WO2025211657A1 - Microorganism comprising nicotinamide nucleotide transhydrogenase variant and method for producing l-amino acid using same - Google Patents

Abstract

The present application relates to: a nicotinamide nucleotide transhydrogenase alpha subunit variant polypeptide; a polynucleotide encoding the variant polypeptide; a microorganism comprising same; and a use of the microorganism for producing L-amino acids.

Description

Microorganism comprising a variant of nicotinamide nucleotide transhydrogenase and method for producing L-amino acid using the same

The present application relates to a microorganism comprising at least one selected from among a nicotinamide nucleotide transhydrogenase alpha subunit variant polypeptide; and a polynucleotide encoding the variant polypeptide; a method for producing an L-amino acid, the method comprising the step of culturing the microorganism in a medium; a composition for producing an L-amino acid, the composition comprising at least one selected from among the microorganism, a culture of the microorganism, and a fermented product of the microorganism; and a use of the microorganism for producing an L-amino acid.

Coryneform microorganisms are Gram-positive microorganisms frequently used industrially to produce a variety of materials, including L-amino acids and various nucleic acids, for use in feed, pharmaceuticals, and food. Recently, coryneform microorganisms have also been used to produce diamines and keto acids.

To produce useful products through microbial fermentation, the demand for energy sources or reducing power increases along with the strengthening of the biosynthetic pathway of the target product in microorganisms. Among these, NADPH (nicotinamide adenine dinucleotide phosphate) is an essential element for supplying reducing power. The oxidized form, NADP+, and the reduced form, NADPH, act as electron transport substances in the body and are involved in various synthetic processes. Among the central metabolic pathways, NADPH is mainly known to be produced by 1) the oxidative pentose phosphate pathway and 2) the NADP-dependent isocitrate dehydrogenase (Icd gene) of the tricarboxylic acid (TCA) pathway. In addition, various alternative pathways for supplying NADPH in various microorganisms include malate enzyme and glucose dehydrogenase. It has nonphosphorylation glyceraldehyde-3-phosphate dehydrogenase.

Additionally, enzymes that produce NADPH independent of the central metabolic pathway include transhydrogenase and ferredoxin:NADP+oxidoredutase.

With the coexistence of various pathway enhancements and the demand for energy sources or reducing power, there is still a growing need for methods to increase the productivity of the desired L-amino acid or its derivatives.

[Prior Art Literature]

[Patent Document]

(Patent Document 1) 1. U.S. Patent Publication No. US 7629142 B2

The problem to be solved by the present application is to provide a microorganism comprising a variant of nicotinamide nucleotide transhydrogenase and a method for producing L-amino acid using the same.

One aspect of the present application provides a nicotinamide nucleotide transhydrogenase alpha subunit mutant polypeptide, wherein the amino acid corresponding to position 437 of SEQ ID NO: 10 is replaced with another amino acid.

In one specific example, the mutant polypeptide may have the amino acid corresponding to position 437 of SEQ ID NO: 10 substituted with alanine.

In another specific example, the mutant polypeptide may be composed of the amino acid sequence of SEQ ID NO: 14.

In another specific example, the nicotinamide nucleotide transhydrogenase alpha subunit may be encoded by the pntA gene.

Another aspect of the present application provides a nicotinamide nucleotide transhydrogenase comprising the mutant polypeptide.

Another aspect of the present application provides a polynucleotide encoding the mutant polypeptide.

Another aspect of the present application provides a microorganism comprising at least one selected from the group consisting of the mutant polypeptide; a polynucleotide encoding the mutant polypeptide; or a vector comprising the same.

A microorganism according to any one of the preceding specific examples, wherein the microorganism may be a microorganism of the genus Corynebacterium.

As a microorganism according to any one of the preceding specific examples, the microorganism of the genus Corynebacterium may be Corynebacterium glutamicum.

A microorganism according to any one of the preceding specific examples, wherein the microorganism may have increased L-amino acid production ability compared to a microorganism comprising a polypeptide of SEQ ID NO: 10 or a polynucleotide encoding the same.

A microorganism according to any one of the preceding specific examples, wherein said microorganism may have increased L-threonine.

Another aspect of the present application provides a method for producing L-amino acid, comprising the step of culturing the microorganism in a medium.

As one specific example, the method may further comprise a step of recovering L-amino acid from the cultured microorganism; a culture of the microorganism; a fermented product of the microorganism; or the culture medium.

As another specific example, the method may be a method for producing L-threonine.

Another aspect of the present application provides a composition for producing L-amino acids, comprising at least one selected from the group consisting of a mutant polypeptide; a polynucleotide encoding the mutant polypeptide; a vector comprising the same; a microorganism comprising the vector; a culture of the microorganism; or a fermentation product of the microorganism.

As a specific example, the composition may be a composition for producing L-threonine.

When culturing a microorganism containing the nicotinamide nucleotide transhydrogenase alpha subunit mutant polypeptide of the present application, high yield L-amino acid production is possible compared to a microorganism having an existing unmodified polypeptide.

This is explained in detail as follows. Meanwhile, each description and embodiment disclosed in this application can also be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in this application fall within the scope of this application. Furthermore, the scope of this application is not limited by the specific descriptions described below. Furthermore, numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated into this specification in their entirety by reference to more clearly explain the level of the technical field to which this application belongs and the contents of this application.

One aspect of the present application provides a nicotinamide nucleotide transhydrogenase alpha subunit mutant polypeptide, wherein the amino acid corresponding to position 437 of SEQ ID NO: 10 is replaced with another amino acid.

In the present application, the term "nicotinamide nucleotide transhydrogenase" may mean a transmembrane pyridine nucleotide transhydrogenase, which is an enzyme that maintains the redox balance of a cell through the reaction [H ⁺ + NADP ⁺ + NADH ↔ H ⁺ + NADPH + NAD ⁺ ], exists as a homodimer with each protomer including two soluble nucleotide binding domains that mediate the transfer of hydride between NAD(H) and NADP(H) and a proton-translocating transmembrane domain, and which catalyzes the reduction of NADP+ to NADPH through the oxidation of NADH to NAD+, and is composed of two subunits, alpha and beta, which are encoded by the pntA and pntB genes, respectively.

The nicotinamide nucleotide transhydrogenase of the present application can be used interchangeably with pntAB. Specifically, the nicotinamide nucleotide transhydrogenase of the present application can be used interchangeably with pntAB ( pntA and pntB ). It may be a protein having nicotinamide nucleotide transhydrogenase activity encoded by a gene, but is not particularly limited in type as long as it has an activity corresponding to nicotinamide nucleotide transhydrogenase.

The nicotinamide nucleotide transhydrogenase encoded by the above pntAB gene is known in the art, and the amino acid and polynucleotide sequences of the nicotinamide nucleotide transhydrogenase can be obtained from known data databases (e.g., GenBank), but are not limited thereto.

For example, the nicotinamide nucleotide transhydrogenase may be composed of an alpha subunit PntA and a beta subunit PntB.

The nicotinamide nucleotide transhydrogenase may include an amino acid sequence of SEQ ID NO: 10 or an amino acid sequence having 60% or more homology or identity therewith; and an amino acid sequence of SEQ ID NO: 11 or an amino acid sequence having 60% or more homology or identity therewith, but is not limited thereto as long as it has nicotinamide nucleotide transhydrogenase activity. Specifically, even if it includes a sequence in which some sequences in the amino acid sequences of SEQ ID NOs: 10 and 11 are deleted, modified, substituted or added, as long as it is a protein that exhibits an effect corresponding to nicotinamide nucleotide transhydrogenase, it may be included in the nicotinamide nucleotide transhydrogenase. Additionally, it may have, include, consist of, or consist essentially of an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology or identity with the amino acid sequences of SEQ ID NOs: 10 and 11.

The protein to be subjected to mutation in the present application may have nicotinamide nucleotide transhydrogenase activity. Specifically, it may be composed of or include the amino acid sequence of SEQ ID NO: 10, but is not limited thereto. This does not exclude meaningless sequence additions before or after the amino acid sequence described in SEQ ID NO: 10, mutations that may occur naturally, or silent mutations thereof. If it has the same or corresponding activity as a protein including the amino acid sequence of SEQ ID NO: 10, it may be considered a protein to be subjected to mutation in the present application. For example, the protein that is the target of mutation introduction in the present application may be a protein composed of an amino acid sequence that has 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.9% or more homology or identity with the amino acid sequence of SEQ ID NO: 10. In addition, it is obvious that a polypeptide that has an amino acid sequence in which a part of the sequence is deleted, modified, substituted, conservatively substituted, or added is also included within the scope of the polypeptide that is the target of mutation in the present application, as long as it has such homology or identity and the nicotinamide nucleotide transhydrogenase alpha subunit is an amino acid sequence that exhibits an effect corresponding to the polypeptide.

As used herein, the term "variant polypeptide", "variant protein" or "variant" refers to a polypeptide in which one or more amino acids are conservatively substituted and/or modified, thereby differing from the amino acid sequence of the variant before the mutation, but retaining functions or properties. Such variants can generally be identified by modifying one or more amino acids in the amino acid sequence of the polypeptide and evaluating the properties of the modified polypeptide. That is, the ability of the variant may be increased, unchanged, or decreased compared to the polypeptide before the mutation. Additionally, some variants may include variants in which one or more portions, such as the N-terminal leader sequence or the transmembrane domain, are deleted. Other variants may include variants in which portions are deleted from the N- and/or C-terminus of the mature protein. The above term "variant" may be used interchangeably with terms such as variant, variant polypeptide, variant protein, variant polypeptide, variant protein, variant polypeptide, and variant protein (in English, modified polypeptide, modified protein, mutant, mutein, etc.), and is not limited thereto if the term is used in a variant sense.

Additionally, variants may include deletions or additions of amino acids that have minimal impact on the properties and secondary structure of the polypeptide. For example, the polypeptide may be conjugated to a signal (or leader) sequence at the N-terminus of the protein that is involved in co-translational or post-translational protein transfer. The polypeptide may also be conjugated to other sequences or linkers to facilitate identification, purification, or synthesis of the polypeptide.

The mutant polypeptide of the present application may be one in which the amino acid corresponding to the 437th position from the N-terminus of SEQ ID NO: 10 is substituted with another amino acid. The “mutant polypeptide” may be referred to as “nicotinamide nucleotide transhydrogenase alpha subunit mutant polypeptide,” “nicotinamide nucleotide transhydrogenase alpha subunit mutant,” “mutant pntA,” “pntA mutant,” etc. The nicotinamide nucleotide transhydrogenase alpha subunit polypeptide to which the mutation is introduced in the present application may be used interchangeably with the term “PntA,” and may be encoded by the pntA gene, but is not particularly limited thereto, and may be encoded by the pntA gene derived from Corynebacterium variabile, but is not limited thereto.

In the present application, the amino acid sequence before modification of the nicotinamide nucleotide transhydrogenase alpha subunit to be modified, i.e., the amino acid before modification corresponding to position 437 of SEQ ID NO: 10 in the sequence that can be the parent sequence, may be valine (V), but is not limited thereto.

The nicotinamide nucleotide transhydrogenase alpha subunit mutant polypeptide of the present application may have an amino acid at position 437 in the amino acid sequence of SEQ ID NO: 10 substituted with an amino acid different from the amino acid before substitution.

In one specific example, the mutant polypeptide may be one in which the amino acid corresponding to position 437 in the amino acid sequence of SEQ ID NO: 10 is substituted with an amino acid other than valine, which is the amino acid before substitution.

In another specific example, the variant polypeptide may be one in which the amino acid corresponding to position 437 of SEQ ID NO: 10 is substituted with an amino acid selected from the group consisting of asparagine, serine, glycine, leucine, arginine, alanine, methionine, threonine, glutamine, proline, isoleucine, tryptophan, phenylalanine, histidine, cysteine, tyrosine, lysine, aspartate, and glutamic acid.

In any one of the above-described embodiments, the variant polypeptide provided in the present application may be one in which the amino acid corresponding to position 437 from the N-terminus of SEQ ID NO: 10 is substituted with alanine.

In another embodiment of the above-described embodiments, the variant polypeptide provided in the present application may comprise an amino acid sequence having at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.8% homology or identity with SEQ ID NO: 10, or less than 100%.

For example, the variant polypeptide of the present application may include an amino acid sequence in which the amino acid corresponding to position 437 in the amino acid sequence set forth in SEQ ID NO: 10 is fixed to alanine, and has at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.8% homology or identity with SEQ ID NO: 10. In addition, it is obvious that a variant polypeptide having an amino acid sequence in which a part of the sequence is deleted, modified, substituted, conservatively substituted, or added is also included within the scope of the present application, as long as it has such homology or identity and exhibits an effect corresponding to the variant polypeptide of the present application.

Meanwhile, a person skilled in the art can identify the amino acid corresponding to the 437th position of the amino acid sequence of SEQ ID NO. 10 of the present application in any amino acid sequence through sequence alignment known in the art, and even if not described separately in the present application, when "an amino acid at a specific position in a specific sequence number" is described, it is self-evident that it includes "an amino acid at a corresponding position" in any amino acid sequence.

In another specific example, the mutant polypeptide may be composed of the amino acid sequence of SEQ ID NO: 14.

Specifically, the variant polypeptide of the present application may have, comprise, consist of, or consist essentially of the amino acid sequence of SEQ ID NO: 14 or an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology or identity with the amino acid sequence of SEQ ID NO: 14.

Additionally, the variant polypeptide of the present application may also include sequence additions or deletions, or naturally occurring mutations, silent mutations, or conservative substitutions that do not alter the function of the variant polypeptide at the N-terminus, C-terminus, and/or within the amino acid sequence of the variant polypeptide of the present application.

The above "other amino acid" is not limited to an amino acid different from the amino acid prior to substitution. Furthermore, when the present application states that "a specific amino acid has been substituted," it is self-evident that the amino acid has been substituted with an amino acid different from the amino acid prior to substitution, even if it is not specifically stated that it has been substituted with another amino acid.

Amino acids can generally be classified based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or amphipathic nature of their residues.

Examples of these classifications 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. As another example, amino acids with charged side chains (electrically charged amino acids) include arginine, lysine, histidine, glutamic acid, and aspartic acid; and amino acids with uncharged side chains (neutral amino acids) include glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. As another example, phenylalanine, tryptophan, and tyrosine can be classified as aromatic amino acids. As another example, valine, leucine, and isoleucine can be classified as branched amino acids. As another example, the 20 amino acids can be classified by size into five groups: glycine, alanine, and serine; cysteine, proline, threonine, aspartic acid, and asparagine; valine, histidine, glutamic acid, and glutamine; isoleucine, leucine, methionine, lysine, and arginine; and phenylalanine, tryptophan, and tyrosine. However, this is not necessarily limited to these groups.

For example, if it is described that "the amino acid corresponding to position 437 in SEQ ID NO: 10 is replaced with another amino acid," it may mean that the amino acid is replaced with asparagine, serine, glycine, alanine, glutamate, phenylalanine, arginine, aspartate, cysteine, glutamine, histidine, proline, isoleucine, tyrosine, lysine, tryptophan, methionine, threonine, or leucine, excluding valine.

Even if the present application describes a "protein having an amino acid sequence described by a specific sequence number", it is obvious that a protein having an amino acid sequence in which some sequences are deleted, modified, substituted, conservatively substituted, or added may also be used in the present application, as long as it has the same or corresponding activity as the protein consisting of the amino acid sequence of the corresponding sequence number. For example, if it has the same or corresponding activity as the mutant protein, it does not exclude sequence additions that do not alter the function of the protein before or after the amino acid sequence, mutations that may occur naturally, silent mutations thereof, or conservative substitutions, and it is obvious that even if it has such sequence additions or mutations, it falls within the scope of the present application.

The "N-position" of the present application may include the N-position and an amino acid position corresponding to the N-position. Specifically, it may include an amino acid position corresponding to any amino acid residue in a mature polypeptide disclosed in a specific amino acid sequence. The specific amino acid sequence may be the amino acid sequence of SEQ ID NO: 10.

As used herein, the term "corresponding to" refers to an amino acid residue at a position listed in a polypeptide, or an amino acid residue that is similar, identical, or homologous to the residue listed in the polypeptide. Identifying an amino acid at a corresponding position may be determining a specific amino acid in a sequence that references a particular sequence. As used herein, "corresponding region" generally refers to a similar or corresponding position in a related or reference protein.

For example, any amino acid sequence can be aligned with SEQ ID NO: 10, and based on this, each amino acid residue of the amino acid sequence can be numbered by referring to the numerical position of the amino acid residue corresponding to the amino acid residue in SEQ ID NO: 10. For example, a sequence alignment algorithm such as that described in the present application can identify the position of an amino acid, or the position at which a modification such as a substitution, insertion, or deletion occurs, by comparing it to a query sequence (also referred to as a “reference sequence”).

For such alignment, for example, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453), the Needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000), Trends Genet. 16: 276-277) can be used, but is not limited thereto, and any sequence alignment program known in the art, pairwise sequence comparison algorithm, etc. can be appropriately used.

The term "conservative substitution" in this application refers to the replacement of one amino acid with another amino acid having similar structural and/or chemical properties. Such amino acid substitutions may generally occur based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or amphipathic nature of the residues. For example, positively charged (basic) amino acids include arginine, lysine, and histidine; negatively charged (acidic) amino acids include glutamic acid and aspartate; aromatic amino acids include phenylalanine, tryptophan, and tyrosine; and hydrophobic amino acids include alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan. Additionally, amino acids can be classified into those with electrically charged side chains and those with uncharged side chains. Charged side chain amino acids include aspartic acid, glutamic acid, lysine, arginine, and histidine. Uncharged side chain amino acids can be further classified into nonpolar amino acids or polar amino acids. Nonpolar amino acids include glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline. Polar amino acids include serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Typically, conservative substitutions have little or no effect on the activity of the resulting polypeptide. Typically, conservative substitutions may have little or no effect on the activity of a protein or polypeptide.

Another aspect of the present application provides a nicotinamide nucleotide transhydrogenase comprising the mutant polypeptide.

The above nicotinamide nucleotide transhydrogenase may comprise the mutant polypeptides PntA; and PntB.

That is, the nicotinamide nucleotide transhydrogenase of the present application may be composed of the nicotinamide nucleotide transhydrogenase alpha subunit mutant polypeptide and the nicotinamide nucleotide transhydrogenase beta subunit of the present application.

Another aspect of the present application provides a polynucleotide encoding the mutant polypeptide.

In this application, the term "polynucleotide" means a polymer of nucleotides in which nucleotide units (monomers) are covalently bonded to form a long chain, a DNA or RNA strand of a certain length or longer, and more specifically, a polynucleotide fragment encoding the mutant polypeptide.

The polynucleotide encoding the nicotinamide nucleotide transhydrogenase alpha subunit variant polypeptide of the present application may include, without limitation, any polynucleotide sequence capable of forming nicotinamide nucleotide transhydrogenase together with the beta subunit.

For example, the polynucleotide encoding the nicotinamide nucleotide transhydrogenase alpha subunit variant polypeptide of the present application may be, but is not limited to, a polynucleotide sequence encoding an amino acid sequence in which the amino acid corresponding to position 437 of SEQ ID NO: 10 is substituted with alanine.

For example, the polynucleotide of the present application may comprise a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 14. Furthermore, as an example, the polynucleotide of the present application may have or comprise the sequence of SEQ ID NO: 15. Furthermore, the polynucleotide of the present application may consist of, or consist essentially of, the sequence of SEQ ID NO: 15.

The polynucleotide of the present application may be modified in various ways in the coding region without altering the amino acid sequence of the variant polypeptide of the present application, taking into account codon degeneracy or preferred codons in the organism that is intended to express the variant polypeptide of the present application. Therefore, it is self-evident that the polynucleotide of the present application may also be translated into a polypeptide consisting of the amino acid sequence of the variant polypeptide of the present application or a polypeptide having homology or identity therewith due to codon degeneracy. For example, the polynucleotide of the present application may be composed of or include the sequence of SEQ ID NO: 15; or a degenerated sequence thereof.

For example, the polynucleotide of the present application may include, but is not limited to, a base sequence having a homology or identity of 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.8% or more with respect to the sequence of SEQ ID NO: 12, in which the codon corresponding to the codon at positions 1309 to 1311 of SEQ ID NO: 12 is substituted with a codon encoding an amino acid other than the original amino acid, for example, alanine. In addition, it is obvious that a variant having a polynucleotide sequence having a deletion, modification, substitution, conservative substitution or addition of a part of a sequence that has such homology or identity and encodes the amino acid sequence of the nicotinamide nucleotide transhydrogenase alpha subunit variant polypeptide of the present application is also included within the scope of the present application.

As another example, the polynucleotide of the present application may have or include a nucleic acid sequence having at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, and less than 100% homology or identity with the sequence of SEQ ID NO: 15, or may consist of, consist essentially of, or include a nucleic acid sequence having at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, and less than 100% homology or identity with the sequence of SEQ ID NO: 15.

Additionally, the polynucleotide of the present application may include, without limitation, a probe that can be prepared from a known genetic sequence, for example, a sequence that can hybridize under stringent conditions with a complementary sequence to all or part of the polynucleotide sequence of the present application.

The above "stringent conditions" refer to conditions that allow specific hybridization between polynucleotides. These conditions are specifically described in the literature (see J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989; F.M. Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, 9.50-9.51, 11.7-11.8). For example, conditions in which polynucleotides having high homology or identity hybridize with each other, 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 hybridize with each other, and polynucleotides having lower homology or identity do not hybridize with each other, or conditions in which washing is performed once, specifically twice or three times, at a salt concentration and temperature equivalent to 60°C, 1ХSSC, 0.1% SDS, specifically 60°C, 0.1ХSSC, 0.1% SDS, and more specifically 68°C, 0.1ХSSC, 0.1% SDS, which are washing conditions of typical southern hybridization, are performed.

Hybridization requires that two nucleic acids have complementary sequences, although mismatches between bases are possible depending on the stringency of hybridization. The term "complementary" is used to describe the relationship between nucleotide bases that can hybridize with each other. For example, in DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Therefore, the polynucleotides of the present application may also include isolated nucleic acid fragments that are complementary in their entirety, as well as substantially similar nucleic acid sequences.

Specifically, a polynucleotide having homology or identity with the polynucleotide of the present application can be detected using hybridization conditions including a hybridization step at a Tm value of 55°C and using the conditions described above. In addition, the Tm value may be, but is not limited to, 60°C, 63°C, or 65°C, and can be appropriately adjusted by a person skilled in the art depending on the purpose.

The appropriate stringency for hybridizing the polynucleotides depends on the length and degree of complementarity of the polynucleotides, variables which are well known in the art (e.g., J. Sambrook et al., supra).

As used herein, the terms "homology" or "identity" refer to the degree of similarity between two given amino acid sequences or base sequences, which may be expressed as a percentage. The terms homology and identity are often used interchangeably.

Sequence homology or identity of conserved polynucleotides or polypeptides is determined by standard alignment algorithms, and may be combined with default gap penalties established by the program being used. In practice, homologous or identical sequences can generally hybridize with all or part of the sequence under moderate or high stringency conditions. It should be appreciated that hybridization also includes hybridization with polynucleotides containing common codons or codons that take codon degeneracy into account.

Whether any two polynucleotide or polypeptide sequences are homologous, similar or identical can be determined using known computer algorithms such as the "FASTA" program using default parameters, for example as in Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444. Alternatively, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277) (version 5.0.0 or later) can be determined using the GCG program package (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 ETA/.](1988) SIAM J Applied Math 48: 1073). For example, homology, similarity, or identity can be determined using BLAST or ClustalW of the National Center for Biotechnology Information Database.

Homology, similarity, or identity of polynucleotides or polypeptides can be determined by comparing sequence information, for example, using a GAP computer program such as that of Needleman et al. (1970), J Mol Biol. 48:443, as disclosed, for example, in Smith and Waterman, Adv. Appl. Math (1981) 2:482. In brief, the GAP program can be defined as the total number of symbols in the shorter of the two sequences divided by the number of similarly arranged symbols (i.e., nucleotides or amino acids). Default parameters for the GAP program include (1) a binary comparison matrix (containing values of 1 for identity and 0 for non-identity) and (2) a comparison matrix as disclosed by Gribskov et al. (1986) Nucl. Acids Res. 48:443, as disclosed by Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353-358 (1979). 14: 6745 weighted comparison matrix (or EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix); (2) a penalty of 3.0 for each gap and an additional penalty of 0.10 for each symbol in each gap (or a gap opening penalty of 10 and a gap extension penalty of 0.5); and (3) no penalty for terminal gaps. Accordingly, the term "homology" or "identity" as used herein refers to the relevance between sequences.

Another aspect of the present application provides a vector comprising the polynucleotide of the present application. The vector may be, but is not limited to, an expression vector for expressing the polynucleotide in a microorganism.

The term "vector" in this application may include a DNA construct comprising a base sequence of a polynucleotide encoding a target polypeptide operably linked to a suitable expression control region (or expression control sequence) so as to enable expression of the target polypeptide in a suitable host. The expression control region may include a promoter capable of initiating transcription, an optional operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosome binding site, and sequences regulating the termination of transcription and translation. After being transformed into a suitable microorganism, the vector may replicate or function independently of the host genome, or may be integrated into the genome itself.

The vector used in the present application is not particularly limited, and any vector known in the art can be used. Examples of commonly used vectors include plasmids, cosmids, viruses, and bacteriophages in a natural or recombinant state. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A can be used as phage vectors or cosmid vectors, and pDZ series, pBR series, pUC series, pBluescriptII series, pGEM series, pTZ series, pCL series, and pET series can be used as plasmid vectors. Specifically, pDZ, pDC, pDCM2, pDC24, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vectors can be used.

For example, a polynucleotide encoding a target polypeptide can be inserted into a chromosome via a vector for intracellular chromosomal insertion. The insertion of the polynucleotide into the chromosome can be achieved by any method known in the art, for example, homologous recombination, but is not limited thereto. A selection marker for confirming the chromosomal insertion can be additionally included. The selection marker is used to select cells transformed with the vector, i.e., to confirm the insertion of the target nucleic acid molecule. Markers that confer a selectable phenotype, such as drug resistance, nutrient requirement, cytotoxic agent resistance, or expression of a surface polypeptide, can be used. In an environment treated with a selective agent, only cells expressing the selection marker survive or exhibit other phenotypic traits, so that transformed cells can be selected.

The term "transformation" in this application refers to introducing a vector containing a polynucleotide encoding a target polypeptide into a microorganism or into a microorganism, so that the polypeptide encoded by the polynucleotide can be expressed in the microorganism. The transformed polynucleotide can be located either within the chromosome of the microorganism or outside the chromosome, as long as it can be expressed in the microorganism. In addition, the polynucleotide includes DNA and/or RNA encoding the target polypeptide. The polynucleotide can be introduced in any form as long as it can be introduced into the microorganism and expressed. For example, the polynucleotide can be introduced into the microorganism in the form of an expression cassette, which is a genetic construct containing all elements necessary for autonomous expression. The expression cassette can typically include a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal, all of which are operably linked to the polynucleotide. The expression cassette can be in the form of a self-replicating expression vector. Additionally, the polynucleotide may be introduced into a microorganism in its own form and operably linked to a sequence required for expression in the microorganism, but is not limited thereto.

Additionally, the term "operably linked" as used herein means that the polynucleotide sequence is functionally linked to a promoter sequence that initiates and mediates transcription of the polynucleotide encoding the target variant polypeptide of the present application.

The method for transforming the vector of the present application includes any method for introducing nucleic acids into cells, and can be performed by selecting an appropriate standard technique known in the art depending on the host cell. Examples thereof include, but are not limited to, electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, and lithium acetate-DMSO method.

Another aspect of the present application provides a microorganism comprising at least one selected from the group consisting of the mutant polypeptide; a polynucleotide encoding the mutant polypeptide; and a vector comprising the same.

Another aspect of the present application provides a microorganism further comprising at least one selected from the group consisting of a nicotinamide nucleotide transhydrogenase beta subunit; a polynucleotide encoding the beta subunit; and a vector comprising the same.

In one specific example, the microorganism of the present application may be a microorganism having the ability to produce L-amino acid or a derivative thereof.

In this application, the term "microorganism (or strain)" includes both wild-type microorganisms and microorganisms that have undergone natural or artificial genetic modification. It may be a microorganism that has a specific mechanism weakened or strengthened due to causes such as the insertion of an external gene or the enhancement or inactivation of the activity of an endogenous gene, and may be a microorganism that includes genetic modification for the production of a desired polypeptide, protein, or product. In this application, "microorganism," "strain," and "microorganism" may be used interchangeably without limitation with the same meaning.

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 nucleic acid sequence coding of the microorganism), and may include all progeny or potential progeny of the microorganism. The terms "recombinant microorganism," "genetically modified microorganism," "recombinant host cell," "recombinant cell," and "recombinant strain" may be used interchangeably in this application. The recombinant microorganism may, for example, express a gene not found in its native (non-recombinant) form; may not express a gene expressed in its native form; or may express a native gene in a manner different from that in which it is expressed in its native form.

In the present application, the term "microorganism producing L-amino acid" refers to a prokaryotic or eukaryotic microbial strain capable of producing L-amino acid within the organism, and may include both a microorganism in which the ability to produce L-amino acid has been conferred on a parent strain that does not have the ability to produce L-amino acid, or a microorganism that inherently has the ability to produce L-amino acid. The ability to produce L-amino acid may be conferred or enhanced by species improvement.

In one embodiment, the microorganism of the present application may be a microorganism that naturally has the ability to produce a nicotinamide nucleotide transhydrogenase alpha subunit variant polypeptide or an L-amino acid; or a microorganism that has been introduced into a parent strain that does not have the ability to produce a nicotinamide nucleotide transhydrogenase alpha subunit variant polypeptide or an L-amino acid by a variant polypeptide of the present application or a polynucleotide encoding the same (or a vector including the polynucleotide) and/or has been given the ability to produce an L-amino acid, but is not limited thereto.

In one embodiment, the microorganism of the present application includes, but is not limited to, a microorganism having a chromosomal gene encoding a nicotinamide nucleotide transhydrogenase alpha subunit polypeptide mutated to include a nicotinamide nucleotide transhydrogenase alpha subunit variant polypeptide sequence of the present application and/or a polynucleotide encoding a nicotinamide nucleotide transhydrogenase alpha subunit variant polypeptide of the present application; or a vector including the same, thereby introducing a microorganism including a nicotinamide nucleotide transhydrogenase alpha subunit variant polypeptide of the present application.

In this application, the term "unmodified microorganism" does not exclude a strain that contains a mutation that can occur naturally in a microorganism, and may refer to a wild-type strain or a natural strain itself, or a strain before its characteristics are changed by genetic mutation caused by natural or artificial factors. For example, the unmodified microorganism may refer to a strain into which the nicotinamide nucleotide transhydrogenase alpha subunit mutant polypeptide described herein is not introduced or before it is introduced. The "unmodified microorganism" may be used interchangeably with "pre-modified strain," "pre-modified microorganism," "unmutated strain," "unmodified microorganism," "unmutated microorganism," or "reference microorganism."

The microorganism having L-amino acid production ability of the present application may be, but is not limited to, a microorganism comprising at least one of the variant polypeptide of the present application, the polynucleotide of the present application, and a vector comprising the polynucleotide of the present application; a microorganism modified to express the variant polypeptide of the present application or the polynucleotide of the present application; a microorganism (e.g., a recombinant strain) expressing the variant polypeptide of the present application or the polynucleotide of the present application; or a microorganism (e.g., a recombinant strain) having the activity of the variant polypeptide of the present application.

For example, the microorganism of the present application is a cell or microorganism that is transformed with a polynucleotide encoding the mutant polypeptide of the present application or a vector containing the same, and expresses the mutant polypeptide of the present application, and the strain of the present application may include all microorganisms capable of producing L-amino acids or derivatives thereof, including the mutant polypeptide of the present application. For example, the microorganism of the present application may be a recombinant strain in which a polynucleotide encoding the mutant polypeptide of the present application is introduced into a natural wild-type microorganism or a microorganism having the ability to produce L-amino acids, thereby expressing a nicotinamide nucleotide transhydrogenase alpha subunit mutant polypeptide, or expressing a mutant nicotinamide nucleotide transhydrogenase containing the mutant polypeptide of the present application, thereby increasing the ability to produce L-amino acids. The recombinant strain with increased L-amino acid productivity may be a microorganism with increased L-amino acid productivity compared to a natural wild-type microorganism or a non-modified microorganism (e.g., a microorganism expressing a wild-type nicotinamide nucleotide transhydrogenase or a microorganism that does not express the mutant polypeptide of the present application), but is not limited thereto. For example, the microorganism with increased L-amino acid productivity of the present application may be a microorganism with increased L-amino acid productivity compared to a microorganism comprising the polypeptide of SEQ ID NO: 10 or a polynucleotide encoding the same, but is not limited thereto. For example, the non-modified microorganism, which is a target strain for comparing whether the L-amino acid productivity is increased, may be, but is not limited to, KCCM 12502P (CA09-0903), a wild-type Corynebacterium glutamicum strain.

The microorganism of the present application may include any microorganism capable of expressing the nicotinamide nucleotide transhydrogenase alpha subunit mutant polypeptide of the present application by various known methods in addition to the above nucleic acid or vector introduction.

For example, the L-amino acid production ability of the microorganism with increased L-amino acid production ability is about 1% or more, specifically, about 1% or more, about 2% or more, about 3% or more, about 4% 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 11% or more, about 12% or more, about 13% or more, about 14% or more, about 15% or more, about 16% or more, about 17% or more, about 18% or more, about 19% or more, about 20% or more, about 25% or more, about 30% or more, about 35% or more, about 40% or more, about 50% or more (the upper limit is not particularly limited, for example, about 200% or less, about 150% or less, about It may be increased by 100% or less, about 90% or less, about 80% or less, about 70% or less, about 60% or less, about 55% or less, or about 50% or less), but is not limited thereto, as long as it has a positive value of increase compared to the productivity of the parent strain or unmodified microorganism before the mutation. In another example, the recombinant strain with increased L-amino acid production ability has an L-amino acid production ability of about 1.01 times or more, about 1.02 times or more, about 1.03 times or more, about 1.04 times or more, about 1.05 times or more, about 1.06 times or more, about 1.07 times or more, about 1.08 times or more, about 1.09 times or more, about 1.1 times or more, about 1.11 times or more, about 1.12 times or more, about 1.13 times or more, about 1.14 times or more, about 1.15 times or more, about 1.16 times or more, about 1.17 times or more, about 1.18 times or more, about 1.19 times or more, about 1.20 times or more, about 1.25 times or more, about 1.30 times or more, about 1.35 times or more, or about It may be increased by, but is not limited to, 1.40 times or more, about 1.45 times or more, or about 1.50 times or more (the upper limit is not particularly limited, and may be, for example, about 10 times or less, about 5 times or less, about 3 times or less, about 2 times or less, or about 1.5 times or less). The term “about” includes, but is not limited to, a range including all of ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and includes all values in a range equal to or similar to the value following the term “about.”

As a microorganism according to any one of the preceding specific examples, the microorganism of the present application may be a microorganism belonging to the genus Corynebacteria sp ., Escherichia sp ., Erwinia sp ., Serratia sp ., Providencia sp., Pseudomonas sp ., Leptospira sp ., Salmonella sp., Brevibacteria sp ., Hypomononas sp ., Chromobacterium sp. , and Norcardia sp . , or a fungus or yeast, specifically, a microorganism belonging to the genus Corynebacterium, but is not limited thereto.

As an example of the present application, the microorganism of the present application is Corynebacterium glutamicum , Corynebacterium crudilactis , Corynebacterium deserti , Corynebacterium efficiens , Corynebacterium callunae, Corynebacterium stationis , Corynebacterium singulare, Corynebacterium halotolerans , Corynebacterium striatum , Corynebacterium ammoniagenes, It may be Corynebacterium pollutisoli , Corynebacterium imitans , Corynebacterium testudinoris, or Corynebacterium flavescens . Specifically, the microorganism of the present application may be a microorganism of the genus Corynebacterium, more specifically, Corynebacterium glutamicum, but is not limited thereto.

Specifically, the microorganism of the present application may be a microorganism of the genus Corynebacterium, more specifically, Corynebacterium glutamicum, but is not limited thereto.

Meanwhile, the Corynebacterium genus microorganism with improved L-amino acid productivity of the present application may include a natural wild-type microorganism itself, a Corynebacterium genus microorganism with improved L-amino acid productivity by increasing or decreasing the activity of a gene related to the L-amino acid production mechanism, or a Corynebacterium genus microorganism with improved L-amino acid productivity by introducing or increasing the activity of an external gene. As a specific example, the present application may include, but is not limited to, a Corynebacterium genus microorganism with improved L-amino acid productivity by introducing the present mutant polypeptide or a mutant nicotinamide nucleotide transhydrogenase comprising the same.

In this application, the term "increase" of protein (polypeptide) activity means that the activity of the protein (polypeptide) within a host cell (microorganism) is increased compared to its intrinsic activity. The increase may be used interchangeably with terms such as activation, up-regulation, overexpression, and enhancement. The host cell (microorganism) may be a prokaryotic or eukaryotic microorganism.

The increase in the above protein (polypeptide) activity may include the display of a protein (polypeptide) activity that the host cell (microorganism) did not inherently possess, or the display of an enhanced protein (polypeptide) activity compared to the inherent activity or activity before modification.

For example, the above “exhibiting a protein (polypeptide) activity that was not inherently present” or “exhibiting an improved protein (polypeptide) activity” may be due to, but is not limited to, “introduction of a protein (polypeptide).”

In this application, the term "introduction" of a protein (polypeptide) means that a gene that a microorganism did not originally possess is expressed within the microorganism, thereby causing the activity of a specific protein to be exhibited, or that the activity of the polypeptide is strengthened, increased, or improved compared to the intrinsic activity of the protein or the activity before modification. For example, this may be due to the introduction of a gene encoding the protein (polypeptide) into a host cell (microorganism). For example, a polynucleotide encoding a specific protein (polypeptide) may be introduced into the chromosome of a host cell (microorganism), or a vector containing a polynucleotide encoding a specific protein (polypeptide) may be introduced into the host cell (microorganism), thereby causing the activity of the protein (polypeptide) to be exhibited or improved.

The above "intrinsic activity" refers to the activity of a specific protein (polypeptide) originally possessed by a host cell (microorganism) or an untransformed host cell (microorganism) before transformation, when the trait changes due to genetic mutation caused by natural or artificial factors. This may be used interchangeably with "activity before transformation."

An increase in the activity of a protein (polypeptide) compared to the intrinsic activity means that the activity and/or concentration (expression amount) of the protein (polypeptide) of the host cell (microorganism) is enhanced compared to the activity and/or concentration (expression amount) of the protein (polypeptide) originally present in the host cell (microorganism) before transformation or in the non-transformed host cell (microorganism).

For example, the increase may be, but is not limited to, an increase in the activity or concentration of the corresponding protein (polypeptide) from a previous state, or an increase in the activity or concentration thereof, typically by at least about 1%, at least about 10%, at least about 25%, at least about 50%, at least about 75%, at least about 100%, at least about 150%, at least about 200%, at least about 300%, at least about 400%, or at least about 500%, up to at least about 1000% or at least about 2000%, relative to the activity or concentration in the host cell (microorganism) before transformation or in the untransformed host cell (microorganism).

An increase in the activity of the above protein (polypeptide) can be achieved by introducing an exogenous protein (polypeptide) or by increasing the activity of an endogenous protein (polypeptide). Whether the activity of the above protein (polypeptide) has increased can be confirmed by an increase in the activity level of the protein (polypeptide), the expression level, or the amount of a product resulting from the activity of the protein (polypeptide).

The increase in the activity of the above protein (polypeptide) can be achieved by various methods well known in the art, and is not limited thereto, as long as the activity of the target protein (polypeptide) can be increased compared to the host cell (microorganism) before transformation. Specifically, it may be achieved by using genetic engineering and/or protein engineering, which are routine methods of molecular biology and are well known to those skilled in the art, but 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.).

Specifically, the increase in activity of the protein (polypeptide) of the present application is

1) Increase in the intracellular copy number of a polynucleotide encoding a protein (polypeptide);

2) Modification of the gene expression control region on the chromosome that codes for a protein (polypeptide) (e.g., introduction of a mutation in the expression control region, replacement with a sequence having stronger activity, or insertion of a sequence having stronger activity);

3) Modification of the base sequence encoding the initiation codon or 5'-UTR region of a gene transcript encoding a protein (polypeptide);

4) Modification of the amino acid sequence of the protein (polypeptide) so as to increase the activity of the protein (polypeptide);

5) Modification of a polynucleotide sequence encoding a protein (polypeptide) such that the activity of the protein (polypeptide) is increased (e.g., modification of a polynucleotide sequence of a protein (polypeptide) encoding gene such that the protein (polypeptide) is modified such that the activity of the protein (polypeptide) is increased);

6) Introduction of a foreign protein (polypeptide) that exhibits the activity of a protein (polypeptide) or a foreign polynucleotide encoding the same;

7) Codon optimization of polynucleotides encoding proteins (polypeptides);

8) Analyzing the tertiary structure of a protein (polypeptide) and selecting the exposed portion to modify or chemically modify; or

9) Control of cellular localization of proteins (polypeptides); or

10) It may be a combination of two or more selected from 1) to 9), but is not particularly limited thereto.

for example,

The increase in the intracellular copy number of the polynucleotide encoding the protein (polypeptide) described above 1) may be caused by introducing a vector containing a polynucleotide encoding the protein (polypeptide) operably linked to an appropriate regulatory sequence into a host cell (microorganism). Alternatively, one copy or two or more copies of the polynucleotide encoding the protein (polypeptide) operably linked to an appropriate regulatory sequence may be introduced into a chromosome within the host cell (microorganism). The introduction into the chromosome may be performed by introducing a vector capable of inserting the polynucleotide into the chromosome within the host cell (microorganism), but is not limited thereto. The vector is as described above. The regulatory sequence may be a natural sequence (same in origin) or a foreign sequence (derived from another gene) to the polynucleotide sequence it encodes, or a mutant sequence thereof, or another artificial sequence, and may induce expression of the polynucleotide within the host cell (microorganism).

2) The replacement of the gene expression control region (or expression control sequence) on the chromosome encoding the protein (polypeptide) with a sequence having strong activity may be, for example, introducing a mutation in the sequence by deletion, insertion, substitution, or a combination thereof to further increase the activity of the expression control region, or replacement with a sequence having stronger activity. The expression control region may include, but is not particularly limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence regulating the termination of transcription and translation. As an example, the original promoter may be replaced with a strong promoter, but is not limited thereto.

Examples of known strong promoters include, but are not limited to, the cj1 to cj7 promoters (US Patent No. US 7662943 B2), the lac promoter, the trp promoter, the trc promoter, the tac promoter, the lambda phage PR promoter, the PL promoter, the tet promoter, the gapA promoter, the SPL7 promoter, the SPL13 (sm3) promoter (US Patent No. US 10584338 B2), the O2 promoter (US Patent No. US 10273491 B2), the tkt promoter, and the yccA promoter.

The above 3) modification of the base sequence of the region encoding the initiation codon or 5'-UTR of the gene encoding the protein (polypeptide) may be, for example, a modification that encodes another initiation codon having a higher protein (polypeptide) expression rate than the endogenous initiation codon, or an RBS sequence having a higher protein (polypeptide) expression rate than the endogenous RBS (ribosome binding site) sequence, but is not limited thereto.

The modification of the amino acid sequence or polynucleotide sequence of the protein (polypeptide) of the above 4) and 5) may be, but is not limited to, introducing a sequence mutation by deletion, insertion, substitution, or a combination thereof in the amino acid sequence of the protein (polypeptide) or the polynucleotide sequence encoding the protein (polypeptide) so as to increase the activity of the protein (polypeptide), or replacing it with an amino acid sequence or polynucleotide sequence modified to increase the activity. The replacement may be performed, for example, by inserting a polynucleotide into a chromosome by homologous recombination, but is not limited thereto.

The introduction of the foreign polynucleotide exhibiting the activity of the above 6) protein (polypeptide) may be the introduction into the host cell (microorganism) of a foreign polynucleotide encoding a protein (polypeptide) exhibiting the same/similar activity as the protein (polypeptide). The foreign polynucleotide is not limited in its origin or sequence as long as it exhibits the same/similar activity as the protein (polypeptide). The method used for the above introduction can be performed by a person skilled in the art appropriately selecting a known transformation method, and the protein (polypeptide) can be produced by expressing the introduced polynucleotide in the host cell, thereby increasing its activity.

The above 7) codon optimization of a polynucleotide encoding a protein (polypeptide) may be codon optimization of an endogenous polynucleotide to increase transcription or translation within a host cell (microorganism), or codon optimization of a foreign polynucleotide to achieve optimized transcription or translation within a host cell (microorganism).

The above 8) analyzing the tertiary structure of a protein (polypeptide) and selecting an exposed portion to modify or chemically modify may be done by, for example, comparing the sequence information of the protein (polypeptide) to be analyzed with a database storing the sequence information of known proteins, determining a template protein candidate based on the degree of sequence similarity, confirming the structure based on this, and selecting an exposed portion to modify or chemically modify, and modifying or modifying it.

The above 9) regulation of the intracellular location of a protein (polypeptide) may target the protein (polypeptide) to a specific organelle or specific intracellular space within the cell. For example, targeting to the periplasm or cytoplasm may be achieved by adding or removing a leader sequence that functions in targeting the protein (polypeptide), but is not limited thereto.

Such an increase in protein (polypeptide) activity may be, but is not limited to, an increase in the activity or concentration of the corresponding protein (polypeptide) relative to the activity or concentration of the protein (polypeptide) expressed in the wild type or pre-transformed host cell (microorganism), or an increase in the amount of a product resulting from the activity of the corresponding protein (polypeptide).

Modification of part or all of the polynucleotide in the microorganism of the present application may be induced by, but is not limited to, (a) a method using homologous recombination using a vector for chromosome insertion into the microorganism, or genome editing using genetic scissors (engineered nuclease, e.g., CRISPR-Cas9), and/or (b) treatment with light and/or chemicals such as ultraviolet rays and radiation.

The microorganism of the present application may have improved L-amino acid production ability.

In the present application, “L-amino acid” includes all L-amino acids that can be produced by microorganisms through metabolic processes from various carbon sources, and specifically, may include L-threonine, but is not limited thereto.

In one specific example of the present application, the microorganism of the genus Corynebacterium may have an increased L-threonine production ability compared to a non-modified microorganism.

Another aspect of the present application provides a method for producing an L-amino acid, comprising the step of culturing a microorganism comprising at least one selected from the group consisting of a variant polypeptide of the present application; a polynucleotide encoding the variant polypeptide; and a vector comprising the same in a medium.

The above microorganisms are as described in other aspects.

In this application, the term "cultivation" refers to growing the microorganism of this application under appropriately controlled environmental conditions. The culturing process in this application can be performed using any suitable medium and culture conditions known in the art. Such culturing process can be easily adjusted and used by those skilled in the art depending on the selected strain. Specifically, the culturing process may be batch, continuous, and/or fed-batch, but is not limited thereto.

The microorganism of the present application can be cultured under aerobic conditions in a conventional medium containing appropriate carbon sources, nitrogen sources, phosphorus, inorganic compounds, amino acids, and/or vitamins, while controlling temperature, pH, etc.

In the present application, the carbon source 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.; amino acids such as glutamic acid, methionine, lysine, etc. In addition, natural organic nutrients such as starch hydrolysate, molasses, blackstrap molasses, rice winter, cassava, sugarcane bagasse, and corn steep liquor may be used, and specifically, carbohydrates such as glucose and sterilized pretreated molasses (i.e., molasses converted to reducing sugar) may be used, and other appropriate amounts of carbon sources may be used in various ways without limitation. These carbon sources may be used alone or in combination of two or more, but are not limited thereto.

The nitrogen source may include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, ammonium nitrate, etc.; organic nitrogen sources such as amino acids such as glutamic acid, methionine, glutamine, etc.; peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolysate, fish or its decomposition product, defatted soybean cake or its decomposition product, etc. These nitrogen sources may be used alone or in combination of two or more, but are not limited thereto.

The above-mentioned components may include potassium phosphate monobasic, potassium phosphate dibasic, or their corresponding sodium-containing salts. Inorganic compounds may include sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, etc. In addition, amino acids, vitamins, and/or suitable precursors may be included. These components or precursors may be added to the medium in batch or continuous manner, but are not limited thereto.

During the cultivation of the microorganism of the present invention, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid, etc. can be added to the medium in an appropriate manner to adjust the pH of the medium. In addition, during the cultivation, an antifoaming agent such as fatty acid polyglycol ester can be used to suppress bubble formation. In addition, to maintain the aerobic state of the medium, oxygen or an oxygen-containing gas can be injected into the medium, or to maintain anaerobic and microaerobic states, nitrogen, hydrogen, or carbon dioxide gas can be injected without gas injection, but the present invention is not limited thereto.

Additionally, the culture medium may contain metal salts, such as magnesium sulfate or iron sulfate, necessary for growth. Finally, in addition to the above substances, essential growth substances, such as amino acids and vitamins, may be used. Appropriate precursors may also be used in the culture medium. The above-mentioned raw materials may be added to the culture in a batch or continuous manner during the culture process, but are not limited thereto.

In the present application, during the cultivation of microorganisms, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid, etc. can be appropriately added to the culture to adjust the pH of the culture. In addition, during the cultivation, an antifoaming agent such as fatty acid polyglycol ester can be used to suppress bubble formation. Furthermore, to maintain the aerobic state of the culture, oxygen or an oxygen-containing gas can be injected into the culture, or to maintain anaerobic or microaerobic states, nitrogen, hydrogen, or carbon dioxide gas can be injected without gas injection, but is not limited thereto.

In the culture of the present application, the culture temperature can be maintained at 20 to 35°C, specifically 25 to 35°C, and the culture period can be continued until the amount of useful material produced is obtained, and the culture can be performed for about 10 to 160 hours, about 20 to 130 hours, about 24 to 120 hours, about 36 to 120 hours, about 48 to 120 hours, about 48 hours or more, or about 48 hours, about 72 hours, or about 120 hours, but is not limited thereto.

L-amino acids produced by the culture of the present invention may be secreted into the medium or remain within the cells.

In one specific example of the present application, the L-amino acid may be, but is not limited to, L-threonine.

In one specific example, the method for producing L-amino acids of the present application may further include, for example, prior to the culturing step, a step of preparing a microorganism of the present application, a step of preparing a medium for culturing the strain, or a combination thereof (in any order).

The L-amino acid production method of the present application may further include a step of recovering a target substance, specifically an L-amino acid or a derivative thereof, from the cultured microorganism, a culture of the microorganism, a fermented product of the microorganism, or the culture medium. The recovering step may be additionally included after the culturing step.

The above recovery may be performed by collecting the target L-amino acid or its derivative using a suitable method known in the art according to the culture method of the microorganism of the present application, for example, a batch, continuous or fed-batch culture method. For example, various chromatographies such as centrifugation, filtration, treatment with a crystallized protein precipitant (salting out method), extraction, ultrasonic disruption, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, HPLC or a combination thereof may be used, and the target substance, specifically, L-amino acid, may be recovered from the medium or microorganism using a suitable method known in the art.

Additionally, the L-amino acid production method of the present application may additionally include a purification step. The purification may be performed using any suitable method known in the art. In one example, if the L-amino acid production method of the present application includes both a recovery step and a purification step, the recovery step and the purification step may be performed sequentially or discontinuously, regardless of order, or may be performed simultaneously or integrated into a single step, but is not limited thereto.

In the method of the present application, the mutant polypeptide, the introduction and L-amino acid or derivative thereof, etc. are as described in the other embodiments above.

Another aspect of the present application provides a composition for producing L-amino acids, comprising at least one selected from the group consisting of: a variant polypeptide of the present application; a polynucleotide encoding the variant polypeptide; a vector comprising the same; a microorganism comprising the vector; a culture of the microorganism; and a fermented product of the microorganism.

The composition of the present application may further comprise any suitable excipient commonly used in compositions for producing L-amino acids, and such excipients may be, for example, but are not limited to, preservatives, wetting agents, dispersing agents, suspending agents, buffering agents, stabilizers, or isotonic agents.

In one specific example, each component present in the composition of the present application may be included in a microbiologically effective amount, or an amount that can be suitably present in the composition for production.

In the composition of the present application, the mutant polypeptide, introduction and L-amino acid, etc. are as described in the other embodiments above.

Another aspect of the present application provides a use for producing L-amino acid or a derivative thereof by a microorganism having improved L-amino acid production ability, wherein at least one selected from the variant polypeptide of the present application and a polynucleotide encoding the variant polypeptide is introduced.

In the purposes of the present application, the mutant polypeptide, introduction and L-amino acid, etc. are as described in the other embodiments above.

Another aspect of the present application provides a method for producing a microorganism with improved L-amino acid production ability, comprising the step of introducing at least one selected from the mutant polypeptide of the present application and a polynucleotide encoding the mutant polypeptide into a Corynebacterium microorganism having L-amino acid production ability.

Another aspect of the present application provides a method for increasing L-amino acid production capacity, comprising the step of introducing at least one selected from the group consisting of a variant polypeptide of the present application and a polynucleotide encoding the variant polypeptide into a Corynebacterium genus microorganism having L-amino acid production capacity.

In the method of the present application, the mutant polypeptide, introduction and L-amino acid, etc. are as described in the other embodiments above.

Hereinafter, this application will be described in more detail through examples and experimental examples. However, these examples and experimental examples are intended to exemplify this application and the scope of this application is not limited to these examples and experimental examples.

Example 1: Construction of a recombinant vector for introducing exogenous nicotinamide nucleotide transhydrogenase (pntAB).

Example 1-1: Construction of plasmids for gene insertion

To insert a foreign nicotinamide nucleotide transhydrogenase (pntAB) gene into the Corynebacterium glutamicum chromosome, NCgl1287, known as a gene encoding a transposon, was used as an insertion site (J Bacteriol. 2011 Mar; 193(5): 1237-1249). To replace the NCgl1287 gene with the foreign pntAB, NCgl1287 deletion and target gene insertion vectors were constructed. To construct the vectors, PCR was performed using the chromosome of Corynebacterium glutamicum ATCC13032 as a template and primer pairs of SEQ ID NO: 1 and SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, respectively. The primer sequences used to perform each PCR are shown in Table 1 below.

PfuUltra ^™ high-fidelity DNA polymerase (Stratagene) was used as the polymerase for the PCR reaction. The PCR conditions were denaturation at 95°C for 30 seconds; denaturation at 55°C for 30 seconds; and polymerization at 72°C for 1 minute. These denaturation, annealing, and polymerization cycles were repeated 28 times. As a result, DNA fragments of 720 bp and 854 bp were obtained, respectively. The obtained DNA products were purified using a PCR purification kit (PCR Purification kit, QUIAGEN). The purified amplified product and the pDC24 vector (SEQ ID NO: 16), which was heat-treated at 65°C for 20 minutes after treatment with the restriction enzyme smaI, were cloned using the Infusion Cloning Kit (TaKaRa) according to the provided manual to construct the vector pDC24△N1287 (13032) for NCgl1287 deletion and target gene insertion.

Sequence number designation order 1 Primer AATTCGAGCTCGGTACCCAATGGAGCTGAAAGAAT 2 Primer CTTCCTGATAGTCCCGGGACATTGTTTTTC 3 Primer GAAAAAACAATGTCCCGGGACTATCAGGAAG 4 Primer GTCGACTCTAGAGGATCCCCATAAAAACAGGCAGAGGA

Example 1-2: Construction of a plasmid for pntAB introduction

To further enhance the activity of the introduced pntAB, a plasmid was constructed in which the endogenous promoter of the introduced pntAB gene was replaced with PgapA.

First, using the chromosomal DNA of Corynebacterium glutamicum 13032 as a template, PCR was performed using the primer pair of SEQ ID NO: 5 and SEQ ID NO: 6 to obtain a PgapA promoter fragment. In addition, the amino acid sequence of PntAB derived from Corynebacterium variabile and the base sequence encoding it were obtained from the National Institutes of Health (NIH GenBank) in the United States, and based on this, the pntAB gene derived from Corynebacterium variabile was synthesized using the gene synthesis service of Cosmogenetech. PCR was performed using the synthesized pntAB gene as a template and the primers of SEQ ID NO: 7 and SEQ ID NO: 8, respectively. The primer sequences used to perform each PCR are as shown in Table 2 below.

Sequence number designation order 5 Primer CCTGAAAAACAATGTCCCAACGACCGAGCCTATTG 6 Primer GTTGTGTCTCCTCTAAAGAT 7 Primer TCTTTAGAGGAGACACAACATGCTTATTGGTATTCCGAG 8 Primer AACCAACTTCCTGATAGTCCCCTACAGGTGGGGAGATGATCT

PfuUltra ^™ high-fidelity DNA polymerase (Stratagene) was used as the polymerase for the PCR reaction, and the PCR conditions were denaturation at 95°C for 30 seconds; denaturation at 55°C for 30 seconds; and polymerization at 72°C for 1 minute. These denaturation, annealing, and polymerization reactions were repeated 28 times. As a result, a 472-bp DNA fragment of the PgapA promoter region and a 3,041-bp DNA fragment of the pntAB gene of Corynebacterium variabilis were obtained, respectively.

PCR was performed using the amplified promoter and the pntAB DNA fragment of Corynebacterium variabilis as templates with the primer pairs SEQ ID NO: 5 and SEQ ID NO: 8. PCR was performed under the following conditions: denaturation at 95°C for 5 minutes, followed by 28 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 2 minutes, followed by polymerization at 72°C for 5 minutes.

As a result, a pntAB DNA fragment of approximately 3.5 Kb encoding the nicotinamide nucleotide transhydrogenase of Corynebacterium variabilis linked to the PgapA promoter was amplified.

The amplified product was purified using a PCR purification kit (PCR Purification kit, QUIAGEN) and used as an insert DNA fragment for vector construction. The molar concentration (M) ratio of the purified amplified product and the pDC24△N1287(13032) vector, which was treated with the restriction enzyme smaI and heat-treated at 65°C for 20 minutes, was made 2:1, and the vector pDC24△N1287(13032)::PgapA_pntAB was constructed to introduce the pntAB gene of Corynebacterium variabilis into the Corynebacterium glutamicum chromosome using an Infusion Cloning Kit (TaKaRa) according to the provided manual.

Example 2: Production of a strain introducing pntAB random mutations

Example 2-1: Construction of a library plasmid for introducing pntAB random mutations

Error-prone PCR was performed using a diversify PCR random mutagenesis kit (Takara) to induce random mutagenesis in the wild-type pntAB (C.va) gene encoding the nicotinamide nucleotide transhydrogenase protein. To select the mutation rate condition, error-prone PCR was performed under two conditions according to the amount of MnSO4 added, as shown below. pDC24△N1287(13032)::PgapA_pntAB was used as the DNA template to introduce mutations, and the primer pairs of SEQ ID NO: 7 and SEQ ID NO: 8 were used as primers to perform PCR. PCR was performed under the following conditions: denaturation at 95°C for 30 seconds, 25 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, polymerization at 68°C for 2 minutes, and then polymerization at 68°C for 3 minutes.

The composition of the composition for performing error-inducing PCR was as shown in Table 3 below.

case # case #1 (MnSO ₄ 1 μl) case #2 (MnSO ₄ 2 μl) 10X Titanium taq Buffer 5 5 MnSO ₄ (8mM) 1 2 dGTP (2 mM) 1 1 50 X dNTP Mix 1 1 Titanium Taq Polymerase 1 1 Forward primer (5 pmol) 2 2 Reverse primer (5 pmol) 2 2 Template DNA 1 1 dH ₂ O 36 35 Total 50 50

The error-induced PCR product obtained above was treated with DpnI to remove the template plasmid, and the DNA was cloned into the pDC24△N1287(13032) vector cut with the SmaI restriction enzyme using the Gibson assembly method to obtain a recombinant mutant plasmid library pDC24△N1287(13032)::PgapA_pntAB(mt).

Example 2-2: Screening of pntAB random mutant library

The pntAB mutant library plasmid produced in the above Example 2-1 and the control group pDC24△N1287(13032)::PgapA_pntAB were transformed into the threonine-producing strain Corynebacterium glutamicum KCCM 12502P (CA09-0903, Republic of Korea Patent No. 10-2126951), and the threonine production was analyzed.

Specifically, the control groups CA09-0903:: △N1287::PgapA_pntAB((WT)) and CA09-0903:: △N1287::PgapA_pntAB((mt)) libraries were each inoculated into a 96-Deep Well Plate-Dome (Bioneer) containing 400 μl of seed medium and cultured in a plate shaking incubator (TAITEC) at 32°C and 12,000 rpm for approximately 120 hr.

The threonine concentrations of approximately 3,000 cultured strains were individually confirmed through NIR, and pntAB mutations were confirmed through sequencing in six strains with increased threonine concentrations compared to the wild-type pntAB expression strain. As a result, the strains expressed PntAB with the mutant sequences of PntA(V437A)B, PntA(S476P)B(D14V), PntA(I306S)B, PntA(N41I)B, PntAB(E119G), or PntA(L342S, I486T)B(L199P, S346T), respectively.

Example 3: Production of an L-amino acid producing strain with the mutant pntAB introduced and evaluation of amino acid production capacity.

Example 3-1: Production of an L-threonine-producing strain with a mutant pntAB and evaluation of threonine production capacity.

To confirm the threonine productivity of the six strains screened in Example 2-2 above at the flask level, they were cultured using the following method.

First, each strain was inoculated into a 250 ml corner-baffle flask containing 25 ml of seed medium and cultured at 30°C for 20 hours with shaking at 200 rpm. Then, 1 ml of the seed culture was inoculated into a 250 ml corner-baffle flask containing 24 ml of production medium and cultured at 32°C for 24 hours with shaking at 200 rpm.

<Seed medium (pH 7.0)>

Glucose 20 g, peptone 10 g, yeast extract 5 g, urea 1.5 g, KH2PO4 4 g, K2HPO4 8 g, MgSO4 7H2O 0.5 g, biotin 100 μg, thiamine HCl 1000 μg, calcium-pantothenic acid 2000 μg, nicotinamide 2000 μg (based on 1 liter of distilled water)

<Production medium (pH 7.2)>

Glucose 30g, KH2PO4 2g, Urea 3g, (NH4)2SO4 40g, Peptone 2.5g, CSL(Sigma) 5g(10 ml), MgSO4.7H2O 0.5g, Leucine 400mg, CaCO3 20g (based on 1 liter of distilled water)

After the culture was completed, the amount of L-threonine produced was measured using high-performance liquid chromatography (HPLC), and the concentration is shown in Table 4 below.

Threonine production capacity of microorganisms expressing PntAB mutants Strain name L-threonine concentration (g/L) Percentage of concentration compared to wild-type pntAB strain (%) CA09-0903 △N1287::PgapA_pntAB 5.6 100 CA09-0903 △N1287::PgapA_pntA(V437A)B 5.8 104 CA09-0903 △N1287::PgapA_pntA(S476P)B(D14V) 5.3 95 CA09-0903 △N1287::PgapA_pntA(I306S)B 5.0 89 CA09-0903 △N1287::PgapA_pntA(N41I)B 5.2 93 CA09-0903 △N1287::PgapA_pntA(E119G) 4.6 82 CA09-0903 △N1287::PgapA_pntA(L342S, I486T)B(L199P, S346T) 4.9 88

As a result, as shown in Table 4 above, it was confirmed that the parent strain, Corynebacterium glutamicum CA09-0903 △N1287::PgapA_pntAB, produced about 5.6 g/L of threonine. Among the strains selected in Example 2-2, the threonine production ability of CA09-0903 △N1287::PgapA_pntA(V437A)B, a strain expressing PntAB (V437A), was confirmed to increase by 4% compared to the control group.

The above results suggest that a strain expressing pntA (V437A) with a modification of the 437th amino acid of PntA in the amino acid sequence of PntAB derived from Corynebacterium variabile, for example, valine changed to alanine, can produce L-amino acids more efficiently by increasing pntAB activity than the wild type.

From the above description, those skilled in the art will understand that the present application can be implemented in other specific forms without altering its technical concept or essential characteristics. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of this application should be interpreted to include all changes or modifications derived from the meaning and scope of the following claims and their equivalents, rather than the detailed description above.

Claims (15)

A nicotinamide nucleotide transhydrogenase alpha subunit mutant polypeptide in which the amino acid corresponding to position 437 of sequence number 10 is substituted with another amino acid. In claim 1, the mutant polypeptide is a mutant polypeptide in which the amino acid corresponding to position 437 of SEQ ID NO: 10 is substituted with alanine. A mutant polypeptide according to claim 1, wherein the amino acid sequence of the mutant polypeptide has at least 90% identity with the amino acid sequence of SEQ ID NO: 10. A mutant polypeptide according to claim 1, wherein the mutant polypeptide comprises an amino acid sequence of SEQ ID NO: 14. A nicotinamide nucleotide transhydrogenase comprising a mutant polypeptide according to any one of claims 1 to 4. A polynucleotide encoding a mutant polypeptide of any one of claims 1 to 4. A microorganism comprising a mutant polypeptide according to any one of claims 1 to 4; and at least one polynucleotide encoding the mutant polypeptide. In claim 7, the microorganism is a microorganism of the genus Corynebacterium. In claim 8, the microorganism of the genus Corynebacterium is Corynebacterium glutamicum. In claim 7, the microorganism has an increased ability to produce L-amino acids compared to a microorganism comprising a polypeptide of sequence number 10 or a polynucleotide encoding the same. A microorganism in claim 10, wherein the L-amino acid is L-threonine. A method for producing L-amino acid, comprising a step of culturing the microorganism of claim 7 in a medium. A method according to claim 12, further comprising a step of recovering L-amino acid from the cultured microorganism; a culture of the microorganism; a fermented product of the microorganism; or the culture medium. A method according to claim 13, wherein the L-amino acid is L-threonine. A use of a microorganism for producing L-amino acids, comprising a variant polypeptide according to any one of claims 1 to 4; or a polynucleotide encoding the variant polypeptide or the variant polypeptide.