RU2793442C1 - New option of asparagine synthase and a method for obtaining l-valine using it - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: polypeptide involved in the production of L-valine is proposed, it consists of the amino acid sequence represented by SEQ ID NO: 1 in which proline being the amino acid corresponding to position 192 of SEQ ID NO: 3, replaced by leucine. Also the following are proposed: a polynucleotide encoding the said polypeptide, Corynebacterium glutamicum producing L-valine and containing the said polypeptide or polynucleotide encoding the said polypeptide, and a method for producing of L-valine.

EFFECT: increasing the yield of L-valine.

5 cl, 1 dwg, 3 tbl, 3 ex

Description

Cross reference to related inventions

This application claims priority from Korean Patent Application No. 10-2021-0011011, filed January 26, 2021, the full disclosure of which is incorporated herein as part of this disclosure.

The present disclosure relates to a new variant of asparagine synthase, a strain of Corynebacterium glutamicum containing this variant, and to a method for producing L-valine using this strain.

PRIOR ART

Conduct various research to develop highly efficient microorganisms and fermentation process technologies for the production of L-amino acids and other beneficial substances. For example, a target-substance-specific approach is generally used in which the expression of a gene encoding an enzyme involved in L-valine biosynthesis is increased, or in which genes not required for biosynthesis are removed (US 8465962 BT).

However, research is still needed to effectively increase the L-valine producing capacity as the demand for L-valine increases.

DESCRIPTION OF THE INVENTION

Technical problem

The purpose of the present disclosure is to provide a variant of asparagine synthase consisting of the amino acid sequence shown in SEQ ID NO: 1, in which proline (Pro, P), which is the amino acid corresponding to position 192 of the amino acid sequence of SEQ ID NO: 3, is replaced by leucine (Leu, L).

Another object of the present disclosure is to provide a polynucleotide encoding a variant of the present disclosure.

Yet another object of the present disclosure is to provide a strain of Corynebacterium glutamicum that contains a variant of the present disclosure, or a polynucleotide encoding the variant, and has the ability to produce L-valine.

Yet another object of the present disclosure is to provide a method for producing L-valine, which comprises culturing in a medium a strain of Corynebacterium glutamicum that contains a variant of the present disclosure or a polynucleotide encoding the variant and has the ability to produce L-valine.

The best way to implement the invention

The present disclosure will be described in detail as follows. In the meantime, each of the descriptions and embodiments disclosed in this disclosure may apply to other descriptions and embodiments. In other words, all combinations of different elements disclosed in the present disclosure are within the scope of the present disclosure. In addition, the scope of the present disclosure should not be considered to be limited by the specific description provided below. In addition, a number of articles and patent documents are referenced and cited throughout the present specification. The entire content disclosed in the cited articles and patent documents is hereby incorporated by reference in order to more clearly describe the level of the technical field to which the present invention belongs and the content of the present invention.

According to one aspect of the present disclosure, a variant of asparagine synthase is provided, consisting of the amino acid sequence shown in SEQ ID NO: 1, in which proline (Pro, P), which is the amino acid corresponding to position 192 of the amino acid sequence of SEQ ID NO: 3, is replaced by leucine (Leu , L).

The variant of the present disclosure may have, contain, or essentially consist of the amino acid sequence represented by SEQ ID NO: 1.

In a variant of the present disclosure, the amino acid corresponding to position 192 based on the amino acid sequence of SEQ ID NO: 3 in the amino acid sequence shown by SEQ ID NO: 1 is leucine, and the variant may contain an amino acid sequence having at least 70% , 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% 99.7% or 99.9% or greater homology or identity to the amino acid sequence represented by SEQ ID NO: 1. Obviously, variants having amino acid sequences in which some sequences are deleted, modified, substituted, conservatively substituted or added are also included within the scope of this disclosure, provided that the amino acid sequences have such homology or identity and exhibit potency corresponding to that of the variant of the present disclosure.

Examples thereof include variants having a sequence addition or deletion that does not change the function of the variant of the present disclosure, at the N-terminus, C-terminus of an amino acid sequence and/or within that amino acid sequence, a naturally occurring mutation, a silent mutation, or a conservative substitution.

The term "conservative substitution" means the replacement of one amino acid with another amino acid having similar structural and/or chemical properties. Such an amino acid substitution can typically exist based on similarities in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or amphipathic nature of the residues. Typically, a conservative substitution may or may not affect the activity of proteins or polypeptides.

In the present disclosure, the term "variant" refers to a polypeptide that has an amino acid sequence different from the amino acid sequence of the variant prior to modification by conservative substitution and/or modification of one or more amino acids, but retains functions or properties. Such a variant can usually be identified by modifying one or more amino acids of the amino acid sequence of a given polypeptide and evaluating the properties of that modified polypeptide. In other words, the ability of a given variant can be increased, left unchanged, or reduced compared to the ability of the polypeptide before the change. Some variants may include variants in which one or more than one portion, such as the N-terminal leader sequence or the transmembrane domain, has been deleted. Other variants may include variants in which a portion of the N- and/or C-terminus of the mature protein has been removed. The term "variant" can be used interchangeably with and is not limited to terms such as modification, modified polypeptide, modified protein, mutant, mutein, and divergent, as long as it is a term used with the meaning of variation. For the purposes of this disclosure, this variant may be a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1, in which proline (Pro, P), which is the amino acid corresponding to position 192 of the amino acid sequence of SEQ ID NO: 3, has been replaced by leucine ( Leu, L).

This variant may contain deletions or additions of amino acids that have minimal effect on the properties and secondary structure of the polypeptide. For example, a signal (or leader) sequence that co-translationally or post-translationally participates in protein translocation can be conjugated to the N-terminus of this variant. This variant can be conjugated to other sequences or linkers in such a way as to be identified, purified or synthesized.

In the present disclosure, the term "homology" or "identity" means the degree of similarity between two given amino acid or base sequences, and may be expressed as a percentage. The terms "homology" and "identity" can often be used interchangeably.

Homology or sequence identity of a conserved polynucleotide a or polypeptide is determined by standard alignment algorithms and the default gap penalty set by the application program can be shared. Substantially homologous or identical sequences are generally capable of hybridizing to all or part of the sequence under conditions of moderate to high stringency. Obviously, hybridization also includes the hybridization of polynucleotide a with a polynucleotide containing a common type codon or a degenerate codon.

Whether any two polynucleotide or polypeptide sequences share homology, similarity or identity can be determined using known computer algorithms such as the FASTA program, for example using default parameters as in Pearson et at, (1988) Proc. Natl. Acad. sci. USA 85:2444. Alternatively, homology, similarity, or identity can be determined using the Needleman and Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277) (version 5.0.0 or later) (including GCG software package (Devereux, J., et al, Nucleic Acids Research 12: 387 (1984)), BLASTP, BLASTN, FASTA (Atschul, S. F., et al, JMOLEC 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 from the National Center for Biotechnology Information or ClustalW can be used to determine homology, similarity, or identity.

Homology, similarity, or identity of polynucleotides or polypeptides can be determined by comparing sequence information using, for example, the GAP computer program, such as Needleman et al. (1970), J Mol Biol. 48:443, as announced, for example, in Smith and Waterman, Adv. Appl. Math (1981) 2:482. Finally, the result of the GAP program can be defined as the value obtained by dividing the number of similar aligned characters (namely, nucleotides or amino acids) by the total number of characters in the shorter of the two sequences. The default parameters of the GAP program may include (1) a binary comparison matrix (including values of 1 for identity and 0 for non-identity) and a weighted comparison matrix of Gribskov et al, (1986) Nucl. Acids Res. 14:6745 (or the EDNAFULL substitution matrix (EMBOSS version NCBI NUC4.4)), as disclosed in Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp.353-358 (1979); (2) a penalty of 3.0 for each gap and an additional penalty of 0.10 for each character in each gap (or a penalty of 10 for opening a gap, a penalty of 0.5 for extending a gap); and (3) no end gap penalty.

As an example of the present disclosure, a variant of the present disclosure may exhibit asparagine synthase activity. A variant of the present disclosure may exhibit activity such that it has an increased ability to form L-valine compared to a wild-type polypeptide.

In the present disclosure, the term "asparagine synthase" is a polypeptide that may have glutamine hydrolyzing activity and convert aspartate and glutamine to asparagine. The term "asparagine synthase" of the present disclosure may be used interchangeably with "asparagine synthesis enzyme", "asparagine synthase (eg Asparagine synthase B)", "glutamine-dependent asparagine synthase or LtsA". In the present disclosure, the sequence of asparagine synthase can be obtained from GenBank NCBI, a known database (eg, WP_003859651.1, WP_060564818.1, WP_040967635.1, etc.). In particular, it may be a polypeptide showing asparagine synthase activity encoded by the ItsA gene, but is not limited thereto.

In the present disclosure, the term "corresponding" refers to amino acid residues at the positions listed in the polypeptide, or to amino acid residues that are similar, identical or homologous to the residues listed in this polypeptide. The identification of an amino acid at an appropriate position may be determined by the specific amino acid in the sequence that refers to the specific sequence. The term "corresponding region" as used here, usually refers to a similar or corresponding position in a related protein or reference protein.

For example, an arbitrary amino acid sequence is aligned with SEQ ID NO: 3, and based on this, each amino acid residue of that amino acid sequence can be numbered with respect to the amino acid residue of SEQ ID NO: 3 and the numerical position of the corresponding amino acid residue. For example, a sequence alignment algorithm as described herein can determine the position of an amino acid or the position at which a modification occurs, such as a substitution, insertion, or deletion, by comparison with a position in a requested sequence (also referred to as a "reference sequence").

For such alignments, 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) and the like, but the program and algorithm are not limited thereto, and a sequence alignment program, a pairwise sequence comparison algorithm, and the like known in the art may be suitably used.

Another aspect of the present disclosure is to provide a polynucleotide a encoding a variant of the present disclosure.

In the present disclosure, the term "polynucleotide" is a strand of DNA or RNA, having a certain or greater length, as a polymer of nucleotides, in which nucleotide monomers are joined into a long chain by covalent bonds, and, more specifically, means a polynucleotide fragment a encoding a given variant.

A polynucleotide encoding a variant of the present disclosure may comprise a base sequence encoding the amino acid sequence represented by SEQ ID NO: 1. As an example of the present disclosure, a polynucleotide of the present disclosure may have or contain the sequence of SEQ ID NO: 2. A polynucleotide of the present disclosure may consist or essentially consist of the sequence of SEQ ID NO: 2.

In another embodiment, in a polynucleotide of the present disclosure, the base corresponding to position 575 based on the nucleic acid sequence of SEQ ID NO: 4 in the nucleic acid sequence represented by SEQ ID NO: 2 is T, and the polynucleotide may comprise a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%- 99.5%, 99.7% or 99.9% or greater homology or identity with the nucleic acid sequence represented by SEQ ID NO: 2. Obviously, polynucleotides having amino acid sequences in which certain sequences deleted, modified, substituted, conservatively substituted, or added are also included within the scope of this disclosure, provided that the nucleic acid sequences have such homology or identity and encode a polypeptide or protein exhibiting an efficacy corresponding to that of the variant of the present disclosure.

In a polynucleotide of the present disclosure, various modifications can be made in the coding region, provided that the amino acid sequence of the variant of the present disclosure is not altered when considering the degeneracy of the preferred codons in the organisms that are intended to express the variant of the present disclosure. In particular, the polynucleotide of the present disclosure has or contains a base sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95 % or greater, 96% or greater, 97% or greater, 98% or greater, but less than 100% homology or identity with the sequence of SEQ ID NO: 2 or may consist of or essentially consists of a base sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96 % or greater, 97% or greater, 98% or greater, but less than 100% homology or identity with the sequence of SEQ ID NO: 2, but not limited to it. Here, in a sequence having homology or identity, the codon encoding the amino acid corresponding to position 192 in SEQ ID NO: 1 may be one of the codons encoding leucine.

The polynucleotide of the present disclosure may comprise a probe that can be derived from a sequence of a known gene, such as a sequence without limitation, provided that it is a sequence that can hybridize to a complementary sequence of all or part of the sequence of the polynucleotide a of the present disclosure under stringent conditions. "Stringent conditions" means conditions that allow for specific hybridization between polynucleotides. These conditions are specifically described in documents (see J. Sambrook et al, Molecular Cloning, A Laboratory Manual, ^2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989; FM Ausubel et al, Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, 9.50-9.51, 11.7-11.8). Examples thereof include conditions under which polynucleotides having higher homology or identity, namely: polynucleotides having 70% or more, 75% or more, 80% or more, 85% or more, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater homology or identity, hybridize with each other , while polynucleotides having less homology or identity do not hybridize with each other, or conditions under which washing is carried out once, in particular two to three times at a salt concentration and temperature equivalent to 60 ° C, 1 × SSC ( citrate and sodium chloride solution), 0.1% SDS (sodium dodecyl sulfate), in particular at 60°C, 0.1 × SSC, 0.1% SDS, more specifically at 68°C, 0.1 × SSC , 0.1% SDS, which are wash conditions for conventional Southern hybridization.

Hybridization requires that the two nucleic acids have complementary sequences, although mismatches between bases are tolerated, depending on the stringency of the hybridization. The term "complementary" is used to describe the relationship between nucleotide bases capable of hybridizing with each other. For example, in relation to DNA, adenine is complementary to thymine and cytosine is complementary to guanine. Therefore, a polynucleotide of the present disclosure may also contain substantially similar nucleic acid sequences, as well as isolated nucleic acid fragments that are complementary to the entire sequence.

In particular, a polynucleotide having homology or identity with a polynucleotide of the present disclosure can be detected using hybridization conditions, including a hybridization step at a T _m value of 55°C and the conditions described above. The T _m value may be 60° C., 63° C. or 65° C., but is not limited to, and may be appropriately adjusted by those skilled in the art according to purpose.

The appropriate stringency for hybridization of a polynucleotide depends on the length and degree of complementarity of the given polynucleotide, and the variables are well known in the art (e.g., J. Sambrook et al, Molecular Cloning, A Laboratory Manual, ^2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989).

Another aspect of the present disclosure is the provision of a vector containing the polynucleotide of the present disclosure. This vector may be an expression vector for carrying out the expression of a polynucleotide in a host cell, but is not limited to it.

The vector of the present disclosure may include a DNA construct comprising a polynucleotide sequence encoding a polypeptide of interest operably linked to a suitable expression control region (or expression control sequence) such that the polypeptide of interest can be expressed in a suitable host. The regulatory region of expression may comprise a promoter capable of initiating transcription, any operator sequence for regulating transcription, a sequence encoding a suitable mRNA binding site for the ribosome, and a sequence regulating transcription and translation termination. This vector can be transformed into a suitable host cell and then can replicate or function independently of the host genome, or can integrate itself into the genome.

The vector used in the present disclosure is not specifically limited, but any vector known in the art can be used. Examples of commonly used vectors include natural or recombinant plasmids, cosmids, viruses, and bacteriophages. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, Charon21A and the like can be used as the phage vector or cosmid vector, and the pDZ system, pBR system, pUC, pBluescript II system, pGEM system, pTZ system, pCL system, pET system, and the like. In particular, pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118 and pCC1BAC vectors, and the like can be used.

For example, a polynucleotide encoding a polypeptide of interest can be inserted into a chromosome via an intracellular chromosomal insertion vector. Insertion of a given polynucleotide into a chromosome can be accomplished by any method known in the art, such as, but not limited to, homologous recombination. This vector may further contain a selectable marker to confirm the insertion into the chromosome. This selectable marker serves to select cells transformed with vectors, i.e. to confirm the insertion of a nucleic acid molecule of interest, and markers that confer selectable phenotypes such as drug resistance, auxotrophy, resistance to cytotoxic agents, or expression of surface polypeptides can be used. In the medium treated with the selective agent, only cells expressing the selectable marker survive or exhibit other phenotypic characteristics, and thus transformed cells can be selected.

In the present disclosure, the term "transformation" means that a vector containing a polynucleotide encoding a target polypeptide is introduced into a host cell or microorganism such that the polypeptide encoded by the polynucleotide can be expressed in the host cell. The transformed polynucleotide may be localized by insertion into the chromosome of the host cell, or localized off the chromosome, so long as it can be expressed in the host cell. This polynucleotide contains DNA and/or RNA encoding the polypeptide of interest. This polynucleotide can be inserted in any form, provided that it can be introduced into the host cell and can be expressed. For example, a given polynucleotide can be introduced into a host cell as an expression cassette, which is a genetic construct containing all the elements required for autonomous expression. This expression cassette typically may contain a promoter operably linked to the polynucleotide, a transcription termination signal, a ribosome binding site, and a translation termination signal. This expression cassette may be in the form of an expression vector capable of autonomous replication. A polynucleotide can be introduced into a host cell by itself and is operably linked to a sequence required for expression in the host cell, but is not limited to.

As used above, "operably linked" means that a polynucleotide sequence is operably linked to a promoter sequence that initiates and mediates transcription of a polynucleotide encoding a variant of interest according to the present disclosure.

Yet another aspect of the present disclosure is to provide a strain of Corynebacterium glutamicum that contains a variant of the present disclosure or a polynucleotide of the present disclosure.

The strain of the present disclosure may contain a modified polypeptide of the present disclosure, a polynucleotide encoding the polypeptide, or a vector containing the polynucleotide of the present disclosure.

In the present disclosure, a "strain (or microorganism)" includes all wild-type or naturally or artificially genetically modified microorganisms, and may be a microorganism in which a specific mechanism is attenuated or enhanced due to the insertion of an external gene or enhancement of activity, or inactivation of an endogenous a gene, and it may be a microorganism containing a genetic modification to produce a polypeptide, protein or product of interest.

The strain of the present disclosure may be a strain containing any one or more variants of the present disclosure, a polynucleotide of the present disclosure, or a vector containing a polynucleotide of the present disclosure; a strain modified to express a variant of the present disclosure or a polynucleotide of the present disclosure; a strain (eg, a recombinant strain) expressing a variant of the present disclosure or a polynucleotide of the present disclosure; or a strain (eg, a recombinant strain) that exhibits, but is not limited to, activity of the variant of the present disclosure.

The strain of the present disclosure may be a strain having the ability to produce L-valine.

The strain of the present disclosure may be a microorganism having naturally asparagine synthase activity and/or the ability to produce L-valine, or a microorganism in which a variant of the present disclosure or a polynucleotide encoding the variant (or a vector containing the polynucleotide) is introduced into a parent strain that does not have asparagine synthase activity or L-valine producing ability, and/or in which the L-valine producing ability is conferred to, but is not limited to, the parent strain.

For example, the strain of the present disclosure is a cell or microorganism that has been transformed with a vector containing a polynucleotide of the present disclosure or a polynucleotide encoding a variant of the present disclosure and expresses the variant of the present disclosure. For the purposes of the present disclosure, the strain of the present disclosure may include all microorganisms that contain the variant of the present disclosure and can produce L-valine. For example, the strain of the present disclosure may be a recombinant strain in which a polynucleotide encoding a variant of the present disclosure is introduced into a natural wild type microorganism or an L-valine producing microorganism, in order to thereby express an asparagine synthase variant and have an increased the ability to produce L-valine. The recombinant strain having an increased ability to produce L-valine may be a microorganism having an increased ability to produce L-valine compared to a natural wild-type microorganism or a microorganism not modified for asparagine synthase (namely, a microorganism expressing wild-type asparagine synthase ( SEQ ID NO: 3), or a microorganism that does not express the modified (SEQ ID NO: 1) protein), but is not limited to it. For example, the strain of the present disclosure having an increased ability to produce L-valine may be a microorganism having an increased ability to produce L-valine compared to Corynebaterium glutamicum, which contains a polypeptide of SEQ ID NO: 3 or a polynucleotide encoding this polypeptide, but not limited to them. As an example, an asparagine synthase-unmodified microorganism that is a target strain for comparing the increase in L-valine producing ability may be, but is not limited to, ATCC14067 strain and/or Corynebaterium glutamicum strain CA08-0072 (KCCM1120 IP).

For example, a recombinant strain having an increased production capacity may have an L-valine production capacity increased by about 1% or more, by about 2.5% or more, by about 5% or more, by 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, about 14.5%, or more, about 14.6% or more, about 14.7% or more, about 14.8% or more, about 14.9% or more, about 15% or more, about 15.1 % (the upper limit is not particularly limited and may 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, about 25% or less, or about 20% or less) as compared to the L-valine-producing ability of the parent strain before modification or with the unmodified microorganism, but the increased value is not limited to it, when provided that the ability to produce has an increased value plus values compared to the ability to produce the parental strain before the change or with the unmodified microorganism. In another example, a recombinant strain having an increased production capacity may have an L-valine production capacity increased by about 1.1 times or more, about 1.12 times or more, about 1.13 times or more, about 1.15 times or more (the upper limit is not particularly limited and may, for example, be about 10 times or less, about 5 times or less, about 3 times or less, or about 2 times or less) compared to with the ability to produce L-valine of the parent strain before the change or with an unmodified microorganism.

More specifically, a recombinant strain having an increased production capacity may have an L-valine production capacity increased by about 15% (or about 1.15 times) compared to the L-valine production capacity of the parental strain before or after the change. unmodified microorganism, but the increase rate is not limited to it. The term "about" is an interval including all of plus/minus 0.5; plus/minus 0.4; plus/minus 0.3; plus/minus 0.2; plus/minus 0.1 and the like, and includes all values in the range equal to or similar to the value after the term "about", but is not limited to them.

In the present disclosure, "non-modified microorganism" does not exclude strains containing a mutation that may occur in microorganisms in nature, and may be a wild-type strain or a natural strain itself, or may be a strain before a trait is changed through genetic variation due to natural or artificial factors. For example, the unmodified microorganism may be a strain that has not or has not yet been introduced the protein variant described herein. The term "unmodified microorganism" can be used interchangeably with the phrases "pre-modification strain", "pre-modification microorganism", "unchanged strain", "unmodified strain", "unchanged microorganism", or "reference microorganism".

В другом примере настоящего раскрытия микроорганизм по настоящему раскрытию может представлять собой Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium effwiens, Corynebacterium callunae, Corynebacterium stationis, Corynebacterium singulare, Corynebacterium halotolerans, Corynebacterium striatum, Corynebacterium ammoniagenes, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium testudinoris or Corynebacterium flavescens.

In the present disclosure, "weakening" of the activity of the polypeptide includes both cases where the activity is reduced compared to endogenous activity or absent. The term "attenuation" can be used interchangeably with terms such as inactivation, insufficiency, down regulation, reduction, reduction and attenuation.

Attenuation may also include the case where the activity of the polypeptide itself is reduced or eliminated compared to the activity of the polypeptide originally possessed by the microorganism by changing the polynucleotide encoding the polypeptide and the like, the case where the overall level of activity and/or concentration (expression level) of the polypeptide in the cell is lower than the level of activity or concentration of the natural strain by inhibiting the expression of the gene of the polynucleotide encoding the polypeptide or inhibiting translation into the polypeptide, the case where the polynucleotide is not expressed at all, and/or the case where the activity of the polypeptide is not shown even when expressed polynucleotide. The term "endogenous activity" means the activity of a particular polypeptide, which was originally possessed by the parental strain before changing the trait, or a wild-type microorganism, or an unmodified microorganism when the trait is changed by genetic variation due to natural or artificial factors. The phrase "endogenous activity" can be used interchangeably with the phrase "activity before modification". The fact that the activity of a polypeptide is "inactivated, deficient, reduced, down-regulated, reduced or attenuated" compared to endogenous activity means that the activity of the polypeptide is reduced relative to the activity of the particular polypeptide that the parent strain originally had before the trait change or a wild-type microorganism, or an unmodified microorganism.

Such weakening of the activity of the polypeptide can be carried out by any method known in this field, but this method is not limited to them, and weakening can be achieved using various methods well known in this field (for example, Nakashima N. et al, Bacterial cellular engineering by genome editing and gene silencing Int J Mol Sci 2014; 15(2): 2773-2793, Sambrook et al, Molecular Cloning 2012, et al.).

In particular, the weakening of the activity of the polypeptide in the present disclosure may be:

1) deletion of all or part of the gene encoding the polypeptide;

2) modification of the regulatory region of expression (or regulatory sequence of expression) to reduce the expression of the gene encoding the polypeptide;

3) modifying the amino acid sequence constituting the polypeptide to eliminate or reduce the activity of the polypeptide (eg, deletion/substitution/addition of one or more amino acids in the amino acid sequence);

4) modification of the sequence of the gene encoding the polypeptide to eliminate or weaken the activity of this polypeptide (for example, deletion/substitution/addition of one or more nucleic acid bases in the nucleic acid base sequence of the polypeptide gene to encode a polypeptide that has been modified to eliminate or weaken activity of the given polypeptide);

5) modification of the initiating codon of the transcript of the gene encoding the polypeptide, or the base sequence encoding the 5'-UTR (5'-untranslated region) region;

6) introducing an antisense oligonucleotide (eg, antisense RNA) that binds complementarily to the transcript of the gene encoding the polypeptide;

7) adding a sequence complementary to the Shine-Dalgarno sequence before the Shine-Dalgarno sequence of the gene encoding the polypeptide in order to form a secondary structure to which the ribosome cannot attach;

8) adding a promoter to be transcribed in the opposite direction to the 3' end of the open reading frame (ORF) of the gene sequence encoding the polypeptide (reverse transcription engineering, RTE); or

9) a combination of two or more than two selected from (1) (8), but is not particularly limited to them.

For example:

1) The deletion of part or all of the gene encoding the polypeptide may be the removal of the entire polynucleotide encoding the endogenous polypeptide of interest on the chromosome, or replacement with a polynucleotide in which some nucleotides are deleted, or replacement with a marker gene.

2) Modification of the expression regulatory region (or expression regulatory sequence) may be a deletion, insertion, non-conservative or conservative substitution, or the appearance of a change in the expression regulatory region (or expression regulatory sequence) due to their combination, or replacement by a sequence showing weaker activity . The expression regulatory region contains, but is not limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence regulating transcription and translation termination.

3) Modification of the start codon of the transcript of the gene encoding the polypeptide, or the base sequence encoding the 5'-UTR region, may be, for example, replacing the base sequence encoding a different start codon having a lower expression rate of the polypeptide compared to the endogenous start codon, but not limited to her.

4) and 5) A modification of the amino acid sequence or sequence of a polynucleotide can be a deletion, insertion, or a non-conservative or conservative substitution of the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide, or the appearance of a change in sequence due to their combination, or a substitution by an amino acid sequence or sequence a polynucleotide modified to exhibit weaker activity, or an amino acid sequence, or a polynucleotide sequence modified to be inactive such that the activity of the polypeptide is reduced, but is not limited to. For example, gene expression can be inhibited or attenuated by introducing a change in the sequence of a polynucleotide and the formation of a stop codon, but the modification is not limited thereto.

6) To introduce an antisense oligonucleotide (eg, antisense RNA) that binds complementarily to the transcript of the gene encoding the polypeptide, reference can be made to documents such as Weintraub, H. et al, Antisense-RNA as a molecular tool for genetic analysis, Reviews - Trends in Genetics, Vol. 1(1) 1986.

7) Addition of a sequence complementary to the Shine-Dalgarno sequence before the Shine-Dalgarno sequence of the gene encoding the polypeptide in order to form a secondary structure to which the ribosome cannot attach, in order to make it impossible to translate the mRNA or to slow down the rate of translation mRNA.

8) The addition of a promoter to be transcribed in the opposite direction to the 3' end of the open reading frame (ORF) of the sequence of the gene encoding the polypeptide (reverse transcription engineering, RTE) can serve to attenuate the activity by obtaining an antisense nucleotide complementary to the transcript of the gene encoding the polypeptide .

In the present disclosure, the term "enhancement" of the activity of the polypeptide means that the activity of the polypeptide is increased compared to endogenous activity. The term "enhancement" can be used interchangeably with terms such as activation, upregulation, overexpression and increase. Here, activation, amplification, upregulation, overexpression and increase can include both showing an activity that was not originally possessed, and showing an improved activity compared to endogenous activity or activity before modification. The term "endogenous activity" means the activity of a particular polypeptide, which was originally possessed by the parental strain before changing the trait or an unmodified microorganism when the trait is changed through genetic variation due to natural or artificial factors. This can be used interchangeably with "activity before modification". The fact that the activity of a polypeptide is "upregulated", "upregulated", "overexpressed" or "increased" compared to endogenous activity means that the activity of the polypeptide is improved relative to the activity and/or concentration (expression level) of a particular polypeptide, which the parent strain originally possesses before the trait change or unmodified microorganism.

This enhancement can be achieved by introducing a foreign polypeptide or by increasing the endogenous activity and/or concentration (expression level) of the polypeptide. An increase in the activity of a polypeptide can be evidenced by an increase in the degree of activity and level of expression of the polypeptide or the amount of product produced from the polypeptide.

Various methods well known in the art can be used to enhance the activity of a polypeptide, and this method is not limited, provided that the activity of the polypeptide of interest can be enhanced relative to the activity of the microorganism prior to modification. In particular, genetic engineering and/or protein engineering well known to those skilled in the art can be used, which are traditional molecular biology techniques, but this technique is not limited to them (e.g., Sitnicka et al, Functional Analysis of Genes. Advances in Cell Biology 2010, Vol. 2. 1-16; Sambrook et al, Molecular Cloning 2012; and the like).

In particular, enhancing the activity of a polypeptide of the present disclosure may be:

1) an increase in the number of intracellular copies of the polynucleotide encoding the polypeptide;

2) replacing the gene expression regulatory region on the chromosome encoding the polypeptide with a sequence showing strong activity;

3) modification of the initiating codon of the transcript of the gene encoding the polypeptide, or the base sequence encoding the 5'-UTR region;

4) modification of the amino acid sequence of the polypeptide to increase the activity of this polypeptide;

5) modifying a polynucleotide sequence encoding a polypeptide to enhance the activity of that polypeptide (eg, modifying the polynucleotide sequence of a polypeptide gene to encode a polypeptide that has been modified to enhance the activity of that polypeptide);

6) introduction of a foreign polypeptide demonstrating the activity of this polypeptide, or a foreign polynucleotide encoding this polypeptide;

7) codon optimization of the polynucleotide encoding the polypeptide;

8) analysis of the tertiary structure of the polypeptide to select and modify or chemically modify the exposed site; or

9) a combination of two or more than two selected from (1) (8), but not specifically limited to them.

More specific:

1) An increase in the intracellular copy number of a polynucleotide encoding a polypeptide can be achieved by introducing into the host cell a vector that can replicate and function independently of the host and to which the polynucleotide encoding the polypeptide is operably linked. Alternatively, the increase can be achieved by introducing one copy or two or more than two copies of the polynucleotide encoding the polypeptide into the chromosome of the host cell. The introduction into the chromosome can be carried out by the introduction of a vector capable of inserting the polynucleotide into the chromosome of the host cell, into the host cell, but is not limited to. This vector is as described above.

2) Replacement of a gene expression regulatory region (or an expression regulatory sequence) on a chromosome encoding a polypeptide with a sequence exhibiting strong activity may be, for example, a deletion, an insertion, a non-conservative or conservative substitution, or the occurrence of a change in sequence due to a combination thereof , or replacement with a sequence showing stronger activity, such that the activity of the regulatory region of expression is further enhanced. The regulatory region of expression is not specifically limited thereto, but may include a promoter, an operator sequence, a sequence encoding a ribosome binding site, a sequence regulating transcription and translation termination, and the like. For example, the replacement may be, but is not limited to, replacing the original promoter with a strong promoter.

Examples of known strong promoters include CJ1-CJ7 promoters (US 7662943 B2), lac promoter, trp promoter, trc promoter, tac promoter, lambda phage PR promoter, PL promoter, tet promoter, gapA promoter, SPL7 promoter, SPL13 (sm3) promoter ( US 10584338 B2), promoter 02 (US 10273491 B2), tkt promoter and ucsA promoter, but are not limited to them.

3) Modification of the initiation codon of the transcript of the gene encoding the polypeptide, or the base sequence encoding the 5'-UTR region, may be, for example, a replacement with a base sequence encoding a different initiation codon having a higher expression rate of the polypeptide compared to the endogenous initiation codon, but not limited to it.

4) and 5) Modification of the amino acid sequence or polynucleotide sequence can be a deletion, insertion, non-conservative or conservative substitution of the amino acid sequence of the polypeptide or the polynucleotide sequence encoding this polypeptide, or the appearance of a change in this sequence due to their combination or replacement by an amino acid sequence or sequence a polynucleotide modified to exhibit stronger activity, or an amino acid sequence or polynucleotide sequence modified to be more active such that the activity of the polypeptide is enhanced, but is not limited to. This replacement can be specifically carried out by inserting a polynucleotide into the chromosome by homologous recombination, but not limited to it. Used here, the vector may additionally contain a selectable marker to confirm insertion into the chromosome. The selectable marker is as described above.

6) The introduction of a foreign polypeptide showing the activity of the polypeptide may be the introduction into the host cell of a foreign polynucleotide encoding a polypeptide showing the same or similar activity to this polypeptide. This foreign polynucleotide is not limited in its origin or sequence, provided that it exhibits the same or similar activity to this polypeptide. The introduction can be carried out by a suitable choice of transformation method, known to experts in this field. Since the introduced polynucleotide is expressed in the host cell, the polypeptide can be produced and its activity can be increased.

7) Codon optimization of a polynucleotide encoding a polypeptide can be codon optimization of an endogenous polynucleotide so as to increase transcription or translation in the host cell, or codon optimization of a foreign polynucleotide so as to achieve optimized transcription and translation in the host cell.

8) Analysis of the tertiary structure of a polypeptide for selection and modification or chemical modification of an exposed site can be performed, for example, to determine a template candidate protein according to the degree of sequence similarity by comparing the sequence information of the polypeptide to be analyzed with a repository of information on the sequences of known database proteins for confirming the structure based on this and for selecting and modifying or chemically modifying the exposed portion to be modified or chemically modified.

Such an increase in the activity of a polypeptide can be an increase in the activity or concentration or level of expression of the corresponding polypeptide based on the activity or concentration of the polypeptide expressed in the wild-type microbial strain or in the microbial strain before modification, or an increase in the amount of product produced from the polypeptide, but is not limited to. .

In the microorganism of the present disclosure, partial or complete modification (e.g., modification to encode a protein variant as described above) of a polynucleotide can be induced by (a) homologous recombination using a vector to insert into a chromosome in the microorganism, or by editing the genome using a genetically modified nuclease (e.g., CRISPR-Cas9), and/or (b) treatment with light such as ultraviolet rays and radiation, and/or chemical agents, but not limited to. The method for modifying part or all of a gene may include a method using recombinant DNA technology. For example, by introducing into the microorganism a nucleotide sequence or a vector containing a nucleotide sequence homologous to a gene of interest, part or all of the gene can be removed to induce homologous recombination. The introduced nucleotide sequence or vector may contain, but is not limited to, a dominant selectable marker.

In the microorganism of the present disclosure, the variant, polynucleotide, L-valine, and the like are as described in other aspects.

According to still another aspect of the present disclosure, a method for producing an L-amino acid is provided, which comprises culturing in a medium a strain of Corynebacterium glutamicum containing a variant of the present disclosure or a polynucleotide of the present disclosure.

The method for producing an L-amino acid of the present disclosure may include culturing in a medium a strain of Corynebacterium glutamicum containing a variant of the present disclosure or a polynucleotide of the present disclosure or a vector of the present disclosure.

In addition, the L-amino acid of the present disclosure may be L-valine.

In the present disclosure, the term "cultivation" means the cultivation of the Corynebacterium glutamicum strain of the present disclosure under suitably controlled environmental conditions. The culturing method of the present disclosure can be carried out in accordance with a suitable medium and culturing conditions known in the art. Such a culture method can be easily adjusted and used by those skilled in the art according to the selected strain. In particular, the cultivation may be of batch type, continuous type, and/or fed-batch type, but is not limited to them.

In the present disclosure, the term "medium" means a mixed substance containing the nutrients required for cultivating the strain of Corynebacterium glutamicum of the present disclosure as the main component, and this medium supplies nutrients, growth factors and the like, including water, which are indispensable for survival. and development. In particular, as the medium and other culture conditions used for culturing the strain of Corynebacterium glutamicum of the present disclosure, any one can be used without particular limitation, as long as it is a medium used for conventional cultivation of microorganisms. The Corynebacterium glutamicum strain of the present disclosure can be cultivated in an ordinary medium containing proper carbon sources, nitrogen sources, phosphorus sources, inorganic compounds, amino acids and/or vitamins, and the like, while controlling temperature, pH, and the like under aerobic conditions.

In particular, a culture medium for a strain of the genus Corynebacterium can be found in "Manual of Methods for General Bacteriology" by the American Society for Bacteriology (Washington, D.C., USA, 1981).

In the present disclosure, carbon sources include carbohydrates such as glucose, sucrose, lactose, fructose, sucrose, and maltose; sugar alcohols such as mannitol and sorbitol; organic acids such as pyruvic acid, lactic acid and citric acid; amino acids such as glutamic acid, methionine and lysine; etc. Natural organic nutrients such as hydrolysed starch, molasses, raw molasses, rice bran, cassava, sugar cane residue and liquid corn extract can be used. In particular, carbohydrates such as glucose and sterilized pre-treated molasses (namely, molasses converted to reducing sugar) can be used, and suitable amounts of other carbon sources can be used in a variety of ways without limitation. These carbon sources can be used alone or in combination with, but not limited to, two or more.

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

Phosphorus sources may include monopotassium phosphate, dipotassium phosphate, or their respective sodium salts. As inorganic compounds, sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate and the like can be used. In addition to these, amino acids, vitamins and/or suitable precursors and the like may be contained. These components or precursors may be added to the medium in batches or continuously, but the method of addition is not limited to them.

During cultivation of the Corynebacterium glutamicum strain of the present disclosure, the pH of the medium can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid to the medium in the correct manner. During culture, foaming can be suppressed by using an antifoam such as a fatty acid polyglycol ester. Oxygen or an oxygen-containing gas may be injected into the environment to maintain the aerobic state of the environment, or the gas may not be injected, or nitrogen, hydrogen, or carbon dioxide gas may be injected in order to maintain the anaerobic and microaerobic conditions, but the method of maintaining this state is not limited to them.

When cultivating according to the present disclosure, it is possible to maintain the culturing temperature from 20°C to 45°C, in particular, from 25°C to 40°C, and this strain can be cultivated for about 10 to 160 hours, but the culturing conditions are not limited to them. .

The L-amino acid produced by the cultivation of the present disclosure may be secreted into the medium or may remain in the cells.

The method for producing the L-amino acid of the present disclosure may further include the step of obtaining the strain of Corynebacterium glutamicum of the present disclosure, the step of preparing a culture medium for the strain, or a combination thereof (in any order), for example, before the cultivation step.

The method for producing an L-amino acid according to the present disclosure may further include the step of isolating the L-amino acid from a medium according to cultivation (media exposed to culture) or from a strain of Corynebacterium glutamicum. After the culture step, an isolation step may be further included.

The isolation may serve to collect the L-amino acid of interest by a suitable method known in the art according to the microorganism culture method of the present disclosure, for example, a batch, continuous or fed-batch culture method. For example, centrifugation, filtration, precipitant treatment of the crystallized protein (salting out), extraction, ultrasonic disruption, ultrafiltration, dialysis, various types of chromatography such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, and affinity chromatography can be used, HPLC (high performance liquid chromatography), or a combination of both. The L-amino acid of interest can be isolated from the medium or microorganism by a suitable method known in the art.

The method for producing an L-amino acid according to the present disclosure may further include a purification step. Purification can be carried out by a suitable method known in this field. In one example, when the process for producing an L-amino acid of the present disclosure includes both a isolation step and a purification step, these isolation and purification steps may be performed continuously or discontinuously regardless of order, or may be performed simultaneously, or by being combined into one step, but the manner in which these steps are carried out is not limited thereto.

In the method of the present disclosure, the variant, polynucleotide, vector, strain, and the like are as described in other aspects.

Yet another aspect of the present disclosure is to provide a composition for producing an L-amino acid, which comprises a strain of Corynebacterium glutamicum containing a variant of the present disclosure, a polynucleotide encoding the variant, a vector containing the polynucleotide, or a polynucleotide of the present disclosure; the medium in which this strain of Corynebacterium glutamicum was cultivated; or a combination of two or more than two of them.

The composition of the present disclosure may further contain any suitable excipients, which are usually used in compositions for the production of amino acids. Such excipients can be, for example, but not limited to a preservative, humectant, dispersing agent, suspending agent, buffering agent, stabilizer or isotonic agent.

In the composition of the present disclosure, the variant, polynucleotide, vector, strain, medium, L-amino acid, and the like are as described in other aspects.

Useful effects

In the case of cultivating a strain of Corynebacterium glutamicum containing the asparagine synthase variant of the present disclosure, it is possible to produce L-valine in a higher yield compared to the case of existing microorganisms having unmodified polypeptides.

Brief description of graphic materials

FIG. 1. Schematic diagram of plasmid pDCM2.

Method for carrying out the invention

Below, the present disclosure will be described in more detail by way of Examples. However, the following Examples are merely preferred embodiments for illustrating the present disclosure, and thus are not intended to limit the scope of the present disclosure. However, technical issues not described in the present description of the invention can be sufficiently understood and easily implemented by experts in the technical field of the present disclosure or in similar technical fields. Example 1: construction of a plasmid

A plasmid (pDCM2, FIG. 1, SEQ ID NO: 11) for inserting and replacing genes in the Corynebacterium chromosome was constructed and synthesized using the Gene-synthesis service from BIONICS Co., Ltd. This plasmid was designed to contain a restriction enzyme site for easy application for cloning with reference to the well-known article on the sacB system (Gene, 145 (1994) 69-73). The pDCM2 plasmid synthesized in this way has the following properties:

1) Plasmid pDCM2 has a replicator that only works in E. coli, and autonomous replication is thus possible in E. coli but not in Corynebacterium.

2) Plasmid pDCM2 has a kanamycin resistance gene as a selectable marker.

3) Plasmid pDCM2 has the levansucrase (sacB) gene as a secondary positive selection marker.

4) In the final engineered strain, no information about the gene derived from the pDCM2 plasmid remains.

Example 2: Construction of a vector for expression in a microorganism of a protein variant

In order to confirm the effect of a variant (P192L; SEQ ID NO: 1) in which the proline (Pro, P) at position 192 of the protein consisting of the amino acid sequence of SEQ ID NO: 3 was replaced by leucine (Leu, L), on production of L-valine, a vector was constructed to construct a strain expressing this variant as follows.

PCR (Polymerase Chain Reaction) was performed using wild-type Corynebacterium glutamicum gDNA ATCC14067 as a template, a primer pair having sequences of SEQ ID NOs: 5 and 6, and a primer pair having sequences of SEQ ID NOs: 7 and 8. PCR was again performed with overlapping primers using a mixture of the two fragments obtained above as a template and a pair of primers having the sequences of SEQ ID NO: 5 and SEQ ID NO: 8 to obtain a fragment. PCR was performed as follows: denaturation at 94°C for 5 minutes; 30 cycles at 94°C for 30 seconds, 55°C for 30 seconds and 72°C for 1 minute 30 seconds; and 72°C for 5 minutes. The pDCM2 vector was treated with smaI and the PCR product obtained above was cloned into it by fusion. Fusion cloning was performed using the hi-Fusion® HD cloning kit (Clontech). The resulting plasmid was named pDCM2-ltsA(P192L). The primer sequences used in this example are described in the following Table 1:

Example 3 Evaluation of the L-Valine Producing Ability of a Microorganism Expressing a Variant Protein

3-1. Construction of a strain expressing a protein variant

The vector constructed in Example 2 was transformed into Corynebacterium glutamicum CA08-0072 (KCCM11201P).

The strain in which this variant was introduced was selected from strains in which homologous recombination occurred using SEQ ID NOs: 9 and 10. The selected strain was named CA08-0072 Its A P192L. The primer sequences used in this example are described in the following Table 2:

3-2. Comparison of L-valine-producing ability of strains expressing a protein variant

The ability to produce L-valine was analyzed by evaluating the flask fermentation titer of each strain constructed in Example 3-1 and the parental control strain.

First, each colony was subcultured in nutrient medium, and then each strain was inoculated into a 250 ml baffled flask containing 25 ml of production medium, and subjected to shaking culture at 200 rpm at 30° C. for 72 hours. Then, the concentration of L-valine was analyzed by HPLC, and the analyzed concentration of L-valine is shown in Table 3 below.

Nutrient medium (pH 7.2)

10 g glucose, 5 g beef extract, 10 g polypeptone, 2.5 g sodium chloride, 5 g yeast extract, 20 g agar, 2 g urea (based on 1 liter of distilled water).

Production medium (pH 7.0)

100 g glucose, 40 g ammonium sulfate, 2.5 g soy protein, 5 g corn extract solids, 3 g urea, 1 g dipotassium phosphate, 0.5 g magnesium sulfate heptahydrate, 100 mcg biotin, 1 mg thiamine-HCl, 2 mg calcium pantothenate, 3 mg nicotinamide, 30 g calcium carbonate (based on 1 liter of distilled water).

This experiment was repeated 3 times, and the average values of the results of its analysis are presented in Table 3 below.

As shown in Table 3, strain CA08-0072_ltsA_P192L showed an increased ability to produce L-valine compared to the ability to produce L-valine of the control group.

The modified CA08-0072_ItsA_P192L strain, named Corynebacterium glutamicum CA08-1747, was deposited with the Korea Microorganism Culture Center, a depository institution operating under the Budapest Agreement, on December 2, 2020, and was assigned accession number KCCM12863P.

From the foregoing description, those skilled in the technical field to which the present disclosure belongs will appreciate that the present disclosure may be embodied in other specific forms without altering its technical nature and essential characteristics. In this regard, it should be understood that the embodiments described above are in all respects illustrative and not restrictive. The scope of the present disclosure should be construed to include all changes or modified forms deriving from the meaning and scope of the claims to be described below, and not from the above detailed description and equivalent ideas.

Depository Name: Korea Microorganism Culture Center

Access Number: KCCM12863P

Deposit date: 02.12.2020

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<110> CJ CheilJedang Corporation

<120> A new variant of asparagine synthase and a method for obtaining L-valine

with its application

<130>OPP20211117KR

<150> KR 10-2021-0011011

<151> 2021-01-26

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Met Arg His Arg Gly Pro Asp Asp Ala Gly Thr Trp His Asp Ala Asp

1 5 10 15

Ala Ala Phe Gly Phe Asn Arg Leu Ser Ile Ile Asp Ile Ala His Ser

20 25 30

His Gln Pro Leu Arg Trp Gly Pro Thr Asp Glu Pro Asp Arg Tyr Ala

35 40 45

Met Thr Phe Asn Gly Glu Ile Tyr Asn Tyr Val Glu Leu Arg Lys Glu

50 55 60

Leu Ser Asp Leu Gly Tyr Thr Phe Asn Thr Ser Gly Asp Gly Glu Pro

65 70 75 80

Ile Val Val Gly Phe His His Trp Gly Glu Ser Val Val Glu His Leu

85 90 95

Arg Gly Met Phe Gly Ile Ala Ile Trp Asp Thr Lys Glu Lys Ser Leu

100 105 110

Phe Leu Ala Arg Asp Gln Phe Gly Ile Lys Pro Leu Phe Tyr Ala Thr

115 120 125

Thr Glu His Gly Thr Val Phe Ser Ser Glu Lys Lys Thr Ile Leu Glu

130 135 140

Met Ala Glu Glu Met Asn Leu Asp Leu Gly Leu Asp Lys Arg Thr Ile

145 150 155 160

Glu His Tyr Val Asp Leu Gln Tyr Val Pro Glu Pro Asp Thr Leu His

165 170 175

Ala Gln Ile Ser Arg Leu Glu Ser Gly Cys Thr Ala Thr Val Arg Leu

180 185 190

Gly Gly Lys Leu Glu Gln Lys Arg Tyr Phe Lys Pro Gln Phe Pro Val

195 200 205

Gln Lys Val Val Lys Gly Lys Glu Gln Asp Leu Phe Asp Arg Ile Ala

210 215 220

Gln Val Leu Glu Asp Ser Val Glu Lys His Met Arg Ala Asp Val Thr

225 230 235 240

Val Gly Ser Phe Leu Ser Gly Ile Asp Ser Thr Ala Ile Ala Ala

245 250 255

Leu Ala Lys Arg His Asn Pro Asp Leu Leu Thr Phe Thr Thr Gly Phe

260 265 270

Glu Arg Glu Gly Tyr Ser Glu Val Asp Val Ala Ala Glu Ser Ala Ala

275 280 285

Ala Ile Gly Thr Glu His Ile Val Lys Ile Val Ser Pro Glu Glu Tyr

290 295 300

Ala Asn Ala Ile Pro Lys Ile Met Trp Tyr Leu Asp Asp Pro Val Ala

305 310 315 320

Asp Pro Ser Leu Val Pro Leu Tyr Phe Val Ala Ala Glu Ala Arg Lys

325 330 335

His Val Lys Val Val Leu Ser Gly Glu Gly Ala Asp Glu Leu Phe Gly

340 345 350

Gly Tyr Thr Ile Tyr Lys Glu Pro Leu Ser Leu Ala Pro Phe Glu Lys

355 360 365

Ile Pro Ser Pro Leu Arg Lys Gly Leu Gly Lys Leu Ser Lys Val Leu

370 375 380

Pro Asp Gly Met Lys Gly Lys Ser Leu Leu Glu Arg Gly Ser Met Thr

385 390 395 400

Met Glu Glu Arg Tyr Tyr Gly Asn Ala Arg Ser Phe Asn Phe Glu Gln

405 410 415

Met Gln Arg Val Ile Pro Trp Ala Lys Arg Glu Trp Asp His Arg Glu

420 425 430

Val Thr Ala Pro Ile Tyr Ala Gln Ser Arg Asn Phe Asp Pro Val Ala

435 440 445

Arg Met Gln His Leu Asp Leu Phe Thr Trp Met Arg Gly Asp Ile Leu

450 455 460

Val Lys Ala Asp Lys Ile Asn Met Ala Asn Ser Leu Glu Leu Arg Val

465 470 475 480

Pro Phe Leu Asp Lys Glu Val Phe Lys Val Ala Glu Thr Ile Pro Tyr

485 490 495

Asp Leu Lys Ile Ala Asn Gly Thr Thr Thr Lys Tyr Ala Leu Arg Arg Ala

500 505 510

Leu Glu Gln Ile Val Pro Pro His Val Leu His Arg Lys Lys Leu Gly

515 520 525

Phe Pro Val Pro Met Arg His Trp Leu Ala Gly Asp Glu Leu Phe Gly

530 535 540

Trp Ala Gln Asp Ala Ile Lys Glu Ser Gly Thr Glu Asp Ile Phe Asn

545 550 555 560

Lys Gln Ala Val Leu Asp Met Leu Asn Glu His Arg Asp Gly Val Ser

565 570 575

Asp His Ser Arg Arg Leu Trp Thr Val Leu Ser Phe Met Val Trp His

580 585 590

Gly Ile Phe Val Glu Asn Arg Ile Asp Pro Gln Ile Glu Asp Arg Ser

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Tyr Pro Val Glu Leu

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atgcgccacc gtggtcctga cgatgccggc acttggcatg acgccgatgc agcgtttgga 60

ttcaaccgcc tctccatcat tgatattgca cactcccacc aaccactgcg ttggggacct 120

acggatgaac ccgaccgcta cgcaatgact ttcaacggtg agatctacaa ctacgttgag 180

ctgcgtaaag agctctcgga tttgggatat acctttaata cttctggcga tggcgagcca 240

attgttgtcg gtttccacca ctggggcgag tccgtggtcg agcatctccg cggaatgttc 300

ggcattgcca tttgggatac aaaggaaaag tcgcttttcc ttgcgcgtga tcagttcggc 360

atcaagccac tgttctacgc aaccaccgag catggcaccg tgttctcctc agagaagaag 420

accatcttgg agatggccga ggagatgaat ctagatctgg gccttgataa gcgcaccatt 480

gagcactacg tggacttgca gtacgtgccc gagccagata cccttcacgc gcagatttcc 540

cgcttggagt caggctgcac cgcaacagtt cgtctgggcg gcaagctgga acagaagcgt 600

tacttcaagc ctcagttccc agtacagaag gtcgtaaagg gtaaggagca ggacctcttc 660

gatcgcattg cccaggtgtt ggaggatagc gtcgaaaagc atatgcgtgc cgacgtgacc 720

gtaggctcgt tcctttccgg cggcattgat tcaaccgcaa ttgcggcgct tgcaaagcgc 780

cacaaccctg acctgctcac cttcaccacc ggtttcgagc gtgaaggcta ctcggaggtc 840

gatgtggctg cggagtccgc cgctgcgatt ggcactgagc acatcgtgaa gattgtctcg 900

cctgaggaat acgccaacgc gattcctaag atcatgtggt acttggatga tcctgtagct 960

gacccatcat tggtcccgct gtacttcgtg gcagcggaag cacgtaagca cgtcaaggtt 1020

gtgctgtctg gcgagggcgc agatgagctg ttcggtggat acaccattta caaggagccg 1080

ctatcgcttg ctccatttga gaagatccct tccccactac gtaaaaggcct gggaaagctc 1140

agcaaggttc tgccagacgg catgaagggc aagtcccttc ttgagcgtgg ctccatgacc 1200

atggaagagc gctactacgg caacgctcgc tccttcaatt tcgagcagat gcaacgcgtt 1260

attccatggg caaagcgcga atgggaccac cgcgaagtca ctgcgccgat ctacgcacag 1320

tcccgcaact ttgatccagt agcccgcatg caacacctgg atctgttcac ctggatgcgc 1380

ggcgacatcc tggtcaaggc tgacaagatc aacatggcga actcccttga gctgcgagtt 1440

ccattcttgg ataaagaagt tttcaaggtt gcagagacca ttccttacga tctgaagatt 1500

gccaacggta ccaccaagta cgcgctgcgc agggcactcg agcagattgt tccgcctcac 1560

gttttgcacc gcaagaagct gggcttccct gttcccatgc gccactggct tgccggcgat 1620

gagctgttcg gttgggcgca ggacgccatc aaggaatccg gtactgaaga tatcttcaac 1680

aagcaggctg tgctggatat gctgaacgag caccgcgatg gcgtgtcaga tcattcccgt 1740

cgactgtgga ctgttctgtc atttatggtg tggcacggca tttttgtgga aaaccgcatt 1800

gatccacaga ttgaggaccg ctcctaccca gtcgagcttt aa 1842

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Met Arg His Arg Gly Pro Asp Asp Ala Gly Thr Trp His Asp Ala Asp

1 5 10 15

Ala Ala Phe Gly Phe Asn Arg Leu Ser Ile Ile Asp Ile Ala His Ser

20 25 30

His Gln Pro Leu Arg Trp Gly Pro Thr Asp Glu Pro Asp Arg Tyr Ala

35 40 45

Met Thr Phe Asn Gly Glu Ile Tyr Asn Tyr Val Glu Leu Arg Lys Glu

50 55 60

Leu Ser Asp Leu Gly Tyr Thr Phe Asn Thr Ser Gly Asp Gly Glu Pro

65 70 75 80

Ile Val Val Gly Phe His His Trp Gly Glu Ser Val Val Glu His Leu

85 90 95

Arg Gly Met Phe Gly Ile Ala Ile Trp Asp Thr Lys Glu Lys Ser Leu

100 105 110

Phe Leu Ala Arg Asp Gln Phe Gly Ile Lys Pro Leu Phe Tyr Ala Thr

115 120 125

Thr Glu His Gly Thr Val Phe Ser Ser Glu Lys Lys Thr Ile Leu Glu

130 135 140

Met Ala Glu Glu Met Asn Leu Asp Leu Gly Leu Asp Lys Arg Thr Ile

145 150 155 160

Glu His Tyr Val Asp Leu Gln Tyr Val Pro Glu Pro Asp Thr Leu His

165 170 175

Ala Gln Ile Ser Arg Leu Glu Ser Gly Cys Thr Ala Thr Val Arg Pro

180 185 190

Gly Gly Lys Leu Glu Gln Lys Arg Tyr Phe Lys Pro Gln Phe Pro Val

195 200 205

Gln Lys Val Val Lys Gly Lys Glu Gln Asp Leu Phe Asp Arg Ile Ala

210 215 220

Gln Val Leu Glu Asp Ser Val Glu Lys His Met Arg Ala Asp Val Thr

225 230 235 240

Val Gly Ser Phe Leu Ser Gly Ile Asp Ser Thr Ala Ile Ala Ala

245 250 255

Leu Ala Lys Arg His Asn Pro Asp Leu Leu Thr Phe Thr Thr Gly Phe

260 265 270

Glu Arg Glu Gly Tyr Ser Glu Val Asp Val Ala Ala Glu Ser Ala Ala

275 280 285

Ala Ile Gly Thr Glu His Ile Val Lys Ile Val Ser Pro Glu Glu Tyr

290 295 300

Ala Asn Ala Ile Pro Lys Ile Met Trp Tyr Leu Asp Asp Pro Val Ala

305 310 315 320

Asp Pro Ser Leu Val Pro Leu Tyr Phe Val Ala Ala Glu Ala Arg Lys

325 330 335

His Val Lys Val Val Leu Ser Gly Glu Gly Ala Asp Glu Leu Phe Gly

340 345 350

Gly Tyr Thr Ile Tyr Lys Glu Pro Leu Ser Leu Ala Pro Phe Glu Lys

355 360 365

Ile Pro Ser Pro Leu Arg Lys Gly Leu Gly Lys Leu Ser Lys Val Leu

370 375 380

Pro Asp Gly Met Lys Gly Lys Ser Leu Leu Glu Arg Gly Ser Met Thr

385 390 395 400

Met Glu Glu Arg Tyr Tyr Gly Asn Ala Arg Ser Phe Asn Phe Glu Gln

405 410 415

Met Gln Arg Val Ile Pro Trp Ala Lys Arg Glu Trp Asp His Arg Glu

420 425 430

Val Thr Ala Pro Ile Tyr Ala Gln Ser Arg Asn Phe Asp Pro Val Ala

435 440 445

Arg Met Gln His Leu Asp Leu Phe Thr Trp Met Arg Gly Asp Ile Leu

450 455 460

Val Lys Ala Asp Lys Ile Asn Met Ala Asn Ser Leu Glu Leu Arg Val

465 470 475 480

Pro Phe Leu Asp Lys Glu Val Phe Lys Val Ala Glu Thr Ile Pro Tyr

485 490 495

Asp Leu Lys Ile Ala Asn Gly Thr Thr Thr Lys Tyr Ala Leu Arg Arg Ala

500 505 510

Leu Glu Gln Ile Val Pro Pro His Val Leu His Arg Lys Lys Leu Gly

515 520 525

Phe Pro Val Pro Met Arg His Trp Leu Ala Gly Asp Glu Leu Phe Gly

530 535 540

Trp Ala Gln Asp Ala Ile Lys Glu Ser Gly Thr Glu Asp Ile Phe Asn

545 550 555 560

Lys Gln Ala Val Leu Asp Met Leu Asn Glu His Arg Asp Gly Val Ser

565 570 575

Asp His Ser Arg Arg Leu Trp Thr Val Leu Ser Phe Met Val Trp His

580 585 590

Gly Ile Phe Val Glu Asn Arg Ile Asp Pro Gln Ile Glu Asp Arg Ser

595 600 605

Tyr Pro Val Glu Leu

610

<210> 4

<211> 1842

<212> DNA

<213> Artificial Sequence

<220>

<223> Gene encoding wild-type asparagine synthase

<400> 4

atgcgccacc gtggtcctga cgatgccggc acttggcatg acgccgatgc agcgtttgga 60

ttcaaccgcc tctccatcat tgatattgca cactcccacc aaccactgcg ttggggacct 120

acggatgaac ccgaccgcta cgcaatgact ttcaacggtg agatctacaa ctacgttgag 180

ctgcgtaaag agctctcgga tttgggatat acctttaata cttctggcga tggcgagcca 240

attgttgtcg gtttccacca ctggggcgag tccgtggtcg agcatctccg cggaatgttc 300

ggcattgcca tttgggatac aaaggaaaag tcgcttttcc ttgcgcgtga tcagttcggc 360

atcaagccac tgttctacgc aaccaccgag catggcaccg tgttctcctc agagaagaag 420

accatcttgg agatggccga ggagatgaat ctagatctgg gccttgataa gcgcaccatt 480

gagcactacg tggacttgca gtacgtgccc gagccagata cccttcacgc gcagatttcc 540

cgcttggagt caggctgcac cgcaacagtt cgtccgggcg gcaagctgga acagaagcgt 600

tacttcaagc ctcagttccc agtacagaag gtcgtaaagg gtaaggagca ggacctcttc 660

gatcgcattg cccaggtgtt ggaggatagc gtcgaaaagc atatgcgtgc cgacgtgacc 720

gtaggctcgt tcctttccgg cggcattgat tcaaccgcaa ttgcggcgct tgcaaagcgc 780

cacaaccctg acctgctcac cttcaccacc ggtttcgagc gtgaaggcta ctcggaggtc 840

gatgtggctg cggagtccgc cgctgcgatt ggcactgagc acatcgtgaa gattgtctcg 900

cctgaggaat acgccaacgc gattcctaag atcatgtggt acttggatga tcctgtagct 960

gacccatcat tggtcccgct gtacttcgtg gcagcggaag cacgtaagca cgtcaaggtt 1020

gtgctgtctg gcgagggcgc agatgagctg ttcggtggat acaccattta caaggagccg 1080

ctatcgcttg ctccatttga gaagatccct tccccactac gtaaaaggcct gggaaagctc 1140

agcaaggttc tgccagacgg catgaagggc aagtcccttc ttgagcgtgg ctccatgacc 1200

atggaagagc gctactacgg caacgctcgc tccttcaatt tcgagcagat gcaacgcgtt 1260

attccatggg caaagcgcga atgggaccac cgcgaagtca ctgcgccgat ctacgcacag 1320

tcccgcaact ttgatccagt agcccgcatg caacacctgg atctgttcac ctggatgcgc 1380

ggcgacatcc tggtcaaggc tgacaagatc aacatggcga actcccttga gctgcgagtt 1440

ccattcttgg ataaagaagt tttcaaggtt gcagagacca ttccttacga tctgaagatt 1500

gccaacggta ccaccaagta cgcgctgcgc agggcactcg agcagattgt tccgcctcac 1560

gttttgcacc gcaagaagct gggcttccct gttcccatgc gccactggct tgccggcgat 1620

gagctgttcg gttgggcgca ggacgccatc aaggaatccg gtactgaaga tatcttcaac 1680

aagcaggctg tgctggatat gctgaacgag caccgcgatg gcgtgtcaga tcattcccgt 1740

cgactgtgga ctgttctgtc atttatggtg tggcacggca tttttgtgga aaaccgcatt 1800

gatccacaga ttgaggaccg ctcctaccca gtcgagcttt aa 1842

<210> 5

<211> 36

<212> DNA

<213> Artificial Sequence

<220>

<223>ltsA_1F

<400> 5

tcgagctcgg tacccctcca tcattgatat tgcaca 36

<210> 6

<211> 44

<212> DNA

<213> Artificial Sequence

<220>

<223>ltsA_2R

<400> 6

ttctgttcca gcttgccgcc cagacgaact gttgcggtgc agcc 44

<210> 7

<211> 44

<212> DNA

<213> Artificial Sequence

<220>

<223>ltsA_3F

<400> 7

ggctgcaccg caacagttcg tctgggcggc aagctggaac agaa 44

<210> 8

<211> 36

<212> DNA

<213> Artificial Sequence

<220>

<223>ltsA_4R

<400> 8

ctctagagga tcccccttga cgtgcttacg tgcttc 36

<210> 9

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223>ltsA_5F

<400> 9

ctccatcatt gatattgcac a 21

<210> 10

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223>ltsA_6R

<400> 10

cttgacgtgc ttacgtgctt c 21

<210> 11

<211> 5803

<212> DNA

<213> Artificial Sequence

<220>

<223>pDCM2

<400> 11

gttcgcttgc tgtccataaa accgcccagt ctagctatcg ccatgtaagc ccactgcaag 60

ctacctgctt tctctttgcg cttgcgtttt cccttgtcca gatagcccag tagctgacat 120

tcatccgggg tcagcaccgt ttctgcggac tggctttcta cgtgttccgc ttcctttagc 180

agcccttgcg ccctgagtgc ttgcggcagc gtgaagctag cttttatcgc cattcgccat 240

tcaggctgcg caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc 300

360

cacgacgttg taaaacgacg gccagtgaat tcgagctcgg tacccgggga tcctctagag 420

tcgacctgca ggcatgcaag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat 480

tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg 540

ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag 600

tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt 660

ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg 720

ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg 780

gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag 840

gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga 900

cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct 960

ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc 1020

tttctccctt cgggaagcgt ggcgctttct caatgctcac gctgtaggta tctcagttcg 1080

gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc 1140

tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca 1200

ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag 1260

ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct 1320

ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc 1380

accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga 1440

tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca 1500

1560

tgggcgaaga actccagcat gagatccccg cgctggagga tcatccagcc ctgatagaaa 1620

cagaagccac tggagcacct caaaaacacc atcatacact aaatcagtaa gttggcagca 1680

tcacccgacg cactttgcgc cgaataaata cctgtgacgg aagatcactt cgcagaataa 1740

ataaatcctg gtgtccctgt tgataccggg aagccctggg ccaacttttg gcgaaaatga 1800

gacgttgatc ggcacgtaag aggttccaac tttcaccata atgaaataag atcactaccg 1860

ggcgtatttt ttgagttatc gagattttca ggagctgata gaaacagaag ccactggagc 1920

acctcaaaaa caccatcata cactaaatca gtaagttggc agcatcaccc gacgcacttt 1980

gcgccgaata aatacctgtg acggaagatc acttcgcaga ataaataaat cctggtgtcc 2040

ctgttgatac cgggaagccc tgggccaact tttggcgaaa atgagacgtt gatcggcacg 2100

taagaggttc caactttcac cataatgaaa taagatcact accgggcgta ttttttgagt 2160

tatcgagatt ttcaggagct ctttggcatc gtctctcgcc tgtcccctca gttcagtaat 2220

ttcctgcatt tgcctgtttc cagtcggtag atattccaca aaacagcagg gaagcagcgc 2280

ttttccgctg cataaccctg cttcggggtc attatagcga ttttttcggt atatccatcc 2340

tttttcgcac gatatacagg attttgccaa agggttcgtg tagactttcc ttggtgtatc 2400

caacggcgtc agccggggcag gataggtgaa gtaggcccac ccgcgagcgg gtgttccttc 2460

ttcactgtcc cttattcgca cctggcggtg ctcaacggga atcctgctct gcgaggctgg 2520

ccggctaccg ccggcgtaac agatgagggc aagcggatgg ctgatgaaac caagccaacc 2580

aggaagggca gcccacctat caaggtgtac tgccttccag acgaacgaag agcgattgag 2640

gaaaaggcgg cggcggccgg catgagcctg tcggcctacc tgctggccgt cggccaggggc 2700

tacaaaatca cgggcgtcgt ggactatgag cacgtccgcg agggcgtccc ggaaaacgat 2760

tccgaagccc aacctttcat agaaggcggc ggtggaatcg aaatctcgtg atggcaggtt 2820

gggcgtcgct tggtcggtca tttcgaaaaa ggttaggaat acggttagcc atttgcctgc 2880

ttttatatag ttcantatgg gattcacctt tatgttgata agaaataaaa gaaaatgcca 2940

ataggatatc ggcattttct tttgcgtttt tatttgttaa ctgttaattg tccttgttca 3000

aggatgctgt ctttgacaac agatgttttc ttgcctttga tgttcagcag gaagctcggc 3060

gcaaacgttg attgtttgtc tgcgtagaat cctctgtttg tcatatagct tgtaatcacg 3120

acattgtttc ctttcgcttg aggtacagcg aagtgtgagt aagtaaaggt tacatcgtta 3180

ggcggatcaa gatccatttt taacacaagg ccagttttgt tcagcggctt gtatgggcca 3240

gttaaagaat tagaaacata accaagcatg taaatatcgt tagacgtaat gccgtcaatc 3300

gtcatttttg atccgcggga gtcagtgaac aggtaccatt tgccgttcat tttaaagacg 3360

3420

ttcagtgtgt aatcatcgtt tagctcaatc ataccgagag cgccgtttgc taactcagcc 3480

3540

ttgccatagt atgctttgtt aaataaagat tcttcgcctt ggtagccatc ttcagttcca 3600

gtgtttgctt caaatactaa gtatttgtgg cctttatctt ctacgtagtg aggatctctc 3660

agcgtatggt tgtcgcctga gctgtagttg ccttcatcga tgaactgctg tacattttga 3720

tacgtttttc cgtcaccgtc aaagattgat ttataatcct ctacaccgtt gatgttcaaa 3780

3840

ttaccggaga aatcagtgta gaataaacgg atttttccgt cagatgtaaa tgtggctgaa 3900

3960

tctttaaaga cgcggccagc gtttttccag ctgtcaatag aagtttcgcc gactttttga 4020

tagaacatgt aaatcgatgt gtcatccgca tttttaggat ctccggctaa tgcaaagacg 4080

atgtggtagc cgtgatagtt tgcgacagtg ccgtcagcgt tttgtaatgg ccagctgtcc 4140

caaacgtcca ggccttttgc agaagagata tttttaattg tggacgaatc aaattcagaa 4200

acttgatatt tttcattttt ttgctgttca gggatttgca gcatatcatg gcgtgtaata 4260

tgggaaatgc cgtatgtttc cttatatggc ttttggttcg tttctttcgc aaacgcttga 4320

gttgcgcctc ctgccagcag tgcggtagta aaggttaata ctgttgcttg ttttgcaaac 4380

tttttgatgt tcatcgttca tgtctccttt tttatgtact gtgttagcgg tctgcttctt 4440

ccagccctcc tgtttgaaga tggcaagtta gttacgcaca ataaaaaaag acctaaaata 4500

tgtaaggggt gacgccaaag tatacacttt gccctttaca cattttaggt cttgcctgct 4560

4620

gattgcaact ggtctatttt cctcttttgt ttgatagaaa atcataaaag gattgcaga 4680

ctacggggcct aaagaactaa aaaatctatc tgtttctttt cattctctgt attttttata 4740

gtttctgttg catgggcata aagttgcctt tttaatcaca attcagaaaa tatcataata 4800

tctcatttca ctaaataata gtgaacggca ggtatatgtg atgggttaaa aggatcacc 4860

ccagagtccc gctcagaaga actcgtcaag aaggcgatag aaggcgatgc gctgcgaatc 4920

gggagcggcg ataccgtaaa gcacgaggaa gcggtcagcc cattcgccgc caagctcttc 4980

agcaatatca cgggtagcca acgctatgtc ctgatagcgg tccgccacac cggccggcc 5040

acagtcgatg aatccagaaa agcggccatt ttccaccatg atattcggca agcaggcatc 5100

gccatgggtc acgacgagat cctcgccgtc gggcatccgc gccttgagcc tggcgaacag 5160

ttcggctggc gcgagcccct gatgctcttc gtccagatca tcctgatcga caagaccggc 5220

ttccatccga gtacgtgctc gctcgatgcg atgtttcgct tggtggtcga atgggcaggt 5280

agccggatca agcgtatgca gccgccgcat tgcatcagcc atgatggata ctttctcggc 5340

aggagcaagg tgagatgaca ggagatcctg ccccggcact tcgcccaata gcagccagtc 5400

ccttcccgct tcagtgacaa cgtcgagaca gctgcgcaag gaacgcccgt cgtggccagc 5460

cacgatagcc gcgctgcctc gtcttggagt tcattcaggg caccggacag gtcggtcttg 5520

acaaaaagaa cggggcgccc ctgcgctgac agccggaaca cggcggcatc agagcagccg 5580

attgtctgtt gtgcccagtc atagccgaat agcctctcca cccaagcggc cggagaacct 5640

gcgtgcaatc catcttgttc aatcatgcga aacgatcctc atcctgtctc ttgatcagat 5700

cttgatcccc tgcgccatca gatccttggc ggcaagaaag ccatccagtt tactttgcag 5760

ggcttcccaa ccttaccaga gggcgcccca gctggcaatt ccg 5803

<---

Claims (5)

1. A polypeptide involved in the production of L-valine, consisting of the amino acid sequence shown by SEQ ID NO: 1, where proline, which is the amino acid corresponding to position 192 of SEQ ID NO: 3, is replaced by leucine. 2. A polynucleotide encoding a polypeptide according to claim 1. 3. The microorganism Corynebacterium glutamicum for producing L-valine, containing the polypeptide according to claim 1 or a polynucleotide encoding this polypeptide. 4. The microorganism according to claim 3, which has an increased ability to produce L-valine compared to Corynebacterium glutamicum containing a polypeptide of SEQ ID NO: 3 or a polynucleotide encoding this polypeptide. 5 . A method for producing L-valine, comprising cultivating in a medium a microorganism Corynebacterium glutamicum containing a polypeptide according to claim 1 or a polynucleotide encoding this polypeptide.

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