Microorganisms introducing trehalase and use thereof

Abstract

The present disclosure provides microorganisms that incorporate trehalase and uses thereof.

Description

Technical Field

[0001] This disclosure relates to microorganisms that introduce trehalase and their uses.

[0002] Cross-references to related applications This application claims the benefit of priority based on Korean Patent Application No. 10-2025-0025095, filed on February 26, 2025, the entire contents of which are incorporated herein by reference. Background Technology

[0003] L-Lysine is an essential amino acid, crucial for physiological functions in humans and animals. Particularly in the feed and food industries, lysine is used as a key component in protein synthesis. Lysine is primarily produced through microbial fermentation. Since lysine production is highly dependent on the carbon metabolism capacity of the microorganisms, the efficient utilization of energy sources is a significant factor in productivity. Therefore, it is necessary to investigate effective methods to increase lysine production.

[0004] [Existing technical documents] (Patent Document 1) US Patent Publication No. 2011-0008864 A1 Summary of the Invention

[0005] [Technical Issues] One object of this disclosure is to provide a Corynebacterium that produces lysine ( Corynebacterium It belongs to the microorganisms, among which trehalase derived from Escherichia coli was introduced.

[0006] Another object of this disclosure is to provide a composition comprising the aforementioned Corynebacterium microorganisms for the production of lysine.

[0007] Another object of this disclosure is to provide a method for producing lysine, comprising culturing the Corynebacterium genus microorganism in a culture medium.

[0008] Another object of this disclosure is to provide a method for increasing lysine production, comprising culturing the Corynebacterium genus microorganisms in a culture medium.

[0009] [Technical Solution] This will be described in detail below. Furthermore, each description and embodiment disclosed in this disclosure can be applied to every other description and embodiment. That is, all combinations of the various elements disclosed in this disclosure fall within the scope of this disclosure. Moreover, the scope of this disclosure should not be considered limited to the specific descriptions below. In addition, numerous papers and patent documents are cited throughout the specification, and their sources are indicated. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to more clearly explain the level of the technical field to which this disclosure pertains and the content of this disclosure. Furthermore, those skilled in the art can identify or determine multiple equivalents of specific aspects of this disclosure described herein using only conventional experiments. Furthermore, these equivalents are intended to be included in this disclosure.

[0010] This disclosure will be described in more detail below.

[0011] One approach provides a Corynebacterium microorganism for producing lysine, in which trehalase derived from Escherichia coli is introduced.

[0012] In this disclosure, trehalase can refer to an enzyme that degrades trehalose into two glucose molecules. Trehalase can be cytoplasmic trehalase (treF) and / or periplasmic trehalase (treA).

[0013] Trehalase, a cytoplasmic enzyme, is mainly distributed in the cytoplasm of microorganisms. It can degrade intracellular trehalose into glucose, which serves as an essential energy source for microbial metabolism.

[0014] Periplasmic trehalase is mainly distributed in the periplasm, which is the space between the cell membrane (inner membrane) and outer membrane of microorganisms. It can degrade extracellular trehalose to serve as an energy source for microorganisms.

[0015] The amino acid sequence of trehalase from E. coli or the nucleic acid sequence encoding the enzyme can be obtained from the known database NCBI.

[0016] In one implementation, *E. coli*-derived trehalase can refer to a protein derived from *E. coli* that has trehalase activity.

[0017] In one embodiment, the cytoplasmic trehalase derived from *E. coli* may comprise, have, consist of, or consist substantially of the following sequence: the amino acid sequence of SEQ ID NO: 1 or having at least 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 93.8%, 93.9%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97%. Amino acid sequences with sequence homology or sequence identity of 4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%, but not limited to these.

[0018] In one embodiment, the cytoplasmic trehalase derived from Escherichia coli may be an enzyme encoded by a gene containing the nucleic acid sequence of SEQ ID NO: 2.

[0019] In one embodiment, the Corynebacterium genus microorganism may be a microorganism further incorporating periplasmic trehalase derived from Escherichia coli.

[0020] In one implementation, periplasmic trehalase of Escherichia coli can refer to a protein of Escherichia coli that has periplasmic trehalase activity.

[0021] In one embodiment, the periplasmic trehalase derived from *E. coli* may comprise, have, consist of, or consist substantially of the following sequence: the amino acid sequence of SEQ ID NO: 3, or have periplasmic trehalase activity and have at least 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 85.5%, 86%, 86.5%, 87%, 87.5%, 88%, 88.5%, 89%, 89.5%, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.4% of the amino acid sequence of SEQ ID NO: 3. Amino acid sequences with sequence homology or identity of 96.5%, 97%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%, but not limited to these.

[0022] In one embodiment, the periplasmic trehalase derived from Escherichia coli may be encoded by a gene containing the nucleic acid sequence of SEQ ID NO: 4.

[0023] Furthermore, trehalases may also include variants of amino acid sequences with deletions, modifications, substitutions, conserved substitutions, or additions, provided that the variants possess such homology or identity and exhibit corresponding efficacy to trehalases (e.g., cytoplasmic trehalases and / or periplasmic trehalases). For example, this includes cases involving sequence additions or deletions that do not alter the N-terminus, C-terminus, and / or the trehalase activity within the amino acid sequence, or cases involving naturally occurring mutations, silent mutations, or conserved substitutions.

[0024] "Conservative substitution" refers to the replacement of one amino acid with another amino acid that has similar structure and / or chemical properties. Such amino acid substitutions can typically occur based on the similarity of residue polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphiphilicity. Generally, conservative substitutions have little or no effect on the activity of proteins or peptides.

[0025] In this disclosure, the statement that a polynucleotide or polypeptide “has a specific nucleic acid sequence (or nucleotide sequence) or amino acid sequence, contains a specific nucleic acid sequence (or nucleotide sequence) or amino acid sequence, is composed of a specific nucleic acid sequence (or nucleotide sequence) or amino acid sequence, or is substantially composed of a specific nucleic acid sequence (or nucleotide sequence) or amino acid sequence” can be interpreted to include “substantially equivalent sequences”, wherein mutations (deletions, substitutions, modifications and / or additions) are introduced into the specific nucleic acid sequence or amino acid sequence (or such mutations are not excluded) to the extent that the original function and / or desired function of the polynucleotide or polypeptide is maintained. In one embodiment, the expression "having a specific nucleic acid sequence or amino acid sequence, comprising a specific nucleic acid sequence or amino acid sequence, consisting of a specific nucleic acid sequence or amino acid sequence, or substantially consisting of a specific nucleic acid sequence or amino acid sequence" can refer to a polynucleotide or polypeptide (i) comprising a specific nucleic acid sequence or amino acid sequence, or (ii) consisting of a component having a specific nucleic acid sequence or amino acid sequence ... It consists of nucleic acid or amino acid sequences that are 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% homologous or identical, or substantially contains the above sequences, and retains the original and / or desired functions.

[0026] In this disclosure, "homology" or "identity" refers to the degree of similarity between two given amino acid sequences or nucleic acid sequences, expressed as a percentage. The terms homology and identity are often used interchangeably.

[0027] Sequence homology or identity of conserved polynucleotides or polypeptides is determined by standard alignment algorithms and can be used together with default gap penalties established by the program used. Essentially, homologous or identical sequences can generally hybridize with whole or partial sequences under moderately or highly stringent conditions. Clearly, hybridization also includes hybridization with polynucleotides containing universal codons or codons that take into account codon degeneracy in polynucleotides.

[0028] Whether any two polynucleotide or polypeptide sequences are homologous, similar, or identical can be determined using known computer algorithms, such as the “FASTA” program with default parameters, for example, as in Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444. Alternatively, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) can be used to determine this, as in the Needleman program of the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277) (version 5.0.0 or later) (including the GCG package (Devereux, J., et al, Nucleic Acids Research 12: 387 (1984)), BLASTP, BLASTN, FASTA (Atschul, [S.] [F.,] [ET AL, J MOLEC BIOL 215]: 403 (1990); Guideto Huge Computers, Martin J. Bishop, [ED.,] Academic Press, San Diego, 1994 and [CARILLO This is performed as described in ETA / .](1988) SIAM J Applied Math 48: 1073). For example, homology, similarity, or identity can be determined using BLAST or ClustalW from the National Center for Biotechnology Information.

[0029] Homology, similarity, or identity of polynucleotides or polypeptides can be determined by comparing sequence information using the GAP computer program, as known in: e.g., Smith and Waterman, Adv. Appl. Math (1981) 2:482, e.g., Needleman et al. (1970), J Mol Biol. 48:443. In summary, the value calculated by the GAP program can be defined as the number of similarly arranged symbols (i.e., nucleotides or amino acids) divided by the total number of symbols in the shorter sequence of the two sequences. The default parameters of the GAP procedure may include (1) a binary comparison matrix (containing a value of 1 for identity and a value of 0 for non-identity) and a weighted comparison matrix (or an EDNAFULL (EMBOSS version of NCBI NUC4.4) replacement matrix) as disclosed in Atlas of Protein Sequence And Structure, edited by Schwartz and Dayhoff, National Biomedical Research Foundation, pp. 353-358 (1979); (2) a penalty of 3.0 for each vacancy and an additional penalty of 0.10 for each symbol in each vacancy (or a penalty of 10 for vacancy opening and 0.5 for vacancy extension); and (3) no penalty for terminal vacancy.

[0030] In one embodiment, the Corynebacterium genus microorganism may be one or more selected from the group consisting of: Corynebacterium glutamicum, Corynebacterium lactis (… Corynebacterium crudilactis ), Corynebacterium desertis ( Corynebacterium deserti ), effective corynebacteria ( Corynebacterium efficiens ), Corynebacterium tumefaciens ( Corynebacterium callunae ), and stationary corynebacterium ( Corynebacterium stationis ), single rod-shaped bacteria ( Corynebacterium singular ), salt-tolerant Corynebacterium ( Corynebacterium halotolerans ), Corynebacterium bandingii ( Corynebacterium striatum ), contaminating Corynebacterium ( Corynebacterium polluted soil ), mimicking Corynebacterium ( Corynebacterium imitans ), Corynebacterium testis ( Corynebacterium testudinoris ) and Corynebacterium fulvidracosum ( Corynebacterium flavescens (but not limited to this).

[0031] Corynebacteria can include both wild-type microorganisms and naturally or artificially genetically modified microorganisms. Corynebacteria are microorganisms in which a specific mechanism is enhanced or weakened due to reasons such as the insertion of a foreign gene or the enhancement or weakening of endogenous gene activity, and can be recombinant microorganisms that include genetic modifications for the production of desired peptides, proteins, or products (e.g., lysine).

[0032] Corynebacterium microorganisms can be microorganisms that have been introduced with trehalases (cytoplasmic trehalase (treF)) and / or periplasmic trehalase (treA)) derived from Escherichia coli or the genes encoding them.

[0033] The introduction of a gene encoding trehalase from *E. coli* can be achieved by introducing a vector into a host cell in which the gene is operatively linked and can replicate and function independently of the host cell (Corynebacterium), or by introducing one or two or more copies into the chromosome of the host cell. Chromosomal introduction can be performed by introducing a vector capable of inserting polynucleotides into the chromosome into the host cell, but is not limited to this.

[0034] The gene encoding trehalase from *E. coli* can be inserted into a location that does not affect the expression of other genes in the host cell, such as a genomic safe harbor. In one embodiment, the genomic safe harbor may be the NCgl2284 gene region of *Corynebacterium glutamicum*, the region between the NCgl2195 and NCgl2196 genes of *Corynebacterium glutamicum*, or the region between the NCgl0866 and NCgl0867 genes of *Corynebacterium glutamicum*.

[0035] As used in this disclosure, the term "vector" refers to a DNA construct for delivering a target polynucleotide to a suitable host or host cell. The vector can be a recombinant vector or an expression vector. For example, it may contain a nucleotide sequence encoding a target polypeptide, operatively linked to a suitable expression control region (or expression control sequence) such that the target polypeptide can be expressed in a suitable host. The expression control region may include a promoter capable of initiating transcription, any operator sequence controlling such transcription, a sequence encoding a suitable mRNA ribosome binding site, and sequences controlling the termination of transcription and translation. After transformation into a suitable host cell, the vector can replicate or function independently of the host genome, or it may integrate into the genome itself. For example, the target polynucleotide may be inserted into a chromosome via a chromosome insertion vector. The polynucleotide can be inserted into a chromosome by any method known in the art, such as homologous recombination, but not limited to this. It may also contain a selection marker for confirming whether the polynucleotide has been inserted into the chromosome. The selection marker is used to select cells transformed with the vector, i.e., to confirm whether the target nucleic acid molecule has been inserted, and may use markers conferring selectable phenotypes such as drug resistance, auxotrophic phenotype, cytotoxic agent resistance, or surface polypeptide expression. In an environment treated with a selector, only cells expressing the selector marker survive or exhibit other phenotypic traits, thus allowing for the selection of transformed cells.

[0036] There are no particular limitations on the vectors used in this disclosure, and any vector known in the art may be used. Examples of commonly used vectors include natural or recombinant plasmids, granules, viruses, and bacteriophages. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A can be used as phage vectors or granule vectors, and pDZ-based, pDC-based, pBR-based, pUC-based, pBluescriptII-based, pGEM-based, pTZ-based, pCL-based, and pET-based vectors can be used as plasmid vectors. For example, pDZ, pDC, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, and pCC1BAC vectors may be used.

[0037] The introduction of vectors into microorganisms can be performed by known transformation methods appropriately selected by those skilled in the art. Transformation methods can be carried out by any method of introducing nucleic acids into host cells (microorganisms), and can be performed by appropriately selecting transformation techniques known in the art based on the host cell. Examples of known transformation methods include, but are not limited to, electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol-mediated uptake, DEAE-dextran method, cationic liposome method, liposome transfection, lithium acetate-DMSO method, and genome editing using engineered nucleases (e.g., CRISPR-Cas9).

[0038] Corynebacterium microorganisms incorporating trehalase from *E. coli* can be microorganisms in which trehalase activity is enhanced by operatively linking a strong promoter to the gene encoding trehalase. As mentioned above, operatively linking a strong promoter to the gene encoding trehalase may mean replacing (substituting) the original promoter of trehalase with a strong promoter, or positioning the strong promoter before the gene encoding trehalase so that the expression of the gene encoding trehalase can be controlled by the strong promoter. The original promoter of trehalase may refer to a promoter operatively linked to trehalase in the chromosome of a wild-type microorganism.

[0039] In this disclosure, the term "operably ligated" means that the promoter of this disclosure is functionally ligated to a target gene sequence (e.g., a gene encoding trehalase) to initiate and mediate the transcription of that gene. Operable ligations can be prepared using gene recombination techniques known in the art of this disclosure, and site-specific DNA cutting and ligation can be performed using cutting and ligating enzymes known in the art of this disclosure.

[0040] A strong promoter can be a promoter of an endogenous or exogenous gene from a microorganism. In one embodiment, the strong promoter can be derived from a Corynebacterium genus. In a specific embodiment, the strong promoter can be derived from Corynebacterium glutamicum.

[0041] In one implementation, the strong promoter may be CJ1 to CJ7 promoters (US Patent No. 7,662,943 B2), lac promoter, trp promoter, trc promoter, tac promoter, λ phage PR promoter, PL promoter, tet promoter, gapA promoter, SPL7 promoter, SPL13 (sm3) promoter (US Patent No. 10,584,338 B2), O2 promoter (US Patent No. 10,273,491 B2), tkt promoter, yccA promoter, etc., but is not limited to these.

[0042] The fact that trehalase activity is enhanced means that the activity of trehalase is increased compared to its endogenous activity. The term "enhanced" can be used interchangeably with terms such as "activated," "upregulated," "overexpressed," and "increased." In this context, "activated," "enhanced," "upregulated," "overexpressed," and "increased" can all include exhibiting activity not initially present, or exhibiting activity that is improved compared to endogenous activity or activity before modification. The term "endogenous activity" refers to the activity of a specific polypeptide that the parental strain or unmodified microorganism originally possessed before the trait change, when the trait is altered due to genetic mutation caused by natural or artificial factors. This can be used interchangeably with "activity before modification." The fact that the activity of a polypeptide is "enhanced," "upregulated," "overexpressed," or "increased" compared to endogenous activity means that it is improved compared to the activity and / or concentration (expression level) of the specific polypeptide that the parental strain or unmodified microorganism originally possessed before the trait change.

[0043] The enhancement can be achieved by introducing exogenous peptides or by enhancing the activity and / or increasing the concentration (expression level) of endogenous peptides. Whether the peptide activity is enhanced can be confirmed by an increase in the activity level, the expression level of the peptide, or the amount of product generated by the peptide activity.

[0044] The Corynebacterium spp. microorganisms to which trehalase from Escherichia coli is introduced can be microorganisms with lysine production capacity or with increased lysine production capacity (or yield). These microorganisms can be naturally deficient in lysine production, or they can be microorganisms that have been endowed or have their lysine production capacity conferred or enhanced by introducing or enhancing trehalase activity into lysine-producing microorganisms, but are not limited thereto.

[0045] The presence of enhanced lysine production capacity in the microorganism may mean that the microorganism has enhanced lysine production capacity compared to parental strains without E. coli-derived trehalase, unmodified microorganisms, pre-recombination cells, and / or wild-type strains, or is conferred lysine production capacity unlike parental strains without E. coli-derived trehalase, unmodified microorganisms without lysine production capacity, pre-recombination cells, and / or wild-type strains.

[0046] The microorganism can be one whose lysine production capacity is increased compared to a microorganism without the introduction of trehalase from *E. coli* (i.e., an unmodified microorganism of the same species). In this disclosure, "unmodified microorganism" does not exclude strains that may naturally occur in the microorganism, and can refer to the wild-type strain or the naturally occurring strain itself, or the strain before its traits were altered due to genetic mutations caused by natural or artificial factors. For example, according to one embodiment, an unmodified microorganism can refer to a strain before the introduction of trehalase. "Unmodified microorganism" can be used interchangeably with "pre-modified strain," "pre-modified microorganism," "non-mutant strain," "unmodified strain," "non-mutant microorganism," or "reference microorganism." In one embodiment, the unmodified microorganism used as the target strain for comparing whether lysine production capacity is increased may be a wild-type Corynebacterium glutamicum ATCC13032 strain, wherein one or more amino acid substitution mutations selected from the group consisting of pyc (P458S), hom (V59A) and lysC (T311I) have been introduced into the wild-type Corynebacterium glutamicum ATCC13032 strain, and / or Corynebacterium glutamicum CJ3P strain (US 9556463 ​​B2).

[0047] The microorganism may additionally contain mutations that increase lysine production, and there are no restrictions on the location of the mutation and / or the type of gene and / or protein targeted by the mutation, as long as the mutation increases lysine production. The use of the recombinant cell is unrestricted, provided it is a transformable cell.

[0048] In one embodiment, the lysine production capacity of the microorganism may be increased by about 5% or more, about 10% or more, about 11% 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 15% or more, about 16% or more, about 17% or more, about 18% or more, about 19% or more, about 20% or more, about 25% or more, about 30% or more, about 35% or more, about 40% or more, about 50% or more, about 100% or more, about 200% or more, about 300% or more, about 400% or more, about 500% or more, about 600% or more, about 700% or more, about 800% or more, about 900% or more, or about 1000% or more, but is not limited thereto (there is no particular upper limit, but it can be about 1000%).

[0049] In another embodiment, the microorganism may have a lysine production capacity of about 1.1 times or more, about 1.11 times or more, about 1.12 times or more, about 1.125 times or more, about 1.13 times or more, about 1.135 times or more, about 1.14 times or more, about 1.145 times or more, about 1.15 times or more, about 1.2 times or more, about 1.25 times or more, about 1.3 times or more, about 1.35 times or more, about 1.4 times or more, about 1.45 times or more, about 1.5 times or more, about 2 times or more, about 3 times or more, about 4 times or more, about 5 times or more, about 6 times or more, about 7 times or more, about 8 times or more, about 9 times or more, or about 10 times or more (the upper limit is not particularly limited, for example, it can be about 10 times) compared with the parental strain before mutation or the unmodified microorganism, but is not limited thereto.

[0050] In another embodiment, the lysine production capacity of the microorganism can be increased by about 0.5 g / L or more, about 0.6 g / L or more, about 0.7 g / L or more, about 0.75 g / L or more, about 0.8 g / L or more, about 0.85 g / L or more, about 0.9 g / L or more, about 1.0 g / L or more, about 1.11 g / L or more, about 1.12 g / L or more, about 1.13 g / L or more, about 1.14 g / L or more, about 1.15 g / L or more, about 1.2 g / L or more, about 1.5 g / L or more, about 2.0 g / L or more, about 2.5 g / L or more, about 3 g / L or more, about 3.5 g / L or more, about 4 g / L or more, about 4.5 g / L or more, about 5 g / L or more, about 5.5 g / L or more, about 6 g / L or more, about 7 g / L or more, compared with the parental strain before mutation and the unmodified microorganism. g / L or more, about 8 g / L or more, about 9 g / L or more, or about 10 g / L or more (there is no particular upper limit, for example it can be about 10 g / L), but is not limited thereto.

[0051] Compared to the parental strain before mutation and / or the unmodified microorganism, the microorganism may have an increased trehalose degradation capacity. The microorganism can improve lysine production capacity because it can utilize more glucose through efficient trehalose degradation.

[0052] In one embodiment, the microorganism may have an increased trehalose degradation capacity compared to the parental strain before mutation or the unmodified microorganism, wherein the capacity is increased by 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or within the range of 30 to 99%, 30 to 95%, 30 to 90%, 35 to 99%, 35 to 95%, 35 to 90%, 40 to 99%, 40 to 95%, 40 to 90%, 45 to 99%, 45 to 95%, 45 to 90%, 50 to 99%, 50 to 95%, 50 to 90%, 52.5 to 99%, 52.5 to 95%, 52.5 to 90%, 55 to 99%, 55 to 95%, or 55 to 90%, but is not limited thereto.

[0053] In another embodiment, the microorganism, compared with the parental strain before mutation or the unmodified microorganism, showed an increase in trehalose degradation capacity measured over the same time period of 50 mg / L or more, 60 mg / L or more, 70 mg / L or more, 80 mg / L or more, 90 mg / L or more, 100 mg / L or more, 110 mg / L or more, 120 mg / L or more, 130 mg / L or more, 60 to 200 mg / L, 60 to 190 mg / L, 60 to 180 mg / L, 60 to 170 mg / L, 60 to 160 mg / L, 60 to 150 mg / L, 60 to 140 mg / L, 65 to 200 mg / L, 65 to 190 mg / L, 65 to 180 mg / L, 65 to 170 mg / L, 65 to 160 mg / L, 65 to 150 mg / L, 65 to 140 mg / L, 70 to 200 mg / L. mg / L, 70 to 190 mg / L, 70 to 180 mg / L, 70 to 170 mg / L, 70 to 160 mg / L, 70 to 150 mg / L, 70 to 140 mg / L, 75 to 200 mg / L, 75 to 190 mg / L, 75 to 180 mg / L, 75 to 170 mg / L, 75 to 160 mg / L, 75 to 150 mg / L, or 75 to 140 mg / L, but not limited to these.

[0054] The term “approximately” includes all of the ranges of ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and includes, but is not limited to, all values ​​in the range that are equivalent to or similar to the value following the term “approximately”.

[0055] On the other hand, methods for producing lysine or increasing lysine production are provided, including culturing Corynebacterium microorganisms in a culture medium.

[0056] The method for producing lysine or increasing lysine yield disclosed herein may further include, for example, preparing microorganisms, preparing a culture medium for culturing microorganisms, or a combination thereof (in any order) prior to the culturing step.

[0057] Methods for producing lysine or increasing lysine production may include culturing microorganisms in a culture medium.

[0058] In this disclosure, "culture" can refer to the cultivation of microorganisms under appropriately controlled environmental conditions. The cultivation process of this disclosure can be carried out according to suitable culture media and cultivation conditions known in the art. Those skilled in the art can readily adapt and use such a cultivation process based on the selected strain. Specifically, cultivation can be batch, continuous, and / or fed-batch, but is not limited thereto.

[0059] In this disclosure, the term "culture medium" refers to a substance in which nutrients required for the cultivation of microorganisms are mixed as the main components, and which can provide nutrients and growth factors, including water necessary for survival and growth. Specifically, any culture medium and other culture conditions for culturing the microorganisms of this disclosure can be used without particular limitation, as long as it is a culture medium for culturing common microorganisms. However, the microorganisms of this disclosure can be cultured in a common culture medium containing suitable carbon sources, nitrogen sources, phosphorus sources, inorganic compounds, amino acids, and / or vitamins, under aerobic conditions, while controlling temperature, pH, etc. In one embodiment, the culture medium for Corynebacterium strains can be found in the literature [Manual of Methods for General Bacteriology, American Bacteriological Society (Washington, D.C., USA, 1981)].

[0060] Carbon sources can 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. In addition, natural organic nutrients such as starch hydrolysate, molasses, brown molasses, rice bran, cassava, bagasse, and corn steep liquor can be used. Specifically, carbohydrates such as glucose and sterilized pre-treated molasses (i.e., molasses converted to reducing sugars) can be used, and other appropriate amounts of carbon sources can be used in various ways without limitation. These carbon sources can be used alone or in combination of two or more, but are not limited to this.

[0061] Nitrogen sources can be selected from the group consisting of: 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 (including glutamic acid, methionine, and glutamine), peptone, NZ-amines, meat extracts, yeast extracts, malt extracts, corn steep liquor, casein hydrolysate, fish or its degradation products, and defatted soybean meal or its degradation products. These nitrogen sources can be used alone or in combination of two or more, but are not limited thereto.

[0062] Phosphorus sources may include potassium dihydrogen phosphate, dipotassium hydrogen phosphate, or corresponding sodium-containing salts. As inorganic compounds, sodium chloride, calcium chloride, ferric chloride, magnesium sulfate, ferric sulfate, manganese sulfate, calcium carbonate, etc., may be used. In addition, amino acids, vitamins, and / or suitable precursors may be included. These components or precursors may be added to the culture medium in batches or continuously. However, the method is not limited to these.

[0063] During microbial culture, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid can be added to the culture medium in an appropriate manner to adjust the pH of the culture medium.

[0064] In the cultivation of this disclosure, the pH of the culture medium can be 5 to 9, 5 to 8.5, 5 to 8, 5 to 7.5, 5 to 7, 5 to 6.5, 5 to 6, 5 to 5.5, 5.5 to 9, 5.5 to 8.5, 5.5 to 8, 5.5 to 7.5, 5.5 to 7, 5.5 to 6.5, 5.5 to 6, 6 to 9, 6 to 8.5, 6 to 8, 6 to 7.5, 6 to 7, 6 to 6.5, 6.5 to 9, 6.5 to 8.5, 6.5 to 8, 6.5 to 7.5, 6.5 to 7, 7 to 9, 7 to 8.5, 7 to 8, 7 to 7.5, 7.5 to 9, 7.5 to 8.5, 7.5 to 8, 8 to 9, 8 to 8.5, or 8.5 to 9, but is not limited thereto. In one embodiment, the pH of the culture medium can be 6 to 8, or 7, but is not limited thereto.

[0065] Furthermore, to maintain aerobic conditions in the culture medium, oxygen or oxygen-containing gas can be injected into the medium; or to maintain anaerobic and microaerobic conditions, gas injection can be omitted, or nitrogen, hydrogen, or carbon dioxide gas can be injected, but this is not limited to these methods. In one embodiment, the oxygen concentration in the culture medium can be maintained within a range where dissolved oxygen is not restricted during microbial culture, allowing the microorganisms to grow normally. In one embodiment, the oxygen concentration in the culture medium can be from 0.1 to 20 ppm, but is not limited to this. Air can be supplied to the culture medium in an appropriate amount and at an appropriate rate to maintain the oxygen concentration within this range. In one embodiment, air can be supplied to the culture medium at a rate of 0.1 to 2 vvm (air volume per minute / culture medium volume) to maintain the oxygen concentration within this range, but is not limited to this.

[0066] In the cultivation process disclosed herein, the cultivation temperature can be maintained between 20°C and 45°C, specifically between 25°C and 40°C, and the cultivation can be carried out for approximately 10 to 160 hours, but is not limited thereto.

[0067] Lysine produced through the culture of this disclosure can be secreted into the culture medium or retained in the cells. The method for producing lysine or increasing lysine yield of this disclosure may further include a step of recovering lysine from the culture medium (the medium in which the culture is performed) or from the microorganism (e.g., a strain of Corynebacterium). A recovery step may also be included after the culture step.

[0068] Recovery can be achieved by collecting the desired lysine using suitable methods known in the art, according to the microbial culture methods of this disclosure (e.g., batch, continuous, or fed-batch culture methods). For example, various methods such as centrifugation, filtration, crystallization, treatment with a protein precipitant (salting out), extraction, ultrasonic disruption, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, HPLC, or combinations of these methods can be used, and the desired lysine can be recovered from the culture medium or microorganisms using suitable methods known in the art.

[0069] Furthermore, the method for producing lysine or increasing lysine yield of this disclosure may additionally include a step of lysine purification. Purification can be performed using suitable methods known in the art. In one embodiment, when the method for producing lysine or increasing lysine yield of this disclosure includes both a recovery step and a purification step, the recovery step and the purification step may be performed continuously or discontinuously, regardless of the order, or may be performed simultaneously or integrated into a single step, but are not limited thereto.

[0070] On the other hand, it provides a composition for the production of lysine, the composition comprising a Corynebacterium genus microorganism, a culture medium in which the microorganism has been cultured, or a combination thereof.

[0071] The composition may also contain any suitable excipient commonly used in compositions for the production of lysine, and such excipient may be, for example, a preservative, wetting agent, dispersant, suspending agent, buffer, stabilizer or isotonic agent, but is not limited thereto.

[0072] On the other hand, it provides the use of Corynebacterium microorganisms for the production of lysine, for increasing its yield, or for the manufacture of compositions for the production of lysine.

[0073] This disclosure is summarized as follows: 1. A Corynebacterium microorganism that produces lysine, wherein a cytoplasmic trehalase (treF) derived from Escherichia coli is introduced.

[0074] 2. The Corynebacterium genus microorganism according to Project 1, wherein the cytoplasmic trehalase derived from Escherichia coli contains an amino acid sequence having at least 94% identity with the amino acid sequence of SEQ ID NO: 1.

[0075] 3. The Corynebacterium genus microorganism according to item 1 or 2, wherein the cytoplasmic trehalase derived from said Escherichia coli is encoded by a gene containing the nucleic acid sequence of SEQ ID NO: 2.

[0076] 4. A Corynebacterium microorganism according to any one of the preceding items, wherein a periplasmic trehalase (treA) derived from Escherichia coli is also introduced.

[0077] 5. The Corynebacterium genus microorganism according to Project 4, wherein the periplasmic trehalase derived from said Escherichia coli contains an amino acid sequence having at least 85% identity with the amino acid sequence of SEQ ID NO: 3.

[0078] 6. The Corynebacterium genus microorganism according to item 4 or 5, wherein the periplasmic trehalase derived from said Escherichia coli is encoded by a gene containing the nucleic acid sequence of SEQ ID NO: 4.

[0079] 7. A Corynebacterium genus microorganism according to any one of the preceding items, wherein the Corynebacterium genus microorganism is Corynebacterium glutamicum.

[0080] 8. A Corynebacterium microorganism according to any one of the preceding items, wherein the microorganism has increased lysine production capacity compared with a parent strain or wild-type microorganism without the introduction of cytoplasmic trehalase from Escherichia coli.

[0081] 9. A Corynebacterium microorganism according to any one of the preceding items, which has increased trehalose degradation capacity compared with a parent strain or wild-type microorganism without the introduction of cytoplasmic trehalase from Escherichia coli.

[0082] 10. A composition for producing lysine comprising a Corynebacterium microorganism according to any one of the preceding items.

[0083] 11. A method for producing lysine, comprising culturing a Corynebacterium microorganism according to any one of the preceding items in a culture medium.

[0084] 12. The method for producing lysine according to item 11 further includes recovering lysine from a culture medium produced by cultivation or from Corynebacterium microorganisms.

[0085] 13. A method for increasing lysine production, comprising culturing Corynebacterium microorganisms according to any one of the preceding items in a culture medium.

[0086] [Beneficial Effects] According to this disclosure, the microorganisms that introduce trehalase have excellent lysine production capacity and reduce by-products, and therefore can be effectively used for the large-scale production of lysine. Detailed Implementation

[0087] The present disclosure will be described in more detail below through the following embodiments. However, these are for illustrative purposes only, and the scope of the present disclosure is not limited to these embodiments.

[0088] Example 1. Preparation of plasmids for gene insertion To insert the *E. coli*-derived *treF* and *treA* genes into the chromosome of *Corynebacterium glutamicum*, the gene NCgl2284, known to encode transposons, was used as the insertion site. To replace the NCgl2284 gene with *E. coli* *treF* and / or *treA*, NCgl2284 deletion and target gene insertion vectors were prepared. For vector preparation, the chromosome of *Corynebacterium glutamicum* ATCC13032 was used as a template, and PCR was performed using primer pairs SEQ ID NO: 5 and SEQ ID NO: 6, and SEQ ID NO: 7 and SEQ ID NO: 8, respectively. The primer sequences used for each PCR are shown in Table 1 below.

[0089] Solg™ Pfu-X DNA polymerase (Solgent) was used as the polymerase for the PCR reaction. The PCR conditions were as follows: denaturation at 95°C for 30 seconds, denaturation at 55°C for 30 seconds, and polymerization at 68°C for 1 minute; these denaturation, annealing, and polymerization reactions were repeated 28 times. As a result, a 500 bp DNA fragment was obtained. The obtained DNA product was purified using a PCR purification kit (QIAGEN), and the purified amplified product was cloned into the pDC24 vector (Korean Patent Publication No. KR 10-2024-0167588 A) using an In-Fusion cloning kit (TaKaRa) according to the manufacturer's instructions. This vector had been treated with the restriction enzyme SmaI at 37°C for 1 hour, followed by heat treatment at 65°C for 20 minutes, thus preparing the vector pDC24_△NCgl2284 for NCgl2284 deletion and target gene insertion.

[0090] Example 2. Preparation of recombinant plasmids for introducing treF and treA from Escherichia coli. To introduce the treF and treA genes from Escherichia coli, plasmids for introducing the treF and treA genes from E. coli were prepared using the gapA promoter (PgapA) of Corynebacterium glutamicum.

[0091] Specifically, the chromosome of *Corynebacterium glutamicum* ATCC13032 containing the PgapA nucleic acid sequence was used as a template, and the PgapA promoter fragment was obtained by PCR using primers SEQ ID NO: 9 and SEQ ID NO: 10. The treF fragment (protein sequence: SEQ ID NO: 1; gene sequence: SEQ ID NO: 2) was obtained by PCR using the chromosome of *Escherichia coli* (*E. coli* strain K-12 substrain MG1655) (GenBank: CP032667.1) as a template, and primers SEQ ID NO: 11 and SEQ ID NO: 12. Furthermore, the treA fragment (protein sequence: SEQ ID NO: 3; gene sequence: SEQ ID NO: 4) was obtained by PCR using primers SEQ ID NO: 13 and SEQ ID NO: 14. PfuUltra TM High-fidelity DNA polymerase (Stratagene) was used as the polymerase for the PCR reaction. The PCR conditions were: denaturation at 95°C for 30 seconds, denaturation at 55°C for 30 seconds, and polymerization at 68°C for 3 minutes. The denaturation, annealing, and polymerization reactions were repeated 28 times. As a result, a 488 bp DNA fragment from the PgapA promoter region, a 1650 bp DNA fragment from the *E. coli* treF region, and a 1698 bp DNA fragment from the treA region were obtained.

[0092] Using the In-Fusion Cloning Kit (TaKaRa), each obtained PCR fragment was cloned using the pDC24_△NCgl2284 vector according to the provided manual. This vector had been treated with restriction enzyme SmaI at 37°C for 1 hour and then heat-treated at 65°C for 20 minutes to prepare two types of vectors: pDC24_△NCgl2284::PgapA_treF(E.co) and pDC24_△NCgl2284::PgapA_treA(E.co).

[0093] To simultaneously express *E. coli*-derived treF and treA, the *PgapA_treF*(*E.co*) fragment was obtained by PCR using SEQ ID NO: 9 and SEQ ID NO: 15, with the previously prepared pDC24_△NCgl2284::PgapA_treF(*E.co*) as a template. Similarly, the *PgapA_treA*(*E.co*) fragment was obtained by PCR using SEQ ID NO: 14 and SEQ ID NO: 16, with pDC24_△NCgl2284::PgapA_treA(*E.co*) as a template. The amplification products were purified using a PCR purification kit (QIAGEN) and used as the insert DNA fragment in vector construction. Each obtained PCR fragment was cloned using the In-Fusion Cloning Kit (TaKaRa) according to the provided manual, using the pDC24_△NCgl2284 vector, which had been treated with restriction enzyme SmaI at 37°C for 1 hour and then heat-treated at 65°C for 20 minutes to prepare pDC24_△NCgl2284::PgapA_treF-PgapA(E.co)_treA(E.co). The primer sequences used for each PCR and the treF and treA sequences from *E. coli* are shown in Table 1 below.

[0094] [Table 1] Example 3. Construction of strains infused with Escherichia coli treF and treA and evaluation of their lysine production capacity. The three types of vectors (pDC24_△NCgl2284::PgapA_treF(E.co), pDC24_△NCgl2284::PgapA_treA(E.co), pDC24_△NCgl2284::PgapA_treA(E.co), pDC24_△NCgl2284::PgapA_treF-PgapA(E.co)_treA(E.co)) prepared in Example 2 were transformed into the lysine-producing strain Corynebacterium glutamicum CJ3P (US 9556463 ​​B2) using electroporation via the electrical pulse method (Vander Rest et al., Appl. Microbiol. Biotecnol. 52:541-545, 1999). Each strain was named CJ3P_treF(E.co), CJ3P_treA(E.co), and CJ3P_treF-treA(E.co).

[0095] To confirm the L-lysine production capacity and trehalose degradation capacity of the prepared strain and the parental strain used as a control group, a shake-flask fermentation titer evaluation was conducted.

[0096] First, each strain was inoculated into a 250 ml baffled Erlenmeyer flask containing 25 ml of seed culture medium and cultured at 37°C and 200 rpm with shaking for 20 hours. Then, 1 ml of seed culture was inoculated into a 250 ml baffled Erlenmeyer flask containing 24 ml of production culture medium and cultured at 37°C and 200 rpm with shaking for 40 hours.

[0097] Parental strains CJ3P, CJ3P_treF(E.co), CJ3P_treA(E.co), and CJ3P_treF-treA(E.co) were cultured in flasks using production medium, with treF and treA strains derived from E. coli introduced. Lysine and trehalose concentrations were measured. The experiment was repeated three times, and the average values ​​of the results are shown in Table 2 below.

[0098] Seed Culture Medium 20 g crude sugar, 10 g peptone, 5 g yeast extract, 1.5 g urea, 4 g KH2PO4, 8 g K2HPO4, 0.5 g MgSO4 · 7H2O, 0.1 mg biotin, 1 mg thiamine hydrochloride, 2 mg calcium pantothenate and 2 mg nicotinamide (based on 1 L of distilled water) <Production Culture Medium> 100 g crude sugar, 40 g (NH4)2SO4, 2.5 g soybean protein, 5 g corn steep liquor solids, 3 g urea, 1 g KH2PO4, 0.5 g MgSO4 4· 7H2O, 100 μg biotin, 1000 μg thiamine hydrochloride, 2000 μg calcium pantothenate, 3000 μg nicotinamide and 30 g CaCO3 (based on 1 L of distilled water).

[0099] [Table 2] As shown in Table 2 above, compared with the parent strain, the lysine concentrations of the CJ3P_treF(E.co) strain introduced with *E. coli*-derived *treF* and the CJ3P_treA(E.co) strain introduced with *E. coli*-derived *treA* increased by approximately 14.5% and 12.1%, respectively. Furthermore, the introduction of these genes reduced the trehalose accumulation in the culture medium by 55.6% and 48.2%, respectively. In addition, for the CJ3P_treF-treA(E.co) strain introduced with both genes, the lysine concentration increased by approximately 20.3% compared with the parent strain, and the trehalose accumulation in the culture medium was reduced by as much as 97.7%. Based on these results, it is confirmed that the introduction of *E. coli*-derived *treF* not only increased the lysine concentration but also reduced the trehalose accumulation in the culture medium. Moreover, it is confirmed that the simultaneous introduction of *E. coli*-derived *treF* and *treA* is more effective in reducing trehalose, and it is believed that the reduction of trehalose in the culture medium contributes to improving fermentation purity.

[0100] Based on the above description, those skilled in the art to which this disclosure pertains will understand that this disclosure can be implemented in other specific forms without altering its technical spirit or essential characteristics. In this regard, it should be understood that the above embodiments are illustrative in all respects and not restrictive. The scope of this disclosure should be interpreted to include all variations or modifications derived from the meaning and scope of the appended claims and their equivalents, rather than the specific embodiments described above.

Claims

1. A Corynebacterium microorganism that produces lysine, wherein a cytoplasmic trehalase (treF) derived from Escherichia coli is introduced.

2. The Corynebacterium genus microorganism according to claim 1, wherein the cytoplasmic trehalase derived from Escherichia coli comprises an amino acid sequence having at least 94% identity with the amino acid sequence of SEQ ID NO:

1.

3. The Corynebacterium genus microorganism according to claim 1, wherein the cytoplasmic trehalase derived from Escherichia coli is encoded by a gene containing the nucleic acid sequence of SEQ ID NO:

2.

4. The Corynebacterium genus microorganism according to claim 1, wherein a periplasmic trehalase (treA) derived from Escherichia coli is further introduced.

5. The Corynebacterium genus microorganism according to claim 4, wherein the periplasmic trehalase derived from Escherichia coli comprises an amino acid sequence having at least 85% identity with the amino acid sequence of SEQ ID NO:

3.

6. The Corynebacterium microorganism according to claim 4, wherein the periplasmic trehalase derived from Escherichia coli is encoded by a gene containing the nucleic acid sequence of SEQ ID NO:

4.

7. The Corynebacterium genus microorganism according to claim 1, wherein the Corynebacterium genus microorganism is Corynebacterium glutamicum.

8. The Corynebacterium microorganism according to claim 1, wherein the microorganism has increased lysine production capacity compared with a parent strain or wild-type microorganism without the introduction of cytoplasmic trehalase from Escherichia coli.

9. The Corynebacterium microorganism according to claim 1, wherein the microorganism has increased trehalose degradation capacity compared with a parent strain or wild-type microorganism without the introduction of cytoplasmic trehalase from Escherichia coli.

10. A composition for producing lysine, comprising a Corynebacterium microorganism according to any one of claims 1 to 9.

11. A method for producing lysine, comprising culturing a Corynebacterium microorganism according to any one of claims 1 to 9 in a culture medium.

12. The method for producing lysine according to claim 11, further comprising recovering lysine from a culture medium produced by cultivation or from Corynebacterium spp. microorganisms.

13. A method for increasing lysine production, comprising culturing a Corynebacterium microorganism according to any one of claims 1 to 9 in a culture medium.

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