program can use ClustalW's EMBOSS EMMA (version 1.83 or later; Thompson et al., 1994, Nucleic Acids Research 22: 4673-4680), and can use their respective default parameters, but is not limited to them.

[0046] Furthermore, when peptides derived from the mature peptide of SEQ ID NO: 1 cannot be correlated using traditional sequence-based comparisons, alternative pairwise sequence comparison algorithms can be used (Lindahl and Elofsson, 2000, J. Mol. Biol. 295: 613-615). Higher sensitivity for sequence-based searches can be achieved by using search procedures that utilize probabilistic representations of peptide families (spectrals) to search databases. For example, the PSI BLAST procedure generates spectra through an iterative database search process and is capable of detecting distant homologs (Atschul et al., 1997, Nucleic Acids Res. 25: 3389-3402). Even higher sensitivity can be achieved if the peptide family or superfamily has one or more representatives in a protein structure database. Programs such as GenTHREADER (Jones, 1999, J. Mol. Biol. 287:797-815; McGuffin and Jones, 2003, Bioinformatics 19: 874-881) utilize information from various sources (such as PSI-BLAST, secondary structure prediction, structure alignment spectra, and solvation potential) as input to neural networks that predict the structural folding of query sequences. Similarly, the method of Gough et al., 2000, J. Mol. Biol. 313: 903-919 can be used to align sequences of unknown structures with superfamily models existing in the SCOP database. These alignments can then be used to generate homology models of peptides, and the accuracy of these models can be evaluated using various tools developed for this purpose.

[0047] For proteins with known structures, several tools and resources are available for retrieving and generating structure alignments. For example, the SCOP superfamily of proteins has been structurally aligned, and these alignments are accessible and downloadable. Two or more protein structures can be aligned using various algorithms, such as distance alignment matrices (Holm and Sander, 1998, Proteins 33: 88-96) or combinatorial extension (CE) (Shindyalov and Bourne, 1998, Protein Engineering 11: 739-747). Implementations of these algorithms can also be used to query structure databases with target structures to discover potential structural homologs (Holm and Park, 2000, Bioinformatics 16: 566-567).

[0048] The methods described above are illustrative and not limited thereto.

[0049] In this disclosure, regarding amino acid sequences, it is clear that a polypeptide or protein "comprising," "composed of," or "having" an amino acid sequence represented by a specific sequence number may also include polypeptides or proteins with missing, modified, substituted, or added amino acids, provided that they have the same or corresponding activity as the polypeptide or protein composed of the amino acid sequence represented by the corresponding sequence number. For example, a polypeptide or protein may also include polypeptides or proteins with amino acid additions or deletions that do not alter protein function, naturally occurring mutations, their silent mutations, or conserved substitutions located within, upstream of, or downstream of (N-terminus or C-terminus) the polypeptide or protein, provided that they have the same or corresponding activity.

[0050] In addition, for example, peptides or proteins conjugated with an N-terminal signal (or leader) sequence that is co-translated or post-translational involved in protein (peptide) translocation, or peptides or proteins conjugated with other sequences or linkers for identification, purification or synthesis of the peptide or protein, may also be included in the scope of peptides or proteins with amino acid sequences represented by a specific sequence number.

[0051] As used herein, the term "conservative substitution" refers to the replacement of one amino acid with another amino acid having similar structure and / or chemical properties. Such amino acid substitutions can typically occur based on the similarity of the residues' polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphiphilic properties. For example, positively charged (basic) amino acids include arginine, lysine, and histidine; negatively charged (acidic) amino acids include glutamic acid and aspartic acid; amino acids with nonpolar side chains (nonpolar amino acids) include glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline; and amino acids with polar or hydrophilic side chains (polar amino acids) include serine, threonine, cysteine, tyrosine, asparagine, and glutamine. For example, amino acids can be classified into amino acids with charged side chains (charged amino acids) such as arginine, lysine, histidine, glutamic acid, and aspartic acid, and amino acids with uncharged side chains (uncharged amino acids; also known as neutral amino acids) such as glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. For example, phenylalanine, tryptophan, and tyrosine can be classified as aromatic amino acids. For example, valine, leucine, and isoleucine can be classified as branched-chain amino acids. For example, classifying the 20 amino acids based on size, starting with the smaller groups, amino acids can be divided into five groups: glycine, alanine, serine; cysteine, proline, threonine, aspartic acid, asparagine; valine, histidine, glutamic acid, glutamine; isoleucine, leucine, methionine, lysine, arginine; and phenylalanine, tryptophan, tyrosine. However, they are not necessarily limited to this. Generally, conserved substitutions may have little or no effect on the activity of polypeptides or proteins.

[0052] As used herein, the term "another amino acid" is not limited, as long as it is an amino acid different from the amino acid before the substitution. Furthermore, in this disclosure, the statement "a specific amino acid has been substituted" clearly indicates that the amino acid has been replaced by an amino acid different from the amino acid before the substitution, even if it is not specifically stated that the amino acid has been substituted by another amino acid.

[0053] In this disclosure, the term "change / modification" means alteration or modification. This can be a naturally occurring change. For example, a polypeptide can be altered in a way that makes the polypeptide different from a parental sequence or a reference sequence.

[0054] In this disclosure, the modified polypeptide can be a polypeptide that does not exist in nature, i.e., a polypeptide that does not exist naturally.

[0055] As used herein, the term "modified" means, for example, a modification derived from a naturally occurring form. Modified polypeptides of this disclosure include polypeptides that are not naturally occurring or variants of naturally occurring polypeptides. For example, the modified polypeptides of this disclosure are modified polypeptides not found in nature. For example, the modified polypeptides of this disclosure may not arise spontaneously, but are not limited thereto.

[0056] When used in conjunction with amino acid / base (nucleic acid) sequences, the term "modification" as used herein may include replacing an amino acid / nucleotide of the parent sequence with another amino acid / nucleic acid at one or more sites in the parent amino acid / base (nucleotide) sequence, deleting an amino acid / nucleotide (or a series of amino acids / nucleotides) at one or more sites in the parent amino acid / base (nucleotide) sequence, adding an amino acid / nucleotide (or a series of amino acids / nucleotides) at one or more sites in the parent amino acid / base (nucleotide) sequence, or any combination thereof. For example, deletion may include, but is not limited to, truncating an N-terminal and / or C-terminal amino acid or a 5' and / or 3' nucleotide.

[0057] As used herein, the term "variant" or "variant polypeptide" refers to a polypeptide that differs from its parental sequence due to the presence of one or more conserved substitutions and / or modifications (substitution, addition, or deletion, etc.) of amino acids in the parental sequence. Compared to the native polypeptide, the function or properties of such a variant polypeptide may be increased, unchanged, or decreased. For example, some variant polypeptides may contain one or more regions, such as the N-terminal leader sequence or transmembrane domain, that have been removed. Furthermore, for example, another variant polypeptide may contain a variant in which a region has been removed from the N- and / or C-terminus of the mature protein. The term "variant polypeptide" may be used interchangeably with, and is not limited to, terms such as modification, modified protein, mutant, mutated protein, dismutation, variant, etc., as long as it is a term with the meaning of variation.

[0058] Furthermore, variant peptides can contain the deletion or addition of amino acids that have minimal impact on the properties and secondary structure of the peptide. For example, the peptide can be conjugated to the N-terminal signal (or leader) sequence of a peptide involved in co-translation or post-translational protein translocation. Additionally, the peptide can be conjugated to other sequences or linkers for identification, purification, or synthesis of the peptide.

[0059] The variant polypeptides disclosed herein can be in isolated form.

[0060] As used herein, the term "isolated" refers to a substance that exists in a non-naturally occurring environment or in a non-naturally occurring form. This includes substances (sequences or nucleic acids) that are at least substantially free of at least other components having that substance (e.g., sequence or nucleic acid), that are naturally associated with and found in nature.

[0061] For example, the isolated sequences or nucleic acids provided in this disclosure may be provided in a form that is substantially free of one or more contaminants.

[0062] Examples of isolated substances may include, but are not limited to, i) any substance that is not naturally occurring, ii) any substance from which one or more or all of its naturally occurring components associated with it in nature have been removed (e.g., enzymes, variants, nucleic acids, proteins, peptides, or cofactors), iii) any substance artificially modified from a substance found in nature, or iv) a substance modified to alter the amount of that substance relative to other components naturally associated with it (e.g., by increasing the copy number of the gene encoding the particular substance; by modifying a promoter naturally associated with the gene encoding the particular substance with a promoter that has a stronger activity).

[0063] The variant peptides disclosed herein may contain bioactive fragments of the variant peptides.

[0064] In this disclosure, the term "bioactive fragment" can refer to a "functional fragment" in relation to an amino acid or base (nucleic acid) sequence. A "functional fragment" can also refer to an active fragment, which is a polypeptide containing fewer amino acids than a full-length protein but possessing at least one biological activity of the corresponding full-length protein. For example, a functional fragment of an enzyme can contain the enzyme's catalytic site.

[0065] The bioactive fragment of the variant polypeptide disclosed herein may comprise a portion of the full length of the natural polypeptide. For example, it may comprise at least about 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, or 98% or more, and less than 100% of the amino acids of the full length of the natural polypeptide, but is not limited thereto.

[0066] Throughout this instruction manual, standard single-letter and three-letter codes for naturally occurring amino acids are used. Furthermore, the amino acids mentioned herein are abbreviated according to the IUPAC-IUB naming convention.

[0067] Alanine (Ala), Arginine (Arg), R

[0068] Asparagine Asn, N-aspartic acid Asp, D

[0069] Cysteine ​​(Cys), Glutamate (Glu), E

[0070] Glutamine (Gln), Glycine (Gly), G

[0071] Histidine (His, H), Isoleucine (Ile, I)

[0072] Leucine (Leu), L-lysine (Lys), K

[0073] Methionine (Met), M-phenylalanine (Phe), F

[0074] Proline (Pro), Serine (S),

[0075] Threonine (Thr), T-tryptophan (Trp), W

[0076] Tyrosine (Tyr), Y-valine (Val), V

[0077] At the same time, any amino acid can be described as Xaa, X.

[0078] In addition to naturally occurring amino acids, three-letter codes that are usually allowed for other amino acids, such as 2-aminoisobutyric acid (Aib), N-methylglycine (Sar), and α-methylglutamic acid, can also be used.

[0079] Amino acids can usually be classified based on the similarity of their residue polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphiphilic properties.

[0080] Examples of this classification can include positively charged (basic) amino acids, such as arginine, lysine, and histidine; negatively charged (acidic) amino acids, such as glutamic acid and aspartic acid; amino acids with nonpolar side chains (nonpolar amino acids), such as glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline; and amino acids with polar or hydrophilic side chains (polar amino acids), such as serine, threonine, cysteine, tyrosine, asparagine, and glutamine. For example, amino acids can be classified as having charged side chains (charged amino acids), such as arginine, lysine, histidine, glutamic acid, and aspartic acid, and having uncharged side chains (uncharged amino acids; also called neutral amino acids), such as glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. For example, phenylalanine, tryptophan, and tyrosine can be classified as aromatic amino acids. For example, valine, leucine, and isoleucine can be classified as branched-chain amino acids. For example, classifying the 20 amino acids according to size, starting with the smaller groups, amino acids can be divided into five groups: glycine, alanine, serine; cysteine, proline, threonine, aspartic acid, asparagine; valine, histidine, glutamic acid, glutamine; isoleucine, leucine, methionine, lysine, arginine; and phenylalanine, tryptophan, and tyrosine. However, they are not necessarily limited to these categories.

[0081] The following nomenclature is used to describe the variations provided in this disclosure.

[0082] In this disclosure, referring to a specific position in an amino acid sequence may include referring to the amino acid present or substituted at that position. Referring to an amino acid at a specific position can be described in different ways. For example, “position 003” can be described as “position 3”, “amino acid 3”, or “the third amino acid”. Furthermore, for example, when the amino acid at position 3 is serine (S), it can be described as “S3” or “Ser3”.

[0083] Amino acid substitutions can be represented by describing the amino acid before the substitution, its position, and the amino acid to be substituted in sequence. Amino acids can be represented using traditional single-letter and three-letter codes. For example, when alanine, an amino acid at position 6 of a specific sequence, is substituted with valine, it can be described as "A8V" or "Ala8Val".

[0084] Any amino acid at a specific position can be designated as "X". For example, X6 refers to any amino acid at position 6. Furthermore, when the amino acid to be substituted is represented as X, it means that the amino acid is replaced by an amino acid different from the one present before the substitution. For example, "V6X" means that the V at position 6 is replaced by any amino acid other than V.

[0085] Genes, polynucleotides

[0086] As used herein, the term "gene" narrowly refers to a polynucleotide that encodes a functional molecule, and broadly refers to a polynucleotide that contains a polynucleotide encoding a functional molecule as well as upstream and downstream regions of that polynucleotide. In one specific embodiment, the functional molecule may be RNA or a protein, and the gene may have sequences (introns) inserted between its respective coding regions (exons).

[0087] As used herein, the terms “polynucleotide,” “nucleic acid,” or “nucleic acid molecule” refer to a chain of DNA (e.g., cDNA or genomic DNA) or RNA (e.g., mRNA) of a certain length or longer, which is a polymer of nucleotides, wherein nucleotide monomers are linked together in a long chain by covalent bonds. In this disclosure, “polynucleotide,” “nucleic acid,” and “nucleic acid molecule” are used interchangeably.

[0088] Homology, Identity

[0089] As used herein, the term “identity” or “homology” refers to the degree of correlation between two given amino acid or nucleotide sequences, expressed as a percentage. In this disclosure, “homology” and “identity” are generally used interchangeably.

[0090] Sequence homology or identity of conserved polynucleotides or polypeptides can be determined using standard alignment algorithms, and can be used together with default gap penalties established by the program used.

[0091] Whether any two polynucleotide or polypeptide sequences are homologous, similar, or identical can be determined using known computer algorithms such as the “FASTA” program, for example, with default parameters (as in Pearson et al. (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444). Alternatively, sequence information can be determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453), such as in the Needleman program (version 5.0.0 or later) of the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), or by aligning sequence information using GAP computer programs such as the Smith-Waterman algorithm (Smith and Waterman, Adv. Appl. Math (1981) 2:482) (including GCG program packages (Devereux, J. et al., Nucleic Acids Research 12: 387(1984)), BLASTP, BLASTN, FASTA (Atschul, [S.] [F.,] [ET AL, J MOLEC BIOL)). 215]:403 (1990); Guide to Huge Computers, Martin J. Bishop, [ED.,] Academic Press, San Diego, 1994 and [CARILLO et al.] (1988) SIAM J Applied Math 48: 1073). For example, BLAST or ClustalW from the National Center for Biotechnology Information can be used to determine homology, similarity, or identity.

[0092] Furthermore, whether any two polynucleotide sequences are homologous, similar, or identical can be determined by Southern hybridization experiments under appropriate hybridization conditions, which can be determined by methods well known to those skilled in the art (e.g., J. Sambrook et al., Molecular Cloning, A Laboratory Manual; FMAusubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York), but are not limited thereto. For example, under stringent conditions, homologous or identical polynucleotide sequences can typically hybridize along the entire sequence or at least about 50%, 60%, 70%, 80%, or 90% of the full-length sequence.

[0093] As used herein, the term “strict conditions” refers to conditions that allow specific hybridization between polynucleotides. These conditions are described in detail in the literature (see Sambrook et al., above, 9.50–9.51, 11.7–11.8). For example, stringent conditions may include polynucleotides having high homology or identity, i.e., polynucleotides having 60% or more, 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 99% or more homology or identity, hybridizing with each other, while polynucleotides having lower homology or identity do not hybridize with each other, or washing conditions for ordinary Southern hybridization, i.e., washing once at a salt concentration and temperature corresponding to 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, specifically, washing two to three times.

[0094] Hybridization can occur between nucleotides with complementary base sequences, but depending on the strictness of the hybridization process, hybridized polynucleotides can contain some mismatches between bases. The term "complementary" is used to describe the relationship between nucleotide bases that are capable of hybridizing with each other. For example, in the case of DNA, adenosine is complementary to thymine, and cytosine is complementary to guanine. Therefore, the polynucleotides of this disclosure can also contain substantially similar nucleotide sequences as well as separate nucleic acid fragments complementary to the whole sequence.

[0095] For example, polynucleotides homologous to or identical with the polynucleotides disclosed herein can be detected by hybridization at a Tm value of 55°C. Furthermore, the Tm value can be 60°C, 63°C, or 65°C, but is not limited thereto, and can be appropriately adjusted by those skilled in the art.

[0096] The appropriate stringency of hybrid polynucleotides depends on the length and complementarity of the polynucleotides, and the variables are well known in the art (e.g., J. Sambrook et al., ibid.).

[0097] Nucleic acid constructs, vectors, and transformation

[0098] As used herein, the term "nucleic acid construct" refers to an artificially designed single- or double-stranded nucleic acid molecule contained in a vector, which can be used to integrate target genetic material into a suitable host or host cell. For example, a nucleic acid construct may contain a transgene delivered via a transformation vector that allows the inserted sequence to be replicated and / or expressed in a host cell. For example, the transgene may be replicated from an existing sequence or may be artificially synthesized.

[0099] As used herein, the term "vector" refers to a DNA construct used to deliver desired polynucleotides into a suitable host or host cell.

[0100] For example, a vector can contain a nucleotide sequence of a polynucleotide encoding a desired polypeptide, which is operatively linked to a suitable expression regulatory region (regulatory sequence) to express the desired polypeptide in a suitable host cell. The expression regulatory region may contain a promoter capable of initiating transcription, any operon sequence regulating transcription, a sequence encoding a suitable mRNA ribosome binding site, and sequences regulating transcription and translation termination. Once transformed into a suitable host cell (microorganism), the vector can replicate or function independently of the host genome, or it can integrate into its genome to replicate or function.

[0101] Furthermore, for example, the vector of this disclosure may contain a sequence for inserting the desired polynucleotide into a chromosome. Insertion of the polynucleotide into a chromosome using the vector can be performed by any method known in the art, such as homologous recombination, but is not limited thereto.

[0102] There are no particular limitations on the vectors used in this disclosure, but any vector known in the art may be used. Examples of commonly used vectors may include natural or recombinant plasmids, granules, viruses, and bacteriophages. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, Charon21A, etc., can be used as phage vectors or granule vectors, and the pDZ system, pDC system, pBR system, pUC system, pBluescript II system, pGEM system, pTZ system, pCL system, pET system, etc., can be used as plasmid vectors. For example, pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vectors, etc., may be used.

[0103] The vector may further include selection markers to confirm transformation into host cells, or further, insertion into the host cell's chromosome. Selection markers are used to select cells transformed with the vector, or to confirm the insertion of desired polynucleotides into the chromosome, and may use markers that provide selectable phenotypes (e.g., drug resistance, auxotrophic phenotypes, resistance to cytotoxic agents, or expression of surface peptides). Only cells expressing the selection markers are able to survive or exhibit different phenotypes under conditions treated with the selection agent, thus allowing for the selection of transformed cells.

[0104] As used herein, the term "transformation" refers to the introduction of a desired polynucleotide or a vector containing it into a host cell (microorganism) to alter the genetic traits of the host cell (microorganism). The transformed polynucleotide may be inserted into the chromosome of the host cell (microorganism) or may be located outside the chromosome. Furthermore, the polynucleotide may contain DNA or RNA. Depending on the purpose of the introduction, the polynucleotide may be introduced in a suitable form. For example, a polynucleotide for expressing a desired polypeptide may be introduced into the host cell (microorganism) in the form of an expression cassette, which is a gene construct containing all the elements required for self-expression. The expression cassette may typically contain a promoter, transcription termination signal, ribosome binding site, and translation termination signal operatively linked to the coding sequence of the desired polypeptide. The expression cassette may be in the form of a self-replicating expression vector. Furthermore, the polynucleotide may be introduced into the host cell (microorganism) in its own form and operatively linked to the sequence required for expression in the host cell (microorganism), but is not limited thereto.

[0105] As used herein, the term "operably linked" refers to an arrangement in which a regulatory sequence is positioned such that it directs the expression of a coding sequence. Therefore, the term "operably linked" includes attachment or connection between a regulatory region (such as a promoter, terminator, signal sequence, or enhancer) with a functional domain having known or desired activity and a target (gene or polypeptide), thereby regulating the expression, secretion, or function of the target according to known or desired activity. For example, the term implies a functional link between a polynucleotide sequence and a promoter sequence that initiates and mediates the transcription of a polynucleotide encoding a polypeptide.

[0106] As used herein, the term “expression” includes, but is not limited to, any process involved in peptide production, such as transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0107] As used herein, the term "expression vector" refers to a linear or circular nucleic acid molecule containing a desired polynucleotide sequence and a operatively linked regulatory sequence for its expression. For example, an expression vector may contain a nucleotide sequence of a polynucleotide encoding a desired polypeptide, which is operatively linked to a suitable expression regulatory region (expression regulatory sequence) to enable the expression of the desired polypeptide in a suitable host cell.

[0108] As used herein, the term "regulatory sequence" refers to a polynucleotide sequence required to regulate the expression of a desired polynucleotide sequence. Each regulatory sequence can be a natural (from the same source) or exogenous (from a different gene) sequence of the coding sequence, or a mutant or other artificial sequence thereof. Examples of regulatory sequences can include leader sequences, polyadenylated sequences, propeptide sequences, promoters, signal peptide sequences, operon sequences, sequences encoding ribosome-binding domains, and sequences regulating transcription and translation termination. The smallest unit of a regulatory sequence can contain a promoter and sequences that terminate transcription and translation.

[0109] As used in this article, the term “genetic recombination” refers to a natural or artificial process in which elements of a gene, such as DNA or RNA, are altered from their original sequence during the process of breakdown and recombination.

[0110] As used herein, the term "recombinant gene" refers to a gene having a novel genetic structure resulting from genetic recombination (such as chemical synthesis or genetic engineering techniques). In this disclosure, the terms "recombinant gene," "recombinant DNA," and "recombinant polynucleotide" are used interchangeably. For example, a recombinant gene can comprise an artificial combination of nucleic acid fragments, such as regulatory sequences, that are not found together in nature.

[0111] As used in this article, the term "recombinant protein" refers to a protein produced by genetic recombination.

[0112] microorganism

[0113] As used herein, the term "microorganism (or strain)" includes all naturally occurring or artificially genetically modified wild-type microorganisms or prokaryotic or eukaryotic microorganisms, and can be a microorganism in which a particular mechanism is weakened or enhanced due to the insertion of a foreign gene or the enhancement or inactivation of an endogenous gene, and can be a microorganism containing genetic modifications for the production of desired polypeptides, proteins, or products. As used herein, the terms "microorganism," "strain," "host," and "host cell" are used interchangeably.

[0114] As used herein, the term "recombinant microorganism" refers to a microorganism that has been genetically modified to exhibit a different genotype and / or phenotype compared to a naturally occurring microorganism (e.g., when the genetic modification affects the coding of the microorganism's base (nucleic acid) sequence), and may include any offspring or potential offspring of that microorganism. As used herein, the terms "recombinant microorganism," "genetically modified microorganism," "recombinant host cell," "recombinant cell," and "recombinant strain" are used interchangeably. For example, a recombinant microorganism may express genes not present in its natural (non-recombinant) form; may not express genes expressed in their natural form; or may express natural genes in a manner different from their natural expression.

[0115] For example, the microorganisms disclosed herein may be any one or more of the variant peptides, polynucleotides, and vectors containing the polynucleotides disclosed herein; microorganisms modified to express the variant peptides or polynucleotides disclosed herein; recombinant microorganisms expressing the variant peptides or polynucleotides disclosed herein; or recombinant microorganisms having the variant peptide activity disclosed herein, but are not limited thereto.

[0116] As used herein, the term "microorganism with L-amino acid production capacity" refers to a prokaryotic or eukaryotic microorganism capable of producing L-amino acids in a living organism, and may include all microorganisms inherently possessing L-amino acid production capacity, as well as microorganisms that do not inherently possess L-amino acid production capacity but are endowed with it. L-amino acid production capacity may be endowed or enhanced by expressing variant peptides of this disclosure or by species modification.

[0117] As used herein, the term "unmodified microorganism (strain)" does not exclude the inclusion of microorganisms (strains) that may have naturally occurring mutations, and can be a wild-type microorganism (strain) or a natural microorganism (strain) itself, or a microorganism (strain) before its traits are altered by genetic variation due to natural or artificial factors. As used herein, the term "unmodified microorganism (strain)" may be used interchangeably with "pre-modified microorganism (strain)," "unmutated microorganism (strain)," "parental microorganism," "parental strain," "wild-type microorganism (strain)," "reference microorganism (strain)," or "standard microorganism (strain)." In this disclosure, an unmodified microorganism may refer to a microorganism (strain) in which no variant polypeptide of this disclosure has been introduced or has not yet been introduced; or a microorganism (strain) containing a wild-type polypeptide, but is not limited thereto. Furthermore, in this disclosure, an unmodified microorganism may be a microorganism containing a polypeptide consisting of SEQ ID NO: 1 or a polynucleotide consisting of SEQ ID NO: 2, but is not limited thereto.

[0118] Increased protein (peptide) activity

[0119] As used herein, the term “increased protein (peptide) activity” refers to an increase in protein (peptide) activity in the host cell (microbe) compared to its endogenous activity. This increase can be used interchangeably with terms such as activation, upregulation, overexpression, and enhancement. The host cell (microbe) can be a prokaryotic or eukaryotic microorganism.

[0120] Increased protein (peptide) activity may include exhibiting protein (peptide) activity not present in the host cell (microorganism) endogenous form, and exhibiting protein (peptide) activity that is enhanced compared to endogenous activity or pre-modified activity.

[0121] For example, “exhibiting protein (peptide) activity not present in endogenous proteins” or “exhibiting enhanced protein (peptide) activity” can be caused by “the introduction of proteins (peptides),” but is not limited to this.

[0122] As used herein, the term "introduction" of a protein (peptide) refers to the expression of a specific protein's activity, or an enhanced, increased, or elevated peptide activity compared to the corresponding protein's endogenous activity or its unmodified activity, when a gene not originally present in the microorganism is expressed. For example, this can be caused by the introduction of a gene encoding a protein (peptide) into a host cell (microorganism). For instance, a polynucleotide encoding a specific protein (peptide) can be introduced into the chromosome of a host cell (microorganism), or a vector containing a polynucleotide encoding a specific protein (peptide) can be introduced into a host cell (microorganism) to express or enhance its activity.

[0123] "Endogenous activity" refers to the activity of a specific protein (peptide) that was originally present in the host cell (microorganism) before the trait change, or in the unmodified host cell (microorganism), when the trait is altered due to genetic variation caused by natural or artificial factors. This can be used interchangeably with "activity before modification".

[0124] The fact that the activity of a protein (peptide) is increased compared to its endogenous activity means that the activity and / or concentration (expression level) of the protein (peptide) in the host cell (microorganism) is higher than the activity and / or concentration (expression level) of the protein (peptide) originally present in the host cell (microorganism) before the trait change or in the unmodified host cell (microorganism).

[0125] For example, an increase indicates the presence of activity of a corresponding protein (peptide) that was not originally present, or its activity or concentration based on the activity or concentration in the host cell (microorganism) before the phenotypic change or in the unmodified host cell (microorganism), typically increasing to about 1% or more, about 10% or more, about 25% or more, about 50% or more, about 75% or more, about 100% or more, about 150% or more, about 200% or more, about 300% or more, about 400% or more, or about 500% or more, up to about 1000% or about 2000% or more, but not limited thereto.

[0126] Increased protein (peptide) activity can be achieved by introducing exogenous proteins (peptides) or increasing the activity of endogenous proteins (peptides). This increase can be confirmed by an increase in the activity level and expression level of the corresponding protein (peptide), or by an increase in the amount of product released from the activity of the corresponding protein (peptide).

[0127] Increased protein (peptide) activity can be achieved by a variety of methods well known in the art, and such methods are not limited, as long as the desired protein (peptide) activity can be increased compared to the activity of the host cell (microbe) before modification. Specifically, genetic engineering and / or protein engineering well known to those skilled in the art can be used, as these are routine methods in molecular biology, but the methods are not limited thereto (e.g., Sitnicka et al., Functional Analysis of Genes. Advances in Cell Biology. 2010, Vol. 2. 1-16, Sambrook et al., Molecular Cloning 2012, etc.).

[0128] Specifically, the increase in protein (peptide) activity disclosed herein can be:

[0129] 1) Increase the intracellular copy number of the polynucleotide encoding the protein (peptide);

[0130] 2) Modify the gene expression regulatory region on the chromosome that encodes the protein (peptide) (e.g., introduce a mutation into the expression regulatory region, replace it with a more active sequence, or insert a more active sequence).

[0131] 3) Modify the nucleotide sequence of the start codon or 5'-UTR region of the gene transcript (which encodes the protein (polypeptide));

[0132] 4) Modify the amino acid sequence of the protein (peptide) to increase its activity;

[0133] 5) Modify the polynucleotide sequence encoding the protein (peptide) to increase the activity of the protein (peptide) (e.g., modify the polynucleotide sequence of the gene encoding the protein (peptide) to encode a protein (peptide) that has been modified to increase the activity of the protein (peptide)).

[0134] 6) Introduce an exogenous protein (peptide) exhibiting the activity of the stated protein (peptide) or an exogenous polynucleotide encoding it;

[0135] 7) Codon optimization of the polynucleotides encoding the protein (peptide);

[0136] 8) Analyze the tertiary structure of the protein (peptide) to select and modify or chemically modify exposed sites;

[0137] 9) Controlling the intracellular localization of the protein (peptide); or

[0138] 10) A combination of two or more of 1) to 9), but not limited thereto.

[0139] For example,

[0140] 1) Increasing the intracellular copy number of the polynucleotide encoding the protein (peptide) can be achieved by introducing a vector containing the polynucleotide encoding the protein (peptide) into a host cell (microorganism), the polynucleotide being operatively linked to a suitable regulatory sequence. Alternatively, this increase can be achieved by introducing one or two or more copies of the polynucleotide encoding the protein (peptide) into the chromosome of the host cell (microorganism), the polynucleotide being operatively linked to a suitable regulatory sequence. Chromosomal introduction can be performed by introducing a vector capable of inserting the polynucleotide into the host cell (microorganism) chromosome, but is not limited thereto. The vector is as described above. The regulatory sequence can be a natural (from the same source) or exogenous (from a different gene) sequence encoding the polynucleotide sequence, or a mutant or other artificial sequence thereof, and can induce the expression of the polynucleotide in the host cell (microorganism).

[0141] 2) Replacing the gene expression regulatory region (or expression regulatory sequence) on the chromosome encoding the protein (peptide) with a sequence of greater activity can, for example, involve introducing mutations into the sequence through deletion, insertion, substitution, or a combination thereof, or by replacing it with a sequence of greater activity to further increase the activity of the expression regulatory region. The expression regulatory region may include, but is not limited to, promoters, operon sequences, sequences encoding ribosome binding sites, sequences controlling transcription and translation termination, etc. For example, the original promoter can be replaced with a stronger promoter, but it is not limited to this.

[0142] Examples of known strong promoters include, but are not limited to, the CJ1 to CJ7 promoters (US Patent No. 7,662,943 B2), the lac promoter, the trp promoter, the trc promoter, the tac promoter, the λ phage PR promoter, the PL promoter, the tet promoter, the gapA promoter, the SPL7 promoter, the SPL13(sm3) promoter (US Patent No. 10,584,338 B2), the O2 promoter (US Patent No. 10,273,491 B2), the tkt promoter, and the yccA promoter.

[0143] 3) Modifying the nucleotide sequence of the start codon or 5'-UTR region of the gene encoding the protein (peptide) may, for example, be replacing it with another start codon having a higher expression rate than the endogenous start codon, or modifying it to encode an RBS sequence having a higher expression rate than the endogenous ribosome binding site (RBS) sequence, but is not limited thereto.

[0144] 4) and 5) Modification of the amino acid sequence or polynucleotide sequence of the protein (peptide) may be performed by introducing mutations in the amino acid sequence of the protein (peptide) or the polynucleotide sequence encoding the protein (peptide) through deletion, insertion, substitution, or a combination thereof to increase the activity of the protein (peptide), or by replacing it with a modified amino acid sequence or polynucleotide sequence to increase activity, but is not limited thereto. Substitution may be performed, for example, by inserting a polynucleotide into the chromosome through homologous recombination, but is not limited thereto.

[0145] 6) The introduction of exogenous polynucleotides exhibiting the activity of the protein (peptide) can be achieved by introducing exogenous polynucleotides encoding a protein (peptide) exhibiting the same or similar activity as the protein (peptide) into a host cell (peptide). There are no restrictions on their source or sequence, as long as the exogenous polynucleotide exhibits the same or similar activity as the protein (peptide). The method used for introduction can be carried out by transformation methods known to those skilled in the art, appropriately selected. As the introduced polynucleotide is expressed in the host cell, the protein (peptide) can be produced and its activity can be increased.

[0146] 7) Codon optimization of the polynucleotide encoding the protein (peptide) may be codon optimization of endogenous polynucleotides to enhance transcription or translation in the host cell (microorganism), or codon optimization of exogenous polynucleotides to optimize transcription and translation in the host cell (microorganism).

[0147] 8) Analyzing the tertiary structure of the protein (peptide) to select and modify or chemically modify the exposed sites can, for example, be done by comparing the sequence information of the protein (peptide) to be analyzed with a database storing the sequence information of known proteins, determining template protein candidates based on sequence similarity, confirming the structure based on this, and modifying or chemically modifying the exposed sites to be modified or chemically modified.

[0148] 9) Controlling the cellular localization of the protein (peptide) can be achieved by targeting the protein (peptide) to specific organelles or specific intracellular spaces. For example, targeting can be achieved by adding or removing a leader sequence that functions in the target protein (peptide), but is not limited thereto.

[0149] This increase in protein (peptide) activity may be based on the activity or concentration of the protein (peptide) expressed in the wild type or host cell (microorganism) before modification, an increase in the activity or concentration of the corresponding protein (peptide), or an increase in the amount of product generated by the activity of the corresponding protein (peptide), but is not limited thereto.

[0150] The modification of some or all of the polynucleotides in the host cell (microorganism) disclosed herein can be induced by: (a) homologous recombination using a vector for chromosome insertion or by genome editing using engineered nucleases (e.g., CRISPR-Cas9), and / or (b) treatment with light (e.g., ultraviolet light and radiation) and / or chemicals, but is not limited thereto.

[0151] nourish

[0152] As used herein, the term "culture" refers to the cultivation of microorganisms under appropriately regulated environmental conditions. Cultivation procedures can be performed using suitable culture media or cultivation conditions known in the art. Depending on the selected microorganisms, those skilled in the art can readily adapt and use such cultivation methods. Specifically, cultivation can be batch, continuous, and / or fed-batch, but is not limited to these.

[0153] As used herein, "culture medium" refers to a mixture containing nutrients necessary for the cultivation of microorganisms as its main component, wherein the culture medium provides nutrients such as water and growth factors necessary for survival and growth. Specifically, for the culture medium and other culture conditions used to cultivate the microorganisms of this disclosure, any culture medium commonly used for culturing microorganisms may be used without particular limitation. For example, the microorganisms of this disclosure may be cultured under aerobic conditions in a common culture medium containing suitable carbon sources, nitrogen sources, phosphorus sources, inorganic compounds, amino acids, and / or vitamins, while controlling temperature, pH, etc. For example, culture media for Corynebacterium spp. can be found in the literature [Manual of Methods for General Bacteriology, American Bacteriological Society (Washington DC, USA, 1981)].

[0154] In this disclosure, carbon sources may include carbohydrates such as glucose, saccharose, lactose, fructose, sucrose, maltose, etc.; sugar alcohols such as mannitol, sorbitol, etc.; organic acids such as pyruvic acid, lactic acid, citric acid, etc.; and amino acids such as glutamic acid, methionine, lysine, etc. In addition, natural organic nutrient sources such as starch hydrolysate, molasses, brown molasses, rice bran, cassava, bagasse, and corn steep liquor may be used. Specifically, carbohydrates such as glucose and aseptically pretreated molasses (i.e., molasses converted to reducing sugars) may be used, and various other carbon sources may be used in unlimited quantities. These carbon sources may be used alone or in combination of two or more, but are not limited thereto.

[0155] For nitrogen sources, inorganic nitrogen sources can be used, such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, ammonium nitrate, etc.; amino acids, such as glutamic acid, methionine, glutamine, etc.; and organic nitrogen sources, such as peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolysate, fish or its decomposition products, defatted soybean meal or its degradation products, etc. These nitrogen sources can be used alone or in combination of two or more, but are not limited to these.

[0156] Phosphorus sources may include potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and their corresponding sodium-containing salts. For inorganic compounds, sodium chloride, calcium chloride, ferric chloride, magnesium sulfate, ferric sulfate, manganese sulfate, calcium carbonate, etc., may be used. In addition, amino acids, vitamins, and / or suitable precursors may be included. These components or precursors may be added to the culture medium in batches or continuously. However, this disclosure is not limited thereto.

[0157] Furthermore, the pH of the culture medium can be adjusted during the cultivation of the microorganisms disclosed herein by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid to the medium in an appropriate manner. Additionally, antifoaming agents, such as polyethylene glycol esters of fatty acids, can be used to inhibit foam formation during cultivation. Furthermore, oxygen or oxygen-containing gases can be injected into the culture medium to maintain an aerobic state, or no gas can be injected, or nitrogen, hydrogen, or carbon dioxide gas can be injected to maintain an anaerobic or non-anaerobic state, but these are not limited to these methods.

[0158] In the cultivation process disclosed herein, the cultivation temperature can be maintained between 20°C and 45°C, specifically between 25°C and 40°C, and the cultivation can be carried out for approximately 10 hours to 160 hours, but is not limited thereto.

[0159] As used herein, the term "culture" refers to a culture medium, concentrated culture medium, dried culture medium, culture filtrate, concentrated culture filtrate, or dried culture filtrate obtained by culturing specific microorganisms in a culture medium, and a culture medium refers to those containing specific microorganisms, while a culture filtrate refers to those substantially not containing specific microorganisms (meaning that the specific microorganisms to be separated by filtration, etc., are substantially excluded, but not necessarily that the microorganisms are completely excluded from the filtrate). The dosage form of a culture is not limited, but it can be, for example, a liquid, emulsion, or solid.

[0160] As used in this article, the term "fermentation" refers to the process by which microorganisms use their own enzymes to break down organic matter, excluding putrefaction. Fermentation and putrefaction are similar processes, but when the decomposition produces useful substances, it is called fermentation; when it produces foul odors or harmful substances, it is called putrefaction.

[0161] In this disclosure, the methods for obtaining fermentation products from microorganisms are not particularly limited, and the fermentation products can be obtained according to methods commonly used in the art or similar fields.

[0162] As used herein, the term "fermentation product" includes not only the fermentation material itself, but also all types of materials containing fermentation products produced by microorganisms, such as materials containing fermenting microorganisms, cultures produced by fermenting microorganisms, fermentation products of cultures, concentrated fermentation products, dried products of fermentation products, filtrates of fermentation products, filtrates of concentrated fermentation products, or dried products of filtrates of fermentation products, extracts of fermentation products, or dilutes of fermentation products, etc.

[0163] Detailed description of this disclosure

[0164] The specific implementation of this disclosure will be described in more detail below.

[0165] One aspect of this disclosure provides a variant polypeptide of ribophosphate diphosphate kinase, wherein the amino acid at position 239 of the amino acid sequence corresponding to SEQ ID NO: 1 is replaced by another amino acid.

[0166] As used herein, the term "ribose-phosphobisphosphate kinase (PRSA)" refers to an enzyme that converts ribose 5-phosphate into phosphoribosyl pyrophosphate. The ribose-phosphobisphosphate kinase of this disclosure is interchangeable with PRSA (phosphoribosyl pyrophosphate synthase).

[0167] Specifically, the ribophosphate diphosphate kinase protein disclosed herein may be a protein having the activity of ribophosphate diphosphate kinase encoded by the prsA gene, but is not limited to this type, as long as it has the activity corresponding to ribophosphate diphosphate kinase. Ribophosphate diphosphate kinase proteins encoded by the prsA gene are known in the art, and the amino acid and polynucleotide sequences of ribophosphate diphosphate kinase proteins can be obtained from known databases, such as NCBI's GenBank, etc., but are not limited thereto.

[0168] For example, the ribophosphate diphosphate kinase protein targeted for mutation introduction in this disclosure may comprise, but is not limited to, the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having 60% or more homology or identity with it, as long as it possesses ribophosphate diphosphate kinase activity. Specifically, it is apparent that any protein containing a deletion, modification, substitution, or addition to a portion of the amino acid sequence of SEQ ID NO: 1 may also be included in the ribophosphate diphosphate kinase protein, provided that it is a protein exhibiting the corresponding efficacy of ribophosphate diphosphate kinase. Furthermore, any protein may be included in the ribophosphate diphosphate kinase protein, provided that it has an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher homology or identity with the amino acid sequence of SEQ ID NO: 1, contains that amino acid sequence, is composed of that amino acid sequence, or is substantially composed of that amino acid sequence, while exhibiting the efficacy corresponding to the efficacy of ribophosphate diphosphate kinase.

[0169] Furthermore, for example, based on codon information known in the art, a polynucleotide sequence encoding a ribophosphate diphosphate kinase protein having the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having 60% or more homology or identity with it can be obtained. For example, the ribophosphate diphosphate kinase protein may be encoded by a polynucleotide having or comprising the sequence of SEQ ID NO: 2 or a nucleotide sequence having 60% or more, 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 99% or more homology or identity with the sequence of SEQ ID NO: 2, or consisting of or substantially consisting of said nucleotide sequence, but not limited thereto. Furthermore, the nucleotide sequence of SEQ ID NO: 2 can be obtained from known databases, such as NCBI's GenBank, but is not limited thereto.

[0170] In this disclosure, a polynucleotide (gene) containing the nucleotide sequence of SEQ ID NO: 2 may be used interchangeably with a polynucleotide (gene) having the nucleotide sequence of SEQ ID NO: 2, a polynucleotide (gene) composed of the nucleotide sequence of SEQ ID NO: 2, or prsA.

[0171] The variant polypeptide of the ribophosphate diphosphate kinase disclosed herein may be a variant polypeptide in which the amino acid at position 239 of the amino acid sequence corresponding to SEQ ID NO: 1 is replaced by another amino acid.

[0172] The variant polypeptides of ribophosphobisphosphonate kinase disclosed herein may include polypeptides in which, in the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.9% or higher homology or identity with the amino acid sequence of SEQ ID NO: 1, the amino acid corresponding to position 239 from the N-terminus of the amino acid sequence of SEQ ID NO: 1 is replaced by another amino acid. Furthermore, it is apparent that any variant having partially deleted, substituted, conserved substituted, or added amino acid sequences may also fall within the scope of this disclosure, provided that it is an amino acid sequence containing the aforementioned amino acid substitutions, has the aforementioned homology or identity, and exhibits efficacy corresponding to the variants of this disclosure.

[0173] The term "another amino acid" is not limited, as long as it is an amino acid different from the amino acid before the substitution. Furthermore, in this disclosure, when it is stated that "a specific amino acid has been substituted," it is clear that the amino acid has been replaced by an amino acid different from the amino acid before the substitution, even if it is not stated that the amino acid has been replaced by another amino acid.

[0174] In this disclosure, when it is stated that "the amino acid at position 239 of the amino acid sequence corresponding to SEQ ID NO: 1 has been replaced by another amino acid", it may mean that the amino acid has been replaced by glutamic acid, phenylalanine, glycine, arginine, aspartic acid, cysteine, asparagine, glutamine, histidine, proline, serine, tyrosine, isoleucine, lysine, tryptophan, valine, methionine, threonine, or leucine (excluding alanine), but is not limited thereto.

[0175] In any of the above embodiments, the variant polypeptide of ribophosphate diphosphate kinase provided in this disclosure may be a variant polypeptide in which the amino acid corresponding to the N-terminus of the amino acid sequence of SEQ ID NO: 1 is replaced by any one of the amino acids selected from glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, and glutamine (these amino acids are amino acids with uncharged side chains).

[0176] In any of the above embodiments, the variant polypeptide of ribophosphate diphosphate kinase disclosed herein may be a variant polypeptide in which the amino acid corresponding to position 239 of the amino acid sequence of SEQ ID NO: 1 is replaced by valine, proline, asparagine, methionine, glycine or tyrosine.

[0177] For example, the variant polypeptide of ribophosphate diphosphate kinase disclosed herein may comprise an amino acid sequence having at least 60% or higher, 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, 96% or higher, 97% or higher, or 98% or higher and less than 100% homology or identity with the amino acid sequence shown in SEQ ID NO: 1, wherein the amino acid corresponding to position 239 of the amino acid sequence shown in SEQ ID NO: 1 is fixed as valine, proline, asparagine, methionine, glycine, or tyrosine. Furthermore, it is apparent that any variant polypeptide of ribophosphate diphosphate kinase having amino acid sequences with partial deletions, modifications, substitutions, conservative substitutions, or additions may also fall within the scope of this disclosure, provided that it has an amino acid sequence having the aforementioned homology or identity and exhibiting efficacy corresponding to the efficacy of the variant polypeptide of ribophosphate diphosphate kinase disclosed herein.

[0178] In another specific embodiment, the variant polypeptide may consist of any amino acid sequence selected from SEQ ID NO: 15 to 20.

[0179] Specifically, the variant polypeptides of this disclosure may have or contain any of the amino acid sequences selected from SEQ ID NO: 15 to 20, or have at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher homology or identity with any of the sequences selected from SEQ ID NO: 15 to 20, or may be composed of said amino acid sequences, or may be substantially composed of said amino acid sequences.

[0180] Another aspect of this disclosure provides a polynucleotide encoding a variant polypeptide of ribose-phosphobisphosphonate kinase.

[0181] The polynucleotide encoding the variant polypeptide of the ribophosphate diphosphate kinase disclosed herein may include any polynucleotide sequence without limitation, as long as it is a polynucleotide sequence encoding the variant polypeptide disclosed herein. The polynucleotide may be prepared based on codon information known in the art, but is not limited thereto. For example, the polynucleotide may comprise any nucleotide sequence selected from SEQ ID NO: 47 to 52, and may consist of or substantially consist of any nucleotide sequence selected from SEQ ID NO: 47 to 52.

[0182] In the polynucleotides of this disclosure, various modifications can be made to the coding region, as long as the amino acid sequence of the variant polypeptide of this disclosure is not altered due to codon degeneracy or consideration of preferred codons in the organism intended to express the variant polypeptide of this disclosure. Therefore, it is apparent that, due to codon degeneracy, the polynucleotide may also contain a polypeptide to be translated into a polypeptide consisting of the amino acid sequence of the variant polypeptide of this disclosure, or a polypeptide homologous to or identical with it. For example, the polynucleotide of this disclosure may be any nucleotide sequence selected from SEQ ID NO: 47 to 52 or a degenerate sequence thereof.

[0183] In another example, the polynucleotide of this disclosure may have or comprise any nucleotide sequence selected from SEQ ID NO: 47 to 52, or a nucleotide sequence having 60% or more, 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 99% or more homology or identity with it, and may consist of or substantially consist of said nucleotide sequences, but is not limited thereto. For example, the polynucleotide of this disclosure may comprise a nucleotide sequence in which the codon encoding the amino acid at position 239 corresponding to any one of SEQ ID NO: 15 to 20 is fixed as any one of the codons encoding valine, proline, asparagine, methionine, glycine, or tyrosine.

[0184] Furthermore, the polynucleotides disclosed herein may comprise probes prepared from known gene sequences; for example, they may comprise any sequence without limitation, as long as it is a sequence capable of hybridizing under stringent conditions with a sequence wholly or partially complementary to the polynucleotide sequence of this disclosure and encoding a variant polypeptide of this disclosure. The stringent conditions are as described above.

[0185] Another aspect of this disclosure provides a vector comprising the said polynucleotide. The vector may be an expression vector for expressing polynucleotides in microorganisms, but is not limited thereto.

[0186] Another aspect of this disclosure provides microorganisms comprising a variant polypeptide of ribose-phosphodiesterase or a polynucleotide encoding said variant polypeptide.

[0187] In one specific embodiment, the microorganism disclosed herein may be a microorganism capable of producing L-amino acids.

[0188] In this disclosure, specifically, the L-amino acid can be any one or more selected from the group consisting of L-tryptophan and L-histidine.

[0189] The microorganisms disclosed herein include, but are not limited to, all microorganisms that contain variant polypeptide sequences of ribophosphate diphosphate kinase of the present disclosure due to a mutation on the chromosome of the gene encoding the polypeptide encoding the polypeptide of the present disclosure; and / or microorganisms that contain variant polypeptide sequences of ribophosphate diphosphate kinase of the present disclosure by introducing a polynucleotide encoding the variant polypeptide of the present disclosure or a vector containing the variant polypeptide of the present disclosure.

[0190] Regarding the purposes of this disclosure, the microorganisms of this disclosure may include any microorganism containing a variant polypeptide of the ribophosphate-bisphosphonate kinase of this disclosure and capable of producing the desired L-amino acids. For example, the microorganisms of this disclosure may be microorganisms transformed with a vector containing a polynucleotide encoding a variant polypeptide of the ribophosphate-bisphosphonate kinase of this disclosure to express the variant polypeptide of this disclosure, thereby having an increased L-amino acid production capacity, but are not limited thereto. In another example, the microorganisms of this disclosure may be microorganisms that have an increased L-amino acid production capacity by expressing the variant polypeptide of this disclosure through the introduction of a polynucleotide encoding a variant polypeptide of the ribophosphate-bisphosphonate kinase of this disclosure into a naturally wild-type microorganism or an L-amino acid-producing microorganism, but are not limited thereto. Microorganisms with increased L-amino acid production capacity may be genetically engineered microorganisms or recombinant microorganisms, and microorganisms with increased L-amino acid production capacity may be microorganisms with increased L-amino acid production capacity compared to naturally wild-type microorganisms or unmodified microorganisms (e.g., microorganisms expressing wild-type polypeptides or polypeptides containing parental sequences; or microorganisms not expressing variant polypeptides of the ribophosphate-bisphosphonate kinase of this disclosure), but are not limited thereto.

[0191] For example, the microorganisms with L-amino acid production capabilities disclosed herein may include any microorganism transformed with a vector to express a variant polypeptide of the ribophosphate diphosphate kinase disclosed herein and to produce L-amino acids.

[0192] In addition to introducing polynucleotides or vectors, the microorganisms disclosed herein may also include any microorganism capable of expressing variant polypeptides of the ribophosphate diphosphate kinase disclosed herein by various known methods.

[0193] For example, the microorganisms with increased L-amino acid production capacity disclosed herein may be microorganisms with increased L-amino acid production capacity compared to parental microorganisms (parental strains) before modification or unmodified microorganisms (e.g., microorganisms expressing wild-type peptides, peptides containing parental sequences, or peptides of SEQ ID NO: 1; or microorganisms not expressing variant peptides of the ribophosphate diphosphate kinase disclosed herein), but are not limited thereto. For example, the parental microorganisms (parental strains) before modification or unmodified microorganisms used to compare whether L-amino acid production capacity is increased may be strains such as CM05-9157 (Korean Patent No. 10-2278000), CA14-0809 (KCCM12489P, Korean Patent No. 10-2019-0046934), CM05-9157β, or CA14-0809βprsA(B.su), but are not limited thereto.

[0194] For example, microorganisms with increased L-amino acid production capacity may have an increase of about 1% or more compared to the parent microorganism before modification (parent strain) or the unmodified microorganism, specifically, about 1% or more, about 2.5% or more, about 5% or more, about 6% or more, about 7% or more, about 8% or more, about 9% or more, about 10% or more, about 10.5% or more, about 11% or more, about 11.5% or more, about 12% or more, about 12.5% ​​or more, about 13% or more, about 13.5% or more, about 14% or more, or about 14% or more. More, about 14.5% or more, about 15% or more, or about 15.5% or more (there is no particular upper limit, but it can be, for example, about 200% or less, about 150% or less, about 100% or less, about 50% or less, about 45% or less, about 40% or less, about 35% or less, about 30% or less, or about 25% or less) of L-amino acid production capacity, but the increase is not limited to this, as long as the production capacity has an increased + value compared to the production capacity of the parent microorganism before modification (parent strain) or the unmodified microorganism. In another instance, a recombinant microorganism with increased L-amino acid production capacity may have an L-amino acid production capacity of approximately 1.1 times or more, approximately 1.12 times or more, approximately 1.13 times or more, approximately 1.14 times or more, or approximately 1.15 times or more (with no particular upper limit, but may be, for example, approximately 10 times or less, approximately 5 times or less, approximately 3 times or less, approximately 2 times or less, approximately 1.5 times or less, approximately 1.45 times or less, approximately 1.4 times or less, approximately 1.35 times or less, approximately 1.3 times or less, or approximately 1.25 times or less) compared to the unmodified parental microorganism (parental strain) or the unmodified microorganism, but is not limited thereto.

[0195] For example, microorganisms capable of producing L-amino acids can be prokaryotic or eukaryotic cells, specifically prokaryotic cells. Prokaryotic cells can include, but are not limited to, microorganisms of the genera *Escherichia*, *Erwinia*, *Serratia*, *Providencia*, *Corynebacteria*, *Pseudomonas*, *Leptospira*, *Salmonella*, *Brevibacteria*, *Hypomononas*, *Chromobacterium*, and *Norcardia*. Specifically, prokaryotic cells can be microorganisms of the genus *Escherichia* or *Corynebacteria*. More specifically, prokaryotic cells can be microorganisms of the genus *Corynebacteria*.

[0196] Corynebacterium species can include *Corynebacterium glutamicum*, *Corynebacterium crudilactis*, *Corynebacterium deserti*, *Corynebacterium efficiens*, *Corynebacterium callunae*, *Corynebacterium stationis*, *Corynebacterium singulare*, *Corynebacterium halotolerans*, *Corynebacterium striatum*, *Corynebacterium ammoniagenes*, *Corynebacterium pollutisoli*, *Corynebacterium imitans*, *Corynebacterium testudinoris*, or *Corynebacterium flavescens*. Specifically, the microorganisms disclosed herein can be *Corynebacterium glutamicum*, but are not limited thereto.

[0197] Meanwhile, it is known that Corynebacterium species can produce L-amino acids, but the genes involved in the production mechanism or its underlying principles have not been fully elucidated. Therefore, the Corynebacterium species with L-amino acid production capacity disclosed herein may include all naturally occurring wild-type microorganisms themselves, Corynebacterium species with enhanced L-amino acid production capacity by enhancing or weakening the activity of genes related to the L-amino acid production mechanism, or Corynebacterium species with enhanced L-amino acid production capacity by introducing or enhancing the activity of exogenous genes.

[0198] Another aspect of this disclosure provides a method for producing L-amino acids, the method comprising culturing in a culture medium a variant polypeptide of the present disclosure containing ribose-2,5-phosphate kinase or a polynucleotide encoding the variant polypeptide, wherein the amino acid at position 239 of the amino acid sequence corresponding to SEQ ID NO: 1 in the variant polypeptide is replaced by another amino acid.

[0199] Regarding the methods of this disclosure, variant peptides, polynucleotides, microorganisms, and L-amino acids of the ribophosphate diphosphate kinase are as described in other aspects.

[0200] Regarding the methods of this disclosure, the cultivation of the microorganisms can be carried out using any culture conditions and methods known in the art. Depending on the selected microorganisms, those skilled in the art can readily adapt and use this culture method.

[0201] The L-amino acids produced by the culture method disclosed herein can be released into the culture medium or retained in the cells.

[0202] In one specific embodiment, the method for producing L-amino acids disclosed herein may further include the steps of preparing the microorganisms of the present disclosure, preparing a culture medium for culturing the microorganisms, or a combination thereof (in any order, regardless of the sequence), for example, prior to the culturing step.

[0203] The method for producing L-amino acids disclosed herein may further include the step of recovering the target substance, particularly L-amino acids, from cultured microorganisms, the culture medium of said microorganisms, the fermentation product of said microorganisms, or the culture medium. A recovery step may be further included after the culturing step.

[0204] Recovery can be achieved by culturing microorganisms according to the methods disclosed herein, such as batch, continuous, or fed-batch cultures, by collecting the desired L-amino acids using suitable methods known in the art. For example, centrifugation, filtration, treatment with a crystalline protein precipitant (salting out), extraction, ultrasonic disruption, ultrafiltration, dialysis, various chromatography methods (such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, and affinity chromatography), HPLC, and combinations of these methods can be used to recover the target substance, particularly L-amino acids, from the culture medium or microorganisms using suitable methods known in the art.

[0205] Furthermore, the method for producing L-amino acids disclosed herein may further include a purification step. Purification can be performed using suitable methods known in the art. In exemplary embodiments, when the method for producing L-amino acids disclosed herein includes both a recovery step and a purification step, the recovery step and the purification step may be performed sequentially or discontinuously without regard to the order, or they may be performed simultaneously or integrated into a single step, but are not limited thereto.

[0206] Another aspect of this disclosure provides a composition for producing L-amino acids, the composition comprising: a variant polypeptide of the ribophosphate diphosphate kinase of this disclosure; a polynucleotide encoding the variant polypeptide; a microorganism comprising the variant polypeptide or the polynucleotide encoding the variant polypeptide; a culture of the microorganism; and a combination of two or more thereof.

[0207] Regarding the compositions disclosed herein, the variant peptides, polynucleotides, microorganisms, cultures, and L-amino acids of the ribophosphate diphosphate kinase are as described in other aspects.

[0208] The compositions disclosed herein may further comprise any suitable excipients commonly used in compositions for the production of L-amino acids, and such excipients may comprise, for example, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers, isotonic agents, etc., but are not limited thereto.

[0209] In one specific embodiment, each component present in the compositions of this disclosure may be included in a microbially effective amount or in an amount that may be suitably present in the compositions used for production.

[0210] Another aspect of this disclosure provides the use of a variant polypeptide of the ribophosphate diphosphate kinase of this disclosure, a microorganism comprising the variant polypeptide or a polynucleotide encoding the variant polypeptide, or a culture of the microorganism for the production of L-amino acids.

[0211] Regarding the uses of this disclosure, the variant peptides of the ribophosphate diphosphate kinase, microorganisms, L-amino acids, etc., are as described in other respects.

[0212] Methods of implementing the present invention

[0213] The present disclosure will be described in more detail below by way of exemplary embodiments. However, the following exemplary embodiments are merely preferred embodiments for illustrating the present disclosure and are therefore not intended to limit the scope of the disclosure thereto. Meanwhile, technical matters not described in this specification can be fully understood and readily implemented by those skilled in the art or similar art.

[0214] Example 1. Preparation of L-tryptophan-producing microorganisms infused with ribophosphate diphosphokinase derived from Bacillus subtilis

[0215] Example 1-1. Construction of a vector for gene insertion

[0216] To insert the gene into the chromosome of Corynebacterium, plasmid pDCM2 (Korean Patent No. 10-2278000) was used as the parental vector, and to enhance the activity of ribose-phosphodiesterase, a plasmid for further insertion of the prsA gene was constructed using the Pcj7 promoter (Korean Patent No. 10-0620092).

[0217] In detail, using chromosomal DNA from wild-type Corynebacterium glutamicum ATCC13869 as a template, the upstream and downstream regions of the chromosome where homologous recombination occurred were amplified using primer pairs SEQ ID NO: 3 and SEQ ID NO: 4, and primer pairs SEQ ID NO: 5 and SEQ ID NO: 6, respectively, to obtain their respective gene fragments. The sequences of the primers used are shown in Table 1 below.

[0218] [Table 1]

[0219]

[0220] To obtain the above fragment, PCR was performed. Solg was used. TM Pfu-X DNA polymerase was used as the polymerase for PCR amplification under the following conditions: denaturation at 95°C for 4 minutes, followed by 27 cycles of denaturation at 95°C, annealing at 55°C, and polymerization at 72°C, and then polymerization at 72°C for 5 minutes.

[0221] Using the Gibson assembly method (DG Gibson et al., NATURE METHODS, VOL.6 NO.5, MAY2009, NEBuilder HiFi DNA Assembly Master Mix), the upstream and downstream fragments of the region on the chromosome where homologous recombination occurred, obtained through the above process, were cloned into the chromosome transformation vector pDCM2, which was digested with EcoRI and SalI restriction enzymes, to obtain a recombinant plasmid, which was named pDCM2-ΔTn.

[0222] Examples 1-2. Construction of vectors for introducing ribophosphate diphosphate kinase derived from Bacillus subtilis

[0223] First, to obtain the Pcj7 promoter, PCR was performed using p117-cj7-gfp (US 7662943 B2) as a template and primers SEQ ID NO: 7 and SEQ ID NO: 8 from Table 2 below. Solg... TM Pfu-X DNA polymerase (SolGent co.) was used as the polymerase. The PCR amplification conditions were: denaturation at 95°C for 2 minutes, followed by 27 cycles of denaturation at 95°C, annealing at 55°C, and polymerization at 72°C, and then polymerization at 72°C for 5 minutes.

[0224] The ribophosphate-diphosphate kinase derived from Bacillus subtilis has the amino acid sequence shown in SEQ ID NO: 1. The gene encoding ribophosphate-diphosphate kinase and its surrounding nucleotide sequence (accession number NC_000964.3, SEQ ID NO: 2) were obtained from the National Institutes of Health (NIH) GenBank. Based on the obtained nucleotide sequence, primers for inserting the gene derived from Bacillus subtilis into the genomic DNA of Corynebacterium glutamicum were synthesized.

[0225] The ribose-phosphobisphosphate kinase gene of Bacillus subtilis was synthesized using the gene synthesis service of Bionics Co., Ltd., and PCR was performed using primers for SEQ ID NO: 9 and SEQ ID NO: 10 in Table 2 below. Solg... TM Pfu-X DNA polymerase was used as the polymerase. The PCR amplification conditions were as follows: denaturation at 95°C for 2 minutes, followed by 27 cycles of denaturation at 95°C for 20 seconds, annealing at 55°C, polymerization at 72°C for 1 minute, and then polymerization at 72°C for 5 minutes.

[0226] [Table 2]

[0227]

[0228] Subsequently, the amplified Pcj7 promoter region, the gene fragment derived from Bacillus subtilis, and the chromosome transformation vector pDCM2-ΔTn digested with the ScaI restriction enzyme were cloned using the Gibson assembly method (DG Gibson et al., NATURE METHODS, VOL.6 NO.5, MAY 2009, NEBuilder HiFi DNA Assembly Master Mix) to obtain the recombinant plasmid, which was named pDCM2-β. Cloning was performed by mixing the Gibson assembly reagent and each gene fragment in calculated molar amounts, and then storing the mixture at 50°C for 1 hour.

[0229] Examples 1-3. Preparation of L-tryptophan-producing microorganisms infused with ribophosphate diphosphate kinase derived from Bacillus subtilis.

[0230] The pDCM2-β vector constructed in Examples 1-2 was transformed into the L-tryptophan-producing strain CM05-9157 (Korean Patent No. 10-2278000) via electroporation (Appl. Microbiol. Biotechnol. (1999) 52:541-545), followed by a secondary crossover process to obtain a strain in which one copy of the Pcj7-prsA(B.su) gene was inserted between transposon genes on the chromosome. Subsequently, the genetic manipulation was detected by PCR and genome sequencing using primers listed in Table 3 below, which amplified the outer regions of the upstream and downstream homologous recombination regions of the inserted gene.

[0231] [Table 3]

[0232]

[0233] The strain obtained by the above method was named CM05-9157β.

[0234] Example 2. Evaluation of L-tryptophan production capacity of introducing L-tryptophan-producing microorganisms containing ribophosphate diphosphokinase derived from Bacillus subtilis.

[0235] To test the L-tryptophan production capacity of the CM05-9157β strain and the parental strain CM05-9157 prepared in Examples 1-3, these strains were cultured using the following methods and culture medium compositions.

[0236] First, each strain was inoculated into a 250 ml corner-baffled flask containing 25 ml of seed culture medium and cultured at 30°C with shaking at 200 rpm for 20 hours. Then, 1 ml of the seed culture was inoculated into a 250 ml corner-baffled flask containing 25 ml of production culture medium and cultured at 30°C with shaking at 200 rpm for 24 hours. After culturing, the L-tryptophan yield was measured by HPLC.

[0237] Seed culture medium (pH 7.0)

[0238] 20 g glucose, 10 g peptone, 5 g yeast extract, 1.5 g urea, 4 g KH2PO4, 8 g K2HPO4, 0.5 g MgSO47H2O, 100 μg biotin, 1000 μg thiamine hydrochloride, 2000 μg calcium pantothenate, 2000 μg nicotinamide (based on 1 liter of distilled water)

[0239] <Production medium (pH 7.0)>

[0240] 30 g glucose, 15 g (NH4)2SO4, 1.2 g MgSO4·7H2O, 1 g KH2PO4, 5 g yeast extract, 900 μg biotin, 4500 μg thiamine hydrochloride, 4500 μg calcium pantothenate, 30 g CaCO3 (based on 1 liter of distilled water)

[0241] The L-tryptophan production of each strain is shown in Table 4 below.

[0242] [Table 4]

[0243]

[0244] The CM05-9157β strain, infused with a ribose-9157 phosphokinase gene derived from Bacillus subtilis, produced a final L-tryptophan concentration of 2.29 g / L in shake-flask culture, representing a 21% increase in fermentation yield compared to the control strain CM05-9157. This indicates that the L-tryptophan production capacity of Corynebacterium glutamicum strains can be significantly enhanced by introducing a ribose-9157 phosphokinase gene derived from Bacillus subtilis.

[0245] Example 3. Preparation of L-tryptophan-producing microorganisms incorporating a variant of wild-type ribophosphate diphosphokinase derived from Bacillus subtilis with amino acid 239 replaced.

[0246] To further enhance the activity of ribophosphate diphosphate kinase derived from Bacillus subtilis, a variant was constructed in which the amino acid alanine at position 239 (hereinafter referred to as alanine at position 239 or the 239th amino acid) was replaced by another amino acid. To mutate to an amino acid other than alanine, site-directed mutagenesis was performed using pDCM2-β from Example 1 as a template. Site-directed mutagenesis was performed according to the PCR composition and PCR cycles shown in Tables 5 and 6 below.

[0247] [Table 5]

[0248]

[0249] [Table 6]

[0250]

[0251] To replace alanine at position 239 of the prsA (B.su) amino acid sequence with other amino acids, namely valine (V) (SEQ ID NO: 15), proline (P) (SEQ ID NO: 16), asparagine (N) (SEQ ID NO: 17), methionine (M) (SEQ ID NO: 18), glycine (G) (SEQ ID NO: 19), and tyrosine (Y) (SEQ ID NO: 20), the PCR mixtures in Table 5 were prepared using each set of mutagenic primers listed in Table 7, and PCR was performed using the cycles in Table 6. After PCR, 1 μl of DpnI restriction enzyme was added, and the mixture was incubated at 37°C for 1 hour. 3 μl of DpnI-treated DNA was transformed into DH5α competent cells to obtain pDCM2-β plasmids of each variant, and the substitutions with the mutations shown in Table 7 were identified by sequencing.

[0252] [Table 7]

[0253]

[0254] The pDCM2-βA239V, pDCM2-βA239P, pDCM2-βA239N, pDCM2-βA239M, pDCM2-βA239G, and pDCM2-βA239Y vectors prepared as shown in Table 7 were transformed into the L-tryptophan-producing strain CM05-9157 prepared in Examples 1-3 via electroporation. Six strain types were then obtained through a two-stage crossover process, in which the variant prsA(B.su) gene was inserted into the chromosome. Genetic manipulation was detected by PCR and genome sequencing using primers of SEQ ID NO: 33 and SEQ ID NO: 34, which amplified the external regions of the upstream and downstream homologous recombination regions of the inserted gene.

[0255] The transformed strains obtained in this way were named CM05-9157βprsA(B.su) A239V), CM05-9157βprsA(B.su) A239P), CM05-9157βprsA(B.su) A239N), CM05-9157βprsA(B.su) A239M), CM05-9157βprsA(B.su) A239G, and CM05-9157βprsA(B.su) A239Y, respectively.

[0256] Example 4. Evaluation of the L-tryptophan production capacity of an L-tryptophan-producing microorganism with a variant of wild-type ribophosphate diphosphokinase derived from Bacillus subtilis, in which the amino acid at position 239 was substituted.

[0257] To compare the L-tryptophan yield of the six strains prepared in Example 3 and CM05-9157β, they were cultured using the same method as in Example 2. After culturing, the L-tryptophan yield was measured using HPLC.

[0258] As shown in Table 8 below, the results of the experiment confirmed that the L-tryptophan production of the CM05-0157β strain with the prsA(B.su) A239V mutation was 8.98 g / L, which was about 15.6% higher than that of the control CM05-9157β strain with the wild-type prsA(B.su) protein. It was confirmed that the yield of strain CM05-9157βprsA(B.su) A239P) was increased by approximately 14.3%, strain CM05-9157βprsA(B.su) A239N) by approximately 13.9%, strain CM05-9157βprsA(B.su) A239M) by approximately 12.6%, strain CM05-9157βprsA(B.su) A239G) by approximately 11.7%, and strain CM05-9157βprsA(B.su) A239Y) by approximately 10.4%.

[0259] [Table 8]

[0260]

[0261] The above results indicate that the L-tryptophan production capacity of Corynebacterium glutamicum strains can be further increased by introducing a variant of ribophosphate diphosphate kinase at position 239 from Bacillus subtilis.

[0262] Example 5. Preparation of L-histidine-producing microorganisms infused with ribophosphate diphosphate kinase derived from Bacillus subtilis

[0263] Example 5-1. Construction of a vector for gene insertion

[0264] To insert the gene into the chromosome of Corynebacterium, plasmid pDCM2 (Korean Patent No. 10-2278000) was used as the parental vector, and to enhance the activity of ribose-phosphodiesterase, a plasmid for further insertion of the prsA gene was constructed using the Pm4ddh promoter (Korean Patent No. 10-0987281).

[0265] In detail, using chromosomal DNA from wild-type Corynebacterium glutamicum ATCC13032 as a template, the upstream and downstream regions of the chromosome where homologous recombination occurred were amplified using primer pairs SEQ ID NO: 35 and SEQ ID NO: 36 and primer pairs SEQ ID NO: 37 and SEQ ID NO: 38, respectively, to obtain their respective gene fragments. The sequences of the primers used are shown in Table 9 below.

[0266] [Table 9]

[0267]

[0268] To obtain the above fragment, PCR was performed. Solg was used. TM Pfu-X DNA polymerase was used as the polymerase for PCR amplification under the following conditions: denaturation at 95°C for 4 minutes, followed by 27 cycles of denaturation at 95°C, annealing at 55°C, and polymerization at 72°C, and then polymerization at 72°C for 5 minutes.

[0269] Using the Gibson assembly method (DG Gibson et al., NATURE METHODS, VOL.6 NO.5, MAY2009, NEBuilder HiFi DNA Assembly Master Mix), the upstream and downstream fragments of the region on the chromosome where homologous recombination occurred, obtained through the above process, were cloned into the chromosome transformation vector pDCM2, which was digested with SalI restriction enzyme, to obtain a recombinant plasmid, named pDCM2-β.

[0270] Example 5-2. Construction of a vector for introducing ribophosphate diphosphate kinase derived from Bacillus subtilis.

[0271] First, to obtain the ddhP1 promoter, PCR was performed using pDZ-ddhP1 (Korean Patent No. 10-0987281) as a template and primers from SEQ ID NO: 41 and SEQ ID NO: 42 in Table 10 below. Solg... TM Pfu-X DNA polymerase (SolGent co.) was used as the polymerase. The PCR amplification conditions were: denaturation at 95°C for 2 minutes, followed by 27 cycles of denaturation at 95°C, annealing at 55°C, and polymerization at 72°C, and then polymerization at 72°C for 5 minutes.

[0272] The ribophosphate-diphosphate kinase derived from Bacillus subtilis has the amino acid sequence shown in SEQ ID NO: 1. Information on the gene encoding ribophosphate-diphosphate kinase and its surrounding nucleotide sequence (accession number NC_000964.3, SEQ ID NO: 2) was obtained from the National Institutes of Health (NIH) GenBank. Based on the obtained nucleotide sequence, primers for inserting the gene derived from Bacillus subtilis into the genomic DNA of Corynebacterium glutamicum were synthesized.

[0273] To amplify the gene derived from Bacillus subtilis, the ribophosphate-bisphosphate kinase gene of the Bacillus subtilis strain was synthesized using the gene synthesis service of Bionics Co., Ltd., and PCR was performed using primers from SEQ ID NO: 39 and SEQ ID NO: 40 in Table 10 below. Solg... TMPfu-X DNA polymerase was used as the polymerase. The PCR amplification conditions were as follows: denaturation at 95°C for 2 minutes, followed by 27 cycles of denaturation at 95°C for 20 seconds, annealing at 55°C, polymerization at 72°C for 1 minute, and then polymerization at 72°C for 5 minutes.

[0274] [Table 10]

[0275]

[0276] Subsequently, the amplified Pcj7 promoter region, the gene fragment derived from Bacillus subtilis, and the chromosome transformation vector pDCM2-ΔTn2 digested with the ScaI restriction enzyme were cloned using the Gibson assembly method (DG Gibson et al., NATURE METHODS, VOL.6 NO.5, MAY 2009, NEBuilder HiFi DNA Assembly Master Mix) to obtain the recombinant plasmid, which was named pDCM2-β. Cloning was performed by mixing the Gibson assembly reagent and each gene fragment in calculated molar amounts, and then storing the mixture at 50°C for 1 hour.

[0277] Example 5-3. Preparation of L-histidine-producing microorganisms infused with ribophosphate diphosphate kinase derived from Bacillus subtilis.

[0278] The pDCM2-β vector constructed in Example 5-2 was transformed into the L-histidine-producing strain CA14-0809 (Korean Patent No. 10-2019-0065984) by electroporation (Appl. Microbiol. Biotechnol. (1999) 52:541-545), followed by a second crossover process to obtain a strain in which one copy of the ddhP1_prsA(B.su) gene was inserted between transposon genes on the chromosome. The genetic manipulation was detected by PCR and genome sequencing using primers listed in Table 11 below, which amplify the outer regions of the upstream and downstream regions of the homologous recombination inserted into the corresponding gene.

[0279] [Table 11]

[0280]

[0281] The strain obtained in this way was named CA14-0809β.

[0282] Example 6. Evaluation of L-histidine production capacity of introducing L-histidine-producing microorganisms containing ribophosphate diphosphokinase derived from Bacillus subtilis.

[0283] To test the L-histidine production capacity of the CA14-0809β strain prepared in Examples 5-3 and infused with ribophosphate diphosphokinase from Bacillus subtilis, the histidine-producing strain CA14-0809, which served as the parent strain, was used as the control strain and cultured using the following method.

[0284] First, each strain was inoculated into a 250 ml baffled Erlenmeyer flask containing 25 ml of seed culture medium and cultured at 30°C with shaking at 200 rpm for 20 hours. Then, 1 ml of the seed culture was inoculated into a 250 ml baffled Erlenmeyer flask containing 25 ml of production culture medium and cultured at 30°C with shaking at 200 rpm for 24 hours. The composition of the culture medium used in this example is as follows. After cultivation, the yield of L-histidine was measured by HPLC.

[0285] Seed culture medium (pH 7.0)

[0286] 5% glucose, 1% bacterial peptone, 0.25% sodium chloride, 1% yeast extract, 0.4% urea, pH 7.2

[0287] <Production medium (pH 7.0)>

[0288] 5% glucose, 2% ammonium sulfate, 0.1% potassium dihydrogen phosphate, 0.05% magnesium sulfate heptahydrate, 2.0% corn steep liquor (CSL), 200 μg / L biotin, 30 g / L calcium carbonate, pH 7.2

[0289] The L-histidine production of each strain is shown in Table 12 below as the results of the experiment.

[0290] [Table 12]

[0291]

[0292] The CA14-0809β strain, infused with a ribose-6-phosphate kinase gene from Bacillus subtilis, produced a final L-histidine yield of 5.7 g / L in shake-flask culture, representing an approximately 12% increase in fermentation yield compared to the control strain CA14-0809. This indicates that the L-histidine production capacity of Corynebacterium glutamicum strains can be significantly enhanced by introducing a ribose-6-phosphate kinase gene from Bacillus subtilis.

[0293] Example 7. Preparation of L-histidine-producing microorganisms infused with a variant of wild-type ribophosphate diphosphokinase derived from Bacillus subtilis, in which the amino acid at position 239 was substituted.

[0294] To further increase the activity of ribophosphate diphosphate kinase derived from Bacillus subtilis, an attempt was made to increase the production of L-histidine by introducing the A239V variant. In Example 4, the A239V variant most significantly increased the production of L-tryptophan.

[0295] To mutate alanine to valine, pDCM2-β used in Example 5-2 was used as a template, and site-directed mutagenesis was employed. The site-directed mutagenesis method was performed according to the PCR composition and PCR cycles in Tables 13 and 14 below.

[0296] [Table 13]

[0297]

[0298] [Table 14]

[0299]

[0300] To replace alanine, the amino acid at position 239 of the prsA (B.su) amino acid sequence, with another amino acid, valine (V) (SEQ ID NO: 15), the PCR mixtures in Table 13 were prepared using each set of mutagenic primers listed in Table 15, and PCR was performed using the cycles in Table 14. After PCR, 1 μl of DpnI restriction enzyme was added, and the mixture was treated at 37°C for 1 hour. Following the same procedure as in Example 4, 3 μl of DpnI-treated DNA was transformed into DH5α competent cells to obtain the variant pDCM2-β plasmid, and the substitutions with the mutations shown in Table 15 were identified by sequencing.

[0301] [Table 15]

[0302]

[0303] The pDCM2-βA239V vector prepared in Table 15 was transformed into the L-histidine-producing strain CA14-0809 (KCCM12489P, Korean Patent No. 10-2019-0046934) via electroporation. A second crossover process was then performed to obtain a strain in which the variant prsA(B.su) gene was inserted into the chromosome. Genetic manipulation was detected by PCR and genome sequencing using primers specified in Table 11 (SEQ ID NO: 43 and SEQ ID NO: 44), which amplified the outer regions of the upstream and downstream homologous recombination regions of the inserted gene. The transformed strain thus obtained was named CA14-0809βprsA(B.su)A239V.

[0304] Example 8. Evaluation of the L-histidine production capacity of an L-histidine-producing microorganism with a variant of wild-type ribophosphate diphosphokinase derived from Bacillus subtilis, in which the amino acid at position 239 was substituted.

[0305] To compare the histidine production of the CA14-0809βprsA(B.su)A239V strain and the CA14-0809βprsA(B.su) strain prepared in Example 7, they were cultured using the same method as in Example 6. After culturing, the L-histidine production was measured using HPLC.

[0306] As shown in Table 16 below, the results of the experiment confirmed that the L-histidine production of the CA14-0809βprsA(B.su)A239V strain with the prsA(B.su) A239V mutation was 6.2 g / L, which was about 12.7% higher than that of the control CA14-0809βprsA(B.su) strain with wild-type prsA(B.su) protein.

[0307] [Table 16]

[0308]

[0309] The above results indicate that the L-histidine production capacity of *Corynebacterium glutamicum* strains can be further increased by introducing a variant of ribophosphate diphosphate kinase at position 239 from *Bacillus subtilis*.

[0310] Based on the foregoing description, those skilled in the art will understand that this disclosure can be implemented in different specific forms without altering its technical spirit or essential characteristics. In this regard, it should be understood that the above embodiments are not restrictive but illustrative in all respects. The scope of this disclosure is defined by the appended claims and not by the description that follows them; therefore, all variations and modifications falling within the boundaries and scope of the claims, or equivalents of such boundaries and scope, are included in the claims.

Claims

1. A variant polypeptide of ribose phosphate diphosphokinase, wherein the amino acid corresponding to position 239 in the amino acid sequence of SEQ ID NO: 1 is substituted with another amino acid.

2. The variant polypeptide according to claim 1, wherein the amino acid corresponding to position 239 in the amino acid sequence of SEQ ID NO: 1 is substituted with valine, proline, asparagine, methionine, glycine or tyrosine.

3. The variant polypeptide according to claim 1, wherein the variant polypeptide consists of any one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 15 to 20.

4. A polynucleotide encoding the variant polypeptide of ribose phosphate diphosphokinase according to any one of claims 1 to 3.

5. A microorganism comprising the variant polypeptide of ribose phosphate diphosphokinase according to any one of claims 1 to 3, or a polynucleotide encoding the variant polypeptide.

6. The microorganism according to claim 5, wherein the microorganism has increased L-amino acid production capacity compared to an unmodified microorganism.

7. The microorganism according to claim 5, wherein the microorganism is a microorganism of the genus Corynebacterium.

8. The microorganism according to claim 7, wherein the microorganism of the genus Corynebacterium is Corynebacterium glutamicum.

9. The microorganism according to claim 6, wherein the L-amino acid is any one or more selected from the group consisting of L-tryptophan and L-histidine.

10. A method of producing an L-amino acid, the method comprising the step of culturing the microorganism according to claim 5 in a culture medium.

11. The method according to claim 10, further comprising the step of recovering a target substance from the cultured microorganism, the culture of the microorganism, the fermentation product of the microorganism or the culture medium.

12. A composition for producing an L-amino acid, the composition comprising: the variant polypeptide of ribose phosphate diphosphokinase according to any one of claims 1 to 3; a polynucleotide encoding the variant polypeptide; a microorganism comprising the variant polypeptide or the polynucleotide encoding the variant polypeptide; a culture of the microorganism; a combination of two or more thereof.

13. Use of the variant polypeptide of ribose phosphate diphosphokinase according to any one of claims 1 to 3, a polynucleotide encoding the variant polypeptide, a microorganism comprising the variant polypeptide or the polynucleotide encoding the variant polypeptide, or a culture of the microorganism for producing an L-amino acid.

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