Lysine decarboxylase derived from pantoea alhagi and method for producing cadaverine using microorganism comprising same

Abstract

The present application relates to a lysine decarboxylase variant and a method for producing cadaverine using a microorganism comprising same.

Description

Lysine decarboxylase derived from Pantoea alhaji and method for producing cadaverine using microorganisms containing the same

[0001] The present application relates to a lysine decarboxylase variant and a method for producing cadaverine (PMDA) in a microorganism containing the same.

[0002]

[0003] Cadaverine is a diamine organic compound composed of five carbons with the molecular formula NH2(CH2)5NH2, and can serve as a raw material for nylon 5,6. If bio-based cadaverine is manufactured, it is expected that various types of nylon can be produced while satisfying the demands of the bio-based market.

[0004] Regarding the bio-based production of cadaverine, research on the biotransformation of lysine was widely known prior to the 1940s. Lysine decarboxylase, a key step in this biotransformation, is an enzyme that catalyzes the decarboxylation reaction from lysine to produce cadaverine. The activity of lysine decarboxylase has been reported in various microorganisms, and the lysine decarboxylases with known specific activity (mmol / min / mg) are four species: Escherichia coli, Bacterium cadaveris, Glycine max, and Selenomonas ruminantium. Among these, the lysine decarboxylase derived from E. coli is evaluated as exhibiting the highest activity, and the enzyme utilized in actual production is limited to CadA derived from E. coli (European Patent Publication EP 1482055 A1, etc.).

[0005] It is efficient and economical to secure lysine decarboxylase with high activity at the same microbial biomass. Mass production technology for biocatalysts can be a significant influencing factor on the purification efficiency and manufacturing cost economics of the final product.

[0006]

[0007] The object of the present application is to provide a lysine decarboxylase variant in which the amino acid corresponding to the 451st position of the amino acid sequence of SEQ ID NO. 1 is substituted with another amino acid.

[0008]

[0009] One objective of the present application is to provide a lysine decarboxylase variant in which the amino acid corresponding to the 451st position of the amino acid sequence of SEQ ID NO. 1 is substituted with another amino acid.

[0010] Another object of the present application is to provide a polynucleotide encoding the lysine decarboxylase variant.

[0011] Another object of the present application is to provide a microorganism comprising one or more of the lysine decarboxylase variant and the polynucleotide encoding the variant.

[0012] Another object of the present application is to provide a method for producing cadaverine, comprising the step of converting lysine to cadaverine using a microorganism comprising the lysine decarboxylase variant or a polynucleotide encoding the variant.

[0013] Another object of the present application is to provide a composition for producing cadaverine comprising one or more of the above-mentioned lysine decarboxylase variant and microorganisms expressing it.

[0014]

[0015] The lysine decarboxylase variant of the present application has the effect of converting lysine to cadaverine at a high level.

[0016]

[0017] This is explained in detail as follows. Meanwhile, each description and embodiment disclosed in this application may be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in this application fall within the scope of this application. Furthermore, the scope of this application should not be considered limited by the specific descriptions provided below.

[0018] In addition, a person skilled in the art can recognize or identify a number of equivalents to the specific embodiments of the present application described herein by using only ordinary experiments. In addition, such equivalents are intended to be included in the present application.

[0019] Furthermore, 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 state of the art to which this application pertains and the content of this application.

[0020]

[0021] One aspect of the present application provides a lysine decarboxylase variant in which the amino acid corresponding to the 451st position of the amino acid sequence of SEQ ID NO. 1 is substituted with another amino acid.

[0022]

[0023] In the present application, "a lysine decarboxylase variant in which the amino acid corresponding to the 451st position of the amino acid sequence of SEQ ID NO. 1 is substituted with another amino acid" may mean a lysine decarboxylase variant comprising one or more amino acid substitutions in the amino acid sequence of a parent lysine decarboxylase, and may be used interchangeably with terms such as "lysine decarboxylase variant," "variant," and "variant lysine decarboxylase."

[0024]

[0025] In this application, the term "lysine decarboxylase" refers to a protein having the activity to decarboxylate lysine to produce cadaverine and carbon dioxide by catalyzing the decarboxylation reaction of lysine.

[0026] In this application, "parent lysine decarboxylase" refers to a protein that is subject to mutation introduction as a lysine decarboxylase, which is modified to produce the lysine decarboxylase variant of this application. Specifically, the parent lysine decarboxylase or parent sequence may be a naturally occurring polypeptide or a wild-type polypeptide, may be a mature polypeptide thereof, and may include a variant or functional fragment thereof, but is not limited thereto as long as it is a polypeptide that has lysine decarboxylase activity and can serve as the parent of the variant.

[0027] In the present application, the parent lysine decarboxylase may be a protein having lysine decarboxylase activity encoded by the cadA gene, but is not particularly limited to any type of polypeptide that has activity corresponding to lysine decarboxylase and can be the parent of a variant.

[0028] Specifically, the above-mentioned parent lysine decarboxylase protein may include, for example, SEQ ID NO. 1 or an amino acid sequence having 70% or more homology or identity therewith, but is not limited thereto as long as it has lysine decarboxylase activity. Specifically, meaningless sequence additions before or after the amino acid sequence described in SEQ ID NO. 1, naturally occurring mutations, or silent mutations thereof are not excluded, and if it has the same or corresponding activity as the protein containing the amino acid sequence of SEQ ID NO. 1, it may correspond to the protein subject to the introduction of mutation in the present application.

[0029] Specifically, the parent lysine decarboxylase of the present application may comprise the amino acid sequence of SEQ ID NO. 1; or an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more homology or identity with the amino acid sequence of SEQ ID NO. 1. In addition, it is obvious that a protein having an amino acid sequence in which some sequences are deleted, modified, substituted, or added, provided that it has such homology or identity and exhibits efficacy corresponding to the protein, is also included within the scope of the parent lysine decarboxylase of the present application. The amino acid sequence of SEQ ID NO. 1 can be obtained from known databases such as NCBI’s GenBank or KEGG (Kyoto Encyclopedia of Genes and Genomes).

[0030] In one embodiment, the parent lysine decarboxylase sequence of the present application is NCBI Accession No. WP_085071266.1. In another embodiment, the parent lysine decarboxylase sequence of the present application is NCBI Accession No. WP_250650242.1.

[0031]

[0032] In the present application, the parent lysine decarboxylase may be a lysine decarboxylase derived from Pantoea alhagi. The microorganism is an example of a microorganism from which the parent lysine decarboxylase of the present application may be derived, and includes microorganisms derived from which are taxonomically homologous, regardless of the name of the microorganism.

[0033] In this application, the sequence "derived from" a specific microorganism is not limited to those naturally produced or produceable in that microorganism, but also includes sequences encoded by genes produced and isolated from microorganisms containing that gene.

[0034]

[0035] In this application, the terms “variant,” “variant polypeptide,” or “variant protein” refer to a polypeptide in which one or more amino acids are conservatively substituted and / or modified, resulting in a sequence of amino acids different from that of the variant prior to mutation, while retaining functions or properties. Such a variant can generally be identified by modifying one or more amino acids in the amino acid sequence of the polypeptide and evaluating the properties of the modified polypeptide. That is, the capabilities of the variant may be increased, unchanged, or decreased compared to the polypeptide prior to mutation. Additionally, some variants may include variants in which one or more parts, such as an N-terminal leader sequence or a transmembrane domain, have been removed. Other variants may include variants in which a portion of the N-terminus and / or C-terminus of a mature protein has been removed. The term "variant" mentioned above may be used interchangeably with terms such as variant, variant polypeptide, variant protein, variant polypeptide, variant protein, variant polypeptide, and variant protein (in English expressions such as modified polypeptide, modified protein, mutant, mutein, etc.), and is not limited to these terms as long as they are used with the meaning of being mutated.

[0036] Additionally, variants may include deletions or additions of amino acids that have minimal effect on the properties and secondary structure of the polypeptide. For example, the polypeptide may be conjugated with a signal (or leader) sequence at the N-terminus of a protein involved in the co-translational or post-translational transfer of the protein. Additionally, the polypeptide may be conjugated with another sequence or linker to enable identification, purification, or synthesis of the polypeptide.

[0037]

[0038] In this application, the terms "lysine decarboxylase variant" or "variant lysine decarboxylase" refer to a protein in which one or more amino acids differ from the amino acid sequence of parent lysine decarboxylase.

[0039] In one embodiment, the lysine decarboxylase variant of the present application has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity with the amino acid sequence of SEQ ID NO. 1, and the amino acid corresponding to position 451 from the N-terminus of the amino acid sequence of SEQ ID NO. 1 may be substituted with another amino acid.

[0040] In another embodiment, the lysine decarboxylase variant comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO. 40, and the amino acid corresponding to position 451 from the N-terminus of the amino acid sequence of SEQ ID NO. 1 may be substituted with another amino acid.

[0041] In any one of the aforementioned embodiments, the lysine decarboxylase variant may comprise the amino acid sequence of SEQ ID NO. 40. The amino acid sequence of SEQ ID NO. 40 is an amino acid sequence in which the tyrosine corresponding to the 451st position in the amino acid sequence from the N-terminus of the amino acid sequence described in SEQ ID NO. 1 is substituted with another amino acid.

[0042] In any one of the aforementioned embodiments, the lysine decarboxylase variant may have at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more, or 100%, sequence identity with the amino acid sequence of SEQ ID NO. 2, SEQ ID NO. 26, SEQ ID NO. 27, or SEQ ID NO. 35.

[0043] In any one of the aforementioned embodiments, the lysine decarboxylase variant may comprise the amino acid sequence of the following general formula 1:

[0044] [General Formula 1]

[0045] X1GPHKEA EEYIARVFNA ERSYMVTNGT STANKIVGMY SAPAGSTILI DRNCHKSLTH LMMMSDVTPI YFRPTRNAYG ILGGIPQSEF QHETIAKRVK ATPNASWPTH AVITNSTYDG LLYNTDYIKK TLDVKSIHFD SAWVPYTNFH PIYX2GKSGMS GX3RVEGKVIY ETQSTHKLLA AFSQASMIHV KGDVNQETFN EAYMMHTTTS PHYGIVASTE TAAAMMRGNA GKRLIGESIE RALRFRKEIK RLNAESEGWF IDKTKALSLL RALTDFKRAF DLNLRVKNIL PSLWREAPDF YKDMRIQELA ANIHQLIQRH NLPDLMYRAF ENLPAMVLTP YHAFQKELHG EVEEVYLEEM VGRVSANMIL PYPPGVPLIM PGEMITDETL PVLEFLQLLC EIGSHYPGFE TDIHGAYRQE DGRYTVKVLK

[0046] In the above general formula 1, X1 is serine or threonine, X2 is serine or threonine, X3 is glycine or aspartic acid, X4 is an amino acid other than tyrosine, X5 is glycine or glutamic acid, and X6 is glutamic acid or aspartic acid.

[0047] In any one of the aforementioned embodiments, the lysine decarboxylase variant may be one in which the amino acid corresponding to the 451st position of the amino acid sequence of SEQ ID NO. 1 is substituted with a polar or nonpolar amino acid.

[0048] In any one of the aforementioned embodiments, the lysine decarboxylase variant may be one in which the amino acid corresponding to the 451st position of the amino acid sequence of SEQ ID NO. 1 is substituted with glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, or glutamine.

[0049] In any one of the aforementioned embodiments, the lysine decarboxylase variant may be one in which the amino acid corresponding to the 451st position of the amino acid sequence of SEQ ID NO. 1 is substituted with an amino acid selected from phenylalanine, serine, and asparagine.

[0050] In any one of the aforementioned embodiments, the lysine decarboxylase variant may be one in which the amino acid corresponding to the 451st position of the amino acid sequence of SEQ ID NO. 1 is substituted with phenylalanine.

[0051]

[0052] In the amino acid sequence of lysine decarboxylase that is subject to mutation in the present application, that is, the sequence that can be the parent sequence, the amino acid before modification corresponding to the 451st amino acid of SEQ ID NO. 1 may be tyrosine (Y), but is not limited thereto.

[0053] For example, the lysine decarboxylase variant of the present application may comprise an amino acid sequence in which the amino acid corresponding to position 451 from the N-terminus of the amino acid sequence of SEQ ID NO. 1 is fixed as an amino acid other than tyrosine, and which has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more homology or identity with SEQ ID NO. 1. Furthermore, it is obvious that a variant polypeptide having an amino acid sequence in which some sequences are deleted, modified, substituted, conservatively substituted, or added is also included within the scope of this application, provided that the amino acid sequence has such homology or identity and exhibits efficacy corresponding to the variant polypeptide of this application.

[0054] Meanwhile, a person skilled in the art can identify the amino acid corresponding to the 451st position of the amino acid sequence of SEQ ID NO. 1 of the present application in any amino acid sequence through sequence alignment known in the art, and it is obvious that when "an amino acid at a specific position in a specific SEQ ID NO" is described in the present application, even if not separately stated, it means including "an amino acid at a corresponding position" in any amino acid sequence.

[0055]

[0056] In this application, the term “consisting of” means that the proportion of the specific feature, step, component, or other component(s) described below the term is 100%. The feature, step, component, or other component described below the term “consisting of” may be essential or mandatory. For example, any other feature, step, component, or other component, or non-essential feature, step, component, or other component, in addition to the feature, step, component, or other component described below the term “consisting of,” may be excluded.

[0057] In this application, the term “consisting essentially of” means that one or more unspecified features, steps, components, or other components may be present, where the features, steps, components, or other components of the subject matter claimed in this application are not substantially affected by the presence of said unspecified features, steps, components, or other components.

[0058] In this application, the term "comprising" means the presence of the features, steps, components, or other components described below the above term, and does not exclude the presence of one or more additional features, steps, components, or other components. The features, steps, components, or other components described below the "comprising" in this application may be essential or mandatory, but some embodiments may further include other optional or non-essential features, steps, components, or other components.

[0059]

[0060] Even if the present application is described as "a protein having an amino acid sequence described by a specific sequence number" or "a protein of a specific sequence number," it is obvious that a protein having an amino acid sequence in which some sequences are deleted, modified, substituted, conservatively substituted, or added may also be used in the present application if it has the same or corresponding activity as the protein composed of the amino acid sequence number. For example, if it has the same or corresponding activity as the variant protein, it does not exclude the addition of sequences before or after the amino acid sequence that do not alter the function of the protein, naturally occurring mutations, silent mutations, or conservative substitutions, and it is obvious that such sequence additions or mutations fall within the scope of the present application.

[0061] The "position N" of the present application may include position N and an amino acid position corresponding to position N. Specifically, it may include an amino acid position corresponding to any amino acid residue in a mature polypeptide disclosed in a specific amino acid sequence. The specific amino acid sequence may be the amino acid sequence of SEQ ID NO. 1.

[0062] In this application, the term “corresponding to” refers to an amino acid residue at a position listed in the polypeptide, or an amino acid residue that is similar, identical, or homologous to a residue listed in the polypeptide. Identifying the amino acid at the corresponding position may involve determining a specific amino acid of a sequence that references a specific sequence. As used in this application, “corresponding region” generally refers to a similar or corresponding position in a related protein or a reference protein.

[0063] For example, any amino acid sequence can be aligned with sequence number 1, and based on this, each amino acid residue of the said amino acid sequence can be numbered by referring to the numerical position of the amino acid residue corresponding to the amino acid residue of sequence number 1. For example, a sequence alignment algorithm such as that described in the present application can identify the position of an amino acid, or the position where modifications such as substitution, insertion, or deletion occur, by comparing with a query sequence (also referred to as a "reference sequence").

[0064] For such alignment, examples such as the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453), the Needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000), Trends Genet. 16: 276-277) can be used, but are not limited thereto, and sequence alignment programs and pairwise sequence comparison algorithms known in the art can be appropriately used.

[0065] For example, for the purposes of this application, any amino acid sequence aligned with SEQ ID NO. 1 may be an amino acid sequence of lysine decarboxylase, and the description of parent lysine decarboxylase may be referenced.

[0066]

[0067] In this application, the term “conservative substitution” means substituting one amino acid with another amino acid having similar structural and / or chemical properties. Such amino acid substitutions may generally occur based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues. For example, positively charged (basic) amino acids include arginine, lysine, and histidine; negatively charged (acidic) amino acids include glutamic acid and aspartic acid; aromatic amino acids include phenylalanine, tryptophan, and tyrosine; and hydrophobic amino acids include alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan. In addition, amino acids can be classified into those with electrically charged side chains and those with uncharged side chains; amino acids with electrically charged side chains include aspartic acid, glutamic acid, lysine, arginine, and histidine; amino acids with uncharged side chains can be further classified into nonpolar amino acids or polar amino acids; nonpolar amino acids include glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline; and polar amino acids include serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Typically, conservative substitutions have little to no effect on the activity of the resulting polypeptide. Typically, conservative substitutions have little to no effect on the activity of a protein or polypeptide.

[0068]

[0069] Another aspect of the present application provides a polynucleotide encoding the lysine decarboxylase variant.

[0070] The terms used in this embodiment are as described above.

[0071] In this application, the term "polynucleotide" refers to a polymer of nucleotides in which nucleotide monomers are linked together in a long chain by covalent bonds, such as a DNA or RNA strand of a certain length or longer, and more specifically, a polynucleotide fragment encoding the variant.

[0072] The polynucleotide encoding the lysine decarboxylase variant of the present application may undergo various modifications to its coding region within the scope of not altering the amino acid sequence of the variant of the present application, taking into account codon degeneracy or the codons preferred by the organism intended to express the variant of the present application. Therefore, it is evident that the polynucleotide may also include a polypeptide consisting of the amino acid sequence of the variant of the present application or a polypeptide having homology or identity therewith, due to codon degeneracy.

[0073] The polynucleotide encoding the lysine decarboxylase variant of the present application may be such that, in SEQ ID NO. 3 or its degenerated sequence, the codon encoding the amino acid corresponding to the 451st position of the amino acid sequence of SEQ ID NO. 1 is substituted with a codon encoding an amino acid other than tyrosine.

[0074] For example, the polynucleotide of the present application may include, but is not limited to, a base sequence having homology or identity of 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more with respect to the sequence of SEQ ID NO. 3, wherein the codon encoding threonine, which is the 451st amino acid of the amino acid sequence of SEQ ID NO. 1, is substituted with an amino acid other than tyrosine.

[0075] Furthermore, it is obvious that if a polynucleotide sequence has such homology or identity and codes for the amino acid sequence of a variant of the present application, a polynucleotide sequence in which some sequences are deleted, modified, substituted, conservatively substituted, or added is also included within the scope of the polynucleotide of the present application.

[0076]

[0077] In addition, the polynucleotide of the present application may be included without limitation as long as it is a probe that can be prepared from a known gene sequence, for example, a sequence that can be hybridized under strict conditions with a sequence complementary to all or part of the polynucleotide sequence of the present application.

[0078] The above "stringent condition" refers to a condition that enables specific hybridization between polynucleotides. Such conditions are specifically described in the literature (see J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989; FM Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, 9.50-9.51, 11.7-11.8). For example, conditions may be listed in which polynucleotides with high homology or identity are hybridized with each other, having homology or identity of 45% or more, 50% or more, 55% or more, 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, and polynucleotides with homology or identity of less than that are not hybridized, or conditions in which the polynucleotides have homology or identity of 60°C, 1XSSC, 0.1% SDS, specifically 60°C, 0.1XSSC, 0.1% SDS, more specifically 68°C, 0.1XSSC, 0.1% SDS, which are washing conditions of normal southern hybridization, are washed once, specifically two to three times, at a salt concentration and temperature equivalent to 60°C, 1XSSC, 0.1% SDS, more specifically 68°C, 0.1XSSC, 0.1% SDS.

[0079] Hybridization requires that two nucleic acids have complementary sequences, even though a mismatch between bases may be possible depending on the degree of hybridization. The term "complementary" is used to describe the relationship between nucleotide bases that can hybridize with each other. For example, regarding DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Accordingly, the polynucleotides of this application may also include isolated nucleic acid fragments that are complementary to the entire sequence, as well as substantially similar nucleic acid sequences.

[0080] Specifically, a polynucleotide having homology or identity with the polynucleotide of the present application can be detected using hybridization conditions including a hybridization step at a Tm value of 55°C and using the conditions described above. Additionally, the Tm value may be 60°C, 63°C, or 65°C, but is not limited thereto and can be appropriately adjusted by a person skilled in the art according to the purpose.

[0081] The appropriate strictness for hybridizing the above polynucleotides depends on the length and degree of complementarity of the polynucleotides, and the variables are well known in the art (e.g., J. Sambrook et al., i.e.).

[0082] In this application, the terms "homology" or "identity" refer to the degree of similarity between two given amino acid sequences or base sequences and may be expressed as a percentage. The terms homology and identity may often be used interchangeably.

[0083] 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. Practically, homologous or identical sequences can generally be hybridized with the entire sequence or a part thereof under moderate or high stringent conditions. It is evident that hybridization also includes hybridization with polynucleotides containing common codons or codons that account for codon degeneracy.

[0084] 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 ET AL.](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.

[0085] 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 unitary 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. Thus, the terms “homology” or “identity” as used in this application refer to the relevance between the sequences.

[0086]

[0087] Another aspect of the present application provides a vector comprising the polynucleotide of the present application.

[0088] The above vector may be an expression vector for expressing the above polynucleotide in a microorganism, but is not limited thereto.

[0089] In this application, the term “vector” may comprise a DNA product comprising a sequence of nucleotides of a polynucleotide encoding said target polypeptide, which is operably linked to a suitable expression control region (or expression control sequence) to enable the expression of said 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 microorganism, the vector may replicate or function independently of the host genome and may be incorporated into the genome itself.

[0090] The vectors used in this application 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, pBR-based, pUC-based, pBluescriptII-based, pGEM-based, pTZ-based, pCL-based, and pET-based vectors may be used as plasmid vectors. Specifically, pDZ, pDC, pDCM2, pDC24, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vectors may be used.

[0091] For example, a polynucleotide encoding a target polypeptide can be inserted into a chromosome using a vector for intracellular chromosome insertion. The insertion of said 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 by 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.

[0092] In this application, the term "transformation" means introducing a vector containing a polynucleotide encoding a target polypeptide into a microorganism or into a microorganism so that the polypeptide encoded by said polynucleotide can be expressed within the microorganism. The transformed polynucleotide may include either inserted into or located outside the chromosomes of the microorganism, as long as it can be expressed within the microorganism. Additionally, said polynucleotide includes DNA and / or RNA encoding the target polypeptide. said polynucleotide may be introduced in any form that can be introduced into the microorganism and expressed. For example, said polynucleotide may be introduced into the microorganism in the form of an expression cassette, which is a genetic structure containing all the elements necessary for self-expression. said expression cassette may typically include a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal operably linked to said polynucleotide. said expression cassette may be in the form of a self-replicating expression vector. In addition, the polynucleotide may be introduced into the microorganism in its own form and operably linked to a sequence required for expression in the microorganism, but is not limited thereto.

[0093] In addition, the term "operably connected" above means that a promoter sequence and a polynucleotide sequence are functionally connected to initiate and mediate the transcription of a polynucleotide encoding the target variant of the present application.

[0094] The method of transforming the vector of the present application includes any method of introducing nucleic acid into a cell, and depending on the host cell, a suitable standard technique as known in the art may be selected and performed. Examples include, but are not limited to, electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, and lithium acetate-DMSO method.

[0095]

[0096] Another aspect of the present application provides a microorganism comprising one or more of the lysine decarboxylase variant and the polynucleotide encoding the variant.

[0097] The terms used in this embodiment are as described above.

[0098] In this application, the term "microorganism (or strain)" includes both wild-type microorganisms and microorganisms that have undergone natural or artificial genetic modification. It may be a microorganism in which specific mechanisms are weakened or strengthened due to causes such as the insertion of external genes or the enhancement or inactivation of the activity of endogenous genes, and may be a microorganism that includes genetic modification for the production of a desired polypeptide, protein, or product. In this application, "strain" and "microorganism" may be used interchangeably without limitation as having the same meaning.

[0099] In this application, the term “recombinant microorganism” refers to a microorganism that is genetically modified to exhibit a different genotype and / or phenotype compared to a naturally occurring microorganism (e.g., when the genetic modification affects the nucleic acid sequence coding of the microorganism), and may include both offspring and potential offspring of said microorganism. In this application, the terms “recombinant microorganism,” “genetically modified microorganism,” “recombinant host cell,” “recombinant cell,” and “recombinant strain” may be used interchangeably. The recombinant microorganism may, for example, express a gene not found in its natural (non-recombinant) form; not express a gene expressed in its natural form; or express a natural gene in a manner different from that expressed in its natural form.

[0100]

[0101] In one embodiment, the microorganism of the present application may be a microorganism capable of producing cadaverine.

[0102] In another embodiment, the microorganism further includes a modification that increases the cadaverine production capacity.

[0103] In this application, the term "microorganism capable of producing cadaverine" refers to a microbial strain capable of producing cadaverine by converting lysine into cadaverine, and may include both microorganisms to which cadaverine-producing ability has been conferred upon a parent strain that lacks cadaverine-producing ability, and microorganisms that inherently possess cadaverine-producing ability. Cadaverine-producing ability may be conferred or enhanced by species improvement.

[0104]

[0105] In this application, the term "non-mutated microorganism" does not exclude strains containing mutations that may naturally occur in microorganisms, and may refer to wild-type strains or natural-type strains themselves, or strains prior to genetic mutations caused by natural or artificial factors.

[0106] For example, the above-mentioned non-modified microorganism may refer to a strain that does not express or has not been modified to express the lysine decarboxylase variant described herein. 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."

[0107]

[0108] The microorganism of the present application may be, but is not limited to, a microorganism comprising one or more of the variant of the present application, the polynucleotide of the present application, and a vector comprising the polynucleotide of the present application; a microorganism modified to express the variant of the present application or the polynucleotide of the present application; a microorganism expressing the variant of the present application or the polynucleotide of the present application (e.g., a recombinant strain); or a microorganism having the activity of the variant of the present application (e.g., a recombinant strain).

[0109] For example, the strain of the present application is a cell or microorganism that expresses the variant of the present application, and the strain of the present application may include all microorganisms capable of producing cadaverine, including the variant of the present application.

[0110] For example, the microorganism of the present application may be a recombinant strain in which a lysine decarboxylase variant is expressed by introducing a polynucleotide encoding the variant of the present application into a natural wild-type microorganism or a microorganism having cadaverine production ability, thereby increasing cadaverine production ability. The recombinant strain with increased cadaverine production ability may be a microorganism with increased cadaverine production ability compared to a natural wild-type microorganism or a non-modified microorganism (e.g., a microorganism expressing wild-type lysine decarboxylase or a microorganism not expressing the variant lysine decarboxylase of the present application), but is not limited thereto. As an example, a microorganism with increased cadaverine production ability including the variant of the present application may be a microorganism with increased cadaverine production ability compared to a microorganism containing a polypeptide consisting of the amino acid sequence of SEQ ID NO. 1 or SEQ ID NO. 34 or a polynucleotide encoding it, but is not limited thereto.

[0111] In one embodiment, the cadaverine production ability of the microorganism of the present application can be evaluated by measuring the conversion rate of lysine to cadaverine.

[0112]

[0113] The microorganisms of the present application may include all microorganisms capable of expressing the variant of the present application by various known methods in addition to the introduction of a polynucleotide encoding the variant or a vector containing the polynucleotide.

[0114] For example, the microorganism with increased cadaverine production capacity is about 1% or more compared to the cadaverine production capacity of the parent strain before mutation or the non-mutated microorganism, specifically about 1% or more, about 2% or more, about 3% or more, about 4% or more, about 5% or more, about 6% or more, about 7% or more, about 8% or more, about 9% or more, about 10% or more, about 11% or more, about 12% or more, about 13% 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 (there is no special restriction on the upper limit value, for example, about 200% or less, about 150% or less, about 100% or less, about 90% or less, about 80% or less, about 70% or less, about It may be increased by 60% or less, about 55% or less, or about 50% or less, but is not limited thereto as long as it has a positive increase compared to the production capacity of the parent strain or non-mutated microorganism before mutation. In another example, the recombinant strain with increased cadaverine production capacity has a cadaverine production capacity of about 1.01 times or more, about 1.02 times or more, about 1.03 times or more, about 1.04 times or more, 1.05 times or more, 1.06 times or more, 1.07 times or more, 1.08 times or more, 1.09 times or more, about 1.1 times or more, about 1.11 times or more, about 1.12 times or more, about 1.13 times or more, about 1.14 times or more, about 1.15 times or more, about 1.16 times or more, about 1.17 times or more, about 1.18 times or more, about 1.19 times or more, about 1.20 times or more, about 1.25 times or more, or about 1.3 times or more compared to the parent strain before mutation or the non-mutated microorganism. (There is no specific limitation on the upper limit, for example, about It may be increased by 10 times or less, about 5 times or less, about 3 times or less, about 2 times or less, or about 1.5 times or less, but is not limited thereto.

[0115] The above term "about" is a range that includes all of ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and includes all values ​​within a range equivalent to or similar to the value following the term "about," but is not limited thereto.

[0116]

[0117] In another embodiment, the microorganism of the present application may be a microorganism that expresses a lysine decarboxylase variant. The microorganism expresses the lysine decarboxylase variant without the ability to produce cadaverine, and the expressed lysine decarboxylase may be recovered from the microorganism and used.

[0118]

[0119] For example, the microorganism of the present application may be either a prokaryotic cell or a eukaryotic cell, but specifically may be a prokaryotic cell.

[0120] For example, the microorganisms of the present application may include, but are not limited to, microorganisms belonging to the genera Escherichia sp., Erwinia sp., Serratia sp., Providencia sp., Corynebacterium sp., Pseudomonas sp., Leptospira sp., Salmonella sp., Brevibacteria sp., Hypomononas sp., Chromobacterium sp., Norcardia sp., Bacillus sp., or fungi or yeasts.

[0121] In any one of the embodiments described above, the microorganism of the present application may be selected from the genus Escherichia, the genus Corynebacterium, and the genus Bacillus.

[0122] For example, the microorganism of the genus Escherichia may be selected from Escherichia coli, Escherichia albertii, Escherichia fergusonii, Escherichia hermannii, Escherichia vulneris, and Escherichia blattae.

[0123] For example, the above-mentioned microorganisms of the genus Bacillus include Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus brews, Bacillus stearothermophilus, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus circulans, Bacillus lentus, Bacillus thuringiensis, Bacillus alcalophilus, Bacillus brevis, Bacillus clausii, Bacillus firmus, and Bacillus It may be selected from Bacillus lautus, Bacillus megaterium, and Bacillus pumilus.

[0124] For example, the above-mentioned 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 ammoniagenes, and Corynebacterium It may be selected from Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium testudinoris, and Corynebacterium flavescens.

[0125] In any one of the embodiments described above, the microorganism of the present application may be selected from Escherichia coli, Corynebacterium glutamicum, and Bacillus subtilis.

[0126] In any one of the embodiments described above, the microorganism of the present application may be Escherichia coli.

[0127]

[0128] Another aspect of the present application provides a method for producing cadaverine, comprising the step of converting lysine to cadaverine using a lysine decarboxylase variant of the present application or a microorganism expressing the same.

[0129] The terms used in this embodiment are as described above.

[0130] In this application, the term "Cadaverine (CAD)" is represented by the chemical formula NH2(CH2)5NH2. Cadaverine may also be named 1,5-pentanediamine or pentamethylenediamine.

[0131] In this application, lysine and cadaverine may be in a free-from form, neutral, or as salts. Since lysine and cadaverine exist mostly in salt form under normal aqueous conditions, it should be understood that in this application, lysine includes the lysine salt form and cadaverine includes the cadaverine salt form.

[0132] Lysine salts are salts formed from lysine and conjugate anions bonded to it. Examples of conjugate anions may include inorganic anions such as carbonate ions, chloride ions, phosphate ions, and nitrate ions, and organic anions such as dicarboxylate ions. Examples of lysine salts with dicarboxylate ions as conjugate anions include, but are not limited to, lysine succinate, lysine glutarate, lysine adipate, lysine suberate, lysine azelaate, lysine sebate, lysine undecandioicate, lysine dodecanedicarboxylate, lysine isophthalate, and lysine terephthalate.

[0133] Cadaverine salts are salts formed from cadaverine and a conjugate anion bonded thereto. Examples of conjugate anions may include inorganic anions such as carbonate ions, chloride ions, phosphate ions, and nitrate ions, and organic anions such as dicarboxylate ions. Examples of cadaverine salts having a dicarboxylate ion as the conjugate anion include, but are not limited to, cadaverine succinate, cadaverine glutarate, cadaverine adipate, cadaverine suberate, cadaverine azelaate, cadaverine sebate, cadaverine undecandioicate, cadaverine dodecanedicarboxylate, cadaverine isophthalate, and cadaverine terephthalate.

[0134] In one embodiment, the method for producing cadaverine may include the step of contacting the lysine decarboxylase variant of the present application or a microorganism expressing the same with lysine.

[0135] In another embodiment, the method for producing cadaverine may include the step of contacting a lysine decarboxylase variant or a microorganism expressing it with lysine in the presence of a cofactor. As an example, the cofactor may be pyridoxal-phosphate (PLP), but is not limited thereto.

[0136] In one embodiment, the lysine decarboxylase variant of the present application can convert lysine to cadaverine in a solution containing a lysine salt and a conjugate anion, and the produced cadaverine can be obtained in the form of a salt.

[0137] In any one of the embodiments described above, the method for producing cadaverine may further include the step of culturing an L-lysine-producing microorganism to obtain a lysine salt. The obtained lysine salt may be converted into a cadaverine salt by reacting with the lysine decarboxylase variant of the present application.

[0138] In any one of the aforementioned embodiments, a salt addition or substitution reaction may be performed on the cadaverine salt obtained through the conversion reaction of lysine.

[0139]

[0140] In one embodiment, the method for producing cadaverine according to the present application may include the step of converting lysine into cadaverine using a protein having lysine decarboxylase activity produced from a recombinant microorganism expressing a lysine decarboxylase variant of the present application or said microorganism.

[0141] In one embodiment, the lysine decarboxylase of the present application may be used in a form extracted from a microorganism expressing it.

[0142] In any one of the embodiments described above, the method may include the step of extracting and purifying the lysine decarboxylase from a microorganism expressing the lysine decarboxylase variant of the present application. Such extraction and purification methods may use methods known in the art.

[0143] In another embodiment, the method for producing cadaverine according to the present application comprises the step of culturing a microorganism expressing lysine decarboxylase in a medium containing lysine. In one embodiment, the lysine decarboxylase according to the present application is not recovered from the microorganism, and the microorganism expressing it itself may be used for the production of cadaverine.

[0144] Cadaberine produced by the culture of the present application may be secreted into the culture medium or remain within the cell.

[0145] In the method of the present application, any culture conditions and methods known in the art may be used for the culture of microorganisms. Such a culture process can be easily adjusted and used by those skilled in the art depending on the microorganism selected.

[0146] For example, the culture medium of the microorganism of the present application may contain lysine. For example, the culture medium of the microorganism of the present application may contain a cofactor, such as pyridoxal-phosphate (PLP).

[0147]

[0148] In one embodiment, the method for producing cadaverine of the present application may additionally include the step of preparing a microorganism of the present application, the step of preparing a medium for culturing said strain, or a combination thereof (in any order), for example, prior to the culturing step.

[0149] The method for producing cadaverine according to the present application may further include a step of recovering a target substance, specifically cadaverine, from the cultured microorganism, the culture of the microorganism, the fermented product of the microorganism, or the culture medium. The recovery step may be additionally included after the culture step.

[0150] The above recovery may involve collecting the desired cadaverine using a suitable method known in the art according to the culture method of the microorganism of the present application, such as a batch, continuous, or fed-batch culture method. For example, various chromatographic methods 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 target substance, specifically cadaverine, can be recovered from the culture medium or microorganism using a suitable method known in the art.

[0151]

[0152] The method for manufacturing cadaverine according to the present application may additionally include a purification step. The purification may be performed using a suitable method known in the art. In one example, where the method for manufacturing cadaverine according to the present application includes both a recovery step and a purification step, the recovery step and the purification step may be performed continuously or discontinuously regardless of the order, or simultaneously or integrated into a single step, but are not limited thereto.

[0153]

[0154] Another aspect of the present application provides a composition for producing cadaverine comprising one or more of the lysine decarboxylase variant of the present application and microorganisms expressing the same.

[0155] The terms used in this embodiment are as described above.

[0156] In one embodiment, the composition may comprise a microorganism into which the lysine decarboxylase of the present application or a polynucleotide encoding the same has been introduced, a culture of the microorganism, a fermented product of the microorganism, or a combination of two or more of these.

[0157] In any one of the aforementioned embodiments, the composition may include lysine.

[0158] In any one of the aforementioned embodiments, the composition may include pyridoxal-phosphate (PLP).

[0159] The composition of the present application may further include any suitable excipients commonly used in compositions for the production of cadaverine, and such excipients may be, for example, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers or isotonic agents, but are not limited thereto.

[0160] In one embodiment, each component present in the composition of the present application may be included in a microbiologically effective amount or in an amount that can be appropriately present in a composition for production.

[0161]

[0162] Another aspect of the present application provides the use of the lysine decarboxylase variant of the present application for the production of cadaverine.

[0163] Another aspect of the present application provides a use for cadaverine production of a microorganism expressing the lysine decarboxylase variant of the present application.

[0164] The terms used in this embodiment are as described above.

[0165]

[0166] The present application will be explained in more detail below through examples and experimental examples. However, these examples and experimental examples are intended to illustrate the present application, and the scope of the present application is not limited to these examples and experimental examples.

[0167]

[0168] Example 1: Search and screening of foreign lysine decarboxylase genes for cadaverine production

[0169]

[0170] In this example, lysine decarboxylase (hereinafter cadA) genes presumed to be capable of converting lysine into cadaverine (Pentamethylenediamine, PMDA) and microorganisms possessing them were selected and summarized in Table 1 below.

[0171]

[0172] Microorganism origin Unitor Accession IDPrimer Sequence Number Vector Strain 1 Escherichia coli W3110P0A9H3 (Sequence No. 28) 7, 8 pECCG117_ParaB-cadA(Eco)W3110 / pECCG117_ParaB-cadA(Eco) 2 Pantoea alhagi strain LTYR-11ZARJ43210.1 (Sequence No. 1) 9, 10 pECCG117_ParaB-cadA(Pal)W3110 / pECCG117_ParaB-cadA(Pal) 3 Acinetobacter sp. ANC 4558A0A241VSL9 (SEQ No. 29)11, 12pECCG117_ParaB-cadA(Aci)W3110 / pECCG117_ParaB-cadA(Aci)4Sutterella seckiiA0A6I1EP29 (SEQ No. 30)13, 14pECCG117_ParaB-cadA(Sse)W3110 / pECCG117_ParaB-cadA(Sse)5Pyramidobacter porciA0A6L5YBP5 (SEQ No. 31)15, 16pECCG117_ParaB-cadA(Ppo)W3110 / pECCG117_ParaB-cadA(Ppo)6Lactobacillus plantarumBBA81366.1 (SEQ No. 32)17, 18pECCG117_ParaB-cadA(Lpl)W3110 / pECCG117_ParaB-cadA(Lpl)7Clostridium acetobutylicumAAK78278.1 (SEQ ID NO: 33) 19, 20pECCG117_ParaB-cadA(Cac)W3110 / pECCG117_ParaB-cadA(Cac)

[0173] Gene synthesis was performed based on the genetic information of the obtained microorganisms, and seven types of template DNA encoding them were obtained. A vector was constructed to increase the expression of the selected cadA, and the method is as follows.

[0174] PCR was performed using the primer sequences in Table 1 with the cadA DNA fragments of each obtained microorganism as templates. PCR was performed using PfuUltra-™ high-reliability DNA polymerase (Stratagene), and the PCR conditions consisted of denaturation at 95°C for 30 seconds; annealing at 55°C for 30 seconds; and polymerization at 72°C for 1 minute 30 seconds, repeated 30 times. As a result, each cadA DNA fragment was obtained. To obtain the araB promoter derived from Escherichia coli (W3110), primers of SEQ ID NO. 5 and SEQ ID NO. 6 were used with the E. coli genomic DNA as a template, and the DNA fragment was obtained by performing PCR on the promoter in the same manner as above. A plasmid was obtained by cloning the pECCG117 vector (Korean Registered Patent No. 10-0057684), which had been treated with the restriction enzyme BamHI at 37°C for 1 hour and then heat-treated at 65°C for 20 minutes, and DNA fragments (ParaB, each cadA) at a molar concentration (M) of 2:1:1 using the Takara Infusion Cloning Kit according to the provided manual. The names of the vector and the information of the introduced genes are indicated in Table 1 above.

[0175] Seven types of vectors were each transformed into Escherichia coli (W3110) by electroporation to produce strains expressing foreign cadA, and the information on the strains is indicated in Table 1 above.

[0176]

[0177] Example 2: Comparison of Cadaverine Production Capacity of Microorganisms Expressing Foreign Lysine Decarboxylase (cadA)

[0178]

[0179] To confirm the cadaverine production ability of the strains obtained in Example 1 above, the parent strain and the strains prepared in Table 1 were each inoculated into a 250 ml Corner-Barpool flask containing 25 ml of production medium with the composition as follows, and then cultured at 37°C for 16 hours. After culture, 1% (v / v) xylene (mix of isomer, ≥98.5%) was added and reacted at 37°C for 1 hour to lyse the microorganisms (hereinafter referred to as the enzyme solution).

[0180] Then, 2.5% (v / v) enzyme solution was added to 1 ml of lysine (200 g / ℓ (pH 7.0)) containing 0.5 g / ℓ of the cofactor Pyridoxal 5'-phosphate (PLP). The conversion reaction to cadaverine was carried out using a thermomixer at 40°C and 1500 rpm for 10 minutes, and the cadaverine production was compared.

[0181]

[0182] Production Medium

[0183] Glucose 1%, Yeast extract 1.0%, Sodium chloride 0.5%, Bactroptone 1.6%, pH 7.2

[0184]

[0185] After the enzymatic reaction was completed, the cadaverine concentration was measured by High Performance Liquid Chromatography (HPLC), and the cadaverine conversion rate (mole% = mole of cadaverine produced / mole of lysine added) was calculated. The cadaverine conversion rates of foreign cadA are summarized in Table 2 below.

[0186] Strain-added Lysine Concentration (g / ℓ) Cadaverine Concentration (g / ℓ) Conversion Rate (mole%) W3110200 g / ℓ (pH 7.0) 0.08 0.0% W3110 / pECCG117_ParaB-cadA(Eco) 8 1.06 57.9% W3110 / pECCG117_ParaB-cadA(Pal) 12 1.8 287.1% W3110 / pECCG117_ParaB-cadA(Aci) 0.5 3.5% W3110 / pECCG117_ParaB-cadA(Sse) 9.1 26.5% W3110 / pECCG117_ParaB-cadA(Ppo) 2.08 1.48% W3110 / pECCG117_ParaB-cadA(Lpl)4.323.0%W3110 / pECCG117_ParaB-cadA(Cac)6.024.3%

[0187] As shown in Table 2 above, the strain with overexpressed E. coli cadA gene showed a conversion rate of about 57.9%, but the strain with overexpressed cadA derived from Pantoea alhagi showed a conversion rate of about 87.1%. This result is about 1.5 times higher than the conversion rate of E. coli cadA.

[0188] The above results confirmed that among the seven types of lysine decarboxylases selected in this application, cadA derived from Pantoea alhagi most efficiently increased the production capacity of cadaverine.

[0189]

[0190] Example 3: Construction of a variant lysine decarboxylase (cadA) library vector

[0191]

[0192] In this example, a variant library of the gene encoding cadA derived from Pantoea alhagi was constructed to obtain a variant lysine decarboxylase with a more improved cadaverine conversion rate. The library was constructed using an error-prone PCR kit (Clontech Diversify® PCR Random Mutagenesis Kit), and PCR was performed using primers of SEQ ID NO. 9 and SEQ ID NO. 10 with DNA of cadA derived from Pantoea alhagi as a template under conditions where mutations could occur. Specifically, under conditions where 0 to 3 mutations occur per 1000 bp, the process was performed 25 times by pre-heating at 94°C for 30 seconds, followed by 94°C for 30 seconds and 68°C for 1 minute and 30 seconds. As a result, the cadA(Pal, library) fragment was obtained.

[0193] PCR was performed using the pECCG117_ParaB-cadA (Pal) prepared in Example 1 as a template and primers of SEQ ID NO. 6 and SEQ ID NO. 21. PCR was performed using PfuUltra-™ high-reliability DNA polymerase (Stratagene), and the PCR conditions were repeated 30 times: denaturation at 95°C for 30 seconds; annealing at 55°C for 30 seconds; and polymerization at 72°C for 5 minutes. As a result, the pECCG117_ParaB vector fragment was obtained.

[0194] Subsequently, the cadA(Pal, library) fragment obtained by the above error-prone PCR and the pECCG117_ParaB vector fragment were cloned using the Takara Infusion Cloning Kit according to the provided manual at a molar concentration (M) of 2:1 to obtain a plasmid.

[0195] The constructed plasmid was transformed into Escherichia coli (W3110) by electroporation and plated on LB solid medium containing kanamycin (25 mg / L). After selecting 20 transformed colonies, the plasmid was obtained and its polynucleotide sequence was analyzed. It was confirmed that mutations were introduced at different positions with a frequency of 2 mutations / kb, and the obtained plasmid was named pECCG117_ParaB-cadA(Pal)-library.

[0196]

[0197] Example 4: Preparation of a cadaverine-converted strain with a variant lysine decarboxylase (cadA) library

[0198]

[0199] After transforming the pECCG11_ParaB-cadA(Pal)-library produced in Example 3 above into E. coli W3110 by electroporation, 5,000 colonies of strains with the inserted mutant gene were obtained by plating on LB solid medium containing kanamycin (25 mg / L), and each colony was named from W3110 / pECCG117_ParaB-cadA(Pal)-01 to W3110 / pECCG117_ParaB-cadA(Pal)-5000.

[0200] To determine the cadaverine conversion rate of the 5,000 secured strains, the parent strain and the strains were each inoculated into a 15 ml test tube containing 3 ml of production medium with the composition as follows, and then cultured at 37°C for 16 hours. After culture, 1% (v / v) xylene (mix of isomer, ≥98.5%) was added and reacted at 37°C for 1 hour to lyse the microorganisms (hereinafter referred to as the enzyme solution).

[0201] Then, 2.5% (v / v) enzyme solution was added to 1 ml of lysine (200 g / ℓ (pH 7.0)) containing 0.5 g / ℓ of the cofactor Pyridoxal 5'-phosphate (PLP). The conversion reaction to cadaverine was carried out using a thermomixer at 40°C and 1500 rpm for 10 minutes, and the cadaverine production was compared.

[0202]

[0203] Production Medium

[0204] Glucose 1%, Yeast extract 1.0%, Sodium chloride 0.5%, Bactroptone 1.6%, pH 7.2

[0205]

[0206] H decrease during the conversion of lysine to cadaverine +To select mutant strains with improved pH based on the concentration, the supernatant of the reaction solution after the enzymatic reaction was separated and reacted with the indicator 0.1% (v / v) cresol red. Cresol red undergoes structural changes depending on pH, and the concentration of cadaverine could be measured by analyzing the absorbance at 575 nm. These conditions are specifically described in the literature (Xi Y, Ye LD, Yu HW, et al. Enhanced thermal and alkaline stability of L-lysine decarboxylase CadA by combining directed evolution and computation-guided virtual screening. Bioresour Bioprocess. 2022;9(1):24. doi: 10.1186 / s40643-022-00510-w.). Absorbance was measured at a wavelength of 575 nm, and mutant strains with the greatest increase in absorbance were selected. To confirm the cadaverine production ability of one selected mutant strain, W3110 / pECCG17-ParaB-cadA (Pal, Wild type), and the parent strain W3110, culture and enzymatic reaction were carried out in the same manner as in Example 2.

[0207] After the enzymatic reaction was completed, the cadaverine concentration was measured using High Performance Liquid Chromatography (HPLC) to calculate the cadaverine conversion rate (mole%), and each cadaverine conversion rate is summarized in Table 3 below.

[0208] Genetically Added Lysine Concentration (g / ℓ) Cadaverine Concentration (g / ℓ) Conversion Rate (mole%) W3110200 g / ℓ (pH 7.0)0.050.04%W3110 / pECCG117_ParaB-cadA(Pal, Wild type)118.084.3%W3110 / pECCG117_ParaB-cadA(Pal)-3218139.198.9%

[0209] As shown in Table 3 above, it was confirmed that the cadaverine conversion rate of W3110 / pECCG117_ParaB-cadA(Pal)-3218 increased to a high level compared to W3110 / pECCG117_ParaB-cadA(Pal, Wild type). Gene sequencing was performed on the W3110 / pECCG117_ParaB-cadA(Pal)-3218 strain with improved cadaverine conversion rate, and the gene encoding cadA derived from Pantoea alhagi contained in the strain was compared with the gene encoding cadA derived from wild type Pantoea alhagi. As a result, it was confirmed that the variant strain contains a mutation in the cadA(Pal) gene.

[0210] Specifically, it was confirmed that the W3110 / pECCG117_ParaB-casA(Pal)-3218 strain contains a mutation in which the 451st amino acid, tyrosine (Y), of wild-type Pantoea alhagi cadA, which consists of the amino acid sequence of SEQ ID NO. 2, is substituted with phenylalanine (F) (Y451F).

[0211]

[0212] Example 5: Construction of a site-mutated vector for Pantoea alhagi-derived lysine decarboxylase (cadA)

[0213]

[0214] In order to confirm through Example 4 above that the 451st position of cadA derived from Pantoea alhagi is an important position for increasing the cadaverine conversion rate, a mutant strain in which the 451st position was substituted with an amino acid different from the original amino acid was produced and the effect was confirmed.

[0215] PCR was performed using the primer sequences in Table 4 below with pECCG117_ParaB-cadA(Pal) produced in Example 1 as a template to amplify DNA fragments of the gene encoding cadA derived from Pantoea alhagi. The PCR was performed using PfuUltra™ high-reliability DNA polymerase (Stratagene) under the conditions of denaturation at 95°C for 30 seconds; annealing at 55°C for 30 seconds; and polymerization at 72°C for 1 minute 30 seconds, repeated 30 times. As a result, a 1,374 bp DNA fragment in the 5' upstream region and an 808 bp DNA fragment in the 3' downstream region were obtained, centered around the mutation (residue 451) of the gene.

[0216] In order to obtain the araB promoter from Escherichia coli W3110, PCR was performed in the same manner as described above using primers of SEQ ID NO. 5 and SEQ ID NO. 6 with the genomic DNA of Escherichia coli W3110 as a template, and a promoter DNA fragment was obtained.

[0217] Mutant Plasmid-Substituted Amino Acid Primer Sequence No. 1 (451st amino acid residue of cadA derived from wild-type Pantoea alhagi) Y451S (Sequence No. 26) Sequence No. 9, 22 / 10, 23 Y451N (Sequence No. 27) Sequence No. 9, 24 / 10, 25 Y451F (Sequence No. 2)

[0218] Plasmids were obtained by cloning the pECCG117 vector (Korean Registered Patent No. 10-0057684), which was treated with the restriction enzyme BamHI at 37°C for 1 hour and then heat-treated at 65°C for 20 minutes, and the obtained DNA fragments (each cadA upstream region, cadA downstream region, araB promoter) in a molar concentration (M) of 2:1:1:1 (pECCG117 vector : cadA(Pal) upstream region : cadA(Pal) downstream region : araB promoter) using the Takara Infusion Cloning Kit according to the provided manual, and the names of the 18 types of plasmids obtained and the introduced gene information are listed in Table 5.

[0219] Mutant Plasmid Substitution Amino Acid Mutant Plasmid constructed to induce amino acid substitution Sequence No. 1 (451st amino acid residue of cadA derived from wild-type Pantoea alhagi) Y451SpECCG117_ParaB-cadA(Pal, Y451S) Y451NpECCG117_ParaB-cadA(Pal, Y451N) Y451FpECCG117_ParaB-cadA(Pal, Y451F)

[0220]

[0221] Example 6: Evaluation of Cadaverine Production Capacity of Variant Pantoea Alhaji-Derived Lysine Decarboxylase (cadA)

[0222]

[0223] After introducing 18 variant plasmids produced in Example 5, pECCG117_ParaB-cadA (Pal, wild type) produced in Example 1, and pECCG117_ParaB-cadA (Pal, Y451F) produced in Example 4 into wild-type Escherichia coli W3110 strains by electroporation, the transformed strains were obtained by plating them onto LB solid medium containing kanamycin (25 mg / L). Subsequently, flask evaluation and conversion reactions were carried out in the same manner as in Example 2, and the results are shown in Table 6 below.

[0224] Strain-added Lysine Concentration (g / ℓ) Cadaverine Concentration (g / ℓ) Average Conversion Rate (mole%) Batch 1 Batch 2 W 3 1 1 0 200 g / ℓ 0.0 3 0.0 3 0.0% W 3 1 1 0 / pECCG117_ParaB-cadA(Pal, Y451S) 12 1.8 1 19.9 86.4% W 3 1 1 0 / pECCG117_ParaB-cadA(Pal, Y451N) 12 2.1 1 19 86.1% W 3 1 1 0 / pECCG117_ParaB-cadA(Pal, Y451F) 13 9.0 1 39.4 99.5% W 3 1 1 0 / pECCG117_ParaB-cadA(Pal, wild type) 11 9.9 1 1 7.8 85.0%

[0225] As a result, as shown in Table 6, it was confirmed that mutant strains into which the Y451S, Y451N, and Y451F variants of Pantoea alhagi-derived cadA were introduced converted lysine to cadaverine at a higher level than the wild type. In particular, it was confirmed that the E. coli W3110 strain containing the variant Pantoea alhagi-derived cadA in which the 451st amino acid is substituted with phenylalanine converted lysine to cadaverine at a significantly higher level than W3110 / pECCG117_ParaB-cadA (Pal, wild type).

[0226]

[0227] As a result, it was confirmed that the 451st position of cadA derived from wild-type Pantoea alhagi is an important position for activity, and that it is an important position that affects cadaverine production capacity when substituted with other amino acids.

[0228]

[0229] Example 7: Construction of a mutant vector of lysine decarboxylase (cadA) derived from a variant Pantoea alhaji and evaluation of cadaverine production capacity

[0230]

[0231] Through Example 6 above, it was confirmed that substituting tyrosine (Y) at the 451st position of cadA derived from Pantoea alhagi (WP_085071266.1) with phenylalanine (F) increased the cadaverine conversion rate the most. To confirm that this site is an important location, the importance of the 451st position was also confirmed in another Pantoea alhagi (Pantoea alhagi NX-11, WP_250650242.1, SEQ ID No. 34) listed on the National Center for Biotechnology Information (NCBI).

[0232] Gene synthesis was performed based on the genetic information of Pantoea alhagi (WP_250650242.1) specified in NCBI, and one type of template DNA encoding them was obtained. A vector was constructed to increase the expression of the obtained Pantoea alhagi (WP_250650242.1) cadA, and the method is as follows.

[0233] PCR was performed using the obtained Pantoea alhagi (NX-11, WP_250650242.1) cadA DNA fragment as a template and the primer sequences in Table 7. PCR was performed using PfuUltra-™ high-reliability DNA polymerase (Stratagene), and the PCR conditions consisted of denaturation at 95°C for 30 seconds; annealing at 55°C for 30 seconds; and polymerization at 72°C for 1 minute 30 seconds, repeated 30 times. As a result, the Pantoea alhagi (NX-11, WP_250650242.1) cadA DNA fragment was obtained. To obtain the araB promoter derived from Escherichia coli (W3110), primers of SEQ ID NO. 5 and SEQ ID NO. 6 were used with the genomic DNA of Escherichia coli as a template, and DNA fragments were obtained by performing PCR on the promoter in the same manner as above. After treating with the restriction enzyme BamHI at 37°C for 1 hour and then heat-treating at 65°C for 20 minutes, the pECCG117 vector (Korean Registered Patent No. 10-0057684) and DNA fragments (ParaB, each cadA) were prepared at a molar concentration (M) of 2:1:1 and cloned according to the provided manual using the Takara Infusion Cloning Kit to obtain plasmids, and the names of the vectors and information on the introduced genes are indicated in Table 7.

[0234] Microorganism origin NCBI NO. Substituted amino acid Primer Sequence number Construction vector 1 Pantoea alhagi strain NX-11WP_250650242.1(Sequence number 34)Wild type 36, 37 pECCG117_ParaB-cadA(Pal2) 2 Pantoea alhagi strain NX-11WP_250650242.1(Sequence number 35)Y45IF 36,38 / 37, 39 pECCG117_ParaB-cadA(Pal2, Y451F)

[0235] PCR was performed using the primer sequences of Table 7 and pECCG117_ParaB-cadA(Pal2) prepared in Table 7 to amplify the variant Pantoea alhagi (NX-11, WP_250650242.1) cadA(Y451F) DNA fragment. The PCR was performed using PfuUltra™ high-reliability DNA polymerase (Stratagene) under the conditions of denaturation at 95°C for 30 seconds; annealing at 55°C for 30 seconds; and polymerization at 72°C for 1 minute 30 seconds, repeated 30 times. As a result, a 1,374 bp DNA fragment in the 5' upstream region and an 808 bp DNA fragment in the 3' downstream region were obtained, centered around the mutation (residue 451) of the gene.

[0236] In order to obtain the araB promoter from Escherichia coli W3110, PCR was performed in the same manner as described above using primers of SEQ ID NO. 5 and SEQ ID NO. 6 with the genomic DNA of Escherichia coli W3110 as a template, and a promoter DNA fragment was obtained. A plasmid was obtained by cloning the pECCG117 vector (Korean Registered Patent No. 10-0057684), which was treated with the restriction enzyme BamHI at 37°C for 1 hour and then heat-treated at 65°C for 20 minutes, and the obtained DNA fragments (each cadA upstream region, cadA downstream region, and araB promoter) in a molar concentration (M) of 2:1:1:1 (pECCG117 vector: cadA(Pal) upstream region: cadA(Pal) downstream region: araB promoter) according to the provided manual using the Takara Infusion Cloning Kit, and the name of the plasmid and the introduced gene information are indicated in Table 7.

[0237]

[0238] Example 8: Evaluation of Cadaverine Production Capacity of Variant Pantoea Alhaji-Derived Lysine Decarboxylase (cadA)

[0239]

[0240] Two types of plasmids prepared in Example 7 were introduced into wild-type Escherichia coli W3110 by electroporation, and each transformed strain was obtained by plating them on LB solid medium containing kanamycin (25 mg / L). Subsequently, flask evaluation and conversion reactions were carried out in the same manner as in Example 2, and the results are shown in Table 8 below.

[0241] Strain-added Lysine Concentration (g / ℓ) Cadaverine Concentration (g / ℓ) Average Conversion Rate (mole%) Batch 1 Batch 2W 3110 200 g / ℓ 0.02 0.01 0.0% W 3110 / pECCG117_ParaB-cadA(Pal2) 114.01 10.38 0.2% W 3110 / pECCG117_ParaB-cadA(Pal2, Y451F) 139.01 40.19 9.8%

[0242] As a result, as shown in Table 8, it was confirmed that the mutant strain introduced with the Y451F mutation of cadA from Pantoea alhagi (NX-11, WP_250650242.1) converts lysine to cadaverine at a higher level than the wild type.

[0243] As a result, it was confirmed that the 451st position of cadA derived from wild-type Pantoea alhagi (Pantoea alhagi NX-11, WP_250650242.1) is an important position for activity.

[0244]

[0245] As a result, it was confirmed that the 451st position of cadA derived from wild-type Pantoea alhagi is an important position for activity, and that it is an important position that affects cadaverine production capacity when substituted with other amino acids.

[0246]

[0247] From the foregoing description, those skilled in the art to which this application pertains will understand that this application may be implemented in other specific forms without altering its technical concept or essential features. In this regard, the embodiments described above should be understood as illustrative in all respects and not restrictive. The scope of this application 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

A lysine decarboxylase variant in which the amino acid corresponding to the 451st position of the amino acid sequence of SEQ ID NO. 1 is substituted with another amino acid. In claim 1, the lysine decarboxylase variant is a lysine decarboxylase variant having at least 85% sequence identity with the amino acid sequence of SEQ ID NO.

1. In claim 1, the variant is a lysine decarboxylase variant comprising the amino acid sequence of SEQ ID NO.

40. In claim 1, the variant is a lysine decarboxylase variant having 90% or more sequence identity with the amino acid sequence of SEQ ID NO. 2, SEQ ID NO. 26, SEQ ID NO. 27, or SEQ ID NO.

35. In claim 1, the variant comprises a polypeptide described by the amino acid sequence of the following general formula 1: [General Formula 1] X1GPHKEA EEYIARVFNA ERSYMVTNGT STANKIVGMY SAPAGSTILI DRNCHKSLTH LMMMSDVTPI YFRPTRNAYG ILGGIPQSEF QHETIAKRVK ATPNASWPTH AVITNSTYDG LLYNTDYIKK TLDVKSIHFD SAWVPYTNFH PIYX2GKSGMS GX3RVEGKVIY ETQSTHKLLA AFSQASMIHV KGDVNQETFN EAYMMHTTTS PHYGIVASTE TAAAMMRGNA GKRLIGESIE RALRFRKEIK RLNAESEGWF IDKTKALSLL RALTDFKRAF DLNLRVKNIL PSLWREAPDF YKDMRIQELA ANIHQLIQRH NLPDLMYRAF ENLPAMVLTP YHAFQKELHG EVEEVYLEEM VGRVSANMIL PYPPGVPLIM PGEMITDETL PVLEFLQLLC EIGSHYPGFE TDIHGAYRQE DGRYTVKVLK Here, X1 of the above general formula 1 is serine or threonine, and X2 is serine or threonine, and X3 is glycine or aspartic acid, and X4 is an amino acid other than tyrosine, and X5 is glycine or glutamic acid, and X6 is glutamic acid or aspartic acid. In claim 1, the lysine decarboxylase is a lysine decarboxylase variant derived from Pantoea alhagi. A lysine decarboxylase variant according to claim 1, wherein the other amino acid is a polar or non-polar amino acid. A lysine decarboxylase variant according to claim 1, wherein the other amino acid is selected from phenylalanine, serine, and asparagine. A polynucleotide encoding a lysine decarboxylase variant of any one of claims 1 to 8. A microorganism comprising any one or more of a lysine decarboxylase variant of any one of claims 1 to 8 and a polynucleotide encoding said variant. In paragraph 10, the microorganism is selected from microorganisms of the genus Escherichia, the genus Corynebacterium, and the genus Bacillus. In claim 11, the microorganism is selected from Escherichia coli, Corynebacterium glutamicum, and Bacillus subtilis. A method for producing cadaverine, comprising the step of converting lysine into cadaverine using a lysine decarboxylase variant of any one of claims 1 to 8 or a microorganism expressing the same. A method for manufacturing cadaverine according to claim 13, comprising the step of recovering the converted cadaverine. A composition for producing cadaverine comprising one or more of a lysine decarboxylase variant of any one of claims 1 to 8 and a microorganism expressing the same. Use of a lysine decarboxylase variant of any one of claims 1 to 8 or a microorganism expressing the same for the production of cadaverine.