CN113906044B

CN113906044B - Transcription regulatory factor variant and method for producing L-valine using the same

Info

Publication number: CN113906044B
Application number: CN202180002626.4A
Authority: CN
Inventors: 朴暠夽; 金善慧; 尹炳勋; 李羲奭; 尹效真; 吴海娜; 许兰; 柳慧连; 金斐那; 孙晟光
Original assignee: CJ CheilJedang Corp
Current assignee: CJ CheilJedang Corp
Priority date: 2021-04-07
Filing date: 2021-04-28
Publication date: 2022-05-31
Anticipated expiration: 2041-04-28
Also published as: EP4092123A1; EP4092123A4; CN113906044A

Abstract

The present disclosure relates to a novel transcription regulatory factor variant, a Corynebacterium glutamicum (Corynebacterium glutamicum) strain comprising the same, and a method for producing L-valine using the same.

Description

Transcription regulatory factor variant and method for producing L-valine using the same

Technical Field

Background

Various studies are being conducted to develop efficient production microorganisms and fermentation process technologies for producing L-amino acids and other useful substances. For example, a substance-specific approach method in which the expression of a gene encoding an enzyme involved in L-valine biosynthesis is increased or in which a gene unnecessary for biosynthesis is removed is mainly used (US 8465962B 2).

However, as the demand for L-valine increases, studies are still required to effectively increase L-valine productivity.

[ Prior art documents ]

[ patent document ]

US 8465962B 2 (published 2013 in 06 months and 18 days)

[ non-patent document ]

JOURNAL OF BACTERIOLOGY,2011 10 months, 5155-

Disclosure of Invention

Technical problem

It is an object of the present disclosure to provide a transcription regulator (transcriptional regulator) variant consisting of SEQ ID NO:1, wherein the amino acid sequence as a sequence corresponding to SEQ ID NO:3 by substitution of glutamic acid (Glu, E) at the 247 th amino acid of the amino acid sequence with lysine (Lys, K).

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

It is still another object of the present disclosure to provide a corynebacterium glutamicum strain, which comprises the variant of the present disclosure or a polynucleotide encoding the variant and has L-valine-producing ability.

It is still another object of the present disclosure to provide a method for producing L-valine, comprising the step of culturing a Corynebacterium glutamicum strain, which comprises the variant of the present disclosure or a polynucleotide encoding the variant and has L-valine productivity, in a medium.

Means for solving the problems

The present disclosure will be described in detail as follows. Meanwhile, each description and embodiment disclosed in the present disclosure is applicable to other descriptions and embodiments. In other words, all combinations of the various elements disclosed in this disclosure are within the scope of this disclosure. Furthermore, the scope of the present disclosure should not be considered limited by the detailed description set forth below. Further, throughout this specification, a number of papers and patent documents are cited and cited. The entire contents disclosed in the cited papers and patent documents are incorporated by reference in this specification to more clearly describe the level of skill in the art to which this invention pertains and the contents of this invention.

One aspect of the disclosure provides a transcription regulator variant consisting of SEQ ID NO:1, wherein the amino acid sequence as shown as SEQ ID NO:3 by substituting glutamic acid (Glu, E) at the 247 th amino acid of the amino acid sequence of the above amino acid sequence with lysine (Lys, K).

Variants of the present disclosure may have, comprise, or consist essentially of the amino acid sequence represented by SEQ ID NO 1.

In the variant of the present disclosure, the amino acid corresponding to position 247 of the amino acid sequence based on SEQ ID No. 3 in the amino acid sequence represented by SEQ ID No. 1 is lysine, and the variant may comprise 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 more homology or identity to the amino acid sequence represented by SEQ ID No. 1. It is apparent that variants of amino acid sequences having deletions, modifications, substitutions, conservative substitutions, or additions of sequences are also included in the scope of the present disclosure, as long as the amino acid sequences have such homology or identity and exhibit efficacy corresponding to the efficacy of the variants of the present disclosure.

Examples thereof include variants having sequence additions or deletions, naturally occurring mutations, silent mutations (silent mutations) or conservative substitutions at the N-terminus, C-terminus and/or within the amino acid sequence that do not alter the function of the variants of the disclosure.

"conservative substitution" means the substitution of one amino acid by another amino acid having similar structural and/or chemical properties. Such amino acid substitutions may typically occur based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues. In general, conservative substitutions may have little or no effect on the activity of the protein or polypeptide.

In the present disclosure, the term "variant" refers to a polypeptide whose amino acid sequence differs from that of the variant prior to variation by conservative substitution and/or modification of one or more amino acids, but which maintains function or properties. Such variants can generally be identified by modifying one or more amino acids of the amino acid sequence of the polypeptide and evaluating the properties of the modified polypeptide. In other words, the ability of a variant may be increased, unchanged, or decreased as compared to the ability of the polypeptide prior to the variation. Some variants may include variants with one or more portions removed, such as an N-terminal leader sequence or transmembrane domain. Other variants may include variants in which a portion of the N-terminus and/or C-terminus is removed from the mature protein. The term "variant" may be used interchangeably with terms such as modification, modified polypeptide, modified protein, mutant, mutein, and divergence, and is not limited thereto, as long as it is a term used in the meaning of variation. For the purposes of this disclosure, a variant may be a polypeptide comprising the sequence set forth by SEQ ID NO:1, wherein the polypeptide is a polypeptide having an amino acid sequence corresponding to SEQ ID NO:3 by substitution of glutamic acid (Glu, E) at the 247 th amino acid of the amino acid sequence with lysine (Lys, K).

Variants may include 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 is co-or post-translationally involved in protein translocation (translocation) may be conjugated to the N-terminus of the variant. Variants may be conjugated to other sequences or linkers for identification, purification, or synthesis.

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

Sequence homology or identity for conserved polynucleotides or polypeptides is determined by standard alignment algorithms and default gap penalties established by the program used can be used together. Basically, sequences that are homologous (homologus) or identical (identifying) are generally capable of hybridizing to all or part of the sequence under conditions of moderate or high stringency. Obviously, hybridization also includes hybridization of a polynucleotide to a polynucleotide comprising universal codons or codons that take into account codon degeneracy.

Whether any two polynucleotide or polypeptide sequences have 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 al, (1988) proc. natl.acad.sci.usa 85: 2444. Furthermore, homology, similarity or identity may be determined using The Needleman-Wunsch algorithm (Needleman and Wunsch,1970, J.Mol.Biol.48: 443) as performed 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, including The GCG package (Deverux, J.et al, Nucleic Acids Research 12:387(1984), J MOLEC BIOL 215:403 (Guide to computer, mart.J.Biol.48: 443), BLASTN, FASTA (Atschul, S.F. et al, J MOLEC BIOL 215: 403) (Guide to Computers, Martin J.Bishediting, Acchop, priority, S.F. et al, Saneg., USA, Sanego, USA, Sanremo, USA, see BLAST.Som.48, USA information such as homology, USA information, homology, and SLAM 1078, USA information may be determined using The American Molecular Biology Package, USA information, and BLASTL.S.S.M 1078.

The homology, similarity or identity of a polynucleotide or polypeptide 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 published, for example, in Smith and Waterman, adv.Appl.Math (1981)2: 482. In general, the GAP program can be defined as the value obtained by dividing the total number of symbols (i.e., nucleotides or amino acids) in the shorter of the two sequences by the number of similarly aligned symbols. Default parameters for the GAP program may include (1) a binary comparison matrix (including the same value Of 1 And not the same value Of 0) And the weighted comparison matrix Of Gribsko et al, (1986) nucleic Acids res.14:6745 (or EDNAFULL (EMBOSS version Of NCBI NUC 4.4)) as disclosed in Schwartz And dayoff, Atlas Of Protein Sequence And Structure, National biological Research Foundation, p. 353-358 (1979); (2) a penalty of 3.0 per gap and an additional penalty of 0.10 per symbol per gap (or a gap opening penalty of 10 and a gap extension penalty of 0.5); and (3) no penalty for end gaps.

As an example of the disclosure, a variant of the disclosure may exhibit transcriptional regulator activity. The variants of the present disclosure may exhibit an activity such that the L-valine-producing ability is increased as compared to the wild-type polypeptide.

In the present disclosure, the term "transcription regulator (transcriptional regulator), iolR" is a transcription regulator involved in the use of Myo-inositol (Myo-inositol). The transcriptional regulator of the present disclosure may be used interchangeably with IolR. In the present disclosure, the sequence of the transcription regulatory factor can be obtained from GenBank of NCBI (e.g., WP _003857140.1, etc.) of a known database. Specifically, the transcription regulatory factor may be a polypeptide that shows the activity of the transcription regulatory factor encoded by the iolR gene, but is not limited thereto.

In the present disclosure, 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 a residue listed in a polypeptide. Identifying the amino acid at the corresponding position can be identifying a particular amino acid in the sequence that references a particular sequence. As used herein, "corresponding region" generally refers to a similar or corresponding position in a related protein or a reference (reference) protein.

For example, any amino acid sequence is aligned with SEQ ID NO:3 (align), and based thereon, each amino acid residue of the above amino acid sequences can be numbered with reference to the numerical positions of the amino acid residue and the corresponding amino acid residue of SEQ ID NO: 3. For example, a sequence alignment algorithm as described in the present disclosure can determine amino acid positions or where modifications such as substitutions, insertions, or deletions occur by comparison to a query sequence (also referred to as a "reference sequence").

For such alignment, for example, a Needleman-Wunsch algorithm (Needleman and Wunsch,1970, J.mol.biol.48: 443-.

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

In the present disclosure, the term "polynucleotide" is a DNA or RNA strand of more than a certain length in the form of a polymer of nucleotides in which nucleotide monomers are covalently linked into a long chain, and more specifically refers to a polynucleotide fragment encoding a variant.

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

In another embodiment, in the polynucleotide of the invention, the nucleotide sequence of SEQ ID NO:2 corresponding to a sequence based on SEQ ID NO: 4 is a, 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 more homology or identity to the nucleic acid sequence represented by SEQ ID No. 2. It is apparent that polynucleotides having a nucleic acid sequence in which some sequence is deleted, modified, substituted, conservatively substituted or added are also included within the scope of the disclosure, provided that the nucleic acid sequence has such homology or identity and encodes a polypeptide or protein that exhibits an efficacy corresponding to the efficacy of the variants of the disclosure.

In the polynucleotides of the present disclosure, various modifications may be made in the coding region within a range that does not change the amino acid sequence of the variants of the present application, taking into account codon degeneracy (degeneration) or codons preferred in organisms expressing the variants of the present application. Specifically, the polynucleotides of the present disclosure have or comprise, but are not limited to, a base sequence having 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 but less than 100% homology or identity to the sequence of SEQ ID No. 2, or may consist of or consist essentially 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 more, 97% or more, 98% or more but less than 100% homology or identity to the sequence of SEQ ID No. 2. Here, in the sequences having homology or identity, the codon encoding the amino acid corresponding to position 247 of SEQ ID NO. 1 may be one of the codons encoding lysine.

The polynucleotides of the present disclosure may comprise probes that can be prepared from known gene sequences, such as sequences without limitation, so long as they are sequences that can hybridize under stringent conditions to the complement of all or a portion of the polynucleotide sequences of the present disclosure. "stringent conditions" means conditions under which specific hybridization between polynucleotides is achieved. These conditions are explicitly described in the literature (see J. Sambrook et al, Molecular Cloning, A Laboratory Manual, 2 nd edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989; F.M. Autosubel et al, Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, 9.50-9.51, 11.7-11.8). Examples thereof include a condition in which polynucleotides having higher homology or identity (i.e., polynucleotides having 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) hybridize to each other, while polynucleotides having lower homology or identity compared thereto do not hybridize to each other, or a condition in which washing is performed once, specifically two to three times, under a salt concentration and temperature equivalent to 60 ℃,1 khss SSC, 0.1% SDS, specifically 60 ℃, 0.1 khss SSC, 0.1% SDS, more specifically 68 ℃, 0.1 khss SSC, 0.1% SDS, which are washing conditions for common Southern hybridization.

Hybridization requires that the two nucleic acids have complementary sequences, but that mismatches are allowed between the bases, depending on the stringency of the hybridization (mismatch). The term "complementary" is used to describe the relationship between nucleotide bases capable of hybridizing to each other. For example, with respect to DNA, adenine is complementary to thymine and cytosine is complementary to guanine. Thus, the polynucleotides of the present disclosure may also comprise substantially similar nucleic acid sequences as well as isolated nucleic acid fragments that are complementary to the entire sequence.

In particular, polynucleotides having homology or identity to the polynucleotides of the present disclosure can be detected using hybridization conditions that include a hybridization step at a Tm of 55 ℃ and the conditions described above. The Tm value may be 60 ℃, 63 ℃ or 65 ℃, but is not limited thereto, and may be appropriately adjusted by those skilled in the art according to the purpose.

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

Another aspect of the present disclosure is to provide a vector comprising a polynucleotide of the present disclosure. The vector may be an expression vector for expressing a polynucleotide in a host cell, but is not limited thereto.

The vectors of the present disclosure may include DNA constructs comprising a base sequence of a polynucleotide encoding a polypeptide of interest operably linked to a suitable expression control region (or expression control sequence) such that the polypeptide of interest may be expressed in a suitable host. The expression control region may contain a promoter capable of initiating transcription, any operator sequence for regulating transcription, a sequence encoding a suitable mRNA ribosome binding site, and sequences which regulate termination of transcription and translation. The vector may be transformed into a suitable host cell and then replicated and functions independently of the host genome, or the vector may be integrated into the genome itself.

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

For example, a polynucleotide encoding a polypeptide of interest may be inserted into a chromosome via a vector for intracellular chromosomal insertion. The insertion of the polynucleotide into the chromosome may be performed by any method known in the art, for example, homologous recombination (homologus recombination), but is not limited thereto. The vector may further contain a selection marker (selection marker) for confirming the presence or absence of chromosomal insertion. Selection markers are used to select vector-transformed cells, i.e., to confirm the absence or insertion of a nucleic acid molecule of interest, and markers that confer a selectable phenotype such as drug resistance, auxotrophy, cytotoxic agent resistance, or expression of a surface polypeptide may be used. In the context of treatment with a selection agent (selective agent), only cells expressing the selection marker survive or exhibit other phenotypic traits and thus transformed cells can be selected.

In the present disclosure, the term "transformation" means the introduction of a vector comprising a polynucleotide encoding a target polypeptide into a host cell or microorganism so that the polypeptide encoded by the polynucleotide can be expressed in the host cell. The transformed polynucleotide may be located by insertion into the chromosome of the host cell, or located extrachromosomally, so long as it can be expressed in the host cell. The polynucleotide comprises DNA and/or RNA encoding the polypeptide of interest. The polynucleotide may be introduced in any form as long as it can be introduced into a host cell and expressed. For example, the polynucleotide may be introduced into the host cell in the form of an expression cassette, which is a genetic construct containing all the elements required for self-expression. The expression cassette can generally comprise a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal operably linked to the polynucleotide. The expression cassette may be in the form of an expression vector capable of self-replication. The polynucleotide may be introduced into the host cell in its own form and operably linked to sequences required for expression in the host cell, but is not limited thereto.

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

Another aspect of the present disclosure is to provide a corynebacterium glutamicum strain comprising a variant of the present disclosure or a polynucleotide of the present disclosure.

The strain of the disclosure may comprise a modified polypeptide of the disclosure, a polynucleotide encoding the polypeptide, or a vector comprising a polynucleotide of the disclosure.

In the present disclosure, "strain (or microorganism)" includes all wild-type microorganisms or natural or artificial genetically modified microorganisms, and the strain (or microorganism) may be a microorganism in which a specific mechanism is weakened or strengthened due to insertion of an external gene or activity enhancement or inactivation of an endogenous gene, and the strain (or microorganism) may be a microorganism comprising a genetic modification for production of a polypeptide, protein or product of interest.

The strains of the present disclosure may be: a strain comprising any one or more of a variant of the disclosure, a polynucleotide of the disclosure, or a vector comprising a polynucleotide of the disclosure; a strain modified to express a variant of the disclosure or a polynucleotide of the disclosure; a strain (e.g., a recombinant strain) expressing a variant of the disclosure or a polynucleotide of the disclosure; or a strain (e.g., a recombinant strain) that exhibits the activity of a variant of the disclosure, but is not limited thereto.

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

The strain of the present disclosure may be a microorganism naturally having a transcription regulator activity and/or an L-valine-producing ability, or a microorganism in which a variant or a polynucleotide encoding the variant of the present disclosure (or a vector comprising the above-mentioned polynucleotide) is introduced into a parent strain having no transcription regulator activity or an L-valine-producing ability and/or an L-valine-producing ability is imparted to the parent strain, but is not limited thereto.

For example, a strain of the disclosure is a cell or microorganism transformed with a vector comprising a polynucleotide of the disclosure or a polynucleotide encoding a variant of the disclosure and expressing the variant of the disclosure. For the purposes of the present disclosure, the strains of the present disclosure may include all microorganisms that comprise the variants of the present disclosure and can produce L-valine. For example, the strain of the present disclosure may be a recombinant strain that expresses a transcription regulatory factor variant and has increased L-valine productivity by introducing a polynucleotide encoding the variant of the present disclosure into a natural wild-type microorganism or a L-valine-producing microorganism. The recombinant strain having an increased L-valine productivity may be a microorganism having an increased L-valine productivity as compared to a natural wild-type microorganism or a transcription regulator-unmodified microorganism, i.e., a microorganism expressing a wild-type (SEQ ID NO:3) protein or a microorganism not expressing a modified (SEQ ID NO:1) protein, but is not limited thereto. For example, the non-modified microorganism as a transcription regulator of a target strain for comparing an increase in L-valine productivity may be ATCC14067 and/or Corynebacterium glutamicum CA08-0072 strain (KCCM11201P), but is not limited thereto.

For example, the L-valine productivity of a recombinant strain having an increased productivity may be increased by about 1% or more, about 2.5% or more, about 5% or more, about 6% or more, about 7% or more, about 8% or more, about 9% or more, about 10% or more, about 10.5% or more, about 11% or more, about 11.5% or more, about 12% or more, about 12.5% or more, about 13% or more, about 13.5% or more, about 14% or more, about 14.5% or more, about 15% or more, about 15.5% or more, about 16% or more, about 16.5% or more, about 17% or more, about 17.5% or more, about 18% or more, about 18.5% or more, about 19% or more, about 19.5% or more, about 20% or more, as compared to the L-valine productivity of the parent strain or unmodified microorganism prior to mutation, About 20.5% or more, about 21% or more, about 21.5% or more, about 22% or more, about 22.1% or more, or about 22.2% or more (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, or about 33% or less), but the amount of increase is not limited thereto as long as the productivity has an increased amount of + value as compared with the productivity of the parent strain or unmodified microorganism before mutation. In another example, the L-valine productivity of a recombinant strain having an increased productivity may be increased by about 1.1-fold or more, about 1.12-fold or more, about 1.13-fold or more, about 1.15-fold or more, about 1.16-fold or more, about 1.17-fold or more, about 1.18-fold or more, about 1.19-fold or more, about 1.2-fold or more, about 1.21-fold or more, or about 1.22-fold or more (the upper limit is not particularly limited and may be, for example, about 10-fold or less, about 5-fold or less, about 3-fold or less, or about 2-fold or less) as compared to the L-valine productivity of the parent strain or unmodified microorganism before mutation.

More specifically, the L-valine productivity of the recombinant strain having an increased productivity may be increased by about 22.2% (or about 1.22-fold) as compared with the L-valine productivity of the parent strain or unmodified microorganism before mutation, but the increase rate is not limited thereto. The term "about" is intended to include all ranges of ± 0.5, ± 0.4, ± 0.3, ± 0.2, ± 0.1 etc., including all values in ranges equal or similar to the values following the term "about", but not limited thereto.

In the present disclosure, "unmodified microorganism" does not exclude a strain containing a mutation that may naturally occur in the microorganism, and may be a wild-type strain or a natural strain itself, or may be a strain before a trait is changed by genetic variation due to natural or artificial factors. For example, an unmodified microorganism can be a strain into which a protein variant described in this specification has not or has not been introduced. The term "unmodified microorganism" may be used interchangeably with "strain prior to modification", "microorganism prior to modification", "nonvariant strain", "unmodified strain", "nonvariant microorganism" or "reference microorganism".

In another example of the present disclosure, the microorganism of the present disclosure may be Corynebacterium glutamicum, Corynebacterium lactofermentum (Corynebacterium glutamicum), Corynebacterium desertorum (Corynebacterium desugarium), Corynebacterium effectivum (Corynebacterium efficiens), Corynebacterium vitreoides (Corynebacterium calophyllum), Corynebacterium stagnanum (Corynebacterium staphylium), Corynebacterium monomicum (Corynebacterium singulatum), Corynebacterium halodurans (Corynebacterium halodurans), Corynebacterium striatum (Corynebacterium striatum), Corynebacterium ammoniagenes (Corynebacterium ammoniagenes), Corynebacterium contaminans (Corynebacterium lutescens), Corynebacterium iminum (Corynebacterium immungensis), Corynebacterium stomachaetes (Corynebacterium lutescens), or Corynebacterium flavum.

In the present disclosure, "attenuation" of a polypeptide includes both a decrease in activity or a loss of activity as compared to endogenous activity. Attenuation may be used interchangeably with terms such as inactivation, defect, downregulation, reduction, decrease, and attenuation.

Weakening may also include: a case where the activity of the polypeptide itself is reduced or eliminated due to a mutation or the like of a polynucleotide encoding the polypeptide, as compared with the activity of the polypeptide originally possessed by the microorganism; a lower level of activity and/or concentration (expression level) of the polypeptide in the cell as a whole due to inhibition of expression of a gene encoding a polynucleotide of the polypeptide or inhibition of translation into the polypeptide, as compared to the native strain; the case where the polynucleotide is not expressed at all; and/or a case where the polypeptide activity is not exhibited even when the polynucleotide is expressed. "endogenous activity" means the activity of a particular polypeptide originally possessed by a parent strain or a wild-type or unmodified microorganism prior to alteration of a trait in the case where the trait is altered by genetic variation due to natural or artificial factors. Endogenous activity can be used interchangeably with "activity prior to modification". The fact that the activity of a polypeptide is "inactivated, deficient, reduced, down-regulated, reduced or attenuated" as compared to the endogenous activity means that the activity of the polypeptide is reduced as compared to the activity of the particular polypeptide originally possessed by the parent strain or wild-type or unmodified microorganism prior to the alteration in the trait.

Such polypeptide activity attenuation can be carried out by any method known in the art, but the method is not limited thereto, and attenuation can be achieved by applying various methods well known in the art (e.g., Nakashima N. et al, Bacterial cellular engineering by genome editing and gene Cloning. int J Mol Sci.2014; 15(2): 2773) 2793; Sambrook et al, Molecular Cloning 2012, etc.).

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

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

2) modifying an expression regulatory region (or expression regulatory sequence) to reduce expression of a gene encoding a polypeptide;

3) modifying an amino acid sequence that constitutes a polypeptide to eliminate or attenuate polypeptide activity (e.g., deletion/substitution/addition of one or more amino acids in the amino acid sequence);

4) modifying a gene sequence encoding a polypeptide to eliminate or attenuate polypeptide activity (e.g., deletion/substitution/addition of one or more nucleobases in a nucleobase sequence of a polypeptide gene encoding a polypeptide modified to eliminate or attenuate polypeptide activity);

5) modifying the start codon of a gene transcript encoding a polypeptide or a base sequence encoding a 5' -UTR region;

6) introducing an antisense oligonucleotide (e.g., antisense RNA) that binds complementarily to a transcript of a gene encoding a polypeptide;

7) adding a sequence complementary to the SD sequence before the SD sequence (Shine-Dalgarno sequence) of the gene encoding the polypeptide to form a secondary structure of a non-linkable ribosome;

8) adding a promoter that is 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 selected from 1) to 8), but not particularly limited thereto.

For example:

deletion of a portion or the whole of the gene encoding the polypeptide of 1) above may remove the entire polynucleotide encoding the endogenous polypeptide of interest in the chromosome, replace it with a polypeptide lacking some nucleotides, or replace it with a marker gene.

The modification of the above 2) expression regulatory region (or expression regulatory sequence) may be deletion, insertion, non-conservative or conservative substitution, or a variation of the expression regulatory region (or expression regulatory sequence) due to a combination thereof, or a substitution with a sequence exhibiting weaker activity. The expression control region includes, but is not limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, and sequences that control termination of transcription and translation.

The above-mentioned 3) modification of the initiation codon of the gene transcript encoding the polypeptide or the base sequence encoding the 5' -UTR region may be, for example, a substitution with a base sequence encoding another initiation codon having a lower polypeptide expression rate than the endogenous initiation codon, but is not limited thereto.

The modifications of the above-mentioned 4) and 5) amino acid sequence or polynucleotide sequence may be deletion, insertion or non-conservative or conservative substitution of the amino acid sequence of the polypeptide or the nucleotide sequence encoding the polypeptide, or a variation in the sequence due to a combination thereof, or substitution with an amino acid sequence or polynucleotide sequence modified to exhibit weaker activity or an amino acid sequence or polynucleotide sequence modified to be inactivated, so that the activity of the polypeptide is weakened, but not limited thereto. For example, gene expression can be inhibited or attenuated by introducing a variation into the polynucleotide sequence and forming a stop codon, but the modification is not limited thereto.

6) introduction of Antisense oligonucleotides (e.g., Antisense RNA) that complementarily bind to transcripts of a gene encoding a polypeptide, reference is made to, for example, Weintraub, H.et al, Antisense-RNA as a molecular tool for genetic analysis, Reviews-Trends in Genetics, Vol.1 (1) 1986.

The above 7) adding a sequence complementary to the SD sequence before the SD sequence of the gene encoding the polypeptide to form a secondary structure of a non-linkable ribosome may be to make mRNA non-translatable or to slow down the mRNA translation rate.

The above-mentioned 8) promoter (reverse transcription engineering, RTE) added to be transcribed in the opposite direction to the 3' -end of the Open Reading Frame (ORF) of the gene sequence encoding the polypeptide may be an activity attenuated by preparing an antisense nucleotide complementary to the transcript of the gene encoding the polypeptide.

In the present disclosure, the term "enhancement" of the activity of a polypeptide means an increase in the activity of the polypeptide compared to the endogenous activity. Enhancement may be used interchangeably with terms such as activation, upregulation, overexpression, and increase. Herein, activation, enhancement, upregulation, overexpression, and increase can include exhibiting an activity not initially present and exhibiting an improved activity compared to the endogenous activity or activity prior to modification. "endogenous activity" means the activity of a particular polypeptide originally possessed by a parent strain or unmodified microorganism prior to alteration of a trait, in the case where the trait is altered by genetic variation due to natural or artificial factors. This can be used interchangeably with "activity prior to modification". The fact that the activity of a polypeptide is "enhanced", "upregulated", "overexpressed", or "increased" compared to the endogenous activity means that the activity of the polypeptide is improved compared to the activity and/or concentration (expression level) of the particular polypeptide originally possessed by the parent strain or unmodified microorganism prior to the alteration of the trait.

Enhancement can be achieved by introducing a foreign polypeptide or enhancing the activity and/or concentration (expression level) of an endogenous polypeptide. Enhancement of the activity of a polypeptide can be confirmed by an increase in the degree and expression level of the activity of the polypeptide or the amount of a product produced from the polypeptide.

For the enhancement of the activity of the polypeptide, various methods well known in the art can be applied, and the method is not limited as long as the activity of the polypeptide of interest is enhanced as compared with the activity of the microorganism before modification. Specifically, genetic engineering and/or protein engineering, which are routine methods in Molecular biology but are not limited thereto, well known to those skilled in the art, may be used (e.g., Sitnicka et al, Functional Analysis of genes in Cell biology.2010, Vol.2.1-16; Sambrook et al, Molecular Cloning 2012, etc.).

In particular, the enhancement of the activity of the polypeptides of the present disclosure may be:

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

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

3) modifying the start codon of a gene transcript encoding the polypeptide or a base sequence encoding the 5' -UTR region;

4) modifying the amino acid sequence of the polypeptide to enhance the activity of the polypeptide;

5) modifying a polynucleotide sequence encoding a polypeptide to enhance the activity of the polypeptide (e.g., modifying a polynucleotide sequence of a polypeptide gene to encode a polypeptide modified to enhance the activity of the polypeptide);

6) introducing a foreign polypeptide or a foreign polynucleotide encoding a polypeptide that exhibits the activity of the polypeptide;

7) optimizing codons of a polynucleotide encoding the polypeptide;

8) analyzing the tertiary structure of the polypeptide to select and modify or chemically modify the exposed sites; or

9) A combination of two or more selected from the above-mentioned 1) to 8), but not particularly limited thereto.

More particularly:

the above 1) polynucleotide encoding the polypeptide of the intracellular copy number increase can be introduced into the host cells of the vector to achieve, independent of the host replication and function and operatively connected to encoding the polypeptide of the polynucleotide. Alternatively, the increase may be achieved by introducing one copy or two or more copies of the polynucleotide encoding the polypeptide into the chromosome of the host cell. Introduction into the chromosome may be performed by introducing a vector capable of inserting the polynucleotide into the chromosome of the host cell into the host cell, but is not limited thereto. The vector is as described above.

The above 2) replacement of the gene expression regulatory region (or expression regulatory sequence) encoding the polypeptide on the chromosome with a sequence exhibiting strong activity may be, for example, deletion, insertion, non-conservative or conservative substitution, or a variation in sequence due to a combination thereof, or replacement with a sequence exhibiting strong activity, so that the activity of the expression regulatory region is further enhanced. The expression control region is not particularly limited, but may contain a promoter, an operator sequence, a sequence encoding a ribosome binding site, a sequence regulating termination of transcription and translation, and the like. For example, the replacement can be, but is not limited to, replacing the original promoter with a strong promoter.

Examples of known strong promoters include CJ1 to CJ7 promoter (US 7662943B 2), lac promoter, trp promoter, trc promoter, tac promoter, λ phage PR promoter, PL promoter, tet promoter, gapA promoter, SPL7 promoter, SPL13(sm3) promoter (US 10584338B 2), O2 promoter (US 10273491B 2), tkt promoter, and yccA promoter, but are not limited thereto.

The above-mentioned 3) modification of the initiation codon of the gene transcript encoding the polypeptide or the base sequence encoding the 5' -UTR region may be, for example, a substitution with a base sequence encoding another initiation codon having a higher polypeptide expression rate than the endogenous initiation codon, but is not limited thereto.

The modifications of the above-mentioned 4) and 5) amino acid sequence or polynucleotide sequence may be deletion, insertion, non-conservative or conservative substitution of the amino acid sequence of the polypeptide or the nucleotide sequence encoding the polypeptide, or a variation in the sequence due to a combination thereof, or substitution with an amino acid sequence or polynucleotide sequence modified to exhibit stronger activity or an amino acid sequence or polynucleotide sequence modified to be more active, so that the activity of the polypeptide is enhanced, but not limited thereto. The substitution may be specifically performed by inserting the polynucleotide into a chromosome by homologous recombination, but is not limited thereto. The vector used herein may further contain a selection marker for confirming chromosome insertion. The selection marker is as described above.

The above-mentioned 6) introducing a foreign polypeptide exhibiting the activity of the polypeptide may be introducing a foreign polynucleotide encoding a polypeptide exhibiting the same or similar activity as the activity of the polypeptide into a host cell. The origin or sequence of the foreign polynucleotide is not limited as long as it exhibits the same or similar activity as that of the polypeptide. The introduction can be carried out by appropriately selecting a transformation method known to those skilled in the art. When the introduced polynucleotide is expressed in a host cell, the polypeptide may be produced, and its activity may be increased.

The above 7) codon optimizing a polynucleotide encoding a polypeptide can be codon optimizing an endogenous polynucleotide for increased transcription or translation in a host cell or codon optimizing a foreign polynucleotide for optimized transcription and translation in a host cell.

8) analyzing the tertiary structure of the polypeptide to select and modify or chemically modify the exposed site may be, for example, determining a template protein candidate according to the degree of similarity of sequences by comparing the sequence information of the polypeptide to be analyzed with a database storing sequence information of known proteins, confirming the structure based thereon and selecting and modifying or chemically modifying the exposed portion to be subjected to modification or chemical modification.

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

In the microorganisms of the present disclosure, partial or complete modification of a polypeptide (e.g., modification for encoding a protein variant described above) may be induced by: (a) homologous recombination using a vector inserted into a chromosome in a microorganism or genome editing using an engineered nuclease (e.g., CRISPR-Cas 9); and/or (b) treatment with light such as ultraviolet light and radiation and/or chemicals, but is not limited thereto. The method for modifying a part or all of a gene may include a method using a DNA recombination technique. For example, a part or all of a gene may be deleted by introducing a nucleotide sequence or a vector containing a nucleotide sequence having homology with a target gene into a microorganism to cause homologous recombination. The introduced nucleotide sequence or vector may comprise a dominant selection marker, but is not limited thereto.

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

Another aspect of the present disclosure provides a method for producing an L-amino acid, comprising the step of culturing a Corynebacterium glutamicum strain comprising a variant of the present disclosure or a polynucleotide of the present disclosure in a medium.

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

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

In the present disclosure, the term "culturing" means growing the corynebacterium glutamicum strains of the present disclosure under appropriately controlled environmental conditions. The culturing process of the present disclosure can be performed according to suitable media and culture conditions known in the art. Such culturing processes can be readily adapted and used by those skilled in the art depending on the selected strain. Specifically, the culture may be of a batch type, a continuous type and/or a fed-batch type, but is not limited thereto.

In the present disclosure, the term "medium" means a mixed substance containing nutrients required for culturing the corynebacterium glutamicum strain of the present disclosure as a main component, and the medium supplies nutrients, growth factors, and the like, including water, which are indispensable for survival and development. Specifically, as a medium and other culture conditions for culturing the corynebacterium glutamicum strain of the present disclosure, any one may be used without particular limitation as long as it is a medium used for ordinary culture of microorganisms. The corynebacterium glutamicum strain of the present disclosure may be cultured in a common medium containing an appropriate carbon source, nitrogen source, phosphorus source, inorganic compound, amino acid, and/or vitamin, etc., while controlling temperature, pH, etc., under aerobic conditions.

In particular, the culture medium of Corynebacterium strains can be found in the American society for microbiology document "Manual of Methods for General Bacteriology" (Washington, D.C., USA, 1981).

In the present disclosure, carbon sources include: carbohydrates such as glucose, sucrose (saccharose), lactose, fructose, sucrose (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; and the like. Natural organic nutrients such as starch hydrolysate, molasses, brown sugar paste, rice bran, cassava, bagasse and corn steep liquor may be used. Specifically, carbohydrates such as glucose and sterilized pretreated molasses (i.e., molasses converted into reducing sugars) may be used, and appropriate amounts of other carbon sources may be used in various ways without limitation. These carbon sources may be used alone or in a combination of two or more, but are not limited thereto.

As nitrogen sources, it is possible to use: inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate and ammonium nitrate; and organic nitrogen sources such as amino acids (such as glutamic acid, methionine and glutamine), peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolysate, fish or its decomposition product, and defatted soybean cake or its decomposition product. These nitrogen sources may be used alone or in a combination of two or more, but are not limited thereto.

The phosphorus source may include potassium dihydrogen phosphate, dipotassium hydrogen phosphate, or sodium-containing salts corresponding thereto. As the inorganic compound, sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, or 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 manner of addition is not limited thereto.

During the cultivation of the Corynebacterium glutamicum strain of the present disclosure, the pH of the medium may be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid and sulfuric acid to the medium in an appropriate manner. During the culture, foaming can be suppressed by using an antifoaming agent such as fatty acid polyglycol ester. Oxygen or oxygen-containing gas may be injected into the culture medium to maintain the aerobic state of the culture medium, or no gas may be injected or nitrogen, hydrogen or carbon dioxide gas may be injected to maintain the anaerobic and micro-aerobic state, but the method for maintaining the state is not limited thereto.

In the culture of the present disclosure, the culture temperature may be maintained at 20 ℃ to 45 ℃, specifically 25 ℃ to 40 ℃, and the strain may be cultured for about 10 to 160 hours, but the culture conditions are not limited thereto.

The L-amino acid produced by the culture of the present disclosure may be secreted into the culture medium or may be retained in the cell.

The method for producing an L-amino acid of the present disclosure may further include, for example, a step of preparing the Corynebacterium glutamicum strain of the present application, a step of preparing a medium for strain culture, or a combination of these (in any order) prior to the culturing step.

The method for producing an L-amino acid of the present disclosure may further include a step of recovering an L-amino acid from the medium according to the culture (medium in which the culture is performed) or from the Corynebacterium glutamicum strain. A recovery step may also be included after the culturing step.

The recovery may be according to the method for culturing the microorganism of the present disclosure, such as a batch, continuous or fed-batch culture method, by the appropriate methods known in the art to collect the target L-amino acid. For example, centrifugation, filtration, treatment with a crystalline protein precipitant (salting-out method), extraction, sonication, ultrafiltration, dialysis, various forms of chromatography such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography and affinity chromatography, HPLC or combinations thereof may be used. The target L-amino acid can be recovered from the medium or the microorganism by an appropriate method known in the art.

The method for producing an L-amino acid of the present disclosure may further include a purification step. Purification may be carried out by suitable methods known in the art. In one example, when the method for producing an L-amino acid of the present disclosure includes a recovery step and a purification step, the recovery step and the purification step may be continuously or discontinuously performed without being sequentially performed, or may be performed simultaneously or by being combined into one step, but the manner of performing the steps is not limited thereto.

In the methods of the invention, the variants, polynucleotides, vectors, strains, etc. are as described above for the other aspects.

Another aspect of the present disclosure is to provide a composition for L-amino acid production, comprising a corynebacterium glutamicum strain comprising a variant of the present disclosure, a polynucleotide encoding the variant, a vector comprising the polynucleotide, or a polynucleotide of the present disclosure; a medium in which the Corynebacterium glutamicum strain is cultured; or a combination of two or more thereof.

The compositions of the present disclosure may also comprise any suitable excipient commonly used in compositions for amino acid production. Such excipients may be, for example, preservatives, wetting agents, dispersing agents, suspending agents, buffering agents, stabilizing agents, or isotonic agents, but are not limited thereto.

In the compositions of the present disclosure, the variants, polynucleotides, vectors, strains, media, L-amino acids, and the like are as otherwise described.

Effects of the invention

L-valine can be produced in higher yields in the case of culturing a Corynebacterium glutamicum strain comprising the novel transcription regulatory factor variant of the present disclosure, as compared with the case of an existing microorganism having an unmodified polypeptide.

Detailed Description

Hereinafter, the present invention will be described in more detail by way of examples. However, the following examples are only for illustrating preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Meanwhile, technical matters not described in the present specification may be sufficiently understood and easily implemented by those skilled in the technical field of the present invention or the like.

Example 1: construction of vector for expressing transcription regulatory factor variant in microorganism

To confirm the sequence encoded by SEQ ID NO:3 (E247K; SEQ ID NO:1) on L-valine production, a vector for constructing a strain expressing the variant was constructed as follows using plasmid pDCM2 (Korean patent laid-open No. 10-2020-0136813) for inserting and replacing a gene into a chromosome of Corynebacterium (Corynebacterium).

The gDNA (genomic DNA) of wild-type Corynebacterium glutamicum (Corynebacterium glutamicum) ATCC14067 was used as a template, and the DNA having the sequence of SEQ ID NO: 5 and 6 and the primer pair of SEQ ID NO: the primer pairs of 7 and 8 sequences were subjected to PCR, respectively. The mixture of the two fragments obtained above was used as a template with the sequence shown in SEQ ID NO: 5 and SEQ ID NO: the primer pair of 8 sequences was again subjected to overlap PCR to obtain fragments. After 5 minutes of denaturation at 94 ℃, PCR was performed at 94 ℃ for 30 seconds, 55 ℃ for 30 seconds, and 72 ℃ for 1 min and 30 seconds for 30 cycles, and then PCR was performed at 72 ℃ for 5 minutes. pDCM2 vector was treated with smaI, and the PCR product obtained above was subjected to fusion cloning. Use of

The HD cloning kit (Clontech) performs fusion cloning. The resulting plasmid was designated pDCM2-iolR (E247K). The sequences of the primers used in this example are described in table 1 below:

[ TABLE 1 ]

Example 2: for expressionEvaluation of L-valine-producing ability of microorganism of transcription regulator variant

2-1 construction of Strain expressing transcription regulatory factor variant

The vector constructed in example 1 was transformed into Corynebacterium glutamicum CA08-0072(KCCM11201P) (cf. US 8,465,962).

Using SEQ ID NO: 9 and 10, a strain which underwent homologous recombination was selected from the above-mentioned transformed strains and designated CA08-0072_ iolR _ E247K. The sequences of the primers used in this example are described in table 2 below:

[ TABLE 2 ]

Name (R)	Sequence (5' ->3’)	SEQ ID NO
			iolR_5F	CCAAGAAGTCGTAGACAAAG	SEQ ID NO:9
iolR_6R	TTGAGCTAAGTATCCTATGA	SEQ ID NO:10

2-2 comparison of L-valine Productivity of Strain expressing transcription regulatory factor variant

L-valine productivity was analyzed by evaluation of flask fermentation titer of each strain constructed in example 2-1 and the control parent strain.

First, each colony was subcultured in a nutrient medium, and then each strain was inoculated into a 250mL corner baffle flask containing 25mL of a production medium and cultured with shaking at 200rpm at 30 ℃ for 72 hours. Thereafter, 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 of glucose, 5 g of meat extract, 10 g of peptone, 2.5 g of sodium chloride, 5 g of yeast extract, 20 g of agar and 2 g of urea (based on 1 liter of distilled water).

< production Medium (pH7.0) >

100 g of glucose, 40 g of ammonium sulfate, 2.5 g of soybean protein, 5 g of Corn Steep liquor (Corn Stem Solids), 3 g of urea, 1 g of dipotassium hydrogen phosphate, 0.5 g of magnesium sulfate heptahydrate, 100 mu g of biotin, 1mg of thiamine hydrochloride, 2mg of calcium pantothenate, 3mg of nicotinamide and 30g of calcium carbonate (based on 1 liter of distilled water).

The experiment was performed in 3 replicates, and the average values of the analytical results are shown in table 3 below.

[ Table 3]

Bacterial strains	L-valine concentration (g/L)	L-valine concentration increase Rate (%)
			CA08-0072	1.8	-
CA08-0072_iolR_E247K	2.2	22.2

As shown in Table 3, the CA08-0072_ iolR _ E247K strain exhibited increased L-valine-producing ability as compared with the control group.

From the above description, those skilled in the art to which the present invention pertains will appreciate that the present invention can be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are exemplary in all respects, rather than limiting. The scope of the present invention should be construed to include all changes or modifications derived from the meaning and scope of the following claims, rather than the above detailed description and its equivalent concepts.

Claims

1. A transcription regulatory factor variant consisting of the amino acid sequence represented by SEQ ID NO. 1, wherein glutamic acid as the amino acid corresponding to position 247 of SEQ ID NO. 3 is substituted with lysine.

2. A polynucleotide encoding the variant of claim 1.

3. A corynebacterium glutamicum strain comprising the variant of claim 1, or a polynucleotide encoding the variant.

4. The strain of claim 3, which has an increased L-valine productivity as compared to Corynebacterium glutamicum comprising the polypeptide of SEQ ID NO. 3 or a polynucleotide encoding the polypeptide.

5. A method for producing L-valine comprising the step of culturing a corynebacterium glutamicum strain comprising the variant of claim 1 or a polynucleotide encoding the variant in a medium.