5'UTR mutant sequences and their uses

Abstract

The present application relates to a 5'UTR mutant sequence of atpB and uses thereof.

Description

[Technical Field]

[0001] The present application relates to a 5'UTR mutant sequence, a microorganism containing the same, and a method for producing L-lysine using the same. [Background technology]

[0002] In order to produce L-amino acids and other useful substances, various studies have been conducted to develop highly efficient production microorganisms and fermentation process technologies. For example, target substance-specific approaches such as increasing the expression of genes encoding enzymes involved in L-lysine biosynthesis and removing genes unnecessary for biosynthesis have been mainly used (Patent Document 1).

[0003] However, as the demand for L-lysine is increasing, research is still needed to effectively improve L-lysine production capacity. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. 2008 / 082181 [Patent Document 2] U.S. Patent No. 9,593,354 [Patent Document 3] Korean Patent Publication No. 10-2020-0136813 [Patent Document 4] U.S. Patent No. 9,556,463 [Non-patent literature]

[0005] [Non-Patent Document 1] Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453 [Non-patent document 2] Rice et al., 2000, Trends Genet. 16: 276-277 [Non-patent document 3] Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

Non-licensed Document 7

Non-licensed literature 9

Non-licensed literature 10

Non-licensed Document 11

Non-licensed Document 12

[0006] The present application relates to a 5'UTR mutant sequence, a microorganism containing the same, and a method for producing L-lysine using the same. [Means for solving the problem]

[0007] The present application aims to provide a mutant polynucleotide encoding a mutant 5' untranslated region (5'-UTR) in which the bases at positions corresponding to the 10th to 13th bases in the base sequence of SEQ ID NO: 13 are substituted with TCGG.

[0008] Another object of the present application is to provide a mutant polynucleotide comprising a base sequence encoding F0F1-ATPase or a subunit thereof, wherein the mutant polynucleotide further comprises a base sequence encoding a mutant 5'UTR in which the bases at positions corresponding to the 10th to 13th bases in the base sequence of SEQ ID NO: 13 are substituted with TCGG.

[0009] Furthermore, the present application aims to provide an L-lysine-producing microorganism comprising a mutant polynucleotide encoding a mutant 5' untranslated region (5'-UTR) in which the bases at positions 10 to 13 in the base sequence of SEQ ID NO: 13 are substituted with TCGG, or a mutant polynucleotide comprising a base sequence encoding F0F1-ATPase or a subunit thereof, wherein the mutant polynucleotide further comprises a base sequence encoding the mutant 5'UTR.

[0010] Furthermore, the present application aims to provide a method for producing L-lysine, comprising the step of culturing in a medium a microorganism containing a mutant polynucleotide encoding a mutant 5' untranslated region (5'-UTR) in which the bases at positions 10 to 13 in the base sequence of SEQ ID NO: 13 are substituted with TCGG, or a mutant polynucleotide comprising a base sequence encoding F0F1-ATPase or a subunit thereof, wherein the mutant polynucleotide further comprises a base sequence encoding the mutant 5'UTR. [Effects of the Invention]

[0011] By regulating the gene expression level with the 5' untranslated region (5'-UTR) mutant sequence of the present application, L-lysine productivity can be improved. DETAILED DESCRIPTION OF THE INVENTION

[0012] These will be described in detail below. Note that each description and embodiment disclosed in this application also applies to other descriptions and embodiments. In other words, all combinations of various elements disclosed in this application are included in this application. Furthermore, this application is not limited to the following specific descriptions.

[0013] Additionally, those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the present application described herein which equivalents are intended to be encompassed by this application.

[0014] Furthermore, throughout this specification, many papers and patent documents are referenced and citations are provided, the disclosures of which are incorporated herein by reference in their entirety to more clearly explain the state of the art to which this application pertains and the contents of this application.

[0015] One aspect of the present application is a mutant polynucleotide encoding a mutant 5' untranslated region (5'-UTR) in which the bases at positions corresponding to the 10th to 13th bases in the base sequence of SEQ ID NO: 13 are substituted with TCGG.

[0016] In this application, the term "5' untranslated region (5'-UTR)" refers to a region that is present at the 5' end of an mRNA transcript but is not translated into amino acids. The process of mRNA translation into protein begins when the 30S subunit of the ribosome binds to the 5'UTR. Specifically, translation into protein begins when 16S rRNA (16S ribosomal RNA) in the 30S subunit of the ribosome binds to the RBS in the 5'UTR and tRNA recognizes and binds to the start codon (AUG) of the mRNA. The ribosome binding site and the start codon in the 5'UTR are known to be located approximately 6 to 8 nucleotides apart.

[0017] In one embodiment, the 5'UTR of the present application may include a ribosome binding site (RBS).

[0018] In this application, the term "ribosome binding site (RBS)" refers to a region (structure) within an mRNA chain where a ribosome directly binds and initiates translation, or a region (structure) of a DNA chain that forms such a region upon transcription. Most prokaryotes have an RBS, a short sequence that facilitates ribosome recognition and binding to an mRNA, located near the 5' upstream of the start codon (commonly "AUG") on the mRNA containing the open reading frame (ORF). The RBS contains a sequence complementary to the 3' end of 16S rRNA, known as the Shine-Dalgarno sequence.

[0019] In any of the above-described embodiments, the 5'UTR may contain a sequence complementary to the 3' end sequence of 16S rRNA, i.e., a Shine-Dalgarno sequence, and the sequences before and after it may contain regulated sequences.

[0020] "5'UTR sequence adjusted" or "adjusted 5'UTR" means that at least one base in the base sequence constituting the 5'UTR has been altered compared to the wild-type sequence, and such alterations include base substitution, addition, deletion, or inversion.

[0021] For example, the Shine-Dalgarno sequence is conserved in the 5'UTR, and the sequences before and after it include sequences in which at least one base has been modified by substitution, addition, deletion, inversion, etc., but are not limited to these.

[0022] The 5'UTR that serves as the parent sequence of the mutant 5'UTR of the present application may be a sequence contained in the 5'UTR of a gene encoding F0F1-ATPase or a subunit thereof.

[0023] Therefore, another aspect of the present application is a mutant polynucleotide comprising a base sequence encoding an F0F1-ATPase or a subunit thereof, wherein the mutant polynucleotide further comprises a base sequence encoding a mutant 5'UTR in which the bases at positions corresponding to the 10th to 13th bases in the base sequence of SEQ ID NO: 13 are substituted with TCGG.

[0024] In one embodiment, the 5'UTR that serves as the parent sequence of the mutant 5'UTR of the present application may contain, have, or consist essentially of the base sequence of SEQ ID NO: 13.

[0025] In any of the above-described Examples, the 5'UTR of the gene encoding the F0F1-ATPase or a subunit thereof may comprise SEQ ID NO: 13 or a nucleotide sequence having at least 70%, 75%, 76%, 80%, 84%, 85%, 88%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% or more homology or identity to the nucleotide sequence represented by SEQ ID NO: 13. Needless to say, the present application also includes nucleotide sequences in which a portion of the sequence has been deleted, modified, substituted, conservatively substituted, or added, so long as the nucleotide sequence has such homology or identity and exhibits the same efficacy as the 5'UTR sequence of SEQ ID NO: 13.

[0026] On the other hand, SEQ ID NO: 1 of the present application is a sequence including a part of the sequence encoding atpB, a subunit of F0F1-ATPase (ATPase), and its 5'UTR, and bases 556 to 558 of SEQ ID NO: 1 correspond to the start codon. Bases 1 to 555 of SEQ ID NO: 1 correspond to the 5'UTR sequence, and include SEQ ID NO: 13.

[0027] In any of the above-mentioned Examples, the 5'UTR sequence of the gene encoding the F0F1-ATPase or a subunit thereof of the present application may be the nucleotide sequence of SEQ ID NO: 1, which includes SEQ ID NO: 13.

[0028] In any of the above-described embodiments, the 5'UTR sequence of the gene encoding the F0F1-ATPase or a subunit thereof of the present application may comprise SEQ ID NO: 1 or a nucleotide sequence having at least 70%, 75%, 76%, 80%, 84%, 85%, 88%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% or more homology or identity to the nucleotide sequence represented by SEQ ID NO: 1.

[0029] The mutant 5'UTR of the present application contains a mutation in the ribosome binding site in the 5'UTR. Therefore, the mutant 5'UTR of the present application is also referred to as a "mutant RBS-containing sequence."

[0030] The mutant 5'UTR of the present application may be a 5'UTR sequence in which the base corresponding to the 10th base of the base sequence of SEQ ID NO: 13 is substituted with T, the base corresponding to the 11th base is substituted with C, the base corresponding to the 12th base is substituted with G, and the base corresponding to the 13th base is substituted with G, i.e., the bases at the positions corresponding to the 10th to 13th bases are substituted with TCGG.

[0031] In one embodiment, the mutant 5'UTR of the present application may comprise, have, or consist essentially of the base sequence of SEQ ID NO: 14.

[0032] In any of the above-mentioned examples, the mutant 5'UTR of the present application has, compared to SEQ ID NO: 13, bases at positions corresponding to bases 10 to 13 of SEQ ID NO: 13 fixed to TCGG, and has a base sequence that is at least 70% or more, 75% or more, 76% or more, 80% or more, 81% or more, 82% or more, or 83% or more of the entire sequence, or comprises said base sequence, or is substantially composed of said base sequence, but is not limited to these.

[0033] Furthermore, it goes without saying that mutant 5'UTRs having a base sequence in which a portion of the sequence has been deleted, modified, substituted, conservatively substituted, or added are also included in the present application, as long as the base sequence has such homology or identity and exhibits efficacy equivalent to that of the mutant 5'UTR of the present application.

[0034] In this application, the term "polynucleotide" refers to a nucleotide polymer in which nucleotide monomers are linked in a long chain by covalent bonds, and refers to a DNA or RNA chain longer than a certain length.

[0035] In the present application, the term "mutant" refers to a polynucleotide in which at least one base has been modified, resulting in a difference from the base sequence of the mutant polynucleotide before the mutation, but which maintains its functions or properties. Such variants can generally be identified by modifying at least one base in the base sequence of the polynucleotide and evaluating the properties of the modified polynucleotide. That is, the ability of the mutant polynucleotide may be improved, unchanged, or decreased compared to the polynucleotide before the mutation. The term "mutant polynucleotide" is often used interchangeably with terms such as variant, modification, mutated polynucleotide, mutated gene, mutation, and variant (in English, variant, modification, modified polynucleotide, modified gene, mutant, mutein, divergent, etc.), but any term meaning a mutation may be used.

[0036] The variant polynucleotide may be attached to other sequences or linkers to allow identification, purification or synthesis.

[0037] In this application, "corresponding to" means the base at the recited position in the polynucleotide, or a base similar, identical, or equivalent to the recited base in the polynucleotide. Identifying the base at the corresponding position determines the specific base of the sequence to which the particular sequence refers. In this application, "corresponding region" generally refers to a similar or corresponding position in a related or reference polynucleotide sequence.

[0038] For example, when any base sequence is aligned with SEQ ID NO: 13 or SEQ ID NO: 1, each base in the base sequence can be numbered based on the number and position of the base corresponding to the base in SEQ ID NO: 13 or SEQ ID NO: 1. For example, the sequence alignment algorithm in the present application can identify the positions of bases or positions where modifications such as substitutions, insertions, deletions, etc. occur when compared with a query sequence (also referred to as a "reference sequence").

[0039] For such alignment, for example, the Needleman-Wunsch algorithm (Non-Patent Document 1) or the Needle program in the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Non-Patent Document 2) can be used, but the present invention is not limited to these. Sequence alignment programs, pairwise sequence comparison algorithms, and the like known in the technical field can also be used as appropriate.

[0040] In this application, "homology" or "identity" refers to the degree of similarity between two given amino acid or nucleotide sequences, expressed as a percentage. Homology and identity are often used interchangeably.

[0041] Sequence homology or identity of conserved polynucleotides or polypeptides can be determined by standard sequence algorithms, optionally with default gap penalties established by the program used. Substantially homologous or identical sequences will generally hybridize to all or part of the sequence under moderately or highly stringent conditions. Hybridization, of course, also includes hybridization to polynucleotides containing common codons or codons that take into account codon degeneracy in the polynucleotide.

[0042] Whether any two polynucleotide or polypeptide sequences have homology, similarity, or identity can be determined using known computer algorithms, such as the "FASTA" program, with default parameters, as described in, for example, Non-Patent Document 3. Alternatively, the Needleman-Wunsch algorithm (Non-Patent Document 1) can be used, as implemented in the Needle program (EMBOSS: The European Molecular Biology Open Software Suite, Non-Patent Document 2) (version 5.0.0 or later) of the EMBOSS package (which includes the GCG program package (Non-Patent Document 4), BLASTP, BLASTN, and FASTA (Non-Patent Documents 5, 6, and 7)). For example, BLAST or ClustalW from the National Center for Biotechnology Information can be used to determine homology, similarity, or identity.

[0043] Homology, similarity, or identity of polynucleotides or polypeptides can be determined by comparing sequence information using a GAP computer program such as that disclosed in Non-Patent Document 1, as disclosed in, for example, Non-Patent Document 8. Briefly, the GAP program defines the number of similar sequence symbols (i.e., nucleotides or amino acids) divided by the total number of symbols in the shorter of the two sequences. Default parameters for the GAP program include: (1) a unitary matrix (identity takes a value of 1, non-identity takes a value of 0) and a weighted comparison matrix (or EDNAFULL (the EMBOSS version of NCBINUC4.4) substitution matrix) as disclosed in Non-Patent Document 9; (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 terminal gaps.

[0044] The polynucleotide of the present application may undergo various modifications in the coding region to the extent that the base sequence of the mutant gene containing the mutant 5'UTR of the present application is not changed, taking into account codon degeneracy or preferred codons in the organism in which the mutant gene containing the mutant 5'UTR of the present application is to be expressed.

[0045] Furthermore, the polynucleotide of the present application may be any sequence that hybridizes under stringent conditions with a probe prepared from a known gene sequence, for example, a sequence complementary to all or part of the polynucleotide sequence of the present application. The term "stringent conditions" refers to conditions that allow specific hybridization between polynucleotides. Such conditions are specifically described in the literature (see Non-Patent Documents 11 and 12). For example, conditions include those under which polynucleotides having high homology or identity, such as 70% or more, 75% or more, 76% or more, 80% or more, 84% or more, 85% or more, 88% or more, 90% or more, 92% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, 99.7% or more, 99.8% or more, or 99.9% or more, hybridize with each other, while polynucleotides having lower homology or identity do not hybridize with each other; or conditions for washing once, specifically two to three times, at a salt concentration and temperature equivalent to those used in conventional Southern hybridization, i.e., 60°C, 1×SSC, 0.1% SDS, specifically 60°C, 0.1×SSC, 0.1% SDS, more specifically 68°C, 0.1×SSC, 0.1% SDS.

[0046] Hybridization requires that two nucleic acids have complementary sequences, even if mismatches between bases are possible depending on the stringency of the hybridization. "Complementary" is used to describe the relationship between nucleotide bases that can hybridize to each other. For example, in DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Thus, the polynucleotides of the present application may include not only substantially similar nucleic acid sequences, but also isolated nucleic acid fragments that are complementary to the entire sequence.

[0047] Specifically, polynucleotides having homology or identity to the polynucleotides of the present application can be detected using the hybridization conditions described above, in which the hybridization step is performed at a Tm value of 55° C. The Tm value may be, but is not limited to, 60° C., 63° C., or 65° C., and can be appropriately adjusted by those skilled in the art depending on the purpose.

[0048] The appropriate stringency for hybridizing the polynucleotides depends on the length of the polynucleotides and the degree of complementation, variables known in the art (eg, Non-Patent Document 11).

[0049] The mutant polynucleotide encoding the mutant 5' UTR of the present application may regulate the translation of a polynucleotide encoding a target protein operably linked thereto, which may be any target protein whose expression is regulated by the mutant 5' UTR of the present application.

[0050] In one embodiment, the mutant polynucleotide encoding the 5'UTR may be included upstream of a base sequence encoding F0F1-ATPase or a subunit thereof.

[0051] In any of the above-described embodiments, the mutant polynucleotide encoding the mutant 5'UTR may be operably linked to the atpBEFHAGDC operon and regulate the activity of at least one of the subunit proteins (subunits) that make up the F0F1-ATPase complex.

[0052] In any of the above-described embodiments, the mutant polynucleotide encoding the mutant 5'UTR may be operably linked to the atpB gene.

[0053] In any of the above-described embodiments, the mutant polynucleotide encoding the mutant 5'UTR may regulate expression of the F0F1-ATP hydrolase operon.

[0054] In this application, "F0F1-ATPase" refers to an enzyme that synthesizes ATP in the cell membrane using proton motive force. Because it has the function of synthesizing ATP in a general cellular environment, it is also called ATP synthase. The F0F1-ATPase of Corynebacterium genus microorganisms is known to contain eight subunits and be encoded by the atpBEFHAGDC operon, which contains eight genes.

[0055] In this application, the term "operon" refers to a functional unit of DNA containing a group of genes whose expression is regulated by a single expression control sequence, specifically a single promoter. The mRNA transcribed by the operon may be polycistronic mRNA, in which one mRNA molecule encodes one or more proteins, or monocistronic mRNA, in which one mRNA molecule encodes one protein.

[0056] The operon of the present application may include an expression regulatory region and a structural gene. The "expression regulatory region" of an operon refers to a site that is located upstream of the structural gene that constitutes the operon and is capable of regulating the expression of the structural gene. The expression regulatory region of an operon may include a 5'UTR region excluding the structural gene. The expression regulatory region may include an RBS region.

[0057] Yet another aspect of the present application is a vector comprising a mutant polynucleotide encoding a mutant 5' untranslated region (5'-UTR) in which the bases at positions corresponding to the 10th to 13th bases in the base sequence of SEQ ID NO: 13 are substituted with TCGG.

[0058] Yet another aspect of the present application is a vector comprising a mutant polynucleotide comprising a nucleotide sequence encoding F0F1-ATPase or a subunit thereof, wherein the vector further comprises a nucleotide sequence encoding a mutant 5'UTR in which the nucleotides at positions corresponding to the 10th to 13th nucleotides in the nucleotide sequence of SEQ ID NO: 13 are substituted with TCGG.

[0059] The mutant polynucleotide, the base sequence of SEQ ID NO: 13, and the mutant 5'UTR are as described above.

[0060] The vector is an expression vector for expressing a polynucleotide of interest in a host cell, but is not limited to this.

[0061] The vector of the present application includes a DNA product containing the base sequence of the target polynucleotide operably linked to a suitable expression control region (or expression control sequence) so as to enable expression of the target polynucleotide in a suitable host. The expression control region includes a promoter that initiates transcription, an optional operator sequence for regulating the transcription, a sequence encoding a suitable RBS, and a sequence that regulates the termination of transcription and translation.

[0062] For purposes of the present application, the expression regulatory region may be a mutant 5'UTR in the above-described embodiment. For example, the target polynucleotide may be a nucleotide sequence encoding an F0F1-ATPase or a subunit thereof. For example, the target polynucleotide may be a nucleotide sequence encoding the α subunit of the F0 part of the F0F1-ATPase. For example, the target polynucleotide may be the atpBEFHAGDC operon. For example, the target polynucleotide may be the atpB gene or NCgl1159. However, any gene whose expression is regulated by the mutant 5'UTR of the present application may be used.

[0063] When a vector is transformed into a suitable host cell, it can replicate and function independently of the host genome and is integrated into the genome itself.

[0064] 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 naturally occurring or recombinant plasmids, cosmids, viruses, and bacteriophages. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A may be used as phage or cosmid vectors. Examples of plasmid vectors that may be used include pDZ, pBR, pUC, pBluescriptII, pGEM, pTZ, pCL, and pET. Specifically, pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, and pCC1BAC vectors may be used.

[0065] For example, a target polynucleotide can be inserted into a chromosome using a vector for intracellular chromosome introduction. The insertion of the polynucleotide into a chromosome can be achieved by any method known in the art, including, but not limited to, homologous recombination. A selection marker for determining whether or not the polynucleotide has been inserted into the chromosome may be further included. The selection marker is used to select cells transformed with the vector, i.e., to determine whether or not the target nucleic acid molecule has been inserted. Markers that confer selectable phenotypes, such as drug resistance, auxotrophy, resistance to cytotoxic agents, and expression of surface polypeptides, are used. In an environment treated with a selective agent, only cells expressing the selection marker survive or exhibit a different phenotype, allowing the selection of transformed cells.

[0066] In this application, "transformation" refers to the introduction of a vector containing a target polynucleotide into a host cell or microorganism, thereby expressing the polynucleotide in the host cell. A transformed polynucleotide may be any polynucleotide that is expressed in the host cell, regardless of whether it is located intrachromosomally or extrachromosomally. The polynucleotide may also include DNA and / or RNA encoding a target polypeptide. The polynucleotide may be introduced into the host cell in any form that allows it to be expressed in the host cell. For example, the polynucleotide may be introduced into the host cell in the form of an expression cassette, which is a genetic construct containing all elements necessary for its own expression. Typically, the expression cassette contains a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal operably linked to the polynucleotide. The expression cassette may be in the form of a self-replicating expression vector. The polynucleotide may also be introduced into the host cell in its own form and operably linked to sequences necessary for expression in the host cell, but this is not limited thereto.

[0067] Furthermore, the term "operably linked" refers to a functional linkage between an expression regulatory sequence comprising a nucleotide sequence encoding the mutant 5'UTR of the present application and a nucleotide sequence encoding a target protein. For example, the nucleotide sequence encoding the target protein may be a nucleotide sequence encoding F0F1-ATPase or a subunit thereof. For example, the nucleotide sequence encoding the target protein may be the atpBEFHAGDC operon. For example, the nucleotide sequence encoding the target protein may be a nucleotide sequence encoding the α subunit of the F0 part of F0F1-ATPase. For example, the nucleotide sequence encoding the target protein may be the atpB gene or NCgl1159.

[0068] For example, but not limited to, the mutant 5'UTR of the present application may regulate the translation of a polynucleotide encoding a target protein operably linked thereto into a protein.

[0069] In yet another aspect, the present application provides an L-lysine-producing microorganism comprising a mutant polynucleotide encoding a mutant 5' untranslated region (5'-UTR) in which the bases at positions 10 to 13 in the base sequence of SEQ ID NO: 13 are substituted with TCGG, or a mutant polynucleotide comprising a base sequence encoding F0F1-ATPase or a subunit thereof, wherein the mutant polynucleotide further comprises a base sequence encoding the mutant 5'UTR.

[0070] In one embodiment, the microorganism of the present application comprises a mutant polynucleotide encoding a 5'UTR in which the bases at positions corresponding to the 10th to 13th bases in the base sequence of SEQ ID NO: 13 are substituted with TCGG, and may further comprise a polynucleotide encoding an F0F1-ATPase or a subunit thereof.

[0071] In any of the above embodiments, the microorganism of the present application may comprise a vector comprising the polynucleotide.

[0072] In any of the above-mentioned Examples, the microorganism of the present application may comprise at least one selected from a mutant polynucleotide encoding a mutant 5' untranslated region (5'-UTR) in which the bases at positions corresponding to the 10th to 13th bases in the base sequence of SEQ ID NO: 13 are substituted with TCGG, or a mutant polynucleotide comprising a base sequence encoding F0F1-ATPase or a subunit thereof, which further comprises a base sequence encoding the mutant 5'UTR, and a vector comprising the polynucleotide.

[0073] In the microorganism of the present application, the 5'UTR, the F0F1-ATPase, the mutant polynucleotide, the vector, and the like are as described above.

[0074] The term "microorganism (or strain)" in this application includes all wild-type microorganisms and microorganisms that have been genetically modified, either naturally or artificially, and may also be a microorganism in which a specific mechanism has been weakened or strengthened by inserting an exogenous gene or by strengthening or inactivating the activity of an endogenous gene, and may also be a microorganism that has been genetically modified to express a polypeptide of interest.

[0075] The microorganism may be a Corynebacterium microorganism.

[0076] As an example of the present application, the microorganism of the present application is Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium stationis, Corynebacterium singulare, Corynebacterium halotolerans, Corynebacterium striatum, Corynebacterium ammoniagenes, Corynebacterium ammoniagenes, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium testudinoris or Corynebacterium flavescens.

[0077] Specifically, the microorganism of the present application is a microorganism of the genus Corynebacterium, more specifically Corynebacterium glutamicum, but is not limited thereto.

[0078] The microorganism of the present application may be capable of producing L-lysine.

[0079] The microorganism of the present application may be a naturally occurring wild-type microorganism, an unmodified microorganism, or a recombinant microorganism into which a mutant polynucleotide transcribed by the mutant 5' UTR of the present application, or a mutant polynucleotide encoding an F0F1-ATP hydrolase or a subunit thereof comprising the mutant polynucleotide transcribed by the mutant 5' UTR of the present application, has been introduced into an L-lysine-producing microorganism.

[0080] The term "unmodified microorganism" in the present application does not exclude strains containing naturally occurring mutations in microorganisms, but refers to a wild-type strain or a naturally occurring strain itself, or a strain before its traits are changed due to genetic mutation caused by natural or artificial factors. For example, the unmodified microorganism refers to a strain into which a mutant polynucleotide encoding a mutant 5' UTR as used herein, or a mutant polynucleotide encoding an F0F1-ATP hydrolase or a subunit thereof comprising a mutant polynucleotide transcribed by the mutant 5' UTR, has not been introduced or has not yet been introduced. For example, the unmodified microorganism refers to a strain containing the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 13. The term "unmodified microorganism" is also used interchangeably with "strain before modification," "microorganism before modification," "non-mutated strain," "non-modified strain," "non-mutated microorganism," or "reference microorganism."

[0081] In one embodiment, the microorganism of the present application may further enhance or attenuate the activity of a protein or gene involved in the L-lysine biosynthetic pathway. In any of the above-mentioned embodiments, the microorganism of the present application may contain the pyc(P458S), hom(V59A), and lysC(T311I) mutations (Non-Patent Document 13, Patent Document 2).

[0082] In any of the above-mentioned embodiments, the microorganism of the present application may further have enhanced activity of at least one selected from aspartate kinase, transketolase, and pyruvate carboxylase.

[0083] In any of the above-mentioned embodiments, the microorganism of the present application may further have enhanced activity of at least one gene selected from the group consisting of the lysC gene, the tkt gene, and the pyc gene. In any of the above-mentioned embodiments, the microorganism of the present application may have the start codon of at least one gene selected from the group consisting of the lysC gene, the tkt gene, and the pyc gene substituted with ATG.

[0084] Yet another aspect of the present application is a method for producing L-lysine, comprising the step of culturing in a medium an L-lysine-producing microorganism comprising a mutant polynucleotide encoding a mutant 5' untranslated region (5'-UTR) in which the bases at positions 10 to 13 in the base sequence of SEQ ID NO: 13 are substituted with TCGG, or a mutant polynucleotide comprising a base sequence encoding F0F1-ATPase or a subunit thereof, the mutant polynucleotide further comprising a base sequence encoding the mutant 5'UTR.

[0085] In the method of the present application, the 5'UTR, F0F1-ATPase, mutant polynucleotide, microorganism, etc. are as described above.

[0086] The term "culturing" as used herein means growing the microorganism of the present application under appropriately controlled environmental conditions. The culturing process of the present application can be carried out using a suitable medium and culture conditions known in the art. Those skilled in the art can easily adjust such a culturing process depending on the selected strain. Specifically, the culturing may be batch, continuous, and / or fed-batch culture, but is not limited thereto.

[0087] The term "culture medium" as used herein refers to a mixture of nutrients necessary for culturing the microorganism of the present application as its main components, and supplies nutrients such as water essential for survival and growth, growth factors, etc. Specifically, the medium and other culture conditions used to culture the microorganism of the present application may be any medium used for culturing ordinary microorganisms, and the microorganism of the present application can be cultured in an ordinary medium containing a suitable carbon source, nitrogen source, phosphorus source, inorganic compounds, amino acids, and / or vitamins under aerobic conditions by adjusting the temperature, pH, etc.

[0088] In the present application, examples of carbon sources that can be used include carbohydrates such as glucose, saccharose, lactose, fructose, sucrose, and maltose; sugar alcohols such as mannitol and sorbitol; organic acids such as pyruvic acid, lactic acid, and citric acid; and amino acids such as glutamic acid, methionine, and lysine. Natural organic nutrient sources such as starch hydrolysates, molasses, blackstrap molasses, rice bran, cassava, bagasse, and corn steeping liquid can also be used. Specifically, carbohydrates such as glucose and sterilized pretreated molasses (i.e., molasses converted into reducing sugars) can be used. Any other carbon source can also be used as long as it is present in an appropriate amount. These carbon sources can be used alone or in combination of two or more, but are not limited to these.

[0089] Examples of the nitrogen source that can be used include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate, and organic nitrogen sources such as amino acids such as glutamic acid, methionine, and glutamine, peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steeping liquid, casein hydrolysate, fish or its degradation products, and defatted soybean cake or its degradation products. These nitrogen sources can be used alone or in combination of two or more, but are not limited to these.

[0090] Examples of the phosphorus source that can be used include potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and sodium-containing salts corresponding thereto. Examples of inorganic compounds that can be used include sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, and calcium carbonate. Other examples include amino acids, vitamins, and / or suitable precursors. These components or precursors can be added to the medium in a batch or continuous manner. However, the present invention is not limited to these.

[0091] Furthermore, the pH of the medium can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid to the medium in a suitable manner during cultivation of the microorganism of the present application. Furthermore, during cultivation, foam formation can be suppressed using an antifoaming agent such as a fatty acid polyglycol ester. Furthermore, oxygen or an oxygen-containing gas may be injected into the medium to maintain an aerobic state, and nitrogen, hydrogen, or carbon dioxide gas may be injected, or no gas may be injected, to maintain anaerobic and microaerobic states, but these are not limiting.

[0092] In the culture of the present application, the culture temperature is maintained at 20 to 45°C, specifically 25 to 40°C, and the culture is carried out for about 10 to 160 hours, but is not limited thereto.

[0093] The L-lysine produced by the culture of the present application is either secreted into the medium or remains intracellularly.

[0094] The L-lysine production method of the present application may further include a step of providing a microorganism of the present application, a step of preparing a medium for culturing the strain, or a combination thereof (in any order), e.g., prior to the culturing step.

[0095] The method for producing L-lysine of the present application may further include a step of recovering L-lysine from the culture medium or the strain used for the culture. The recovery step may be further included after the culture step.

[0096] The target L-lysine may be collected using a suitable method known in the art depending on the culture method of the microorganism of the present application, such as batch, continuous, or fed-batch culture. For example, centrifugation, filtration, crystallization, treatment with a protein precipitant (salting out), extraction, ultrasonic disruption, ultrafiltration, dialysis, various types of chromatography such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, and affinity chromatography, HPLC, or a combination thereof can be used. The target L-lysine can be collected from the medium or the microorganism using a suitable method known in the art.

[0097] The L-lysine production method of the present application may further include a purification step. The purification can be performed by any suitable method known in the art. For example, when the L-lysine production method of the present application includes both a recovery step and a purification step, the recovery step and the purification step may be performed continuously or discontinuously in any order, simultaneously, or integrated into a single step, but are not limited thereto.

[0098] Yet another aspect of the present application provides an L-lysine-producing microorganism, a culture medium in which the microorganism has been cultured, or a composition for producing L-lysine, comprising a mutant polynucleotide encoding a mutant 5' untranslated region (5'-UTR) in which the bases corresponding to the 10th to 13th bases in the base sequence of SEQ ID NO: 13 have been substituted with TCGG, or a mutant polynucleotide comprising a base sequence encoding F0F1-ATP hydrolase or a subunit thereof, and further comprising a base sequence encoding the mutant 5'UTR;

[0099] The compositions of the present application may further contain any suitable excipients commonly used in compositions for producing L-lysine, including, but not limited to, preservatives, wetting agents, dispersing agents, suspending agents, buffers, stabilizers, isotonicity agents, etc.

[0100] Yet another aspect of the present application provides use of a microorganism comprising a mutant polynucleotide encoding a mutant 5' untranslated region (5'-UTR) in which the bases at positions 10 to 13 in the base sequence of SEQ ID NO: 13 are substituted with TCGG, or a mutant polynucleotide comprising a base sequence encoding F0F1-ATPase or a subunit thereof, wherein the mutant polynucleotide further comprises a base sequence encoding the mutant 5'UTR, for the production of L-lysine.

[0101] The 5'UTR, F0F1-ATPase, mutant polynucleotide, microorganism, etc. are as described above. [Example]

[0102] The present application will be described in more detail below with reference to examples and experimental examples. However, these examples and experimental examples are merely illustrative of the present application, and the present application is not limited to these examples and experimental examples. [Example]

[0103] Identification of atpB upstream sequence variants using UTR Designer (RBS prediction program) Three RBS sequence mutations were designed to predict and regulate protein expression levels, and the expression levels were predicted using UTRDesigner (Non-Patent Document 14). To predict the current protein expression level of the target gene, atpB, the upstream 25-nucleotide sequence of the atpB gene and the 35-nucleotide sequence including the target gene initiation codon were input, along with the Corynebacterium 16s rRNA sequence [CCUCCUUUC]. The program predicted the atpB protein expression level to be approximately 6 (Table 1). The three mutant sequences designed and their predicted expression levels are shown in Table 2.

[0104] They were named atpBRBS1(56) / atpB RBS2(974) / atpB RBS3(1802) in order of decreasing protein expression level.

[0105] [Table 1]

[0106] [Table 2] [Example]

[0107] Construction of vectors containing predicted sequences and transfection of strains To construct vectors containing each predicted RBS sequence, primers were designed based on SEQ ID NO: 1. To construct vectors containing the atpBRBS1 sequence, template PCR was performed using the chromosome of Corynebacterium glutamicum ATCC 13032 as a template, using primer pairs SEQ ID NOs: 2 and 3 and SEQ ID NOs: 4 and 5. To construct vectors containing the atpBRBS2 / 3 sequence, template PCR was performed using primer pairs SEQ ID NOs: 2 and 6, SEQ ID NOs: 7 and 5, SEQ ID NOs: 2 and 8, and SEQ ID NOs: 9 and 5. The PCR conditions were denaturation at 94°C for 5 minutes, followed by 30 cycles of 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 1 minute 30 seconds, followed by 5 minutes at 72°C. Each template PCR fragment was digested with the restriction enzyme SmaI into the plasmid pDCM2 (Patent Document 3), which is used for gene insertion and replacement into the Corynebacterium chromosome, and then fusion cloned. The resulting vector was transformed into Escherichia coli DH5α using the In-Fusion® HD Cloning Kit (Clontech) and then spread onto LB solid medium containing kanamycin (25 mg / L). Colonies transformed with the vector containing the target gene were selected by PCR using primers SEQ ID NOs: 10 and 11, and then plasmids were isolated using standard plasmid extraction methods. The resulting plasmids were designated pDC_atpBRBS1, pDC_atpB RBS2, and pDC_atpB RBS3. The sequences of the primers used in this example are shown in Table 3.

[0108] [Table 3]

[0109] The vectors pDC_atpB RBS1, pDC_atpB RBS2, pDC_atpB RBS3, and pDC_atpB RBS4 prepared in Example 1 were transformed into Corynebacterium glutamicum CJ3P (Patent Document 4) by the electric pulse method (Non-Patent Document 15). Strains in which a mutation had been introduced upstream of the atpB gene by homologous chromosomal recombination were identified using the primer pair of SEQ ID NOs: 2 and 5. These were designated CJ3P_atpB-RBS1, CJ3P_atpB-RBS2, and CJ3P_atpB-RBS3. [Example]

[0110] Comparison of L-lysine production ability of the constructed atpBRBS mutant strains To confirm the L-lysine-producing ability of the strains prepared in Example 2 and the control parent strain, a flask fermentation titer evaluation was carried out.

[0111] First, each strain was inoculated into a 250 mL corner-baffled flask containing 25 mL of seed medium and cultured with shaking at 37°C and 200 rpm for 20 hours. One mL of the seed culture was inoculated into a 250 mL corner-baffled flask containing 24 mL of production medium and cultured with shaking at 37°C and 200 rpm for 36 hours. To accurately measure the glycogen consumption point, 700 μL of culture medium was sampled after 18 hours of culture and the L-lysine concentration was measured. The productivity of each strain was then calculated. Table 4 shows the productivity (g / L / h), productivity improvement rate (%), and lysine concentration (g / L) after culture completion for each strain tested.

[0112] The above experiment was repeated three times, and the average values ​​of the analytical results are shown in Table 4. <Seed medium (pH 7.0)> Glucose 20g, peptone 10g, yeast extract 5g, urea 1.5g, KH2PO4 4g, K2HPO4 8g, MgSO4·7H2O 0.5g, biotin 0.1mg, thiamine HCl 1mg, calcium pantothenate 2mg, nicotinamide 2mg (in 1 liter of distilled water) <Production medium (pH 7.0)> 100g glucose, 40g (NH4)2SO4, 2.5g soy protein, 5g corn steep solids, 3g urea, 1g KH2PO4, 0.5g MgSO4·7H2O, 100μg biotin, 1000μg thiamine hydrochloride, 2000μg calcium pantothenate, 3000μg nicotinamide, 3000μg CaCO3 (in 1 liter of distilled water).

[0113] [Table 4]

[0114] As shown in Table 4, of the three atpBRBS mutations, only the CJ3P_atpB-RBS1 strain, which contains the atpB RBS1 mutation, was found to have an L-lysine productivity increase of approximately 103.8% compared to the control CJ3P. The other mutations resulted in a decrease in lysine concentration and lysine productivity, and in particular, the CJ3P_atpB-RBS3 strain was found to completely inhibit strain growth. These results confirmed that atpB expression, regulated by a specific sequence in atpBRBS1, is necessary for improved lysine productivity.

[0115] From the above description, those skilled in the art to which the present application pertains will understand that the present application can be implemented in other specific forms without changing the technical idea or essential features thereof. It should be understood that the above examples are merely illustrative and not limiting. The present application should be construed as including all modifications and variations derived from the meaning and scope of the claims, rather than the specification, and their equivalent concepts.

Claims

1. It encodes a mutant 5' untranslated region (5'-UTR) in which the bases at positions corresponding to the 10th to 13th bases in the base sequence of SEQ ID NO: 13 are substituted with TCGG. Mutant polynucleotides.

2. F ０ F １ - a mutant polynucleotide comprising a base sequence encoding an ATPase or a subunit thereof, the mutant polynucleotide further comprises a base sequence encoding a mutant 5'UTR in which bases at positions corresponding to the 10th to 13th bases in the base sequence of SEQ ID NO: 13 are substituted with TCGG; Mutant polynucleotides.

3. The mutant 5'UTR comprises the base sequence of SEQ ID NO:

14. The mutant polynucleotide of claim 1 or 2.

4. the mutant polynucleotide encoding the mutant 5'UTR is operably linked to a polynucleotide encoding a target protein; The mutant polynucleotide of claim 1.

5. the mutant polynucleotide encoding the mutant 5'UTR is operably linked to the atpB gene; The mutant polynucleotide of claim 1.

6. The mutant polynucleotide encoding the mutant 5'UTR is ０ F １ - regulates the expression of the ATP hydrolase operon, The mutant polynucleotide of claim 1.

7. The base sequence of SEQ ID NO: 13 contains a ribosome binding site (RBS). The mutant polynucleotide of claim 1 or 2.

8. A mutant polynucleotide encoding a mutant 5' untranslated region (5'-UTR) in which the bases at positions corresponding to the 10th to 13th bases in the base sequence of SEQ ID NO: 13 are substituted with TCGG; or F ０ F １ - A mutant polynucleotide comprising a base sequence encoding an ATPase or a subunit thereof, further comprising a base sequence encoding the mutant 5'UTR, A microorganism that produces L-lysine.

9. A mutant polynucleotide encoding a mutant 5' untranslated region (5'-UTR) in which the bases at positions corresponding to the 10th to 13th bases in the base sequence of SEQ ID NO: 13 are substituted with TCGG; or F ０ F １ - comprising a step of culturing in a medium a microorganism containing a mutant polynucleotide comprising a nucleotide sequence encoding an ATPase or a subunit thereof, the mutant polynucleotide further comprising a nucleotide sequence encoding the mutant 5'UTR; Methods for producing L-lysine.

10. The method further comprises recovering L-lysine from the cultured microorganism, the culture of the microorganism, the fermentation product of the microorganism, or the culture medium.

10. The method of claim 9.

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