WO2022231042A1

WO2022231042A1 - Novel variant and method for producing l-valine using same

Info

Publication number: WO2022231042A1
Application number: PCT/KR2021/005522
Authority: WO
Inventors: 박고운; 김선혜; 윤병훈; 이희석; 윤효진; 오해나; 허란; 유혜련; 김비나; 손성광
Original assignee: 씨제이제일제당 (주)
Priority date: 2021-04-30
Filing date: 2021-04-30
Publication date: 2022-11-03

Abstract

The present application relates to a novel L-valine-producing Corynebacterium glutamicum strain and a method for producing L-valine using the strain.

Description

Novel variant and L-valine production method using same

In order to produce L-amino acids and other useful substances, various studies are being conducted for the development of high-efficiency production microorganisms and fermentation process technology. For example, a target substance-specific approach such as increasing the expression of a gene encoding an enzyme involved in L-valine biosynthesis or removing a gene unnecessary for biosynthesis is mainly used (US8465962 B2).

As the demand for L-valine increases, there is a need for research to effectively increase the production capacity of L-valine.

One object of the present application is one or more or two or more selected from the group consisting of the following (1) to (17) (eg, 1, 2, 3, 4, 5, 6, 7) , 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17) protein variants:

(1) Serine (Serine, Ser, S), which is an amino acid corresponding to position 109 of the amino acid sequence of SEQ ID NO: 3, is substituted with phenylalanine (Phenylalanine, Phe, F), consisting of the amino acid sequence shown in SEQ ID NO: 1, protein (NCgl1391) variant;

(2) serine (serine, Ser, S), which is an amino acid corresponding to position 130 of the amino acid sequence of SEQ ID NO: 13, is substituted with phenylalanine (Phenylalanine, Phe, F), consisting of the amino acid sequence shown in SEQ ID NO: 11, ATP an ATP phosphoribosyltransferase variant;

(3) Asparagine, consisting of the amino acid sequence set forth in SEQ ID NO: 21, in which proline (Proline, Pro, P), which is an amino acid corresponding to position 192 of the amino acid sequence of SEQ ID NO: 23, is substituted with leucine (Leucine, Leu, L) Asparagine synthase variants;

(4) Alanine (Alanine, Ala, A), which is an amino acid corresponding to position 213 of the amino acid sequence of SEQ ID NO: 33, is substituted with valine (Valine, Val, V), consisting of the amino acid sequence set forth in SEQ ID NO: 31, spur Spermidine synthase variants;

(5) cysteine consisting of the amino acid sequence set forth in SEQ ID NO: 41, in which proline (Proline, Pro, P), which is an amino acid corresponding to position 302 of the amino acid sequence of SEQ ID NO: 43, is substituted with serine (Serine, Ser, S) Cysteine sulfinate desulfinase variants

(6) Proline (Proline, Pro, P), which is an amino acid corresponding to the 56th position of the amino acid sequence of SEQ ID NO: 53, is substituted with leucine (Leucine, Leu, L), consisting of the amino acid sequence set forth in SEQ ID NO: 51, urease Urease accessory protein variants;

(7) glycine (Glycine, Gly, G), which is an amino acid corresponding to position 834 of the amino acid sequence of SEQ ID NO: 63, is substituted with serine (Serine, Ser, S), consisting of the amino acid sequence set forth in SEQ ID NO: 61, proline Proline dehydrogenase variants;

(8) Arginine (Arginine, Arg, R), which is an amino acid corresponding to the 140th position of the amino acid sequence of SEQ ID NO: 73, is substituted with cysteine (Cysteine, Cys, C), consisting of the amino acid sequence set forth in SEQ ID NO: 71, tetra Hydrodipicolinate N-succinyltransferase (Tetrahydrodipicolinate N-succinyltransferase) variant;

(9) Arginine (Arginine, Arg, R), which is an amino acid corresponding to the 4th position of the amino acid sequence of SEQ ID NO: 83, is substituted with histidine (Histidine, His, H), consisting of the amino acid sequence set forth in SEQ ID NO: 81, 5 ,10-methylenetetrahydrofolate reductase (5,10-methylenetetrahydrofolate reductase) variant;

(10) Glutamic acid (Glutamic acid, Glu, E), which is an amino acid corresponding to position 253 of the amino acid sequence of SEQ ID NO: 93, is substituted with lysine (Lysine, Lys, K), consisting of the amino acid sequence set forth in SEQ ID NO: 91, protein (NCgl2805) variant;

(11) Alanine (Alanine, Ala, A), which is an amino acid corresponding to position 315 of the amino acid sequence of SEQ ID NO: 103, is substituted with valine (Valine, Val, V), consisting of the amino acid sequence shown in SEQ ID NO: 101, sugar Porter family MFS transporter (sugar porter family MFS transporter) variant;

(12) Glutamic acid (Glu, E), which is an amino acid corresponding to the 247th position of the amino acid sequence of SEQ ID NO: 113, is substituted with lysine (Lysine, Lys, K), consisting of the amino acid sequence set forth in SEQ ID NO: 111, transcriptional regulator variants;

(13) The amino acid sequence shown in SEQ ID NO: 121 in which glycine (Glycine, Gly, G), which is an amino acid corresponding to the 40th position of the amino acid sequence of SEQ ID NO: 123, is substituted with aspartic acid (Asp, D) consisting of a WhiB family transcriptional regulator WhcA (WhcA) variant;

(14) The amino acid sequence shown in SEQ ID NO: 131 in which glycine (Glycine, Gly, G), which is an amino acid corresponding to the 77th position of the amino acid sequence of SEQ ID NO: 133, is substituted with aspartic acid (Asp, D) Consisting of, dihydrolipoyl acetyltransferase (dihydrolipoamide acetyltransferase) variant;

(15) Proline (Proline, Pro, P), which is an amino acid corresponding to the 87th position of the amino acid sequence of SEQ ID NO: 143, is substituted with leucine (Leucine, Leu, L), consisting of the amino acid sequence set forth in SEQ ID NO: 141, 2 -isopropylmalate synthase (2-isopropylmalate synthase) variant;

(16) Alanine (Alanine, Ala, A), which is an amino acid corresponding to the 112th position of the amino acid sequence of SEQ ID NO: 153, is substituted with Threonine (Threonine, Thr, T), consisting of the amino acid sequence set forth in SEQ ID NO: 151, branched branched-chain amino acid permease variants; and

(17) glycine (Glycine, Gly, G), which is an amino acid corresponding to the 134th position of the amino acid sequence of SEQ ID NO: 163, is substituted with serine (Serine, Ser, S), consisting of the amino acid sequence set forth in SEQ ID NO: 161, glycine A glyceraldehyde-3-phosphate dehydrogenase variant.

Another object of the present application is to provide a polynucleotide encoding the above-described protein variant.

Another object of the present application is, (i) one or more protein variants selected from the group consisting of protein variants of (1) to (17), (ii) protein variants of (1) to (17) above At least one polynucleotide selected from the group consisting of a polynucleotide encoding is to provide

Another object of the present application is, (i) one or more protein variants selected from the group consisting of protein variants of (1) to (17), (ii) protein variants of (1) to (17) above Containing one or more polynucleotides selected from the group consisting of a polynucleotide encoding, or a combination of (i) and (ii), and having L-valine-producing ability, a Corynebacterium glutamicum strain is cultured in a medium It is to provide a method for producing L-valine, comprising the step of:

This will be described in detail as follows. Meanwhile, each description and embodiment disclosed in the present application may be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in this application fall within the scope of this application. In addition, it cannot be seen that the scope of the present application is limited by the detailed description described below. In addition, a number of papers and patent documents are referenced throughout this specification and their citations are indicated. The disclosure contents of the cited papers and patent documents are incorporated herein by reference in their entirety to more clearly describe the level of the technical field to which the present invention pertains and the content of the present invention.

protein variant and polynucleotides

(1) protein variant (SEQ ID NO: 1) and polynucleotide encoding the same

One aspect of the present application is the amino acid sequence set forth in SEQ ID NO: 1 in which serine (Serine, Ser, S), which is an amino acid corresponding to position 109 of the amino acid sequence of SEQ ID NO: 3, is substituted with phenylalanine (Phenylalanine, Phe, F) It provides a protein variant consisting of.

In one example, the variant of the present application may have or include the amino acid sequence set forth in SEQ ID NO: 1, or may consist essentially of the amino acid sequence.

In addition, in the variant of the present application, the amino acid corresponding to position 109 based on the amino acid sequence of SEQ ID NO: 3 in the amino acid sequence set forth in SEQ ID NO: 1 is phenylalanine, and the amino acid sequence described in SEQ ID NO: 1 and at least 70 may comprise an amino acid sequence having at least %, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% homology or identity. have. In addition, as long as it is an amino acid sequence having such homology or identity and exhibiting efficacy corresponding to the variant of the present application, variants having an amino acid sequence in which some sequences are deleted, modified, substituted, conservatively substituted or added are also included within the scope of the present application. is self-evident

For example, sequence additions or deletions, naturally occurring mutations, silent mutations or conservation within the N-terminus, C-terminus and/or within the amino acid sequence that do not alter the function of the variants of the present application It is a case of having an enemy substitution.

As an example of the present application, the mutant of the present application may have an activity to increase L-valine production capacity compared to the wild-type polypeptide.

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

The polynucleotide encoding the variant of the present application may include a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 1. As an example of the present application, the polynucleotide of the present application may have or include the sequence of SEQ ID NO: 2. In addition, the polynucleotide of the present application may consist of, or consist essentially of, the sequence of SEQ ID NO: 2.

In another example, in the polynucleotide of the present application, the base corresponding to position 326 based on the nucleic acid sequence of SEQ ID NO: 4 in the nucleic acid sequence set forth in SEQ ID NO: 2 is T, and at least the nucleic acid sequence set forth in SEQ ID NO: 2 and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% or more homology or identity of a nucleic acid sequence. can In addition, if a sequence encoding a polypeptide or protein having such homology or identity and exhibiting efficacy corresponding to the variant of the present application is a polynucleotide having a nucleic acid sequence in which some sequences are deleted, modified, substituted, conservatively substituted, or added It is obvious that they are included within the scope of the present application.

In consideration of codon degeneracy or preferred codons in organisms that want to express the variants of the present application, the polynucleotides of the present application are various in the coding region within the range that does not change the amino acid sequence of the variants of the present application. Deformation can be made. Specifically, the polynucleotide of the present application has 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% homology or identity to the sequence of SEQ ID NO: 2 Having or including a nucleotide sequence that is more than, 98% or more, and less than 100%, or homology or identity with the sequence of SEQ ID NO: 2 is 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, and less than 100% of the nucleotide sequence may consist of or consist essentially of, but is not limited thereto. In this case, in the sequence having the homology or identity, the codon encoding the amino acid corresponding to the 109th position of SEQ ID NO: 1 may be one of the codons encoding phenylalanine.

(2) a protein variant (SEQ ID NO: 11) and a polynucleotide encoding the same

Another aspect of the present application is the amino acid set forth in SEQ ID NO: 11 in which serine (Serine, Ser, S), which is an amino acid corresponding to position 130 of the amino acid sequence of SEQ ID NO: 13, is substituted with phenylalanine (Phenylalanine, Phe, F) An ATP phosphoribosyltransferase variant consisting of the sequence is provided.

The variant of the present application may have or include the amino acid sequence set forth in SEQ ID NO: 11, or may consist essentially of the amino acid sequence.

In addition, in the variant of the present application, the amino acid corresponding to position 130 based on the amino acid sequence of SEQ ID NO: 13 in the amino acid sequence set forth in SEQ ID NO: 11 is phenylalanine, and the amino acid sequence set forth in SEQ ID NO: 11 and at least 70 may comprise an amino acid sequence having at least %, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% homology or identity. have. In addition, as long as it is an amino acid sequence having such homology or identity and exhibiting efficacy corresponding to the variant of the present application, variants having an amino acid sequence in which some sequences are deleted, modified, substituted, conservatively substituted or added are also included within the scope of the present application. is self-evident

As an example of the present application, the variant may have ATP phosphoribosyltransferase activity. In addition, the mutant may have an activity to increase the ability to produce L-valine compared to the wild-type polypeptide.

In the present application, the term "ATP phosphoribosyltransferase" refers to 1-(5-phospho-D-ribosyl)-ATP and diphosphate using ATP and 5-phospho -alpha-D-ribose 1-diphosphate producing polypeptide. ATP phosphoribosyltransferase of the present application may be used in combination with phosphoribosyl-ATP diphosphorylase (Phosphoribosyl-ATP diphosphorylase), phosphoribosyl-ATP pyrophosphorylase (Phosphoribosyl-ATP pyrophosphorylase) or HisG. have. In the present application, the ATP phosphoribosyltransferase sequence can be obtained from GenBank of NCBI, a known database (eg, WP_003860009.1, WP_003856149.1, WP_063967509.1, WP_074495858.1, etc.). Specifically, it may be a polypeptide having ATP phosphoribosyltransferase activity encoded by the hisG gene, but is not limited thereto.

The polynucleotide encoding the variant may include a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 11. As an example of the present application, the polynucleotide of the present application may have or include the sequence of SEQ ID NO: 12. In addition, the polynucleotide of the present application may consist of, or consist essentially of, the sequence of SEQ ID NO: 12.

In another example, in the polynucleotide of the present application, the base corresponding to position 389 based on the nucleic acid sequence of SEQ ID NO: 14 in the nucleic acid sequence set forth in SEQ ID NO: 2 is T, and at least the nucleic acid sequence set forth in SEQ ID NO: 12 and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% or more homology or identity of a nucleic acid sequence. can In addition, if a sequence encoding a polypeptide or protein having such homology or identity and exhibiting efficacy corresponding to the variant of the present application is a polynucleotide having a nucleic acid sequence in which some sequences are deleted, modified, substituted, conservatively substituted, or added It is obvious that they are included within the scope of the present application.

In consideration of codon degeneracy or preferred codons in organisms that want to express the variants of the present application, the polynucleotides of the present application are various in the coding region within the range that does not change the amino acid sequence of the variants of the present application. Deformation can be made. Specifically, the polynucleotide of the present application has 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more homology or identity to the sequence of SEQ ID NO: 12 It has or contains a nucleotide sequence that is more than, 98% or more, and less than 100%, or has 70% or more, 75% or more, 80% or more, 85% or more, 90% or more of homology or identity with the sequence of SEQ ID NO: 12; 95% or more, 96% or more, 97% or more, 98% or more, and less than 100% of the nucleotide sequence may consist of or consist essentially of, but is not limited thereto. In this case, in the sequence having the homology or identity, the codon encoding the amino acid corresponding to the 130th position of SEQ ID NO: 11 may be one of the codons encoding phenylalanine.

(3) protein variant (SEQ ID NO: 21) and polynucleotides encoding the same

Another aspect of the present application is the amino acid set forth in SEQ ID NO: 21 in which proline (Proline, Pro, P), which is an amino acid corresponding to the 192th position of the amino acid sequence of SEQ ID NO: 23, is substituted with leucine (Leucine, Leu, L) An asparagine synthase variant consisting of the sequence is provided.

The variant of the present application may have or include the amino acid sequence set forth in SEQ ID NO: 21, or may consist essentially of the amino acid sequence.

In addition, in the variant of the present application, the amino acid corresponding to position 192 based on the amino acid sequence of SEQ ID NO: 23 in the amino acid sequence set forth in SEQ ID NO: 21 is leucine, and the amino acid sequence set forth in SEQ ID NO: 21 and at least 70 may comprise an amino acid sequence having at least %, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% homology or identity. have. In addition, as long as it is an amino acid sequence having such homology or identity and exhibiting efficacy corresponding to the variant of the present application, variants having an amino acid sequence in which some sequences are deleted, modified, substituted, conservatively substituted or added are also included within the scope of the present application. is self-evident

As an example of the present application, the variant may have asparagine synthase activity. In addition, the mutant may have an activity to increase the ability to produce L-valine compared to the wild-type polypeptide.

As used herein, the term “asparagine synthase” refers to glutamine-hydrolysing activity, and aspartate and glutamine are used as asparagine and glutamate. It is a polypeptide that converts Asparagine synthase of the present application may be used in combination with asparagine synthetase, asparagine synthetase (eg, Asparagine synthetase B), glutamine-dependent asparagine synthetase (glutamine-dependent asparagine synthetase) or LtsA. In the present application, the asparagine synthase sequence can be obtained from GenBank of NCBI, a known database (eg, WP_003859651.1, WP_060564818.1, WP_040967635.1, etc.). Specifically, it may be a polypeptide having asparagine synthase activity encoded by the ltsA gene, but is not limited thereto.

The polynucleotide encoding the variant may include a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 21. As an example of the present application, the polynucleotide of the present application may have or include the sequence of SEQ ID NO: 22. In addition, the polynucleotide of the present application may consist of, or consist essentially of, the sequence of SEQ ID NO: 22.

In another example, in the polynucleotide of the present application, the base corresponding to position 575 based on the nucleic acid sequence of SEQ ID NO: 24 in the nucleic acid sequence set forth in SEQ ID NO: 22 is T, and at least the nucleic acid sequence set forth in SEQ ID NO: 22 and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% or more homology or identity of a nucleic acid sequence. can In addition, if a sequence encoding a polypeptide or protein having such homology or identity and exhibiting efficacy corresponding to the variant of the present application is a polynucleotide having a nucleic acid sequence in which some sequences are deleted, modified, substituted, conservatively substituted, or added It is obvious that they are included within the scope of the present application.

In consideration of codon degeneracy or preferred codons in organisms that want to express the variants of the present application, the polynucleotides of the present application are various in the coding region within the range that does not change the amino acid sequence of the variants of the present application. Deformation can be made. Specifically, the polynucleotide of the present application has 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more homology or identity to the sequence of SEQ ID NO: 22 Having or including a nucleotide sequence that is more than, 98% or more, and less than 100%, or homology or identity with the sequence of SEQ ID NO: 2 is 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, and less than 100% of the nucleotide sequence may consist of or consist essentially of, but is not limited thereto. In this case, in the sequence having the homology or identity, the codon encoding the amino acid corresponding to the 192th position of SEQ ID NO: 21 may be one of the codons encoding leucine.

(4) protein variant (SEQ ID NO: 31) and polynucleotides encoding the same

One aspect of the present application is the amino acid sequence set forth in SEQ ID NO: 31 in which alanine (Alanine, Ala, A), which is an amino acid corresponding to position 213 of the amino acid sequence of SEQ ID NO: 33, is substituted with valine (Valine, Val, V) It provides a spermidine synthase variant consisting of.

The variant of the present application may have or include the amino acid sequence set forth in SEQ ID NO: 31, or may consist essentially of the amino acid sequence.

In addition, in the variant of the present application, the amino acid corresponding to position 213 based on the amino acid sequence of SEQ ID NO: 33 in the amino acid sequence set forth in SEQ ID NO: 31 is valine, and the amino acid sequence set forth in SEQ ID NO: 31 and at least 70 may comprise an amino acid sequence having at least %, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% homology or identity. have. In addition, as long as it is an amino acid sequence having such homology or identity and exhibiting efficacy corresponding to the variant of the present application, variants having an amino acid sequence in which some sequences are deleted, modified, substituted, conservatively substituted or added are also included within the scope of the present application. is self-evident

As an example of the present application, the variant may have spermidine synthase activity. In addition, the mutant may have an activity to increase the ability to produce L-valine compared to the wild-type polypeptide.

As used herein, the term "Spermidine synthase" is a polypeptide belonging to the aminopropyl transferase group, and during spermidine biosynthesis, the putre of the propylamine group of S-adenosylmethioninamine. It is a polypeptide that catalyzes the transition to Putrescine. The spermidine synthase of the present application may be used in combination with spermidine synthase, aminopropyltransferase, putrescine aminopropyltransferase, polyamine aminopropyltransferase, or SpeE. . In the present application, the spermidine synthase sequence can be obtained from GenBank of NCBI, which is a known database. Specifically, it may be a polypeptide having spermidine synthase activity encoded by the speE gene, but is not limited thereto.

The polynucleotide encoding the variant may include a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 31. As an example of the present application, the polynucleotide of the present application may have or include the sequence of SEQ ID NO: 32. In addition, the polynucleotide of the present application may consist of, or consist essentially of, the sequence of SEQ ID NO: 32.

In another example, in the polynucleotide of the present application, in the nucleic acid sequence set forth in SEQ ID NO: 32, the base corresponding to position 638 based on the nucleic acid sequence of SEQ ID NO: 34 is T, and at least the nucleic acid sequence set forth in SEQ ID NO: 32 and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% or more homology or identity of a nucleic acid sequence. can In addition, if a sequence encoding a polypeptide or protein having such homology or identity and exhibiting efficacy corresponding to the variant of the present application is a polynucleotide having a nucleic acid sequence in which some sequences are deleted, modified, substituted, conservatively substituted, or added It is obvious that they are included within the scope of the present application.

In consideration of codon degeneracy or preferred codons in organisms that want to express the variants of the present application, the polynucleotides of the present application are various in the coding region within the range that does not change the amino acid sequence of the variants of the present application. Deformation can be made. Specifically, the polynucleotide of the present application has 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more homology or identity to the sequence of SEQ ID NO: 32 Having or including a nucleotide sequence that is greater than or equal to 98%, and less than 100%, or homology or identity with the sequence of SEQ ID NO: 32 is 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, and less than 100% of the nucleotide sequence may consist of or consist essentially of, but is not limited thereto. In this case, in the sequence having the homology or identity, the codon encoding the amino acid corresponding to the 213th position of SEQ ID NO: 31 may be one of the codons encoding valine.

(5) protein variant (SEQ ID NO: 41) and polynucleotides encoding the same

Another aspect of the present application is the amino acid set forth in SEQ ID NO: 41 in which proline (Proline, Pro, P), which is an amino acid corresponding to position 302 of the amino acid sequence of SEQ ID NO: 43, is substituted with serine (Serine, Ser, S) A cysteine sulfinate disulfinase variant consisting of the sequence is provided.

The variant of the present application may have or include the amino acid sequence set forth in SEQ ID NO: 41, or may consist essentially of the amino acid sequence.

In addition, in the variant of the present application, the amino acid corresponding to position 302 based on the amino acid sequence of SEQ ID NO: 43 in the amino acid sequence set forth in SEQ ID NO: 41 is Serine, and the amino acid sequence set forth in SEQ ID NO: 1 and at least 70 may comprise an amino acid sequence having at least %, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% homology or identity. have. In addition, as long as it is an amino acid sequence having such homology or identity and exhibiting efficacy corresponding to the variant of the present application, variants having an amino acid sequence in which some sequences are deleted, modified, substituted, conservatively substituted or added are also included within the scope of the present application. is self-evident

As an example of the present application, the variant may have cysteine sulfinate disulfinase activity. In addition, the mutant may have an activity to increase the ability to produce L-valine compared to the wild-type polypeptide.

As used herein, the term "Cysteine sulfinate desulfinase (Cysteine sulfinate desulfinase)" is also called cysteine desulfurase or NadS, L-cysteine and receptor (acceptor; eg, [enzyme]-cysteine) as a substrate It is a polypeptide that catalyzes a reaction that produces L-alanine and S-sulfanyl-acceptor (eg, [enzyme]-S-sulfanylcysteine). In the present application, the cysteine sulfinate disulfinase sequence can be obtained from GenBank of NCBI, a known database (eg, WP_004568050.1, etc.). Specifically, it may be a polypeptide having cysteine sulfinate disulfinase activity encoded by the nadS gene, but is not limited thereto.

The polynucleotide encoding the variant may include a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 41. As an example of the present application, the polynucleotide of the present application may have or include the sequence of SEQ ID NO: 42. In addition, the polynucleotide of the present application may consist of, or consist essentially of, the sequence of SEQ ID NO: 42.

In another example, in the polynucleotide of the present application, in the nucleic acid sequence set forth in SEQ ID NO: 2, the base corresponding to position 904 based on the nucleic acid sequence of SEQ ID NO: 44 is T, and the nucleic acid sequence set forth in SEQ ID NO: 42 and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% or more homology or identity of a nucleic acid sequence. can In addition, if a sequence encoding a polypeptide or protein having such homology or identity and exhibiting efficacy corresponding to the variant of the present application is a polynucleotide having a nucleic acid sequence in which some sequences are deleted, modified, substituted, conservatively substituted, or added It is obvious that they are included within the scope of the present application.

In consideration of codon degeneracy or preferred codons in organisms that want to express the variants of the present application, the polynucleotides of the present application are various in the coding region within the range that does not change the amino acid sequence of the variants of the present application. Deformation can be made. Specifically, the polynucleotide of the present application has 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more homology or identity to the sequence of SEQ ID NO: 42 It has or contains a nucleotide sequence that is at least 98%, and less than 100%, or has 70% or more, 75% or more, 80% or more, 85% or more, 90% or more of homology or identity with the sequence of SEQ ID NO: 42; 95% or more, 96% or more, 97% or more, 98% or more, and less than 100% of the nucleotide sequence may consist of or consist essentially of, but is not limited thereto. In this case, in the sequence having the homology or identity, the codon encoding the amino acid corresponding to the 302th position of SEQ ID NO: 41 may be one of the codons encoding serine.

(6) protein variant (SEQ ID NO: 51) and polynucleotides encoding the same

Another aspect of the present application is the amino acid set forth in SEQ ID NO: 51 in which proline (Proline, Pro, P), which is an amino acid corresponding to the 56th position of the amino acid sequence of SEQ ID NO: 53, is substituted with leucine (Leucine, Leu, L) Provided is a urease accessory protein variant consisting of the sequence.

The variant of the present application may have or include the amino acid sequence set forth in SEQ ID NO: 51, or may consist essentially of the amino acid sequence.

In addition, in the variant of the present application, the amino acid corresponding to position 56 based on the amino acid sequence of SEQ ID NO: 53 in the amino acid sequence set forth in SEQ ID NO: 51 is leucine, and the amino acid sequence set forth in SEQ ID NO: 1 and at least 70 may comprise an amino acid sequence having at least %, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% homology or identity. have. In addition, as long as it is an amino acid sequence having such homology or identity and exhibiting efficacy corresponding to the variant of the present application, variants having an amino acid sequence in which some sequences are deleted, modified, substituted, conservatively substituted or added are also included within the scope of the present application. is self-evident

As an example of the present application, the variant may have urease accessory protein activity. In addition, the mutant may have an activity to increase the ability to produce L-valine compared to the wild-type polypeptide.

As used herein, the term "Urease accessory protein (Ure)" is a Ni-containing urea hydrolase involved in nitrogen recycling from arginine, ureide, and purine and , is involved in activating urease by delivering Ni to proteins. The urease accessory protein of the present application may be urease accessory protein UreE (UreE). In the present application, the sequence of the urease accessory protein UreE can be obtained from GenBank of NCBI, a known database (eg, WP_003859936.1, WP_038581987.1, WP_074492623.1, etc.). Specifically, it may be a polypeptide having a urease accessory protein activity encoded by the ureE gene, but is not limited thereto.

The polynucleotide encoding the variant may include a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 51. As an example of the present application, the polynucleotide of the present application may have or include the sequence of SEQ ID NO: 52. In addition, the polynucleotide of the present application may consist of, or consist essentially of, the sequence of SEQ ID NO: 52.

In another example, in the polynucleotide of the present application, in the nucleic acid sequence set forth in SEQ ID NO: 52, the base corresponding to position 167 based on the nucleic acid sequence of SEQ ID NO: 54 is T, and the nucleic acid sequence set forth in SEQ ID NO: 52 and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% or more homology or identity of a nucleic acid sequence. can In addition, if a sequence encoding a polypeptide or protein having such homology or identity and exhibiting efficacy corresponding to the variant of the present application is a polynucleotide having a nucleic acid sequence in which some sequences are deleted, modified, substituted, conservatively substituted, or added It is obvious that they are included within the scope of the present application.

In consideration of codon degeneracy or preferred codons in organisms that want to express the variants of the present application, the polynucleotides of the present application are various in the coding region within the range that does not change the amino acid sequence of the variants of the present application. Deformation can be made. Specifically, the polynucleotide of the present application has 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more homology or identity to the sequence of SEQ ID NO: 52 Having or including a nucleotide sequence that is more than, 98% or more, and less than 100%, or homology or identity with the sequence of SEQ ID NO: 2 is 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, and less than 100% of the nucleotide sequence may consist of or consist essentially of, but is not limited thereto. In this case, in the sequence having the homology or identity, the codon encoding the amino acid corresponding to the 56th position of SEQ ID NO: 51 may be one of the codons encoding leucine.

(7) protein variant (SEQ ID NO: 61) and polynucleotides encoding the same

Another aspect of the present application is the amino acid set forth in SEQ ID NO: 61 in which glycine (Glycine, Gly, G), which is an amino acid corresponding to the 834th position of the amino acid sequence of SEQ ID NO: 63, is substituted with serine (Serine, Ser, S) A proline dehydrogenase variant comprising the sequence is provided.

The variant of the present application may have or include the amino acid sequence set forth in SEQ ID NO: 61, or may consist essentially of the amino acid sequence.

In addition, in the variant of the present application, the amino acid corresponding to position 834 based on the amino acid sequence of SEQ ID NO: 63 in the amino acid sequence set forth in SEQ ID NO: 61 is serine, and the amino acid sequence set forth in SEQ ID NO: 61 and at least 70 may comprise an amino acid sequence having at least %, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% homology or identity. have. In addition, as long as it is an amino acid sequence having such homology or identity and exhibiting efficacy corresponding to the variant of the present application, variants having an amino acid sequence in which some sequences are deleted, modified, substituted, conservatively substituted or added are also included within the scope of the present application. is self-evident

As an example of the present application, the variant may have proline dehydrogenase activity. In addition, the mutant may have an activity to increase the ability to produce L-valine compared to the wild-type polypeptide.

As used herein, the term "proline dehydrogenase" is a polypeptide belonging to the oxidoreductase family, and (S)-1-pyrroline-5-carboxylate [ (S)-1-pyrroline-5-carboxylate] and a polypeptide that catalyzes a reaction to produce ubiquinol. In the present application, the proline dehydrogenase can be used in combination with PutA, and its sequence can be obtained from GenBank of NCBI, a known database (eg, WP_034983428.1, etc.). Specifically, it may be a polypeptide having proline dehydrogenase activity encoded by the putA gene, but is not limited thereto.

The polynucleotide encoding the variant of the present application may include a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 61. As an example of the present application, the polynucleotide of the present application may have or include the sequence of SEQ ID NO: 62. In addition, the polynucleotide of the present application may consist of, or consist essentially of, the sequence of SEQ ID NO: 62.

In another example, in the polynucleotide of the present application, the base corresponding to position 2500 based on the nucleic acid sequence of SEQ ID NO: 64 in the nucleic acid sequence set forth in SEQ ID NO: 62 is A, and at least the nucleic acid sequence set forth in SEQ ID NO: 62 and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% or more homology or identity of a nucleic acid sequence. can In addition, if a sequence encoding a polypeptide or protein having such homology or identity and exhibiting efficacy corresponding to the variant of the present application is a polynucleotide having a nucleic acid sequence in which some sequences are deleted, modified, substituted, conservatively substituted, or added It is obvious that they are included within the scope of the present application.

In consideration of codon degeneracy or preferred codons in organisms that want to express the variants of the present application, the polynucleotides of the present application are various in the coding region within the range that does not change the amino acid sequence of the variants of the present application. Deformation can be made. Specifically, the polynucleotide of the present application has 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more homology or identity to the sequence of SEQ ID NO: 62 It has or includes a nucleotide sequence that is greater than or equal to 98%, and less than 100%, or has 70% or more, 75% or more, 80% or more, 85% or more, 90% or more of homology or identity to the sequence of SEQ ID NO: 62; 95% or more, 96% or more, 97% or more, 98% or more, and less than 100% of the nucleotide sequence may consist of or consist essentially of, but is not limited thereto. In this case, in the sequence having the homology or identity, the codon encoding the amino acid corresponding to the 834th position of SEQ ID NO: 61 may be one of the codons encoding serine.

(8) protein variant (SEQ ID NO: 71) and polynucleotides encoding the same

Another aspect of the present application is an amino acid set forth in SEQ ID NO: 71 in which arginine (Arginine, Arg, R), which is an amino acid corresponding to the 140th position of the amino acid sequence of SEQ ID NO: 73, is substituted with cysteine (Cysteine, Cys, C) Provided is a tetrahydrodipicolinate N-succinyltransferase variant consisting of the sequence.

The variant of the present application may have or include the amino acid sequence set forth in SEQ ID NO: 71, or may consist essentially of the amino acid sequence.

In addition, in the variant of the present application, the amino acid corresponding to position 140 based on the amino acid sequence of SEQ ID NO: 73 in the amino acid sequence set forth in SEQ ID NO: 71 is cysteine, and the amino acid sequence set forth in SEQ ID NO: 71 and at least 70 may comprise an amino acid sequence having at least %, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% homology or identity. have. In addition, as long as it is an amino acid sequence having such homology or identity and exhibiting efficacy corresponding to the variant of the present application, variants having an amino acid sequence in which some sequences are deleted, modified, substituted, conservatively substituted or added are also included within the scope of the present application. is self-evident

As an example of the present application, the variant may have tetrahydrodipicolinate N-succinyltransferase activity. In addition, the mutant may have an activity to increase the ability to produce L-valine compared to the wild-type polypeptide.

As used herein, the term "Tetrahydrodipicolinate N-succinyltransferase" is an acyltransferase-based polypeptide, succinyl-CoA, (S)-2,3,4,5-tetra Hydropyridine-2,6-dicarboxylate, and CoA and N-succinyl-L- ₂ -amino-6-oxoheptanedioate (N-succinyl-L-2-amino- It is a polypeptide that catalyzes a reaction that produces 6-oxoheptanedioate). Tetrahydrodipicolinate N-succinyltransferase of the present application is tetrahydropicolinate succinylase, succinyl-CoA:tetrahydrodipicolinate N-succinyltransferase (succinyl-CoA:tetrahydrodipicolinate) N-succinyltransferase) or DapD2 may be used in combination. In the present application, the tetrahydrodipicolinate N-succinyltransferase sequence can be obtained from GenBank of NCBI, a known database (eg, WP_003863792.1, etc.). Specifically, it may be a polypeptide having tetrahydrodipicolinate N-succinyltransferase activity encoded by the dapD2 gene, but is not limited thereto.

The polynucleotide encoding the variant may include a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 71. As an example of the present application, the polynucleotide of the present application may have or include the sequence of SEQ ID NO: 72. In addition, the polynucleotide of the present application may consist of, or consist essentially of, the sequence of SEQ ID NO: 72.

In another example, in the polynucleotide of the present application, in the nucleic acid sequence set forth in SEQ ID NO: 72, the base corresponding to position 418 based on the nucleic acid sequence of SEQ ID NO: 74 is T, and at least the nucleic acid sequence set forth in SEQ ID NO: 72 and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% or more homology or identity of a nucleic acid sequence. can In addition, if a sequence encoding a polypeptide or protein having such homology or identity and exhibiting efficacy corresponding to the variant of the present application is a polynucleotide having a nucleic acid sequence in which some sequences are deleted, modified, substituted, conservatively substituted, or added It is obvious that they are included within the scope of the present application.

In consideration of codon degeneracy or preferred codons in organisms that want to express the variants of the present application, the polynucleotides of the present application are various in the coding region within the range that does not change the amino acid sequence of the variants of the present application. Deformation can be made. Specifically, the polynucleotide of the present application has 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more homology or identity to the sequence of SEQ ID NO: 72 It has or contains a nucleotide sequence of at least 98%, and less than 100%, or homology or identity with the sequence of SEQ ID NO: 72 is 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, and less than 100% of the nucleotide sequence may consist of or consist essentially of, but is not limited thereto. In this case, in the sequence having the homology or identity, the codon encoding the amino acid corresponding to the 140th position of SEQ ID NO: 71 may be one of the codons encoding cysteine.

(9) protein variant (SEQ ID NO: 81) and polynucleotides encoding the same

One aspect of the present application is an amino acid sequence set forth in SEQ ID NO: 81 in which arginine (Arginine, Arg, R), which is an amino acid corresponding to the 4th position of the amino acid sequence of SEQ ID NO: 83, is substituted with histidine (Histidine, His, H) It provides a 5,10-methylenetetrahydrofolate reductase variant consisting of.

The variant of the present application may have or include the amino acid sequence set forth in SEQ ID NO: 81, or may consist essentially of the amino acid sequence.

In addition, in the variant of the present application, the amino acid corresponding to position 4 based on the amino acid sequence of SEQ ID NO: 83 in the amino acid sequence set forth in SEQ ID NO: 81 is histidine, and the amino acid sequence set forth in SEQ ID NO: 1 and at least 70 may comprise an amino acid sequence having at least %, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% homology or identity. have. In addition, as long as it is an amino acid sequence having such homology or identity and exhibiting efficacy corresponding to the variant of the present application, variants having an amino acid sequence in which some sequences are deleted, modified, substituted, conservatively substituted or added are also included within the scope of the present application. is self-evident

As an example of the present application, the variant may have 5,10-methylenetetrahydrofolate reductase activity. In addition, the mutant may have an activity to increase the ability to produce L-valine compared to the wild-type polypeptide.

As used herein, the term "5,10-methylenetetrahydrofolate reductase" refers to a compound that catalyzes the conversion of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate. is a polypeptide. In the present application, the 5,10-methylenetetrahydrofolate reductase can be used in combination with MetF, and its sequence can be obtained from GenBank of NCBI, a known database (eg, WP_003863792.1, etc.). Specifically, it may be a polypeptide having 5,10-methylenetetrahydrofolate reductase activity encoded by the metF gene, but is not limited thereto.

The polynucleotide encoding the variant of the present application may include a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 81. As an example of the present application, the polynucleotide of the present application may have or include the sequence of SEQ ID NO: 82. In addition, the polynucleotide of the present application may consist of, or consist essentially of, the sequence of SEQ ID NO: 82.

In another example, in the polynucleotide of the present application, in the nucleic acid sequence set forth in SEQ ID NO: 82, the base corresponding to position 11 based on the nucleic acid sequence of SEQ ID NO: 84 is A, and the nucleic acid sequence set forth in SEQ ID NO: 82 and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% or more homology or identity of a nucleic acid sequence. can In addition, if a sequence encoding a polypeptide or protein having such homology or identity and exhibiting efficacy corresponding to the variant of the present application is a polynucleotide having a nucleic acid sequence in which some sequences are deleted, modified, substituted, conservatively substituted, or added It is obvious that they are included within the scope of the present application.

In consideration of codon degeneracy or preferred codons in organisms that want to express the variants of the present application, the polynucleotides of the present application are various in the coding region within the range that does not change the amino acid sequence of the variants of the present application. Deformation can be made. Specifically, the polynucleotide of the present application has 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more homology or identity to the sequence of SEQ ID NO: 82 or more, 98% or more, and less than 100% of the base sequence, or the homology or identity with the sequence of SEQ ID NO: 82 is 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, and less than 100% of the nucleotide sequence may consist of or consist essentially of, but is not limited thereto. In this case, in the sequence having the homology or identity, the codon encoding the amino acid corresponding to the 4th position of SEQ ID NO: 81 may be one of the codons encoding histidine.

(10) protein variant (SEQ ID NO: 91) and polynucleotides encoding the same

Another aspect of the present application is that glutamic acid (Glutamic acid, Glu, E), which is an amino acid corresponding to position 253 of the amino acid sequence of SEQ ID NO: 93, is substituted with lysine (Lysine, Lys, K), described in SEQ ID NO: 91 Protein variants, consisting of an amino acid sequence, are provided.

The variant of the present application may have or include the amino acid sequence set forth in SEQ ID NO: 91, or may consist essentially of the amino acid sequence.

In addition, in the variant of the present application, the amino acid corresponding to position 253 based on the amino acid sequence of SEQ ID NO: 93 in the amino acid sequence set forth in SEQ ID NO: 91 is lysine, and the amino acid sequence set forth in SEQ ID NO: 91 and at least 70 may comprise an amino acid sequence having at least %, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% homology or identity. have. In addition, as long as it is an amino acid sequence having such homology or identity and exhibiting efficacy corresponding to the variant of the present application, variants having an amino acid sequence in which some sequences are deleted, modified, substituted, conservatively substituted or added are also included within the scope of the present application. is self-evident

The polynucleotide encoding the variant may include a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 91. As an example of the present application, the polynucleotide of the present application may have or include the sequence of SEQ ID NO: 92. In addition, the polynucleotide of the present application may consist of, or consist essentially of, the sequence of SEQ ID NO: 92.

In another example, in the polynucleotide of the present application, in the nucleic acid sequence set forth in SEQ ID NO: 92, the base corresponding to position 757 based on the nucleic acid sequence of SEQ ID NO: 94 is A, and the nucleic acid sequence set forth in SEQ ID NO: 92 and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% or more homology or identity of a nucleic acid sequence. can In addition, if a sequence encoding a polypeptide or protein having such homology or identity and exhibiting efficacy corresponding to the variant of the present application is a polynucleotide having a nucleic acid sequence in which some sequences are deleted, modified, substituted, conservatively substituted, or added It is obvious that they are included within the scope of the present application.

In consideration of codon degeneracy or preferred codons in organisms that want to express the variants of the present application, the polynucleotides of the present application are various in the coding region within the range that does not change the amino acid sequence of the variants of the present application. Deformation can be made. Specifically, the polynucleotide of the present application has 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more homology or identity to the sequence of SEQ ID NO: 92 It has or contains a nucleotide sequence of at least 98%, and less than 100%, or homology or identity with the sequence of SEQ ID NO: 92 is 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, and less than 100% of the nucleotide sequence may consist of or consist essentially of, but is not limited thereto. In this case, in the sequence having the homology or identity, the codon encoding the amino acid corresponding to the 253th position of SEQ ID NO: 91 may be one of the codons encoding the lysine.

(11) protein variant (SEQ ID NO: 101) and polynucleotides encoding the same

One aspect of the present application is the amino acid sequence set forth in SEQ ID NO: 101 in which alanine (Alanine, Ala, A), which is an amino acid corresponding to position 315 of the amino acid sequence of SEQ ID NO: 103, is substituted with valine (Valine, Val, V) It provides a sugar porter family MFS transporter variant consisting of.

The variant of the present application may have or include the amino acid sequence set forth in SEQ ID NO: 101, or may consist essentially of the amino acid sequence.

In addition, in the variant of the present application, the amino acid corresponding to position 315 based on the amino acid sequence of SEQ ID NO: 103 in the amino acid sequence set forth in SEQ ID NO: 101 is valine, and the amino acid sequence set forth in SEQ ID NO: 101 and at least 70 may comprise an amino acid sequence having at least %, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% homology or identity. have. In addition, as long as it is an amino acid sequence having such homology or identity and exhibiting efficacy corresponding to the variant of the present application, variants having an amino acid sequence in which some sequences are deleted, modified, substituted, conservatively substituted or added are also included within the scope of the present application. is self-evident

As an example of the present application, the mutant may have sugar porter-based MFS transporter activity. In addition, the mutant may have an activity to increase the ability to produce L-valine compared to the wild-type polypeptide.

As used herein, the term "sugar porter family MFS transporter" is one of inositol transporters. In the present application, the sugar porter-based MFS transporter can be used in combination with IolT1, and its sequence can be obtained from GenBank of NCBI, a known database (eg, WP_003862502.1). Specifically, it may be a polypeptide having sugar porter-type MFS transporter activity encoded by the iolT1 gene, but is not limited thereto.

The polynucleotide encoding the variant may include a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 101. As an example of the present application, the polynucleotide of the present application may have or include the sequence of SEQ ID NO: 102. In addition, the polynucleotide of the present application may consist of, or consist essentially of, the sequence of SEQ ID NO: 102.

In another example, in the polynucleotide of the present application, the base corresponding to position 944 based on the nucleic acid sequence of SEQ ID NO: 104 in the nucleic acid sequence set forth in SEQ ID NO: 102 is T, and at least the nucleic acid sequence set forth in SEQ ID NO: 102 and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% or more homology or identity of a nucleic acid sequence. can In addition, if a sequence encoding a polypeptide or protein having such homology or identity and exhibiting efficacy corresponding to the variant of the present application is a polynucleotide having a nucleic acid sequence in which some sequences are deleted, modified, substituted, conservatively substituted, or added It is obvious that they are included within the scope of the present application.

In consideration of codon degeneracy or preferred codons in organisms that want to express the variants of the present application, the polynucleotides of the present application are various in the coding region within the range that does not change the amino acid sequence of the variants of the present application. Deformation can be made. Specifically, the polynucleotide of the present application has 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more homology or identity to the sequence of SEQ ID NO: 102 Having or including a nucleotide sequence that is greater than or equal to 98%, and less than 100%, or homology or identity with the sequence of SEQ ID NO: 102 is 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, and less than 100% of the nucleotide sequence may consist of or consist essentially of, but is not limited thereto. In this case, in the sequence having the homology or identity, the codon encoding the amino acid corresponding to the 315th position of SEQ ID NO: 101 may be one of the codons encoding valine.

(12) protein variant (SEQ ID NO: 111) and polynucleotide encoding the same

One aspect of the present application is an amino acid set forth in SEQ ID NO: 111 in which glutamic acid (Glu, E), which is an amino acid corresponding to the 247th position of the amino acid sequence of SEQ ID NO: 113, is substituted with lysine (Lysine, Lys, K) A transcriptional regulator variant, consisting of the sequence, is provided.

The variant of the present application may have or include the amino acid sequence set forth in SEQ ID NO: 111, or may consist essentially of the amino acid sequence.

In addition, in the variant of the present application, the amino acid corresponding to position 247 based on the amino acid sequence of SEQ ID NO: 113 in the amino acid sequence set forth in SEQ ID NO: 111 is lysine, and the amino acid sequence described in SEQ ID NO: 111 and at least 70 may comprise an amino acid sequence having at least %, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% homology or identity. have. In addition, as long as it is an amino acid sequence having such homology or identity and exhibiting efficacy corresponding to the variant of the present application, variants having an amino acid sequence in which some sequences are deleted, modified, substituted, conservatively substituted or added are also included within the scope of the present application. is self-evident

As an example of the present application, the mutant may have transcriptional regulator activity. In addition, the mutant may have an activity to increase the ability to produce L-valine compared to the wild-type polypeptide.

As used herein, the term "iolR, a transcriptional regulator," is a transcriptional regulator involved in Myo-inositol utilization. In the present application, the transcription regulator can be used in combination with IolR, and its sequence can be obtained from GenBank of NCBI, a known database (eg, WP_003857140.1). Specifically, it may be a polypeptide having a transcriptional regulator activity encoded by the iolR gene, but is not limited thereto.

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

The polynucleotide encoding the variant of the present application may include a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 111. As an example of the present application, the polynucleotide of the present application may have or include the sequence of SEQ ID NO: 112. In addition, the polynucleotide of the present application may consist of, or consist essentially of, the sequence of SEQ ID NO: 112.

In another example, in the polynucleotide of the present application, in the nucleic acid sequence set forth in SEQ ID NO: 112, the base corresponding to position 739 based on the nucleic acid sequence of SEQ ID NO: 114 is A, and the nucleic acid sequence set forth in SEQ ID NO: 112 and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% or more homology or identity of a nucleic acid sequence. can In addition, if a sequence encoding a polypeptide or protein having such homology or identity and exhibiting efficacy corresponding to the variant of the present application is a polynucleotide having a nucleic acid sequence in which some sequences are deleted, modified, substituted, conservatively substituted, or added It is obvious that they are included within the scope of the present application.

In consideration of codon degeneracy or preferred codons in organisms that want to express the variants of the present application, the polynucleotides of the present application are various in the coding region within the range that does not change the amino acid sequence of the variants of the present application. Deformation can be made. Specifically, the polynucleotide of the present application has 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more homology or identity to the sequence of SEQ ID NO: 112 It has or contains a nucleotide sequence that is more than, 98% or more, and less than 100%, or has 70% or more, 75% or more, 80% or more, 85% or more, 90% or more of homology or identity with the sequence of SEQ ID NO: 112; 95% or more, 96% or more, 97% or more, 98% or more, and less than 100% of the nucleotide sequence may consist of or consist essentially of, but is not limited thereto. In this case, in the sequence having the homology or identity, the codon encoding the amino acid corresponding to the 247th position of SEQ ID NO: 111 may be one of the codons encoding the lysine.

(13) protein variant (SEQ ID NO: 121) and polynucleotides encoding the same

Another aspect of the present application is that glycine (Glycine, Gly, G), which is an amino acid corresponding to the 40th position of the amino acid sequence of SEQ ID NO: 123, is substituted with aspartic acid (Aspartic acid, Asp, D), SEQ ID NO: 121 It provides a variant of the WhiB family transcriptional regulator WhcA, consisting of the amino acid sequence described as .

The variant of the present application may have or include the amino acid sequence set forth in SEQ ID NO: 121, or may consist essentially of the amino acid sequence.

In addition, in the variant of the present application, the amino acid corresponding to position 40 based on the amino acid sequence of SEQ ID NO: 123 in the amino acid sequence set forth in SEQ ID NO: 121 is aspartic acid, and the amino acid sequence set forth in SEQ ID NO: 121 an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.9% homology or identity with may include In addition, as long as it is an amino acid sequence having such homology or identity and exhibiting efficacy corresponding to the variant of the present application, variants having an amino acid sequence in which some sequences are deleted, modified, substituted, conservatively substituted or added are also included within the scope of the present application. is self-evident

As an example of the present application, the mutant may have a WhiB-series transcriptional regulator WhcA activity. In addition, the mutant may have an activity to increase the ability to produce L-valine compared to the wild-type polypeptide.

In the present application, the term "WhcA (WhiB family transcriptional regulator WhcA)" may be used interchangeably with WhcA in this application, and the sequence can be obtained from GenBank of NCBI, a known database (eg, WP_003863319.1). Specifically, it may be a polypeptide having a WhiB family transcriptional regulator WhcA activity encoded by the whcA gene, but is not limited thereto.

The polynucleotide encoding the variant of the present application may include a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 121. As an example of the present application, the polynucleotide of the present application may have or include the sequence of SEQ ID NO: 122. In addition, the polynucleotide of the present application may consist of, or consist essentially of, the sequence of SEQ ID NO: 122.

In another example, in the polynucleotide of the present application, in the nucleic acid sequence set forth in SEQ ID NO: 122, the base corresponding to position 119 based on the nucleic acid sequence of SEQ ID NO: 124 is A, and the nucleic acid sequence set forth in SEQ ID NO: 122 and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% or more homology or identity of a nucleic acid sequence. can In addition, if a sequence encoding a polypeptide or protein having such homology or identity and exhibiting efficacy corresponding to the variant of the present application is a polynucleotide having a nucleic acid sequence in which some sequences are deleted, modified, substituted, conservatively substituted, or added It is obvious that they are included within the scope of the present application.

In consideration of codon degeneracy or preferred codons in organisms that want to express the variants of the present application, the polynucleotides of the present application are various in the coding region within the range that does not change the amino acid sequence of the variants of the present application. Deformation can be made. Specifically, the polynucleotide of the present application has 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more homology or identity to the sequence of SEQ ID NO: 122 It has or contains a nucleotide sequence that is at least 98%, and less than 100%, or has 70% or more, 75% or more, 80% or more, 85% or more, 90% or more of homology or identity to the sequence of SEQ ID NO: 122; 95% or more, 96% or more, 97% or more, 98% or more, and less than 100% of the nucleotide sequence may consist of or consist essentially of, but is not limited thereto. In this case, in the sequence having the homology or identity, the codon encoding the amino acid corresponding to the 40th position of SEQ ID NO: 121 may be one of the codons encoding aspartic acid.

(14) protein variant (SEQ ID NO: 131) and polynucleotides encoding the same

Another aspect of the present application is that glycine (Glycine, Gly, G), which is an amino acid corresponding to the 77th position of the amino acid sequence of SEQ ID NO: 133, is substituted with aspartic acid (Aspartic acid, Asp, D), SEQ ID NO: 131 It provides a dihydrolipoyl acetyltransferase variant, consisting of the amino acid sequence described as.

The variant of the present application may have or include the amino acid sequence set forth in SEQ ID NO: 131, or may consist essentially of the amino acid sequence.

In addition, in the variant of the present application, the amino acid corresponding to position 77 based on the amino acid sequence of SEQ ID NO: 133 in the amino acid sequence set forth in SEQ ID NO: 131 is aspartic acid, and the amino acid sequence set forth in SEQ ID NO: 131 an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.9% homology or identity with may include In addition, as long as it is an amino acid sequence having such homology or identity and exhibiting efficacy corresponding to the variant of the present application, variants having an amino acid sequence in which some sequences are deleted, modified, substituted, conservatively substituted or added are also included within the scope of the present application. is self-evident

As an example of the present application, the variant may have dihydrolipoyl acetyltransferase activity. In addition, the mutant may have an activity to increase the ability to produce L-valine compared to the wild-type polypeptide.

As used herein, the term "dihydrolipoamide acetyltransferase" is an enzyme component of the multi-enzyme pyruvate dehydrogenase complex. The pyruvate dehydrogenase complex is responsible for the pyruvate decarboxylation step that links glycolysis to the citric acid cycle. In the present application, the dihydrolipoyl acetyltransferase can be used in combination with AceF, and its sequence can be obtained from GenBank of NCBI, a known database (eg, WP_034983836.1). Specifically, it may be a polypeptide having a dihydrolipoyl acetyltransferase activity encoded by the aceF gene, but is not limited thereto.

The polynucleotide encoding the variant of the present application may include a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 131. As an example of the present application, the polynucleotide of the present application may have or include the sequence of SEQ ID NO: 132. In addition, the polynucleotide of the present application may consist of, or consist essentially of, the sequence of SEQ ID NO: 132.

In another example, in the polynucleotide of the present application, the base corresponding to position 230 based on the nucleic acid sequence of SEQ ID NO: 134 in the nucleic acid sequence set forth in SEQ ID NO: 132 is A, and at least the nucleic acid sequence set forth in SEQ ID NO: 132 and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% or more homology or identity of a nucleic acid sequence. can In addition, if a sequence encoding a polypeptide or protein having such homology or identity and exhibiting efficacy corresponding to the variant of the present application is a polynucleotide having a nucleic acid sequence in which some sequences are deleted, modified, substituted, conservatively substituted, or added It is obvious that they are included within the scope of the present application.

In consideration of codon degeneracy or preferred codons in organisms that want to express the variants of the present application, the polynucleotides of the present application are various in the coding region within the range that does not change the amino acid sequence of the variants of the present application. Deformation can be made. Specifically, the polynucleotide of the present application has 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% homology or identity to the sequence of SEQ ID NO: 132 or more, 98% or more, and less than 100% of the base sequence, or homology or identity with the sequence of SEQ ID NO: 132 is 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, and less than 100% of the nucleotide sequence may consist of or consist essentially of, but is not limited thereto. In this case, in the sequence having the homology or identity, the codon encoding the amino acid corresponding to the 77th position of SEQ ID NO: 131 may be one of the codons encoding aspartic acid.

(15) protein variant (SEQ ID NO: 141) and polynucleotides encoding the same

Another aspect of the present application is the amino acid set forth in SEQ ID NO: 141 in which proline (Proline, Pro, P), which is an amino acid corresponding to the 87th position of the amino acid sequence of SEQ ID NO: 143, is substituted with leucine (Leucine, Leu, L) Provided is a 2-isopropylmalate synthetase variant consisting of the sequence.

The variant of the present application may have or include the amino acid sequence set forth in SEQ ID NO: 141, or may consist essentially of the amino acid sequence.

In addition, in the variant of the present application, the amino acid corresponding to position 87 based on the amino acid sequence of SEQ ID NO: 143 in the amino acid sequence set forth in SEQ ID NO: 141 is leucine, and the amino acid sequence set forth in SEQ ID NO: 141 and at least 70 may comprise an amino acid sequence having at least %, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% homology or identity. have. In addition, as long as it is an amino acid sequence having such homology or identity and exhibiting efficacy corresponding to the variant of the present application, variants having an amino acid sequence in which some sequences are deleted, modified, substituted, conservatively substituted or added are also included within the scope of the present application. is self-evident

As an example of the present application, the variant may have 2-isopropylmalate synthetase activity. In addition, the mutant may have an activity to increase the ability to produce L-valine compared to the wild-type polypeptide.

As used herein, the term "2-isopropylmalate synthase" refers to a chemical reaction of acetyl-CoA + 3-methyl-2-oxobutanoate + H2O ↔ (2S)-2-isopropylmalate + CoA. It is an enzyme that catalyzes In the present application, the 2-isopropyl malate synthetase can be used in combination with LeuA, and its sequence can be obtained from GenBank of NCBI, a known database (eg, WP_172768021.1). Specifically, it may be a polypeptide having 2-isopropyl malate synthase activity encoded by the leuA gene, but is not limited thereto.

The polynucleotide encoding the variant may include a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 141. As an example of the present application, the polynucleotide of the present application may have or include the sequence of SEQ ID NO: 142. In addition, the polynucleotide of the present application may consist of, or consist essentially of, the sequence of SEQ ID NO: 142.

In another example, in the polynucleotide of the present application, in the nucleic acid sequence set forth in SEQ ID NO: 142, the base corresponding to position 260 based on the nucleic acid sequence of SEQ ID NO: 144 is T, and the nucleic acid sequence set forth in SEQ ID NO: 142 and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% or more homology or identity of a nucleic acid sequence. can In addition, if a sequence encoding a polypeptide or protein having such homology or identity and exhibiting efficacy corresponding to the variant of the present application is a polynucleotide having a nucleic acid sequence in which some sequences are deleted, modified, substituted, conservatively substituted, or added It is obvious that they are included within the scope of the present application.

In consideration of codon degeneracy or preferred codons in organisms that want to express the variants of the present application, the polynucleotides of the present application are various in the coding region within the range that does not change the amino acid sequence of the variants of the present application. Deformation can be made. Specifically, the polynucleotide of the present application has 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more of homology or identity to the sequence of SEQ ID NO: 142. It has or contains a nucleotide sequence that is more than, 98% or more, and less than 100%, or has 70% or more, 75% or more, 80% or more, 85% or more, 90% or more of homology or identity with the sequence of SEQ ID NO: 142; 95% or more, 96% or more, 97% or more, 98% or more, and less than 100% of the nucleotide sequence may consist of or consist essentially of, but is not limited thereto. In this case, in the sequence having the homology or identity, the codon encoding the amino acid corresponding to the 87th position of SEQ ID NO: 141 may be one of the codons encoding leucine.

(16) protein variant (SEQ ID NO: 151) and polynucleotides encoding the same

Another aspect of the present application is the amino acid set forth in SEQ ID NO: 151 in which alanine (Alanine, Ala, A), which is an amino acid corresponding to the 112th position of the amino acid sequence of SEQ ID NO: 153, is substituted with threonine (Threonine, Thr, T) Provided is a branched chain amino acid permease variant consisting of the sequence.

The variant of the present application may have or include the amino acid sequence set forth in SEQ ID NO: 151, or may consist essentially of the amino acid sequence.

In addition, in the variant of the present application, the amino acid corresponding to position 112 based on the amino acid sequence of SEQ ID NO: 153 in the amino acid sequence set forth in SEQ ID NO: 151 is Threonine, and the amino acid sequence set forth in SEQ ID NO: 151 and at least 70 may comprise an amino acid sequence having at least %, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% homology or identity. have. In addition, as long as it is an amino acid sequence having such homology or identity and exhibiting efficacy corresponding to the variant of the present application, variants having an amino acid sequence in which some sequences are deleted, modified, substituted, conservatively substituted or added are also included within the scope of the present application. is self-evident

As an example of the present application, the variant may have branched-chain amino acid permease activity. In addition, the mutant may have an activity to increase the ability to produce L-valine compared to the wild-type polypeptide.

As used herein, the term "branched-chain amino acid permease" is a component of a branched-chain amino acid (leucine, isoleucine, valine) transport system that operates by a proton motive force (PMF). . In the present application, the branched-chain amino acid permease can be used in combination with BrnQ, and its sequence can be obtained from GenBank of NCBI, a known database (eg, WP_080708840.1). Specifically, it may be a polypeptide having a branched-chain amino acid permease activity encoded by the brnQ gene, but is not limited thereto.

The polynucleotide encoding the variant may include a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 151. As an example of the present application, the polynucleotide of the present application may have or include the sequence of SEQ ID NO: 152. In addition, the polynucleotide of the present application may consist of or consist essentially of the sequence of SEQ ID NO: 152.

In another example, in the polynucleotide of the present application, in the nucleic acid sequence set forth in SEQ ID NO: 152, the base corresponding to position 334 based on the nucleic acid sequence of SEQ ID NO: 154 is A, and the nucleic acid sequence set forth in SEQ ID NO: 152 and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% or more homology or identity of a nucleic acid sequence. can In addition, if a sequence encoding a polypeptide or protein having such homology or identity and exhibiting efficacy corresponding to the variant of the present application is a polynucleotide having a nucleic acid sequence in which some sequences are deleted, modified, substituted, conservatively substituted, or added It is obvious that they are included within the scope of the present application.

In consideration of codon degeneracy or preferred codons in organisms that want to express the variants of the present application, the polynucleotides of the present application are various in the coding region within the range that does not change the amino acid sequence of the variants of the present application. Deformation can be made. Specifically, the polynucleotide of the present application has 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more homology or identity to the sequence of SEQ ID NO: 152 It has or contains a nucleotide sequence that is greater than or equal to 98%, and less than 100%, or homology or identity with the sequence of SEQ ID NO: 152 is 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, and less than 100% of the nucleotide sequence may consist of or consist essentially of, but is not limited thereto. In this case, in the sequence having the homology or identity, the codon encoding the amino acid corresponding to the 112th position of SEQ ID NO: 151 may be one of the codons encoding threonine.

(17) protein variant (SEQ ID NO: 161) and polynucleotides encoding the same

Another aspect of the present application is that glycine (Glycine, Gly, G), which is an amino acid corresponding to the 134th position of the amino acid sequence of SEQ ID NO: 163, is substituted with serine (Serine, Ser, S), the amino acid set forth in SEQ ID NO: 161 A glyceraldehyde-3-phosphate dehydrogenase variant comprising the sequence is provided.

The variant of the present application may have or include the amino acid sequence set forth in SEQ ID NO: 161, or may consist essentially of the amino acid sequence.

In addition, in the variant of the present application, the amino acid corresponding to position 134 based on the amino acid sequence of SEQ ID NO: 163 in the amino acid sequence set forth in SEQ ID NO: 161 is Serine, and the amino acid sequence set forth in SEQ ID NO: 161 and at least 70 may comprise an amino acid sequence having at least %, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% homology or identity. have. In addition, as long as it is an amino acid sequence having such homology or identity and exhibiting efficacy corresponding to the variant of the present application, variants having an amino acid sequence in which some sequences are deleted, modified, substituted, conservatively substituted or added are also included within the scope of the present application. is self-evident

As an example of the present application, the variant may have glyceraldehyde-3-phosphate dehydrogenase activity. In addition, the mutant may have an activity to increase the ability to produce L-valine compared to the wild-type polypeptide.

As used herein, the term "glyceraldehyde-3-phosphate dehydrogenase" is an enzyme of about 37 kDa that catalyzes the sixth step of glycolysis, and converts glyceraldehyde 3-phosphate to 1 It catalyzes the reversible reaction of conversion to ,3-bisphosphoglyceric acid. In the present application, the glyceraldehyde-3-phosphate dehydrogenase can be used in combination with GapA, and its sequence can be obtained from GenBank of NCBI, a known database (eg, WP_003862250.1). Specifically, it may be a polypeptide having glyceraldehyde-3-phosphate dehydrogenase activity encoded by the gapA gene, but is not limited thereto.

The polynucleotide encoding the variant may include a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 161. As an example of the present application, the polynucleotide of the present application may have or include the sequence of SEQ ID NO: 162. In addition, the polynucleotide of the present application may consist of, or consist essentially of, the sequence of SEQ ID NO: 162.

In another example, in the polynucleotide of the present application, in the nucleic acid sequence set forth in SEQ ID NO: 162, the base corresponding to position 400 based on the nucleic acid sequence of SEQ ID NO: 164 is A, and the nucleic acid sequence set forth in SEQ ID NO: 162 and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% or more homology or identity of a nucleic acid sequence. can In addition, if a sequence encoding a polypeptide or protein having such homology or identity and exhibiting efficacy corresponding to the variant of the present application is a polynucleotide having a nucleic acid sequence in which some sequences are deleted, modified, substituted, conservatively substituted, or added It is obvious that they are included within the scope of the present application.

In consideration of codon degeneracy or preferred codons in organisms that want to express the variants of the present application, the polynucleotides of the present application are various in the coding region within the range that does not change the amino acid sequence of the variants of the present application. Deformation can be made. Specifically, the polynucleotide of the present application has 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% homology or identity to the sequence of SEQ ID NO: 162 It has or contains a nucleotide sequence of at least 98%, and less than 100%, or has 70% or more, 75% or more, 80% or more, 85% or more, 90% or more of homology or identity to the sequence of SEQ ID NO: 162; 95% or more, 96% or more, 97% or more, 98% or more, and less than 100% of the nucleotide sequence may consist of or consist essentially of, but is not limited thereto. In this case, in the sequence having the homology or identity, the codon encoding the amino acid corresponding to the 134th position of SEQ ID NO: 161 may be one of the codons encoding serine.

As used herein, the term "conservative substitution" means substituting one amino acid for another amino acid having similar structural and/or chemical properties. Such amino acid substitutions may generally occur based on similarity in the polarity, charge, solubility, hydrophobicity, hydrophilicity and/or amphipathic nature of the residues. Typically, conservative substitutions may have little or no effect on the activity of the protein or polypeptide.

In the present application, the term "variant" means that one or more amino acids are conservatively substituted and/or modified so that they differ from the amino acid sequence before the mutation of the variant but have functions or properties. refers to a polypeptide that is maintained. Such variants can generally be identified by modifying one or more amino acids in the amino acid sequence of the polypeptide and evaluating the properties of the modified polypeptide. That is, the ability of the variant may be increased, unchanged, or decreased compared to the polypeptide before the mutation. In addition, some variants may include variants in which one or more portions, such as an N-terminal leader sequence or a transmembrane domain, have been removed. Other variants may include variants in which a portion is removed from the N- and/or C-terminus of the mature protein. The term "variant" may be used interchangeably with terms such as mutant, modified, mutant polypeptide, mutated protein, mutant and mutant (in English, modified, modified polypeptide, modified protein, mutant, mutein, divergent, etc.) and, as long as it is a term used in a mutated sense, it is not limited thereto.

In addition, 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 involved in protein translocation may be conjugated to the N-terminus of the mutant, either co-translationally or post-translationally. The variants may also be conjugated with other sequences or linkers for identification, purification, or synthesis.

As used herein, the term 'homology' or 'identity' refers to the degree of similarity between two given amino acid sequences or nucleotide sequences and may be expressed as a percentage. The terms homology and identity can often be used interchangeably.

Sequence homology or identity of a conserved polynucleotide or polypeptide is determined by standard alignment algorithms, with default gap penalties established by the program used may be used. Substantially homologous or identical sequences are generally capable of hybridizing with all or part of a sequence under moderate or high stringent conditions. It is apparent that hybridization also includes hybridization with polynucleotides containing common codons or codons taking codon degeneracy into account in the polynucleotide.

Whether any two polynucleotide or polypeptide sequences have homology, similarity or identity can be determined, for example, by Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444, using a known computer algorithm such as the "FASTA" program. or, 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), Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) can be used to determine (GCG program package (Devereux, J., et al, Nucleic Acids) Research 12: 387 (1984)), BLASTP, BLASTN, FASTA (Atschul, [S.] [F.,] [ET AL, J MOLEC BIOL 215]: 403 (1990); Guide to Huge Computers, Martin J. Bishop , [ED.,] Academic Press, San Diego, 1994, and [CARILLO ETA/.] (1988) SIAM J Applied Math 48: 1073).For example, BLAST of the National Center for Biotechnology Information Database, or ClustalW can be used to determine homology, similarity or identity.

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

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

For example, any amino acid sequence can be replaced with the amino acid sequence of a subtype polypeptide provided herein (e.g., SEQ ID NOs: 3, 13, 23, 33, 43, 53, 63, 73, 83, 93, 103, 113, 123 , 133, 143, 153, and/or 163), and based on this, each amino acid residue in the amino acid sequence is identified by reference to the numerical position of the amino acid residue corresponding to the amino acid residue in the amino acid sequence of the isotype polypeptide. can be numbered. For example, a sequence alignment algorithm such as that described in this application can identify the position of an amino acid, or a position at which modifications, such as substitutions, insertions, or deletions, occur compared to a query sequence (also referred to as a "reference sequence").

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

As used herein, the term "polynucleotide" refers to a DNA or RNA strand of a certain length or longer as a polymer of nucleotides in which nucleotide monomers are linked in a long chain by covalent bonds, and more specifically, provided in the present application. It refers to a polynucleotide fragment encoding a variant.

The polynucleotide of the present application may be included without limitation as long as it is a probe that can be prepared from a known gene sequence, for example, a sequence that can hybridize under stringent conditions with a sequence complementary to all or part of the polynucleotide sequence of the present application. . The "stringent condition" means a condition that enables specific hybridization between polynucleotides. These conditions are described in J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989; F.M. Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, 9.50-9.51, 11.7-11.8). For example, polynucleotides with high homology or identity, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or a condition in which polynucleotides having 99% or more homology or identity hybridize with each other and polynucleotides with lower homology or identity do not hybridize, or a washing condition of conventional Southern hybridization at 60°C, 1 ХSSC, 0.1% SDS, specifically 60° C., 0.1ХSSC, 0.1% SDS, more specifically 68° C., 0.1ХSSC, 0.1% SDS at a salt concentration and temperature equivalent to one wash, specifically two to three times conditions can be enumerated.

Hybridization requires that two nucleic acids have complementary sequences, although mismatch between bases is possible depending on the stringency of hybridization. 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. Accordingly, the polynucleotides of the present application may also include substantially similar nucleic acid sequences as well as isolated nucleic acid fragments complementary to the overall sequence.

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

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

recombinant vector

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

The vector of the present application may include a DNA preparation comprising a nucleotide sequence of a polynucleotide encoding the target polypeptide operably linked to a suitable expression control region (or expression control sequence) so that the target polypeptide can be expressed in a suitable host. have. The expression control region may include a promoter capable of initiating transcription, an optional operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence regulating the termination of transcription and translation. After transformation into an appropriate host cell, the vector can replicate or function independently of the host genome, and can be integrated into the genome itself.

The vector used in the present application is not particularly limited, and any vector known in the art may be used. Examples of commonly used vectors include plasmids, cosmids, viruses and bacteriophages in a natural or recombinant state. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A may be used as phage vectors or cosmid vectors, and pDZ-based, pBR-based, and pUC-based plasmid vectors may be used. , pBluescript II-based, pGEM-based, pTZ-based, pCL-based, pET-based and the like can be used. 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 target polypeptide may be inserted into a chromosome through a vector for intracellular chromosome insertion. The insertion of the polynucleotide into the chromosome may be performed by any method known in the art, for example, homologous recombination, but is not limited thereto. It may further include a selection marker (selection marker) for confirming whether the chromosome is inserted. The selection marker is used to select cells transformed with the vector, that is, to determine whether a target nucleic acid molecule is inserted, and selectable phenotypes such as drug resistance, auxotrophy, resistance to cytotoxic agents, or surface polypeptide expression. Markers to be given can be used. In an environment treated with a selective agent, only the cells expressing the selectable marker survive or exhibit other expression traits, so that the transformed cells can be selected.

As used herein, the term "transformation" refers to introducing a vector including a polynucleotide encoding a target polypeptide into a host cell or microorganism so that the polypeptide encoded by the polynucleotide can be expressed in the host cell. The transformed polynucleotide may include all of them regardless of whether they are inserted into the chromosome of the host cell or located outside the chromosome, as long as they can be expressed in the host cell. In addition, the polynucleotide includes DNA and/or RNA encoding a target polypeptide. The polynucleotide may be introduced in any form as long as it can be introduced and expressed into a host cell. For example, the polynucleotide may be introduced into a host cell in the form of an expression cassette, which is a gene construct including all elements necessary for self-expression. The expression cassette may include a promoter operably linked to the polynucleotide, a transcription termination signal, a ribosome binding site, and a translation termination signal. The expression cassette may be in the form of an expression vector capable of self-replication. In addition, the polynucleotide may be introduced into a host cell in its own form and operably linked to a sequence required for expression in the host cell, but is not limited thereto.

In addition, the term “operably linked” as used herein means that a promoter sequence that initiates and mediates transcription of a polynucleotide encoding the target variant of the present application and the polynucleotide sequence are functionally linked.

Recombinant microorganisms

Another aspect of the present application is (i) one or more or two or more selected from the above-described variants (1) to (17) (eg, 1, 2, 3, 4, 5, 6) dog, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17) variants, (ii) polynucleotides encoding said variants; one or more or two or more (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14) , 15, 16 or 17) polynucleotides, or (iii) a combination thereof, Corynebacterium glutamicum glutamicum ) to provide a strain.

The strain of the present application may include a variant polypeptide of the present application, a polynucleotide encoding the polypeptide, a vector including the polynucleotide, or a combination thereof.

As used herein, the term "strain (or microorganism)" includes both wild-type microorganisms and microorganisms in which genetic modification has occurred naturally or artificially. As a result of which a specific mechanism is weakened or enhanced, it may be a microorganism including genetic modification for the production of a desired polypeptide, protein or product.

The strain of the present application includes a strain comprising any one or more of one or more variants of the present application, one or more polynucleotides of the present application, and one or more vectors comprising one or more polynucleotides of the present application; strains modified to express one or more variants of the present application or one or more polynucleotides of the present application; a strain expressing one or more variants of the present application or one or more polynucleotides of the present application (eg, a recombinant strain); Or it may be a strain (eg, a recombinant strain) having one or more variant activities of the present application, but is not limited thereto.

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

The strain of the present application is a microorganism having naturally L-valine-producing ability, or a mutant of the present application or a polynucleotide (or a vector containing the polynucleotide) encoding the same is introduced into a parent strain without L-valine-producing ability. and/or L-valine-producing ability may be endowed, but is not limited thereto.

As an example, the strain of the present application is transformed with a vector containing the polynucleotide of the present application or a polynucleotide encoding the variant of the present application, and expresses the variant of the present application as a cell or microorganism, The strains of the application may include all microorganisms capable of producing L-valine, including the variants of the present application. For example, the strain of the present application may be a recombinant strain in which the polynucleotide encoding the variant of the present application is increased in L-valine-producing ability in a natural wild-type microorganism or a microorganism producing L-valine.

The recombinant strain with increased L-amino acid production ability is a natural wild-type microorganism or unmodified microorganism (ie, wild-type (SEQ ID NO: 3, 13, 23, 33, 43, 53, 63, 73, 83, 93, 103, 113) , 123, 133, 143, 153, and/or 163) a microorganism or variant (SEQ ID NO: 1, 11, 21, 31, 41, 51, 61, 71, 81, 91, 101, 111, 121) expressing the protein. , 131, 141, 151, and/or 161) microorganisms that do not express proteins) may be microorganisms having an increased ability to produce L-valine compared to microorganisms that do not express proteins, but is not limited thereto. For example, the non-modified microorganism, which is the target strain for comparing the increase in L-valine production ability, may be the ATCC14067 strain and/or the Corynebacterium glutamicum CA08-0072 strain (KCCM11201P), but is not limited thereto. .

As an example, the recombinant strain having increased L-valine production capacity may include a strain comprising any one or more of a mutant (NCgl1391 protein mutant) of SEQ ID NO: 1, a polynucleotide encoding the same, and a vector including the polynucleotide; a strain modified to express a variant of SEQ ID NO: 1 or a polynucleotide encoding the same; a strain expressing the variant of SEQ ID NO: 1 or a polynucleotide encoding the same (eg, a recombinant strain); Or it may be a strain having a variant activity of SEQ ID NO: 1 (eg, a recombinant strain).

The recombinant strain having an increased L-valine production capacity has an L-valine production capacity of about 1% or more, about 2.5% or more, about 5% or more, about 6% or more, or about 7%, compared to the parent strain or unmodified microorganism before mutation. 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% at least about 19%, at least about 19.5%, at least about 20%, at least about 20.5%, at least about 21%, at least about 21.5%, at least about 22%, at least about 22.5%, at least about 23%, or at least about 23.5 % or more (the upper limit is not particularly limited, for example, about 200% or less, about 150% or less, about 100% or less, about 50% or less, about 45% or less, about 40% or less, about 35% or less, about 30% or less or about 25% or less), but is not limited thereto, as long as it has an increased amount of + value compared to the production capacity of the parent strain or unmodified microorganism before mutation. In another example, the recombinant strain having an increased L-valine production capacity has an L-valine production capacity of about 1.1 times or more, about 1.12 times or more, about 1.13 times or more, 1.15 times or more, compared to the parent strain or unmodified microorganism before mutation, 1.16 times or more, 1.17 times or more, 1.18 times or more, 1.19 times or more, about 1.2 times or more, 1.21 times or more, 1, 22 or about 1.23 times or more (the upper limit is not particularly limited, for example, about 10 times or less, about 5 times or more may be up to fold, up to about 3 fold, or up to about 2 fold) may be increased.

More specifically, the recombinant strain having an increased L-valine production capacity may have an L-valine production capacity increased by about 25.0% (or about 1.25 times) compared to the parent strain or unmodified microorganism before mutation, but is not limited thereto. does not

As another example, the recombinant strain having an increased L-valine production ability is a strain comprising at least one of a mutant of SEQ ID NO: 11 (ATP phosphoribosyltransferase mutant), a polynucleotide encoding the same, and a vector including the polynucleotide ; a strain modified to express a variant of SEQ ID NO: 11 or a polynucleotide encoding the same; a strain expressing the variant of SEQ ID NO: 11 or a polynucleotide encoding the same (eg, a recombinant strain); Or it may be a strain having the mutant activity of SEQ ID NO: 11 (eg, a recombinant strain).

The recombinant strain having an increased L-valine production capacity has an L-valine production capacity of about 1% or more, about 2.5% or more, about 5% or more, about 6% or more, or about 7%, compared to the parent strain or unmodified microorganism before mutation. 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, about 20.5% or more, about 20.6% or more, about 20.7% or more, about 20.8% or more, about 20.9% or more, or about 21% or more (the upper limit is There is no particular limitation, for example, about 200% or less, about 150% or less, about 100% or less, about 50% or less, about 45% or less, about 40% or less, about 35% or less, about 30% or less, or about 25% or less. % or less), but is not limited thereto, as long as it has an increase of + value compared to the production capacity of the parent strain or unmodified microorganism before mutation. In another example, the recombinant strain having an increased L-valine production capacity has an L-valine production capacity of about 1.1 times or more, about 1.12 times or more, about 1.13 times or more, 1.15 times or more, compared to the parent strain or unmodified microorganism before mutation, 1.16 times or more, 1.17 times or more, 1.18 times or more, 1.19 times or more, or about 1.2 times or more (the upper limit is not particularly limited, for example, about 10 times or less, about 5 times or less, about 3 times or less, or about 2 times or more. may be below) may be increased.

More specifically, the recombinant strain having an increased L-valine production capacity may have an L-valine production capacity increased by about 21% (or about 1.21 times) compared to the parent strain or unmodified microorganism before mutation, but is not limited thereto. does not

As another example, the recombinant strain having an increased L-valine production ability is a strain comprising at least one of a mutant (asparagine synthase mutant) of SEQ ID NO: 21, a polynucleotide encoding the same, and a vector including the polynucleotide; a strain modified to express a variant of SEQ ID NO: 21 or a polynucleotide encoding the same; a strain expressing the variant of SEQ ID NO: 21 or a polynucleotide encoding the same (eg, a recombinant strain); Or it may be a strain having a variant activity of SEQ ID NO: 21 (eg, a recombinant strain).

The recombinant strain having an increased L-valine production capacity has an L-valine production capacity of about 1% or more, about 2.5% or more, about 5% or more, about 6% or more, or about 7%, compared to the parent strain or unmodified microorganism before mutation. 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, or about 15.1% or more (the upper limit is not particularly limited, 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 % or less), but is not limited thereto, as long as it has an increase of + value compared to the production capacity of the parent strain or unmodified microorganism before mutation. In another example, the recombinant strain having an increased L-valine production capacity has an L-valine production capacity of about 1.1 times or more, about 1.12 times or more, about 1.13 times or more, about 1.14 times or more, compared to the parent strain or unmodified microorganism before mutation. , or about 1.15 times or more (the upper limit is not particularly limited, for example, it may be about 10 times or less, about 5 times or less, about 3 times or less, or about 2 times or less).

More specifically, the recombinant strain having an increased L-valine production capacity may have an L-valine production capacity increased by about 15% (or about 1.15 times) compared to the parent strain or unmodified microorganism before mutation, but is not limited thereto. does not

As another example, the recombinant strain having an increased L-valine production ability is a strain comprising at least one of a mutant (spermidine synthase mutant) of SEQ ID NO: 31, a polynucleotide encoding the same, and a vector including the polynucleotide; a strain modified to express a variant of SEQ ID NO: 31 or a polynucleotide encoding the same; a strain expressing a variant of SEQ ID NO: 31 or a polynucleotide encoding the same (eg, a recombinant strain); Or it may be a strain having a variant activity of SEQ ID NO: 31 (eg, a recombinant strain).

The recombinant strain having an increased L-valine production capacity has an L-valine production capacity of about 1% or more, about 2.5% or more, about 5% or more, about 6% or more, or about 7%, compared to the parent strain or unmodified microorganism before mutation. 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 13.1% or more, about 13.2% or more, or about 13.3% or more (the upper limit is not particularly limited, 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, about 20% or less, or about 15% or less) may be increased, but a + value compared to the production capacity of the parent strain or unmodified microorganism before mutation As long as it has an increasing amount of , it is not limited thereto. In another example, the recombinant strain having an increased L-valine production capacity has an L-valine production capacity of about 1.1 times or more, about 1.12 times or more, or about 1.13 times or more (the upper limit is a special There is no limitation, for example, about 10 times or less, about 5 times or less, about 3 times or less, or about 2 times or less) may be increased.

More specifically, the recombinant strain having an increased L-valine production capacity may have an L-valine production capacity increased by about 13.2% (or about 1.13 times) compared to the parent strain or unmodified microorganism before mutation, but is not limited thereto. does not

As another example, the recombinant strain having an increased L-valine production ability includes any one or more of a mutant of SEQ ID NO: 41 (cysteine sulfinate disulfinase mutant), a polynucleotide encoding the same, and a vector including the polynucleotide strain; a strain modified to express a variant of SEQ ID NO: 41 or a polynucleotide encoding the same; a strain expressing the variant of SEQ ID NO: 41 or a polynucleotide encoding the same (eg, a recombinant strain); Or it may be a strain having the mutant activity of SEQ ID NO: 41 (eg, a recombinant strain).

The recombinant strain having an increased L-valine production capacity has an L-valine production capacity of about 1% or more, about 2.5% or more, about 5% or more, about 6% or more, or about 7%, compared to the parent strain or unmodified microorganism before mutation. 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, about 20.5% or more, about 21% or more, about 21.5% or more, about 22% or more, about 22.5% or more, about 23% or more, about 23.5% or more, about 24% or more, about 24.5% or more, about 25% or more, about 25.5% or more, about 25.7% or more, about 26% or more, or about 26.1% or more (the upper limit is not particularly limited, 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 30% or less) Or, as long as it has an increased amount of + value compared to the production capacity of the unmodified microorganism, it is not limited thereto. In another example, the recombinant strain having an increased L-valine production capacity has an L-valine production capacity of about 1.1 times or more, about 1.12 times or more, about 1.13 times or more, about 1.15 times or more, compared to the parent strain or unmodified microorganism before mutation. , about 1.16 times or more, about 1.17 times or more, about 1.18 times or more, about 1.19 times or more, about 1.2 times or more, about 1.21 times or more, about 1.22 times or more, about 1.23 times or more, about 1.24 times or more, about 1.25 times or more , or about 1.26 times or more (the upper limit is not particularly limited, for example, it may be about 10 times or less, about 5 times or less, about 3 times or less, or about 2 times or less).

More specifically, the recombinant strain having an increased L-valine production capacity may have an L-valine production capacity increased by about 26% (or about 1.26 times) compared to the parent strain or unmodified microorganism before mutation, but is not limited thereto. does not

As another example, the recombinant strain having an increased L-valine production ability is a strain comprising at least one of a mutant (urease accessory protein mutant) of SEQ ID NO: 51, a polynucleotide encoding the same, and a vector including the polynucleotide; a strain modified to express a variant of SEQ ID NO: 51 or a polynucleotide encoding the same; a strain expressing a variant of SEQ ID NO: 51 or a polynucleotide encoding the same (eg, a recombinant strain); Or it may be a strain having a variant activity of SEQ ID NO: 51 (eg, a recombinant strain).

The recombinant strain having an increased L-valine production capacity has an L-valine production capacity of about 1% or more, about 2.5% or more, about 5% or more, about 6% or more, or about 7%, compared to the parent strain or unmodified microorganism before mutation. 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, about 20.5% or more, about 21% or more, about 21.5% or more, about 22% or more, about 22.5% or more, about 23% or more, about 23.5% or more, about 24% or more, about 24.5% or more, about 25% or more, about 25.5% or more, about 26% or more, about 26.5% or more, about 27% or more, about 27.1% or more, about 27.2% or more, about 27.3% or more, about 27.4% or more, about 27.5% or more, or about 27.6% or more (the upper limit is not particularly limited, 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 30% or less) may be increased) . In another example, the recombinant strain having an increased L-valine production capacity has an L-valine production capacity of about 1.1 times or more, about 1.12 times or more, about 1.13 times or more, about 1.15 times or more, compared to the parent strain or unmodified microorganism before mutation. , about 1.16 times or more, about 1.17 times or more, about 1.18 times or more, about 1.19 times or more, about 1.2 times or more, about 1.21 times or more, about 1.22 times or more, about 1.23 times or more, about 1.24 times or more, about 1.25 times or more , about 1.26 times or more, or about 1.27 times or more (the upper limit is not particularly limited, and may be, for example, about 10 times or less, about 5 times or less, about 3 times or less, or about 2 times or less).

More specifically, the recombinant strain having an increased L-valine production capacity may have an L-valine production capacity increased by about 27.5% (or about 1.28 times) compared to the parent strain before mutation or an unmodified microorganism, but is not limited thereto. does not

As another example, the recombinant strain having an increased L-valine production ability is a strain comprising at least one of a mutant (proline dehydrogenase mutant) of SEQ ID NO: 61, a polynucleotide encoding the same, and a vector including the polynucleotide; a strain modified to express a variant of SEQ ID NO: 61 or a polynucleotide encoding the same; a strain expressing the variant of SEQ ID NO: 61 or a polynucleotide encoding the same (eg, a recombinant strain); Or it may be a strain having a variant activity of SEQ ID NO: 61 (eg, a recombinant strain).

The recombinant strain having an increased L-valine production capacity has an L-valine production capacity of about 1% or more, about 2.5% or more, about 5% or more, about 6% or more, or about 7%, compared to the parent strain or unmodified microorganism before mutation. 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, about 20.5% or more, about 21% or more, about 21.5% or more, about 22% or more, about 22.5% or more, about 23% or more, about 23.5% or more, about 24% or more, about 24.5% or more, about 25% or more, about 25.5% or more, about 26% or more, about 26.5% or more, about 27% or more, about 27.5% or more, about 28% or more, about 28.1% or more, about 28.2% or more, about 28.3% or more, about 28.4% or more, or about 28.5% or more (the upper limit is not particularly limited, 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) may be increased, but as long as it has an increase in + value compared to the production capacity of the parent strain or unmodified microorganism before mutation , but not limited thereto. In another example, the recombinant strain having an increased L-valine production capacity has an L-valine production capacity of about 1.1 times or more, about 1.12 times or more, about 1.13 times or more, 1.15 times or more, compared to the parent strain or unmodified microorganism before mutation, 1.16 times or more, 1.17 times or more, 1.18 times or more, 1.19 times or more, about 1.2 times or more, 1.25 times or more, 1.26 times or more, 1.27 times or more, or about 1.28 times or more (the upper limit is not particularly limited, for example, about 10 times It may be up to a fold, up to about 5 times, up to about 3 times, or up to about 2 times) may be increased.

More specifically, the recombinant strain having an increased L-valine production capacity may have an L-valine production capacity increased by about 28.6% (or about 1.29 times) compared to the parent strain before mutation or an unmodified microorganism, but is not limited thereto. does not

As another example, the recombinant strain with increased L-valine production ability is a variant of SEQ ID NO: 71 (tetrahydrodipicolinate N-succinyltransferase mutant), a polynucleotide encoding the same, and a vector comprising the polynucleotide. strains comprising one or more; a strain modified to express a variant of SEQ ID NO: 71 or a polynucleotide encoding the same; a strain expressing the variant of SEQ ID NO: 71 or a polynucleotide encoding the same (eg, a recombinant strain); Or it may be a strain having the mutant activity of SEQ ID NO: 71 (eg, a recombinant strain).

The recombinant strain having an increased L-valine production capacity has an L-valine production capacity of about 1% or more, about 2.5% or more, about 5% or more, about 6% or more, or about 7%, compared to the parent strain or unmodified microorganism before mutation. 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 18.6% or more, about 18.7% or more, about 18.8% or more, about 18.9% or more, or about 19% or more (the upper limit is not particularly limited, for example, about 200% or less, about 150% or less, about 100% or less, about 50% or less, about 45% or less, about 40% or less, about 35% or less, about 30% or less, or about 25% or less) It is not limited thereto, as long as it has an increase in the + value compared to the productivity. In another example, the recombinant strain having an increased L-valine production capacity has an L-valine production capacity of about 1.1 times or more, about 1.12 times or more, about 1.13 times or more, about 1.15 times or more, compared to the parent strain or unmodified microorganism before mutation. , about 1.16 times or more, about 1.17 times or more, about 1.18 times or more, or about 1.19 times or more (the upper limit is not particularly limited, for example, about 10 times or less, about 5 times or less, about 3 times or less, or about 2 times may be below) may be increased.

More specifically, the recombinant strain having increased L-valine production capacity may have an L-valine production capacity of about 19% (or about 1.19 times) increased compared to the parent strain or unmodified microorganism before mutation, but is not limited thereto. does not

As another example, the recombinant strain having an increased L-valine production ability is a mutant (5,10-methylenetetrahydrofolate reductase mutant) of SEQ ID NO: 81, a polynucleotide encoding the same, and a vector comprising the polynucleotide. strains comprising one or more; a strain modified to express a variant of SEQ ID NO: 81 or a polynucleotide encoding the same; a strain expressing a variant of SEQ ID NO: 81 or a polynucleotide encoding the same (eg, a recombinant strain); Or it may be a strain having the mutant activity of SEQ ID NO: 81 (eg, a recombinant strain).

The recombinant strain having an increased L-valine production capacity has an L-valine production capacity of about 1% or more, about 2.5% or more, about 5% or more, about 6% or more, or about 7%, compared to the parent strain or unmodified microorganism before mutation. 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, about 20.5% or more, about 21% or more, about 21.5% or more, about 22% or more, about 22.5% or more, about 23% or more, about 23.5% or more, about 24% or more, about 24.5% or more, about 25% or more, about 25.5% or more, about 26% or more, about 26.5% or more, about 27% or more, about 27.5% or more, about 28% or more, about 28.5% or more, about 29% or more, about 29.5% or more, about 30% or more, about 30.5% or more, about 31% or more, about 31.5% or more, about 32% or more, about 32.5% or more, about 33% or more, about 33.1% or more, about 33.2% or more, or about 33.3% or more, (the upper limit is not particularly limited, 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, or about 35% or less) may be increased, but is not limited thereto, as long as it has an increase in + value compared to the production capacity of the parent strain or unmodified microorganism before mutation. In another example, the recombinant strain having an increased L-valine production capacity has an L-valine production capacity of about 1.1 times or more, about 1.12 times or more, about 1.13 times or more, 1.15 times or more, compared to the parent strain or unmodified microorganism before mutation, 1.16 times or more, 1.17 times or more, 1.18 times or more, 1.19 times or more, about 1.2 times or more, 1.25 times or more, about 1.3 times or more, about 1.31 times or more, about 1.32 times or more, or about 1.33 times or more (the upper limit is a special limitation is not, for example, about 10 times or less, about 5 times or less, about 3 times or less, or about 2 times or less) may be increased.

More specifically, the recombinant strain having an increased L-valine production capacity may have an L-valine production capacity increased by about 32.1% (or about 1.32 times) compared to the parent strain before mutation or an unmodified microorganism, but is not limited thereto. does not

As another example, the recombinant strain having increased L-valine production capacity may include a strain comprising at least one of a mutant of SEQ ID NO: 91 (NCgl2805 protein mutant), a polynucleotide encoding the same, and a vector including the polynucleotide; a strain modified to express a variant of SEQ ID NO: 91 or a polynucleotide encoding the same; a strain expressing the variant of SEQ ID NO: 91 or a polynucleotide encoding the same (eg, a recombinant strain); Or it may be a strain having the mutant activity of SEQ ID NO: 91 (eg, a recombinant strain).

The recombinant strain having an increased L-valine production capacity has an L-valine production capacity of about 1% or more, about 2.5% or more, about 5% or more, about 6% or more, or about 7%, compared to the parent strain or unmodified microorganism before mutation. 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, or about 15% or more (the upper limit is not particularly limited, 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) increased However, it is not limited thereto, as long as it has an increased amount of + value compared to the production capacity of the parent strain or unmodified microorganism before mutation. In another example, the recombinant strain having an increased L-valine production capacity has an L-valine production capacity of about 1.1 times or more, about 1.12 times or more, about 1.13 times or more, about 1.14 times or more, compared to the parent strain or unmodified microorganism before mutation. , or about 1.15 times or more (the upper limit is not particularly limited, and may be, for example, about 10 times or less, about 5 times or less, about 3 times or less, about 2 times or less, about 2 times or less).

As another example, the recombinant strain having an increased L-valine production ability includes any one or more of a mutant of SEQ ID NO: 101 (sugar porter MFS transporter mutant), a polynucleotide encoding the same, and a vector including the polynucleotide strain; a strain modified to express a variant of SEQ ID NO: 101 or a polynucleotide encoding the same; a strain expressing a variant of SEQ ID NO: 101 or a polynucleotide encoding the same (eg, a recombinant strain); Or it may be a strain having a variant activity of SEQ ID NO: 101 (eg, a recombinant strain).

The recombinant strain having an increased L-valine production capacity has an L-valine production capacity of about 1% or more, about 2.5% or more, about 5% or more, about 6% or more, or about 7%, compared to the parent strain or unmodified microorganism before mutation. 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, about 20.5% or more, about 21% or more, about 21.5% or more, about 22% or more, about 22.5% or more, about 23% or more, about 23.5% or more, about 24% or more, about 24.5% or more, about 25% or more, about 25.5% or more, about 26% or more, about 26.5% or more, about 27% or more, about 27.5% or more, about 28% or more, about 28.5% or more, about 29% or more, about 29.5% or more, about 30% or more, about 30.5% or more, about 31% or more, about 31.5% or more, about 32% or more, about 32.5% or more, about 32.6% or more, about 32.7% or more, about 32.8% or more, about 32.9% or more, or about 33% or more (the upper limit is not particularly limited, 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, or about 35% or less) may be increased, but is not limited thereto, as long as it has an increase in + value compared to the production capacity of the parent strain or unmodified microorganism before mutation. In another example, the recombinant strain having an increased L-valine production capacity has an L-valine production capacity of about 1.1 times or more, about 1.12 times or more, about 1.13 times or more, 1.15 times or more, compared to the parent strain or unmodified microorganism before mutation, about 1.16 times or more, about 1.17 times or more, about 1.18 times or more, about 1.19 times or more, about 1.2 times or more, about 1.25 times or more, about 1.26 times or more, about 1.27 times or more, about 1.28 times or more, about 1.29 times or more, About 1.3 times or more, about 1.31 times or more, about 1.32 times or more, or about 1.33 times or more (the upper limit is not particularly limited, for example, about 10 times or less, about 5 times or less, about 3 times or less, or about 2 times or less. may be increased).

More specifically, the recombinant strain having an increased L-valine production capacity may have an L-valine production capacity increased by about 33% (or about 1.33 times) compared to the parent strain before mutation or an unmodified microorganism, but is not limited thereto. does not

As another example, the recombinant strain having an increased L-valine production ability is a strain comprising any one or more of a mutant (transcriptional regulator mutant) of SEQ ID NO: 111, a polynucleotide encoding the same, and a vector including the polynucleotide; a strain modified to express a variant of SEQ ID NO: 111 or a polynucleotide encoding the same; a strain expressing the variant of SEQ ID NO: 111 or a polynucleotide encoding the same (eg, a recombinant strain); Or it may be a strain having a variant activity of SEQ ID NO: 111 (eg, a recombinant strain).

The recombinant strain having an increased L-valine production capacity has an L-valine production capacity of about 1% or more, about 2.5% or more, about 5% or more, about 6% or more, or about 7%, compared to the parent strain or unmodified microorganism before mutation. 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, 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 There is no particular limitation, for example, it may be 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) It may be increased, but it is not limited thereto as long as it has an increased amount of + value compared to the production capacity of the parent strain or unmodified microorganism before mutation. In another example, the recombinant strain having an increased L-valine production capacity has an L-valine production capacity of about 1.1 times or more, about 1.12 times or more, about 1.13 times or more, about 1.15 times or more, compared to the parent strain or unmodified microorganism before mutation. , about 1.16 times or more, about 1.17 times or more, about 1.18 times or more, about 1.19 times or more, about 1.2 times or more, about 1.21 times or more, or about 1.22 times or more (the upper limit is not particularly limited, for example, about 10 times or less , about 5 times or less, about 3 times or less, or about 2 times or less) may be increased.

More specifically, the recombinant strain with increased L-valine production capacity may have an L-valine production capacity increased by about 22.2% (or about 1.22 times) compared to the parent strain or unmodified microorganism before mutation, but is not limited thereto. does not

As another example, the recombinant strain having an increased L-valine production ability includes any one or more of a mutant of SEQ ID NO: 121 (WhcA variant of the WhiB family transcriptional regulator), a polynucleotide encoding the same, and a vector including the polynucleotide strain; a strain modified to express a variant of SEQ ID NO: 121 or a polynucleotide encoding the same; a strain expressing the variant of SEQ ID NO: 121 or a polynucleotide encoding the same (eg, a recombinant strain); Or it may be a strain having the mutant activity of SEQ ID NO: 121 (eg, a recombinant strain).

The recombinant strain having an increased L-valine production capacity has an L-valine production capacity of about 1% or more, about 2.5% or more, about 5% or more, about 6% or more, or about 7%, compared to the parent strain or unmodified microorganism before mutation. 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, about 20.5% or more, about 21% or more, about 21.5% or more, about 22% or more, about 22.5% or more, about 23% or more, about 23.5% or more, about 24% or more, about 24.5% or more, about 24.6% or more, about 24.7% or more, about 24.8% or more, about 24.9% or more, or about 25% or more (the upper limit is not particularly limited, 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) Or, as long as it has an increased amount of + value compared to the production capacity of the unmodified microorganism, it is not limited thereto. In another example, the recombinant strain having an increased L-valine production capacity has an L-valine production capacity of about 1.1 times or more, about 1.12 times or more, about 1.13 times or more, about 1.15 times or more, compared to the parent strain or unmodified microorganism before mutation. , about 1.16 times or more, about 1.17 times or more, about 1.18 times or more, about 1.19 times or more, about 1.2 times or more, about 1.21 times or more, about 1.22 times or more, about 1.23 times or more, about 1.24 times or more, or about 1.25 times or more. The upper limit value is not particularly limited, and may be increased by, for example, about 10 times or less, about 5 times or less, about 3 times or less, or about 2 times or less.

More specifically, the recombinant strain having an increased L-valine production capacity may have an L-valine production capacity increased by about 25% (or about 1.25 times) compared to the parent strain before mutation or an unmodified microorganism, but is not limited thereto. does not

As another example, the recombinant strain having an increased L-valine production ability includes any one or more of a mutant (dihydrolipoyl acetyltransferase mutant) of SEQ ID NO: 131, a polynucleotide encoding the same, and a vector including the polynucleotide strain comprising; a strain modified to express a variant of SEQ ID NO: 131 or a polynucleotide encoding the same; a strain expressing the variant of SEQ ID NO: 131 or a polynucleotide encoding the same (eg, a recombinant strain); Or it may be a strain having the mutant activity of SEQ ID NO: 131 (eg, a recombinant strain).

The recombinant strain having an increased L-valine production capacity has an L-valine production capacity of about 1% or more, about 2.5% or more, about 5% or more, about 6% or more, or about 7%, compared to the parent strain or unmodified microorganism before mutation. 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, about 20.5% or more, about 21% or more, about 21.5% or more, about 22% or more, about 22.5% or more, about 23% or more, about 23.5% or more, about 24% or more, about 24.5% or more, about 25% or more, about 25.5% or more, about 26% or more, about 26.5% or more, about 27% or more, about 27.5% or more, about 28% or more, about 28.5% or more, about 29% or more, about 29.5% or more, about 29.6% or more, about 29.7% or more, about 29.8% or more, about 29.9% or more, or about 30% or more (the upper limit is not particularly limited, 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) Or, as long as it has an increased amount of + value compared to the production capacity of the unmodified microorganism, it is not limited thereto. In another example, the recombinant strain having an increased L-valine production capacity has an L-valine production capacity of about 1.1 times or more, about 1.12 times or more, about 1.13 times or more, about 1.15 times or more, compared to the parent strain or unmodified microorganism before mutation. , about 1.16 times or more, about 1.17 times or more, about 1.18 times or more, about 1.19 times or more, about 1.2 times or more, about 1.25 times or more, about 1.26 times or more, about 1.27 times or more, about 1.28 times or more, about 1.9 times or more , or about 1.3 times or more (the upper limit is not particularly limited, and may be, for example, about 10 times or less, about 5 times or less, about 3 times or less, or about 2 times or less).

More specifically, the recombinant strain having increased L-valine production capacity may have an L-valine production capacity increased by about 30% (or about 1.3 times) compared to the parent strain or unmodified microorganism before mutation, but is not limited thereto. does not

As another example, the recombinant strain having an increased L-valine production ability comprises any one or more of a mutant (2-isopropyl maleate synthetase mutant) of SEQ ID NO: 141, a polynucleotide encoding the same, and a vector including the polynucleotide strain comprising; a strain modified to express a variant of SEQ ID NO: 141 or a polynucleotide encoding the same; a strain expressing a variant of SEQ ID NO: 141 or a polynucleotide encoding the same (eg, a recombinant strain); Or it may be a strain having the mutant activity of SEQ ID NO: 141 (eg, a recombinant strain).

The recombinant strain having an increased L-valine production capacity has an L-valine production capacity of about 1% or more, about 2.5% or more, about 5% or more, about 6% or more, or about 7%, compared to the parent strain or unmodified microorganism before mutation. 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, about 20.5% or more, about 21% or more, about 21.5% or more, about 22% or more, about 22.5% or more, about 22.6% or more, or about 22.7% or more % or more (the upper limit is not particularly limited, 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 % or less), but is not limited thereto, as long as it has an increase of + value compared to the production capacity of the parent strain or unmodified microorganism before mutation. In another example, the recombinant strain having an increased L-valine production capacity has an L-valine production capacity of about 1.1 times or more, about 1.12 times or more, about 1.13 times or more, about 1.15 times or more, compared to the parent strain or unmodified microorganism before mutation. , about 1.16 times or more, about 1.17 times or more, about 1.18 times or more, about 1.19 times or more, about 1.2 times or more, about 1.25 times or more, about 1.26 times or more, or about 1.27 times or more (the upper limit is not particularly limited, for example, , about 10 times or less, about 5 times or less, about 3 times or less, or about 2 times or less) may be increased.

More specifically, the recombinant strain having increased L-valine production capacity may have an L-valine production capacity increased by about 22.7% (or about 1.23 times) compared to the parent strain or unmodified microorganism before mutation, but is not limited thereto. does not

As another example, the recombinant strain having increased L-valine production ability is a strain comprising at least one of a mutant (branched-chain amino acid permease mutant) of SEQ ID NO: 151, a polynucleotide encoding the same, and a vector including the polynucleotide ; a strain modified to express a variant of SEQ ID NO: 151 or a polynucleotide encoding the same; a strain expressing the variant of SEQ ID NO: 151 or a polynucleotide encoding the same (eg, a recombinant strain); Or it may be a strain having the mutant activity of SEQ ID NO: 151 (eg, a recombinant strain).

The recombinant strain having an increased L-valine production capacity has an L-valine production capacity of about 1% or more, about 2.5% or more, about 5% or more, about 6% or more, or about 7%, compared to the parent strain or unmodified microorganism before mutation. 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, about 20.5% or more, about 21% or more, about 21.5% or more, about 22% or more, about 22.5% or more, about 23% or more, about 23.5% or more, about 24% or more, about 24.5% or more, about 25% or more, about 25.5% or more, about 26% or more, about 26.5% or more, about 27% or more, about 27.5% or more, about 28% or more, about 28.1% or more, about 28.2% or more, about 28.3% or more, about 28.4% or more, about 28.5% or more, or about 28.6% or more (the upper limit is not particularly limited, 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) may be increased, but a + value compared to the production capacity of the parent strain or unmodified microorganism before mutation As long as it has an increasing amount of , it is not limited thereto. In another example, the recombinant strain having an increased L-valine production capacity has an L-valine production capacity of about 1.1 times or more, about 1.12 times or more, about 1.13 times or more, about 1.15 times or more, compared to the parent strain or unmodified microorganism before mutation. , about 1.16 times or more, about 1.17 times or more, about 1.18 times or more, about 1.19 times or more, about 1.2 times or more, about 1.25 times or more, about 1.26 times or more, about 1.27 times or more, or about 1.28 times or more (the upper limit is a special There is no limitation, for example, about 10 times or less, about 5 times or less, about 3 times or less, or about 2 times or less) may be increased.

As another example, the recombinant strain having increased L-valine production ability is at least one of a mutant of SEQ ID NO: 161 (glyceraldehyde-3-phosphate dehydrogenase mutant), a polynucleotide encoding the same, and a vector including the polynucleotide a strain comprising; a strain modified to express a variant of SEQ ID NO: 161 or a polynucleotide encoding the same; a strain expressing a variant of SEQ ID NO: 161 or a polynucleotide encoding the same (eg, a recombinant strain); Or it may be a strain having the mutant activity of SEQ ID NO: 161 (eg, a recombinant strain).

The recombinant strain having an increased L-valine production capacity has an L-valine production capacity of about 1% or more, about 2.5% or more, about 5% or more, about 6% or more, or about 7%, compared to the parent strain or unmodified microorganism before mutation. 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, about 20.5% or more, about 21% or more, about 21.5% or more, about 22% or more, about 22.5% or more, about 23% or more, about 23.5% or more, about 24% or more, about 24.5% or more, about 25% or more, about 25.5% or more, about 26% or more, about 26.1% or more, about 26.2% or more, or about 26.3% or more (the upper limit is not particularly limited, 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) may be increased , as long as it has an increased amount of + value compared to the production capacity of the parent strain or unmodified microorganism before mutation, but is not limited thereto. In another example, the recombinant strain having an increased L-valine production capacity has an L-valine production capacity of about 1.1 times or more, about 1.12 times or more, about 1.13 times or more, about 1.15 times or more, compared to the parent strain or unmodified microorganism before mutation. , about 1.16 times or more, about 1.17 times or more, about 1.18 times or more, about 1.19 times or more, about 1.2 times or more, about 1.25 times or more, or about 1.26 times or more (the upper limit is not particularly limited, for example, about 10 times or less , about 5 times or less, about 3 times or less, or about 2 times or less) may be increased.

More specifically, the recombinant strain having an increased L-valine production capacity may have an L-valine production capacity increased by about 26.3% (or about 1.26 times) compared to the parent strain or unmodified microorganism before mutation, but is not limited thereto. does not

In the present application, the term “about” includes all ranges including ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and includes all values within a range equal to or similar to the value following the term about. However, it is not limited thereto.

As used herein, the term "unmodified microorganism" does not exclude a strain containing a mutation that can occur naturally in a microorganism, it is a wild-type strain or a natural-type strain itself, or a genetic variation caused by natural or artificial factors. It may mean the strain before being changed. For example, the unmodified microorganism may refer to a strain in which the protein variant described herein has not been introduced or has been introduced. The "unmodified microorganism" may be used interchangeably with "strain before modification", "microbe before modification", "unmodified strain", "unmodified strain", "unmodified microorganism" or "reference microorganism".

In another example of the present application, the microorganism of the present application is Corynebacterium glutamicum ( Corynebacterium glutamicum ), Corynebacterium crudilactis ), Corynebacterium deserti ( Corynebacterium deserti ), Cory Nebacterium efficiens ( Corynebacterium efficiens ), Corynebacterium callunae ), Corynebacterium stationis , Corynebacterium stationis ), Corynebacterium singulare ( Corynebacterium singulare ), Corynebacterium halo Tolerans ( Corynebacterium halotolerans ), Corynebacterium striatum ( Corynebacterium striatum ), Corynebacterium ammoniagenes ( Corynebacterium ammoniagenes ), Corynebacterium pollutisoli ( Corynebacterium pollutisoli ), Corynebacterium imitans imitans ), Corynebacterium testudinoris , or Corynebacterium flavescens .

As used herein, the term “weakened” of a polypeptide is a concept that includes both reduced or no activity compared to intrinsic activity. The attenuation may be used interchangeably with terms such as inactivation, deficiency, down-regulation, decrease, reduce, attenuation, and the like.

The attenuation is when the activity of the polypeptide itself is reduced or eliminated compared to the activity of the polypeptide possessed by the original microorganism due to mutation of the polynucleotide encoding the polypeptide, etc. When the overall polypeptide activity level and/or concentration (expression amount) in the cell is lower than that of the native strain due to (translation) inhibition, etc., when the expression of the polynucleotide is not made at all, and/or when the expression of the polynucleotide is Even if there is no activity of the polypeptide, it may also be included. The “intrinsic activity” refers to the activity of a specific polypeptide originally possessed by the parent strain, wild-type or unmodified microorganism before transformation when the trait is changed due to genetic mutation caused by natural or artificial factors. This may be used interchangeably with “activity before modification”. “Inactivation, deficiency, reduction, downregulation, reduction, attenuation” of the activity of a polypeptide compared to the intrinsic activity means that the activity of the specific polypeptide originally possessed by the parent strain or unmodified microorganism before transformation is lowered.

Attenuation of the activity of such a polypeptide may be performed by any method known in the art, but is not limited thereto, and may be achieved by application of various methods well known in the art (eg, Nakashima N et al., Bacterial cellular engineering by genome editing and gene silencing. Int J Mol Sci. 2014;15(2):2773-2793, Sambrook et al. Molecular Cloning 2012, etc.).

Specifically, the attenuation of the polypeptide of the present application is

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

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

3) modification of the amino acid sequence constituting the polypeptide such that the activity of the polypeptide is eliminated or attenuated (eg, deletion/substitution/addition of one or more amino acids on the amino acid sequence);

4) modification of the gene sequence encoding the polypeptide such that the activity of the polypeptide is eliminated or attenuated (eg, one or more nucleobases on the nucleotide sequence of the polypeptide gene to encode a polypeptide modified such that the activity of the polypeptide is eliminated or attenuated) deletion/replacement/addition of);

5) modification of the nucleotide sequence encoding the initiation codon or 5'-UTR region of the gene transcript encoding the polypeptide;

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

7) adding a sequence complementary to the Shine-Dalgarno sequence in front of the Shine-Dalgarno sequence of the gene encoding the polypeptide to form a secondary structure that cannot be attached to the ribosome;

8) addition of a promoter 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) It may be a combination of two or more selected from 1) to 8) above, but is not particularly limited thereto.

for example,

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

In addition, the above 2) modification of the expression control region (or expression control sequence), deletion, insertion, non-conservative or conservative substitution, or a combination thereof, mutation in the expression control region (or expression control sequence) occurs, or weaker replacement with an active sequence. The expression control region includes, but is not limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence regulating the termination of transcription and translation.

In addition, 3) the base sequence modification encoding the start codon or 5'-UTR region of the gene transcript encoding the polypeptide is, for example, a base encoding another start codon having a lower polypeptide expression rate than the intrinsic start codon It may be substituted with a sequence, but is not limited thereto.

In addition, the modification of the amino acid sequence or polynucleotide sequence of 4) and 5) above is a deletion, insertion, non-conservative or conservative substitution of the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide to weaken the activity of the polypeptide. Or a combination thereof may result in a mutation in sequence, or replacement with an amino acid sequence or polynucleotide sequence improved to have weaker activity or an amino acid sequence or polynucleotide sequence improved to have no activity, but is not limited thereto. For example, by introducing a mutation in the polynucleotide sequence to form a stop codon, the expression of a gene may be inhibited or attenuated, but is not limited thereto.

6) The introduction of an antisense oligonucleotide (eg, antisense RNA) that complementarily binds to the transcript of the gene encoding the polypeptide is described, for example, in 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 in front of the Shine-Dalgarno sequence of the gene encoding the polypeptide to form a secondary structure that cannot be attached to the ribosome is mRNA translation It may make it impossible or slow it down.

8) the addition of a promoter 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) is an antisense complementary to the transcript of the gene encoding the polypeptide It may be to attenuate activity by making nucleotides.

As used herein, the term “enhancement” of a polypeptide activity means that the activity of the polypeptide is increased compared to the intrinsic activity. The reinforcement may be used interchangeably with terms such as activation, up-regulation, overexpression, and increase. Herein, activation, enhancement, up-regulation, overexpression, and increase may include all of those exhibiting an activity that was not originally possessed, or exhibiting an improved activity compared to an intrinsic activity or an activity prior to modification. The “intrinsic activity” refers to the activity of a specific polypeptide originally possessed by the parent strain or unmodified microorganism before the transformation when the trait is changed due to genetic mutation caused by natural or artificial factors. This may be used interchangeably with “activity before modification”. “Enhancement”, “up-regulation”, “overexpression” or “increase” in the activity of a polypeptide compared to its intrinsic activity means that the activity and/or concentration (expression) of a specific polypeptide originally possessed by the parent strain or unmodified microorganism before transformation. amount), which means improved.

The enrichment can be achieved by introducing an exogenous polypeptide, or by enhancing the activity and/or concentration (expression amount) of the endogenous polypeptide. Whether or not the activity of the polypeptide is enhanced can be confirmed from the increase in the level of activity, expression level, or the amount of product excreted from the polypeptide.

The enhancement of the activity of the polypeptide can be applied by various methods well known in the art, and is not limited as long as it can enhance the activity of the target polypeptide compared to the microorganism before modification. Specifically, it may be one using genetic engineering and/or protein engineering well known to those skilled in the art, which is a routine method of molecular biology, but is not limited thereto (eg, Sitnicka et al. Functional Analysis of Genes. Advances in Cell). Biology 2010, Vol. 2. 1-16, Sambrook et al. Molecular Cloning 2012, etc.).

Specifically, the enrichment of the polypeptide of the present application is

1) increasing the intracellular copy number of a polynucleotide encoding the polypeptide;

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

3) modification of the nucleotide sequence encoding the initiation codon or 5'-UTR region of the gene transcript encoding the polypeptide;

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

5) modification of the polynucleotide sequence encoding the polypeptide to enhance the polypeptide activity (eg, modification of the polynucleotide sequence of the polypeptide gene to encode a polypeptide modified to enhance the activity of the polypeptide);

6) introduction of a foreign polypeptide exhibiting the activity of the polypeptide or a foreign polynucleotide encoding the same;

7) codon optimization of the polynucleotide encoding the polypeptide;

8) by analyzing the tertiary structure of the polypeptide to select an exposed site for modification or chemical modification; or

More specifically,

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

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

Examples of known strong promoters include CJ1 to CJ7 promoters (US 7662943 B2), lac promoter, trp promoter, trc promoter, tac promoter, lambda phage PR promoter, PL promoter, tet promoter, gapA promoter, SPL7 promoter, SPL13 (sm3) promoter (US Patent US 10584338 B2), O2 promoter (US Patent US 10273491 B2), tkt promoter, yccA promoter, etc., but is not limited thereto.

3) Modification of the nucleotide sequence encoding the start codon or 5'-UTR region of the gene transcript encoding the polypeptide is, for example, a nucleotide sequence encoding another start codon having a higher expression rate of the polypeptide compared to the intrinsic start codon. It may be a substitution, but is not limited thereto.

The modification of the amino acid sequence or polynucleotide sequence of 4) and 5) above may include deletion, insertion, non-conservative or conservative substitution of the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide to enhance the activity of the polypeptide; A combination thereof may result in sequence mutation, or replacement with an amino acid sequence or polynucleotide sequence improved to have stronger activity or an amino acid sequence or polynucleotide sequence improved to increase activity, but is not limited thereto. The replacement may be specifically performed by inserting a polynucleotide into a chromosome by homologous recombination, but is not limited thereto. In this case, the vector used may further include a selection marker for confirming whether or not the chromosome is inserted. The selection marker is the same as described above.

6) The introduction of the foreign polynucleotide exhibiting the activity of the polypeptide may be the introduction of the foreign polynucleotide encoding the polypeptide exhibiting the same/similar activity as the polypeptide into a host cell. The foreign polynucleotide is not limited in its origin or sequence as long as it exhibits the same/similar activity as the polypeptide. The method used for the introduction can be performed by appropriately selecting a known transformation method by those skilled in the art, and the introduced polynucleotide is expressed in a host cell to generate a polypeptide and increase its activity.

7) Codon optimization of the polynucleotide encoding the polypeptide is codon-optimized so that the transcription or translation of the endogenous polynucleotide is increased in the host cell, or the transcription and translation of the foreign polynucleotide is optimized in the host cell. It may be that its codons are optimized so that the

8) Selecting an exposed site by analyzing the tertiary structure of the polypeptide and modifying or chemically modifying it is, for example, by comparing the sequence information of the polypeptide to be analyzed with a database in which sequence information of known proteins is stored to determine the degree of sequence similarity. Accordingly, it may be to determine a template protein candidate, check the structure based on this, and select an exposed site to be modified or chemically modified and modified or modified.

Such enhancement of polypeptide activity is to increase the activity or concentration of the corresponding polypeptide based on the activity or concentration of the polypeptide expressed in the wild-type or pre-modified microbial strain, or increase the amount of product produced from the polypeptide. However, the present invention is not limited thereto.

Modification of some or all of the polynucleotide in the microorganism of the present application (eg, modification for encoding the above-described protein variant) is (a) homologous recombination using a vector for chromosome insertion in the microorganism or engineered nuclease (e.g., CRISPR) -Cas9) and/or (b) induced by light and/or chemical treatment such as ultraviolet and radiation, but not limited thereto. The method for modifying part or all of the gene may include a method by DNA recombination technology. For example, by injecting a nucleotide sequence or a vector containing a nucleotide sequence homologous to a target gene into the microorganism to cause homologous recombination, a part or all of the gene may be deleted. The injected nucleotide sequence or vector may include a dominant selection marker, but is not limited thereto.

In the microorganism of the present application, variants, polynucleotides, L-valine, and the like are as described in the other aspects above.

Production of L-amino acids

Another aspect of the present application provides a method for producing L-amino acids, comprising the step of culturing a Corynebacterium glutamicum strain comprising the mutant of the present application or the polynucleotide of the present application in a medium.

The L-amino acid production method of the present application may include culturing a Corynebacterium glutamicum strain comprising the mutant of the present application or the polynucleotide of the present application or the vector of the present application in a medium.

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

In the present application, the term "cultivation" means growing the Corynebacterium glutamicum strain of the present application under moderately controlled environmental conditions. The culture process of the present application may be performed according to a suitable medium and culture conditions known in the art. Such a culture process can be easily adjusted and used by those skilled in the art according to the selected strain. Specifically, the culture may be a batch, continuous and/or fed-batch, but is not limited thereto.

As used herein, the term "medium" refers to a material in which nutrients required for culturing the Corynebacterium glutamicum strain of the present application are mixed as a main component, and nutrients including water essential for survival and growth and growth factors. Specifically, any medium and other culture conditions used for culturing the Corynebacterium glutamicum strain of the present application may be used without any particular limitation as long as it is a medium used for culturing conventional microorganisms, but the Corynebacterium glutamicum of the present application Lium glutamicum strain can be cultured while controlling the temperature, pH, etc. under aerobic conditions in a conventional medium containing an appropriate carbon source, nitrogen source, phosphorus, inorganic compound, amino acid and / or vitamin and the like.

Specifically, the culture medium for the Corynebacterium sp. strain can be found in the literature ["Manual of Methods for General Bacteriology" by the American Society for Bacteriology (Washington D.C., USA, 1981)].

In the present application, the carbon source includes carbohydrates such as glucose, saccharose, lactose, fructose, sucrose, maltose, and the like; sugar alcohols such as mannitol and sorbitol; organic acids such as pyruvic acid, lactic acid, citric acid and the like; amino acids such as glutamic acid, methionine, lysine, and the like may be included. In addition, natural organic nutrient sources such as starch hydrolyzate, molasses, blackstrap molasses, rice winter, cassava, sugar cane offal and corn steep liquor can be used, specifically glucose and sterilized pre-treated molasses (i.e., converted to reducing sugar). molasses) may be used, and other appropriate amounts of carbon sources may be variously used without limitation. These carbon sources may be used alone or in combination of two or more, but is not limited thereto.

Examples of the nitrogen source include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, anmonium carbonate, and ammonium nitrate; Amino acids such as glutamic acid, methionine, glutamine, and organic nitrogen sources such as peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolyzate, fish or degradation products thereof, defatted soybean cake or degradation products thereof, etc. can be used These nitrogen sources may be used alone or in combination of two or more, but is not limited thereto.

The phosphorus may include potassium first potassium phosphate, second potassium phosphate, or a sodium-containing salt corresponding thereto. As the inorganic compound, sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, etc. may be used, and in addition, amino acids, vitamins and/or suitable precursors may be included. These components or precursors may be added to the medium either batchwise or continuously. However, the present invention is not limited thereto.

In addition, during the culture of the Corynebacterium glutamicum strain of the present application, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid, etc. may be added to the medium in an appropriate manner to adjust the pH of the medium. In addition, during culturing, an antifoaming agent such as fatty acid polyglycol ester may be used to suppress bubble formation. In addition, in order to maintain the aerobic state of the medium, oxygen or oxygen-containing gas may be injected into the medium, or nitrogen, hydrogen or carbon dioxide gas may be injected without or without gas to maintain anaerobic and microaerobic conditions, it is not

In the culture of the present application, the culture temperature may be maintained at 20 to 45° C., specifically, 25 to 40° C., and may be cultured for about 10 to 160 hours, but is not limited thereto.

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

The L-amino acid production method of the present application includes the steps of preparing the Corynebacterium glutamicum strain of the present application, preparing a medium for culturing the strain, or a combination thereof (regardless of the order, in any order) ), for example, prior to the culturing step, may further include.

The method for producing L-amino acids of the present application may further include recovering L-amino acids from the culture medium (the culture medium) or the Corynebacterium glutamicum strain. The recovering step may be further included after the culturing step.

The recovery may be to collect the desired L-amino acid using a suitable method known in the art according to the culture method of the microorganism of the present application, for example, a batch, continuous or fed-batch culture method, etc. . For example, centrifugation, filtration, treatment with a crystallized protein precipitating agent (salting out method), extraction, ultrasonic disruption, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity Various chromatography such as island chromatography, HPLC, or a combination thereof may be used, and a desired L-amino acid may be recovered from a medium or a microorganism using a suitable method known in the art.

In addition, the L-amino acid production method of the present application may include an additional purification step. The purification may be performed using a suitable method known in the art. In one example, when the L-amino acid production method of the present application includes both the recovery step and the purification step, the recovery step and the purification step are performed continuously or discontinuously, regardless of the order, or integrated into one step may be performed, but is not limited thereto.

In the method of the present application, variants, polynucleotides, vectors, strains, and the like are as described in the other aspects above.

Another aspect of the present application is a variant of the present application, a polynucleotide encoding the variant, a vector including the polynucleotide or a Corynebacterium glutamicum strain comprising the polynucleotide of the present application; the culture medium; Or to provide a composition for the production of L- amino acids comprising a combination of two or more of them.

The composition of the present application may further include any suitable excipients commonly used in compositions for the production of amino acids, and these excipients may be, for example, preservatives, wetting agents, dispersing agents, suspending agents, buffering agents, stabilizing agents or isotonic agents, etc. However, the present invention is not limited thereto.

In the composition of the present application, variants, polynucleotides, vectors, strains, media and L-amino acids are the same as those described in the other aspects above.

In the case of culturing the Corynebacterium glutamicum strain, including the novel protein variant of the present application, it is possible to produce L-valine in a high yield compared to a microorganism having an existing unmodified polypeptide.

1 is a schematic diagram of a pDCM2 plasmid.

Hereinafter, the present application will be described in more detail by way of Examples. However, the following examples are merely preferred embodiments for illustrating the present application, and therefore, are not intended to limit the scope of the present application thereto. On the other hand, technical matters not described in this specification can be sufficiently understood and easily implemented by those skilled in the art or similar technical fields of the present application.

실시예 1: NCgl1391 단백질 변이체를 발현하는 미생물의 제작 및 이를 이용한 L-발린 생산Example 1: Production of microorganisms expressing NCgl1391 protein mutant and L-valine production using the same

1.1. 플라스미드의 제작1.1. Construction of the plasmid

A plasmid (pDCM2, FIG. 1, SEQ ID NO: 171) for the insertion and replacement of genes in the Corynebacterium chromosome was designed, and the plasmid was synthesized using the Gene-synthesis service of Bionics Co., Ltd. A plasmid was designed to include a restriction enzyme that is easy to use for cloning with reference to a generally known sacB system related paper [Gene, 145 (1994) 69-73]. The thus synthesized pDCM2 plasmid has the following characteristics.

1) Since it has a replication origin that works only in E. coli, self-replication is possible in E. coli, but self-replication is impossible in Corynebacterium.

2) It has a kanamycin resistance gene as a selectable marker.

3) It has a Levan sucrase gene (sacB) as a secondary positive-selection marker.

4) No genetic information derived from the pDCM2 plasmid is left in the finally constructed strain.

1.2. 미생물내 단백질 변이체 발현을 위한 벡터 제작1.2. Vector construction for expression of protein variants in microorganisms

To determine the effect of a mutant (S109F; SEQ ID NO: 1) in which serine (Ser, S) at position 109 of the protein consisting of the amino acid sequence of SEQ ID NO: 3 is substituted with phenylalanine (Phe, F) on L-valine production, its A vector for constructing an expression strain was prepared as follows.

Using the gDNA (genomic DNA) of wild-type Corynebacterium glutamicum ATCC14067 as a template, PCR was performed using a primer pair of sequences of SEQ ID NOs: 5 and 6 and a pair of primers of sequences of SEQ ID NOs: 7 and 8, respectively. Using the mixture of the two fragments obtained above as a template, overlapping PCR was performed again using the primer pair of SEQ ID NO: 5 and SEQ ID NO: 8 to obtain a fragment. After denaturing at 94°C for 5 minutes, PCR was repeated 30 times for 30 seconds at 94°C for 30 seconds, at 55°C for 30 seconds, and at 72°C for 1 minute and 30 seconds, and then at 72°C for 5 minutes. The pDCM2 vector was treated with smal and the PCR product obtained above was fusion cloned. Fusion cloning was performed using the In-Fusion® HD cloning kit (Clontech). The resulting plasmid was named pDCM2-NCgl1391 (S109F). The sequences of the primers used in this Example are shown in Table 1 below.

명칭designation	서열 (5’->3’)sequence (5'->3')	서열번호SEQ ID NO:
NCgl1391_1FNCgl1391_1F	TCGAGCTCGGTACCCACGGCCTCATGGATGACGTCTTCGAGCTCGGTACCCACGGCCTCATGGATGACGCTCT	서열번호 5SEQ ID NO: 5
NCgl1391_2RNCgl1391_2R	TCGTGGATGTAGCGCACAGAGAACCCACGGCGTTGCATATAGGATCGTGGATGTAGCGCACAGAGAACCCACGGCGTTGCATATAGGA	서열번호 6SEQ ID NO: 6
NCgl1391_3FNCgl1391_3F	TCCTATATGCAACGCCGTGGGTTCTCTGTGCGCTACATCCACGATCCTATATGCAACGCCGTGGGTTCTCTGTGCGCTACATCCACGA	서열번호 7SEQ ID NO: 7
NCgl1391_4RNCgl1391_4R	CTCTAGAGGATCCCCTTTGCTCGTTCAGACAGCTCGCTCTAGAGGATCCCCTTTGCTCGTTCAGACAGCTCG	서열번호 8SEQ ID NO: 8

1.3. 단백질 변이체를 발현하는 미생물의 L-발린 생산능 평가1.3. Evaluation of L-valine-producing ability of microorganisms expressing protein variants

1.3.1. 단백질1.3.1. protein 변이체 발현 균주 제작Production of mutant expression strains

The vector prepared in Example 1.2 was transformed into Corynebacterium glutamicum CA08-0072 (KCCM11201P).

In the transformed strains, a strain in which homologous recombination occurred using the primer pair of SEQ ID NOs: 9 and 10 was selected and named CA08-0072_NCgl1391_S109F. The sequences of the primers used in this Example are shown in Table 2 below.

명칭designation	서열 (5’->3’)sequence (5'->3')	서열번호SEQ ID NO:
NCgl1391_5FNCgl1391_5F	ACGGCCTCATGGATGACGTCTACGGCTCATGGATGACGTCT	서열번호 9SEQ ID NO: 9
NCgl1391_6RNCgl1391_6R	TTTGCTCGTTCAGACAGCTCGTTTGCTCGTTCAGACAGCTCG	서열번호 10SEQ ID NO: 10

1.3.2. 단백질 1.3.2. protein 변이체variant 발현 균주의 L-발린 L-valine of the expression strain 생산능productivity 비교 compare

The L-valine production ability was analyzed by evaluating the flask fermentation titer of each strain and the control parent strain prepared in Example 1.3.1.

First, each colony was subcultured in a nutrient medium, and then each strain was inoculated in a 250 ml corner-baffle flask containing 25 ml of the production medium, and cultured with shaking at 30° C. for 72 hours at 200 rpm. Thereafter, the concentration of L-valine was analyzed using HPLC, and the analyzed concentrations of L-valine are shown in Table 3 below.

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

Glucose 100 g, Ammonium Sulfate 40 g, Soy Protein 2.5 g, Corn Steep Solids 5 g, Urea 3 g, Potassium Dibasic 1 g, Magnesium Sulfate 0.5 g, Biotin 100 μg, Thiamine-HCl 1 mg, calcium pantothenate 2 mg, nicotinamide 3 mg, calcium carbonate 30 g (based on 1 liter of distilled water).

The experiment was repeated 3 times, and the average value of the analysis results is shown in Table 3 below.

균 주strain	L-발린 농도 (g/L)L-valine concentration (g/L)	L-발린 농도 증가율(%)L-valine concentration increase rate (%)
CA08-0072CA08-0072	2.82.8	--
CA08-0072_NCgl1391_S109FCA08-0072_NCgl1391_S109F	3.53.5	25.025.0

As shown in Table 3, the CA08-0072_NCgl1391_S109F strain exhibited increased L-valine-producing ability compared to the control.

The mutant strain CA08-0072_NCgl1391_S109F was named Corynebacterium glutamicum CA08-1743, and was deposited with the Korean Culture of Microorganisms (KCCM), a microbial depository under the Budapest Treaty, with an accession number KCCM12860P on December 02, 2020.

실시예 2: ATP 포스포리보실트랜스퍼라제 변이체를 발현하는 미생물의 제작 및 이를 이용한 L-발린 생산Example 2: Production of microorganisms expressing ATP phosphoribosyltransferase mutants and L-valine production using the same

2.1. 플라스미드의 제작2.1. Construction of the plasmid

2) It has a kanamycin resistance gene as a selectable marker.

3) It has a Levan sucrase gene (sacB) as a secondary positive-selection marker.

2.2. 미생물내 단백질 변이체 발현을 위한 벡터 제작2.2. Vector construction for expression of protein variants in microorganisms

To determine the effect of a mutant (S130F; SEQ ID NO: 11) in which serine (Ser, S) at position 130 of the protein consisting of the amino acid sequence of SEQ ID NO: 13 is substituted with phenylalanine (Phe, F) on L-valine production, its A vector for constructing an expression strain was prepared as follows.

Using the gDNA (genomic DNA) of wild-type Corynebacterium glutamicum ATCC14067 as a template, PCR was performed using a primer pair of sequences of SEQ ID NOs: 15 and 16 and a pair of primers of sequences of SEQ ID NOs: 17 and 18, respectively. Using the mixture of the two fragments obtained above as a template, overlapping PCR was performed again using the primer pair of SEQ ID NO: 15 and SEQ ID NO: 18 to obtain a fragment. After denaturing at 94°C for 5 minutes, PCR was repeated 30 times at 94°C for 30 seconds, at 55°C for 30 seconds, and at 72°C for 1 minute and 30 seconds, and then at 72°C for 5 minutes. The pDCM2 vector was treated with smal and the PCR product obtained above was fusion cloned. Fusion cloning was performed using the In-Fusion® HD cloning kit (Clontech). The resulting plasmid was named pDCM2-hisG(S130F). The sequences of the primers used in this Example are shown in Table 4 below.

명칭designation	서열 (5’->3’)sequence (5'->3')	서열번호SEQ ID NO:
hisG_1FhisG_1F	TCGAGCTCGGTACCCATGTTGAAAATCGCTGTCCCATCGAGCTCGGTACCCATGTTGAAAATCGCTGTCCCA	서열번호 15SEQ ID NO: 15
hisG_2RhisG_2R	GTCGAGGCGGAGCACCTCAGCGAAAAGCCCACGTGCTGCGAGGTGTCGAGGCGGAGCACCTCAGCGAAAAGCCCACGTGCTGCGAGGT	서열번호 16SEQ ID NO: 16
hisG_3FhisG_3F	ACCTCGCAGCACGTGGGCTTTTCGCTGAGGTGCTCCGCCTCGACACCTCGCAGCACGTGGGCTTTTCGCTGAGGTGCTCCGCCTCGAC	서열번호 17SEQ ID NO: 17
hisG_4RhisG_4R	CTCTAGAGGATCCCCCTAGATGCGGGCGATGCGGATCTCTAGAGGATCCCCCTAGATGCGGGCGATGCGGAT	서열번호 18SEQ ID NO: 18

2.3. 단백질 변이체를 발현하는 미생물의 L-발린 생산능 평가2.3. Evaluation of L-valine-producing ability of microorganisms expressing protein variants

2.3.1. 단백질2.3.1. protein 변이체 발현 균주 제작Production of mutant expression strains

The vector prepared in Example 2.2 was transformed into Corynebacterium glutamicum CA08-0072 (KCCM11201P).

In the transformed strains, a strain in which homologous recombination occurred using the primer pair of SEQ ID NOs: 19 and 20 was selected and named CA08-0072_hisG_S130F. The sequences of the primers used in this Example are shown in Table 5 below.

명칭designation	서열 (5’->3’)sequence (5'->3')	서열번호SEQ ID NO:
hisG_5FhisG_5F	ATGTTGAAAATCGCTGTCCCAATGTTGAAAATCGCTGTCCCA	서열번호 19SEQ ID NO: 19
hisG_6RhisG_6R	CTAGATGCGGGCGATGCGGATCTAGATCGGGCGATGCGGAT	서열번호 20SEQ ID NO: 20

2.3.2. 단백질 2.3.2. protein 변이체variant 발현 균주의 L-발린 L-valine of the expression strain 생산능productivity 비교 compare

The L-valine production ability was analyzed by evaluating the flask fermentation titer of each strain and the control parent strain prepared in Example 2.3.1.

First, each colony was subcultured in a nutrient medium, and then each strain was inoculated in a 250 ml corner-baffle flask containing 25 ml of the production medium, and cultured with shaking at 30° C. for 72 hours at 200 rpm. Thereafter, the concentration of L-valine was analyzed using HPLC, and the analyzed concentrations of L-valine are shown in Table 6 below.

The experiment was repeated 3 times, and the average value of the analysis results is shown in Table 6 below.

균 주strain	L-발린 농도 (g/L)L-valine concentration (g/L)	L-발린 농도 증가율(%)L-valine concentration increase rate (%)
CA08-0072CA08-0072	2.82.8	--
CA08-0072_hisG_S130FCA08-0072_hisG_S130F	3.393.39	21.121.1

As shown in Table 6, the CA08-0072_hisG_S130F strain exhibited increased L-valine-producing ability compared to the control.

The mutant strain CA08-0072_hisG_S130F was named Corynebacterium glutamicum CA08-1744, and was deposited with the accession number KCCM12861P on December 02, 2020 at the Korean Culture of Microorganisms (KCCM), a microbial depository under the Budapest Treaty.

실시예 3: 아스파라긴 신타제 변이체를 발현하는 미생물의 제작 및 이를 이용한 L-발린 생산Example 3: Preparation of microorganisms expressing asparagine synthase variants and L-valine production using the same

3.1. 플라스미드의 제작3.1. Construction of the plasmid

2) It has a kanamycin resistance gene as a selectable marker.

3) It has a Levan sucrase gene (sacB) as a secondary positive-selection marker.

3.2. 미생물내 단백질 변이체 발현을 위한 벡터 제작3.2. Vector construction for expression of protein variants in microorganisms

To determine the effect of the mutant (P192L; SEQ ID NO: 21) in which proline (Pro, P) at position 192 of the protein consisting of the amino acid sequence of SEQ ID NO: 23 is substituted with leucine (Leu, L) on L-valine production, its A vector for constructing an expression strain was prepared as follows.

PCR was performed using the primer pair of the sequences of SEQ ID NOs: 25 and 26 and the primer pair of the sequences of SEQ ID NOs: 27 and 28 using gDNA (genomic DNA) of wild-type Corynebacterium glutamicum ATCC14067 as a template, respectively. Using the mixture of the two fragments obtained above as a template, overlapping PCR was performed again using a pair of primers of SEQ ID NO: 25 and SEQ ID NO: 28 to obtain a fragment. After denaturing at 94°C for 5 minutes, PCR was repeated 30 times at 94°C for 30 seconds, at 55°C for 30 seconds, and at 72°C for 1 minute and 30 seconds, and then at 72°C for 5 minutes. The pDCM2 vector was treated with smal and the PCR product obtained above was fusion cloned. Fusion cloning was performed using the In-Fusion® HD cloning kit (Clontech). The resulting plasmid was named pDCM2-ltsA (P192L). The sequences of the primers used in this Example are shown in Table 7 below.

명칭designation	서열 (5’->3’)sequence (5'->3')	서열번호SEQ ID NO:
ltsA_1FltsA_1F	TCGAGCTCGGTACCCCTCCATCATTGATATTGCACATCGAGCTCGGTACCCCTCCATCATTGATATTGCACA	서열번호 25SEQ ID NO: 25
ltsA_2RltsA_2R	TTCTGTTCCAGCTTGCCGCCCAGACGAACTGTTGCGGTGCAGCCTTCTGTTCCAGCTTGCCGCCCAGACGAACTGTTGCGGTGCAGCC	서열번호 26SEQ ID NO: 26
ltsA_3FltsA_3F	GGCTGCACCGCAACAGTTCGTCTGGGCGGCAAGCTGGAACAGAAGGCTGCACCGCAACAGTTCGTCTGGGCGGCAAGCTGGAACAGAA	서열번호 27SEQ ID NO: 27
ltsA_4RltsA_4R	CTCTAGAGGATCCCCCTTGACGTGCTTACGTGCTTCCTCTAGAGGATCCCCCTTGACGTGCTTACGTGCTTC	서열번호 28SEQ ID NO: 28

3.3. 단백질 변이체를 발현하는 미생물의 L-발린 생산능 평가3.3. Evaluation of L-valine-producing ability of microorganisms expressing protein variants

3.3.1. 단백질3.3.1. protein 변이체 발현 균주 제작Production of mutant expression strains

The vector prepared in Example 3.2 was transformed into Corynebacterium glutamicum CA08-0072 (KCCM11201P).

In the transformed strains, a strain in which homologous recombination occurred using the primer pair of SEQ ID NOs: 29 and 30 was selected and named CA08-0072_ltsA_P192L. The sequences of the primers used in this Example are shown in Table 8 below.

명칭designation	서열 (5’->3’)sequence (5'->3')	서열번호SEQ ID NO:
ltsA_5FltsA_5F	CTCCATCATTGATATTGCACACTCCATCATTGATATTGCACA	서열번호 29SEQ ID NO: 29
ltsA_6RltsA_6R	CTTGACGTGCTTACGTGCTTCCTTGACTGCTTACGTGCTTC	서열번호 30SEQ ID NO: 30

3.3.2. 단백질 변이체 발현 균주의 L-발린 생산능 비교3.3.2. Comparison of L-valine production capacity of protein variant expression strains

The L-valine production ability was analyzed by evaluating the flask fermentation titer of each strain and the control parent strain prepared in Example 3.3.1.

First, each colony was subcultured in a nutrient medium, and then each strain was inoculated in a 250 ml corner-baffle flask containing 25 ml of the production medium, and cultured with shaking at 30° C. for 72 hours at 200 rpm. Thereafter, the concentration of L-valine was analyzed using HPLC, and the analyzed concentrations of L-valine are shown in Table 9 below.

The experiment was repeated 3 times, and the average value of the analysis results is shown in Table 9 below.

균 주strain	L-발린 농도 (g/L)L-valine concentration (g/L)	L-발린 농도 증가율(%)L-valine concentration increase rate (%)
CA08-0072CA08-0072	2.82.8	--
CA08-0072_ltsA_P192LCA08-0072_ltsA_P192L	3.233.23	15.415.4

As shown in Table 9, the CA08-0072_ltsA_P192L strain exhibited increased L-valine-producing ability compared to the control.

The mutant strain CA08-0072_ltsA_P192L was named Corynebacterium glutamicum CA08-1747, and was deposited with the Korean Culture of Microorganisms (KCCM), a microbial depository under the Budapest Treaty, as of December 02, 2020 with an accession number KCCM12863P.

실시예 4: 스퍼미딘 신타제 변이체를 발현하는 미생물의 제작 및 이를 이용한 L-발린 생산Example 4: Production of microorganisms expressing spermidine synthase variants and L-valine production using the same

4.1. 플라스미드의 제작4.1. Construction of the plasmid

2) It has a kanamycin resistance gene as a selectable marker.

3) It has a Levan sucrase gene (sacB) as a secondary positive-selection marker.

4.2. 미생물내 단백질 변이체 발현을 위한 벡터 제작4.2. Vector construction for expression of protein variants in microorganisms

To determine the effect of the mutant (A213V; SEQ ID NO: 31) in which alanine (Ala, A) at position 213 of the protein consisting of the amino acid sequence of SEQ ID NO: 33 is substituted with valine (Val, V) on L-valine production, its A vector for constructing an expression strain was prepared as follows.

PCR was performed using the primer pair of SEQ ID NOs: 35 and 36 and the primer pair of SEQ ID NOs: 37 and 38 using gDNA (genomic DNA) of wild-type Corynebacterium glutamicum ATCC14067 as a template, respectively. Using the mixture of the two fragments obtained above as a template, overlapping PCR was performed again using the primer pair of SEQ ID NO: 35 and SEQ ID NO: 38 to obtain a fragment. After denaturing at 94°C for 5 minutes, PCR was repeated 30 times at 94°C for 30 seconds, at 55°C for 30 seconds, and at 72°C for 1 minute and 30 seconds, and then at 72°C for 5 minutes. The pDCM2 vector was treated with smal and the PCR product obtained above was fusion cloned. Fusion cloning was performed using the In-Fusion® HD cloning kit (Clontech). The resulting plasmid was named pDCM2-speE (A213V). The sequences of the primers used in this Example are shown in Table 10 below.

명칭designation	서열 (5’->3’)sequence (5'->3')	서열번호SEQ ID NO:
speE_1FspeE_1F	TCGAGCTCGGTACCCCCTCCCTCATCGTCGCAGGTTTCGAGCTCGGTACCCCCTCCCTCATCGTCGCAGGTT	서열번호 35SEQ ID NO: 35
speE_2RspeE_2R	CACGGTGGCAAGCGCTGCGATCACGAGAAGAAGCGCCACCACGGCACGGTGGCAAGCGCTGCGATCACGAGAAGAAGCGCCACCACGG	서열번호 36SEQ ID NO: 36
speE_3FspeE_3F	CCGTGGTGGCGCTTCTTCTCGTGATCGCAGCGCTTGCCACCGTGCCGTGGTGGCGCTTCTTCTCGTGATCGCAGCGCTTGCCACCGTG	서열번호 37SEQ ID NO: 37
speE_4RspeE_4R	CTCTAGAGGATCCCCCTGGCCATGGTGTCGTTGTTTCTCTAGAGGATCCCCCTGGCCATGGTGTCGTTGTTT	서열번호 38SEQ ID NO: 38

4.3. 단백질 변이체를 발현하는 미생물의 L-발린 생산능 평가4.3. Evaluation of L-valine-producing ability of microorganisms expressing protein variants

4.3.1. 단백질4.3.1. protein 변이체 발현 균주 제작Production of mutant expression strains

The vector prepared in Example 4.2 was transformed into Corynebacterium glutamicum CA08-0072 (KCCM11201P).

In the transformed strains, a strain in which homologous recombination occurred using the primer pair of SEQ ID NOs: 39 and 40 was selected and named CA08-0072_speE_A213V. The sequences of the primers used in this Example are shown in Table 11 below.

명칭designation	서열 (5’->3’)sequence (5'->3')	서열번호SEQ ID NO:
speE_5FspeE_5F	CCTCCCTCATCGTCGCAGGTTCCTCCCTCATCGTCGCAGGTT	서열번호 39SEQ ID NO: 39
speE_6RspeE_6R	CTGGCCATGGTGTCGTTGTTTCTGGCCATGGTGTCGTTGTTTT	서열번호 40SEQ ID NO: 40

4.3.2. 단백질 4.3.2. protein 변이체variant 발현 균주의 L-발린 L-valine of the expression strain 생산능productivity 비교 compare

The L-valine production ability was analyzed by evaluating the flask fermentation titer of each strain and the control parent strain prepared in Example 4.3.1.

First, each colony was subcultured in a nutrient medium, and then each strain was inoculated in a 250 ml corner-baffle flask containing 25 ml of the production medium, and cultured with shaking at 30° C. for 72 hours at 200 rpm. Thereafter, the concentration of L-valine was analyzed using HPLC, and the analyzed concentrations of L-valine are shown in Table 12 below.

The experiment was repeated 3 times, and the average value of the analysis results is shown in Table 12 below.

균 주strain	L-발린 농도 (g/L)L-valine concentration (g/L)	L-발린 농도 증가율 (%)L-valine concentration increase rate (%)
CA08-0072CA08-0072	2.82.8	--
CA08-0072_speE_A213VCA08-0072_speE_A213V	3.173.17	13.213.2

As shown in Table 12, the CA08-0072_speE_A213V strain exhibited increased L-valine-producing ability compared to the control.

The mutant strain CA08-0072_speE_A213V was named Corynebacterium glutamicum CA08-1748, and was deposited with the Korean Culture of Microorganisms (KCCM), a microbial depository under the Budapest Treaty, under the accession number KCCM12864P on December 02, 2020.

실시예 5: 시스테인 설피네이트 디설피나제 변이체를 발현하는 미생물의 제작 및 이를 이용한 L-발린 생산Example 5: Production of microorganisms expressing cysteine sulfinate disulfinase variants and L-valine production using the same

5.1. 플라스미드의 제작5.1. Construction of the plasmid

2) It has a kanamycin resistance gene as a selectable marker.

3) It has a Levan sucrase gene (sacB) as a secondary positive-selection marker.

5.2. 미생물내 단백질 변이체 발현을 위한 벡터 제작5.2. Vector construction for expression of protein variants in microorganisms

To determine the effect of the mutant (P302S; SEQ ID NO: 41) in which proline (Pro, P) at position 302 of the protein consisting of the amino acid sequence of SEQ ID NO: 43 is substituted with serine (Ser, S) on L-valine production, its A vector for constructing an expression strain was prepared as follows.

PCR was performed using the primer pair of the sequences of SEQ ID NOs: 45 and 46 and the primer pair of the sequences of SEQ ID NOs: 47 and 48 using gDNA (genomic DNA) of wild-type Corynebacterium glutamicum ATCC14067 as a template, respectively. Using the mixture of the two fragments obtained above as a template, overlapping PCR was performed again using the primer pair of SEQ ID NO: 45 and SEQ ID NO: 48 to obtain a fragment. After denaturing at 94°C for 5 minutes, PCR was repeated 30 times at 94°C for 30 seconds, at 55°C for 30 seconds, and at 72°C for 1 minute and 30 seconds, and then at 72°C for 5 minutes. The pDCM2 vector was treated with smal and the PCR product obtained above was fusion cloned. Fusion cloning was performed using the In-Fusion® HD cloning kit (Clontech). The resulting plasmid was named pDCM2-nadS (P302S). The sequences of the primers used in this Example are shown in Table 13 below.

명칭designation	서열 (5’->3’)sequence (5'->3')	서열번호SEQ ID NO:
nadS_1FnadS_1F	TCGAGCTCGGTACCCCAGTCAGGCCGGACACCACATTCGAGCTCGGTACCCCAGTCAGGCCGGACACCACAT	서열번호 45SEQ ID NO: 45
nadS_2RnadS_2R	CCGGAACCACAGGCAGATCCAGAGGAGCAGACAATGCCTTGGCGCCGGAACCACAGGCAGATCCAGAGGAGCAGACAATGCCTTGGCG	서열번호 46SEQ ID NO: 46
nadS_3FnadS_3F	CGCCAAGGCATTGTCTGCTCCTCTGGATCTGCCTGTGGTTCCGGCGCCAAGGCATTGTCTGCTCCTCTGGATCTGCCTGTGGTTCCGG	서열번호 47SEQ ID NO: 47
nadS_4RnadS_4R	CTCTAGAGGATCCCCTGATGCCAGCAGTGCAGTCCACTCTAGAGGATCCCCTGATGCCAGCAGTGCAGTCCA	서열번호 48SEQ ID NO: 48

5.3. 단백질 변이체를 발현하는 미생물의 L-발린 생산능 평가5.3. Evaluation of L-valine-producing ability of microorganisms expressing protein variants

5.3.1. 단백질5.3.1. protein 변이체 발현 균주 제작Production of mutant expression strains

The vector prepared in Example 5.2 was transformed into Corynebacterium glutamicum CA08-0072 (KCCM11201P).

In the transformed strains, a strain in which homologous recombination occurred using the primer pair of SEQ ID NOs: 49 and 50 was selected and named CA08-0072_nadS_P302S. The sequences of the primers used in this Example are shown in Table 14 below.

명칭designation	서열 (5’->3’)sequence (5'->3')	서열번호SEQ ID NO:
nadS_5FnadS_5F	CAGTCAGGCCGGACACCACATCAGTCAGGCCGGACACCACAT	서열번호 49SEQ ID NO: 49
nadS_6RnadS_6R	TGATGCCAGCAGTGCAGTCCATGATGCCAGCAGTGCAGTCCA	서열번호 50SEQ ID NO: 50

5.3.2. 단백질 변이체 발현 균주의 L-발린 생산능 비교5.3.2. Comparison of L-valine production capacity of protein variant expression strains

The L-valine production ability was analyzed by evaluating the flask fermentation titer of each strain and the control parent strain prepared in Example 5.3.1.

First, each colony was subcultured in a nutrient medium, and then each strain was inoculated in a 250 ml corner-baffle flask containing 25 ml of the production medium, and cultured with shaking at 30° C. for 72 hours at 200 rpm. Thereafter, the concentration of L-valine was analyzed using HPLC, and the analyzed concentrations of L-valine are shown in Table 15 below.

The experiment was repeated 3 times, and the average value of the analysis results is shown in Table 15 below.

균 주strain	L-발린 농도(g/L)L-valine concentration (g/L)	L-발린 농도 증가율(%)L-valine concentration increase rate (%)
CA08-0072CA08-0072	2.82.8	--
CA08-0072_nadS_P302SCA08-0072_nadS_P302S	3.533.53	26.126.1

As shown in Table 15, the CA08-0072_nadS_P302S strain exhibited increased L-valine-producing ability compared to the control.

The mutant strain CA08-0072_nadS_P302S was named Corynebacterium glutamicum CA08-1751, and was deposited with the Korean Culture of Microorganisms (KCCM), a microbial depository under the Budapest Treaty, with an accession number KCCM12867P as of December 02, 2020.

실시예 6: 우레아제 부속 단백질 변이체를 발현하는 미생물의 제작 및 이를 이용한 L-발린 생산Example 6: Production of microorganisms expressing urease accessory protein variants and L-valine production using the same

6.1. 플라스미드의 제작6.1. Construction of the plasmid

2) It has a kanamycin resistance gene as a selectable marker.

3) It has a Levan sucrase gene (sacB) as a secondary positive-selection marker.

6.2. 미생물내 단백질 변이체 발현을 위한 벡터 제작6.2. Vector construction for expression of protein variants in microorganisms

To determine the effect of the mutant (P56L; SEQ ID NO: 51) in which proline (Pro, P) at position 56 of the protein consisting of the amino acid sequence of SEQ ID NO: 53 is substituted with leucine (Leu, L) on L-valine production, its A vector for constructing an expression strain was prepared as follows.

PCR was performed using the primer pair of sequences of SEQ ID NOs: 55 and 56 and the primer pair of sequences of SEQ ID NOs: 57 and 58, respectively, using gDNA (genomic DNA) of wild-type Corynebacterium glutamicum ATCC14067 as a template. Using the mixture of the two fragments obtained above as a template, overlapping PCR was performed again using the primer pair of SEQ ID NO: 55 and SEQ ID NO: 58 to obtain a fragment. After denaturing at 94°C for 5 minutes, PCR was repeated 30 times at 94°C for 30 seconds, at 55°C for 30 seconds, and at 72°C for 1 minute and 30 seconds, and then at 72°C for 5 minutes. The pDCM2 vector was treated with smal and the PCR product obtained above was fusion cloned. Fusion cloning was performed using the In-Fusion® HD cloning kit (Clontech). The resulting plasmid was named pDCM2-ureE (P56L). The sequences of the primers used in this Example are shown in Table 16 below.

명칭designation	서열 (5’->3’)sequence (5'->3')	서열번호SEQ ID NO:
ureE_1FureE_1F	TCGAGCTCGGTACCCTGGGTTGATGGTCAATTCCCTTCGAGCTCGGTACCCTGGGTTGATGGTCAATTCCCT	서열번호 55SEQ ID NO: 55
ureE_2RureE_2R	CACATCGCCTTCCCGCAGGATGAGGTGCCCGTGATTGAGCCGCACACATCGCCTTCCCGCAGGATGAGGTGCCCGTGATTGAGCCGCA	서열번호 56SEQ ID NO: 56
ureE_3FureE_3F	TGCGGCTCAATCACGGGCACCTCATCCTGCGGGAAGGCGATGTGTGCGGCTCAATCACGGGCACCTCATCCTGCGGGAAGGCGATGTG	서열번호 57SEQ ID NO: 57
ureE_4RureE_4R	CTCTAGAGGATCCCCAAACATCCTCATTGCCAAAGCCTCTAGAGGATCCCCAAACATCCTCATTGCCAAAGC	서열번호 58SEQ ID NO: 58

6.3. 단백질 변이체를 발현하는 미생물의 L-발린 생산능 평가6.3. Evaluation of L-valine-producing ability of microorganisms expressing protein variants

6.3.1. 단백질6.3.1. protein 변이체 발현 균주 제작Production of mutant expression strains

The vector prepared in Example 6.2 was transformed into Corynebacterium glutamicum CA08-0072 (KCCM11201P).

In the transformed strains, a strain in which homologous recombination occurred using the primer pair of SEQ ID NOs: 59 and 60 was selected and named CA08-0072_ureE_P56L. The sequences of the primers used in this Example are shown in Table 17 below.

명칭designation	서열 (5’->3’)sequence (5'->3')	서열번호SEQ ID NO:
ureE_5FureE_5F	TGGGTTGATGGTCAATTCCCTTGGGTTGATGGTCAATTCCCT	서열번호 59SEQ ID NO: 59
ureE_6RureE_6R	AAACATCCTCATTGCCAAAGCAAACATCCTCATTGCCAAAGC	서열번호 60SEQ ID NO: 60

6.3.2. 단백질 변이체 발현 균주의 L-발린 생산능 비교6.3.2. Comparison of L-valine production capacity of protein variant expression strains

L-valine production ability was analyzed through the evaluation of flask fermentation titer of each strain and control parent strain prepared in Example 6.3.1.

First, each colony was subcultured in a nutrient medium, and then each strain was inoculated in a 250 ml corner-baffle flask containing 25 ml of the production medium, and cultured with shaking at 30° C. for 72 hours at 200 rpm. Thereafter, the concentration of L-valine was analyzed using HPLC, and the analyzed concentrations of L-valine are shown in Table 18 below.

The experiment was repeated 3 times, and the average value of the analysis results is shown in Table 18 below.

균 주strain	L-발린 농도 (g/L)L-valine concentration (g/L)	L-발린 농도 증가율(%)L-valine concentration increase rate (%)
CA08-0072CA08-0072	2.82.8	--
CA08-0072_ureE_P56LCA08-0072_ureE_P56L	3.573.57	27.527.5

As shown in Table 18, the CA08-0072_ureE_P56L strain exhibited increased L-valine-producing ability compared to the control.

The mutant strain CA08-0072_ureE_P56L was named Corynebacterium glutamicum CA08-1749, and was deposited with the Korean Culture of Microorganisms (KCCM), a microbial depository under the Budapest Treaty, as of December 02, 2020 with an accession number KCCM12865P.

실시예 7: 프롤린 탈수소효소 변이체를 발현하는 미생물의 제작 및 이를 이용한 L-발린 생산Example 7: Production of microorganisms expressing proline dehydrogenase variants and L-valine production using the same

7.1. 플라스미드의 제작7.1. Construction of the plasmid

2) It has a kanamycin resistance gene as a selectable marker.

3) It has a Levan sucrase gene (sacB) as a secondary positive-selection marker.

7.2. 미생물내 단백질 변이체 발현을 위한 벡터 제작7.2. Vector construction for expression of protein variants in microorganisms

To determine the effect of a mutant (G834S; SEQ ID NO: 61) in which glycine (Gly, G) at position 834 of the protein consisting of the amino acid sequence of SEQ ID NO: 63 is substituted with serine (Ser, S) on L-valine production, its A vector for constructing an expression strain was prepared as follows.

Using the gDNA (genomic DNA) of wild-type Corynebacterium glutamicum ATCC14067 as a template, PCR was performed using a primer pair of sequences of SEQ ID NOs: 65 and 66 and a pair of primers of sequences of SEQ ID NOs: 67 and 68, respectively. Using the mixture of the two fragments obtained above as a template, overlapping PCR was performed again using the primer pair of SEQ ID NO: 65 and SEQ ID NO: 68 to obtain a fragment. After denaturing at 94°C for 5 minutes, PCR was repeated 30 times at 94°C for 30 seconds, at 55°C for 30 seconds, and at 72°C for 1 minute and 30 seconds, and then at 72°C for 5 minutes. The pDCM2 vector was treated with smal and the PCR product obtained above was fusion cloned. Fusion cloning was performed using the In-Fusion® HD cloning kit (Clontech). The resulting plasmid was named pDCM2-putA(G834S). The sequences of the primers used in this Example are shown in Table 19 below.

명칭designation	서열 (5’->3’)sequence (5'->3')	서열번호SEQ ID NO:
putA_1FputA_1F	TCGAGCTCGGTACCCCCGGCGTTCCCCGCGAGGTTCTCGAGCTCGGTACCCCCGGCGTTCCCCGCGAGGTTC	서열번호 65SEQ ID NO: 65
putA_2RputA_2R	GGTTTGACGCCTTCTTTGATGCTGGGTGACCAGAGTCGGCCGGTGGTTTGACGCCTTCTTTGATGCTGGGTGACCAGAGTCGGCCGGT	서열번호 66SEQ ID NO: 66
putA_3FputA_3F	ACCGGCCGACTCTGGTCACCCAGCATCAAAGAAGGCGTCAAACCACCGGCCGACTCTGGTCACCCAGCATCAAAGAAGGCGTCAAACC	서열번호 67SEQ ID NO: 67
putA_4RputA_4R	CTCTAGAGGATCCCCCGGAAGATGTTGGCTTCTACACTCTAGAGGATCCCCCGGAAGATGTTGGCTTCTACA	서열번호 68SEQ ID NO: 68

7.3. 단백질 변이체를 발현하는 미생물의 L-발린 생산능 평가7.3. Evaluation of L-valine-producing ability of microorganisms expressing protein variants

7.3.1. 단백질7.3.1. protein 변이체 발현 균주 제작Production of mutant expression strains

The vector prepared in Example 7.2 was transformed into Corynebacterium glutamicum CA08-0072 (KCCM11201P).

In the transformed strains, a strain in which homologous recombination occurred using the primer pair of SEQ ID NOs: 69 and 70 was selected and named CA08-0072_putA_G834S. The sequences of the primers used in this Example are shown in Table 20 below.

명칭designation	서열 (5’->3’)sequence (5'->3')	서열번호SEQ ID NO:
putA_5FputA_5F	CCGGCGTTCCCCGCGAGGTTCCCGGCGTTCCCCGCGAGGTTC	서열번호 69SEQ ID NO: 69
putA_6RputA_6R	CGGAAGATGTTGGCTTCTACACGGAAGATGTTGGCTTCTACA	서열번호 70SEQ ID NO: 70

7.3.2. 단백질 7.3.2. protein 변이체variant 발현 균주의 L-발린 L-valine of the expression strain 생산능productivity 비교 compare

L-valine production capacity was analyzed through the evaluation of flask fermentation titer of each strain and control parent strain prepared in Example 7.3.1.

First, each colony was subcultured in a nutrient medium, and then each strain was inoculated in a 250 ml corner-baffle flask containing 25 ml of the production medium, and cultured with shaking at 30° C. for 72 hours at 200 rpm. Thereafter, the concentration of L-valine was analyzed using HPLC, and the analyzed concentrations of L-valine are shown in Table 21 below.

The experiment was repeated 3 times, and the average value of the analysis results is shown in Table 21 below.

균 주strain	L-발린 농도 (g/L)L-valine concentration (g/L)	L-발린 농도 증가율(%)L-valine concentration increase rate (%)
CA08-0072CA08-0072	2.82.8	--
CA08-0072_putA_G834SCA08-0072_putA_G834S	3.63.6	28.628.6

As shown in Table 21, the CA08-0072_putA_G834S strain exhibited increased L-valine-producing ability compared to the control.

The mutant strain CA08-0072_putA_G834S was named Corynebacterium glutamicum CA08-1750, and was deposited at the Korean Culture of Microorganisms (KCCM), a microbial depository under the Budapest Treaty, with an accession number KCCM12866P as of December 02, 2020.

실시예 8: 테트라하이드로디피콜리네이트 N-숙시닐트랜스퍼라제 변이체를 발현하는 미생물의 제작 및 이를 이용한 L-발린 생산Example 8: Preparation of microorganisms expressing tetrahydrodipicolinate N-succinyltransferase mutant and L-valine production using the same

8.1. 플라스미드의 제작8.1. Construction of the plasmid

2) It has a kanamycin resistance gene as a selectable marker.

3) It has a Levan sucrase gene (sacB) as a secondary positive-selection marker.

8.2. 미생물내 단백질 변이체 발현을 위한 벡터 제작8.2. Vector construction for expression of protein variants in microorganisms

To determine the effect of a variant (R140C; SEQ ID NO: 71) in which arginine (Arg, R) at the 140th position of the protein consisting of the amino acid sequence of SEQ ID NO: 73 is substituted with cysteine (Cys, C) on L-valine production, its A vector for constructing an expression strain was prepared as follows.

PCR was performed using a pair of primers of SEQ ID NOs: 75 and 76 and a pair of primers of SEQ ID NOs: 77 and 78 using gDNA (genomic DNA) of wild-type Corynebacterium glutamicum ATCC14067 as a template, respectively. Using the mixture of the two fragments obtained above as a template, overlapping PCR was performed again using the primer pair of SEQ ID NO: 75 and SEQ ID NO: 78 to obtain a fragment. After denaturing at 94°C for 5 minutes, PCR was repeated 30 times at 94°C for 30 seconds, at 55°C for 30 seconds, and at 72°C for 1 minute and 30 seconds, and then at 72°C for 5 minutes. The pDCM2 vector was treated with smal and the PCR product obtained above was fusion cloned. Fusion cloning was performed using the In-Fusion® HD cloning kit (Clontech). The resulting plasmid was named pDCM2-dapD2 (R140C). The sequences of the primers used in this Example are shown in Table 22 below.

명칭designation	서열 (5’->3’)sequence (5'->3')	서열번호SEQ ID NO:
dapD2_1FdapD2_1F	TCGAGCTCGGTACCCATGAGTGAAAACATTCGCGGATCGAGCTCGGTACCCATGAGTGAAAACATTCGCGGA	서열번호 75SEQ ID NO: 75
dapD2_2RdapD2_2R	CCGCGGGACCGCAGAGCACCACACACCCACTCAAAGTTTTCAGGCCCGGGGACCGCAGAGGCACCACACACCCACTCAAAGTTTTCAGG	서열번호 76SEQ ID NO: 76
dapD2_3FdapD2_3F	CCTGAAAACTTTGAGTGGGTGTGTGGTGCTCTGCGGTCCCGCGGCCTGAAAACTTTGAGTGGGTGTGTGGTGCTCTGCGGTCCCGCGG	서열번호 77SEQ ID NO: 77
dapD2_4RdapD2_4R	CTCTAGAGGATCCCCCCGGATTCACGCTTGATTGACCTCTAGAGGATCCCCCCGGATTCACGCTTGATTGAC	서열번호 78SEQ ID NO: 78

8.3. 단백질 변이체를 발현하는 미생물의 L-발린 생산능 평가8.3. Evaluation of L-valine-producing ability of microorganisms expressing protein variants

8.3.1. 단백질8.3.1. protein 변이체 발현 균주 제작Production of mutant expression strains

The vector prepared in Example 8.2 was transformed into Corynebacterium glutamicum CA08-0072 (KCCM11201P).

In the transformed strains, a strain in which homologous recombination occurred was selected using the primer pair of SEQ ID NOs: 79 and 80, and was named CA08-0072_dapD2_R140C. The sequences of the primers used in this Example are shown in Table 23 below.

명칭designation	서열 (5’->3’)sequence (5'->3')	서열번호SEQ ID NO:
dapD2_5FdapD2_5F	ATGAGTGAAAACATTCGCGGAATGAGTGAAAACATTCGCGGA	서열번호 79SEQ ID NO: 79
dapD2_6RdapD2_6R	CCGGATTCACGCTTGATTGACCCGGATTCACGCTTGATTGAC	서열번호 80SEQ ID NO: 80

8.3.2. 단백질 변이체 발현 균주의 L-발린 생산능 비교8.3.2. Comparison of L-valine production capacity of protein variant expression strains

L-valine production ability was analyzed through the flask fermentation titer evaluation of each strain and control parent strain prepared in Example 8.3.1.

First, each colony was subcultured in a nutrient medium, and then each strain was inoculated in a 250 ml corner-baffle flask containing 25 ml of the production medium, and cultured with shaking at 30° C. for 72 hours at 200 rpm. Thereafter, the concentration of L-valine was analyzed using HPLC, and the analyzed concentration of L-valine is shown in Table 24 below.

The experiment was repeated 3 times, and the average value of the analysis results is shown in Table 24 below.

균 주strain	L-발린 농도 (g/L)L-valine concentration (g/L)	L-발린 농도 증가율 (%)L-valine concentration increase rate (%)
CA08-0072CA08-0072	2.82.8	--
CA08-0072_dapD2_R140CCA08-0072_dapD2_R140C	3.333.33	18.918.9

As shown in Table 24, the CA08-0072_dapD2_R140C strain exhibited increased L-valine-producing ability compared to the control.

The mutant strain CA08-0072_dapD2_R140C was named Corynebacterium glutamicum CA08-1752, and was deposited at the Korean Culture of Microorganisms (KCCM), a microbial depository under the Budapest Treaty, with an accession number KCCM12868P as of December 02, 2020.

실시예 9: 5,10-메틸렌테트라하이드로폴레이트 리덕타제 변이체를 발현하는 미생물의 제작 및 이를 이용한 L-발린 생산Example 9: Preparation of microorganisms expressing variants of 5,10-methylenetetrahydrofolate reductase and production of L-valine using the same

9.1. 플라스미드의 제작9.1. Construction of the plasmid

2) It has a kanamycin resistance gene as a selectable marker.

3) It has a Levan sucrase gene (sacB) as a secondary positive-selection marker.

9.2. 미생물내 단백질 변이체 발현을 위한 벡터 제작9.2. Vector construction for expression of protein variants in microorganisms

To determine the effect of the mutant (R4H; SEQ ID NO: 81) in which arginine (Arg, R) at the 4th position of the protein consisting of the amino acid sequence of SEQ ID NO: 83 is substituted with histidine (His, H) on L-valine production, its A vector for constructing an expression strain was prepared as follows.

Using the gDNA (genomic DNA) of wild-type Corynebacterium glutamicum ATCC14067 as a template, PCR was performed using a primer pair of sequences of SEQ ID NOs: 85 and 86 and a pair of primers of sequences of SEQ ID NOs: 87 and 88, respectively. Using the mixture of the two fragments obtained above as a template, overlapping PCR was performed again using the primer pair of SEQ ID NO: 85 and SEQ ID NO: 88 to obtain a fragment. After denaturing at 94°C for 5 minutes, PCR was repeated 30 times at 94°C for 30 seconds, at 55°C for 30 seconds, and at 72°C for 1 minute and 30 seconds, and then at 72°C for 5 minutes. The pDCM2 vector was treated with smal and the PCR product obtained above was fusion cloned. Fusion cloning was performed using the In-Fusion® HD cloning kit (Clontech). The resulting plasmid was named pDCM2-metF(R4H). The sequences of the primers used in this Example are shown in Table 25 below.

명칭designation	서열 (5’->3’)sequence (5'->3')	서열번호SEQ ID NO:
metF_1FmetF_1F	TCGAGCTCGGTACCCTTCAGAAGAATTCTTATGGCCTCGAGCTCGGTACCCTTCAGAAGAATTCTTATGGCC	서열번호 85SEQ ID NO: 85
metF_2RmetF_2R	CTCTTCAGCTGCATCATCGTGGGGTGGCATAAACTCGACAGAAACTCTTCAGCTGCATCATCGTGGGGTGGCATAAACTCGACAGAAA	서열번호 86SEQ ID NO: 86
metF_3FmetF_3F	TTTCTGTCGAGTTTATGCCACCCCACGATGATGCAGCTGAAGAGTTTCTGTCGAGTTTATGCCACCCCACGATGATGCAGCTGAAGAG	서열번호 87SEQ ID NO: 87
metF_4RmetF_4R	CTCTAGAGGATCCCCATCAAAGAACATCTGCGTGATCTCTAGAGGATCCCCATCAAAGAACATCTGCGTGAT	서열번호 88SEQ ID NO: 88

9.3. 단백질 변이체를 발현하는 미생물의 L-발린 생산능 평가9.3. Evaluation of L-valine-producing ability of microorganisms expressing protein variants

9.3.1. 단백질9.3.1. protein 변이체 발현 균주 제작Production of mutant expression strains

The vector prepared in Example 9.2 was transformed into Corynebacterium glutamicum CA08-0072 (KCCM11201P).

In the transformed strains, a strain in which homologous recombination occurred using the primer pair of SEQ ID NOs: 89 and 90 was selected and named CA08-0072_metF_R4H. The sequences of the primers used in this Example are shown in Table 26 below.

명칭designation	서열 (5’->3’)sequence (5'->3')	서열번호SEQ ID NO:
metF_5FmetF_5F	TTCAGAAGAATTCTTATGGCCTTCAGAAGAATTCTTATGGCC	서열번호 89SEQ ID NO: 89
metF_6RmetF_6R	ATCAAAGAACATCTGCGTGATATCAAAGAACATCTGCGTGAT	서열번호 90SEQ ID NO: 90

9.3.2. 단백질 9.3.2. protein 변이체variant 발현 균주의 L-발린 L-valine of the expression strain 생산능productivity 비교 compare

The L-valine production ability was analyzed by evaluating the flask fermentation titer of each strain and the control parent strain prepared in Example 9.3.1.

First, each colony was subcultured in a nutrient medium, and then each strain was inoculated in a 250 ml corner-baffle flask containing 25 ml of the production medium, and cultured with shaking at 30° C. for 72 hours at 200 rpm. Thereafter, the concentration of L-valine was analyzed using HPLC, and the analyzed concentrations of L-valine are shown in Table 27 below.

The experiment was repeated 3 times, and the average value of the analysis results is shown in Table 27 below.

균 주strain	L-발린 농도 (g/L)L-valine concentration (g/L)	L-발린 농도 증가율 (%)L-valine concentration increase rate (%)
CA08-0072CA08-0072	2.82.8	--
CA08-0072_metF_R4HCA08-0072_metF_R4H	3.73.7	32.132.1

As shown in Table 27, the CA08-0072_metF_R4H strain exhibited increased L-valine-producing ability compared to the control.

The mutant strain CA08-0072_metF_R4H was named Corynebacterium glutamicum CA08-1746, and was deposited with the Korean Culture of Microorganisms (KCCM), a microbial depository under the Budapest Treaty, with an accession number KCCM12862P as of December 02, 2020.

실시예 10: NCgl2805 단백질 변이체를 발현하는 미생물의 제작 및 이를 이용한 L-발린 생산Example 10: Production of microorganisms expressing NCgl2805 protein variant and L-valine production using the same

10.1. 플라스미드의 제작10.1. Construction of plasmids

2) It has a kanamycin resistance gene as a selectable marker.

3) It has a Levan sucrase gene (sacB) as a secondary positive-selection marker.

10.2. 미생물내 단백질 변이체 발현을 위한 벡터 제작10.2. Vector construction for expression of protein variants in microorganisms

The mutant (E253K; SEQ ID NO: 91) in which the glutamic acid (Glu, E) at position 253 of the protein consisting of the amino acid sequence of SEQ ID NO: 93 is substituted with lysine (Lys, K) on L-valine production To determine the effect thereof A vector for constructing an expression strain was prepared as follows.

Using the gDNA (genomic DNA) of wild-type Corynebacterium glutamicum ATCC14067 as a template, PCR was performed using a primer pair of sequences of SEQ ID NOs: 95 and 96 and a pair of primers of sequences of SEQ ID NOs: 97 and 98, respectively. Using the mixture of the two fragments obtained above as a template, overlapping PCR was performed again using the primer pair of SEQ ID NO: 95 and SEQ ID NO: 98 to obtain a fragment. After denaturing at 94°C for 5 minutes, PCR was repeated 30 times at 94°C for 30 seconds, at 55°C for 30 seconds, and at 72°C for 1 minute and 30 seconds, and then at 72°C for 5 minutes. The pDCM2 vector was treated with smal and the PCR product obtained above was fusion cloned. Fusion cloning was performed using the In-Fusion® HD cloning kit (Clontech). The resulting plasmid was named pDCM2-NCgl2805 (E253K). The sequences of the primers used in this Example are shown in Table 28 below.

명칭designation	서열 (5’->3’)sequence (5'->3')	서열번호SEQ ID NO:
NCgl2805_1FNCgl2805_1F	TCGAGCTCGGTACCCAAGACGCCGTACTCTACGACTCGAGCTCGGTACCCAAGACGCCGTACTCTACGAC	서열번호 95SEQ ID NO: 95
NCgl2805_2RNCgl2805_2R	ACCATGGAATCCAACTCTTTACCCTGGTAGAGCTGTGCTTGACCATGGAATCCAACTCTTTACCCTGGTAGAGCTGTGCTTG	서열번호 96SEQ ID NO: 96
NCgl2805_3FNCgl2805_3F	CAAGCACAGCTCTACCAGGGTAAAGAGTTGGATTCCATGGTCAAGCACAGCTCTACCAGGGTAAAGAGTTGGATTCCATGGT	서열번호 97SEQ ID NO: 97
NCgl2805_4RNCgl2805_4R	CTCTAGAGGATCCCCAGCGCCAGAAGGCATTTCACCTCTAGAGGATCCCCAGCGCCAGAAGGCATTTCAC	서열번호 98SEQ ID NO: 98

10.3. 단백질 변이체를 발현하는 미생물의 L-발린 생산능 평가10.3. Evaluation of L-valine-producing ability of microorganisms expressing protein variants

10.3.1. 단백질10.3.1. protein 변이체 발현 균주 제작Production of mutant expression strains

The vector prepared in Example 10.2 was transformed into Corynebacterium glutamicum CA08-0072 (KCCM11201P).

In the transformed strains, a strain in which homologous recombination occurred using the primer pair of SEQ ID NOs: 99 and 100 was selected and named CA08-0072_NCgl2805_E253K. The sequences of the primers used in this Example are shown in Table 29 below.

명칭designation	서열 (5’->3’)sequence (5'->3')	서열번호SEQ ID NO:
NCgl2805_5FNCgl2805_5F	AAGACGCCGTACTCTACGACAAGACGCCGTACTCTACGAC	서열번호 99SEQ ID NO: 99
NCgl2805_6RNCgl2805_6R	AGCGCCAGAAGGCATTTCACAGCGCCAGAAGGCATTTCAC	서열번호 100SEQ ID NO: 100

10.3.2. 단백질 10.3.2. protein 변이체variant 발현 균주의 L-발린 L-valine of the expression strain 생산능productivity 비교 compare

The L-valine production ability was analyzed by evaluating the flask fermentation titer of each strain and the control parent strain prepared in Example 10.3.1.

First, each colony was subcultured in a nutrient medium, and then each strain was inoculated in a 250 ml corner-baffle flask containing 25 ml of the production medium, and cultured with shaking at 30° C. for 72 hours at 200 rpm. Thereafter, the concentration of L-valine was analyzed using HPLC, and the analyzed concentrations of L-valine are shown in Table 30 below.

The experiment was repeated 3 times, and the average value of the analysis results is shown in Table 30 below.

균 주strain	L-발린 농도 (g/L)L-valine concentration (g/L)	L-발린 농도 증가율(%)L-valine concentration increase rate (%)
CA08-0072CA08-0072	2.82.8	--
CA08-0072_NCgl2805_E253KCA08-0072_NCgl2805_E253K	3.223.22	15%15%

As shown in Table 30, the CA08-0072_NCgl2805_E253K strain exhibited increased L-valine-producing ability compared to the control.

The mutant strain CA08-0072_NCgl2805_E253K was named Corynebacterium glutamicum CA08-1745, and was deposited with the Korean Culture of Microorganisms (KCCM), a microbial depository under the Budapest Treaty, as of December 22, 2020 with an accession number KCCM12909P.

실시예 11: 슈가 포터 계열 MFS 트랜스포터 변이체를 포함하는 재조합 균주의 제조 및 이를 이용한 L-발린 생산Example 11: Preparation of a recombinant strain comprising a sugar porter-based MFS transporter mutant and L-valine production using the same

11.1. 미생물내 단백질 변이체 발현을 위한 벡터 제작11.1. Vector construction for expression of protein variants in microorganisms

To determine the effect of the mutant (A315V; SEQ ID NO: 101) in which alanine (Ala, A) at position 315 of the protein consisting of the amino acid sequence of SEQ ID NO: 103 is substituted with valine (Val, V) on L-valine production, its A vector for constructing an expression strain was prepared as follows using the plasmid pDCM2 (Korea Publication No. 10-2020-0136813) for the insertion and replacement of genes in the Corynebacterium chromosome.

Using the gDNA (genomic DNA) of wild-type Corynebacterium glutamicum ATCC14067 as a template and a primer pair of SEQ ID NOs: 105 and 106 and a pair of primers of SEQ ID NOs: 107 and 108, PCR was performed, respectively. Using the mixture of the two fragments obtained above as a template, overlapping PCR was performed again using the primer pair of SEQ ID NO: 105 and SEQ ID NO: 108 to obtain a fragment. After denaturing at 94°C for 5 minutes, PCR was repeated 30 times at 94°C for 30 seconds, at 55°C for 30 seconds, and at 72°C for 1 minute and 30 seconds, and then at 72°C for 5 minutes. The pDCM2 vector was treated with smal and the PCR product obtained above was fusion cloned. Fusion cloning was performed using the In-Fusion® HD cloning kit (Clontech). The resulting plasmid was named pDCM2-iolT1 (A315V). The sequences of the primers used in this Example are shown in Table 31 below.

명칭designation	서열 (5’->3’)sequence (5'->3')	서열번호SEQ ID NO:
iolT1_1FiolT1_1F	TGAATTCGAGCTCGGTACCCTGTCTACCTGGCTGAACTTGTGAATTCGAGCTCGGTACCCTGTCTACCTGGCTGAACTTG	서열번호 105SEQ ID NO: 105
iolT1_2RiolT1_2R	TCCAGGTGCCACGTTGaCGATCAGAGCTGCATTTCCAGGTGCCACGTTGaCGATCAGAGCTGCATT	서열번호 106SEQ ID NO: 106
iolT1_3FiolT1_3F	AATGCAGCTCTGATCGtCAACGTGGCACCTGGAAATGCAGCTCTGATCGtCAACGTGGCACCTGGA	서열번호 107SEQ ID NO: 107
iolT1_4RiolT1_4R	GTCGACTCTAGAGGATCCCCATATCCTTGTTGAAGATGACGTCGACTCTAGAGGATCCCCATATCCTTGTTGAAGATGAC	서열번호 108SEQ ID NO: 108

11.2. 단백질 변이체를 발현하는 미생물의 L-발린 생산능 평가11.2. Evaluation of L-valine-producing ability of microorganisms expressing protein variants

11.2.1. 단백질 변이체 발현 균주 제작11.2.1. Production of protein mutant expression strains

The vector prepared in Example 11.1 was transformed into Corynebacterium glutamicum CA08-0072 (KCCM11201P) (see US 8,465,962).

In the transformed strains, a strain having homologous recombination was selected using the primer pair of SEQ ID NOs: 109 and 110, and named CA08-0072_iolT1_A315V. The sequences of the primers used in this Example are shown in Table 32 below.

명칭designation	서열 (5’->3’)sequence (5'->3')	서열번호SEQ ID NO:
iolT1_5FiolT1_5F	TGTCTACCTGGCTGAACTTGTGTCTACCTGGCTGAACTTG	서열번호 109SEQ ID NO: 109
iolT1_6RiolT1_6R	ATATCCTTGTTGAAGATGACATATCCTTGTTGAAGATGAC	서열번호 110SEQ ID NO: 110

11.2.2. 단백질 11.2.2. protein 변이체variant 발현 균주의 L-발린 L-valine of the expression strain 생산능productivity 비교 compare

L-valine production ability was analyzed through flask fermentation titer evaluation of each strain and control parent strain prepared in Example 11.2.1.

First, each colony was subcultured in a nutrient medium, and then each strain was inoculated in a 250 ml corner-baffle flask containing 25 ml of the production medium, and cultured with shaking at 30° C. for 72 hours at 200 rpm. Thereafter, the concentration of L-valine was analyzed using HPLC, and the analyzed concentrations of L-valine are shown in Table 33 below.

The experiment was repeated 3 times, and the average value of the analysis results is shown in Table 33 below.

균 주strain	L-발린 농도 (g/L)L-valine concentration (g/L)	L-발린 농도 증가율(%)L-valine concentration increase rate (%)
CA08-0072CA08-0072	2.12.1	--
CA08-0072_iolT1_A315VCA08-0072_iolT1_A315V	2.82.8	33.333.3

As shown in Table 33, the CA08-0072_iolT1_A315V strain exhibited increased L-valine-producing ability compared to the control.

실시예 12: 전사 조절자 변이체를 포함하는 재조합 균주의 제조 및 이를 이용한 L-발린 생산Example 12: Preparation of Recombinant Strain Containing Transcriptional Regulator Mutants and Production of L-Valine Using the Same

12.1. 미생물내 단백질 변이체 발현을 위한 벡터 제작12.1. Vector construction for expression of protein variants in microorganisms

The mutant (E247K; SEQ ID NO: 111) in which the glutamic acid (Glu, E) at the 247th position of the protein consisting of the amino acid sequence of SEQ ID NO: 113 is substituted with lysine (Lys, K) on L-valine production To determine the effect thereof A vector for constructing an expression strain was prepared as follows using the plasmid pDCM2 (Korea Publication No. 10-2020-0136813) for the insertion and replacement of genes in the Corynebacterium chromosome.

Using the gDNA (genomic DNA) of wild-type Corynebacterium glutamicum ATCC14067 as a template, PCR was performed using a primer pair of SEQ ID NOs: 115 and 116 and a pair of primers of SEQ ID NOs: 117 and 118, respectively. Using the mixture of the two fragments obtained above as a template, overlapping PCR was performed again using a pair of primers of SEQ ID NO: 115 and SEQ ID NO: 118 to obtain a fragment. After denaturing at 94°C for 5 minutes, PCR was repeated 30 times for 30 seconds at 94°C for 30 seconds, at 55°C for 30 seconds, and at 72°C for 1 minute and 30 seconds, and then at 72°C for 5 minutes. The pDCM2 vector was treated with smal and the PCR product obtained above was fusion cloned. Fusion cloning was performed using the In-Fusion® HD cloning kit (Clontech). The resulting plasmid was named pDCM2-iolR (E247K). The sequences of the primers used in this Example are shown in Table 34 below.

명칭designation	서열 (5’->3’)sequence (5'->3')	서열번호SEQ ID NO:
iolR_1FiolR_1F	TGAATTCGAGCTCGGTACCCCCAAGAAGTCGTAGACAAAGTGAATTCGAGCTCGGTACCCCAAGAAGTCGTAGACAAAG	서열번호 115SEQ ID NO: 115
iolR_2RiolR_2R	GCCACCAGAGTGGTTTtAAAGTTGTACATATCTGCCACCAGAGTGGTTTtAAAGTTGTACATATCT	서열번호 116SEQ ID NO: 116
iolR_3FiolR_3F	AGATATGTACAACTTTaAAACCACTCTGGTGGCAGATATGTACAACTTTaAAACCACTCTGGTGGC	서열번호 117SEQ ID NO: 117
iolR_4RiolR_4R	GTCGACTCTAGAGGATCCCCTTGAGCTAAGTATCCTATGAGTCGACTCTAGAGGATCCCCTTGAGCTAAGTATCCTATGA	서열번호 118SEQ ID NO: 118

12.2. 단백질 변이체를 발현하는 미생물의 L-발린 생산능 평가12.2. Evaluation of L-valine-producing ability of microorganisms expressing protein variants

12.2.1. 단백질 변이체 발현 균주 제작12.2.1. Production of protein mutant expression strains

The vector prepared in Example 12.1 was transformed into Corynebacterium glutamicum CA08-0072 (KCCM11201P) (see US 8,465,962).

In the transformed strains, a strain having homologous recombination was selected using the primer pair of SEQ ID NOs: 119 and 120 and named CA08-0072_iolR_E247K. The sequences of the primers used in this Example are shown in Table 35 below.

명칭designation	서열 (5’->3’)sequence (5'->3')	서열번호SEQ ID NO:
iolR_5FiolR_5F	CCAAGAAGTCGTAGACAAAGCCAAGAAGTCGTAGACAAAG	서열번호 119SEQ ID NO: 119
iolR_6RiolR_6R	TTGAGCTAAGTATCCTATGATTGAGCTAAGTATCCTATGA	서열번호 120SEQ ID NO: 120

12.2.2. 단백질 12.2.2. protein 변이체variant 발현 균주의 L-발린 L-valine of the expression strain 생산능productivity 비교 compare

The L-valine production ability was analyzed by evaluating the flask fermentation titer of each strain and the control parent strain prepared in Example 12.2.1.

First, each colony was subcultured in a nutrient medium, and then each strain was inoculated in a 250 ml corner-baffle flask containing 25 ml of the production medium, and cultured with shaking at 30° C. for 72 hours at 200 rpm. Thereafter, the concentration of L-valine was analyzed using HPLC, and the analyzed concentrations of L-valine are shown in Table 36 below.

The experiment was repeated 3 times, and the average value of the analysis results is shown in Table 36 below.

균 주strain	L-발린 농도 (g/L)L-valine concentration (g/L)	L-발린 농도 증가율(%)L-valine concentration increase rate (%)
CA08-0072CA08-0072	1.81.8	--
CA08-0072_iolR_E247KCA08-0072_iolR_E247K	2.22.2	22.222.2

As shown in Table 36, the CA08-0072_iolR_E247K strain exhibited increased L-valine-producing ability compared to the control.

실시예 13: WhiB 계열 전사 조절자 WhcA 변이체를 포함하는 재조합 균주의 제조 및 이를 이용한 L-발린 생산Example 13: Preparation of Recombinant Strain Containing WhiB Family Transcriptional Regulator WhcA Variant and Production of L-Valine Using the Same

13.1. 미생물내 단백질 변이체 발현을 위한 벡터 제작13.1. Vector construction for expression of protein variants in microorganisms

The effect of the mutant (G40D; SEQ ID NO: 121) in which glycine (Gly, G) at the 40th position of the protein consisting of the amino acid sequence of SEQ ID NO: 123 is substituted with aspartic acid (Asp, D) on L-valine production A vector for constructing an expression strain thereof was prepared as follows using the plasmid pDCM2 (Korea Publication No. 10-2020-0136813) for the insertion and replacement of genes in the Corynebacterium chromosome.

PCR was performed using the primer pair of SEQ ID NOs: 125 and 126 and the primer pair of SEQ ID NOs: 127 and 128 using gDNA (genomic DNA) of wild-type Corynebacterium glutamicum ATCC14067 as a template, respectively. Using the mixture of the two fragments obtained above as a template, overlapping PCR was performed again using a pair of primers of SEQ ID NO: 125 and SEQ ID NO: 128 to obtain a fragment. After denaturing at 94°C for 5 minutes, PCR was repeated 30 times at 94°C for 30 seconds, at 55°C for 30 seconds, and at 72°C for 1 minute and 30 seconds, and then at 72°C for 5 minutes. The pDCM2 vector was treated with smal and the PCR product obtained above was fusion cloned. Fusion cloning was performed using the In-Fusion® HD cloning kit (Clontech). The resulting plasmid was named pDCM2-whcA (G40D). The sequences of the primers used in this Example are shown in Table 37 below.

명칭designation	서열 (5’->3’)sequence (5'->3')	서열번호SEQ ID NO:
whcA_1FwhcA_1F	TGAATTCGAGCTCGGTACCCTATTCGCAACTGTGCTTACGTGAATTCGAGCTCGGTACCCTATTCGCAACTGTGCTTACG	서열번호 125SEQ ID NO: 125
whcA_2RwhcA_2R	CAATGCATCTGGGTCGtCATTTCGACACTTTGCCAATGCATCTGGGTCGtCATTTCGACACTTTGC	서열번호 126SEQ ID NO: 126
whcA_3FwhcA_3F	GCAAAGTGTCGAAATGaCGACCCAGATGCATTGGCAAAGTGTCGAAATGaCGACCCAGATGCATTG	서열번호 127SEQ ID NO: 127
whcA_4RwhcA_4R	GTCGACTCTAGAGGATCCCCATCTGCTTAGTTGCATGCGTGTCGACTCTAGAGGATCCCCATCTGCTTAGTTGCATGCGT	서열번호 128SEQ ID NO: 128

13.2. 단백질 변이체를 발현하는 미생물의 L-발린 생산능 평가13.2. Evaluation of L-valine-producing ability of microorganisms expressing protein variants

13.2.1. 단백질 변이체 발현 균주 제작13.2.1. Production of protein mutant expression strains

The vector prepared in Example 13.1 was transformed into Corynebacterium glutamicum CA08-0072 (KCCM11201P) (see US 8,465,962).

In the transformed strains, a strain in which homologous recombination occurred using the primer pair of SEQ ID NOs: 129 and 130 was selected and named CA08-0072_whcA_G40D. The sequences of the primers used in this Example are shown in Table 38 below.

명칭designation	서열 (5’->3’)sequence (5'->3')	서열번호SEQ ID NO:
whcA_5FwhcA_5F	TATTCGCAACTGTGCTTACGTATTCGCAACTGTGCTTACG	서열번호 129SEQ ID NO: 129
whcA_6RwhcA_6R	ATCTGCTTAGTTGCATGCGTATTCTGCTTAGTTGCATGCGT	서열번호 130SEQ ID NO: 130

13.2.2. 단백질 13.2.2. protein 변이체variant 발현 균주의 L-발린 L-valine of the expression strain 생산능productivity 비교 compare

L-valine production ability was analyzed through flask fermentation titer evaluation of each strain and control parent strain prepared in Example 13.2.1.

First, each colony was subcultured in a nutrient medium, and then each strain was inoculated in a 250 ml corner-baffle flask containing 25 ml of the production medium, and cultured with shaking at 30° C. for 72 hours at 200 rpm. Thereafter, the concentration of L-valine was analyzed using HPLC, and the analyzed concentrations of L-valine are shown in Table 39 below.

The experiment was repeated 3 times, and the average value of the analysis results is shown in Table 39 below.

균 주strain	L-발린 농도 (g/L)L-valine concentration (g/L)	L-발린 농도 증가율(%)L-valine concentration increase rate (%)
CA08-0072CA08-0072	2.02.0	--
CA08-0072_whcA_G40DCA08-0072_whcA_G40D	2.52.5	25.025.0

As shown in Table 39, the CA08-0072_whcA_G40D strain exhibited increased L-valine-producing ability compared to the control.

실시예 14: 디히드로리포일 아세틸기전이효소 변이체를 포함하는 재조합 균주의 제조 및 이를 이용한 L-발린 생산Example 14: Preparation of a recombinant strain containing a dihydrolipoyl acetyltransferase mutant and L-valine production using the same

14.1. 미생물내 단백질 변이체 발현을 위한 벡터 제작14.1. Vector construction for expression of protein variants in microorganisms

The effect of a mutant (G77D; SEQ ID NO: 131) in which glycine (Gly, G) at position 77 of the protein consisting of the amino acid sequence of SEQ ID NO: 133 is substituted with aspartic acid (Asp, D) on L-valine production A vector for constructing an expression strain thereof was prepared as follows using the plasmid pDCM2 (Korea Publication No. 10-2020-0136813) for the insertion and replacement of genes in the Corynebacterium chromosome.

Using the gDNA (genomic DNA) of wild-type Corynebacterium glutamicum ATCC14067 as a template and a primer pair of SEQ ID NOs: 135 and 136 and a pair of primers of SEQ ID NOs: 137 and 138, PCR was performed, respectively. Using the mixture of the two fragments obtained above as a template, overlapping PCR was performed again using a primer pair of SEQ ID NO: 135 and SEQ ID NO: 138 to obtain a fragment. After denaturing at 94°C for 5 minutes, PCR was repeated 30 times at 94°C for 30 seconds, at 55°C for 30 seconds, and at 72°C for 1 minute and 30 seconds, and then at 72°C for 5 minutes. The pDCM2 vector was treated with smal and the PCR product obtained above was fusion cloned. Fusion cloning was performed using the In-Fusion® HD cloning kit (Clontech). The resulting plasmid was named pDCM2-aceF (G77D). The sequences of the primers used in this Example are shown in Table 40 below.

명칭designation	서열 (5’->3’)sequence (5'->3')	서열번호SEQ ID NO:
aceF_1FaceF_1F	TGAATTCGAGCTCGGTACCCACGACAGACATCTCATTGACTGAATTCGAGCTCGGTACCCACGACAGACATCTCATTGAC	서열번호 135SEQ ID NO: 135
aceF_2RaceF_2R	AGTCTCATCAGCATCGtCGATTATTGCAATGACAGTCTCATCAGCATCGtCGATTATTGCAATGAC	서열번호 136SEQ ID NO: 136
aceF_3FaceF_3F	GTCATTGCAATAATCGaCGATGCTGATGAGACTGTCATTGCAATAATCGaCGATGCTGATGAGACT	서열번호 137SEQ ID NO: 137
aceF_4RaceF_4R	GTCGACTCTAGAGGATCCCCGGTGGAGACCTCAAGAAGTGGTCGACTCTAGAGGATCCCCGGTGGAGACCTCAAGAAGTG	서열번호 138SEQ ID NO: 138

14.2. 단백질 변이체를 발현하는 미생물의 L-발린 생산능 평가14.2. Evaluation of L-valine-producing ability of microorganisms expressing protein variants

14.2.1. 단백질 변이체 발현 균주 제작14.2.1. Production of protein mutant expression strains

The vector prepared in Example 14.1 was transformed into Corynebacterium glutamicum CA08-0072 (KCCM11201P) (see US 8,465,962).

In the transformed strains, a strain in which homologous recombination occurred using the primer pair of SEQ ID NOs: 139 and 140 was selected and named CA08-0072_aceF_G77D. The sequences of the primers used in this Example are shown in Table 41 below.

명칭designation	서열 (5’->3’)sequence (5'->3')	서열번호SEQ ID NO:
aceF_5FaceF_5F	ACGACAGACATCTCATTGACACGACAGACATCTCATTGAC	서열번호 139SEQ ID NO: 139
aceF_6RaceF_6R	GGTGGAGACCTCAAGAAGTGGGTGGAGACCTCAAGAAGTG	서열번호 140SEQ ID NO: 140

14.2.2. 단백질 14.2.2. protein 변이체variant 발현 균주의 L-발린 L-valine of the expression strain 생산능productivity 비교 compare

L-valine production ability was analyzed through the evaluation of flask fermentation titer of each strain and control parent strain prepared in Example 14.2.1.

First, each colony was subcultured in a nutrient medium, and then each strain was inoculated in a 250 ml corner-baffle flask containing 25 ml of the production medium, and cultured with shaking at 30° C. for 72 hours at 200 rpm. Thereafter, the concentration of L-valine was analyzed using HPLC, and the analyzed concentration of L-valine is shown in Table 42 below.

The experiment was repeated 3 times, and the average value of the analysis results is shown in Table 42 below.

균 주strain	L-발린 농도 (g/L)L-valine concentration (g/L)	L-발린 농도 증가율(%)L-valine concentration increase rate (%)
CA08-0072CA08-0072	2.02.0	--
CA08-0072_aceF_G77DCA08-0072_aceF_G77D	2.62.6	30.030.0

As shown in Table 42, the CA08-0072_aceF_G77D strain exhibited increased L-valine-producing ability compared to the control.

실시예 15: 2-이소프로필말레이트합성효소 변이체를 포함하는 재조합 균주의 제조 및 이를 이용한 L-발린 생산Example 15: Preparation of recombinant strain containing 2-isopropylmalate synthetase mutant and production of L-valine using the same

15.1. 미생물내 단백질 변이체 발현을 위한 벡터 제작15.1. Vector construction for expression of protein variants in microorganisms

To determine the effect of the mutant (P87L; SEQ ID NO: 141) in which proline (Pro, P) at position 87 of the protein consisting of the amino acid sequence of SEQ ID NO: 143 is substituted with leucine (Leu, L) on L-valine production, its A vector for constructing an expression strain was prepared as follows using the plasmid pDCM2 (Korea Publication No. 10-2020-0136813) for the insertion and replacement of genes in the Corynebacterium chromosome.

Using the gDNA (genomic DNA) of wild-type Corynebacterium glutamicum ATCC14067 as a template and a primer pair of SEQ ID NOs: 145 and 146 and a pair of primers of SEQ ID NOs: 147 and 148, PCR was performed, respectively. Using the mixture of the two fragments obtained above as a template, overlapping PCR was performed again using a primer pair of SEQ ID NO: 145 and SEQ ID NO: 148 to obtain a fragment. After denaturing at 94°C for 5 minutes, PCR was repeated 30 times at 94°C for 30 seconds, at 55°C for 30 seconds, and at 72°C for 1 minute and 30 seconds, and then at 72°C for 5 minutes. The pDCM2 vector was treated with smal and the PCR product obtained above was fusion cloned. Fusion cloning was performed using the In-Fusion® HD cloning kit (Clontech). The resulting plasmid was named pDCM2-leuA (P87L). The sequences of the primers used in this Example are shown in Table 43 below.

명칭designation	서열 (5’->3’)sequence (5'->3')	서열번호SEQ ID NO:
leuA_1FleuA_1F	TGAATTCGAGCTCGGTACCCTGACAGCACTGGAACTGGGCTGAATTCGAGCTCGGTACCCTGACAGCACTGGAACTGGGC	서열번호 145SEQ ID NO: 145
leuA_2RleuA_2R	GCGGCGCTTACGCTCAaGAGACATCGGATCAATGCGGCGCTTACGCTCAaGAGACATCGGATCAAT	서열번호 146SEQ ID NO: 146
leuA_3FleuA_3F	ATTGATCCGATGTCTCtTGAGCGTAAGCGCCGCATTGATCCGATGTCTCtTGAGCGTAAGCGCCGC	서열번호 147SEQ ID NO: 147
leuA_4RleuA_4R	GTCGACTCTAGAGGATCCCCATTCAATGGAGTCTGCGTAAGTCGACTCTAGAGGATCCCCATTCAATGGAGTCTGCGTAA	서열번호 148SEQ ID NO: 148

15.2. 단백질 변이체를 발현하는 미생물의 L-발린 생산능 평가15.2. Evaluation of L-valine-producing ability of microorganisms expressing protein variants

15.2.1. 단백질 변이체 발현 균주 제작15.2.1. Production of protein mutant expression strains

The vector prepared in Example 15.1 was transformed into Corynebacterium glutamicum CA08-0072 (KCCM11201P) (see US 8,465,962).

In the transformed strains, a strain in which homologous recombination occurred using the primer pair of SEQ ID NOs: 149 and 150 was selected and named CA08-0072_leuA_P87L. The sequences of the primers used in this Example are shown in Table 44 below.

명칭designation	서열 (5’->3’)sequence (5'->3')	서열번호SEQ ID NO:
leuA_5FleuA_5F	TGACAGCACTGGAACTGGGCTGACAGCACTGGAACTGGGC	서열번호 149SEQ ID NO: 149
leuA_6RleuA_6R	ATTCAATGGAGTCTGCGTAAATTCAATGGAGTCTGCGTAA	서열번호 150SEQ ID NO: 150

15.2.2. 단백질 15.2.2. protein 변이체variant 발현 균주의 L-발린 L-valine of the expression strain 생산능productivity 비교 compare

L-valine production capacity was analyzed through flask fermentation titer evaluation of each strain and control parent strain prepared in Example 15.2.1.

First, each colony was subcultured in a nutrient medium, and then each strain was inoculated in a 250 ml corner-baffle flask containing 25 ml of the production medium, and cultured with shaking at 30° C. for 72 hours at 200 rpm. Thereafter, the concentration of L-valine was analyzed using HPLC, and the analyzed concentrations of L-valine are shown in Table 45 below.

The experiment was repeated 3 times, and the average value of the analysis results is shown in Table 45 below.

균 주strain	L-발린 농도 (g/L)L-valine concentration (g/L)	L-발린 농도 증가율(%)L-valine concentration increase rate (%)
CA08-0072CA08-0072	2.22.2	--
CA08-0072_leuA_P87LCA08-0072_leuA_P87L	2.72.7	22.722.7

As shown in Table 45, the CA08-0072_leuA_P87L strain exhibited an increased L-valine-producing ability compared to the control.

실시예 16: 분지쇄아미노산 투과효소 변이체를 포함하는 재조합 균주의 제조 및 이를 이용한 L-발린 생산Example 16: Preparation of a recombinant strain containing a branched-chain amino acid permease mutant and L-valine production using the same

16.1. 미생물내 단백질 변이체 발현을 위한 벡터 제작16.1. Vector construction for expression of protein variants in microorganisms

To determine the effect of a mutant (A112T; SEQ ID NO: 151) in which alanine (Ala, A) at position 112 of the protein consisting of the amino acid sequence of SEQ ID NO: 153 is substituted with threonine (Thr, T) on L-valine production, its A vector for constructing an expression strain was prepared as follows using the plasmid pDCM2 (Korea Publication No. 10-2020-0136813) for the insertion and replacement of genes in the Corynebacterium chromosome.

PCR was performed using the primer pair of the sequences of SEQ ID NOs: 155 and 156 and the primer pair of the sequences of SEQ ID NOs: 157 and 158 using gDNA (genomic DNA) of wild-type Corynebacterium glutamicum ATCC14067 as a template, respectively. Using the mixture of the two fragments obtained above as a template, overlapping PCR was performed again using a primer pair of SEQ ID NO: 155 and SEQ ID NO: 158 to obtain a fragment. After denaturing at 94°C for 5 minutes, PCR was repeated 30 times at 94°C for 30 seconds, at 55°C for 30 seconds, and at 72°C for 1 minute and 30 seconds, and then at 72°C for 5 minutes. The pDCM2 vector was treated with smal and the PCR product obtained above was fusion cloned. Fusion cloning was performed using the In-Fusion® HD cloning kit (Clontech). The resulting plasmid was named pDCM2-brnQ(A112T). The sequences of the primers used in this Example are shown in Table 46 below.

명칭designation	서열 (5’->3’)sequence (5'->3')	서열번호SEQ ID NO:
brnQ_1FbrnQ_1F	TGAATTCGAGCTCGGTACCCTTGATGTGGTTGGACTTCGCTGAATTCGAGCTCGGTACCCTTGATGTGGTTGGACTTCGC	서열번호 155SEQ ID NO: 155
brnQ_2RbrnQ_2R	AAGCCCGAATAAAGCGtATTATCGACGCCAACCAAGCCCGAATAAAGCGtATTATCGACGCCAACC	서열번호 156SEQ ID NO: 156
brnQ_3FbrnQ_3F	GGTTGGCGTCGATAATaCGCTTTATTCGGGCTTGGTTGGCGTCGATAATaCGCTTTATTCGGGCTT	서열번호 7SEQ ID NO: 7
brnQ_4RbrnQ_4R	GTCGACTCTAGAGGATCCCCGCACTGATCAGACCAACTGCGTCGACTCTAGAGGATCCCCGCACTGATCAGACCAACTGC	서열번호 8SEQ ID NO: 8

16.2. 단백질 변이체를 발현하는 미생물의 L-발린 생산능 평가16.2. Evaluation of L-valine-producing ability of microorganisms expressing protein variants

16.2.1. 단백질 변이체 발현 균주 제작16.2.1. Production of protein mutant expression strains

The vector prepared in Example 16.1 was transformed into Corynebacterium glutamicum CA08-0072 (KCCM11201P) (see US 8,465,962).

In the transformed strains, a strain in which homologous recombination occurred using the primer pair of SEQ ID NOs: 159 and 160 was selected and named CA08-0072_brnQ_A112T. The sequences of the primers used in this Example are shown in Table 2 below.

명칭designation	서열 (5’->3’)sequence (5'->3')	서열번호SEQ ID NO:
brnQ_5FbrnQ_5F	TTGATGTGGTTGGACTTCGCTTGATGTGGTTGGACTTCGC	서열번호 159SEQ ID NO: 159
brnQ_6RbrnQ_6R	GCACTGATCAGACCAACTGCGCACTGATCAGACCAACTGC	서열번호 160SEQ ID NO: 160

16.2.2. 단백질 16.2.2. protein 변이체variant 발현 균주의 L-발린 L-valine of the expression strain 생산능productivity 비교 compare

The L-valine production ability was analyzed by evaluating the flask fermentation titer of each strain and the control parent strain prepared in Example 16.2.1.

First, each colony was subcultured in a nutrient medium, and then each strain was inoculated in a 250 ml corner-baffle flask containing 25 ml of the production medium, and cultured with shaking at 30° C. for 72 hours at 200 rpm. Thereafter, the concentration of L-valine was analyzed using HPLC, and the analyzed concentration of L-valine is shown in Table 48 below.

The experiment was repeated 3 times, and the average value of the analysis results is shown in Table 48 below.

균 주strain	L-발린 농도 (g/L)L-valine concentration (g/L)	L-발린 농도 증가율(%)L-valine concentration increase rate (%)
CA08-0072CA08-0072	2.12.1	--
CA08-0072_brnQ_A112TCA08-0072_brnQ_A112T	2.72.7	28.628.6

As shown in Table 48, the CA08-0072_brnQ_A112T strain exhibited an increased L-valine-producing ability compared to the control.

실시예 17: 글리세르알데히드-3-인산탈수소효소 변이체를 포함하는 재조합 균주의 제조 및 이를 이용한 L-발린 생산Example 17: Preparation of a recombinant strain containing a glyceraldehyde-3-phosphate dehydrogenase mutant and L-valine production using the same

17.1. 미생물내 단백질 변이체 발현을 위한 벡터 제작17.1. Vector construction for expression of protein variants in microorganisms

To determine the effect of a mutant (G134S; SEQ ID NO: 161) in which glycine (Gly, G) at position 134 of the protein consisting of the amino acid sequence of SEQ ID NO: 163 is substituted with serine (Ser, S) on L-valine production, its A vector for constructing an expression strain was prepared as follows using the plasmid pDCM2 (Korea Publication No. 10-2020-0136813) for the insertion and replacement of genes in the Corynebacterium chromosome.

Using the gDNA (genomic DNA) of wild-type Corynebacterium glutamicum ATCC14067 as a template, PCR was performed using a pair of primers of SEQ ID NOs: 165 and 166 and a pair of primers of SEQ ID NOs: 167 and 168, respectively. Using the mixture of the two fragments obtained above as a template, overlapping PCR was performed again using a primer pair of SEQ ID NO: 165 and SEQ ID NO: 168 to obtain a fragment. After denaturing at 94°C for 5 minutes, PCR was repeated 30 times for 30 seconds at 94°C for 30 seconds, at 55°C for 30 seconds, and at 72°C for 1 minute and 30 seconds, and then at 72°C for 5 minutes. The pDCM2 vector was treated with smal and the PCR product obtained above was fusion cloned. Fusion cloning was performed using the In-Fusion® HD cloning kit (Clontech). The resulting plasmid was named pDCM2-gapA (G134S). The sequences of the primers used in this Example are shown in Table 49 below.

명칭designation	서열 (5’->3’)sequence (5'->3')	서열번호SEQ ID NO:
gapA_1FgapA_1F	TGAATTCGAGCTCGGTACCCTTCTGTCAGCTCAAGAATTCTGAATTCGAGCTCGGTACCCTTCTGTCAGCTCAGAATTC	서열번호 165SEQ ID NO: 165
gapA_2RgapA_2R	GACTCGTGGTTCACACtGTAAACGAAGGTTGCGGACTCGTGGTTCACACtGTAAACGAAGGTTGCG	서열번호 166SEQ ID NO: 166
gapA_3FgapA_3F	CGCAACCTTCGTTTACaGTGTGAACCACGAGTCCGCAACCTTCGTTTACaGTGTGAACCACGAGTC	서열번호 167SEQ ID NO: 167
gapA_4RgapA_4R	GTCGACTCTAGAGGATCCCCAACTAATTAGAGCTTGGAAGGTCGACTCTAGAGGATCCCCAACTAATTAGAGCTTGGAAG	서열번호 168SEQ ID NO: 168

17.2. 단백질 변이체를 발현하는 미생물의 L-발린 생산능 평가17.2. Evaluation of L-valine-producing ability of microorganisms expressing protein variants

17.2.1. 단백질 변이체 발현 균주 제작17.2.1. Production of protein variant expression strains

The vector prepared in Example 17.1 was transformed into Corynebacterium glutamicum CA08-0072 (KCCM11201P) (see US 8,465,962).

In the transformed strains, a strain in which homologous recombination occurred using the primer pair of SEQ ID NOs: 169 and 170 was selected and named CA08-0072_gapA_G134S. The sequences of the primers used in this Example are shown in Table 50 below.

명칭designation	서열 (5’->3’)sequence (5'->3')	서열번호SEQ ID NO:
gapA_5FgapA_5F	TTCTGTCAGCTCAAGAATTCTTCTGTCAGCTCAAGAATTC	서열번호 169SEQ ID NO: 169
gapA_6RgapA_6R	AACTAATTAGAGCTTGGAAGAACTAATTAGAGCTTGGAAG	서열번호 170SEQ ID NO: 170

17.2.2. 단백질 17.2.2. protein 변이체variant 발현 균주의 L-발린 L-valine of the expression strain 생산능productivity 비교 compare

The L-valine production ability was analyzed through the flask fermentation titer evaluation of the strain prepared in Example 17.2.1 and the control parent strain.

First, each colony was subcultured in a nutrient medium, and then each strain was inoculated in a 250 ml corner-baffle flask containing 25 ml of the production medium, and cultured with shaking at 30° C. for 72 hours at 200 rpm. Then, the concentration of L-valine was analyzed using HPLC, and the analyzed concentration of L-valine is shown in Table 51 below.

The experiment was repeated 3 times, and the average value of the analysis results is shown in Table 51 below.

균 주strain	L-발린 농도 (g/L)L-valine concentration (g/L)	L-발린 농도 증가율(%)L-valine concentration increase rate (%)
CA08-0072CA08-0072	1.91.9	--
CA08-0072_gapA_G134SCA08-0072_gapA_G134S	2.42.4	26.326.3

As shown in Table 51, the CA08-0072_gapA_G134S strain exhibited increased L-valine-producing ability compared to the control.

From the above description, those skilled in the art to which the present application pertains will be able to understand that the present application may be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present application should be construed as including all changes or modifications derived from the meaning and scope of the claims described below, rather than the above detailed description, and equivalent concepts thereof, to be included in the scope of the present application.

Name of deposit institution: Korea Microorganism Conservation Center

Accession number: KCCM12860P

Deposit date: 20201202

Name of deposit institution: Korea Microorganism Conservation Center

Accession number: KCCM12861P

Deposit date: 20201202

Name of deposit institution: Korea Microorganism Conservation Center

Accession number: KCCM12863P

Deposit date: 20201202

Name of deposit institution: Korea Microorganism Conservation Center

Accession number: KCCM12864P

Deposit date: 20201202

Name of deposit institution: Korea Microorganism Conservation Center

Accession number: KCCM12867P

Deposit date: 20201202

Name of deposit institution: Korea Microorganism Conservation Center

Accession number: KCCM12865P

Deposit date: 20201202

Name of deposit institution: Korea Microorganism Conservation Center

Accession number: KCCM12866P

Deposit date: 20201202

Name of deposit institution: Korea Microorganism Conservation Center

Accession number: KCCM12868P

Deposit date: 20201202

Name of deposit institution: Korea Microorganism Conservation Center

Accession number: KCCM12862P

Deposit date: 20201202

Name of deposit institution: Korea Microorganism Conservation Center

Accession number: KCCM12909P

Deposit date: 20201222

Claims

(i) at least one variant selected from the group consisting of the following (1) to (17),

(ii) one or more polynucleotides encoding the variant, or

(iii) combinations thereof;

A Corynebacterium glutamicum strain comprising:

(1) a protein variant, consisting of the amino acid sequence set forth in SEQ ID NO: 1, in which serine, an amino acid corresponding to position 109 of the amino acid sequence of SEQ ID NO: 3, is substituted with phenylalanine;

(2) an ATP phosphoribosyltransferase variant, consisting of the amino acid sequence shown in SEQ ID NO: 11, in which serine, an amino acid corresponding to position 130 of the amino acid sequence of SEQ ID NO: 13, is substituted with phenylalanine;

(3) an asparagine synthase variant, consisting of the amino acid sequence shown in SEQ ID NO: 21, in which proline, an amino acid corresponding to position 192 of the amino acid sequence of SEQ ID NO: 23, is substituted with leucine;

(4) spermidine synthase variant, consisting of the amino acid sequence set forth in SEQ ID NO: 31, in which alanine, an amino acid corresponding to position 213 of the amino acid sequence of SEQ ID NO: 33, is substituted with valine;

(5) a cysteine sulfinate disulfinase variant, consisting of the amino acid sequence set forth in SEQ ID NO: 41, in which proline, an amino acid corresponding to position 302 of the amino acid sequence of SEQ ID NO: 43, is substituted with serine

(6) a urease accessory protein variant, consisting of the amino acid sequence set forth in SEQ ID NO: 51, in which proline, an amino acid corresponding to position 56 of the amino acid sequence of SEQ ID NO: 53, is substituted with leucine;

(7) a proline dehydrogenase variant, consisting of the amino acid sequence set forth in SEQ ID NO: 61, in which glycine, an amino acid corresponding to position 834 of the amino acid sequence of SEQ ID NO: 63, is substituted with serine;

(8) a tetrahydrodipicolinate N-succinyltransferase variant, consisting of the amino acid sequence shown in SEQ ID NO: 71, in which arginine, an amino acid corresponding to position 140 of the amino acid sequence of SEQ ID NO: 73, is substituted with cysteine;

(9) a 5,10-methylenetetrahydrofolate reductase variant, consisting of the amino acid sequence set forth in SEQ ID NO: 81, in which arginine, an amino acid corresponding to the 4th position of the amino acid sequence of SEQ ID NO: 83, is substituted with histidine;

(10) a protein variant, consisting of the amino acid sequence set forth in SEQ ID NO: 91, in which glutamic acid, an amino acid corresponding to position 253 of the amino acid sequence of SEQ ID NO: 93, is substituted with lysine;

(11) a sugar porter family MFS transporter variant, consisting of the amino acid sequence shown in SEQ ID NO: 101, in which alanine, an amino acid corresponding to position 315 of the amino acid sequence of SEQ ID NO: 103, is substituted with valine;

(12) a transcription regulator variant, consisting of the amino acid sequence set forth in SEQ ID NO: 111, in which glutamic acid, an amino acid corresponding to position 247 of the amino acid sequence of SEQ ID NO: 113, is substituted with lysine;

(13) a WhiB family transcriptional regulator WHCA variant, consisting of the amino acid sequence shown in SEQ ID NO: 121, in which glycine, an amino acid corresponding to the 40th position of the amino acid sequence of SEQ ID NO: 123, is substituted with aspartic acid;

(14) a dihydrolipoyl acetyltransferase variant, consisting of the amino acid sequence shown in SEQ ID NO: 131, in which glycine, an amino acid corresponding to position 77 of the amino acid sequence of SEQ ID NO: 133, is substituted with aspartic acid;

(15) a 2-isopropyl malate synthetase variant, consisting of the amino acid sequence set forth in SEQ ID NO: 141, in which proline, an amino acid corresponding to position 87 of the amino acid sequence of SEQ ID NO: 143, is substituted with leucine;

(16) a branched chain amino acid permease variant, consisting of the amino acid sequence set forth in SEQ ID NO: 151, in which alanine, an amino acid corresponding to position 112 of the amino acid sequence of SEQ ID NO: 153, is substituted with threonine; and

(17) A glyceraldehyde-3-phosphate dehydrogenase variant comprising the amino acid sequence set forth in SEQ ID NO: 161, wherein glycine, an amino acid corresponding to position 134 of the amino acid sequence of SEQ ID NO: 163, is substituted with serine.
According to claim 1, wherein the strain,

(i) two or more variants selected from the group consisting of (1) to (17),

(ii) two or more polynucleotides encoding the variant, or

(iii) combinations thereof;

A strain comprising a.
A method for producing L-valine, comprising the step of culturing the Corynebacterium glutamicum strain of claim 1 or 2 in a medium.