Flavin-containing monooxygenase variant and method for producing indigo using same

Abstract

The present invention relates to a flavin-containing monooxygenase (FMO) variant, a polynucleotide encoding same, a microorganism comprising the FMO variant or the polynucleotide encoding same, and a method for producing indigo by using the microorganism. The FMO variant according to the present invention not only produces natural indigo without environmental pollution, but also has an improved ability to produce indigo, and thus will be useful in indigo dyeing or cosmetic ingredient industrial fields.

Description

Flavin-containing monooxygenase mutant and method for producing indigo using the same

[0001] The present invention relates to a flavin-containing monooxygenase (FMO) variant having improved indigo production ability, a polynucleotide encoding the same, a microorganism comprising the FMO variant or a polynucleotide encoding the same, and a method for producing indigo using the microorganism.

[0002]

[0003] Indigo is an organic compound dye that exhibits a characteristic blue color. Traditionally, indigo dyes were extracted from plants such as Polygonum tinctorium and Indigofera tinctoria, but the irregular supply of natural indigo depending on the cultivation environment and the high production cost limited its mass production. In 1882, the Beyer-Drusen indigo synthesis method was developed to produce indigo from 2-hydroxynitrobenzene and acetone, which improved productivity. Currently, BASF has developed a manufacturing method that can synthesize indigo from aniline, making mass production of indigo possible through chemical synthesis. However, chemical synthesis methods have the problem of using toxic by-products (e.g., formaldehyde, HCN, NANH2) during the reaction process and generating a large amount of process wastewater after the reaction, which pollutes the environment.

[0004] Accordingly, a method for producing indigo using environmentally friendly microorganisms is attracting attention. The biological synthesis of indigo largely occurs in three steps. (1) Indole is supplied when tryptophan is broken down into indole, ammonia, and pyruvate by tryptophanase, encoded by the tnaA gene. (2) Indole is converted to indoxyl by flavin-containing monooxygenase (FMO). (3) Indoxyl spontaneously oxidizes under aerobic conditions, dimerizing to produce indigo.

[0005]

[0006]

[0007]

[0008]

[0009] Microbial indigo production was first reported in a recombinant E. coli strain expressing naphthalene dioxygenase (BD Ensley et al. Expression of Naphthalene Oxidation Genes in Escherichia coli Results in the Biosynthesis of Indigo. SCIENCE Vol. 222, Issue 4620 pp. 167-169 (October 14, 1983)), and indigo production is known in recombinant strains that have induced the expression of dioxygenases or monooxygenases such as phenol hydroxylase, FMO, indole oxygenase, and stylene monooxygenase.

[0010] The present invention aims to solve the environmental pollution problem of chemical synthesis methods by producing natural indigo using microorganisms, and to develop a technology for producing natural indigo at an industrial level by overcoming the limitations of productivity through enzyme improvement.

[0011]

[0012] The present invention provides a flavin-containing monooxygenase (FMO) mutant in which the amino acid corresponding to position 246 in the amino acid sequence of wild-type FMO is substituted with histidine.

[0013] The present invention seeks to provide a polynucleotide encoding the above FMO variant.

[0014] The present invention seeks to provide a vector comprising a polynucleotide encoding the above FMO variant.

[0015] The present invention seeks to provide a microorganism comprising the FMO variant or a polynucleotide encoding the FMO variant.

[0016] The present invention seeks to provide a method for producing indigo, comprising a step of culturing a microorganism comprising the FMO variant or a polynucleotide encoding the FMO variant in a medium.

[0017] The present invention seeks to provide a composition for producing indigo, comprising a microorganism comprising the FMO variant or a polynucleotide encoding the FMO variant, a medium in which the microorganism is cultured, or both.

[0018]

[0019] One aspect of the present invention provides a flavin-containing monooxygenase (FMO) mutant in which the amino acid corresponding to position 246 in the amino acid sequence of SEQ ID NO: 1 is substituted with histidine.

[0020] In the present invention, the term "flavin-containing monooxygenase variant (FMO variant)" means any polypeptide having flavin-containing monooxygenase activity or a variant comprising a substitution of an amino acid corresponding to position 246 from the N-terminus of SEQ ID NO: 1 with another amino acid in a flavin-containing monooxygenase.

[0021] The variants of the present invention may be described as “flavin-containing monooxygenase variants”, “(variant) polypeptides having flavin-containing monooxygenase activity”, “FMO variants”.

[0022] In the present invention, the term "flavin-containing monooxygenase (FMO)" refers to a general term for a monooxygenase containing FAD as a prosthetic group, and includes all flavin-containing monooxygenases known in the art. Flavin-containing monooxygenase is involved in the biosynthetic process of indigo and can convert indole into indoxyl. Looking at the biosynthetic pathway of indigo, first, tryptophan is decomposed into indole by tryptophanase, then indole is converted into indoxyl by flavin-containing monooxygenase, and then chemically unstable indoxyl undergoes oxidative dimerization under aerobic conditions to produce indigo.

[0023] The amino acid sequence of the flavin-containing monooxygenase of the present invention can be obtained from the NCBI's GenBank, a known database. The flavin-containing monooxygenase may be a protein comprising the amino acid sequence of SEQ ID NO: 1, but is not limited thereto. As another example, the flavin-containing monooxygenase may be derived from a microorganism of the genus Methylophaga, for example, FMO (abbreviated as “MaFMO”) derived from Methylophaga aminisulfidivorans (HS Choi et al. A novel flavin-containing monooxygenase from Methylophaga sp. strain SK1 and its indigo synthesis in Escherichia coli. Biochemical and Biophysical Research Communications 306 930-936 (2003)), but is not limited thereto, and may include without limitation a sequence having the same flavin-containing monooxygenase activity as the amino acid sequence.

[0024] For example, the flavin-containing monooxygenase of the present invention may be a protein comprising the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or more, or 99% or more homology or identity thereto. In addition, it is obvious that a protein having an amino acid sequence in which a part of the sequence is deleted, modified, substituted or added is also included within the scope of the protein to be modified in the present invention, as long as it has such homology or identity and exhibits an effect corresponding to the protein.

[0025] In addition, although the flavin-containing monooxygenase to be mutated in the present invention is defined as a protein including the amino acid sequence of SEQ ID NO: 1, it does not exclude meaningless sequence additions before and after the amino acid sequence of SEQ ID NO: 1, mutations that may occur naturally, or silent mutations thereof, and it is obvious to those skilled in the art that if it has the same or corresponding activity as the protein consisting of the amino acid sequence of SEQ ID NO: 1, it corresponds to the flavin-containing monooxygenase of the present invention.

[0026] That is, even if the present invention describes a 'protein or polypeptide having an amino acid sequence described by a specific sequence number' or a 'protein or polypeptide comprising an amino acid sequence described by a specific sequence number', it is clear that a protein having an amino acid sequence in which some sequences are deleted, modified, substituted or added can also be used in the present invention, if it has the same or corresponding activity as a polypeptide consisting of the amino acid sequence of the corresponding sequence number.

[0027] As used herein, the terms "homology" or "identity" refer to the degree of similarity between two given amino acid sequences or base sequences, which may be expressed as a percentage. The terms homology and identity are often used interchangeably.

[0028] Sequence homology or identity of conserved polynucleotides or polypeptides is determined by standard alignment algorithms, and may be combined with default gap penalties established by the program being used. In practice, homologous or identical sequences can generally hybridize with all or part of the sequence under moderate or high stringency conditions. It should be appreciated that hybridization also includes hybridization with polynucleotides containing common codons or codons that take codon degeneracy into account.

[0029] Whether any two polynucleotide or polypeptide sequences are homologous, similar or identical can be determined using known computer algorithms such as the "FASTA" program using default parameters, for example as in Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444. Alternatively, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented 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) can be determined using the GCG program package (Devereux, J., et al, Nucleic Acids Research 12: 387(1984)), BLASTP, BLASTN, FASTA (Atschul, [S.] [F.,] [ET AL, J MOLEC BIOL 215]: 403 (1990); Guide to Huge Computers, Martin J. Bishop, [ED.,] Academic Press, San Diego, 1994, and [CARILLO et al.](1988) SIAM J Applied Math 48: 1073). For example, homology, similarity, or identity can be determined using BLAST or ClustalW from the National Center for Biotechnology Information database.

[0030] Homology, similarity, or identity of polynucleotides or polypeptides can be determined by comparing sequence information, for example, using a GAP computer program such as that of Needleman et al. (1970), J Mol Biol. 48:443, as disclosed, for example, in Smith and Waterman, Adv. Appl. Math (1981) 2:482. In brief, the 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). Default parameters for the GAP program include (1) a binary comparison matrix (containing values ​​of 1 for identity and 0 for non-identity) and (2) a comparison matrix as disclosed by Gribskov et al. (1986) Nucl. Acids Res. 48:443, as disclosed by Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353-358 (1979). 14: 6745 weighted comparison matrix (or 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 and a gap extension penalty of 0.5); and (3) no penalty for terminal gaps. Therefore, the term "homology" or "identity" as used herein refers to the relevance between sequences.

[0031] As used herein, the term "variant" or "modified polypeptide" refers to a protein in which one or more amino acids differ from the recited sequence by conservative substitution and / or modification, but the functions or properties of the protein are maintained.

[0032] The above variants differ from the identified sequence by several amino acid substitutions, deletions, or additions. Such variants can generally be identified by modifying one or more amino acids in the amino acid sequence of the protein and evaluating the properties of the modified protein. That is, the ability of the variant may be increased, unchanged, or decreased compared to the native protein. In addition, some variants may include variant polypeptides in which one or more portions, such as the N-terminal leader sequence or the transmembrane domain, are deleted. Other variants may include variants in which portions are deleted from the N- and / or C-termini of the mature protein. The terms "variant" or "variant polypeptide" may be used interchangeably with terms such as variant, modification, mutated protein, and variation (in English, modification, modified protein, mutant, mutein, divergent, variant, etc.), and are not limited thereto as long as the terms are used in the meaning of variation.

[0033] As used herein, the term "conservative substitution" refers to the replacement of one amino acid with another amino acid having similar structural and / or chemical properties. Typically, conservative substitutions have little or no effect on the activity of the resulting polypeptide.

[0034] The above variants may have, for example, one or more conservative substitutions, while still retaining one or more biological activities. Such amino acid substitutions may generally be based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues.

[0035] Examples of these classifications include positively charged (basic) amino acids such as arginine, lysine, and histidine; negatively charged (acidic) amino acids such as glutamic acid and aspartate; amino acids with nonpolar side chains (nonpolar amino acids) such as glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline; and amino acids with polar or hydrophilic side chains (polar amino acids) such as serine, threonine, cysteine, tyrosine, asparagine, and glutamine. As another example, amino acids with charged side chains (electrically charged amino acids) such as arginine, lysine, histidine, glutamic acid, and aspartate, and amino acids with uncharged side chains (also called neutral amino acids) such as glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. As another example, phenylalanine, tryptophan, and tyrosine can be classified as aromatic amino acids. As another example, valine, leucine, and isoleucine can be classified as branched amino acids. As another example, the 20 amino acids can be classified according to size, starting with a group of amino acids with relatively small volume: glycine, alanine, and serine; They can be classified into five groups: cysteine, proline, threonine, aspartate, asparagine; valine, histidine, glutamic acid, glutamine; isoleucine, leucine, methionine, lysine, arginine; and phenylalanine, tryptophan, tyrosine. However, they are not necessarily limited to this.

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

[0037] In one embodiment, the variant of the present invention may be a flavin-containing monooxygenase variant or a variant polypeptide having flavin-containing monooxygenase activity, wherein the amino acid corresponding to position 246 from the N-terminus of SEQ ID NO: 1 among the above-mentioned flavin-containing monooxygenases is substituted with another amino acid.

[0038] In one embodiment, the variant may be capable of increasing indigo production compared to the protein before mutation, the wild type protein, the native polypeptide, or the unmodified polypeptide.

[0039] In the present invention, "substitution with another amino acid" is not limited to an amino acid different from the amino acid prior to substitution. Meanwhile, when the present invention expresses that "a specific amino acid has been substituted," it is self-evident that the amino acid has been substituted with an amino acid different from the amino acid prior to substitution, even if it is not specifically stated that it has been substituted with another amino acid.

[0040] A variant according to the present invention may be a flavin-containing monooxygenase variant in which the amino acid corresponding to position 246 in the amino acid sequence of SEQ ID NO: 1 is substituted with histidine.

[0041] The "N-position" of the present invention may include the N-position and an amino acid position corresponding to the N-position. For example, 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.

[0042] As used herein, the term "corresponding to" refers to an amino acid residue at a position listed in a polypeptide, or an amino acid residue that is similar, identical, or homologous to the residue listed in the polypeptide. Identifying an amino acid at a corresponding position may be determining a specific amino acid in a sequence that references a specific sequence. As used herein, "corresponding region" generally refers to a similar or corresponding position in a related protein or reference protein.

[0043] In the present invention, specific numbering may be used for amino acid residue positions within the protein used in the present invention. For example, by aligning the polypeptide sequences of the target protein to be compared with the protein of the present invention, it is possible to renumber positions corresponding to amino acid residue positions in the protein of the present invention.

[0044] For example, any amino acid sequence can be aligned with SEQ ID NO: 1, and based on this, each amino acid residue of the amino acid sequence can be numbered by referring to the numerical position of the amino acid residue corresponding to the amino acid residue in SEQ ID NO: 1. For example, a sequence alignment algorithm such as that described in the present invention can identify the position of an amino acid, or the position at which a modification such as a substitution, insertion, or deletion occurs, by comparing it with a query sequence (also referred to as a “reference sequence”).

[0045] For such alignment, for example, 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 is not limited thereto, and any sequence alignment program known in the art, pairwise sequence comparison algorithm, etc. can be appropriately used.

[0046] In one embodiment, the flavin-containing monooxygenase variant of the present invention may be a variant comprising a sequence in which the amino acid corresponding to position 246 of SEQ ID NO: 1 is substituted with histidine and has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology or identity with SEQ ID NO: 1. The flavin-containing monooxygenase variant may be a variant having less than 100% homology or identity with SEQ ID NO: 1.

[0047] In one embodiment, the flavin-containing monooxygenase variant of the present invention may be a polypeptide having flavin-containing monooxygenase activity, wherein the amino acid corresponding to position 246 of SEQ ID NO: 1 is substituted with histidine, and has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology or identity with SEQ ID NO: 1.

[0048] In one embodiment, the flavin-containing monooxygenase variant of the present invention may be a polypeptide having flavin-containing monooxygenase activity, wherein the amino acid corresponding to position 246 of SEQ ID NO: 1 is histidine, and has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology or identity with SEQ ID NO: 1.

[0049] In addition, it is obvious that a variant having an amino acid sequence in which some of the sequences are deleted, modified, substituted, conservatively substituted or added is also included within the scope of the present invention, provided that the amino acid sequence has such homology or identity and exhibits an effect corresponding to that of the variant of the present invention.

[0050] In one embodiment, the variant of the present invention may have, comprise, consist essentially of, or consist of the amino acid sequence set forth in SEQ ID NO: 3.

[0051] Even if the present invention describes a "protein having an amino acid sequence described by 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 invention, as long as it has the same or corresponding activity as the protein consisting of the amino acid sequence of the corresponding sequence number. For example, if it has the same or corresponding activity as the mutant protein, it does not exclude sequence additions that do not alter the function of the protein before or after the amino acid sequence, mutations that may occur naturally, silent mutations or conservative substitutions thereof, and it is obvious that even if it has such sequence additions or mutations, it falls within the scope of the present invention.

[0052] In any one of the embodiments described above, the variant of the present invention may comprise a sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99% or more homology or identity with SEQ ID NO: 3, or may be composed of the amino acid sequence. In the variant, the amino acid corresponding to position 246 of SEQ ID NO: 3 may be histidine.

[0053] In any one of the embodiments described above, the flavin-containing monooxygenase variant of the present invention may have enhanced flavin-containing monooxygenase activity, but is not limited thereto.

[0054] In any one of the embodiments described above, the flavin-containing monooxygenase variant of the present invention may have an activity that increases indigo production capacity compared to a wild-type, native or unmodified polypeptide having flavin-containing monooxygenase activity, but is not limited thereto.

[0055]

[0056] Another aspect of the present invention provides a polynucleotide encoding a variant of the present invention. The variant is as described above.

[0057] In the present invention, the term "polynucleotide" means a polymer of nucleotides in which nucleotide units (monomers) are covalently bonded to form a long chain, and is a DNA or RNA strand of a certain length or longer, and more specifically, a polynucleotide fragment encoding the variant.

[0058] The polynucleotide of the present invention can be modified in various ways in the coding region without altering the amino acid sequence of the variant of the present invention, taking into account codon degeneracy or preferred codons in the organism that is to express the variant of the present invention. Therefore, it is self-evident that the polynucleotide can also be translated into a polypeptide comprising the amino acid sequence of the variant of the present invention or a polypeptide having homology or identity therewith due to codon degeneracy.

[0059] For example, the polynucleotide of the present invention may have or include, or consist of or consist essentially of, a base sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology or identity with the polynucleotide sequence of SEQ ID NO: 4, but is not limited thereto. In addition, in the polynucleotide sequence having the homology or identity, the codon encoding the amino acid corresponding to position 246 of SEQ ID NO: 1 may be one of the codons encoding histidine, but is not limited thereto.

[0060] In addition, the polynucleotide of the present invention may include, without limitation, a probe that can be prepared from a known genetic sequence, for example, a sequence that can hybridize under stringent conditions with a complementary sequence to all or part of the polynucleotide sequence of the present invention. The "stringent conditions" above refer to conditions that enable 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 in which polynucleotides having high homology or identity hybridize with each other, polynucleotides having 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 homology or identity hybridize with each other, and polynucleotides having lower homology or identity do not hybridize with each other, or conditions in which washing is performed once, specifically twice or three times, at a salt concentration and temperature equivalent to 60°C, 1×SSC, 0.1% SDS, specifically 60°C, 0.1×SSC, 0.1% SDS, and more specifically 68°C, 0.1×SSC, 0.1% SDS, which are washing conditions of typical southern hybridization, are performed.

[0061] Hybridization requires that two nucleic acids have complementary sequences, although mismatches between bases are possible depending on the stringency of hybridization. The term "complementary" is used to describe the relationship between nucleotide bases that can hybridize with each other. For example, in DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Accordingly, the polynucleotides of the present invention may also include isolated nucleic acid fragments that are complementary in their entirety, as well as substantially similar nucleic acid sequences.

[0062] Specifically, a polynucleotide having homology or identity with the polynucleotide of the present invention can be detected using hybridization conditions including a hybridization step at a Tm value of 55°C and using the conditions described above. In addition, the Tm value may be, but is not limited to, 60°C, 63°C, or 65°C, and can be appropriately adjusted by a person skilled in the art depending on the purpose.

[0063] The appropriate stringency for hybridizing the polynucleotides depends on the length and degree of complementarity of the polynucleotides, variables which are well known in the art (e.g., J. Sambrook et al., supra).

[0064]

[0065] Another aspect of the present invention provides a vector comprising a polynucleotide encoding a variant of the present invention. The variant and polynucleotide are as described in the other aspects above.

[0066] The above vector may be an expression vector for expressing the above polynucleotide in a microorganism, but is not limited thereto.

[0067] The term "vector" as used herein may include a DNA construct comprising a base sequence of a polynucleotide encoding a target polypeptide operably linked to a suitable expression control region (or expression control sequence) so as to enable expression of the target polypeptide in a suitable host. The expression control region may include a promoter capable of initiating transcription, an optional operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosome binding site, and sequences 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 integrated into the genome itself.

[0068] The vector used in the present invention is not particularly limited, and any vector known in the art can be used. Examples of commonly used vectors include plasmids, cosmids, viruses, and bacteriophages in a natural or recombinant state. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A can be used as phage vectors or cosmid vectors, and pDZ series, pBR series, pUC series, pBluescriptII series, pGEM series, pTZ series, pCL series, and pET series can be used as plasmid vectors. Specifically, pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pCL1920, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vectors, etc. can be used.

[0069] For example, a polynucleotide encoding a target polypeptide can be inserted into a chromosome via a vector for intracellular chromosomal insertion. The insertion of the polynucleotide into the chromosome can be achieved by any method known in the art, for example, homologous recombination, but is not limited thereto. A selection marker for confirming the chromosomal insertion can be additionally included. The selection marker is used to select cells transformed with the vector, i.e., to confirm the insertion of the target nucleic acid molecule. Markers that confer a selectable phenotype, such as drug resistance, nutrient requirement, cytotoxic agent resistance, or expression of a surface polypeptide, can be used. In an environment treated with a selective agent, only cells expressing the selection marker survive or exhibit other phenotypic traits, so that transformed cells can be selected.

[0070] The term "transformation" in the present invention refers to introducing a vector containing a polynucleotide encoding a target polypeptide into a microorganism or into a microorganism so that the polypeptide encoded by the polynucleotide can be expressed in the microorganism. The transformed polynucleotide can include both an integrated location within the chromosome of the microorganism or an extrachromosomal location, as long as it can be expressed in the microorganism. In addition, the polynucleotide includes DNA and / or RNA encoding the target polypeptide. The polynucleotide can be introduced in any form as long as it can be introduced into the microorganism and expressed. For example, the polynucleotide can be introduced into the microorganism in the form of an expression cassette, which is a genetic construct containing all elements necessary for autonomous expression. The expression cassette can typically include a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal, which are operably linked to the polynucleotide. The expression cassette can be in the form of an expression vector capable of autonomous replication. Additionally, the polynucleotide may be introduced into a microorganism in its own form and operably linked to a sequence required for expression in the microorganism, but is not limited thereto.

[0071] Additionally, the term "operably linked" as used above means that the polynucleotide sequence is functionally linked to a promoter sequence that initiates and mediates transcription of the polynucleotide encoding the target variant of the present invention.

[0072] In the present invention, the term "introduction" refers to a method of delivering a polynucleotide encoding a variant of the present invention or a vector containing the same to a host cell. Such introduction can be easily performed according to a method commonly used in the art. Common examples include the CaCl2 precipitation method, the Hanahan method, which increases efficiency by using a reducing agent called DMSO (dimethyl sulfoxide) in the CaCl2 method, electroporation, calcium phosphate precipitation, protoplast fusion, a stirring method using silicon carbide fibers, a transformation method using PEG, dextran sulfate, lipofectamine, and desiccation / inhibition-mediated transformation methods. The method for transforming the vector is not limited to the above examples, and any transformation or transfection method commonly used in the art can be used without limitation.

[0073]

[0074] Another aspect of the present invention provides a microorganism comprising a flavin-containing monooxygenase variant of the present invention or a polynucleotide encoding the variant.

[0075] In one embodiment, the microorganism may comprise a vector comprising a polynucleotide encoding the variant.

[0076] In one embodiment, the microorganism of the present invention may be a microorganism having indigo production ability.

[0077] In the present invention, the term "microorganism (or strain)" includes both wild-type microorganisms and microorganisms that have undergone genetic modification naturally or artificially, and may be a microorganism that has a specific mechanism weakened or strengthened due to causes such as the insertion of an external gene or the enhancement or inactivation of the activity of an endogenous gene, and may be a microorganism that includes genetic modification for the production of a desired polypeptide, protein, or product. In the present invention, "microorganism," "strain," and "microorganism" may be used interchangeably without limitation with the same meaning.

[0078] The term "indigo-producing microorganism" in the present invention refers to a prokaryotic or eukaryotic microorganism strain capable of producing indigo within the organism, and may include a microorganism in which indigo-producing ability has been conferred on a parent strain that does not have indigo-producing ability, or a microorganism that inherently possesses indigo-producing ability. Indigo-producing ability may be conferred or enhanced through species improvement.

[0079] In one embodiment, the microorganism of the present invention may be a microorganism that naturally has flavin-containing monooxygenase or indigo-producing ability; or a microorganism that has been introduced with a variant of the present invention or a polynucleotide encoding the same (or a vector including the polynucleotide) into a parent strain that does not have flavin-containing monooxygenase or indigo-producing ability and / or has been endowed with indigo-producing ability, but is not limited thereto.

[0080] In one embodiment, the microorganism of the present invention includes, but is not limited to, a microorganism in which a gene on a chromosome encoding a flavin-containing monooxygenase is mutated to include a sequence of a flavin-containing monooxygenase variant of the present invention and / or a microorganism in which a vector including a polynucleotide encoding a flavin-containing monooxygenase variant of the present invention is introduced to include a flavin-containing monooxygenase variant of the present invention.

[0081] As any one of the above-described embodiments, the microorganism provided in the present invention may be a microorganism genetically modified to express the flavin-containing monooxygenase of the present invention.

[0082] In the present invention, the term "unmodified microorganism" does not exclude a strain that contains a mutation that can occur naturally in a microorganism, and may refer to a wild-type strain or a natural strain itself, or a strain before its characteristics are changed by genetic mutation due to natural or artificial factors. For example, the unmodified microorganism may refer to a strain that is not introduced or before the flavin-containing monooxygenase variant described herein is introduced. The "unmodified microorganism" may be used interchangeably with "pre-modified strain," "pre-modified microorganism," "unmutated strain," "unmodified microorganism," "unmutated microorganism," or "reference microorganism."

[0083] The microorganism having the indigo production ability of the present invention may be, but is not limited to, a microorganism comprising at least one of the variant of the present invention, the polynucleotide of the present invention, and the vector comprising the polynucleotide of the present invention; a microorganism modified to express the variant of the present invention or the polynucleotide of the present invention; a microorganism (e.g., a recombinant strain) expressing the variant of the present invention or the polynucleotide of the present invention; or a microorganism (e.g., a recombinant strain) having the activity of the variant of the present invention.

[0084] For example, the strain of the present invention is a cell or microorganism that is transformed with a vector containing a polynucleotide of the present invention or a polynucleotide encoding a variant of the present invention, and expresses the variant of the present invention. The strain of the present invention may include all microorganisms capable of producing indigo, including the variant of the present invention. For example, the microorganism of the present invention may be a recombinant strain in which a polynucleotide encoding the variant of the present invention is introduced into a natural wild-type microorganism or a microorganism having indigo production ability, thereby expressing a flavin-containing monooxygenase variant, and thus increasing indigo production ability.

[0085] The strain with increased indigo production ability may be a microorganism with increased indigo production ability compared to a natural wild-type microorganism or a flavin-containing monooxygenase-unmodified microorganism (e.g., a microorganism expressing a wild-type flavin-containing monooxygenase or a microorganism not expressing the variant of the present invention), but is not limited thereto.

[0086] In one embodiment, the microorganism having increased indigo production capacity of the present invention may be a microorganism having increased indigo production capacity compared to a microorganism comprising the polypeptide of sequence number 1 or a polynucleotide encoding the same, but is not limited thereto.

[0087] For example, the microorganism with increased indigo production ability may have an increase of about 1% or more, about 2% or more, about 2.5% or more, or about 3% or more (the upper limit is not particularly limited, and may be, for example, about 200% or less, about 150% or less, about 100% or less, about 50% or less, about 45% or less, about 40% or less, about 35% or less, about 30% or less, about 25% or less, or about 20% or less) compared to the indigo production ability of the parent strain before mutation or the non-mutated microorganism, but is not limited thereto as long as it has a positive increase amount compared to the production ability of the parent strain before mutation or the non-mutated microorganism. In another example, the recombinant strain with increased indigo production ability may have an increased indigo production ability of about 1.01 times or more, about 1.02 times or more, or about 1.03 times or more (the upper limit is not particularly limited, and may be, for example, about 10 times or less, about 5 times or less, about 3 times or less, about 2 times or less, or about 1.5 times or less) compared to the parent strain before mutation or the unmodified microorganism, but is not limited thereto.

[0088] Examples of parent strains or unmodified microorganisms before mutation for comparing the increase in the above indigo production ability include, but are not limited to, microorganisms comprising the polypeptide of SEQ ID NO: 1 or a polynucleotide encoding the same.

[0089] The term "about" above includes, but is not limited to, a range including ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and includes all numerical values ​​in a range equal to or similar to the numerical value following the term "about."

[0090] The microorganism of the present invention may include any microorganism capable of expressing the flavin-containing monooxygenase variant of the present invention by various known methods in addition to introducing the nucleic acid or vector.

[0091] In one embodiment, the microorganism of the present invention may be a microorganism of the genus Escherichia, but is not limited thereto.

[0092] The Escherichia genus microorganism having indigo production ability of the present invention includes all of the following: a natural wild-type microorganism itself; an Escherichia genus microorganism having improved indigo production ability by strengthening or weakening the activity of a gene related to the indigo production mechanism; or an Escherichia genus microorganism having improved indigo production ability by introducing or strengthening the activity of an external gene.

[0093] In one embodiment, the microorganism of the present invention may be Escherichia coli.

[0094] The microorganism of the present invention may have enhanced activity of the flavin-containing monooxygenase variant of the present invention.

[0095] In the present invention, the term "enhancement" of polypeptide activity means that the activity of the polypeptide is increased compared to the intrinsic activity. The enhancement may be used interchangeably with terms such as activation, up-regulation, overexpression, and increase. Here, activation, enhancement, up-regulation, overexpression, and increase may all include exhibiting an activity that was not originally present, or exhibiting an enhanced activity compared to the intrinsic activity or the activity before modification. The "intrinsic activity" refers to the activity of a specific polypeptide that a parent strain or an unmodified microorganism originally possessed before the trait change when the trait is changed due to genetic mutation caused by natural or artificial factors. This may be used interchangeably with "activity before modification." "Enhanced," "upregulated," "overexpressed," or "increased" the activity of a polypeptide relative to its intrinsic activity means that the activity and / or concentration (expression amount) of the specific polypeptide is improved compared to the activity and / or concentration (expression amount) that the parent strain or unmodified microorganism originally had prior to the transformation.

[0096] The above enhancement can be achieved by introducing an exogenous polypeptide, or by enhancing the activity and / or concentration (expression level) of an endogenous polypeptide. Whether the activity of the polypeptide is enhanced can be determined by an increase in the level of activity, expression level, or amount of product excreted from the polypeptide.

[0097] Enhancement of the activity of the above polypeptide can be achieved by applying various methods well known in the art, and is not limited as long as the activity of the target polypeptide can be enhanced compared to that of the microorganism before modification. Specifically, it may be achieved by using genetic engineering and / or protein engineering, which are routine methods of molecular biology and are well known to those skilled in the art, but is not limited thereto (e.g., Sitnicka et al. Functional Analysis of Genes. Advances in Cell Biology. 2010, Vol. 2. 1-16, Sambrook et al. Molecular Cloning 2012, etc.).

[0098] Specifically, the enhancement of the polypeptide of the present invention

[0099] 1) Increase in the intracellular copy number of a polynucleotide encoding a polypeptide;

[0100] 2) Replacing the gene expression control region on the chromosome encoding the polypeptide with a highly active sequence;

[0101] 3) Modification of the base sequence encoding the initiation codon or 5'-UTR region of a gene transcript encoding a polypeptide;

[0102] 4) Modification of the amino acid sequence of the polypeptide so as to enhance polypeptide activity;

[0103] 5) Modification of the polynucleotide sequence encoding the polypeptide so as to enhance the activity of the polypeptide (e.g., modification of the polynucleotide sequence of the polypeptide gene so as to encode a polypeptide modified so as to enhance the activity of the polypeptide);

[0104] 6) Introduction of a foreign polypeptide exhibiting the activity of the polypeptide or a foreign polynucleotide encoding the same;

[0105] 7) Codon optimization of a polynucleotide encoding a polypeptide;

[0106] 8) Analyzing the tertiary structure of the polypeptide and selecting the exposed portion to modify or chemically modify; or

[0107] 9) It may be a combination of two or more of the above 1) to 8), but is not particularly limited thereto.

[0108] More specifically,

[0109] The increase in the intracellular copy number of the polynucleotide encoding the polypeptide described above may be achieved by introducing into the host cell a vector capable of replicating and functioning independently of the host, to which the polynucleotide encoding the polypeptide is operably linked. Alternatively, the polynucleotide encoding the polypeptide may be achieved by introducing one copy or two or more copies into the chromosome of the host cell. The introduction into the chromosome may be performed by introducing into the host cell a vector capable of inserting the polynucleotide into the chromosome of the host cell, but is not limited thereto. The vector is as described above.

[0110] 2) Replacing the gene expression control region (or expression control sequence) on the chromosome encoding the polypeptide with a sequence having strong activity may be, for example, a mutation in the sequence such as deletion, insertion, non-conservative or conservative substitution, or a combination thereof to further enhance the activity of the expression control region, or replacement with a sequence having stronger activity. The expression control region may include, but is not particularly limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence regulating the termination of transcription and translation. As an example, it may be, but is not limited to, replacing the original promoter with a strong promoter.

[0111] Examples of known strong promoters include, but are not limited to, the CJ1 to CJ7 promoters (US Patent No. US 7662943 B2), the lac promoter, the trp promoter, the trc promoter, the tac promoter, the lambda phage PR promoter, the PL promoter, the tet promoter, the gapA promoter, the SPL7 promoter, the SPL13 (sm3) promoter (US Patent No. US 10584338 B2), the O2 promoter (US Patent No. US 10273491 B2), the tkt promoter, and the yccA promoter.

[0112] The above 3) modification of the base sequence encoding the initiation codon or 5'-UTR region of the gene transcript encoding the polypeptide may be, for example, a substitution with a base sequence encoding another initiation codon having a higher polypeptide expression rate than the endogenous initiation codon, but is not limited thereto.

[0113] The modification of the amino acid sequence or polynucleotide sequence of the above 4) and 5) may be, but is not limited to, a mutation in the sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof in the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide to enhance the activity of the polypeptide, or replacement with an amino acid sequence or polynucleotide sequence improved to have stronger activity, or an amino acid sequence or polynucleotide sequence improved to have increased activity. The replacement may be specifically performed by inserting the polynucleotide into a chromosome by homologous recombination, but is not limited thereto. The vector used at this time may additionally include a selection marker to confirm whether or not the chromosome has been inserted. The selection marker is as described above.

[0114] The introduction of the foreign polynucleotide exhibiting the activity of the polypeptide as described above 6) may be the introduction into the host cell of a foreign polynucleotide encoding a polypeptide exhibiting the same / similar activity as the polypeptide. The foreign polynucleotide is not limited in its origin or sequence as long as it exhibits the same / similar activity as the polypeptide. The method used for the introduction may be performed by a person skilled in the art appropriately selecting a known transformation method, and the polypeptide may be produced by expressing the introduced polynucleotide in the host cell, thereby increasing its activity.

[0115] The above 7) codon optimization of a polynucleotide encoding a polypeptide may be codon optimization of an endogenous polynucleotide to increase transcription or translation within a host cell, or codon optimization of a foreign polynucleotide to achieve optimized transcription or translation within a host cell.

[0116] The above 8) analyzing the tertiary structure of a polypeptide and selecting an exposed portion to modify or chemically modify may be done by, for example, comparing the sequence information of the polypeptide to be analyzed with a database storing the sequence information of known proteins, determining a template protein candidate based on the degree of sequence similarity, confirming the structure based on this, and selecting an exposed portion to modify or chemically modify, and modifying or modifying it.

[0117] Such enhancement of polypeptide activity may be, but is not limited to, an increase in the activity or concentration or expression level of the corresponding polypeptide relative to the activity or concentration of the polypeptide expressed in the wild type or pre-transformed microbial strain, or an increase in the amount of a product produced from the polypeptide.

[0118] In the microorganism of the present invention, modification of part or all of the polynucleotide may be induced by, but is not limited to, (a) homologous recombination using a vector for chromosome insertion into the microorganism or genome editing using engineered nuclease (e.g., CRISPR-Cas9) and / or (b) treatment with light and / or chemicals such as ultraviolet rays and radiation. The method for modifying part or all of the gene may include a method using DNA recombination technology. For example, a nucleotide sequence or vector including a nucleotide sequence homologous to a target gene may be injected into the microorganism to cause homologous recombination, thereby causing deletion of part or all of the gene. The injected nucleotide sequence or vector may include, but is not limited to, a dominant selection marker.

[0119] In the microorganism of the present invention, the mutant, polynucleotide, and indigo, etc. are as described in the other aspects above.

[0120]

[0121] Another aspect of the present invention provides a method for producing indigo, comprising the step of culturing the microorganism of the present invention in a medium.

[0122] Specifically, the indigo production method of the present invention may include, but is not limited to, a step of culturing a microorganism comprising a variant of the present invention, a polynucleotide of the present invention, or a vector of the present invention in a medium.

[0123] In the present invention, the term "cultivation" refers to growing the microorganism of the present invention under appropriately controlled environmental conditions. The culturing process of the present invention can be performed using appropriate media and culture conditions known in the art. This culturing process can be easily adjusted and used by those skilled in the art depending on the selected strain. Specifically, the culturing process may be batch, continuous, and / or fed-batch, but is not limited thereto.

[0124] In the present invention, the term "medium" refers to a material containing nutrients as main components necessary for culturing the microorganism of the present invention, and supplies nutrients and growth factors, including water essential for survival and growth. Specifically, the medium and other culture conditions used for culturing the microorganism of the present invention may be any medium used for culturing general microorganisms without particular limitation, but the microorganism of the present invention may be cultured under aerobic conditions while controlling temperature, pH, etc. in a general medium containing an appropriate carbon source, nitrogen source, phosphorus source, inorganic compound, amino acid, and / or vitamin.

[0125] In the present invention, the carbon source may include carbohydrates such as glucose, saccharose, lactose, fructose, sucrose, maltose, etc.; sugar alcohols such as mannitol, sorbitol, etc.; organic acids such as pyruvic acid, lactic acid, citric acid, etc.; amino acids such as glutamic acid, methionine, lysine, etc. In addition, natural organic nutrients such as starch hydrolysate, molasses, blackstrap molasses, rice winter, cassava, sugarcane bagasse, and corn steep liquor may be used, and specifically, carbohydrates such as glucose and sterilized pretreated molasses (i.e., molasses converted into reducing sugar) may be used, and other appropriate amounts of carbon sources may be used in various ways without limitation. These carbon sources may be used alone or in combination of two or more, but are not limited thereto.

[0126] The nitrogen source may include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, ammonium nitrate, etc.; 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 liquor, casein hydrolysate, fish or its decomposition product, defatted soybean cake or its decomposition product, etc. These nitrogen sources may be used alone or in combination of two or more, but are not limited thereto.

[0127] The above-mentioned components may include potassium phosphate monobasic, potassium phosphate dibasic, or their corresponding sodium-containing salts. Inorganic compounds may include sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, etc. In addition, amino acids, vitamins, and / or suitable precursors may be included. These components or precursors may be added to the medium in batch or continuous manner, but are not limited thereto.

[0128] In addition, during the cultivation of the microorganism of the present invention, 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. In addition, during the cultivation, foaming can be suppressed by using an antifoaming agent such as fatty acid polyglycol ester. In addition, in order to maintain the aerobic state of the medium, oxygen or an oxygen-containing gas can be injected into the medium, or in order to maintain the anaerobic and microaerobic state, nitrogen, hydrogen, or carbon dioxide gas can be injected without gas injection, but the present invention is not limited thereto.

[0129] In the culture of the present invention, the culture temperature can be maintained at 20 to 45°C, specifically 25 to 40°C, and the culture can be performed for about 10 to 160 hours, but is not limited thereto.

[0130] Indigo produced by the culture of the present invention may be secreted into the medium or remain within the cells.

[0131] The indigo production method of the present invention may additionally include a step of preparing the microorganism of the present invention, a step of preparing a medium for culturing the strain, or a combination thereof (in any order), for example, before the culturing step.

[0132] The indigo production method of the present invention may further include a step of recovering indigo from the culture medium (the medium in which the culture is performed) or the microorganism of the present invention. The recovering step may be additionally included after the culturing step.

[0133] The above recovery may be performed by collecting indigo using a suitable method known in the art according to the culture method of the microorganism of the present invention, such as a batch, continuous or fed-batch culture method. For example, various chromatographies such as centrifugation, filtration, treatment with a crystallized protein precipitant (salting out method), 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 indigo may be recovered from the medium or microorganism using a suitable method known in the art.

[0134] Additionally, the indigo production method of the present invention may additionally include a purification step. This purification may be performed using any suitable method known in the art. In one example, if the indigo production method of the present invention includes both a recovery step and a purification step, the recovery step and the purification step may be performed sequentially or discontinuously, regardless of order, or may be performed simultaneously or integrated into a single step, but this is not limited thereto.

[0135] In the method of the present invention, the mutant, polynucleotide, microorganism, indigo, etc. are as described in the other aspects above.

[0136]

[0137] Another aspect of the present invention provides a composition for producing indigo, comprising a microorganism comprising a flavin-containing monooxygenase variant of the present invention or a polynucleotide encoding the variant; a medium in which the same is cultured; or a combination of two or more thereof.

[0138] The composition of the present invention may further comprise any suitable excipient commonly used in compositions for producing amino acids, and such excipients may be, for example, but are not limited to, preservatives, wetting agents, dispersing agents, suspending agents, buffers, stabilizers, or isotonic agents.

[0139] In the composition of the present invention, the mutant, polynucleotide, microorganism, indigo, etc. are as described in the other aspects above.

[0140]

[0141] A variant of flavin-containing monooxygenase (FMO) according to one aspect of the present invention has improved indigo production ability and can therefore be usefully used in indigo production.

[0142]

[0143] Figure 1 shows the structure of an expression vector of the FMO (WT) wild-type gene according to one embodiment of the present invention.

[0144] Figure 2 shows the structure of an expression vector of an FMO (m7) mutant gene according to one embodiment of the present invention.

[0145] Figure 3 is a graph comparing the indigo production ability of FMO (WT) and FMO (m7) mutant according to one embodiment of the present invention.

[0146]

[0147] Hereinafter, the present invention will be described in more detail through examples. However, these examples are intended to exemplify the present invention, and the scope of the present invention is not limited by these examples.

[0148]

[0149] Example 1. Construction of recombinant vector pCL1920-PgapA-FMO (WT)

[0150]

[0151] 1-1. Preparation of the promoter DNA fragment of the gapA gene

[0152] In order to obtain a DNA fragment of about 0.6 kb containing the promoter sequence of the gapA gene of SEQ ID NO: 5, the chromosomal DNA (gDNA) of the wild type Escherichia coli strain BL21 (DE3) was extracted using the Genomic-tip system of Qiagen, and the gDNA was used as a template for PCR (polymerase chain reaction) using the PCR pfu premix kit (BIONEER). PCR for amplifying the gapA promoter DNA fragment region was performed using primers of SEQ ID NO: 6 and 7, and the cycle consisted of denaturation at 95°C for 30 seconds, annealing at 56°C for 30 seconds, and elongation at 72°C for 1 minute, repeated 30 times. The approximately 0.6 kb PCR product DNA fragment (hereinafter referred to as “PgapA fragment”) was obtained by elution after electrophoresis on a 0.8% agarose gel.

[0153]

[0154] 1-2. Preparation of ORF sequence DNA fragment of FMO gene

[0155] To obtain a DNA fragment of approximately 1.37 kb containing the ORF of the FMO gene, PCR was performed using a PCR pfu premix kit (BIONEER) with pET28a::mafmo (WO 2022 / 097856 A1) as a template. PCR for amplifying the ORF DNA fragment region of the FMO gene was performed using primers of SEQ ID NOs: 8 and 9, and the following 30 cycles were performed: denaturation at 95°C for 30 s, annealing at 56°C for 30 s, and elongation at 72°C for 2 min. The approximately 1.37 kb PCR product DNA fragment was eluted after electrophoresis on a 0.8% agarose gel.

[0156]

[0157] 1-3. Construction of the recombinant vector pCL1920-PgapA_FMO (WT)

[0158] The Escherichia coli expression vector pCL1920 plasmid was treated with SmaI (NEB biolabs, USA), electrophoresed on a 0.8% agarose gel, eluted, and obtained. Then, it was ligated with the DNA fragments obtained in Examples 1-1 and 1-2 using a Gibson assembly kit (NEB biolabs, USA), thereby constructing the pCL1920-PgapA_FMO (WT) plasmid. The structure of the expression vector of the FMO (WT) wild-type gene constructed in this way is shown in Figure 1.

[0159]

[0160] Example 2. Construction of a mutant library using the recombinant vector pCL1920-PgapA-FMO

[0161]

[0162] 2-1. Preparation of mutant FMO fragments using error-prone PCR

[0163] Error-prone PCR (diversify PCR random mutagenesis kit, Clontech) was performed using the wild-type E. coli BL21(DE3) chromosomal DNA as a template and primers of SEQ ID NO: 8 and 9. Specifically, 30 cycles of denaturation at 94°C for 30 s and elongation at 68°C for 90 s were repeated. The PCR product DNA fragment of approximately 1.37 kb (hereinafter referred to as "FMO") m The fragment (called “fragment”) was eluted after electrophoresis on a 0.8% agarose gel.

[0164]

[0165] 2-2. Recombinant vector pCL1920-PgapA-FMO m Library Creation

[0166] The Escherichia coli expression vector pCL1920 plasmid was treated with SmaI (NEB biolabs, USA), eluted after electrophoresis on a 0.8% agarose gel, and ligated with the DNA fragments obtained in Examples 1-1 and 2-1 using a Gibson assembly kit (NEB biolabs, USA), and then transformed using electroporation. Colonies of the transformed strain were selected on LB solid medium (15 ug / ml) containing spectinomycin. The colonies thus obtained were collected, and the plasmid was extracted, and pCL1920-PgapA_FMO m A library was constructed. The structure of the expression vector of the FMO (m7) mutant gene constructed in this way is shown in Figure 2.

[0167]

[0168] Example 3. Introduction of FMO mutant library and selection of Indigo producing strains.

[0169]

[0170] 3-1. pCL1920-PgapA_FMO m Introduction of E. coli BL21(DE3) strain into the library

[0171] pCL1920-PgapA_FMO obtained in Example 2 m The library was introduced into wild-type E. coli BL21 (DE3) by transformation using electroporation.

[0172] pCL1920-PgapA_FMO m As a control group of microorganisms into which the library was introduced, pCL1920-PgapA_FMO (WT) obtained in Example 1 was transformed using the above method to produce an E. coliBL21 (DE3) / pCL1920-PgapA_FMO (WT) strain.

[0173]

[0174] 3-2. Comparison of Indigo Production Ability of Recombinant Microorganisms

[0175] M9 minimal medium containing 1% glucose, 0.2 g / L yeast extract, and 0.2 g / L indole was dispensed into a deep well microplate, and the transformants and control strains prepared in Example 3-1 were inoculated. The transformants prepared in Example 3-1 were cultured in a constant temperature incubator (TAITEC, Japan) at 37°C and 200 rpm for 20 hours to select strains with improved indigo production ability, and finally, the strain with the greatest increase in indigo production was selected. After extraction of the plasmid of the selected FMO mutant (m7), sequence analysis (SEQ ID NO: 4) was performed, and the results are summarized in Table 2.

[0176]

[0177] Information on mutant strains with enhanced indigo production ability Indigo (g / L) Mutant E. coliBL21(DE3) / pCL19200-E. coliBL21(DE3) / pCL1920-PgapA_FMO(WT)0.011-E. coliBL21(DE3) / pCL1920-PgapA_FMO(m7)0.113N246H

[0178] 3-3. Comparison of indigo titers of recombinant microorganisms

[0179] In order to measure the indigo titer of the recombinant microorganism selected in Example 3-2, it was cultured in an indigo titer medium prepared according to the composition in Table 2 below, and it was confirmed that indigo productivity was improved.

[0180]

[0181] Indigo titer medium composition Composition concentration (per liter) Glucose 30 g L-Tryptophan 3 g KH 2 PO 4 0.5 g K 2 HPO 4 1.0 g (NH 4) 2 SO 4 15 g Mg SO 4· 7H2O1 gYeast extract2.5 gFeSO 4· 7H2O5 mgMnSO 4· 4H2O5 mg calcium carbonate 40 g pH 7.0

[0182]

[0183] Specifically, E. coli BL21(DE3) / pCL1920-PgapA_FMO(WT) and E. coli BL21(DE3) / pCL1920-PgapA_FMO(m7) cultured on LB solid medium for 17 hours in a 37℃ incubator were inoculated with one colony each in 3ml of LB and cultured for 2 hours. Then, 0.1% inoculation was performed in 25ml of the titer medium in Table 2 above, and after culturing for 30 hours under conditions of 37℃ and 200rpm, the indigo concentration was compared.

[0184]

[0185] As a result, as described in Table 3 below, it was confirmed that the indigo production ability of the FMO (m7) mutant was approximately 5.4 times improved compared to the wild-type FMO (WT) (see Fig. 3). This suggests that the FMO (m7) mutant is an effective mutation that increases enzyme activity, and thus, it is expected that the indigo production method using microorganisms will be able to achieve an industrially feasible level of productivity.

[0186]

[0187] Comparison of the indigo production capacity of FMO(m7) mutant strains. Tryptophan residue (g / L). Indigo (g / L). E. coli. BL21(DE3) / pCL19203.01-E. coli. BL21(DE3) / pCL1920-PgapA_FMO(WT). 2.8. 10.09. E. coli. BL21(DE3) / pCL1920-PgapA_FMO(m7). 1.5. 3. 0.46.

[0188] The above results suggest that the productivity of the indigo production method using an Escherichia microorganism including the FMO variant of the present invention is greatly improved, and thus has the potential to be economical for mass production.

[0189]

[0190] Sequence number Name Sequence 1 FMO Wild type amino acid sequence MATRIAILGAGPSGMAQLRAFQSAQEKGAEIPELVCFEKQADWGGQWNYTWRTGLDENGEPVHSSMYRYLWSNGPKECLEFADYTFDEHFGKPIASYPPREVLWDYIKGRVEKAGVRKYIRFNTAVRHVEFNEDSQTFTVTVQDHTTDTIYSEEFDYVVCCTGHFSTPYVPEFEGFEKFGGRILHAHDFRDALEFKDKTVLLVGSSYSAEDIGSQCYKYGAKKLISCYR TAPMGYKWPENWDERPNNLVRVDTENAYFADGSSEKVDAIILCTGYIHHFPFLNDDLRLVTNNRLWPLNLYKGVVWEDNPKFFYIGMQDQWYSFNMFDAQAWYARDVIMGRLPLPS KEEMKADSMAWREKELTLVTAEEMYTYQGDYIQNLIDMTDYPSFDIPATNKTFLEWKHHKKENIMTFRDHSYRSSLMTGTMAPKHHTPWIDALDDSLEAYLSDKSEIPVAKEA2FMO Wild-type DNA sequence (WT)cattgccatcaaaagaagagatgaaagccgacagcatggcctggcgtgaaaaagaactgacgctggttacggctgaagaaatgtacacctaccagggtgactacattcagaatctgattgatatgactgactatccgtcatttgatattccggcaaccaacaaaactttcctgga atggaaaacatcacaaaaaagaaaacatcatgactttccgtgaccactcataccgttcactgatgactggcacgatggcaccgaaacatcacaccaccatggatagatgcactggatgattctctggaagcctatctctctgataagagcgaaattcctgtggctaaagaagcttaa3FMO variant amino acids서열MATRIAILGAGPSGMAQLRAFQSAQEKGAEIPELVCFEKQADWGGQWNYTWRTGLDENGEPVHSSMYRYLWSNGPKECLEFADYTFDEHFGKPIASYPPREVLWDYIKGRVEKAGVRKYIRFNTAVRHVEFNEDSQTFTVTVQDHTTDTIYSEEFDYVVCCTGHFSTPYVPEFEGFEKFGGRILHAHDFRDALEFKDKTVLLVGSSYSAEDIGSQCYKYGAKKLISCYRTAPMGYKWPENWDERPHLVRVDTENAYFADGSSEKVDAIILCTGYIHHFPFLNDDLRLVTNNRLWPLNLYKGVVWEDNPKFFYIGMQDQWYSFNMFDAQAWYARDVIMGRLPLPSKEEMKADSMAWREKELTLVTAEEMYTYQGDYIQNLIDMTDYPSFDIPATNKTFLEWKHHKKENIMTFRDHSYRSLMTGTMAPKHHTPWIDALDDSLEAYLSDKSEIPVAKEA4FMO 변이형 DNA 염기서열 (m7)cattgccatcaaaagaagagatgaaagccgacagcatggcctggcgtgaaaaagaactgacgctggttacggctgaagaaatgtacacctaccagggtgactacattcagaatctgattgatatgactgactatccgtcatttgatattccggcaaccaacaaaactttcctggaatggaaacatcacaaaaaagaaaacatcatgactttccgtgaccactcataccgttcactgatgactggcacgatggcaccgaaacatcacacaccatggatagatgcactggatgattctctggaagcctatctctctgataagagcgaaattcctgtggctaaagaagcttaa5Promoter gapA 서열(PgapA)tctatgcgctgcattccatgtgacaagtctttgatataacgaatggattcttcacttaccggttcgtagaaactgggccagccacagccggaatcatactttgtttgggaatgaaacagcggggcatcgcagatcaaacagtgatatacgccgtcacgcttgttatgcagtaaacgacccgtaaatggcggctctgtcccatgattctgcgtcacgtaaaactgcatctcggacaaattttttttcagttcttctgccgaaggtttattagccatttgctcacatctcactttaatcgtgctcacattacgtgactgattctaacaaaacattaacaccaactggcaaaattttgtcctaaacttgatctcgacgaaatggctgcacctaaatcgtgatgaaaatcacatttttatcgtaattgccctttaaaattcggggcgccgaccccatgtggtctcaagcccaaaggaagagtgaggcgagtcagtcgcgtaatgcttaggcacaggattgatttgtcgcaatgattgacacgattccgcttgacgctgcgtaaggtttttgtaattttacaggcaaccttttattcactaacaaatagctggtggaatat6primerctctagaggatcccctctatgcgctgcattccatg7primeraatacgagttgccatatattccaccagctatttgt8primertagctggtggaatatatggcaactcgtattgcgat9primertcgagctcggtacccttaagcttctttagccacag

[0191]

[0192] From the above description, those skilled in the art will understand that the present invention can be implemented in other specific forms without altering its technical spirit or essential characteristics. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be interpreted as encompassing all changes or modifications derived from the meaning and scope of the following claims and their equivalent concepts, rather than the detailed description above.

Claims

1. A flavin-containing monooxygenase (FMO) mutant in which the amino acid corresponding to position 246 in the amino acid sequence of sequence number 1 is substituted with histidine.

2. An FMO variant according to claim 1, comprising an amino acid sequence having sequence number 3 or at least 80% sequence identity therewith.

3. A polynucleotide encoding the FMO variant of claim 1 or 2.

4. A vector comprising a polynucleotide encoding the FMO variant of claim 1 or 2.

5. A microorganism comprising an FMO variant in which the amino acid corresponding to position 246 in the amino acid sequence of sequence number 1 is substituted with histidine, or a polynucleotide encoding the variant.

6. In claim 5, the microorganism is Escherichia coli.

7. A microorganism producing indigo according to claim 5 or 6.

8. A method for producing indigo, comprising the step of culturing a microorganism containing an FMO variant in which the amino acid corresponding to position 246 in the amino acid sequence of sequence number 1 is substituted with histidine or a polynucleotide encoding the variant in a medium.

9. A method for producing indigo, further comprising a step of recovering indigo from the microorganism or medium according to claim 8.

10. A composition for producing indigo, comprising a microorganism comprising an FMO variant in which the amino acid corresponding to position 246 in the amino acid sequence of SEQ ID NO: 1 is substituted with histidine, or a polynucleotide encoding the variant, a medium in which the microorganism is cultured, or both.

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