NOVEL β-CAROTENE 15,15'-OXYGENASE MUTAMIN AND METHOD FOR PRODUCING RETINOID USING THE SAME

Abstract

The present application relates to novel β-carotene 15,15'-oxygenase mutants, microorganisms containing said mutants, methods for producing retinoids using said microorganisms, compositions for producing retinoids, and use of said mutants or microorganisms for producing retinoids.

Description

[Technical field]

[0001] The present application relates to novel β-carotene 15,15'-oxygenase mutants, microorganisms containing said mutants, methods for producing retinoids using said microorganisms, compositions for producing retinoids, and use of said mutants or microorganisms for producing retinoids. [Background technology]

[0002] Vitamin A, an essential nutrient as one of the fat-soluble vitamins, and retinoids, which include vitamin A, are currently produced and sold by chemical synthesis, but due to reasons such as the difficulty in obtaining raw materials, various studies are being conducted on microbial fermentation-based retinoid production, that is, on the development of highly efficient production microorganisms and fermentation process technologies. For example, approaches specific to target substances such as increasing the expression of genes encoding enzymes involved in retinoid biosynthesis and removing genes unnecessary for biosynthesis can be mentioned. One example of an enzyme involved in the retinoid biosynthesis is β-carotene 15,15'-oxygenase (Patent Document 1).

[0003] However, there has been little research on β-carotene 15,15'-oxygenase, and research is still needed to effectively improve vitamin A production capacity in response to increasing demand for vitamin A. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] US Patent Application Publication No. 2020 / 0277644 [Patent Document 2] Korean Patent Publication No. 10-2020-0136813 [Patent Document 3] U.S. Patent No. 7,662,943 [Patent Document 4] U.S. Pat. No. 1,058,438 [Patent Document 5] U.S. Patent No. 10,273,491 Specification

Non-Patent Literature

[0005]

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[0006] An objective of the present application is to provide a β-carotene 15,15'-oxygenase mutant and a method for producing a retinoid using the same. [Means for solving the problem]

[0007] An object of the present application is to provide a β-carotene 15,15'-oxygenase mutant.

[0008] The present application also aims to provide polynucleotides encoding the variants of the present application.

[0009] Furthermore, the present application aims to provide a microorganism comprising the variant of the present application or a polynucleotide encoding said variant.

[0010] Furthermore, the present application aims to provide a method for producing a retinoid, comprising the step of culturing in a medium a microorganism containing the variant of the present application or a polynucleotide encoding said variant.

[0011] Furthermore, the present application aims to provide a composition for producing a retinoid, comprising the variant of the present application, a microorganism containing a polynucleotide encoding the variant, or a culture thereof.

[0012] Furthermore, the present application aims to provide use of the variant of the present application, a polynucleotide encoding said variant, or a microorganism containing same for producing at least one retinoid. Effect of the Invention

[0013] When a microorganism containing a β-carotene 15,15'-oxygenase mutant of the present application is cultured, retinoids can be produced in a higher yield than when a microorganism has a conventional unmodified polypeptide. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] These will be described in detail below. Each description and embodiment disclosed in this application is also applicable to other descriptions and embodiments. That is, all combinations of various elements disclosed in this application are included in this application. In addition, this application is not limited to the following specific description. Furthermore, many papers and patent documents are referenced throughout this specification, and citations thereof are provided. The disclosure contents of the cited papers and patent documents are incorporated in their entirety into this specification by reference, thereby more clearly explaining the state of the art to which the present invention belongs and the contents of the present invention.

[0015] One aspect of the present application provides a β-carotene 15,15'-oxygenase mutant in which at least one of the amino acids alanine, leucine, and isoleucine, which correspond to the 43rd, 58th, and 74th positions from the N-terminus of the amino acid sequence of SEQ ID NO:1, is replaced with another amino acid.

[0016] The β-carotene 15,15'-oxygenase mutant may be a polypeptide having β-carotene 15,15'-oxygenase activity, or a mutant in which at least one of the amino acids corresponding to the 43rd, 58th, and 74th positions from the N-terminus of SEQ ID NO: 1 is substituted with another amino acid. Specifically, the mutant may be one in which one or more, two or more, or three or more of the 43rd, 58th, and 74th positions are substituted, but is not limited thereto.

[0017] In the polypeptide having β-carotene 15,15'-oxygenase activity that is the parent of the mutant, or in the β-carotene 15,15'-oxygenase, the amino acid corresponding to the 43rd position from the N-terminus of the amino acid sequence of SEQ ID NO: 1 is alanine, the amino acid corresponding to the 58th position is leucine, and / or the amino acid corresponding to the 74th position is isoleucine, but is not limited to these.

[0018] As an example, the mutant includes at least one of the following substitutions: substitution of the amino acid corresponding to the 43rd position from the N-terminus of the amino acid sequence of SEQ ID NO:1 with an amino acid other than alanine, substitution of the amino acid corresponding to the 58th position with an amino acid other than leucine, and substitution of the amino acid corresponding to the 74th position with an amino acid other than isoleucine, but is not limited to this.

[0019] The "other amino acid" may be any amino acid different from the amino acid before substitution. It goes without saying that "a specific amino acid is substituted" in the present application means that the amino acid before substitution is substituted with an amino acid different from the amino acid before substitution, even if it is not specified that the amino acid is substituted with another amino acid.

[0020] The substitution with another amino acid may be a substitution with a nonpolar amino acid, a polar amino acid, or a positively charged (basic) amino acid. Specifically, the nonpolar amino acid is selected from the group consisting of glycine, alanine, valine, leucine, isoleucine, methionine, and proline, but is not limited thereto. The polar amino acid is mixed with a hydrophilic amino acid and is selected from the group consisting of serine, threonine, cysteine, tyrosine, asparagine, and glutamine, and the positively charged (basic) amino acid is selected from the group consisting of arginine, lysine, and histidine, but is not limited thereto.

[0021] The variants of the present application are variants in which at least one of the amino acids corresponding to positions 43, 58, and 74 of the amino acid sequence of the reference protein SEQ ID NO:1 is substituted with an amino acid different from the amino acid before substitution, selected from nonpolar amino acids, polar amino acids, and positively charged (basic) amino acids, but are not limited thereto.

[0022] As an example, the substitution of the amino acid corresponding to position 43 with another amino acid is a substitution with a nonpolar amino acid, the substitution of the amino acid corresponding to position 58 with another amino acid is a substitution with a polar amino acid, and the substitution of the amino acid corresponding to position 74 with another amino acid is a substitution with a nonpolar amino acid, but is not limited to this.

[0023] The non-polar amino acid is valine and the polar amino acid is glutamine, but is not limited thereto.

[0024] The other amino acid may be, but is not limited to, valine or glutamine.

[0025] As an example, the variant of the present application has a substitution selected from the group consisting of the substitution of the amino acid corresponding to position 43 of the amino acid sequence of SEQ ID NO:1 with valine, the substitution of the amino acid corresponding to position 58 with glutamine, the substitution of the amino acid corresponding to position 74 with valine, and combinations thereof, but is not limited thereto.

[0026] As an example, the variant of the present application may be, from the N-terminus of the amino acid sequence of SEQ ID NO:1, i) the amino acid alanine corresponding to the 43rd position is replaced with valine, ii) the amino acid leucine corresponding to the 58th position is replaced with glutamine, iii) the amino acid isoleucine corresponding to the 74th position is replaced with valine, iv) the amino acid alanine corresponding to the 43rd position is replaced with valine and the amino acid leucine corresponding to the 58th position is replaced with glutamine, or v) the amino acid leucine corresponding to the 58th position is replaced with glutamine. vi) the amino acid alanine corresponding to the 43rd position is replaced by valine, and the amino acid isoleucine corresponding to the 74th position is replaced by valine; or vii) the amino acid alanine corresponding to the 43rd position is replaced by valine, the amino acid leucine corresponding to the 58th position is replaced by glutamine, and the amino acid isoleucine corresponding to the 74th position is replaced by valine, but is not limited thereto.

[0027] As an example, the variant of the present application is a polypeptide having the amino acid sequence represented by SEQ ID NO: 3, in which the alanine corresponding to the 43rd amino acid position of the amino acid sequence of SEQ ID NO: 1 is replaced with valine, the leucine corresponding to the 58th amino acid position is replaced with glutamine, and the isoleucine corresponding to the 74th amino acid position is replaced with valine, but is not limited to this.

[0028] The variant of the present application may have the amino acid sequence shown in SEQ ID NO:3, may contain said amino acid sequence, may consist of said amino acid sequence, or may essentially consist of said amino acid sequence.

[0029] The variant of the present application may also include an amino acid sequence represented by SEQ ID NO: 3 in which the amino acid corresponding to position 43 is fixed to valine, the amino acid corresponding to position 58 is fixed to glutamine, and the amino acid corresponding to position 74 is fixed to valine, relative to the amino acid sequence of SEQ ID NO: 1, and which has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.9% homology or identity to the amino acid sequence represented by SEQ ID NO: 3. Furthermore, it goes without saying that variants having amino acid sequences in which a portion of the sequence has been deleted, modified, substituted, conservatively substituted, or added are also included in the present application, so long as they have the above-mentioned homology or identity other than at the fixed positions of each of such sequences and exhibit efficacy equivalent to that of the variant of the present application.

[0030] For example, the variants may have additions or deletions of sequences at the N-terminus, C-terminus and / or internally of the amino acid sequence that do not change the function of the variants of the present application, naturally occurring mutations, silent mutations or conservative substitutions.

[0031] The term "conservative substitution" refers to the replacement of an amino acid with another amino acid having similar structural and / or chemical properties. Such amino acid substitutions may generally be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues. Typically, conservative substitutions have little or no effect on the activity of a protein or polypeptide.

[0032] In the present application, the term "variant" refers to a polypeptide that differs from the amino acid sequence of the variant before the mutation by conservative substitution and / or modification of at least one amino acid, but maintains its functions or properties. Such variants can generally be identified by modifying at least one amino acid in the amino acid sequence of the polypeptide and evaluating the properties of the modified polypeptide. That is, the ability of the variant is improved, unchanged, or decreased compared to the polypeptide before the mutation. Some variants also include variants in which at least one portion, such as an N-terminal leader sequence or a transmembrane domain, has been removed. Other variants also include variants in which a portion has been removed from the N- and / or C-terminus of a mature protein. The term "variant" is used interchangeably with mutated, modified, mutant polypeptide, mutated protein, mutation, etc. (in English, modification, modified polypeptide, modified protein, mutant, mutein, divergent, etc.), but may be any term that means a mutation.

[0033] The variant may also include deletions or additions of amino acids that have minimal effect on the properties and secondary structure of the polypeptide. For example, the N-terminus of the variant may be linked to a signal (or leader) sequence involved in co-translationally or post-translationally protein translocation. The variant may also be linked to other sequences or linkers to allow identification, purification, or synthesis.

[0034] In this application, "homology" or "identity" refers to the degree of similarity between two given amino acid or nucleotide sequences, expressed as a percentage. Homology and identity are often used interchangeably.

[0035] Sequence homology or identity of conserved polynucleotides or polypeptides may be determined by standard sequence algorithms, with default gap penalties established by the program used. Substantially homologous or identical sequences will generally hybridize to all or a portion of the sequence under moderate or high stringent conditions. Hybridization, of course, also includes hybridization to polynucleotides having common codons in the polynucleotide or codons that take into account codon degeneracy.

[0036] Whether any two polynucleotide or polypeptide sequences have homology, similarity or identity can be determined using known computer algorithms such as the "FASTA" program with default parameters as in, for example, Non-Patent Document 1. Alternatively, it can be determined using the Needleman-Wunsch algorithm (Non-Patent Document 3), as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Non-Patent Document 2) (version 5.0.0 or later), which includes the GCG program package (Non-Patent Document 4), BLASTP, BLASTN, FASTA (Non-Patent Documents 5, 6 and 7). For example, BLAST or Clustal W from the National Center for Biotechnology Information can be used to determine homology, similarity or identity.

[0037] Homology, similarity or identity of polynucleotides or polypeptides can be determined by comparing sequence information using a GAP computer program such as that disclosed in Non-Patent Document 3, as disclosed in Non-Patent Document 8. In summary, the GAP program defines the number of similar sequence symbols (i.e., nucleotides or amino acids) divided by the total number of symbols in the shorter of the two sequences. Default parameters for the GAP program include (1) a binary comparison matrix (identity takes a value of 1 and non-identity takes a value of 0) and a weighted comparison matrix of Non-Patent Document 10 (or an EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix) as disclosed in Non-Patent Document 9, (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 open penalty of 10 and a gap extension penalty of 0.5), and (3) no penalty for terminal gaps.

[0038] As an example of the present application, the variant of the present application may have β-carotene 15,15'-oxygenase enzyme activity, or may have activity that improves retinoid production ability compared to a wild-type polypeptide having β-carotene 15,15'-oxygenase activity.

[0039] In the present application, "β-carotene 15,15'-oxygenase" means an enzyme that has the function of catalyzing the cleavage of one molecule of β-carotene into two molecules of retinal.

[0040] The β-carotene 15,15'-oxygenase of the present application may be a β-carotene 15,15'-oxygenase that is modified to produce a β-carotene 15,15'-oxygenase mutant provided in the present application, or a polypeptide having β-carotene 15,15'-oxygenase activity. Specifically, it may be a naturally occurring or wild-type polypeptide, a mature polypeptide thereof, or a mutant or functional fragment thereof, and may be any polypeptide that serves as the parent of the β-carotene 15,15'-oxygenase mutant of the present application.

[0041] The β-carotene 15,15'-oxygenase in the present application is, but is not limited to, the polypeptide of SEQ ID NO: 1. It may also be a polypeptide having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity with the polypeptide of SEQ ID NO: 1, and any polypeptide having the same or corresponding activity as the polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 is included in the scope of the β-carotene 15,15'-oxygenase.

[0042] Specifically, the β-carotene 15,15'-oxygenase of the present application is used in combination with BCO or Blh. The sequence of the β-carotene 15,15'-oxygenase in the present application can be obtained from GenBank, a publicly known database, of NCBI. Specifically, the β-carotene 15,15'-oxygenase is a polypeptide having β-carotene 15,15'-oxygenase activity encoded by blh, but is not limited thereto.

[0043] "Corresponding to" in this application means the amino acid residue at the recited position in the polypeptide, or an amino acid residue similar, identical or equivalent to the recited residue in the polypeptide. Identifying the amino acid at the corresponding position will determine the particular amino acid of the sequence to which the particular sequence is referenced. "Corresponding region" in this application generally refers to a similar or corresponding position in a related or reference protein.

[0044] For example, any amino acid sequence can be aligned with SEQ ID NO: 1, and based thereon, each amino acid residue of the amino acid sequence can be numbered with reference to the number and position of the amino acid residues corresponding to the amino acid residues in SEQ ID NO: 1. For example, the sequence alignment algorithm in the present application can identify the positions of amino acids or positions where modifications such as substitutions, insertions, deletions, etc. occur when compared to a query sequence (also referred to as a "reference sequence").

[0045] For such alignment, for example, the Needleman-Wunsch algorithm (Non-Patent Document 3) or the Needle program in the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Non-Patent Document 2) can be used, but is not limited to these. Sequence alignment programs and pairwise sequence comparison algorithms known in the technical field can be used as appropriate.

[0046] Another aspect of the present application provides polynucleotides encoding the variants of the present application.

[0047] In this application, the term "polynucleotide" refers to a nucleotide polymer in which nucleotide monomers are linked in a long chain by covalent bonds, and means a DNA or RNA chain longer than a certain length, and more specifically, means a polynucleotide fragment encoding the mutant.

[0048] A polynucleotide encoding a variant of the present application may, for example, include a base sequence encoding the amino acid sequence shown in SEQ ID NO: 3. As an example of the present application, a polynucleotide of the present application may have or include the sequence of SEQ ID NO: 4. Furthermore, a polynucleotide of the present application may consist of the sequence of SEQ ID NO: 4, or may essentially consist of the sequence of SEQ ID NO: 4.

[0049] The polynucleotide of the present application may be modified in various ways in the coding region, taking into account the codon degeneracy or the codons preferred in the organism in which the variant of the present application is to be expressed, without changing the amino acid sequence of the variant of the present application. Specifically, the polynucleotide of the present application has a base sequence 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, and less than 100% homology or identity to SEQ ID NO: 4, or includes the base sequence, or consists of a base sequence 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, and less than 100% homology or identity to the sequence of SEQ ID NO: 4, or is substantially composed of the base sequence, but is not limited thereto.

[0050] Here, in the sequence having the homology or identity, the codon encoding the amino acid corresponding to the 43rd position of SEQ ID NO: 3 may be one of the codons encoding valine, the codon encoding the amino acid corresponding to the 58th position may be one of the codons encoding glutamine, and the codon encoding the amino acid corresponding to the 74th position may be one of the codons encoding valine.

[0051] Furthermore, the polynucleotide of the present application may be any sequence that hybridizes under stringent conditions with a probe prepared from a known gene sequence, for example, a complementary sequence to all or a part of the polynucleotide sequence of the present application. The term "stringent conditions" refers to conditions that allow specific hybridization between polynucleotides. Such conditions are specifically described in the literature (see Non-Patent Documents 11 and 12). For example, the conditions include conditions under which polynucleotides having high homology or identity, such as polynucleotides having a homology or identity of 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, hybridize with each other, but polynucleotides having lower homology or identity than that do not hybridize with each other, or conditions under which washing is performed once, specifically two to three times, at a salt concentration and temperature equivalent to normal Southern hybridization washing conditions of 60°C, 1 x SSC, 0.1% SDS, specifically 60°C, 0.1 x SSC, 0.1% SDS, more specifically 68°C, 0.1 x SSC, 0.1% SDS.

[0052] Hybridization requires that two nucleic acids have complementary sequences, even though mismatches between bases are possible depending on the stringency of hybridization. "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. Thus, the polynucleotides of the present application may include isolated nucleic acid fragments that are complementary to the entire sequence, as well as substantially similar nucleic acid sequences.

[0053] Specifically, polynucleotides having homology or identity to the polynucleotides of the present application can be detected using the above-mentioned hybridization conditions including a hybridization step at a Tm value of 55° C. The Tm value may be, but is not limited to, 60° C., 63° C., or 65° C., and may be appropriately adjusted by those skilled in the art depending on the purpose.

[0054] The appropriate stringency for hybridizing the polynucleotides depends on the length of the polynucleotides and the degree of complementation, variables known in the art (eg, Non-Patent Document 11).

[0055] Yet another aspect of the present application provides a vector comprising a polynucleotide of the present application, the vector being, but not limited to, an expression vector for expressing the polynucleotide in a host cell.

[0056] The vector of the present application includes a DNA product comprising a base sequence of a polynucleotide encoding a target polypeptide operably linked to a suitable expression control region (or expression control sequence) so that the target polypeptide can be expressed in a suitable host. The expression control region includes a promoter for initiating transcription, an optional operator sequence for regulating the transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence for regulating the termination of transcription and translation. When transformed into a suitable host cell, the vector can replicate and function independently of the host genome, and is integrated into the genome itself.

[0057] The vector used in the present application is not particularly limited, and any vector known in the art may be used. Examples of commonly used vectors include plasmids, cosmids, viruses, and bacteriophages in a natural or recombinant state. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, Charon21A, etc. may be used as phage or cosmid vectors, and pDZ, pBR, pUC, pBluescriptII, pGEM, pTZ, pCL, pET, etc. may be used as plasmid vectors. Specifically, pDZ, pDC, pDCM2 (Patent Document 2), pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC, pIMR53 vectors, etc. may be used.

[0058] For example, a polynucleotide encoding a target polypeptide can be inserted into a chromosome by a vector for intracellular chromosome introduction. The polynucleotide can be inserted into a chromosome by any method known in the art, such as, but not limited to, homologous recombination. A selection marker for confirming whether or not it has been inserted into the chromosome may be further included. The selection marker is for selecting cells transformed with the vector, i.e., for confirming whether or not the target nucleic acid molecule has been inserted, and a marker that confers a selectable phenotype such as drug resistance, auxotrophy, resistance to cytotoxic agents, or expression of a surface polypeptide is used. In an environment treated with a selection agent, only cells expressing the selection marker survive or show a different phenotype, so that transformed cells can be selected.

[0059] In the present application, "transformation" means introducing a vector containing a polynucleotide encoding a target polypeptide into a host cell or a microorganism, thereby expressing the polypeptide encoded by the polynucleotide in the host cell. The transformed polynucleotide may be any polynucleotide that is expressed in the host cell, regardless of whether it is inserted into the chromosome of the host cell or located extrachromosomally. The polynucleotide may include DNA and / or RNA that encodes the target polypeptide. The polynucleotide may be introduced in any form as long as it is introduced into the host cell and expressed. For example, the polynucleotide may be introduced into the host cell in the form of an expression cassette, which is a genetic structure containing all elements necessary for its own expression. Typically, the expression cassette contains a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal operably linked to the polynucleotide. The expression cassette may be in the form of a self-replicating expression vector. The polynucleotide may be introduced into the host cell in its own form and operably linked to a sequence necessary for expression in the host cell, but is not limited thereto.

[0060] Additionally, the term "operably linked" 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 application.

[0061] Yet another aspect of the present application provides a microorganism comprising a variant of the present application, or a polynucleotide of the present application.

[0062] The microorganism of the present application may comprise at least one of the variant of the present application, a polynucleotide encoding the variant, and a vector comprising the polynucleotide. The microorganism of the present application may comprise the variant polypeptide of the present application, a polynucleotide encoding the polypeptide, or a vector comprising the polynucleotide of the present application.

[0063] In the present application, the term "strain" or "microorganism" includes all wild-type microorganisms and microorganisms that have been genetically modified, either naturally or artificially, and in which a specific mechanism has been weakened or enhanced by, for example, inserting an exogenous gene or enhancing or inactivating the activity of an endogenous gene, and which has been genetically modified to produce a desired polypeptide, protein, or product.

[0064] The microorganism of the present application is, but is not limited to, a microorganism comprising at least one of the variants of the present application, the polynucleotides of the present application, and the vectors comprising the polynucleotides of the present application, a microorganism modified to express the variants of the present application or the polynucleotides of the present application, a microorganism (e.g., recombinant microorganism) expressing the variants of the present application or the polynucleotides of the present application, or a microorganism (e.g., recombinant microorganism) having the activity of the variants of the present application. The microorganism may be a microorganism that endogenously contains polynucleotides encoding lycopene cyclase / phytoene synthase (crtYB) and phytoene desaturase (crtI) proteins, or may be a microorganism modified to further contain polynucleotides encoding the proteins and exhibit the protein activity, or the protein activity is enhanced. The microorganism may also be a microorganism in which a parent strain that does not have activity of lycopene cyclase / phytoene synthase (crtYB) and phytoene desaturase (crtI) proteins is modified to contain a polynucleotide encoding the protein, and the microorganism exhibits the activity of the protein. The lycopene cyclase / phytoene synthase or phytoene desaturase is a protein derived from Xanthophyllomyces dendrorhous, but is not limited thereto. As a specific example, the polynucleotide encoding the lycopene cyclase / phytoene synthase or phytoene desaturase may have or include the sequence of SEQ ID NO: 13 or SEQ ID NO: 14, respectively. The polynucleotide may be modified in various ways in the coding region to the extent that the amino acid sequence is not changed, taking into account codon degeneracy or preferred codons in a microorganism in which the mutant of the present application is to be expressed.Specifically, the polynucleotide has a base sequence that is 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and less than 100% homologous or identical to the sequence of SEQ ID NO: 13 or SEQ ID NO: 14, or contains the base sequence, or consists of a base sequence that is 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and less than 100% homologous or identical to the sequence of SEQ ID NO: 13 or SEQ ID NO: 14, or is substantially composed of the base sequence, but is not limited to these.

[0065] The microorganism of the present application may be one having retinoid-producing ability or retinol-producing ability.

[0066] In the present application, the term "retinoid" refers to a substance that acts as or is related to vitamin A. Specifically, the retinoid is at least one selected from the group consisting of retinol, retinal, retinyl ester, and retinoic acid. The microorganism of the present application may be a microorganism capable of producing retinal and retinol, and retinoic acid may be produced by conversion from retinol.

[0067] The microorganism of the present application is a microorganism that naturally has the ability to produce β-carotene 15,15'-oxygenase or a retinoid, or a microorganism into which a mutant of the present application or a polynucleotide encoding the mutant (or a vector containing the polynucleotide) has been introduced into a parent strain that does not have the ability to produce β-carotene 15,15'-oxygenase or a retinoid, and / or which has been conferred the ability to produce a retinoid, but is not limited to these.

[0068] For example, the microorganism of the present application is a cell or microorganism that is transformed with a vector containing the polynucleotide of the present application or a polynucleotide encoding the variant of the present application and expresses the variant of the present application, and for the purposes of this application, the microorganism of the present application may be any microorganism that contains the variant of the present application and produces a retinoid. For example, the microorganism of the present application may be a recombinant microorganism in which a β-carotene 15,15'-oxygenase variant is expressed and retinoid production ability is improved by introducing a polynucleotide encoding the variant of the present application into a naturally occurring wild-type microorganism or a microorganism that produces a retinoid. The recombinant microorganism with improved retinoid production ability is a microorganism with improved retinoid production ability compared to a natural wild-type microorganism or a bifunctional β-carotene 15,15'-oxygenase unmodified microorganism (i.e., a microorganism expressing wild-type β-carotene 15,15'-oxygenase (SEQ ID NO: 1) or a microorganism not expressing a mutant (SEQ ID NO: 3 or the above-mentioned β-carotene 15,15'-oxygenase mutant) protein), but is not limited thereto. For example, a β-carotene 15,15'-oxygenase unmodified microorganism, which is a target microorganism for comparing whether or not the retinoid production ability is improved, is the CC08-1023 strain, but is not limited thereto.

[0069] As an example, the recombinant microorganism with improved production ability has a retinoid production ability of about 1% or more, specifically about 1% or more, about 2.5% 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 11.5% or more, about 12% or more, about 12.5% ​​or more, about 13% or more, about 13.5% or more, about 14% or more, about 14.5% or more, about 15% or more, about 15.5% or more, about 16% or more, about 16.5% or more, about 17% or more, about 17.5% or more, about 18% or more, about 20% or more, about 22% or more, about 24% or more, about 26% or more, about The productivity of the modified microorganism is improved by 28% or more, about 30% or more, about 32% or more, about 34% or more, about 36% or more, about 37% or more, about 38% or more, about 39% or more, about 40% or more, about 41% or more, about 41.5% or more, about 42% or more, about 42.5% or more, about 43% or more, about 43.5% or more, about 44% or more, about 44.5% or more, about 45% or more, about 45.5% or more, or about 46% or more (there is no particular limit to the upper limit, for example, about 200% or less, about 150% or less, about 100% or less, or about 50% or less), but may be any productivity increase of + value compared to the parent strain or unmodified microorganism before mutation. In other examples, the recombinant microorganism with improved production ability has a retinoid production ability that is about 1.01 times or more, about 1.02 times or more, about 1.03 times or more, about 1.05 times or more, about 1.06 times or more, about 1.07 times or more, about 1.08 times or more, about 1.09 times or more, about 1.10 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 1.20 times or more, about The improvement is 1.22 times or more, about 1.24 times or more, about 1.26 times or more, about 1.28 times or more, about 1.30 times or more, about 1.32 times or more, about 1.34 times or more, about 1.36 times or more, about 1.38 times or more, about 1.40 times or more, about 1.41 times or more, about 1.42 times or more, about 1.43 times or more, about 1.44 times or more, about 1.45 times or more, or about 1.46 times or more (there is no particular limit to the upper limit, and it is, for example, about 10 times or less, about 5 times or less, about 3 times or less, or about 2 times or less), but is not limited thereto.The term "about" refers to a range including, but not limited to, ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and may refer to any numerical value that is equal to or in a similar range to the numerical value following the term "about."

[0070] In the present application, the term "unmodified microorganism" does not exclude strains containing mutations that may occur naturally in microorganisms, but refers to a wild-type or naturally occurring strain itself, or a strain before its characteristics are changed due to genetic mutation caused by natural or artificial factors. For example, the unmodified microorganism refers to a strain into which the β-carotene 15,15'-oxygenase mutant described herein has not been introduced or has not been introduced. The term "unmodified microorganism" is used interchangeably with "strain before modification," "microorganism before modification," "non-mutated strain," "non-modified strain," "non-mutated microorganism," or "reference microorganism."

[0071] In another embodiment of the present application, the microorganism of the present application is a Yarrowia sp. microorganism, specifically, but not limited to, Yarrowia lipolytica.

[0072] In the present application, "enhancement" of a polypeptide activity means improving the activity of the polypeptide compared to its endogenous activity. The enhancement is used interchangeably with activation, up-regulation, overexpression, increase, etc. Here, activation, enhancement, up-regulation, overexpression, and increase all include the display of an activity that was not originally present, and the improvement of activity compared to the endogenous activity or activity before modification. The "endogenous activity" means the activity of a specific polypeptide originally possessed by a parent strain or an unmodified microorganism before the trait change when the trait is changed due to genetic mutation caused by natural or artificial factors. This is used interchangeably with "activity before modification." The "enhancement," "up-regulation," "overexpression," or "improvement" of a polypeptide activity compared to its endogenous activity means the improvement of the activity and / or concentration (expression level) of a specific polypeptide originally possessed by a parent strain or an unmodified microorganism before the trait change.

[0073] The enhancement may be achieved by introducing a foreign polypeptide, or by enhancing the activity and / or increasing the concentration (expression level) of an endogenous polypeptide. Whether the activity of the polypeptide has been enhanced can be confirmed by an increase in the level of activity of the polypeptide, the expression level, or the amount of a product produced from the polypeptide.

[0074] To enhance the activity of the polypeptide, various methods well known in the art can be applied, and any method can be used as long as it can enhance the activity of the target polypeptide compared to the microorganism before modification. Specifically, the method is a conventional method in molecular biology that uses genetic engineering and / or protein engineering well known to those skilled in the art, but is not limited to these (e.g., Non-Patent Documents 13 and 14, etc.).

[0075] Specifically, the enhancement of the polypeptide activity of the present application is carried out by 1) increasing the intracellular copy number of a polynucleotide encoding the polypeptide, 2) replacing the gene expression regulatory region on the chromosome encoding the polypeptide with a sequence having stronger activity, 3) modifying the base sequence encoding the start codon or 5'UTR region of the gene transcript encoding the polypeptide, 4) modifying the amino acid sequence of the polypeptide so as to enhance the polypeptide activity, 5) modifying the polynucleotide sequence encoding the polypeptide so as to enhance the polypeptide activity (e.g., modifying the polynucleotide sequence of the polypeptide gene so as to encode a polypeptide modified so as to enhance the polypeptide activity), 6) introducing a foreign polypeptide exhibiting the polypeptide activity or a foreign polynucleotide encoding the same, 7) optimizing the codons of the polynucleotide encoding the polypeptide, 8) analyzing the tertiary structure of the polypeptide and selecting and modifying or chemically modifying the exposed portion, or 9) a combination of two or more selected from 1) to 8) above, but is not particularly limited thereto.

[0076] More specifically, the above 1) increasing the intracellular copy number of the polynucleotide encoding the polypeptide may be achieved by introducing into the host cell a vector to which the polynucleotide encoding the polypeptide is operably linked, the vector replicating and functioning independently of the host. Alternatively, the above may be achieved by introducing one or more copies of the polynucleotide encoding the polypeptide into a chromosome in the host cell. The introduction into the chromosome is achieved by introducing into the host cell a vector capable of inserting the polynucleotide into a chromosome in the host cell, but is not limited thereto. The vector is as described above.

[0077] The expression regulatory region (or expression regulatory sequence) of a gene on a chromosome encoding a polypeptide is replaced with a sequence having a stronger activity, for example, by generating a mutation in the sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, so that the activity of the expression regulatory region is further enhanced, or by replacing the expression regulatory region with a sequence having a higher activity. The expression regulatory region includes, but is not limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, a sequence regulating the termination of transcription and translation, and the like. For example, the expression regulatory region is replaced with a strong promoter, but is not limited to this.

[0078] Examples of known strong promoters include, but are not limited to, the CJ1 to CJ7 promoters (Patent Document 3), lac promoter, trp promoter, trc promoter, tac promoter, lambda phage PR promoter, PL promoter, tet promoter, gapA promoter, SPL7 promoter, SPL13 (sm3) promoter (Patent Document 4), O2 promoter (Patent Document 5), tkt promoter, yccA promoter, TEFINt promoter, EXP1 promoter, and TEF1 promoter.

[0079] The nucleotide sequence encoding the start codon or 5'UTR region of the gene transcription product encoding the polypeptide (3) can be modified, for example, by substituting it with a nucleotide sequence encoding another start codon that has a higher expression rate of the polypeptide compared to the endogenous start codon, but is not limited to this.

[0080] The modification of the amino acid sequence or polynucleotide sequence in 4) and 5) above can be performed by, but is not limited to, generating 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, so as to enhance the activity of the polypeptide, or by replacing the sequence with an amino acid sequence or polynucleotide sequence improved to have a higher activity, or an amino acid sequence or polynucleotide sequence improved to improve the activity. Specifically, the replacement can be performed by, but is not limited to, inserting the polynucleotide into a chromosome by homologous recombination. Here, the vector used may further include a selection marker for checking whether or not it has been inserted into the chromosome. The selection marker is as described above.

[0081] The above 6) introduction of a foreign polynucleotide exhibiting the activity of the polypeptide may be carried out by introducing into a host cell a foreign polynucleotide encoding a polypeptide exhibiting the same / similar activity as the polypeptide. The foreign polynucleotide may be of any origin or sequence as long as it exhibits the same / similar activity as the polypeptide. The introduction can be carried out by a person skilled in the art by appropriately selecting a known transformation method, and the introduced polynucleotide is expressed in the host cell as described above, thereby producing a polypeptide and improving its activity.

[0082] 7) Optimizing the codons of a polynucleotide encoding a polypeptide may be carried out by optimizing the codons of an endogenous polynucleotide so as to increase transcription or translation in a host cell, or by optimizing the codons of an exogenous polynucleotide so as to perform optimized transcription or translation in a host cell.

[0083] 8) Analyzing the tertiary structure of a polypeptide and selecting and altering or chemically modifying an exposed portion may be carried out, for example, by comparing sequence information of the polypeptide to be analyzed with a database in which sequence information of known proteins is stored, determining candidates for template proteins depending on the degree of sequence similarity, confirming the structure based on the candidates, and selecting and altering or modifying the exposed portion to be altered or chemically modified.

[0084] Such enhancement of polypeptide activity can be achieved by, but is not limited to, improving the activity, concentration, or expression level of the corresponding polypeptide compared to the activity or concentration of the polypeptide expressed in a wild-type or unmodified microbial strain, or by increasing the amount of a product produced from the polypeptide.

[0085] In the microorganism of the present application, modification of a part or the whole of a polynucleotide can be induced by (a) homologous recombination using a vector for chromosomal introduction in the microorganism, or genome editing using engineered nuclease (e.g., CRISPR-Cas9), and / or (b) light such as ultraviolet light or radiation and / or chemical treatment, but is not limited thereto. Methods for modifying a part or the whole of the gene include methods using DNA recombination techniques. For example, a nucleotide sequence or vector containing a nucleotide sequence homologous to a target gene is introduced into the microorganism to cause homologous recombination, thereby deleting a part or the whole of the gene. The introduced nucleotide sequence or vector may contain a dominant selection marker, but is not limited thereto.

[0086] The mutants, polynucleotides, retinoids, and the like in the microorganisms of the present application are as described above.

[0087] Yet another aspect of the present application provides a method for producing a retinoid comprising culturing in a medium a microorganism comprising a variant of the present application, or a polynucleotide of the present application.

[0088] The method of producing a retinoid of the present application may comprise the step of culturing in a medium a microorganism comprising a variant of the present application, a polynucleotide of the present application, or a vector of the present application.

[0089] "Culturing" in the present application means growing the microorganism of the present application under appropriately adjusted environmental conditions. The culturing process of the present application can be carried out in a suitable medium and under suitable culture conditions known in the art. Such a culturing process can be easily adjusted and used by a person skilled in the art depending on the strain selected. Specifically, the culturing can be batch, continuous and / or fed-batch culture, but is not limited thereto.

[0090] In the present application, the term "culture medium" refers to a substance that is a mixture of nutrients necessary for culturing the microorganism of the present application as the main components, and that supplies nutrients such as water essential for survival and growth, growth factors, etc. Specifically, the culture medium and other culture conditions used for culturing the microorganism of the present application may be any medium used for culturing ordinary microorganisms, and the microorganism of the present application can be cultured in an ordinary culture medium containing a suitable carbon source, nitrogen source, phosphorus source, inorganic compounds, amino acids and / or vitamins, etc., under aerobic conditions by adjusting the temperature, pH, etc.

[0091] The carbon source in the present application may be 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. Also, natural organic nutrient sources such as starch hydrolysates, molasses, blackstrap molasses, rice bran, cassava, bagasse, corn steeping liquid, etc. may be used, specifically, carbohydrates such as glucose and sterilized pretreated molasses (i.e., molasses converted into reducing sugars) may be used, and any other suitable carbon source may be used. These carbon sources may be used alone or in combination of two or more, but are not limited thereto.

[0092] The nitrogen source may be an inorganic nitrogen source such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, or ammonium nitrate, or an organic nitrogen source such as an amino acid such as glutamic acid, methionine, or glutamine, peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steeping liquid, casein hydrolysate, fish or its decomposition product, or defatted soybean cake or its decomposition product. These nitrogen sources may be used alone or in combination of two or more kinds, but are not limited thereto.

[0093] The phosphorus source may be potassium dihydrogen phosphate, dipotassium hydrogen phosphate, or a sodium-containing salt equivalent thereto. The inorganic compound may be sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, or other suitable compounds, such as amino acids, vitamins, and / or suitable precursors. These components or precursors may be added to the medium in a batch or continuous manner. However, they are not limited thereto.

[0094] In addition, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid may be added to the medium in a suitable manner during the cultivation of the microorganism of the present application to adjust the pH of the medium. Furthermore, foam generation may be suppressed during the cultivation using an antifoaming agent such as fatty acid polyglycol ester. Furthermore, in order to maintain the aerobic state of the medium, oxygen or an oxygen-containing gas may be injected into the medium, and in order to maintain the anaerobic and microaerobic states, no gas may be injected, and nitrogen, hydrogen, or carbon dioxide gas may be injected, but is not limited thereto.

[0095] In the culture of the present application, the culture temperature is maintained at 20 to 45° C., specifically 25 to 40° C., 20 to 35° C., or 25 to 35° C., and the culture is performed for about 10 to 160 hours, but is not limited thereto.

[0096] The retinoids produced by the cultures of the present application are either secreted into the medium or remain intracellularly.

[0097] The retinoid production method of the present application may further include a step of preparing a microorganism of the present application, a step of preparing a medium for culturing said microorganism, or a combination thereof (in any order), e.g., prior to said culturing step.

[0098] The method for producing a retinoid of the present application may further include a step of recovering a retinoid from the medium used for the culture (the medium in which the culture was carried out) or the microorganism of the present application. The recovery step may be further included after the culture step.

[0099] The recovery may involve collecting the target retinoid using a suitable method known in the art depending on the culture method of the microorganism of the present application, such as batch, continuous, or fed-batch culture methods, etc. For example, centrifugation, filtration, crystallization, treatment with a protein precipitant (salting out method), extraction, cell disruption, ultrasonic disruption, ultrafiltration, dialysis, various chromatographies such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, and affinity chromatography, HPLC, or a combination thereof may be used, and the target retinoid can be collected from the medium or the microorganism using a suitable method known in the art.

[0100] In addition, the retinoid production method of the present application may further include a purification step. The purification may be performed by any suitable method known in the art. For example, when the retinoid production method of the present application includes both a recovery step and a purification step, the recovery step and the purification step may be performed continuously or discontinuously regardless of the order, or may be performed simultaneously or integrated into one step, but is not limited thereto.

[0101] In the method for producing a retinol derivative, which is a type of retinoid, of the present application, the culturing step may further include a step of converting retinol into a retinol derivative. In the method for producing a retinol derivative of the present application, the converting step may further be included after the culturing step or the recovering step. The converting step may be performed by a suitable method known in the art (Non-Patent Documents 15 and 16).

[0102] The mutants, polynucleotides, vectors, microorganisms, etc. in the methods of the present application are as described above.

[0103] Yet another aspect of the present application provides a composition for producing a retinoid, comprising a variant of the present application, a polynucleotide encoding the variant, a vector comprising the polynucleotide, or a microorganism comprising a polynucleotide of the present application, or a medium in which the same have been cultured, or a combination of two or more thereof.

[0104] The compositions of the present application may further comprise any suitable excipients commonly used in compositions for producing retinoids, including, but not limited to, preservatives, wetting agents, dispersing agents, suspending agents, buffers, stabilizers, isotonicity agents, etc.

[0105] The mutant, polynucleotide, vector, microorganism, medium, retinoid, and the like in the composition of the present application are as described above.

[0106] Yet another aspect of the present application provides the use of at least one of the variants, polynucleotides, vectors and microorganisms of the present application for the production of a retinoid.

[0107] The mutant, polynucleotide, vector, microorganism, retinoid, etc. are as described above. EXAMPLES

[0108] The present application will be described in more detail below with reference to examples. However, these examples are merely preferred embodiments illustrating the present application, and the present application is not limited thereto. Note that technical matters not described in this specification are fully understood and easily implemented by a skilled engineer in the technical field of the present application or a similar technical field. Example 1

[0109] Construction of BCO mutation vector library To amplify the BCO gene derived from uncultured marine bacterium 66A03, the amino acid sequence of SEQ ID NO: 1 and the polynucleotide sequence of SEQ ID NO: 2 were obtained based on the amino acid sequence (Q4PNI0) registered in UniProtKB (UniProt Knowledgebase). To induce random mutations by error-prone PCR, the diversify PCR random mutagenesis kit (Takara) was used. The PCR conditions and method were optimized and performed under buffer conditions 1 and 2 in the user manual. Specific error-prone PCR conditions are shown in Table 1.

[0110] The wild-type BCO gene (sequence number: 66A03) derived from the marine bacterium Macrogen was synthesized. 2 ) as a template and primers of sequence numbers 5 and 6. PCR conditions were 25 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 68°C for 1 minute. The BCO PCR products amplified by PCR under the two conditions were mixed to prepare a vector library of BCO mutations based on the pIMR53-TEFINtp-CYC1t vector.

[0111] [Table 1]

[0112] The pIMR53-TEFINtp-CYC1t vector was constructed in the following manner.

[0113] To amplify TEFINtp, PCR was performed using primers SEQ ID NO:7 and SEQ ID NO:8 with genomic DNA of Yarrowia lipolytica PO1f as a template, and primers SEQ ID NO:9 and SEQ ID NO:10 with genomic DNA of Saccharomyces cerevisiae as a template. PCR conditions were denaturation at 95°C for 1 minute, annealing at 55°C for 1 minute, and polymerization at 68°C for 1 minute for 32 cycles. Furthermore, PCR was performed using primers SEQ ID NO:11 and SEQ ID NO:12 with pIMR53 vector as a template. PCR conditions were denaturation at 95°C for 1 minute, annealing at 55°C for 1 minute, and polymerization at 68°C for 5 minutes for 32 cycles.

[0114] As a result, 6,774 bp linearized pIMR53, 531 bp TEFINtp, and 248 bp CYC1t were obtained. The PCR-amplified DNA fragments of TEFINtp and CYC1t were ligated to the pIMR53 vector using an Infusion Cloning Kit (Invitrogen), then transformed into E. coli DH5α and spread on LB solid medium containing 30 mg / L ampicillin.

[0115] Colonies transformed with the plasmid containing the desired TEFINt promoter and CYC1 terminator were selected by PCR, and the plasmid was isolated using a well-known plasmid extraction method and named pIMR53-TEFINtp-CYC1t. Example 2

[0116] Construction of Yarrowia lipolytica platform strains for retinoid production Example 2-1. Construction of a strain with crtYB-crtI insertion derived from X. dendrorhous To prepare a platform strain for retinoid production, the lycopene cyclase / phytoene synthase (crtYB) and phytoene desaturase (crtI) genes derived from Xanthophyllomyces dendrorhous were inserted into the genome of the high fat yeast strain Yarrowia lipolytica CC08-0125 (accession number KCCM12972P). For crtYB, a polynucleotide sequence shown in SEQ ID NO: 13 was obtained based on the base sequence registered in the National Center for Biotechnology Information Search database (NCBI) (GenBank: AY177204.1), and for crtI, a polynucleotide sequence shown in SEQ ID NO: 14 was obtained based on the base sequence registered in the NCBI (GenBank: AY177424.1). The polynucleotide sequences of crtYB and crtI were synthesized in the form of TEFINtp-crtYB-CYC1t (SEQ ID NO: 15) and TEFINtp-crtI-CYC1t (SEQ ID NO: 16) at the request of Macrogen. A cassette was designed to be inserted into the MHY1 (YALI0B21582g) gene position using the URA3 gene (SEQ ID NO: 17) of Y. lipolytica as a selection marker. Using the synthesized crtYB and crtI genes and the genomic DNA of KCCM12972P as templates, PCR was performed using primers SEQ ID NO: 18 and SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, SEQ ID NO: 22 and SEQ ID NO: 23, SEQ ID NO: 24 and SEQ ID NO: 25, SEQ ID NO: 26 and SEQ ID NO: 27, and SEQ ID NO: 28 and SEQ ID NO: 29. The PCR conditions were 35 cycles of denaturation at 95°C for 1 minute, annealing at 55°C for 1 minute, and polymerization reaction at 72°C for 3 minutes and 30 seconds. The five resulting DNA fragments were prepared into one cassette by overlap extension PCR.

[0117] The cassette thus prepared was introduced into the KCCM12972P strain by the heat shock method (Non-Patent Document 17), and then colonies formed on a uracil-free solid medium (YLMM1) were obtained. Colonies in which the cassette was confirmed to have been inserted into the genome were spotted on a 5-FOA solid medium using primers of SEQ ID NO: 30 and SEQ ID NO: 31, and cultured at 30°C for 3 days to obtain colonies grown on the 5-FOA solid medium, thereby recovering the URA3 marker.

[0118] Example 2-2. Preparation of HMGR-enhanced strain As described above, a cassette was designed to replace the native promoter (SEQ ID NO: 32) site of the hydroxymethylglutaryl-CoA reductase (HMGR) gene of the strain into which the crtYB and crtI genes prepared in Example 2-1 were inserted with the TEFINt promoter, and PCR was performed using the genomic DNA of KCCM12972P as a template and primers SEQ ID NO: 33 and SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36, SEQ ID NO: 37 and SEQ ID NO: 38, SEQ ID NO: 39 and SEQ ID NO: 40, and SEQ ID NO: 41 and SEQ ID NO: 42. The PCR conditions were 35 cycles of denaturation at 95°C for 1 minute, annealing at 55°C for 1 minute, and polymerization reaction at 72°C for 1 minute and 30 seconds. The five resulting DNA fragments were prepared into one cassette by overlap extension PCR.

[0119] The cassette thus prepared was introduced into the strain prepared in Example 2-1 by the heat shock method, and then colonies formed on a uracil-free solid medium (YLMM1) were obtained. Colonies in which the cassette insertion was confirmed were spotted on a 5-FOA solid medium using primers of SEQ ID NO: 43 and SEQ ID NO: 44, and cultured at 30°C for 3 days to obtain colonies grown on the 5-FOA solid medium, thereby recovering the URA3 marker. The retinoid production platform strain finally prepared was named CC08-1023. <Yarrowia lipolytica minimal media 1(YLMM1)> Glucose 20g / L, Yeast nitrogen base without amino acids 6.7g / L, Yeast Synthetic Drop-out Medium Supplements without uracil 2g / L, Agar 15g / L <5-Fluoroorotic Acid (5-FOA)> Glucose 20g / L, Yeast nitrogen base without amino acids 6.7g / L, Yeast Synthetic Drop-out Medium Supplements without uracil 2g / L, Uracil 50μg / mL, 5-Fluoroorotic acid (5-FOA) 1g / L, Agar 15g / L Example 3

[0120] Primary selection of strains with enhanced BCO activity To select BCO with enhanced activity, the CC08-1023 strain, a platform strain for retinoid production, was transformed by heat shock with the pIMR53 (negative control, NC) vector, the pIMR53-TEFINtp-BCO (wild type, SEQ ID NO: 2)-CYC1t vector (positive control, PC), and the BCO random mutant expression vector library constructed in Example 1. Then, colonies formed on uracil-free solid medium were obtained.

[0121] 350 μl of uracil-free YLMM2 (Yarrowia lipolytica minimal media 2) liquid medium was dispensed into 31 96 deep-well plates, and the obtained colonies were inoculated and cultured at 30° C. for 48 hours. Then, the colonies were spotted onto uracil-free YLMM1 solid medium and cultured at 30° C. for 3 days. After the culture was completed, 17 strains that were lighter in color than the positive control (CC08-1023 / pIMR53-TEFINtp-BCO-CYC1t) were primarily selected from a total of 3,000 cultured colonies. The production method and composition of the YLMM2 are as follows. <YLMM2(pH7.0)> Glucose 40g / L, yeast nitrogen base without amino acids and ammonium sulfate 1.7g / L, uracil 0.5g / L, and ammonium sulfate 2.5g / L dissolved in 0.1M sodium phosphate buffer (pH 7.0). Example 4

[0122] Secondary selection of strains with enhanced BCO activity The 17 species primarily selected in Example 3 were subjected to flask evaluation.

[0123] The 17 strains and strains transformed with PC (positive control) vector were inoculated into a 250 ml corner baffle flask containing 50 ml of uracil-free YLMM2 (Yarrowia lipolytica minimal media 2) so that the initial OD was 1, and cultured with shaking at 30°C and 250 rpm for 48 hours. After the culture was completed, 1 ml of the culture solution was centrifuged to remove the supernatant. Next, 0.5 ml of DMSO (Dimethyl sulfoxide, Sigma) was added, and the cells were disrupted by shaking at 55°C and 2,000 rpm for 10 minutes. Furthermore, 0.5 ml of acetone (Sigma) was added, and the cells were extracted by shaking at 45°C and 2,000 rpm for 15 minutes, and the concentrations were analyzed using HPLC equipment. The analyzed OD, retinol, and retinal concentrations are shown in Table 2.

[0124] [Table 2]

[0125] The retinol concentration extracted from the evaluation sample was then used as the criterion for the secondary selection. It was confirmed that the retinol concentration (Retinol (%)) increased in five of the evaluation strains (numbers 1, 4, 5, 12, and 16) compared to PC (positive control). In particular, it was confirmed that the retinol concentration increased by about 46% compared to PC (positive control) in strain 1. Therefore, the transformed plasmid was extracted from strain 1, which showed the largest increase in retinol concentration, and sequence analysis was performed to confirm the random mutation located in the BCO ORF. The BCO plasmid transformed from the selected strain was extracted using Zymoprep yeast plasmid miniprep kit 2 (Zymo research), and sequence analysis was performed in both directions using sequence numbers 45 and 46 to increase the reliability of the sequence analysis. As a result, it was confirmed that the number of mutations in the BCO ORF was 3. The confirmed mutations are shown in Table 3.

[0126] [Table 3]

[0127] These results confirmed that introduction of the BCO mutant of the present application increases retinoid production.

[0128] From the above description, a person skilled in the art to which the present application pertains will understand that the present application can be implemented in other specific forms without changing its technical ideas or essential features. It should be understood that the above examples are merely illustrative and not limiting. This application should be interpreted as including all modifications or alterations derived from the meaning and scope of the claims and their equivalent concepts, rather than the specification.

[0129] Sequence Catalog The sequences of the present application are shown in Tables 4 to 6.

[0130] [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5] [Table 4-6] [Table 4-7]

Table 4-8

Table 4-9

Table 4-10

Table 4-11

Table 4-12

Table 4-13

Table 4-14

Table 4-15

Table 4-16

[0131]

Table 5-1

Table 5-2

[0132]

Table 6

[0133]

Table 7

Claims

1. A β-carotene 15,15'-oxygenase mutant having at least 90% identity to the amino acid sequence of SEQ ID NO:1, in which the amino acid at the 43rd position from the N-terminus of the amino acid sequence of SEQ ID NO:1 is alanine and replaced with valine, the amino acid at the 58th position is leucine and replaced with glutamine, and the amino acid at the 74th position is isoleucine and replaced with valine.

2. A polynucleotide encoding the variant of claim 1.

3. A microorganism comprising at least one of the mutants according to claim 1 and a polynucleotide encoding said mutant, said microorganism belonging to the genus Yarrowia and having retinoid producing ability.

4. The microorganism of claim 3 , wherein the retinoid is at least one selected from the group consisting of retinal, retinol, retinyl ester, and retinoic acid.

5. The microorganism of claim 3 , wherein the microorganism is Yarrowia lipolytica.

6. 2. A method for producing a retinoid, comprising the step of culturing in a medium a microorganism containing the mutant of claim 1 or a polynucleotide encoding said mutant, wherein said microorganism belongs to the genus Yarrowia and has retinoid-producing ability.

7. The method for producing a retinoid according to claim 6 , further comprising the step of recovering the retinoid from the medium or the microorganism.

8. The method for producing a retinoid according to claim 6 , wherein the retinoid is at least one selected from the group consisting of retinal, retinol, retinyl ester and retinoic acid.

9. A composition for producing a retinoid, comprising the mutant according to claim 1, the microorganism according to claim 4, or a medium containing said microorganism.

10. Use of a β-carotene 15,15'-oxygenase mutant having at least 90% identity to the amino acid sequence of SEQ ID NO: 1 and a microorganism containing at least one polynucleotide encoding the mutant, in the production of a retinoid, wherein the β-carotene 15,15'-oxygenase mutant has the 43rd amino acid from the N-terminus of the amino acid sequence of SEQ ID NO: 1 being alanine and substituted with valine, the 58th amino acid being leucine and substituted with glutamine, and the 74th amino acid being isoleucine and substituted with valine, the microorganism belonging to the genus Yarrowia (Yarrowia sp.) and having retinoid producing ability.

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