Microorganisms capable of producing retinoids and having attenuated chitin transglycosylase activity, and a method for producing retinoids using the same

Abstract

The present application relates to a Yarrowia microorganism having retinoid-producing ability and attenuated chitin transglycosylase activity, a method for producing a retinoid using the same, a composition for producing a retinoid, use of the same for retinoid production, and a method for producing the microorganism.

Description

[Technical Field]

[0001] The present application relates to a Yarrowia microorganism having retinoid-producing ability and attenuated chitin transglycosylase activity, a method for producing a retinoid using the same, a composition for producing a retinoid, use of the same for retinoid production, and a method for producing the microorganism. [Background technology]

[0002] Retinol, a fat-soluble vitamin, is an essential vitamin that contributes to eye health (improving night blindness), strengthening the immune system, and maintaining healthy skin. Currently, it is produced and sold mainly by global leading companies through chemical synthesis, but research is being conducted into the production of retinol through microbial fermentation.

[0003] For this reason, many techniques have been developed to stabilize the retinol compound itself in compositions or products containing retinol (Patent Document 1), but currently there are not many methods developed to stably increase retinol production. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] U.S. Patent No. 6,858,217 [Non-patent literature]

[0005] [Non-Patent Document 1] Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444 [Non-patent document 2] Rice et al., 2000, Trends Genet. 16: 276-277 [Non-patent document 3] Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

Non-licensed Document 7

Non-licensed literature 9

Non-licensed literature 10

Non-licensed Document 11

Non-licensed Document 12

[0006] The problem to be solved by the present application is to provide a Yarrowia microorganism having retinoid-producing ability and attenuated chitin transglycosylase activity, a method for producing a retinoid using the same, a composition for producing a retinoid, use of the same for retinoid production, and a method for producing the microorganism. [Means for solving the problem]

[0007] The present application provides a Yarrowia sp. microorganism having retinoid-producing ability and attenuated chitin transglycosylase activity.

[0008] The present application provides a method for producing a retinoid, comprising culturing the microorganism of the present application in a medium.

[0009] The present application provides a method for producing a microorganism capable of producing a retinoid, the method comprising the step of attenuating chitin transglycosylase activity in a microorganism of the genus Yarrowia having the ability to produce a retinoid.

[0010] The present application provides a method for increasing retinoid excretion, comprising the step of attenuating chitin transglycosylase activity in a Yarrowia microorganism capable of producing retinoids.

[0011] The present application provides a composition for producing a retinoid, comprising the microorganism of the present application or a culture thereof.

[0012] The present application provides the use of the microorganism of the present application for producing a retinoid. [Effects of the Invention]

[0013] The microorganisms of the present application can be used to produce retinoids. [Brief explanation of the drawings]

[0014] [Figure 1] FIG. 1 shows the retinoid concentrations in flask culture evaluations of the control group (CC08-2050) and chitin transglycosylase-attenuated microorganisms (CJ2327, CJ2328, and CJ2329). DETAILED DESCRIPTION OF THE INVENTION

[0015] These will be described in detail below. Note that each description and embodiment disclosed in this application also applies to other descriptions and embodiments. In other words, all combinations of various elements disclosed in this application are included in this application. Furthermore, this application is not limited to the specific descriptions below. Furthermore, many papers and patent documents are referenced throughout this specification, and citations thereof are provided. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety, thereby more clearly explaining the state of the art to which this application pertains and the contents of this application.

[0016] One aspect of the present application provides a Yarrowia sp. microorganism capable of producing retinoids, in which chitin transglycosylase activity is attenuated.

[0017] In the present application, the term "chitin transglycosylase" refers to an enzyme that catalyzes the transfer of chitin in the cell wall to β-1,6 glucan and β-1,3 glucan.

[0018] The chitin transglycosylase of the present application may be a CRH1 protein, a CRH2 protein, or a combination thereof. For example, the combination of CRH1 proteins or CRH2 proteins includes a combination of a CRH1 protein and a CRH2 protein. Furthermore, since two or more CRH1s having different amino acid sequences or two or more CRH2s having different amino acid sequences may exist, the combination of CRH1 proteins or CRH2 proteins also includes a combination of one or more CRH1 proteins (e.g., a combination of a CRH1 having a specific amino acid sequence and a CRH1 having a different amino acid sequence), a combination of CRH2 proteins (e.g., a combination of a CRH2 having a specific amino acid sequence and a CRH2 having a different amino acid sequence), and a combination of one or more CRH1s and one or more CRH2s (e.g., a combination of a CRH1 having a specific amino acid sequence, a CRH1 having a different amino acid sequence, and a CRH2 having a different amino acid sequence).

[0019] For the purposes of this application, the chitin transglycosylase may be any chitin transglycosylase that improves retinoid production and / or secretion. For example, the improvement in retinoid production is due to an improvement in retinoid secretion, but is not limited thereto.

[0020] As one example, the chitin transglycosylase of the present application may have weakened activity in a Yarrowia microorganism compared to endogenous or wild-type chitin transglycosylase, thereby improving the retinoid production and / or excretion ability of the microorganism.

[0021] As an example, the chitin transglycosylase of the present application may include, have, consist of, or consist essentially of an amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, or an amino acid sequence having 60% or more homology or identity to at least one of them. It may also be, but is not limited to, a polypeptide that exhibits chitin transglycosylase activity together with these sequences.

[0022] For example, the amino acid sequence of the chitin transglycosylase of the present application is encoded by the CRH1 gene, the CRH2 gene, or a combination thereof, such as, but not limited to, the CRH1 (YALI0C09680) gene, the CRH1 (YALI0E24673) gene, the CRH2 (YALI0B15510) gene, or a combination thereof. The amino acid sequence can be obtained from various publicly known databases, such as, but not limited to, GenBank of NCBI.

[0023] As an example, but not limited to, the chitin transglycosylase of the present application is derived from Yarrowia lipolytica.

[0024] As an example, SEQ ID NO: 1 may be the CRH1 (YALI0C09680) protein, SEQ ID NO: 2 may be the CRH1 (YALI0E24673) protein, and SEQ ID NO: 3 may be the CRH2 (YALI0B15510) protein.

[0025] Furthermore, even if an example of a chitin transglycosylase of the present application is described as a protein containing SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or a combination thereof, it is clear to those skilled in the art that this does not exclude meaningless addition of sequences before or after the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or a combination thereof, naturally occurring mutations, or silent mutations thereof, and that any protein having the same or equivalent activity as a protein containing the amino acid sequence is included in the chitin transglycosylase of the present application.

[0026] For example, the chitin transglycosylase of the present application may comprise the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or a combination thereof, or may comprise an amino acid sequence having at least 60% or more, 62% or more, 63% or more, 64% or more, 65% or more, 70% or more, 75% or more, 76% or more, 77% or more, 78% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homology or identity to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or a combination thereof. It goes without saying that the present application also encompasses amino acid sequences in which a portion of the sequence has been deleted, modified, substituted, or added, as long as the amino acid sequence has the above-mentioned homology or identity and exhibits an activity equivalent to that of the protein.

[0027] Although the present application describes "a polypeptide or protein comprising an amino acid sequence represented by a specific SEQ ID NO," "a polypeptide or protein consisting of an amino acid sequence represented by a specific SEQ ID NO," or "a polypeptide or protein having an amino acid sequence represented by a specific SEQ ID NO," it goes without saying that any protein having an amino acid sequence in which a portion of the sequence has been deleted, modified, substituted, conservatively substituted, or added is also included in the present application, as long as it has the same or equivalent activity as the polypeptide or protein consisting of the amino acid sequence of the SEQ ID NO. For example, there are proteins having an addition of a sequence at the N-terminus and / or C-terminus of the amino acid sequence that does not change the function of the protein, a naturally occurring mutation, a silent mutation, or a conservative substitution thereof.

[0028] The term "conservative substitution" refers to the substitution of an amino acid with another amino acid having similar structural and / or chemical properties. Such amino acid substitutions may generally be made based on 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.

[0029] In this application, "homology" or "identity" refers to the degree of identity or similarity between two given amino acid or nucleotide sequences, expressed as a percentage. Homology and identity are often used interchangeably.

[0030] Sequence homology or identity of conserved polynucleotides or polypeptides (including proteins) may be determined by standard sequence algorithms, optionally with default gap penalties established by the program used. Substantially homologous or identical sequences will hybridize under moderately or highly stringent conditions, generally to the entire sequence or a portion thereof representing at least about 50%, 60%, 70%, 80%, or 90% of the entire length. Hybridization, of course, also includes hybridization to polynucleotides having codons commonly used in polynucleotides or codons that take into account codon degeneracy.

[0031] Whether any two polynucleotide or polypeptide (including protein) sequences have homology, similarity, or identity can be determined using known computer algorithms such as the "FASTA" program with default parameters, as described 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 (EMBOSS: The European Molecular Biology Open Software Suite, Non-Patent Document 2) (version 5.0.0 or later) in the EMBOSS package (which includes the GCG program package (Non-Patent Document 4), BLASTP, BLASTN, and FASTA (Non-Patent Documents 5, 6, and 7)). For example, BLAST or ClustalW from the National Center for Biotechnology Information can be used to determine homology, similarity, or identity.

[0032] Homology, similarity, or identity of polynucleotides or polypeptides (including proteins) can be determined by comparing sequence information using a GAP computer program such as that disclosed in Non-Patent Document 3, as disclosed in, for example, Non-Patent Document 8. Briefly, 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, non-identity a value of 0) and a weighted comparison matrix (or EDNAFULL (the EMBOSS version of NCBINUC4.4) substitution matrix) as disclosed in Non-Patent Document 9; (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap (or a gap open penalty of 10, a gap extension penalty of 0.5); and (3) no penalty for terminal gaps.

[0033] Furthermore, whether any two polynucleotide or polypeptide (including protein) sequences have homology, similarity, or identity can be confirmed by comparing the sequences in a Southern hybridization experiment under defined stringent conditions, and appropriate defined hybridization conditions are within the skill of the art and can be determined by methods well known to those skilled in the art (e.g., Non-Patent Documents 11 and 12).

[0034] In this application, the term "polynucleotide" refers to a DNA chain longer than a predetermined length, which is a polymer of nucleotides in which nucleotide monomers are linked in a long chain by covalent bonds.

[0035] The polynucleotide sequence encoding the chitin transglycosylase of the present application is represented by the CRH1 gene, the CRH2 gene, or a combination thereof, such as the CRH1 (YALI0C09680) gene, the CRH1 (YALI0E24673) gene, the CRH2 (YALI0B15510) gene, or a combination thereof, and includes polynucleotide sequences encoding the amino acid sequences represented by SEQ ID NO: 1, SEQ ID NO: 2, and / or SEQ ID NO: 3.

[0036] The polynucleotide may have various modifications in its coding region, taking into account codon degeneracy or preferred codons in the organism in which the polypeptide or protein is to be expressed, as long as the amino acid sequence of the polypeptide or protein is not changed. Specifically, the polynucleotide may comprise, consist of, or consist essentially of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or a polynucleotide sequence having at least 60% homology or identity thereto, but is not limited thereto. For example, the polynucleotide may consist of a base sequence that has 60% or more, 62% or more, 63% or more, 64% or more, 65% or more, 70% or more, 76% or more, 77% or more, 78% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homology or identity to SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6, but is not limited to these.

[0037] Furthermore, the polynucleotide of the present application may be any sequence that hybridizes under stringent conditions with a probe, for example, a sequence complementary to all or part of the polynucleotide base sequence. The term "stringent conditions" refers to conditions that allow specific hybridization between polynucleotides. Such conditions are specifically described in the literature (e.g., Non-Patent Document 11). For example, conditions include those under which polynucleotides with high homology or identity, such as polynucleotides with a homology or identity of 40% or more, specifically 90% or more, more specifically 95% or more, 96% or more, 97% or more, 98% or more, and even more specifically 99% or more, hybridize with each other, while polynucleotides with lower homology or identity do not hybridize with each other; or conditions for washing once, specifically two to three times, at a salt concentration and temperature equivalent to those used in conventional Southern hybridization, namely 60°C, 1×SSC, 0.1% SDS, specifically 60°C, 0.1×SSC, 0.1% SDS, more specifically 68°C, 0.1×SSC, 0.1% SDS.

[0038] Hybridization requires that two nucleic acids have complementary sequences, even if mismatches between bases are possible depending on the stringency of the hybridization. "Complementary" is used to describe the relationship between nucleotide bases that can hybridize to 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 not only substantially similar nucleic acid sequences, but also isolated nucleic acid fragments that are complementary to the entire sequence.

[0039] Specifically, polynucleotides having homology or identity can be detected using the hybridization conditions described above, in which the hybridization step is performed 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 can be appropriately adjusted by those skilled in the art depending on the purpose.

[0040] The appropriate stringency for hybridizing polynucleotides depends on the length of the polynucleotides and the degree of complementation, variables known in the art (Non-Patent Document 11).

[0041] In this application, the term "vector" includes a DNA product for inserting a base sequence of a target polynucleotide into a host chromosome, or a DNA product containing a base sequence of a polynucleotide encoding said target polypeptide or protein operably linked to a suitable expression control region (or expression control sequence) so as to enable expression of the target polypeptide or protein in a suitable host. The expression control region includes a promoter that initiates transcription, an optional operator sequence for regulating the transcription, a sequence encoding a suitable mRNA ribosomal binding site, and a sequence that regulates the termination of transcription and translation. When transformed into a suitable host cell, the vector can replicate and function independently of the host genome, or can be integrated into the genome itself.

[0042] The vector of the present application is an insertion vector for inserting a polynucleotide for attenuating the activity of the chitin transglycosylase of the present application into a chromosome, but is not limited thereto. The insertion of the polynucleotide into a chromosome can be performed by any method known in the art, for example, but not limited to, homologous recombination. The vector may further contain a selection marker for confirming whether or not the polynucleotide has been inserted into the chromosome. The selection marker is used to select cells transformed with the vector, i.e., to confirm whether or not the target nucleic acid molecule has been inserted. Markers that confer a selectable phenotype, such as drug resistance, auxotrophy, resistance to cytotoxic agents, or expression of a surface polypeptide or protein, are used. In an environment treated with a selective agent, only cells expressing the selection marker survive or exhibit a different phenotype, allowing the selection of transformed cells. The insertion vector may not contain an origin of replication necessary for replication in transformed cells.

[0043] The vector used in this application is not particularly limited, and any vector known in the art may be used. Examples of commonly used vectors include naturally occurring or recombinant plasmids, cosmids, viruses, and bacteriophages. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A may be used as phage or cosmid vectors. Examples of plasmid vectors that may be used include pDZ, pBR, pUC, pBluescriptII, pGEM, pTZ, pCL, and pET. Specifically, pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, and pCC1BAC vectors may be used.

[0044] In the present application, "transformation" refers to changing the genetic traits of a host cell by introducing into the host cell a vector containing a target polynucleotide (including an insertion vector for inserting a polynucleotide for attenuating the activity of chitin transglycosylase of the present application into a chromosome). The transformed polynucleotide may be inserted into the chromosome of the host cell or may be extrachromosomally located. The polynucleotide may also contain DNA and / or RNA encoding the target protein. The polynucleotide may be introduced in a form appropriate for the purpose of introduction. For example, a polynucleotide for expressing a target protein may be introduced into a host cell in the form of an expression cassette, which is a genetic construct 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. Furthermore, the polynucleotide may be introduced into a host cell in its own form and operably linked to a sequence necessary for expression in the host cell, but is not limited thereto.

[0045] In addition, "operably linked" in this application means that the polynucleotide sequence is functionally linked to a promoter sequence that initiates and mediates transcription of the downstream polynucleotide.

[0046] The method for transforming the vector of the present application may be any method for introducing nucleic acid into cells, and may be carried out by selecting a standard technique suitable for the host cell, as known in the art, including, but not limited to, electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, lithium acetate-DMSO method, etc.

[0047] The term "microorganism" or "strain" as used herein includes all wild-type microorganisms and naturally or artificially genetically modified microorganisms, and refers to microorganisms in which a specific mechanism has been weakened or strengthened by inserting an exogenous gene or by enhancing or inactivating the activity of an endogenous gene, and which have been genetically modified to produce a desired polypeptide, protein, or product.

[0048] The microorganism of the present application may be a microorganism capable of producing a retinoid. The term "microorganism capable of producing a retinoid" is used interchangeably with "microorganisms that produce a retinoid."

[0049] The microorganism of the present application may be a microorganism into which polynucleotides encoding lycopene cyclase / phytoene synthase (crtYB) and phytoene desaturase (crtI) proteins have been introduced so that a microorganism lacking endogenous retinoid-producing ability can be endowed with retinoid-producing ability, or so that the retinoid-producing ability of a microorganism already capable of producing retinoids can be further enhanced, and which exhibits the activity of these proteins, or may be a microorganism in which the activity of these proteins has been enhanced. The lycopene cyclase / phytoene synthase or phytoene desaturase is a protein derived from Xanthophyllomyces dendrorhous, but any protein exhibiting the same or similar activity may be used. As a specific example, the lycopene cyclase / phytoene synthase or phytoene desaturase may consist of or contain the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 8, respectively. Alternatively, the lycopene cyclase / phytoene synthase or phytoene desaturase may consist of or contain an amino acid sequence that has at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology or identity to the amino acid sequence and exhibits activity corresponding to the lycopene cyclase / phytoene synthase or phytoene desaturase. Furthermore, it goes without saying that the lycopene cyclase / phytoene synthase or phytoene desaturase also includes proteins in which a portion of the sequence has been deleted, modified, substituted or added, so long as they have the homology or identity and exhibit activity corresponding to that of the lycopene cyclase / phytoene synthase or phytoene desaturase. Furthermore, as a specific example, the polynucleotide encoding the lycopene cyclase / phytoene synthase or phytoene desaturase may consist of or include the sequence of SEQ ID NO: 9 or SEQ ID NO: 10, respectively.The polynucleotide may be modified in various ways in the coding region, taking into account codon degeneracy or preferred codons in the microorganism of the present application, as long as the amino acid sequence remains unchanged. Specifically, the polynucleotide may consist of or contain a nucleotide sequence that has 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or less than 100% homology or identity to the sequence of SEQ ID NO: 9 or SEQ ID NO: 10, but is not limited to these.

[0050] Furthermore, the microorganism of the present application may be a microorganism that exhibits β-carotene 15,15'-oxygenase activity or a microorganism with enhanced β-carotene 15,15'-oxygenase activity by introducing a polynucleotide encoding a β-carotene 15,15'-oxygenase (BLH) protein into the microorganism so that a microorganism that does not endogenously have the ability to produce retinoids has the ability to produce retinoids, or so that a microorganism that has the ability to produce retinoids has the ability to produce retinoids further enhanced. β-Carotene 15,15'-oxygenase is a protein derived from uncultured marine bacterium 66A03, but any protein that exhibits the same or similar activity may be used. As a specific example, β-carotene 15,15'-oxygenase may consist of or contain the amino acid sequence of SEQ ID NO: 11. Alternatively, it may consist of or contain an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 11 and exhibits activity corresponding to β-carotene 15,15'-oxygenase. Furthermore, proteins with partial deletions, modifications, substitutions, or additions to the sequence are also included in the β-carotene 15,15'-oxygenase, as long as they have the homology or identity and exhibit activity corresponding to β-carotene 15,15'-oxygenase. Furthermore, as a specific example, a polynucleotide encoding β-carotene 15,15'-oxygenase may have or include the sequence of SEQ ID NO: 12. The coding region of the polynucleotide may be variously modified to the extent that the amino acid sequence is not changed, taking into account codon degeneracy or preferred codons in the microorganism of the present application.Specifically, the polynucleotide consists of a base sequence that has a homology or identity of 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or less than 100% to the sequence of SEQ ID NO: 12, or contains the base sequence, but is not limited to these.

[0051] The microorganism of the present application may be a microorganism in which chitin transglycosylase activity has been attenuated and retinoid production and / or excretion ability has been improved.

[0052] In the microorganism of the present application, the improvement in retinoid production ability is due to, but is not limited to, an improvement in retinoid secretion ability.

[0053] The microorganisms of the present application are those that selectively excrete retinoids, but are not limited thereto.

[0054] The microorganism of the present application has improved retinoid secretion ability compared to a microorganism having retinoid-producing ability in which chitin transglycosylase activity is not weakened, but is not limited to this.

[0055] As an example, the microorganism of the present application may selectively excrete retinoid from among β-carotene and retinoid.

[0056] The microorganism of the present application may be a microorganism that naturally has the ability to produce chitin transglycosylase or retinoid, or may be a parent strain that has the ability to produce chitin transglycosylase or retinoid, but in which the chitin transglycosylase of the present application has been further weakened.

[0057] For example, the microorganism of the present application may be any microorganism in which the chitin transglycosylase of the present application is attenuated and which produces a retinoid.

[0058] The microorganism of the present application may have improved retinoid secretion ability compared to a microorganism having retinoid-producing ability in which chitin transglycosylase activity is not attenuated.

[0059] For example, the microorganism of the present application may be a naturally occurring wild-type microorganism, a microorganism having retinoid-producing ability, and / or a recombinant strain of a microorganism containing chitin transglycosylase of the present application in which the activity of the chitin transglycosylase of the present application has been attenuated, thereby improving retinoid-producing ability and / or excretion ability. The recombinant strain with improved retinoid-producing ability and / or excretion ability is a microorganism that has improved retinoid-producing ability and / or excretion ability compared to a naturally occurring wild-type microorganism or a microorganism of the present application in which the chitin transglycosylase activity is not attenuated, but is not limited thereto.

[0060] For example, a microorganism of the present application in which chitin transglycosylase is not attenuated and which is the subject strain for comparison of whether the retinoid production ability and excretion ability are improved is CC08-2050 (KCCM13294P, see Non-Patent Document 13), but is not limited thereto. The deposited strain name of the CJ2050 strain described in the reference (Non-Patent Document 13) is the CC08-2050 strain of the present application, and the CC08-2050 strain of the present application and the CJ2050 strain are the same strain.

[0061] As an example, the recombinant strains and microorganisms with improved retinoid production and / or secretion capabilities have improved retinoid production and / or secretion capabilities by about 1% or more, about 2% or more, about 5% or more, about 7% or more, about 10% or more, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, or about 100% or more (there is no particular upper limit, for example, about 200% or less) compared to the retinoid production and / or secretion capabilities of the parent strain or unmodified microorganism before mutation. The term "about" refers to an increase in retinoid production and / or secretion ability of a microorganism by about 1.01-fold or more, about 1.02-fold or more, about 1.05-fold or more, about 1.07-fold or more, about 1.1-fold or more, about 1.2-fold or more, about 1.3-fold or more, about 1.4-fold or more, about 1.5-fold or more, about 1.6-fold or more, about 1.7-fold or more, about 1.8-fold or more, about 1.9-fold or more, or about 2-fold or more (the upper limit is not particularly limited, for example, about 10-fold or less), but may refer to any increase in the production and / or secretion ability compared to the parent strain or unmodified microorganism before mutation. The term "about" refers to a range that includes ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and may refer to any numerical value in a range that is equal to or similar to the numerical value following the term "about," but is not limited to these.

[0062] For example, the recombinant strain or microorganism has reduced chitin transglycosylase activity compared to that of the parent strain or unmodified microorganism before mutation. If the endogenous chitin transglycosylase activity of the parent strain or unmodified microorganism before mutation is taken as 100%, the recombinant strain or microorganism has chitin transglycosylase activity reduced to 100% or less (e.g., reduced to 99.5%, 99%, 98%, 95%, 90%, 85%, 80%, etc.), but is not limited thereto.

[0063] In the present application, the term "unmodified microorganism" does not exclude strains containing naturally occurring mutations in microorganisms, but refers to a wild-type strain or a naturally occurring strain itself, or a strain before its traits are changed due to genetic mutation caused by natural or artificial factors. For example, the unmodified microorganism refers to a strain in which the chitin transglycosylase of the present application is not attenuated or has not yet been attenuated. The term "unmodified microorganism" is also used interchangeably with "strain before modification," "microorganism before modification," "non-mutated strain," "non-modified strain," "non-mutated microorganism," or "reference microorganism."

[0064] The microorganisms of the present application are, but are not limited to, microorganisms of the genus Yarrowia.

[0065] As an example, the Yarrowia microorganism of the present application may be, but is not limited to, Yarrowia lipolytica.

[0066] In this application, "attenuation" of the activity of chitin transglycosylase (including its polypeptides and proteins, hereinafter the same) is a concept that encompasses all of the following: a decrease in activity compared to the endogenous activity, or the absence of activity. The term "attenuation" is also used interchangeably with terms such as inactivation, deficiency, deletion, down-regulation, decrease, reduce, and attenuation.

[0067] The weakening includes at least one of the following: the activity of the chitin transglycosylase itself is reduced or eliminated compared to the activity of chitin transglycosylase originally possessed by the microorganism due to, for example, a mutation in the polynucleotide encoding the chitin transglycosylase (including its polypeptide and protein); the overall level and / or concentration (expression amount) of chitin transglycosylase activity in the cell is reduced compared to that of a natural strain due to, for example, inhibition of expression of the gene of the polynucleotide encoding it or inhibition of translation into chitin transglycosylase; no expression of the polynucleotide at all; and no chitin transglycosylase activity even if the polynucleotide is expressed.

[0068] The term "endogenous activity" refers to the activity of a specific chitin transglycosylase (including its polypeptide and protein) that a parent strain, wild-type, or unmodified microorganism originally possessed before the trait change, when the trait is changed due to genetic mutation caused by natural or artificial factors. This term is used interchangeably with "activity before transformation." When the chitin transglycosylase activity is "weakened," "inactivated," "deficient," "reduced," "down-regulated," "decreased," or "attenuated" compared to the endogenous activity, it means that the specific chitin transglycosylase activity is lower than that originally possessed by the parent strain or unmodified microorganism before the trait change.

[0069] For the purposes of this application, the microorganism of the present application may have improved retinoid production and / or secretion ability due to attenuation of chitin transglycosylase activity.

[0070] Such attenuation of chitin transglycosylase activity can be achieved by applying various methods well known in the art, including, but not limited to, those described above (for example, Non-Patent Documents 14 and 15).

[0071] Specifically, the activity of chitin transglycosylase of the present application can be attenuated by 1) deleting all or part of the gene encoding the chitin transglycosylase polypeptide, 2) modifying the expression control region (or expression control sequence) so that the expression of the gene encoding the chitin transglycosylase polypeptide is reduced, 3) modifying the amino acid sequence constituting the chitin transglycosylase polypeptide so that the activity of chitin transglycosylase is deleted or attenuated (for example, by deleting / substituting / adding one or more amino acids in the amino acid sequence), or 4) modifying the sequence of the gene (including polynucleotide) encoding the chitin transglycosylase polypeptide so that the activity of chitin transglycosylase is deleted or attenuated (for example, by modifying the sequence of the gene (including polynucleotide) encoding the chitin transglycosylase polypeptide so that the activity of the polypeptide is deleted or attenuated). 5) modifying the base sequence encoding the start codon, Shine-Dalgarno sequence, or 5'UTR region of the gene transcript encoding the chitin transglycosylase polypeptide; 6) introducing an antisense oligonucleotide (e.g., antisense RNA) that binds complementarily to the gene transcript encoding the chitin transglycosylase polypeptide; 7) adding a sequence complementary to the Shine-Dalgarno sequence before the Shine-Dalgarno sequence of the gene encoding the chitin transglycosylase polypeptide so as to form a secondary structure that prevents ribosome attachment; 8) adding a promoter to the 3' end of the ORF (open reading frame) of the gene sequence encoding the chitin transglycosylase polypeptide so as to enable reverse transcription (reverse transcription engineering, RTE); or 9) a combination of two or more selected from 1) to 8) above, but is not limited thereto.

[0072] For example, 1) deleting a part or all of the gene encoding the polypeptide may be carried out by deleting the entire polynucleotide encoding the endogenous target polypeptide in the chromosome, or by substituting a polynucleotide with a partial deletion of nucleotides or a marker gene.

[0073] The modification of the expression regulatory region (or expression regulatory sequence) may be carried out by generating a mutation in the expression regulatory region (or expression regulatory sequence) by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, or by substituting a sequence having a lower activity. The expression regulatory region includes, but is not limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence regulating the termination of transcription and translation.

[0074] The modification of the amino acid sequence or polynucleotide sequence of 3) and 4) above can be carried out 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 attenuate the activity of the polypeptide, or by substituting an amino acid sequence or polynucleotide sequence that has been improved to have lower activity or to eliminate activity. For example, gene expression can be inhibited or attenuated by, but is not limited to, introducing a mutation into a polynucleotide sequence to form a stop codon.

[0075] The base sequence encoding the start codon or 5'UTR region of the gene transcript encoding the polypeptide (5) can be modified, for example, by substituting it with a base sequence encoding another start codon that has a lower polypeptide expression rate than the endogenous start codon, but is not limited to this.

[0076] 6) Introducing an antisense oligonucleotide (e.g., antisense RNA) that binds complementary to the gene transcription product encoding the polypeptide may be performed, for example, as described in Non-Patent Document 16.

[0077] 7) Adding a sequence complementary to the Shine-Dalgarno sequence before the Shine-Dalgarno sequence of a gene encoding a chitin transglycosylase polypeptide so that a secondary structure that makes ribosome attachment impossible is formed may be achieved by making mRNA translation impossible or slowing down the rate.

[0078] Furthermore, the above 8) adding a promoter to the 3' end of the ORF (open reading frame) of a gene sequence encoding a polypeptide so as to induce reverse transcription (reverse transcription engineering, RTE) may be carried out by creating an antisense nucleotide complementary to the gene transcript encoding the polypeptide, thereby attenuating its activity.

[0079] As an example, but not limited to, the microorganism of the present application is one in which at least one gene encoding chitin transglycosylase is deleted.

[0080] In this application, "retinoid" refers chemically to the vitamin A group or to a group of compounds chemically related thereto.

[0081] In one embodiment, the retinoid is any one selected from the group consisting of retinol, retinal, retinoic acid, and retinyl esters, but is not limited thereto.

[0082] As an example, retinol may be converted to other retinoid compounds (eg, retinal, retinoic acid, retinyl esters, etc.) by methods known in the art.

[0083] Another aspect of the present application provides a method for producing a retinoid, comprising culturing the microorganism of the present application in a medium.

[0084] The microorganisms are as described above.

[0085] The term "culturing" as used herein means growing the microorganism of the present application under appropriately controlled environmental conditions. The culturing process of the present application can be carried out using a suitable medium and culture conditions known in the art. Those skilled in the art can easily adjust such a culturing process depending on the selected strain. Specifically, the culturing may be batch, continuous, and / or fed-batch culture, but is not limited thereto.

[0086] The microorganism of the present application can be cultured in a conventional medium containing suitable carbon sources, nitrogen sources, phosphorus sources, inorganic compounds, amino acids and / or vitamins under aerobic conditions by adjusting the temperature, pH, etc.

[0087] In the present application, examples of carbon sources that can be used include carbohydrates such as glucose, saccharose, lactose, fructose, sucrose, and maltose; sugar alcohols such as mannitol and sorbitol; organic acids such as pyruvic acid, lactic acid, and citric acid; and amino acids such as glutamic acid, methionine, and lysine. Natural organic nutrient sources such as starch hydrolysates, molasses, blackstrap molasses, rice bran, cassava, bagasse, and corn steeping liquid can also be used. Specifically, carbohydrates such as glucose and sterilized pretreated molasses (i.e., molasses converted into reducing sugars) can be used. Any other carbon source can also be used as long as it is present in an appropriate amount. These carbon sources can be used alone or in combination of two or more, but are not limited to these.

[0088] Examples of the nitrogen source that can be used include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate, and organic nitrogen sources such as amino acids such as glutamic acid, methionine, and glutamine, peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steeping liquid, casein hydrolysate, fish or its degradation products, and defatted soybean cake or its degradation products. These nitrogen sources can be used alone or in combination of two or more, but are not limited to these.

[0089] Examples of the phosphorus source that can be used include potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and sodium-containing salts corresponding thereto. Examples of inorganic compounds that can be used include sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, and calcium carbonate. Other examples include amino acids, vitamins, and / or suitable precursors. These components or precursors can be added to the medium in a batch or continuous manner. However, the present invention is not limited to these.

[0090] During the cultivation of the microorganism of the present application, the pH of the medium can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, or sulfuric acid to the medium in a suitable manner. Furthermore, during cultivation, foam formation can be suppressed using an antifoaming agent such as a fatty acid polyglycol ester. Furthermore, to maintain an aerobic state in the medium, oxygen or an oxygen-containing gas may be injected into the medium. To maintain anaerobic and microaerobic states, no gas may be injected, and nitrogen, hydrogen, or carbon dioxide gas may be injected, but this is not limited to these.

[0091] Furthermore, the culture medium may contain metal salts necessary for growth, such as magnesium sulfate and iron sulfate. Finally, essential growth substances, such as amino acids and vitamins, may be used in addition to the above substances. Precursors suitable for the culture medium may also be used. The above-mentioned raw materials are added to the culture in a suitable manner, batchwise or continuously, during the culture process, but are not limited thereto.

[0092] In the present application, the pH of the culture can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid to the culture in a suitable manner during the cultivation of the microorganism. Furthermore, foam formation can be suppressed during cultivation using an antifoaming agent such as a fatty acid polyglycol ester. Furthermore, oxygen or an oxygen-containing gas may be injected into the culture to maintain an aerobic state, and nitrogen, hydrogen, or carbon dioxide gas may be injected, or no gas may be injected, to maintain anaerobic and microaerobic states, but these are not limiting.

[0093] In the culture of the present application, the culture temperature is maintained at 20 to 35°C, specifically 25 to 35°C, and the culture period is continued until a sufficient amount of useful substance is produced, which may be about 10 to 160 hours, about 20 to 130 hours, about 24 to 120 hours, about 36 to 120 hours, about 48 to 120 hours, about 48 hours or more, about 48 hours, about 72 hours, or about 120 hours, but is not limited to these.

[0094] The method for producing a retinoid of the present application may further comprise the step of recovering the retinoid from the microorganism or the culture medium.

[0095] The target retinoid can be recovered from the medium 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. For example, centrifugation, filtration, crystallization, treatment with a protein precipitant (salting out), extraction, ultrasonic disruption, ultrafiltration, dialysis, various types of chromatography such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, and affinity chromatography, HPLC, and combinations thereof can be used, but the above examples are not limited to these.

[0096] The method may include a further purification step, which may be carried out by any suitable method known in the art.

[0097] As an example, the retinoid production method of the present application uses a microorganism having retinoid secretion ability in which chitin transglycosylase activity is weakened, and therefore the present application produces retinoids without using microbial cell disruption or dodecane as a solvent, which are widely used in retinoid extraction, but is not limited thereto.

[0098] The method for producing a retinoid of the present application may further include a step of converting the retinol expressed by the microorganism of the present application into a retinoid other than retinol. In the method for producing a retinoid of the present application, the conversion step may be further included after the culturing step or the recovering step. The conversion step can be carried out by a suitable method known in the art. For example, the conversion can be carried out using retinol acyltransferase, but is not limited thereto.

[0099] In one embodiment, the retinoid other than retinol is any one selected from the group consisting of retinal, retinoic acid, and retinyl ester, but may be any one included in the retinoids.

[0100] Yet another aspect of the present application provides a method for producing a Yarrowia microorganism having retinoid-producing ability, the method comprising the step of attenuating chitin transglycosylase activity in the Yarrowia microorganism having retinoid-producing ability.

[0101] Yet another aspect of the present application provides a method for increasing retinoid excretion, comprising the step of attenuating chitin transglycosylase activity in a Yarrowia microorganism capable of producing a retinoid.

[0102] The method for increasing retinoid secretion may be a method for increasing retinoid secretion in a Yarrowia microorganism capable of producing a retinoid.

[0103] The step of attenuating the activity of chitin transglycosylase may be a step of modifying a Yarrowia microorganism so that chitin transglycosylase is attenuated, as described above.

[0104] Yet another aspect of the present application provides a composition for producing a retinoid, comprising at least one of a Yarrowia microorganism in which chitin transglycosylase activity is attenuated and a culture thereof.

[0105] 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.

[0106] The chitin transglycosylase, the Yarrowia microorganism with attenuated chitin transglycosylase activity, the culture, the retinoid, and the like are as described above.

[0107] Yet another aspect of the present application provides use of the Yarrowia microorganism of the present application in which chitin transglycosylase activity is attenuated for the production of a retinoid.

[0108] The chitin transglycosylase, the Yarrowia microorganism with attenuated chitin transglycosylase activity, the retinoid, and the like are as described above. [Example]

[0109] 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 well understood and easily implemented by skilled artisans in the technical field of the present application or a similar technical field. [Example]

[0110] Attenuation of CRH1 (YALI0C09680) in a retinoid-producing Yarrowia strain To attenuate the endogenous CRH1 (YALI0C09680) gene in the retinoid-producing Yarrowia strain CC08-2050 (Non-Patent Document 13), deposited under the designation KCCM13294P, the CRH1 (YALI0C09680) ORF was deleted from the genome. To achieve this, the CRH1 (YALI0C09680) ORF sequence (SEQ ID NO: 4) was obtained based on the nucleotide sequence registered in KEGG (Kyoto Encyclopedia of Genes and Genomes). Furthermore, a CRH1 (YALI0C09680) deletion cassette was constructed using the primers listed in Table 1 and the URA3 gene (SEQ ID NO: 13) of Y. lipolytica as a selection marker. Specifically, PCR was performed using the genomic DNA of CC08-2050 as a template and primers set forth in SEQ ID NOs: 14 and 15, 16 and 17, 18 and 19, and 20 and 21. PCR conditions consisted of 35 cycles of denaturation at 95°C for 1 minute, annealing at 55°C for 1 minute, and polymerization at 72°C for 2 minutes. The resulting DNA fragments were then combined into a single cassette by overlap extension PCR.

[0111] The cassette thus prepared was introduced into the CC08-2050 strain by the heat shock method (Non-Patent Document 17), and colonies formed on uracil-free solid medium (YLMM1) were then isolated. Colonies in which the cassette had been confirmed to have been inserted into the genome were cultured on 5-FOA solid medium at 30°C for 3 days using primers SEQ ID NO: 22 and SEQ ID NO: 23. The URA3 marker was recovered by culturing the colonies formed on 5-FOA solid medium. The final strain thus obtained was designated CJ2327.

[0112] [Table 1]

[0113] The above-mentioned YLMM1 medium and 5-FOA medium used had the following compositions. <Yarrowialipolytica minimal media1(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 20 g / L, Yeast nitrogen base without amino acids 6.7 g / L, Yeast Synthetic Drop-out Medium Supplements without uracil 2 g / L, Uracil 50 μg / mL, 5-fluoroorotic acid (5-FOA) 1 g / L, and agar 15 g / L [Example]

[0114] Attenuation of CRH1 (YALI0E24673) in a retinoid-producing Yarrowia strain To attenuate the endogenous CRH1 (YALI0E24673) gene in the retinoid-producing Yarrowia strain CC08-2050 (KCCM13294P), the CRH1 (YALI0E24673) ORF was deleted from the genome. To do so, the CRH1 (YALI0E24673) ORF sequence (SEQ ID NO: 5) was obtained based on the nucleotide sequence registered in KEGG (Kyoto Encyclopedia of Genes and Genomes). Furthermore, a CRH1 (YALI0E24673) deletion cassette was constructed using the primers listed in Table 2 and the Y. lipolytica URA3 gene (SEQ ID NO: 13) as a selectable marker. Specifically, PCR was performed using the genomic DNA of CC08-2050 as a template and primers SEQ ID NOs: 24 and 25, 26 and 27, 28 and 29, and 30 and 31. The PCR conditions were 35 cycles of denaturation at 95°C for 1 minute, annealing at 55°C for 1 minute, and polymerization at 72°C for 2 minutes. The resulting DNA fragments were then combined into a single cassette by overlap extension PCR.

[0115] [Table 2]

[0116] The cassette thus prepared was introduced into the CC08-2050 strain by the heat shock method, and then colonies formed on uracil-free solid medium (YLMM1; a medium with the same composition as the YLMM1 medium in Example 1) were isolated. Colonies in which the cassette was confirmed to have been inserted into the genome were cultured on 5-FOA solid medium at 30°C for 3 days using primers represented by SEQ ID NOs: 32 and 33, and the URA3 marker was recovered by obtaining colonies formed on the 5-FOA solid medium. The final strain thus obtained was designated CJ2328. [Example]

[0117] Attenuation of CRH2 (YALI0B15510) in a retinoid-producing Yarrowia strain To attenuate the endogenous CRH2 (YALI0B15510) gene in the retinoid-producing Yarrowia strain CC08-2050, the CRH2 (YALI0B15510) ORF was deleted. To do so, the CRH2 (YALI0B15510) ORF sequence (SEQ ID NO: 6) was obtained based on the nucleotide sequence registered in KEGG (Kyoto Encyclopedia of Genes and Genomes). Furthermore, a CRH2 (YALI0B15510) deletion cassette was constructed using the primers listed in Table 3 and the Y. lipolytica URA3 gene (SEQ ID NO: 13) as a selectable marker. Specifically, PCR was performed using the CC08-2050 genomic DNA as a template and primers SEQ ID NOs: 34 and 35, SEQ ID NOs: 36 and 37, SEQ ID NOs: 38 and 39, and SEQ ID NOs: 40 and 41. The PCR conditions were 35 cycles of denaturation at 95°C for 1 minute, annealing at 55°C for 1 minute, and polymerization at 72°C for 2 minutes. The resulting DNA fragments were then combined into a single cassette by overlap extension PCR.

[0118] [Table 3]

[0119] The cassette thus prepared was introduced into the CC08-2050 strain by the heat shock method, and colonies formed on uracil-free solid medium (YLMM1) were then isolated. Colonies in which the cassette had been confirmed to have been inserted into the genome were cultured on 5-FOA solid medium at 30°C for 3 days using primers SEQ ID NO: 42 and SEQ ID NO: 43. The URA3 marker was then recovered by culturing the colonies formed on 5-FOA solid medium. The final strain thus obtained was designated CJ2329. [Example]

[0120] Comparative evaluation of retinoid production and excretion capacity of CRH-attenuated strains Flask evaluation was performed to compare the retinoid production and excretion levels of the strains prepared in Examples 1 to 3. The retinoid-producing Yarrowia strain (CC08-2050 (KCCM13294P); control group), the CRH1 (YALI0C09680)-deficient strain (CJ2327), the CRH1 (YALI0E24673)-deficient strain (CJ2328), and the CRH2 (YALI0B15510)-deficient strain (CJ2329) prepared in Example 1 were each inoculated into 25 ml of YPDLU medium containing 0.05% BHT (3,5-Di-tert-4-butylhydroxytoluene) in a 250 ml corner-baffled flask to an initial OD of 2, and cultured at 30°C and 200 rpm. The YPDLU medium used had the following composition: <ypdlu> Glucose 40g / L, peptone 20g / L, yeast extract 10g / L, uracil 1g / L, leucine 1g / L, 1M phosphate buffer (pH 7.0) 100ml / L

[0121] The degree of growth of each strain was assessed by measuring the OD value at a wavelength of 600 nm using a spectrophotometer.

[0122] In addition, the concentrations of excreted β-carotene, retinol, and retinal were measured by mixing 0.1 ml of the supernatant from which the bacterial cells had been removed after the end of the culture with 0.9 ml of acetone (Sigma) containing 4% BHT, followed by quantitative analysis using an HPLC device.

[0123] The analyzed OD values ​​and concentrations of retinoids and β-carotene are shown in Table 4, and the retinoid concentrations are shown graphically in Figure 1.

[0124] [Table 4]

[0125] As can be seen from the above results, when CRH1 (YALI0C09680g), CRH1 (YALI0E24673g), and CRH2 (YALI0B15510g) were attenuated in a retinoid-producing Yarrowia strain, the retinol / retinal secreted from the cell increased by 2.1-fold / 2.1-fold, 2.4-fold / 2.3-fold, and 2.3-fold / 2.2-fold, respectively, compared to the control group. These results also suggest that even if β-carotene is produced in retinoid-producing microorganisms in which CRH1, CRH2, or a combination thereof is attenuated, only retinoids are selectively secreted.

[0126] From the above description, those 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 the technical idea or essential features thereof. It should be understood that the above examples are merely illustrative and not limiting. The present application should be construed as including all modifications and variations derived from the meaning and scope of the claims, rather than the specification, and their equivalent concepts.

[0127] [Table 5] < / ypdlu>

Claims

1. A microorganism of the genus Yarrowia (Yarrowia sp.) that has retinoid-producing ability and has weakened chitin transglycosylase activity.

2. The microorganism of claim 1 , wherein the chitin transglycosylase is a CRH1 protein, a CRH2 protein, or a combination thereof.

3. The microorganism described in claim 1, wherein the chitin transglycosylase is at least one selected from the group consisting of polypeptides that exhibit chitin transglycosylase activity and have a sequence that is 60% or more identical to the amino acid sequence of SEQ ID NO: 1, polypeptides that exhibit chitin transglycosylase activity and have a sequence that is 60% or more identical to the amino acid sequence of SEQ ID NO: 2, and polypeptides that exhibit chitin transglycosylase activity and have a sequence that is 60% or more identical to the amino acid sequence of SEQ ID NO:

3.

4. 2. The microorganism of claim 1, wherein the chitin transglycosylase is derived from Yarrowia lipolytica.

5. The microorganism according to claim 1, wherein the chitin transglycosylase is encoded by SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or a polynucleotide sequence having 60% or more identity thereto.

6. The microorganism according to claim 1, wherein the microorganism has improved retinoid secretion ability compared to a Yarrowia microorganism having retinoid-producing ability and in which chitin transglycosylase activity is not weakened.

7. The microorganism according to claim 1 , wherein the Yarrowia microorganism is Yarrowia lipolytica.

8. The microorganism according to claim 1 , wherein the retinoid comprises any one selected from the group consisting of retinol, retinal, retinoic acid, and retinyl ester.

9. A method for producing a retinoid, comprising the step of culturing the microorganism according to any one of claims 1 to 8 in a medium.

10. The method for producing a retinoid according to claim 9 , wherein the method comprises a step of recovering the retinoid from the culture medium or the microorganism.

11. 10. The method for producing a retinoid according to claim 9, wherein the method does not involve disrupting microbial cells or using dodecane as a solvent when extracting the retinoid.

12. 10. The method for producing a retinoid according to claim 9, wherein the retinoid comprises any one selected from the group consisting of retinol, retinal, retinoic acid, and retinyl ester.

13. A method for producing a Yarrowia microorganism capable of producing a retinoid, comprising the step of attenuating chitin transglycosylase activity in the Yarrowia microorganism capable of producing a retinoid.

14. A method for increasing retinoid excretion, comprising the step of attenuating chitin transglycosylase activity in a Yarrowia microorganism capable of producing retinoids.

15. A composition for producing a retinoid, comprising at least one of a microorganism of the genus Yarrowia (Yarrowia sp.) having retinoid-producing ability and an attenuated chitin transglycosylase activity, and a culture thereof.

16. Use of a Yarrowia sp. microorganism having retinoid-producing ability and reduced chitin transglycosylase activity for retinoid production.

Images