Microorganism having trehalase introduced thereinto and uses thereof

Abstract

The present disclosure provides a microorganism having trehalase introduced thereinto and uses thereof.

Description

Microorganisms into which trehalose degrading enzymes have been introduced and their uses

[0001] The present disclosure relates to a microorganism into which a trehalose-degrading enzyme has been introduced and the use thereof.

[0002] Cross-citation with related application(s)

[0003] This application claims the benefit of priority based on Korean Patent Application No. 10-2025-0025095 dated February 26, 2025, and all contents disclosed in the document of said Korean patent application are incorporated herein as part of this specification.

[0004]

[0005] L-lysine is an essential amino acid vital for the physiological functions of humans and animals. Particularly in the feed and food industries, lysine serves as a critical component of protein synthesis. Lysine is primarily produced through fermentation processes utilizing microorganisms. Since lysine productivity depends heavily on the carbon metabolic capacity of microorganisms, the efficient use of energy sources is a critical factor in productivity. Therefore, research is necessary to effectively increase lysine production capacity.

[0006]

[0007] [Prior Art Literature]

[0008] (Patent Document 1) U.S. Published Patent Application (US 2011-0008864 A1)

[0009]

[0010] One object of the present disclosure is to provide a lysine-producing microorganism of the genus Corynebacterium into which a trehalose-degrading enzyme derived from Escherichia coli has been introduced.

[0011] Another object of the present disclosure is to provide a composition for producing lysine comprising the microorganism of the genus Corynebacterium.

[0012] Another object of the present disclosure is to provide a method for producing lysine, comprising the step of culturing the microorganism of the genus Corynebacterium in a medium.

[0013] Another object of the present disclosure is to provide a method for increasing lysine production, comprising the step of culturing the microorganism of the genus Corynebacterium in a medium.

[0014]

[0015] This is explained in detail as follows. Meanwhile, each description and embodiment disclosed in this disclosure may be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in this disclosure fall within the scope of this disclosure. Furthermore, the scope of this disclosure is not to be considered limited by the specific descriptions provided below. In addition, numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated by reference into this specification in their entirety to more clearly explain the level of the art to which the present invention pertains and the content of the present invention. Furthermore, a person skilled in the art can recognize or identify numerous equivalents for the specific embodiments of this disclosure described herein using only ordinary experiments. Moreover, such equivalents are intended to be included in this disclosure.

[0016]

[0017] The present disclosure is described in more detail below.

[0018]

[0019] One aspect provides a lysine-producing microorganism of the genus Corynebacterium into which an E. coli-derived trehalose degrading enzyme has been introduced.

[0020] In the present disclosure, trehalase may refer to an enzyme that breaks down trehalose into two molecules of glucose. The trehalase may be a cytoplasmic trehalase (treF) and / or a periplasmic trehalase (treA).

[0021] The above cytoplasmic trehalose degrading enzyme is mainly distributed in the cytoplasm of microorganisms and can break down intracellular trehalose into glucose to supply it as an energy source necessary for the metabolism of microorganisms.

[0022] The above-mentioned periplasmic trehalose degrading enzyme is mainly distributed in the periplasm, which is the space between the inner membrane and the outer membrane of microorganisms, and can degrade extracellular trehalose to be used as an energy source for microorganisms.

[0023] The amino acid sequence of the above-mentioned trehalose degrading enzyme derived from E. coli or the nucleic acid sequence encoding it can be obtained from the known database NCBI.

[0024] In one example, the above E. coli-derived cytoplasmic trehalase may refer to a protein derived from E. coli that has cytoplasmic trehalase activity.

[0025] In one example, the cytoplasmic trehalose degrading enzyme derived from E. coli comprises the amino acid sequence of SEQ ID NO. 1 or the amino acid sequence of SEQ ID NO. 1 and 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%, and 96% It includes, has, is composed of, or is of the said sequence, an amino acid sequence having sequence homology or sequence identity of 96.5% or more, 97% or more, 97.4% or more, 97.5% or more, 97.6% or more, 97.7% or more, 97.8% or more, 97.9% or more, 98% or more, 98.1% or more, 98.2% or more, 98.3% or more, 98.4% or more, 98.5% or more, 98.6% or more, 98.7% or more, 98.8% or more, 98.9% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more. It may be necessary, but is not limited to this.

[0026] In one example, the cytoplasmic trehalose derived from E. coli may be encoded by a gene containing the nucleic acid sequence of SEQ ID NO. 2.

[0027] In one example, the above-mentioned Corynebacterium microorganism may have additionally introduced a cytoplasmic trehalose degrading enzyme derived from E. coli.

[0028] In one example, the above-mentioned E. coli-derived peripheral trehalose degrading enzyme may refer to a protein derived from E. coli that has peripheral trehalose degrading enzyme activity.

[0029] In one example, the E. coli-derived peripheral trehalase has the amino acid sequence of SEQ ID NO. 3 or peripheral trehalase activity, and has 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% or more, 94% or more, 94.5% or more, 95% or more, 95.5% or more, 96% or more, 96.4% or more, 96.5% or more, 97% or more, 97.5% or more, 97.6% or more, 97.7% or more, 97.8% or more, 97.9% or more, 98% or more, 98.1% or more, 98.2% or more, 98.3% or more, 98.4% or more, 98.5% or more, 98.6% or more, 98.7% or more, 98.8% or more, 98.9% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, It may include, have, be composed of, or essentially be composed of an amino acid sequence having 99.8% or more or 99.9% or more of sequence homology or sequence identity, but is not limited thereto.

[0030] In one example, the above-mentioned cytoplasmic trehalose degrading enzyme derived from E. coli may be encoded by a gene containing the nucleic acid sequence of SEQ ID NO. 4.

[0031] Additionally, if the amino acid sequence has such homology or identity and exhibits efficacy corresponding to a trehalase (e.g., cytoplasmic trehalase and / or peripheral cytoplasmic trehalase), variants having amino acid sequences in which some sequences are deleted, modified, substituted, conservatively substituted, or added may also be included in the said trehalase. For example, this includes cases where there are sequence additions or deletions, naturally occurring mutations, silent mutations, or conservatively substituted sequences that do not alter trehalase activity at the N-terminus, C-terminus, and / or within the said amino acid sequence.

[0032] The aforementioned “conservative substitution” refers to the substitution of one amino acid with another amino acid having similar structural and / or chemical properties. Such amino acid substitutions can generally occur based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues. Typically, conservative substitutions have little to no effect on the activity of a protein or polypeptide.

[0033] In the present disclosure, the phrase “a polynucleotide or polypeptide has, comprises, consists of, or is essentially composed of a specific nucleic acid sequence (or base sequence) or amino acid sequence” may be interpreted as comprising (or not excluding) a “substantially equivalent sequence” in which a variation (deletion, substitution, modification, and / or addition) has been applied to the specific nucleic acid sequence or amino acid sequence to maintain the original function and / or intended function of the polynucleotide or polypeptide. In one example, the phrase “a polynucleotide or polypeptide has, includes, is composed of, or is essentially composed of a specific nucleic acid sequence or amino acid sequence” means that the polynucleotide or polypeptide has (i) the specific nucleic acid sequence or amino acid sequence, or (ii) 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%, 94% or more, 94.5% or more, 95% or more, 95.5% or more, 96% or more, 96.5% or more, 97% or more, 97.5% or more, 97.6% or more, 97.7% or more, 97.8% or more, 97.9% or more, 98% or more, 98.1% or more, 98.2% or more, 98.3% or more, 98.4% or more, 98.5% or more, 98.6% or more, 98.7% or more, 98.8% or more, 98.9% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.It may mean that it consists of a nucleic acid sequence or amino acid sequence having 6% or more, 99.7% or more, 99.8% or more, or 99.9% or more homology or identity, or essentially includes such a sequence and maintains its original function and / or intended function.

[0034] In this disclosure, "homology" or "identity" refers to the degree of similarity between two given amino acid sequences or nucleic acid sequences and may be expressed as a percentage. The terms homology and identity may often be used interchangeably.

[0035] The sequence homology or identity of conserved polynucleotides or polypeptides is determined by standard arrangement algorithms, and a default gap penalty established by the program used may be utilized. Substantially, homologous or identical sequences can generally be hybridized with the entire sequence or a part thereof under moderately or highly stringent conditions. It is evident that hybridization also includes hybridization with polynucleotides containing common codons or codons that account for codon degeneracy.

[0036] Whether any two polynucleotide or polypeptide sequences have homology, similarity, or identity can be determined using a known computer algorithm, such as the “FASTA” program, using default parameters as in, for example, Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444. Alternatively, it can be determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453), as performed in the Needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277) (version 5.0.0 or later) (GCG program package (Devereux, J., et al, Nucleic Acids Research 12: 387 (1984)), BLASTP, BLASTN, FASTA (Atschul, [S.] [F.,] [ET AL, J MOLEC BIOL 215]: 403 (1990); Guide to Huge Computers, Martin J. Bishop, [ED.,] (Including Academic Press, San Diego, 1994, and [CARILLO ETA / .](1988) SIAM J Applied Math 48: 1073). For example, homology, similarity, or identity can be determined using BLAST from the National Biotechnology Information Database Center or ClustalW.

[0037] The homology, similarity, or identity of polynucleotides or polypeptides can be determined by comparing sequence information using a GAP computer program, such as that described in, for example, Smith and Waterman, Adv. Appl. Math (1981) 2:482, or Needleman et al. (1970), J Mol Biol. 48:443. In summary, a GAP program can be defined as the total number of symbols in the shorter of the two sequences divided by the number of similarly arranged symbols (i.e., nucleotides or amino acids). The default parameters for a GAP program are (1) a binary comparison matrix (containing values ​​of 1 for identity and 0 for non-identity) and, as disclosed by Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353-358 (1979), or Gribskov et al. (1986) Nucl. Acids Res. 14: A weighted comparison matrix of 6745 (or an EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix); (2) a penalty of 3.0 for each gap and an additional penalty of 0.10 for each symbol in each gap (or a gap opening penalty of 10, a gap extension penalty of 0.5); and (3) no penalty for terminal gaps.

[0038] In one example, microorganisms of the genus Corynebacterium include Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium stationis, Corynebacterium singulare, Corynebacterium halotolerans, Corynebacterium striatum, Corynebacterium pollutisoli, and Corynebacterium imitans It may be one or more species selected from the group consisting of (Corynebacterium imitans), Corynebacterium testudinoris, and Corynebacterium flavescens, but is not limited thereto.

[0039] The above-mentioned microorganisms of the genus Corynebacterium may include both wild-type microorganisms and microorganisms that have undergone natural or artificial genetic modification. The above-mentioned microorganisms of the genus Corynebacterium may be recombinant microorganisms that include genetic modification for the production of a desired polypeptide, protein, or product (e.g., lysine), as they are microorganisms in which a specific mechanism is strengthened or weakened due to causes such as the insertion of an external gene or the strengthening or weakening of the activity of an endogenous gene.

[0040] The above-mentioned microorganisms of the genus Corynebacterium may be those into which trehalase derived from E. coli (cytoplasmic trehalase (treF) and / or periplasmic trehalase (treA)) or genes encoding it have been introduced.

[0041] The gene encoding the trehalose degrading enzyme derived from E. coli may be achieved by introducing a vector into a host cell that can replicate and function independently of the host cell (a microorganism of the genus Corynebacterium) to which the gene is functionally connected, or by introducing one or more copies into the chromosomes of the host cell. The introduction into the chromosomes may be performed by introducing a vector capable of inserting the polynucleotide into the chromosomes of the host cell into the host cell, but is not limited thereto.

[0042] The gene encoding the trehalose degrading enzyme derived from E. coli may be inserted into a location that does not affect the expression of other genes in the host cell, such as within genomic safe harbors. In one example, the genomic safe harbor may be the region between the NCgl2284 gene region of Corynebacterium glutamicum, the NCgl2195 gene and the NCgl2196 gene of Corynebacterium glutamicum, or the region between the NCgl0866 gene and the NCgl0867 gene of Corynebacterium glutamicum.

[0043] As used in this disclosure, the term "vector" refers to a DNA product for delivering a target polynucleotide into a suitable host or host cell. The vector may be a recombinant vector or an expression vector. For example, it may comprise a base sequence of a polynucleotide encoding the target polypeptide operably linked to a suitable expression control region (or expression control sequence) to enable the expression of the target polypeptide within a suitable host. The expression control region may comprise a promoter capable of initiating transcription, any operator sequence for regulating such transcription, a sequence coding for a suitable mRNA ribosome binding site, and a sequence regulating the termination of transcription and translation. After being transformed into a suitable host cell, the vector may replicate or function independently of the host genome and may be incorporated into the genome itself. For example, the target polynucleotide may be inserted into a chromosome via a chromosomal insertion vector. The insertion of the above polynucleotide into the chromosome may be achieved by any method known in the art, for example, homologous recombination, but is not limited thereto. A selection marker may be additionally included to confirm whether the chromosome insertion has occurred. The selection marker is intended to select cells transformed with the vector, that is, to confirm whether the target nucleic acid molecule has been inserted, and markers conferring selectable phenotypes such as drug resistance, nutritional requirements, resistance to cytotoxic agents, or expression of surface polypeptides may be used. Since only cells expressing the selection marker survive or exhibit other phenotypes in an environment treated with a selective agent, the transformed cells can be selected.

[0044] The vectors used in this disclosure are not particularly limited, and any vector known in the art may be used. Examples of commonly used vectors include plasmids, cosmids, viruses, and bacteriophages in their natural or recombinant state. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A may be used as phage vectors or cosmid vectors, and pDZ-based, pDC-based, pBR-based, pUC-based, pBluescriptII-based, pGEM-based, pTZ-based, pCL-based, and pET-based vectors may be used as plasmid vectors. As an example, pDZ, pDC, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vectors may be used.

[0045] The introduction of the above vector into a microorganism can be carried out by a person skilled in the art by appropriately selecting a known transformation method. The above transformation method can be carried out by any method of introducing nucleic acid into a host cell (microorganism), and depending on the host cell, a transformation technique known in the art can be appropriately selected. Examples of the above 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, lipofection, lithium acetate-DMSO method, and genome editing using engineered nucleases (e.g., CRISPR-Cas9).

[0046] Microorganisms of the genus Corynebacterium into which the trehalase derived from E. coli has been introduced may have enhanced activity of the trehalase by operably linking a strong promoter to the gene encoding the trehalase. As described above, the operably linking of a strong promoter to the gene encoding the trehalase may mean replacing (substituting) the original promoter of the trehalase with a strong promoter, or positioning the strong promoter at the front of the gene encoding the trehalase so that the expression of the gene encoding the trehalase can be regulated by the strong promoter. The original promoter of the trehalase may refer to a promoter operably linked to the trehalase within the chromosome of the wild-type microorganism.

[0047] In this disclosure, the term “operatively linked” means that the promoter of this disclosure is functionally linked to the gene sequence to initiate and mediate the transcription of a target gene (e.g., a gene encoding trehalase). An operatively linked link may be produced using gene recombination technology known in the art of this disclosure, and site-specific DNA cleavage and linkage may be produced using cleavage and linkage enzymes known in the art of this disclosure.

[0048] The strong promoter may be a promoter of an endogenous gene or an exogenous gene. In one example, the strong promoter may be of a microorganism of the genus Corynebacterium. In one embodiment, the strong promoter may be of Corynebacterium glutamicum.

[0049] In one example, the strong promoter may be, but is not limited to, CJ1 to CJ7 promoters (US Patent No. 7662943 B2), lac promoter, trp promoter, trc promoter, tac promoter, lambda phage PR promoter, PL promoter, tet promoter, gapA promoter, SPL7 promoter, SPL13 (sm3) promoter (US Patent No. 10584338 B2), O2 promoter (US Patent No. 10273491 B2), tkt promoter, yccA promoter, etc.

[0050] The statement that the activity of the trehalose degrading enzyme has been enhanced means that the activity of the trehalose degrading enzyme has increased compared to its intrinsic activity. The enhancement mentioned above may be used interchangeably with terms such as activation, up-regulation, overexpression, and increase. Here, activation, enhancement, up-regulation, overexpression, and increase may include exhibiting activity that was not originally possessed, or exhibiting improved activity compared to the intrinsic activity or activity prior to modification. The "intrinsic activity" mentioned above refers to the activity of a specific polypeptide originally possessed by the parent strain or the non-modified microorganism prior to the change in traits caused by genetic mutations due to natural or artificial factors. This may be used interchangeably with "activity prior to modification." "Enhancement," "upregulation," "overexpression," or "increase" of polypeptide activity relative to intrinsic activity means that the activity and / or concentration (expression amount) of a specific polypeptide was originally possessed by the parent strain or non-modified microorganism prior to transformation.

[0051] The above enhancement can be achieved by introducing an exogenous polypeptide or by enhancing the activity and / or increasing the concentration (expression amount) of the intrinsic polypeptide. Whether the activity of the polypeptide is enhanced can be confirmed by an increase in the degree of activity, expression amount, or amount of product resulting from the polypeptide activity of said polypeptide.

[0052]

[0053] Microorganisms of the genus Corynebacterium into which the above-mentioned trehalose-degrading enzyme derived from E. coli has been introduced may be microorganisms that have lysine production capabilities or have increased lysine production capabilities (or production volume). The above-mentioned microorganisms may be microorganisms that naturally lack lysine production capabilities, or microorganisms that have lysine production capabilities but have lysine production capabilities conferred or enhanced by the introduction or enhancement of trehalose-degrading enzyme activity, but are not limited thereto.

[0054] The statement that the above microorganism has improved lysine production capacity or possesses lysine production capacity may mean that the above microorganism has improved lysine production capacity compared to the parent strain into which E. coli-derived trehalose-degrading enzyme has not been introduced, the non-modified microorganism, the cell before recombination, and / or the wild-type strain, or that it is endowed with lysine production capacity unlike the parent strain into which E. coli-derived trehalose-degrading enzyme has not been introduced, the non-modified microorganism without lysine production capacity, the cell before recombination, and / or the wild-type strain.

[0055] The above microorganism may have an improved (increased) lysine production capacity compared to a microorganism to which trehalose-degrading enzyme derived from E. coli has not been introduced, i.e., a non-modified microorganism of the same species. In this application, "non-modified microorganism" does not exclude strains containing mutations that may naturally occur in microorganisms, and may refer to a wild-type strain or a natural-type strain itself, or a strain before its traits are changed by genetic variation caused by natural or artificial factors. For example, the above-mentioned non-modified microorganism may refer to a strain before the introduction of trehalose-degrading enzyme, depending on one example. The above-mentioned "non-modified microorganism" may be used interchangeably with "pre-modification strain," "pre-modification microorganism," "non-mutated strain," "non-modified strain," "non-mutated microorganism," or "reference microorganism." In one example, the non-modified microorganism, which is the target strain for comparing whether the lysine production capacity is increased, may be a wild-type Corynebacterium glutamicum ATCC13032 strain, a strain in which one or more amino acid substitution variants selected from the group consisting of pyc (P458S), hom (V59A), and lysC (T311I) are introduced into the wild-type Corynebacterium glutamicum ATCC13032 strain, and / or a Corynebacterium glutamicum CJ3P strain (US 9556463 ​​B2), etc.

[0056] The microorganism may additionally include a mutation that increases lysine production, and the location of the mutation and / or the type of gene and / or protein subject to the mutation may be included without limitation as long as it increases lysine production. The recombinant cell may be used without limitation as long as it is a cell capable of transformation.

[0057] For example, compared to the parent strain before mutation and the non-mutated microorganism, 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 1,000% or more. However, it is not limited to this (there is no specific upper limit, but it may be approximately 1000%).

[0058] In another example, compared to the parent strain before mutation and the non-mutated microorganism, the lysine production capacity of the microorganism may be 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, about 10 times or more (the upper limit is not specifically restricted and may be, for example, about 10 times). However, it is not limited to this.

[0059] In another example, compared to the parent strain before mutation and the non-mutated microorganism, the microorganism has a lysine production capacity of 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, and about 5 g / L. The upper limit may be, but is not limited to, about 5.5 g / L or more, about 6 g / L or more, about 7 g / L or more, about 8 g / L or more, about 9 g / L or more, about 10 g / L or more (there is no special restriction on the upper limit, for example, it may be about 10 g / L).

[0060] The microorganism may have increased trehalose degradation ability compared to the parent strain before mutation and / or the non-mutated microorganism. Since the microorganism can decompose trehalose with high efficiency and utilize more glucose, it can improve lysine production capacity.

[0061] For example, compared to the parent strain before mutation and the non-mutated microorganism, the above microorganism has a trehalose degradation ability of 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, 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%, It may be increased by 52.5 to 95%, 52.5 to 90%, 55 to 99%, 55 to 95%, or 55 to 90%, but is not limited thereto.

[0062] In another example, compared to the parent strain before mutation and the non-mutated microorganism, the trehalose degradation ability measured over the same period of time of the microorganism is 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 It may be increased to 160 mg / L, 65 to 150 mg / L, 65 to 140 mg / L, 70 to 200 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, and 75 to 140 mg / L, but is not limited thereto.

[0063] The above term “about” refers to a range that includes ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and includes, but is not limited to, all numerical values ​​within a range equivalent to or similar to the numerical value following the term “about.”

[0064]

[0065] Another aspect provides a method for producing lysine (or a method for manufacturing) or a method for increasing lysine production, comprising the step of culturing the above-mentioned microorganism of the genus Corynebacterium in a medium.

[0066] The method for producing lysine or the method for increasing lysine production of the present application may additionally include, for example, the step of preparing the microorganism, the step of preparing a medium for culturing the microorganism, or a combination thereof (in any order), prior to the culturing step.

[0067] The above method for producing lysine or increasing lysine production may include the step of culturing the microorganism in a culture medium.

[0068] In the present disclosure, "culture" may mean growing the microorganism under appropriately controlled environmental conditions. The culture process of the present disclosure may be carried out according to suitable media and culture conditions known in the art. Such culture process can be easily adjusted and used by those skilled in the art depending on the strain selected. Specifically, the culture may be batch, continuous, and / or fed-batch, but is not limited thereto.

[0069] In the present disclosure, the term "medium" refers to a substance mixed with nutrients as the main component required to culture the microorganism, and may supply nutrients and growth factors, including water, which is indispensable for survival and growth. Specifically, any medium and other culture conditions used for culturing the microorganism of the present disclosure may be used without special limitations as long as they are media used for culturing ordinary microorganisms; however, the microorganism of the present disclosure may be cultured under aerobic conditions while controlling the temperature, pH, etc., in a conventional medium containing a suitable carbon source, nitrogen source, phosphorus, inorganic compounds, amino acids, and / or vitamins. In one example, a culture medium for a strain of the genus Corynebacterium can be found in the literature ["Manual of Methods for General Bacteriology" by the American Society for Bacteriology (Washington DC, USA, 1981)].

[0070] The above carbon sources may include carbohydrates such as glucose, saccharose, lactose, fructose, sucrose, maltose, etc.; sugar alcohols such as mannitol and sorbitol, organic acids such as pyruvate, lactic acid, and citric acid, etc.; and amino acids such as glutamic acid, methionine, and lysine, etc. In addition, natural organic nutrient sources such as starch hydrolysate, molasses, blackstrap molasses, rice winter, cassava, sugarcane residue, and corn steeping liquid may be used. Specifically, carbohydrates such as glucose and sterilized pre-treated molasses (i.e., molasses converted into reducing sugars) may be used, and other carbon sources in appropriate amounts may be used in various ways without limitation. These carbon sources may be used individually or in combination of two or more types, but are not limited thereto.

[0071] The above nitrogen sources may include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, ammonium nitrate, etc.; and organic nitrogen sources such as amino acids such as glutamic acid, methionine, glutamine, etc., peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquid, casein hydrolysate, fish or its decomposition products, defatted soybean cake or its decomposition products, etc. These nitrogen sources may be used alone or in combination of two or more types, but are not limited thereto.

[0072] The above ingredients may include monopotassium phosphate, dipotassium phosphate, or corresponding sodium-containing salts. Inorganic compounds may include sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, etc., and may also include amino acids, vitamins, and / or suitable precursors. These components or precursors may be added to the culture medium in a batch or continuous manner. However, they are not limited thereto.

[0073] During the cultivation of the above microorganisms, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid, etc., can be added to the medium in an appropriate manner to adjust the pH of the medium.

[0074] In the culture of the present disclosure, the pH in the culture medium is 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 7.5, 6.5 to 7, 7 to 9, 7 to 8.5, 7 to 8, 7 to 7.5, 7.5 It may be up 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 example, the pH in the culture medium may be 6 to 8, or 7, but is not limited thereto.

[0075] In addition, to maintain an aerobic state of the medium, oxygen or an oxygen-containing gas may be injected into the medium, or nitrogen, hydrogen, or carbon dioxide gas may be injected without gas injection to maintain an anaerobic and microaerobic state, but is not limited thereto. In one example, the oxygen concentration in the medium may be maintained within a range where dissolved oxygen is not limited during the cultivation of the microorganism so that the microorganism can grow normally. In one example, the oxygen concentration in the medium may be 0.1 to 20 ppm, but is not limited thereto. Air may be supplied in an appropriate amount and rate to maintain the oxygen concentration in the medium within the above range. In one example, air may be supplied in the medium at a rate of 0.1 to 2 vvm (volume of air per volume of medium per minute) to maintain the oxygen concentration in the medium within the above range, but is not limited thereto.

[0076] In the culture of the present disclosure, the culture temperature may be maintained at 20 to 45°C, specifically 25 to 40°C, and culture may be carried out for about 10 to 160 hours, but is not limited thereto.

[0077] Lysine produced by the culture of the present disclosure may be secreted into the culture medium or remain within the cell. The method for producing lysine or the method for increasing lysine production of the present disclosure may further include a step of recovering lysine from the culture medium (the medium in which the culture is performed) or microorganisms (e.g., strains of the genus Corynebacterium) according to the culture. The recovery step may be further included after the culture step.

[0078] The above recovery may involve collecting the desired lysine using a suitable method known in the art according to the culture method of the microorganism disclosed in this disclosure, for example, batch, continuous, or fed-batch culture methods. For example, various chromatographs such as centrifugation, filtration, treatment with a crystallizing protein precipitating agent (salting out), extraction, ultrasonic disruption, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, HPLC, or a combination thereof may be used, and the desired lysine may be recovered from the culture medium or microorganism using a suitable method known in the art.

[0079] Additionally, the lysine production method or lysine production increase method of the present disclosure may additionally include a lysine purification step. The purification may be performed using a suitable method known in the art. In one example, where the lysine production method or lysine production increase method of the present 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 simultaneously or integrated into a single step, but are not limited thereto.

[0080] Another aspect is to provide a composition for producing lysine comprising the above-mentioned microorganism of the genus Corynebacterium, a culture medium in which the microorganism is cultured, or a combination thereof.

[0081] The above composition may additionally include any suitable excipients commonly used in compositions for lysine production, and such excipients may be, for example, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers, or isotonic agents, but are not limited thereto.

[0082] Another aspect provides a use for the above-mentioned microorganisms of the genus Corynebacterium for the production of lysine, increased production, or the preparation of compositions for lysine production.

[0083]

[0084] The above disclosure is summarized as follows:

[0085] 1. A lysine-producing microorganism of the genus Corynebacterium into which cytoplasmic trehalose degrading enzyme (treF) derived from E. coli has been introduced.

[0086] 2. A microorganism of the genus Corynebacterium according to claim 1, wherein the cytoplasmic trehalose degrading enzyme derived from Escherichia coli comprises an amino acid sequence having at least 94% identity with the amino acid sequence of SEQ ID NO. 1.

[0087] 3. A microorganism of the genus Corynebacterium according to claim 1 or 2 above, wherein the cytoplasmic trehalose degrading enzyme derived from Escherichia coli is encoded by a gene comprising the nucleic acid sequence of SEQ ID NO. 2.

[0088] 4. A microorganism of the genus Corynebacterium, wherein, in any one of the preceding claims, a cytoplasmic trehalose degrading enzyme (treA) derived from E. coli is additionally introduced.

[0089] 5. A microorganism of the genus Corynebacterium, wherein the cytoplasmic trehalose degrading enzyme derived from Escherichia coli according to claim 4 comprises an amino acid sequence having at least 85% identity with the amino acid sequence of SEQ ID NO. 3.

[0090] 6. A microorganism of the genus Corynebacterium, wherein, in claim 4 or 5 above, the cytoplasmic trehalose degrading enzyme derived from Escherichia coli is encoded by a gene containing the nucleic acid sequence of SEQ ID NO. 4.

[0091] 7. In any one of the preceding paragraphs above, the Corynebacterium genus microorganism is Corynebacterium glutamicum.

[0092] 8. A microorganism of the genus Corynebacterium that has increased lysine production capacity compared to a parent strain or wild-type microorganism into which an E. coli-derived cytoplasmic trehalose degrading enzyme has not been introduced, in any one of the preceding claims.

[0093] 9. A microorganism of the genus Corynebacterium that has increased trehalose degradation ability compared to a parent strain or wild-type microorganism into which an E. coli-derived cytoplasmic trehalose degrading enzyme has not been introduced, in any one of the preceding claims.

[0094] 10. A composition for producing lysine comprising a microorganism of the genus Corynebacterium according to any one of the preceding claims.

[0095] 11. A method for producing lysine comprising the step of culturing a microorganism of the genus Corynebacterium according to any one of the preceding claims in a culture medium.

[0096] 12. A method for producing lysine according to claim 11, further comprising the step of recovering lysine from a culture medium or a microorganism of the genus Corynebacterium according to the culture.

[0097] 13. A method for increasing lysine production, comprising the step of culturing a microorganism of the genus Corynebacterium according to any one of the preceding claims in a culture medium.

[0098]

[0099] Microorganisms introduced with the trehalose degrading enzyme of the present disclosure have excellent lysine production capacity and byproduct reduction effects, so they can be efficiently utilized for the mass production of lysine.

[0100]

[0101] The present disclosure is to be explained more specifically below by the following examples. However, these are merely illustrative of the present disclosure, and the scope of the present disclosure is not limited by these examples.

[0102]

[0103] Example 1. Construction of a plasmid for gene insertion

[0104] To insert Escherichia coli-derived treF and treA genes into the chromosome of Corynebacterium glutamicum, NCgl2284, known as a gene encoding a transposon, was used as the insertion site. To replace the NCgl2284 gene with Escherichia coli treF and / or treA, NCgl2284 deletion and target gene insertion vectors were constructed. To construct the vectors, PCR was performed using the chromosome of Corynebacterium glutamicum ATCC13032 as a template and primer pairs of SEQ ID NOs. 5 and 6, and SEQ ID NOs. 7 and 8, respectively. The primer sequences used to perform each of the above PCRs are shown in Table 1 below.

[0105] Solg™ Pfu-X DNA Polymerase (Solgent) was used as the polymerase for the PCR reaction, and the PCR conditions were 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, 500-bp DNA fragments were obtained. The obtained DNA product was purified using a PCR purification kit (QUIAGEN), and the purified amplification product was treated with the restriction enzyme SmaI for 1 hour at 37°C and then heat-treated at 65°C for 20 minutes to produce the pDC24 vector (Korean Published Patent Application KR 10-2024-0167588 A). The vector pDC24_△NCgl2284 for NCgl2284 deletion and target gene insertion was constructed by cloning according to the provided manual using an infusion cloning kit (TaKaRa).

[0106]

[0107] Example 2. Construction of recombinant plasmids for the introduction of E. coli-derived treF and treA

[0108] To introduce the treF and treA genes derived from E. coli, a plasmid was constructed using the gapA promoter (PgapA) of Corynebacterium glutamicum to introduce the E. coli treF and treA genes.

[0109] Specifically, the PgapA promoter fragment was obtained by performing PCR using the primer pair SEQ NO. 9 and SEQ NO. 10 with the chromosome of Corynebacterium glutamicum ATCC13032 containing the PgapA nucleic acid sequence as a template, and the treF fragment (protein sequence: SEQ NO. 1; gene sequence: SEQ NO. 2) was obtained by performing PCR using the primer pair SEQ NO. 11 and SEQ NO. 12 with the chromosome of Escherichia coli (Escherichia coli str. K-12 substr. MG1655) (GenBank: CP032667.1) as a template. Additionally, the treA fragment (protein sequence: SEQ NO. 3; gene sequence: SEQ NO. 4) was obtained using the primer pair SEQ NO. 13 and SEQ NO. 14. PfuUltra was used as the polymerase for the PCR reaction. TM High-reliability DNA polymerase (Stratagene) was used, and 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, and these denaturation, annealing, and polymerization reactions were repeated 28 times. As a result, a 488-bp DNA fragment of the PgapA promoter region, a 1650-bp DNA fragment of the E. coli treF region, and a 1698-bp DNA fragment of the treA region were obtained, respectively.

[0110] Two types of vectors, pDC24_△NCgl2284::PgapA_treF(E.co) and pDC24_△NCgl2284::PgapA_treA(E.co), were constructed by cloning each obtained PCR fragment into the pDC24-△NCgl2284 vector, which was treated with the restriction enzyme SmaI for 1 hour at 37°C and then heat-treated at 65°C for 20 minutes, using the Infusion Cloning Kit (TaKaRa) according to the provided manual.

[0111] To co-express E. coli-derived treF and treA, PCR was performed using SEQ ID NO. 9 and SEQ ID NO. 15 with the previously constructed pDC24_△NCgl2284::PgapA_treF(E.co) as a template to obtain the PgapA_treF(E.co) fragment, and PCR was performed using SEQ ID NO. 14 and SEQ ID NO. 16 with the pDC24_△NCgl2284::PgapA_treA(E.co) as a template to obtain the PgapA_treA(E.co) fragment. The amplification products were purified using a PCR purification kit (QUIAGEN) and used as insertion DNA fragments for vector construction. Each obtained PCR fragment was treated with the restriction enzyme SmaI for 1 hour at 37°C, followed by heat treatment at 65°C for 20 minutes. The pDC24_△NCgl2284::PgapA_treF-PgapA(E.co)_treA(E.co) was then cloned using the Infusion Cloning Kit (TaKaRa) according to the provided manual to construct the pDC24_△NCgl2284::PgapA_treF-PgapA(E.co)_treA(E.co). The primer sequences used to perform each PCR and the E. coli-derived treF and treA sequences are shown in Table 1 below.

[0112]

[0113]

[0114]

[0115]

[0116]

[0117]

[0118]

[0119]

[0120] Example 3. Preparation of Escherichia coli strains introduced with treF and treA and evaluation of lysine production capacity

[0121] The three vectors (pDC24_△NCgl2284::PgapA_treF(E.co), pDC24_△NCgl2284::PgapA_treA(E.co), pDC24_△NCgl2284::PgapA_treF-PgapA(E.co)_treA(E.co)) prepared in Example 2 above were transformed into the lysine-producing strain Corynebacterium glutamicum CJ3P (US 9556463 ​​B2) by electroporation using the electropulse method (Van der Rest et al., Appl. Microbiol. Biotecnol. 52:541-545, 1999), and each strain was transformed into CJ3P_treF(E.co), CJ3P_treA(E.co), and It was named CJ3P_treF-treA(E.co).

[0122] To confirm the L-lysine production ability and trehalose degradation ability of the above-mentioned strain and the control parent strain, a flask fermentation potency evaluation was conducted.

[0123] First, each strain was inoculated into a 250 ml corner-baffle flask containing 25 ml of seed medium and cultured at 37 ℃ for 20 hours with shaking at 200 rpm. 1 ml of seed culture was inoculated into a 250 ml corner-baffle flask containing 24 ml of production medium and cultured at 37 ℃ for 40 hours with shaking at 200 rpm.

[0124] Lysine and trehalose concentrations were measured by flask culturing the parent strain CJ3P and CJ3P_treF(E.co), CJ3P_treA(E.co), and CJ3P_treF-treA(E.co) strains, into which treF and treA derived from E. coli were introduced, using production medium. The above experiment was repeated three times, and the average values ​​of the analysis results are shown in Table 2 below.

[0125] <Bird Badge>

[0126] Raw sugar 20 g, peptone 10 g, yeast extract 5 g, urea 1.5 g, KH2PO44 g, K2HPO48 g, MgSO47H2O 0.5 g, biotin 0.1 mg, thiamine HCl 1 mg, calcium-pantothenic acid 2 mg, nicotinamide 2 mg (based on 1 liter of distilled water)

[0127] Production Badge

[0128] 100 g raw sugar, 40 g (NH4)2SO4, 2.5 g soybean protein, 5 g corn steep solids, 3 g urea, 1 g KH2PO4, 0.5 g MgSO47H2O, 100 µg biotin, 1000 µg thiamine hydrochloride, 2000 µg calcium pantothenic acid, 3000 µg nicotinamide, 30 g CaCO3 (based on 1 liter of distilled water).

[0129] Strain 40hr cell (OD) 600nm Lysine Concentration (g / L) Lysine Concentration Increase Rate (%) Trehalose (mg / L) Trehalose Concentration Decrease Rate (%) CJ3P 43.5 6.2 110 0 138.5 0.0 CJ3P_treF(E.co) 45.7 7.1 11 14.5 6 1.5 55.6 CJ3P_treA(E.co) 44.2 6.9 6 11 2.1 7 1.7 48.2 CJ3P_treF-treA(E.co) 45.1 7.4 7 120.3 3.2 9 7.7

[0130] As can be seen in Table 2 above, it was confirmed that the CJ3P_treF(E.co) strain, into which E. coli-derived treF was introduced, and the CJ3P_treA(e.co) strain, into which E. coli-derived treA was introduced, showed an increase in lysine concentration of approximately 14.5% and 12.1%, respectively, compared to the parent strain, and that the trehalose accumulated in the medium also decreased by 55.6% and 48.2% due to the introduction of these genes. In addition, for the CJ3P_treF-treA(E.co) strain, into which both genes were introduced, the lysine concentration increased by approximately 20.3% compared to the parent strain, and the trehalose in the medium decreased by up to 97.7%. Through these results, it was confirmed that the introduction of E. coli-derived treF has the effect of improving lysine concentration as well as reducing the trehalose accumulated in the medium. In addition, it was confirmed that the simultaneous introduction of treF and treA derived from E. coli more effectively reduces trehalose, and it is believed that the reduced trehalose in the medium will also contribute to the improvement of fermentation purity.

[0131]

[0132] From the foregoing description, those skilled in the art to which the present invention pertains will understand that the present disclosure may be implemented in other specific forms without altering the technical concept or essential features thereof. In this regard, the embodiments described above should be understood as illustrative in all respects and not restrictive. The scope of the present disclosure should be interpreted as including all modifications or variations derived from the meaning and scope of the claims set forth below and their equivalents, rather than from the detailed description above.

Claims

1. A lysine-producing microorganism of the genus Corynebacterium into which cytoplasmic trehalose degrading enzyme (treF) derived from E. coli has been introduced.

2. A microorganism of the genus Corynebacterium according to claim 1, wherein the cytoplasmic trehalose degrading enzyme derived from E. coli comprises an amino acid sequence having at least 94% identity with the amino acid sequence of SEQ ID NO.

1.

3. A microorganism of the genus Corynebacterium, wherein the cytoplasmic trehalose degrading enzyme derived from Escherichia coli is encoded by a gene containing the nucleic acid sequence of SEQ ID NO.

2.

4. A microorganism of the genus Corynebacterium according to claim 1, to which a cytoplasmic trehalose degrading enzyme (treA) derived from Escherichia coli has been additionally introduced.

5. A microorganism of the genus Corynebacterium according to claim 4, wherein the cytoplasmic trehalose degrading enzyme derived from E. coli comprises an amino acid sequence having at least 85% identity with the amino acid sequence of SEQ ID NO.

3.

6. A microorganism of the genus Corynebacterium, wherein the cytoplasmic trehalose degrading enzyme derived from Escherichia coli is encoded by a gene containing the nucleic acid sequence of SEQ ID NO.

4.

7. In paragraph 1, the microorganism of the genus Corynebacterium is Corynebacterium glutamicum.

8. In paragraph 1, a microorganism of the genus Corynebacterium that has increased lysine production capacity compared to a parent strain or wild-type microorganism into which an E. coli-derived cytoplasmic trehalose degrading enzyme has not been introduced.

9. A microorganism of the genus Corynebacterium according to claim 1, having increased trehalose degradation ability compared to a parent strain or wild-type microorganism into which an E. coli-derived cytoplasmic trehalose degrading enzyme has not been introduced.

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

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

12. A method for producing lysine according to claim 11, further comprising the step of recovering lysine from a culture medium or a microorganism of the genus Corynebacterium according to the culture.

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

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