Microorganism having reduced chitin transglycosylase activity and retinoid production capacity, and method for producing retinoid using same

Abstract

The present application relates to a microorganism of the genus Yarrowia having reduced chitin transglycosylase activity and retinoid production capacity, a method for producing retinoid using the microorganism, a composition for producing retinoid, a use for producing retinoid, and a method for producing the microorganism.

Description

Technical Field

[0001] The present disclosure relates to a Yarrowia sp. microorganism having the ability to produce retinoids, in which the activity of chitin transglycosylase is attenuated; a method for producing retinoids using the microorganism; a composition for producing retinoids; uses in the production of retinoids; and a method for preparing the microorganism. Background Art

[0002] Retinol is a fat-soluble vitamin and an essential vitamin related to improving eye health for night blindness, immune enhancement, skin health, etc. Currently, retinol is mainly produced and sold by global advanced companies through chemical synthesis methods, but research on producing retinol based on microbial fermentation is underway.

[0003] To this end, many technologies have been developed to stabilize the retinol compound itself in compositions or products containing retinol (U.S. Patent No. 6858217). However, the development of methods for stably increasing retinol production is still negligible. Summary of the Invention

[0004] [Technical Problem]

[0005] The problem to be solved by the present disclosure is to provide a Yarrowia sp. microorganism having the ability to produce retinoids, in which the activity of chitin transglycosylase is attenuated; a method for producing retinoids using the microorganism; a composition for producing retinoids; uses in the production of retinoids; and a method for preparing the microorganism.

[0006] [Technical Solution]

[0007] The present disclosure provides a Yarrowia sp. microorganism having the ability to produce retinoids, in which the activity of chitin transglycosylase is attenuated.

[0008] The present disclosure provides a method for producing retinoids, the method including the step of culturing the microorganism of the present disclosure in a medium.

[0009] The present disclosure provides a method for preparing a microorganism having the ability to produce retinoids, the method including the step of attenuating the activity of chitin transglycosylase in a Yarrowia sp. microorganism having the ability to produce retinoids.

[0010] The present disclosure provides a method for increasing the secretion of retinoids, the method including the step of attenuating the activity of chitin transglycosylase in a Yarrowia sp. microorganism having the ability to produce retinoids.

[0011] The present disclosure provides a composition for producing retinoids, the composition including the microorganism of the present disclosure or a culture thereof.

[0012] The present disclosure provides the use of the microorganisms of the present disclosure in the production of retinoids.

[0013] [Beneficial effects]

[0014] Retinoids can be produced by using the microorganisms of the present disclosure. Description of the drawings

[0015] Figure 1 Shows the retinoid concentrations in the shake flask culture experiments of the control group (CC08 - 2050) and the microorganisms with attenuated chitin transglycosylase (CJ2327, CJ2328, and CJ2329). Detailed implementation manners

[0016] The present disclosure will be described in detail below. At the same time, the various descriptions and implementation manners disclosed in the present disclosure can also be applied to other descriptions and implementation manners. That is, all combinations of the various elements disclosed in the present disclosure fall within the scope of the present disclosure. In addition, the scope of the present disclosure is not limited by the specific descriptions below. In addition, many papers and patent documents are referenced and cited throughout the specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to further clarify the level and scope of the subject matter to which the present disclosure pertains.

[0017] One aspect of the present disclosure provides a microorganism of the genus Yarrowia with the ability to produce retinoids, in which the activity of chitin transglycosylase is attenuated.

[0018] The "chitin transglycosylase" of the present disclosure is an enzyme capable of catalyzing the transfer of chitin to β(1 - 6) glucan and β(1 - 3) glucan in the cell wall.

[0019] The chitin transglycosylase of the present disclosure can be a CRH1 protein, a CRH2 protein, or a combination thereof. For example, the combination of the CRH1 protein or the CRH2 protein can include the combination of the CRH1 protein and the CRH2 protein. In addition, since there may be two or more CRH1 proteins with different amino acid sequences or two or more CRH2 proteins with different amino acid sequences, the combination of the CRH1 protein or the CRH2 protein can include a combination of one or more CRH1 proteins (for example, the combination of CRH1 with a specific amino acid sequence and CRH1 with an amino acid sequence different from it), a combination of CRH2 proteins (for example, the combination of CRH2 with a specific amino acid sequence and CRH2 with an amino acid sequence different from it), and a combination of one or more CRH1 proteins and one or more CRH2 proteins (for example, the combination of CRH1 with a specific amino acid sequence, CRH1 with an amino acid sequence different from it, and CRH2 with an amino acid sequence different from it), etc.

[0020] For the purposes of the present disclosure, the chitinotransferase can include any one as long as it can enhance the retinol production capacity and / or secretion capacity. For example, the increase in retinol production capacity may be due to, but not limited to, the increase in retinol secretion capacity.

[0021] In one embodiment, compared with the activity of an endogenous or wild-type chitinotransferase, the activity of the chitinotransferase in the Yarrowia yeast microorganism of the present disclosure is weakened, thereby increasing the retinol production capacity and / or secretion capacity of the microorganism.

[0022] In one embodiment, the chitinotransferase of the present disclosure can include, have, or consist of the amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or an amino acid sequence having 60% or higher homology or identity with any one or more of them, or can be substantially composed of the amino acid sequence. The chitinotransferase of the present disclosure can be a polypeptide having chitinotransferase activity, together with the above sequences, but not limited thereto.

[0023] For example, the amino acid sequence of the chitinotransferase of the present disclosure can be encoded by the CRH1 gene, the CRH2 gene, or a combination thereof, and for example, by the CRH1 (YALI0C09680) gene, the CRH1 (YALI0E24673) gene, the CRH2 (YALI0B15510) gene, or a combination thereof, but not limited thereto. The amino acid sequence can be obtained from various databases, such as Genbank of NCBI (which is a known database), etc., but not limited thereto.

[0024] In one embodiment, the chitinotransferase of the present disclosure can be derived from Yarrowia lipolytica, but not limited thereto.

[0025] In one embodiment, SEQ ID NO:1 can be the CRH1 (YALI0C09680) protein, SEQ ID NO:2 can be the CRH1 (YALI0E24673) protein, and SEQ ID NO:3 can be the CRH2 (YALI0B15510) protein.

[0026] In addition, an embodiment of the chitinotransglycosylase of the present disclosure may be described as a protein comprising SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or a combination thereof. However, it will be apparent to those skilled in the art that adding meaningless sequences, naturally occurring mutations, or silent mutations upstream or downstream of the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or a combination thereof is not excluded, and as long as the protein has the same or corresponding activity as the protein comprising the amino acid sequence, it may belong to the chitinotransglycosylase of the present disclosure.

[0027] For example, the chitinotransglycosylase of the present disclosure may comprise the amino acid sequences 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 higher, 62% or higher, 63% or higher, 64% or higher, 65% or higher, 70% or higher, 75% or higher, 76% or higher, 77% or higher, 78% or higher, 80% or higher, 85% or higher, 86% or higher, 87% or higher, 88% or higher, 89% or higher, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher homology or identity with the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or a combination thereof. It is also apparent that a protein having an amino acid sequence in which a partial sequence is deleted, modified, substituted, or added also falls within the scope of the present disclosure, as long as the amino acid sequence has such homology or identity and exhibits the corresponding efficacy of the protein.

[0028] In the present disclosure, although described as "a polypeptide or protein comprising an amino acid sequence described by a specific sequence number", "a polypeptide or protein consisting of an amino acid sequence described by a specific sequence number", or "a polypeptide or protein having an amino acid sequence described by a specific sequence number", it is apparent that any protein having an amino acid sequence in which a partial sequence is deleted, modified, substituted, conservatively substituted, or added can be used in the present disclosure if it has the same or corresponding activity as the polypeptide or protein consisting of the amino acid sequence of the corresponding sequence number. For example, it may be adding a sequence that does not change the protein function, a naturally occurring mutation, its silent mutation, or a conservative substitution at the N-terminus and / or C-terminus of the amino acid sequence.

[0029] "Conservative substitution" refers to the replacement of one amino acid by another amino acid with similar structure and / or chemical properties. Such amino acid substitutions may generally occur based on the similarity of the residues in terms of polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphiphilicity. Generally, conservative substitutions may have little or no effect on the activity of the protein.

[0030] As used herein, the terms "homology" or "identity" refer to the degree of identity or similarity between two given amino acid sequences or nucleotide sequences, which can be expressed as a percentage. The terms "homology" and "identity" are generally used interchangeably with each other.

[0031] The sequence homology or identity of conservative polynucleotides or polypeptides (including proteins) is determined by standard alignment algorithms and the default gap penalties established by the programs to be used can be employed. Basically, homologous or identical sequences can generally hybridize with the full length of the sequence or at least about 50%, about 60%, about 70%, about 80% or about 90% of the full length under moderately or highly stringent conditions. Obviously, hybridization also includes the hybridization of polynucleotides with polynucleotides containing general codons or codons taking into account codon degeneracy.

[0032] 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, for example, using default parameters as in Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444. Alternatively, it can be determined by: the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453), as performed using the Needleman program of the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277) (version 5.0.0 or higher), the GCG program package (Devereux, J., et al, Nucleic Acids Research 12: 387 (1984)), BLASTP, BLASTN, FASTA (Atschul, [S.][F.,][ET AL, J MOLEC BIOL 215]: 403 (1990); Guide to Huge Computers, Martin J. Bishop, [ED.,] Academic Press, San Diego, 1994, and [CARILLOETA / .](1988) SIAM J Applied Math 48: 1073). For example, BLAST or ClustalW from the National Center for Biotechnology Information can be used to determine homology, similarity or identity.

[0033] The homology, similarity or identity of polynucleotides or polypeptides (including proteins) can be determined, for example, by comparing sequence information using, for example, the GAP computer program (such as Needleman et al., (1970), J Mol Biol. 48:443), as disclosed in Smith and Waterman, Adv Appl Math (1981) 2:482. Briefly, the GAP program defines homology, similarity or identity as the value obtained by dividing the number of symbols (i.e., nucleotides or amino acids) that are in similar alignment by the total number of symbols in the shorter of the two sequences. The default parameters of the GAP program may include: (1) a binary comparison matrix (with a value of 1 for identity and a value of 0 for non-identity) and a weighted comparison matrix of Gribskov et al. (1986), Nucl. Acids Res. 14:6745, as disclosed in Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, edited by Schwartz and Dayhoff, pp. 353–358 (1979) (or the EDNAFULL substitution matrix (EMBOSS version of NCBI NUC4.4)); (2) a penalty of 3.0 for each gap and an additional penalty of 0.10 for each symbol in each gap (or a gap opening penalty of 10 and a gap extension penalty of 0.5); and (3) no penalty for terminal gaps.

[0034] In addition, whether any two polynucleotide or polypeptide (including protein) sequences are homologous, similar or identical to each other can be identified by comparing the sequences in a Southern hybridization experiment under defined stringent conditions, and defining appropriate hybridization conditions is within the skill of those in the art and can be determined by methods well known to those in the art (e.g., J Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989; F.M. Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York).

[0035] As used herein, the term "polynucleotide" refers to a DNA strand that is a polymer of nucleotides having a certain length or longer, wherein the nucleotide monomers are covalently linked into a long chain.

[0036] The polynucleotide sequences encoding the chitin transglycosylase of the present disclosure may be referred to as the CRH1 gene, the CRH2 gene, or a combination thereof. In one embodiment, they are referred to as the CRH1 (YALI0C09680) gene, the CRH1 (YALI0E24673) gene, the CRH2 (YALI0B15510) gene, or a combination thereof, which may include polynucleotide sequences encoding the amino acid sequences represented by SEQ ID NO:1, SEQ ID NO:2, and / or SEQ ID NO:3.

[0037] Due to codon degeneracy or considering the preferred codons in the organism expressing the polypeptide or protein, the polynucleotide may undergo various modifications in the coding region within the range of not changing the amino acid sequence of the polypeptide or protein. Specifically, the polynucleotide may include the polynucleotide sequences of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or those having 60% or higher homology or identity therewith, consisting of or substantially consisting of the polynucleotide sequences of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or those having 60% or higher homology or identity therewith, but not limited thereto. For example, the polynucleotide may consist of nucleotide sequences having 60% or higher, 62% or higher, 63% or higher, 64% or higher, 65% or higher, 70% or higher, 76% or higher, 77% or higher, 78% or higher, 80% or higher, 85% or higher, 86% or higher, 87% or higher, 88% or higher, 89% or higher, 90% or higher, 91% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher homology or identity with SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6, but not limited thereto.

[0038] In addition, the polynucleotides of the present disclosure may include probes. For example, without limitation, any sequence may be used as long as it is a sequence capable of hybridizing to a complementary sequence of all or part of a polynucleotide sequence under stringent conditions. "Stringent conditions" refer to conditions that enable specific hybridization between polynucleotides. Such conditions are specifically described in the literature (e.g., J. Sambrook et al., ibid.). For example, stringent conditions may include conditions under which polynucleotides with a high degree of homology or identity of 40% or higher, specifically 90% or higher, more specifically 95% or higher, 96% or higher, 97% or higher, 98% or higher, and even more specifically 99% or higher hybridize to each other, and polynucleotides with a homology or identity lower than the above do not hybridize to each other, or the washing conditions for ordinary Southern hybridization, i.e., washing once, specifically twice or three times, at a salt concentration and temperature corresponding to 60°C, 1X SSC, 0.1% SDS, especially 60°C, 0.1×SSC, 0.1% SDS, and more especially 68°C, 0.1×SSC, 0.1% SDS.

[0039] Hybridization requires that two nucleic acids contain complementary sequences, although mismatches between bases are possible depending on the stringency of the hybridization. The term "complementary" is used to describe the relationship between nucleobases that can hybridize to each other. For example, with respect to DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Thus, the polynucleotides of the present disclosure may include isolated nucleic acid fragments that are complementary to the entire sequence and nucleic acid sequences that are substantially similar thereto.

[0040] Specifically, polynucleotides with homology or identity may be detected using hybridization conditions that include a hybridization step with a Tm value of 55°C under the above conditions. In addition, the Tm value may be 60°C, 63°C, or 65°C, but is not limited thereto, and may be appropriately adjusted by those skilled in the art according to their purposes.

[0041] The appropriate stringency for hybridizing polynucleotides depends on the length and degree of complementarity of the polynucleotides, and these variables are well known in the art (J. Sambrook et al., ibid.).

[0042] As used herein, the term "vector" refers to a DNA construct used to insert a desired nucleotide sequence into a host chromosome, or a DNA construct comprising a nucleotide sequence of a polynucleotide encoding a desired polypeptide or protein, the polynucleotide being operably linked to a suitable expression control region (expression control sequence) so as to be capable of expressing the desired polypeptide or protein in a suitable host. The expression control region may include a promoter capable of initiating transcription, any operator sequence for regulating transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence for regulating transcription and translation termination. Once transformed into a suitable host cell, the vector may replicate or function independently of the host genome, or may integrate into its genome.

[0043] The vector of the present disclosure may be an insertion vector for inserting a polynucleotide for attenuating the chitin transglycosylase activity of the present disclosure into a chromosome, but is not limited thereto. The polynucleotide may be inserted into the chromosome by any method known in the art, such as by homologous recombination, but is not limited thereto. The vector may also include a selection marker to confirm insertion into the chromosome. The selection marker is used to select cells transformed with the vector, i.e., to confirm whether the target nucleic acid molecule has been inserted, and a marker providing a selectable phenotype, such as drug resistance, auxotrophy, resistance to cytotoxic agents, or expression of a surface polypeptide or protein, may be used. Only cells expressing the selection marker can survive or display a different phenotype in an environment treated with a selection agent, and thus the transformed cells can be selected. The insertion vector may not contain an origin of replication necessary for replication within the transformed cell.

[0044] The vectors used in the present disclosure are not particularly limited, and any vectors known in the art may be used. Examples of commonly used vectors may include natural or recombinant plasmids, cosmids, viruses, and phages. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A, etc. may be used as phage vectors or cosmid vectors, while those based on pDZ, pBR, pUC, pBluescriptII, pGEM, pTZ, pCL, and pET, etc. may be used as plasmid vectors. Specifically, pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vectors, etc. may be used.

[0045] As used herein, the term "transformation" refers to the introduction of a vector containing a desired polynucleotide (including an insertion vector for inserting a polynucleotide for attenuating chitinotransglycosylase activity of the present disclosure into a chromosome) into a host cell to change the genetic traits of the host cell. The transformed polynucleotide may be inserted into the chromosome of the host cell or may be located outside the chromosome. In addition, the polynucleotide may contain DNA and / or RNA encoding a desired protein. Depending on the purpose of introduction, the polynucleotide may be introduced in a suitable form. For example, a polynucleotide for expressing a desired protein may be introduced into a host cell in the form of an expression cassette, which is a gene construct containing all the elements required for self-expression. An expression cassette typically may contain a promoter operably linked to the polynucleotide, a transcription termination signal, a ribosome binding site, and a translation termination signal. The expression cassette may be in the form of an expression vector capable of self-replicating. In addition, the polynucleotide may be introduced into the host cell in its own form and operably linked to sequences required for expression in the host cell, but is not limited thereto.

[0046] In addition, as used herein, the term "operably linked" refers to a polynucleotide sequence being functionally linked to a promoter sequence that initiates and mediates the transcription of a downstream polynucleotide.

[0047] Methods for transforming the vectors of the present disclosure include any method for introducing nucleic acids into cells and can be carried out by selecting suitable standard techniques known in the art according to the host cell. For example, transformation methods may include electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) technique, DEAE-dextran technique, cationic liposome technique, lithium acetate-DMSO technique, etc., but are not limited thereto.

[0048] As used herein, the term "microorganism" or "strain" includes all wild-type microorganisms or naturally or artificially genetically modified microorganisms, and it may be a microorganism in which a specific mechanism is attenuated or enhanced due to the insertion of a foreign gene or the enhancement or inactivation of an endogenous gene activity, etc., and it may be a genetically modified microorganism containing a gene for producing a desired polypeptide, protein, or product.

[0049] The microorganisms of the present disclosure may be microorganisms having the ability to produce retinoids. The "microorganisms having the ability to produce retinoids" may be used interchangeably with the "microorganisms producing retinoids".

[0050] The microorganism of the present disclosure can be a microorganism into which a polynucleotide encoding the proteins of lycopene cyclase / phytoene synthase (crtYB) and phytoene desaturase (crtI) has been introduced, thereby exhibiting the activity of the said proteins or having enhanced activity of the said proteins, such that a microorganism that does not endogenously have the ability to produce retinoids acquires the ability to produce retinoids, or the ability of a microorganism that has the ability to produce retinoids to produce retinoids is further enhanced. The lycopene cyclase / phytoene synthase or phytoene desaturase can be a protein derived from Xanthophyllomyces dendrorhous, but is not limited thereto, as long as it is a protein that exhibits the same or similar activity thereto. In a specific embodiment, the lycopene cyclase / phytoene synthase or phytoene desaturase can be composed of the amino acid sequences of SEQ ID NO:7 or SEQ ID NO:8 respectively, or can contain the amino acid sequences of SEQ ID NO:7 or SEQ ID NO:8; it can be composed of an amino acid sequence having 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 with the above amino acids and simultaneously exhibiting an activity equivalent to that of the lycopene cyclase / phytoene synthase or phytoene desaturase, or can contain an amino acid sequence having 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 with the above amino acids and simultaneously exhibiting an activity equivalent to that of the lycopene cyclase / phytoene synthase or phytoene desaturase. It is also obvious that a protein in which a partial sequence has been deleted, modified, substituted or added also belongs to the lycopene cyclase / phytoene synthase or phytoene desaturase, as long as it has such homology or identity and exhibits an activity equivalent to that of the lycopene cyclase / phytoene synthase or phytoene desaturase. In addition, in a specific embodiment, the polynucleotide encoding the lycopene cyclase / phytoene synthase or phytoene desaturase can be composed of the sequences of SEQ ID NO:9 or SEQ ID NO:10 respectively, or can contain the sequences of SEQ ID NO:9 or SEQ ID NO:10. Due to codon degeneracy or considering the preferred codons in the microorganism of the present disclosure, the polynucleotide can undergo various modifications in the coding region within the range of not changing the amino acid sequence.Specifically, the polynucleotide may consist of a nucleotide sequence having 60% or higher, 70% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, and less than 100% homology or identity with the sequence of SEQ ID NO:9 or SEQ ID NO:10, or may comprise a nucleotide sequence having 60% or higher, 70% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, and less than 100% homology or identity with the sequence of SEQ ID NO:9 or SEQ ID NO:10, but is not limited thereto.

[0051] In addition, the microorganism of the present disclosure may be a microorganism into which a polynucleotide encoding a β - carotene 15,15'-oxygenase (BLH) protein has been introduced, thereby exhibiting β - carotene 15,15'-oxygenase activity or having enhanced β - carotene 15,15'-oxygenase activity, enabling a microorganism that does not originally have the ability to produce retinoids to have the ability to produce retinoids, or further enhancing the retinoid production ability of a microorganism that already has the ability to produce retinoids. The β - carotene 15,15'-oxygenase may be a protein derived from an uncultured marine bacterium 66A03, but is not limited thereto, as long as it is a protein exhibiting the same or similar activity. In a specific embodiment, the β - carotene 15,15'-oxygenase may consist of the amino acid sequence of SEQ ID NO:11, or may contain the amino acid sequence of SEQ ID NO:11; it may consist of an amino acid sequence having 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 with the above amino acid sequence and simultaneously exhibiting an activity equivalent to that of β - carotene 15,15'-oxygenase, or may contain an amino acid sequence having 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 with the above amino acid sequence and simultaneously exhibiting an activity equivalent to that of β - carotene 15,15'-oxygenase. It is also obvious that a protein in which some sequences are deleted, modified, substituted or added also belongs to β - carotene 15,15'-oxygenase, as long as it has such homology or identity and exhibits an activity equivalent to that of β - carotene 15,15'-oxygenase. In addition, in a specific embodiment, the polynucleotide encoding β - carotene 15,15'-oxygenase may consist of the sequence of SEQ ID NO:12, or may contain the sequence of SEQ ID NO:12. Due to codon degeneracy or considering the preferred codons in the microorganism of the present disclosure, the polynucleotide may undergo various modifications in the coding region within the range of not changing the amino acid sequence.Specifically, the polynucleotide may consist of a nucleotide sequence having 60% or higher, 70% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, and less than 100% homology or identity with the sequence of SEQ ID NO:12, or may comprise a nucleotide sequence having 60% or higher, 70% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, and less than 100% homology or identity with the sequence of SEQ ID NO:12, but is not limited thereto.

[0052] The microorganism of the present disclosure may be a microorganism in which the activity of chitin transglycosylase is attenuated to increase the retinol production capacity and / or secretion capacity.

[0053] In the microorganism of the present disclosure, the increase in retinol production capacity may be due to the increase in retinol secretion capacity, but is not limited thereto.

[0054] The microorganism of the present disclosure may selectively secrete retinol, but is not limited thereto.

[0055] Compared with a microorganism having the ability to produce retinol and in which the activity of chitin transglycosylase is not attenuated, the microorganism of the present disclosure may have an increased retinol secretion capacity, but is not limited thereto.

[0056] In one embodiment, the microorganism of the present disclosure may selectively secrete retinol from β-carotene and retinol.

[0057] The microorganism of the present disclosure can be obtained by additionally attenuating the chitin transglycosylase of the present disclosure in a microorganism that naturally has chitin transglycosylase or the ability to produce retinol, or in a parental strain having chitin transglycosylase or the ability to produce retinol.

[0058] For example, the microorganism of the present disclosure may include all microorganisms capable of producing retinol, in which the chitin transglycosylase of the present disclosure is attenuated.

[0059] Compared with a microorganism having the ability to produce retinol and in which the activity of chitin transglycosylase is not attenuated, the microorganism of the present disclosure may have an increased retinol secretion capacity.

[0060] For example, the microorganism of the present disclosure may be a recombinant strain in which the retinol production ability and / or secretion ability is increased by attenuating the activity of the chitin transglycosylase of the present disclosure in a natural wild-type microorganism, a microorganism having retinol production ability, and / or a microorganism containing chitin transglycosylase. The recombinant strain with increased retinol production ability and / or secretion ability may be a microorganism in which the retinol production ability and / or secretion ability is increased compared to a natural wild-type microorganism or a microorganism in which the activity of the chitin transglycosylase of the present disclosure is not attenuated, but is not limited thereto.

[0061] For example, the microorganism in which the activity of the chitin transglycosylase of the present disclosure is not attenuated (which is the target strain for comparing whether the retinol production ability and secretion ability are increased) may be CC08-2050 (KCCM13294P, Ref. Park et al., Metabolic engineering 2022; 73:26-37), but is not limited thereto. The strain name of the CJ2050 strain described in the above reference (Park et al., Metabolic engineering 2022; 73:26-37) is the CC08-2050 strain of the present disclosure, and the CC08-2050 strain of the present disclosure and the CJ2050 strain are the same strain.

[0062] For example, compared with the parental strain before modification or an unmodified microorganism, the retinol production capacity and / or secretion capacity of a recombinant strain and microorganism with increased retinol production capacity and / or secretion capacity can be increased 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 limitation on the upper limit, but it can be, for example, about 200% or lower). In another example, the retinol production capacity and / or secretion capacity is 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 (there is no particular limitation on the upper limit, but it can be, for example, about 10-fold or less), but the increased amount is not limited to this, as long as the capacity has an increased + value amount compared with the production capacity and / or secretion capacity of the parental strain before modification or the unmodified microorganism. The term "about" refers to a range including ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and thus includes all values equivalent or similar to those within the range described after the term "about", but is not limited thereto.

[0063] For example, compared with the parental strain before modification or an unmodified microorganism, the recombinant strain or microorganism may have reduced chitin transglycosylase activity, and when the endogenous chitin transglycosylase activity in the parental strain before modification or the unmodified microorganism is regarded as 100%, the chitin transglycosylase activity in the recombinant strain or microorganism can be reduced to 100% or lower (for example, reduced to 99.5%, 99%, 98%, 95%, 90%, 85%, 80%, etc.), but is not limited thereto.

[0064] As used herein, the term "unmodified microorganism" does not exclude strains that may contain mutations naturally occurring in the microorganism, and can be a wild-type strain or the natural strain itself, or can be a strain before its traits are changed by genetic variation due to natural or artificial factors. For example, the unmodified microorganism can refer to a strain in which the chitin transglycosylase of the present disclosure is not attenuated or before the chitin transglycosylase of the present disclosure is attenuated. The "unmodified microorganism" can be used interchangeably with "strain before modification", "microorganism before modification", "unmutated strain", "unmodified strain", "unmutated microorganism", or "reference microorganism".

[0065] The microorganism of the present disclosure can be a microorganism of the genus Yarrowia, but is not limited thereto.

[0066] In one embodiment, the Yarrowia microorganism of the present disclosure can be Yarrowia lipolytica, but is not limited thereto.

[0067] As used herein, the term "attenuation" of chitin transglycosylase (including its polypeptide and its protein, the same hereinafter) activity is a concept that includes two cases: a decrease in activity or the absence of activity compared to the endogenous activity. The attenuation can be used interchangeably with terms such as inactivation, defect / deficiency, deletion, downregulation, reduction, decrease, weakening, etc.

[0068] The attenuation can also include cases where the activity of chitin transglycosylase itself is reduced or eliminated due to variations in the polynucleotide encoding chitin transglycosylase (including its polypeptide and its protein), etc., compared to the activity of chitin transglycosylase initially possessed by the microorganism; cases where the total activity level and / or concentration (expression level) of chitin transglycosylase in the cell is lower due to inhibition of the expression of the polynucleotide encoding chitin transglycosylase or by inhibiting translation into chitin transglycosylase, compared to the natural strain; cases where the polynucleotide is not expressed at all; and / or cases where chitin transglycosylase activity is absent even when the polynucleotide is expressed.

[0069] "Endogenous activity" refers to the activity of a specific chitin transglycosylase (including its polypeptide and its protein) initially possessed by the parental strain, wild type, or unmodified microorganism before the trait is changed when the trait is changed due to genetic variation caused by natural or artificial factors. This can be used interchangeably with "activity before modification". The fact that the activity of chitin transglycosylase is "attenuated, inactivated, defective / deficient, reduced, downregulated, decreased, or weakened" compared to the endogenous activity means that its activity is reduced compared to the activity of the specific chitin transglycosylase initially possessed by the parental strain or unmodified microorganism before the trait is changed.

[0070] For the purposes of the present disclosure, due to the attenuation of chitin transglycosylase activity, the microorganisms of the present disclosure can have enhanced retinol production capacity and / or secretion capacity.

[0071] This attenuation of chitin transglycosylase activity can be carried out by any method known in the art, but the method is not limited thereto, and the attenuation can be achieved by applying various methods well-known in the art (for example, Nakashima N et al., Bacterial cellular engineering by genome editing and gene silencing. Int J Mol Sci. 2014; 15(2): 2773–2793; Sambrook et al. Molecular Cloning 2012, etc.).

[0072] Specifically, the attenuation of chitinotransferase activity in the present disclosure can be:

[0073] 1) Deleting all or part of the gene encoding the chitinotransferase polypeptide;

[0074] 2) Modifying the expression regulatory region (or expression regulatory sequence) to reduce the expression of the gene encoding the chitinotransferase polypeptide;

[0075] 3) Modifying the amino acid sequence constituting the chitinotransferase polypeptide to eliminate or attenuate the activity of the chitinotransferase (for example, deletion / substitution / addition of one or more amino acids in the amino acid sequence);

[0076] 4) Modifying the gene (including polynucleotide) sequence encoding the chitinotransferase polypeptide to eliminate or attenuate the activity of the chitinotransferase (for example, deleting / substituting / adding one or more nucleic acid bases in the nucleic acid sequence of the gene of the polypeptide to encode a polypeptide that has been modified to eliminate or attenuate the polypeptide activity);

[0077] 5) Modifying the start codon of the gene transcript encoding the chitinotransferase polypeptide or the nucleotide sequence encoding the Shine-Dalgarno sequence or the 5'-UTR region;

[0078] 6) Introducing an antisense oligonucleotide (for example, antisense RNA) that binds complementarily to the transcript of the gene encoding the chitinotransferase polypeptide;

[0079] 7) Adding a sequence complementary to the Shine-Dalgarno sequence before the Shine-Dalgarno sequence of the gene encoding the chitinotransferase polypeptide to form a secondary structure to which ribosomes cannot attach;

[0080] 8) Adding a promoter transcribed in the opposite direction (reverse transcription engineering, RTE) to the 3' end of the open reading frame (ORF) of the gene sequence encoding the chitinotransferase polypeptide; or

[0081] 9) A combination of two or more selected from 1) to 8), but not limited thereto.

[0082] For example,

[0083] 1) Deleting all or part of the gene encoding the polypeptide can be removing the complete polynucleotide encoding the endogenous target polypeptide in the chromosome, replacing it with a polynucleotide with some nucleotides deleted, or replacing it with a marker gene.

[0084] 2) A modified expression regulatory region (or expression regulatory sequence) can be a variation that occurs in the expression regulatory region (or expression regulatory sequence) due to deletion, insertion, non-conservative or conservative substitution, or a combination thereof, or substitution with a sequence showing weaker 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 transcription and translation termination.

[0085] 3) and 4) A modified amino acid sequence or polynucleotide sequence can be a variation that occurs in the sequence due to deletion, insertion, non-conservative or conservative substitution of the amino acid sequence of a polypeptide or the polynucleotide sequence encoding the polypeptide, or a combination thereof, or substitution with an amino acid sequence or polynucleotide sequence that has been modified to have weaker activity or an amino acid sequence or polynucleotide sequence that has been modified to be inactive, thereby weakening the activity of the polypeptide, but not limited to this. For example, gene expression can be inhibited or weakened by introducing a variation into the polynucleotide sequence and forming a stop codon, but not limited to this.

[0086] 5) Modifying the start codon of the gene transcript encoding a polypeptide or the nucleotide sequence encoding the 5'-UTR region can be, for example, substituting with a nucleotide sequence encoding another start codon that has a lower polypeptide expression rate compared to the endogenous start codon, but not limited to this.

[0087] 6) Introducing an antisense oligonucleotide (such as antisense RNA) that binds complementarily to the transcript of the gene encoding a polypeptide can refer to references such as [Weintraub, H. et al., Antisense-RNA as a molecular tool for genetic analysis, Reviews-Trends in Genetics, Vol. 1(1) 1986].

[0088] 7) Adding a sequence complementary to the Shine-Dalgarno sequence before the Shine-Dalgarno sequence of the gene encoding a chitin transglycosylase polypeptide to form a secondary structure to which ribosomes cannot attach, which may render mRNA translation impossible or slow down the mRNA translation rate.

[0089] In addition, 8) Adding a promoter transcribed in the opposite direction (reverse transcription engineering, RTE) to the 3'-end of the open reading frame (ORF) of the gene sequence encoding a polypeptide may weaken the activity by making the antisense nucleotide complementary to the transcript of the gene encoding the polypeptide.

[0090] In one embodiment, in the microorganism of the present disclosure, any one or more genes encoding chitin transglycosylase can be deleted, but not limited to this.

[0091] As used herein, the term "retinoid" chemically refers to a group of vitamin A or chemically related compounds.

[0092] In one embodiment, the retinoid can be any one selected from the group consisting of retinol, retinal, retinoic acid, and retinyl esters, but is not limited thereto.

[0093] In one embodiment, retinol can be converted into other retinoid compounds (e.g., retinal, retinoic acid, and retinyl esters) by methods known in the art.

[0094] Another aspect of the present disclosure provides a method for producing a retinoid, the method comprising the step of culturing the microorganism of the present disclosure in a culture medium.

[0095] The microorganism is as described in other aspects.

[0096] As used herein, the term "culturing" refers to culturing the microorganism of the present disclosure under appropriately adjusted environmental conditions. In the present disclosure, the culturing procedure can be carried out according to suitable culture media and culture conditions known in the art. Those skilled in the art can easily adjust and use such a culturing process according to the selected strain. Specifically, the culturing can be of the batch type, continuous type, and / or fed-batch type, but is not limited thereto.

[0097] The microorganism of the present disclosure can be cultured in a common culture medium containing a suitable carbon source, nitrogen source, phosphorus source, inorganic compounds, amino acids, and / or vitamins, etc., while controlling the temperature, pH, etc. under aerobic conditions.

[0098] In the present disclosure, the carbon source can include carbohydrates such as glucose, saccharose, lactose, fructose, sucrose, maltose, etc.; sugar alcohols such as mannitol, sorbitol, etc.; organic acids such as pyruvic acid, lactic acid, citric acid, etc.; and amino acids such as glutamic acid, methionine, lysine, etc. In addition, natural organic nutrient sources such as starch hydrolyzates, molasses, black molasses, rice bran, cassava, bagasse, and corn steep liquor can be used, and specifically, carbohydrates such as glucose and aseptically pre-treated molasses (i.e., molasses converted into reducing sugars) can be used, and various other carbon sources in appropriate amounts can be used without limitation. These carbon sources can be used alone or in combination of two or more, but are not limited thereto.

[0099] For the nitrogen source, inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, ammonium nitrate, etc.; amino acids such as glutamic acid, methionine, glutamine, etc.; and organic nitrogen sources such as peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolysate, fish or its decomposition products, defatted soybean cake or its degradation products, etc. can be used. These nitrogen sources can be used alone or in combination of two or more, but are not limited thereto.

[0100] The phosphorus source can include potassium dihydrogen phosphate, dipotassium hydrogen phosphate and their corresponding sodium-containing salts. For inorganic compounds, sodium chloride, calcium chloride, ferric chloride, magnesium sulfate, ferric sulfate, manganese sulfate, calcium carbonate, etc. can be used. In addition, amino acids, vitamins and / or suitable precursors can also be included. These components or precursors can be added to the culture medium in a batch or continuous manner. However, the present disclosure is not limited thereto.

[0101] During the cultivation of the microorganisms of the present disclosure, the pH of the culture medium can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid, etc. to the culture medium in an appropriate manner. In addition, an antifoaming agent such as fatty acid polyethylene glycol ester can be added to inhibit the formation of foam during the cultivation. In addition, oxygen or an oxygen-containing gas can be injected into the culture medium to maintain the aerobic state of the culture medium, or no gas can be injected or nitrogen, hydrogen or carbon dioxide gas can be injected to maintain the anaerobic or non-aerobic state of the culture medium, but is not limited thereto.

[0102] In addition, the culture medium can contain metal salts required for growth such as magnesium sulfate or ferric sulfate. Finally, in addition to the above substances, essential growth substances such as amino acids and vitamins can also be used. In addition, suitable precursors can be used in the culture medium. The above raw materials can be added to the culture in a suitable manner in batches or continuously during the cultivation process, but are not limited thereto.

[0103] In the present disclosure, during the cultivation of microorganisms, the pH of the culture can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid, etc. to the culture in an appropriate manner. In addition, an antifoaming agent such as fatty acid polyethylene glycol ester can be added to inhibit the formation of foam during the cultivation. In addition, oxygen or an oxygen-containing gas can be injected into the culture to maintain the aerobic state of the culture, or no gas can be injected or nitrogen, hydrogen or carbon dioxide gas can be injected to maintain the anaerobic or non-aerobic state, but is not limited thereto.

[0104] In the cultivation of the present disclosure, the cultivation temperature can be maintained at 20°C to 35°C, specifically 25°C to 35°C, and the cultivation can continue until the desired amount of useful substance is obtained, and it can be carried out for about 10 hours to 160 hours, about 20 hours to 130 hours, about 24 hours to 120 hours, about 36 hours to 120 hours, about 48 hours to 120 hours, about 48 hours or longer, or about 48 hours, about 72 hours, or about 120 hours, but not limited thereto.

[0105] The method for producing retinoids of the present disclosure may further include the step of recovering retinoids from the microorganism or the culture medium.

[0106] Depending on the method for culturing microorganisms of the present disclosure, such as batch, continuous or fed-batch culture methods, suitable methods known in the art can be used to recover the desired retinoids from the culture medium. For example, centrifugation, filtration, treatment with a crystalline protein precipitant (salting out), extraction, sonication, ultrafiltration, dialysis, various types of chromatography (such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography and affinity chromatography), HPLC, and combinations of these methods can be used, but not limited thereto.

[0107] The method may further include an additional purification process. The purification process can be carried out by using suitable methods known in the art.

[0108] In one embodiment, in the method for producing retinoids of the present disclosure, a microorganism with the ability to secrete retinoids is used, and the activity of chitin transglycosylase in the microorganism is attenuated. Therefore, in the present disclosure, retinoids can be produced without using cell disruption of the microorganism or without using dodecane as a solvent (the solvent is widely used in retinoid extraction), but not limited thereto.

[0109] The method for producing retinoids of the present disclosure may further include the step of converting the retinols expressed by the microorganisms of the present disclosure into retinoids other than retinol. In the method for producing retinoids of the present disclosure, the conversion step may also be included after the cultivation step or the recovery step. The conversion step can be carried out by using suitable methods known in the art. For example, retinol acyltransferase can be used for the conversion, but not limited thereto.

[0110] In one embodiment, the retinoid other than retinol can be any one selected from the group consisting of retinal, retinoic acid and retinyl esters, but not limited thereto as long as it is included in retinoids.

[0111] Another aspect of the present disclosure provides a method for preparing a Yarrowia microorganism with the ability to produce retinoids, the method including the step of attenuating the activity of chitin transglycosylase in a Yarrowia microorganism with the ability to produce retinoids.

[0112] Another aspect of the present disclosure provides a method for increasing the secretion of retinoids, the method comprising the step of attenuating the chitin transglycosylase activity in a Yarrowia yeast microorganism having the ability to produce retinoids.

[0113] The method for increasing the secretion of retinoids may be a method for increasing the secretion of retinoids in a Yarrowia yeast microorganism having the ability to produce retinoids.

[0114] The step of attenuating the chitin transglycosylase activity may be a step of modifying the Yarrowia yeast microorganism such that the chitin transglycosylase is attenuated, but as described in other aspects.

[0115] Another aspect of the present disclosure provides a composition for producing retinoids, the composition comprising any one or more Yarrowia yeast microorganisms and their cultures, wherein the activity of chitin transglycosylase in the Yarrowia yeast microorganisms is attenuated.

[0116] The composition of the present disclosure may further comprise any suitable excipients conventionally used in compositions for producing retinoids, and such excipients may include, for example, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers, isotonic agents, etc., but are not limited thereto.

[0117] The chitin transglycosylase of the Yarrowia yeast microorganism (wherein its activity is attenuated), the culture, the retinoids, etc. are as described in other aspects.

[0118] Another aspect of the present disclosure provides the use of a Yarrowia yeast microorganism in the production of retinoids, wherein the activity of the chitin transglycosylase of the present disclosure in the Yarrowia yeast microorganism is attenuated.

[0119] The chitin transglycosylase of the Yarrowia yeast microorganism (wherein its activity is attenuated), the retinoids, etc. are as described in other aspects.

[0120] [Mode for Carrying Out the Invention]

[0121] Hereinafter, the present disclosure will be described in more detail by way of exemplary embodiments. However, the following exemplary embodiments are only preferred embodiments for illustrating the present disclosure, and thus are not intended to limit the scope of the present disclosure thereto. At the same time, technical matters not described in this specification can be fully understood and easily implemented by those skilled in the technical field of the present disclosure or similar technical fields.

[0122] Example 1. Attenuation of CRH1 (YALI0C09680) in a Yarrowia yeast strain producing retinoids

[0123] To attenuate the endogenous CRH1 (YALI0C09680) of the retinol-producing Yarrowia lipolytica strain CC08-2050 (Park et al., Metabolic engineering 2022; 73:26-37) (which was deposited as KCCM13294P), the ORF of CRH1 (YALI0C09680) in the genome was deleted. For this purpose, based on the nucleotide sequence registered in the Kyoto Encyclopedia of Genes and Genomes (KEGG), the ORF sequence of CRH1 (YALI0C09680) (SEQ ID NO: 4) was obtained. In addition, a CRH1 (YALI0C09680) deletion cassette was constructed using the primers shown in Table 1 and using the URA3 gene (SEQ ID NO: 13) of Yarrowia lipolytica as a selection marker. That is, PCR was performed using the genomic DNA of CC08-2050 as a template and the primers of SEQ ID NO: 14 and SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19, and SEQ ID NO: 20 and SEQ ID NO: 21, respectively. The PCR conditions were: denaturation at 95 °C for 1 minute; annealing at 55 °C for 1 minute; and polymerization at 72 °C for 2 minutes, for 35 cycles. The resulting DNA fragments were produced into a cassette by overlap extension PCR.

[0124] The cassette thus constructed was introduced into the CC08-2050 strain by the heat shock method (D.-C. Chen et al., Appl Microbiol Biotechnol, 1997), and then colonies formed on a solid medium (YLMM1) without uracil were obtained. The colonies were cultured on a 5-FOA solid medium at 30 °C for 3 days (where the insertion of the cassette into the genome was confirmed using the primers of SEQ ID NO: 22 and SEQ ID NO: 23), and the URA3 marker was recovered by obtaining colonies that grew on the 5-FOA solid medium. The final strain thus obtained was named CJ2327.

[0125] [Table 1]

[0126]

[0127] Those having the following compositions were used as the above YLMM1 medium and 5-FOA medium.

[0128] <Yarrowia lipolytica minimal medium 1 (YLMM1)>

[0129] 20 g / L glucose, 6.7 g / L yeast nitrogen base without amino acids, 2 g / L yeast synthetic dropout medium supplement without uracil, 15 g / L agar.

[0130] <5-fluoroorotic acid (5-FOA)>

[0131] 20 g / L glucose, 6.7 g / L yeast nitrogen base without amino acids, 2 g / L yeast synthetic dropout medium supplement without uracil, 50 μg / mL uracil, 1 g / L 5-fluoroorotic acid (5-FOA), 15 g / L agar

[0132] Example 2. Weakening of CRH1 (YALI0E24673) in a retinol-producing Yarrowia lipolytica strain

[0133] To weaken the endogenous CRH1 (YALI0E24673) in the retinol-producing Yarrowia lipolytica strain CC08-2050 (KCCM13294P), the ORF of CRH1 (YALI0E24673) in the genome was deleted. For this purpose, based on the nucleotide sequence registered in the Kyoto Encyclopedia of Genes and Genomes (KEGG), the ORF sequence of CRH1 (YALI0E24673) (SEQ ID NO: 5) was obtained. In addition, a CRH1 (YALI0E24673) deletion cassette was constructed using the primers shown in Table 2 and using the URA3 gene of Yarrowia lipolytica (SEQ ID NO: 13) as a selection marker. That is, PCR was performed using the genomic DNA of CC08-2050 as a template and the primers of SEQ ID NO: 24 and SEQ ID NO: 25, SEQ ID NO: 26 and SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29, and SEQ ID NO: 30 and SEQ ID NO: 31, respectively. The PCR conditions were: denaturation at 95 °C for 1 minute; annealing at 55 °C for 1 minute; and polymerization at 72 °C for 2 minutes, for 35 cycles. The resulting DNA fragments were produced into a cassette by overlap extension PCR.

[0134] [Table 2]

[0135]

[0136] The cassette thus constructed was introduced into strain CC08-2050 by heat shock method, and then colonies formed on a solid medium without uracil (YLMM1; a medium having the same composition as the YLMM1 medium in Example 1) were obtained. The colonies (in which the insertion of the cassette into the genome was confirmed using the primers of SEQ ID NO:32 and SEQ ID NO:33) were cultured on a 5-FOA solid medium at 30 °C for 3 days, and the URA3 marker was recovered by obtaining colonies that grew on the 5-FOA solid medium. The final strain thus obtained was named CJ2328.

[0137] Example 3. Weakening of CRH2 (YALI0B15510) in a retinol-producing Yarrowia lipolytica strain

[0138] To weaken the endogenous CRH2 (YALI0B15510) of the retinol-producing Yarrowia lipolytica strain CC08-2050, the ORF of CRH2 (YALI0B15510) was deleted. For this purpose, based on the nucleotide sequence registered in the Kyoto Encyclopedia of Genes and Genomes (KEGG), the ORF sequence of CRH2 (YALI0B15510) (SEQ ID NO:6) was obtained. In addition, a CRH2 (YALI0B15510) deletion cassette was constructed using the primers shown in Table 3 and using the URA3 gene (SEQ ID NO:13) of Yarrowia lipolytica as a selection marker. That is, PCR was performed using the genomic DNA of CC08-2050 as a template and the primers of SEQ ID NO:34 and SEQ ID NO:35, SEQ ID NO:36 and SEQ ID NO:37, SEQ ID NO:38 and SEQ ID NO:39, and SEQ ID NO:40 and SEQ ID NO:41, respectively. The PCR conditions were: denaturation at 95 °C for 1 minute; annealing at 55 °C for 1 minute; and polymerization at 72 °C for 2 minutes, for 35 cycles. The resulting DNA fragments were produced into a cassette by overlap extension PCR.

[0139] [Table 3]

[0140]

[0141]

[0142] The cassette thus constructed was introduced into the CC08-2050 strain by heat shock method, and then colonies formed on a solid medium (YLMM1) without uracil were obtained. The colonies (in which the insertion of the cassette into the genome was confirmed using the primers of SEQ ID NO: 42 and SEQ ID NO: 43) were cultured on a 5-FOA solid medium at 30 °C for 3 days, and the URA3 marker was recovered by obtaining colonies that grew on the 5-FOA solid medium. The final strain thus obtained was named CJ2329.

[0143] Example 4. Comparative evaluation of retinol production and secretion ability of strains with attenuated CRH

[0144] To compare the retinol production and secretion levels of the strains prepared in Examples 1-3, a shake flask test was conducted. The retinol-producing Yarrowia lipolytica strain (CC08-2050 (KCCM13294P); control group), the CRH1 (YALI0C09680)-deficient strain (CJ2327) prepared in Example 1, the CRH1 (YALI0E24673)-deficient strain (CJ2328), and the CRH2 (YALI0B15510)-deficient strain (CJ2329) were each inoculated at an initial OD = 2 into a 250 ml Erlenmeyer flask with baffles containing 25 ml of YPDLU medium supplemented with 0.05% 3,5-di-tert-butyl-4-hydroxytoluene (BHT), and cultured under the conditions of 30 °C and 200 rpm. The following composition of the medium was used as the above YPDLU medium.

[0145] <ypdlu>

[0146] 40 g / L glucose, 20 g / L bacterial peptone, 10 g / L yeast extract, 1 g / L uracil, 1 g / L leucine, 100 ml / L 1 M phosphate buffer (pH 7.0)

[0147] The growth of each strain was evaluated by measuring the OD value at a wavelength of 600 nm using a spectrophotometer.

[0148] In addition, the concentrations of secreted β-carotene, retinol, and retinal were measured by the following steps: 0.1 ml of the supernatant (cells were removed after completion of the culture) was mixed with 0.9 ml of acetone (Sigma) containing 4% BHT, and then it was quantitatively analyzed using an HPLC instrument.

[0149] The analyzed OD values and the concentrations of retinoids and β-carotene are shown in Table 4, and the retinoid concentrations are illustrated and shown in Figure 1 it.

[0150] [Table 4]

[0151]

[0152] As shown by the above results, when CRH1 (YALI0C09680g), CRH1 (YALI0E24673g), and CRH2 (YALI0B15510g) were attenuated in the retinol-producing Yarrowia lipolytica strains, the retinol / retinal secreted from the cells increased by 2.1-fold / 2.1-fold, 2.4-fold / 2.3-fold, and 2.3-fold / 2.2-fold, respectively, compared with the control group. In addition, the above results indicate that even when β-carotene is produced in retinol-producing microorganisms in which CRH1, CRH2, or their combination is attenuated, only retinol will be selectively secreted.

[0153] Based on the above description, those skilled in the art will understand that the present disclosure can be implemented in different specific forms without changing its technical spirit or basic characteristics. In this regard, it should be understood that the above embodiments are not restrictive but illustrative in all aspects. The scope of the present disclosure is defined by the appended claims rather than by the description after them, and thus all changes and modifications that fall within the boundaries and limits of the claims, or the equivalents of these boundaries and limits, are encompassed by the claims.

[0154]

[0155] < / ypdlu>

Claims

1. A Yarrowia yeast microorganism with retinol production ability, wherein the activity of chitin transglycosylase is attenuated.

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

3. The microorganism according to claim 1, wherein the chitin transglycosylase is any one or more selected from the group consisting of: a polypeptide comprising a sequence having 60% or higher identity with the amino acid sequence of SEQ ID NO:1 and showing chitin transglycosylase activity, a polypeptide comprising a sequence having 60% or higher identity with the amino acid sequence of SEQ ID NO:2 and showing chitin transglycosylase activity, and a polypeptide comprising a sequence having 60% or higher identity with the amino acid sequence of SEQ ID NO:3 and showing chitin transglycosylase activity.

4. The microorganism according to 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 higher identity therewith.

6. The microorganism according to claim 1, which has an increased retinol secretion ability compared to a Yarrowia yeast microorganism with retinol production ability but without attenuated chitin transglycosylase activity.

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

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

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

10. The method according to claim 9, which includes the step of recovering retinol from the medium or the microorganism.

11. The method according to claim 9, wherein when extracting retinol, microbial cell disruption is not used or dodecane is not used as a solvent.

12. The method according to claim 9, wherein the retinol includes any one selected from the group consisting of retinol, retinal, retinoic acid, and retinyl ester.

13. A method for preparing a Yarrowia yeast microorganism with retinol production ability, the method comprising the step of attenuating the activity of chitin transglycosylase in a Yarrowia yeast microorganism with retinol production ability.

14. A method for increasing retinol secretion, the method comprising the step of attenuating the activity of chitin transglycosylase in a Yarrowia yeast microorganism with retinol production ability.

15. A composition for producing retinol, the composition comprising any one or more Yarrowia yeast microorganisms with retinol production ability, wherein the activity of chitin transglycosylase in the microorganisms is attenuated, and a culture thereof.

16. Use of a microorganism of the genus Yarrowia with retinol production ability in the production of retinol, wherein the activity of chitin transglycosylase in the microorganism is attenuated.

Images