A Yaroia microorganism for retinoid production with increased PHO84 protein activity and a method for producing retinoids using the same.

Abstract

This application relates to a Yarrowia microorganism having retinoid-producing ability in which the activity of the PHO84 protein is increased compared to its endogenous activity; a method for producing retinoids using the same; a method for producing a Yarrowia microorganism having retinoid-producing ability; and a composition for retinoid production.

Description

Technical Field

[0001] This application relates to a Yarrowia microorganism having retinoid-producing ability, in which the activity of the PHO84 protein is increased compared to the intrinsic activity; a method for producing retinoids using the same; a method for producing a Yarrowia microorganism having retinoid-producing ability; and a composition for retinoid production.

Background Art

[0002] Retinol, a fat-soluble vitamin, is an essential vitamin involved in improving night blindness, eye health, strengthening immunity, skin health, etc. Currently, it is produced and sold by chemical synthesis mainly by global leading companies, but research is underway to produce retinol based on microbial fermentation.

[0003] Therefore, although many technologies for stabilizing the retinol compound itself in compositions or products containing retinol have been developed (U.S. Patent Publication No. 6858217), the method for stably increasing retinol production is in a very limited state of development.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Non-Patent Documents

[0005]

Non-Patent Document 1

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

Non-licensed Document 7

Non-licensed Document 8

Non-licensed literature 9

Non-licensed literature 10

Non-licensed Document 11

[0006] The problem that this application aims to solve is to provide a Yarrowia microorganism having retinoid-producing ability in which the activity of the PHO84 protein is increased compared to its endogenous activity; a method for producing retinoids using the same; a method for producing a Yarrowia microorganism having retinoid-producing ability; and a composition for retinoid production. [Means for solving the problem]

[0007] One objective of this application is to provide a Yarrowia sp. microorganism with retinoid-producing ability in which the activity of the PHO84 protein is increased compared to its endogenous activity.

[0008] Another object of the present application is to provide a method for producing retinoids, which includes the step of culturing the microorganism of the present application in a medium.

[0009] Another object of the present application is to provide a method for producing a Yarrowia microorganism having retinoid-producing ability, which includes the step of increasing the activity of PHO84 protein in a Yarrowia microorganism having retinoid-producing ability compared to its intrinsic activity.

[0010] Another object of the present application is to provide a composition for producing retinoids, which includes one or more of the microorganism of the present application and its culture.

[0011] Another object of the present application is to provide the use of the microorganism of the present application or its culture for producing retinoids.

Advantages of the Invention

[0012] Retinoids can be produced using the microorganism of the present application.

Brief Description of the Drawings

[0013] [Figure 1] It is a diagram that schematizes the retinoid-producing ability of strains.

Modes for Carrying Out the Invention

[0014] Specifically, it is as follows. On the one hand, each explanation and embodiment disclosed in the present application is also applicable to each other explanation and embodiment. That is, all combinations of various elements disclosed in the present application belong to the scope of the present application. Also, it is not considered that the category of the present application is limited by the specific description described below. Also, a number of papers and patent documents are referred to throughout this specification, and their citations are indicated. The disclosure contents of the cited papers and patent documents are incorporated herein by reference in their entirety, and the level of the technical field to which the present application belongs and the content of the present application are more clearly explained.

[0015] One aspect of this application provides a Yarrowia sp. microorganism with retinoid-producing ability, in which the activity of the PHO84 (high-affinity inorganic phosphate (Pi) transporter PHO84) protein is increased compared to its endogenous activity.

[0016] The increased activity of the PHO84 protein can be measured, but is not limited to, by measuring the amount of the PHO84 protein or the polynucleotide encoding it, or by measuring the retinoid production capacity (or yield). For example, if the retinoid production capacity of the microorganism of this application increases compared to the retinoid production capacity of a natural wild-type or non-mutant Yarrowia microorganism (e.g., a Yarrowia microorganism expressing a wild-type polypeptide having the activity of the wild-type PHO84 protein (e.g., the polypeptide of SEQ ID NO: 1)), the increased activity of the PHO84 protein can be measured, but is not limited to, by measuring the increased retinoid production capacity.

[0017] The "PHO84 protein (high-affinity inorganic phosphate (Pi) transporter PHO84)" of this application is a type of inorganic phosphate transporter, and may function as both a high-affinity inorganic phosphate (Pi) transporter and a low-affinity manganese transporter.

[0018] The PHO84 protein of this application may contain one or more of the proteins YALI0A15125, YALI0A21307, and YALI0B19008. The YALI0A15125, YALI0A21307, and YALI0B19008 proteins can be used interchangeably with YALI0A15125p, YALI0A21307p, and YALI0B19008p, respectively.

[0019] The PHO84 protein of this application may include any PHO84 protein that increases retinoid production capacity.

[0020] As one example, the PHO84 protein of this application may increase the activity of endogenous or wild-type PHO84 proteins in microorganisms of the genus Yarrowia, thereby increasing the retinoid production capacity of said microorganisms.

[0021] As an example, the PHO84 protein of this application may contain, have, or be composed of an amino acid sequence having 80% or more homology or identity with one or more of SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 5, or substantially consist of said amino acid sequence.

[0022] As an example of the aforementioned embodiment, the YALI0A15125 protein (i.e., YALI0A15125p), YALI0A21307 protein (i.e., YALI0A21307p), and YALI0B19008 protein (i.e., YALI0B19008p) in the PHO84 protein of this application may contain, have, or be composed of, or substantially consist of, an amino acid sequence having 80% or more homology or identity with SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5.

[0023] For example, the amino acid sequence of the PHO84 protein of this application may, but is not limited to, being encoded by one or more of the YALI0A15125 gene (i.e., YALI0A15125g), the YALI0A21307 gene (i.e., YALI0A21307g), and the YALI0B19008 gene (i.e., YALI0B19008g). The amino acid sequence may, but is not limited to, be obtained from various databases, such as the NCBI's GenBank, which is a known database.

[0024] As an example, the PHO84 protein of this application may, but is not limited to, be derived from Yarrowia lipolytica.

[0025] Furthermore, although it is stated that one embodiment of the PHO84 protein of this application includes one or more of the sequences of SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 5, this does not exclude the addition of meaningless sequences before or after one or more of the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 5, or naturally occurring mutations, or silent mutations thereof. It is obvious to those skilled in the art that any protein having the same or corresponding activity as one or more of the sequences of SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 5 falls under the category of the PHO84 protein of this application.

[0026] For example, the PHO84 protein of this application may include, contain, be composed of, or substantially consist of amino acid sequences that have at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology or identity with one or more amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 5. Furthermore, it is obvious that any amino acid sequence that has the aforementioned homology or identity and exhibits the efficacy corresponding to the protein is included within the scope of this application, even if some sequences are deleted, modified, substituted, or added.

[0027] Even if this application describes a polypeptide (or protein) containing 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 obvious that proteins having amino acid sequences in which some sequences are deleted, modified, substituted, or added may also be used in this application, as long as they have the same or corresponding activity as the polypeptide or protein consisting of the amino acid sequence of the said sequence number. For example, this includes cases where the N-terminus and / or C-terminus of the amino acid sequence have additions of sequences that do not alter the function of the protein, naturally occurring mutations, silent mutations, or conservative substitutions.

[0028] The term "conservative substitution" refers to the replacement of one amino acid with another amino acid having similar structural and / or chemical properties. Such amino acid substitutions generally occur based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues. Typically, conservative substitutions have little to no effect on protein activity.

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

[0030] The homology or identity of sequences of conserved polynucleotides or polypeptides (including proteins) is determined by standard sequencing algorithms, which may also be used in conjunction with a default gap penalty established by the program used. Substantially homologous or identical sequences can generally be hybridized under moderate to high stringent conditions with the entire sequence or with portions representing at least about 50%, 60%, 70%, 80%, or 90% of the total length. It is obvious that hybridization also includes hybridization with polynucleotides containing codons in general or codons considering codon degeneracy in polynucleotides.

[0031] Whether any two polynucleotide or polypeptide (including protein) sequences are homologous, similar, or identical can be determined using known computer algorithms such as the "FASTA" program with default parameters, for example, as described in Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]:2444. Alternatively, it can be determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453), as performed in the Needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277) (version 5.0.0 or later) (GCG program package (Devereux, J., et al, Nucleic Acids Research 12:387 (1984)), BLASTP, BLASTN, FASTA (Atschul, [S.] [F.] [ET AL, J MOLEC BIOL 215]:403 (1990); Guide to Huge Computers, Martin J. Bishop, [ED.] Academic Press, San (Including Diego, 1994, and [CARILLO ETA / .](1988) SIAM J Applied Math 48:1073). For example, homology, similarity, or identity can be determined using BLAST or ClustalW from the National Center for Biotechnology Information Databases.

[0032] The homology, similarity, or identity of polynucleotides or polypeptides (including proteins) can be determined by comparing sequence information using a GAP computer program, such as Needleman et al. (1970), J Mol Biol. 48:443, as is publicly known, for example, in Smith and Waterman, Adv. Appl. Math (1981) 2:482. In summary, the GAP program can be defined as the total number of symbols in the shorter of two sequences divided by the number of similarly sequenced symbols (i.e., nucleotides or amino acids). Default parameters for the GAP program may include: (1) a binary comparison matrix (containing values ​​of 1 for identity and 0 for non-identity) and a weighted comparison matrix of Gribskov et al (1986) Nucl. Acids Res. 14:6745 (or EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix) as disclosed by Schwartz and Dayhoff, eds., Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, pp. 353-358 (1979); (2) a penalty of 3.0 for each gap and an additional penalty of 0.10 for each symbol in each gap (or a gap opening penalty of 10, a gap extension penalty of 0.5); and (3) no penalty for terminal gaps.

[0033] Furthermore, whether any two polynucleotide or polypeptide (including protein) sequences are homologous, similar, or identical can be confirmed by comparing the sequences in a Southern hybridization experiment under appropriate hybridization conditions, and such appropriate hybridization conditions are within the scope of the art and can be determined by methods well known to those skilled 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; FM Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York).

[0034] In this application, the term "polynucleotide" refers to a polymer of nucleotides in which nucleotide monomers are covalently linked together in a long chain, and is a DNA chain of a certain length or longer.

[0035] The polynucleotide sequence encoding the PHO84 protein of this application may be referred to as one or more sequences of the YALI0A15125 gene, the YALI0A21307 gene, and the YALI0B19008 gene, and may include a polynucleotide sequence encoding one or more of the amino acid sequences described in SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 5.

[0036] The polynucleotide may be subjected to various modifications in its coding region, within the limits that do not alter the amino acid sequence of the polypeptide or protein, due to codon degeneracy or taking into consideration the preferred codons in the organism that intends to express the polypeptide or protein. Specifically, the polynucleotide may consist of, but is not limited to, one or more of SEQ ID NOs: 2, SEQ ID NOs: 4, and SEQ ID NOs: 6, or a polynucleotide sequence having 80% or more homology or identity with them. For example, the polynucleotide may consist of a base sequence having 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 with one or more of SEQ ID NOs: 2, SEQ ID NOs: 4, and SEQ ID NOs: 6, and may also be a degenerate sequence thereof.

[0037] Furthermore, the polynucleotides of this application may include, without limitation, any probes, such as sequences that can hybridize under stringent conditions with a complementary sequence to all or part of the polynucleotide base sequence.

[0038] In this application, the term "stringent condition" means conditions that enable specific hybridization between polynucleotides. Such conditions are specifically described in the literature (e.g., J. Sambrook et al., ibid.). For example, the conditions may be such that polynucleotides with high homology or identity hybridize with each other, with 40% or more homology or identity, specifically 90% or more, more specifically 95% or more, 96% or more, 97% or more, 98% or more, and more specifically 99% or more, and do not hybridize with polynucleotides with lower homology or identity, or that the washing conditions are the same as those for normal Southern hybridization, with a salt concentration and temperature equivalent to 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, and the washing is performed once, specifically two to three times.

[0039] The aforementioned hybridization may occur between nucleotides having complementary sequences, but the hybridized polynucleotide may contain some mismatches between the bases depending on the stringency of the hybridization. The term “complementary” is used to describe the relationships between nucleotide bases that can hybridize with one another. For example, with respect to DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Thus, the polynucleotides of this application may also include not only substantially similar nucleic acid sequences, but also isolated nucleic acid fragments that are complementary throughout the sequence.

[0040] Specifically, polynucleotides having homology or identity can be detected using hybridization conditions that include a hybridization step at a Tm value of 55°C, and under the conditions described above. The Tm value may be 60°C, 63°C, or 65°C, but is not limited thereto and can be adjusted as appropriate by those skilled in the art.

[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] For example, homologous or identical polynucleotide sequences can generally be hybridized under stringent conditions by all or at least about 50%, 60%, 70%, 80%, or 90% of the entire sequence.

[0043] In this application, “vector” means a DNA product for delivering a target polynucleotide into a suitable host or host cell. For example, it may include, but is not limited to, a base sequence of a polynucleotide encoding a target polypeptide or protein, which is operably linked to a suitable regulatory region (or regulatory sequence) so as to enable expression of the target polypeptide or protein in a suitable host.

[0044] The expression regulatory region may include a promoter capable of initiating transcription, an optional operator sequence for regulating such transcription, a sequence encoding a suitable mRNA-ribosome binding site, and sequences regulating the termination of transcription and decoding. The vector, after being transformed in a suitable host cell (microorganism), can replicate or function independently of the host genome, or it can replicate or function after integration into the genome itself.

[0045] The vector of this application may be, but is not limited to, an insertion vector for inserting a polynucleotide into a chromosome to increase the activity of the PHO84 protein of this application. The insertion of the polynucleotide into the chromosome may be, but is not limited to, any method known in the art, such as homologous recombination. The vector may further include a selection marker for confirming the presence or absence of transformation of host cells or further the presence or absence of chromosome insertion into host cells. The selection marker is for selecting cells transformed with the vector or for confirming the presence or absence of chromosome insertion of the target polynucleotide, and markers that confer selectable phenotypes such as drug resistance, nutritional requirements, resistance to cytotoxic agents, or expression of surface polypeptides or proteins are used. Transformed cells can be selected so that only cells expressing the selection marker survive or exhibit other phenotypes in an environment treated with the selective agent. The insertion vector may not include origins of replication necessary for replication in transformed cells.

[0046] The vectors used in this application are not particularly limited, and any vector known in the art may be used. Examples of commonly used vectors include plasmids, cosmids, viruses, and bacteriophages in their natural or recombinant state. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A can be used as phage vectors or cosmid vectors, and pDZ, pBR, pUC, pBluescriptII, pGEM, pTZ, pCL, and pET can be used as plasmid vectors. Specifically, pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, and pCC1BAC vectors can be used.

[0047] In this application, the term "transformation" means altering the genetic traits of a host cell (microorganism) by introducing a target polynucleotide and / or a vector containing it into the host cell (microorganism). In this application, transformation may also mean altering the genetic traits of a host cell by introducing a vector containing a polynucleotide for increasing the activity of the PHO84 protein into the host cell. The transformed polynucleotide can be inserted into or located outside the chromosome of the host cell. The polynucleotide may also include DNA and / or RNA encoding the target protein (e.g., the PHO84 protein). The polynucleotide may be introduced in a form appropriate to 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 gene structure containing all the elements necessary for self-expression. The expression cassette may typically include a promoter operably linked to the coding sequence of the target polypeptide, a transcription termination signal, a ribosome binding site, and a translation termination signal. The expression cassette may also 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 to these forms.

[0048] In this application, the term “operatably linked” means a configuration in which a regulatory sequence is positioned appropriately so that the regulatory sequence controls the expression of a coding sequence. Accordingly, “operatably linked” includes a regulatory region of a functional domain known as a promoter, terminator, signal sequence, or enhancer region, or having a desired activity, being attached to or linked to a target (gene or polypeptide) so that the expression, secretion, or function of that target can be regulated by the known or desired activity. For example, it means that a promoter sequence that initiates and mediates the transcription of a polynucleotide encoding a polypeptide is functionally linked to the polynucleotide sequence.

[0049] The method for transforming the vector of this application includes any method for introducing nucleic acids into cells, and can be performed by selecting a suitable standard technique as is known in the art, using a host cell. Examples include, but are not limited to, electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, and lithium acetate-DMSO method.

[0050] In this application, the term "microorganism (or strain)" includes all wild-type microorganisms and prokaryotic or eukaryotic microorganisms that have undergone natural or artificial genetic modification, and which are microorganisms in which a particular mechanism has been weakened or increased due to causes such as the insertion of external genes or the increase or inactivation of endogenous gene activity, and may include microorganisms that undergo genetic modification for the production of a target polypeptide, protein, or product. In this application, the terms "microorganism," "strain," "host," and "host cell" are used interchangeably.

[0051] In this application, the term “recombinant microorganism” means a microorganism that has been genetically modified to exhibit a genotype and / or phenotype different from that of a naturally occurring microorganism (for example, when the genetic modification affects the nucleic acid sequence coding of the microorganism), and may include all offspring or potential offspring of such microorganism. In this application, the terms “recombinant microorganism,” “genetically modified microorganism,” “recombinant host cell,” “recombinant cell,” and “recombinant strain” are used interchangeably. Such recombinant microorganism may, for example, express genes not found in the natural (non-recombinant) form; or may not express genes that are expressed in the natural form, or may express natural genes in a manner different from that expressed in the natural form.

[0052] For example, the microorganism of this application may be, but is not limited to, a recombinant microorganism in which the activity of the PHO84 protein is increased compared to its endogenous activity.

[0053] The microorganisms of this application may be microorganisms that have the ability to produce retinoids. The term "microorganisms that have the ability to produce retinoids" may be used interchangeably with "microorganisms that produce retinoids."

[0054] The microorganisms of this application may be microorganisms into which polynucleotides encoding lycopene cyclase / phytoene synthase (crtYB) and phytoene desaturase (crtI) proteins have been introduced, so that microorganisms that do not inherently lack retinoid production ability acquire retinoid production ability, or so that the retinoid production ability of microorganisms that already have retinoid production ability is further increased, and which exhibit activity of these proteins or have increased activity of these proteins. The lycopene cyclase / phytoene synthase or phytoene desaturase may be proteins derived from Xanthophyllomyces dendrorhous, but are not limited to these as long as they are proteins that exhibit the same or similar activity. As a specific example, the lycopene cyclase / phytoen synthase or phytoendesaturase may consist of or contain the amino acid sequence of SEQ ID NO: 45 or SEQ ID NO: 47, respectively, but may also consist of or contain an amino acid sequence that exhibits activity corresponding to the lycopene cyclase / phytoen synthase or phytoendesaturase while 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 aforementioned amino acid sequence. Furthermore, it is obvious that proteins having the aforementioned homology or identity, while exhibiting activity corresponding to the lycopene cyclase / phytoen synthase or phytoendesaturase, are also included in the lycopene cyclase / phytoen synthase or phytoendesaturase if some sequences are deleted, altered, substituted, or added. As a specific example, the polynucleotide encoding the lycopene cyclase / phytoen synthase or phytoendesaturase may consist of or contain the sequence of SEQ ID NO: 46 or SEQ ID NO: 48, respectively.The polynucleotide may be subjected to various modifications in the coding region, taking into consideration the degeneracy of the codon or the codon preferred in the microorganism of this application, as long as the amino acid sequence is not altered. Specifically, the polynucleotide may consist of, but is not limited to, a base sequence having 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 99% or more homology or identity with the sequence of SEQ ID NO: 46 or SEQ ID NO: 48.

[0055] Furthermore, the microorganisms of this application may be microorganisms in which a polynucleotide encoding a β-carotene 15,15'-oxygenase (BLH) protein has been introduced into the microorganism so that a microorganism that does not inherently 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, thereby exhibiting β-carotene 15,15'-oxygenase activity, or microorganisms in which β-carotene 15,15'-oxygenase activity has been increased. The β-carotene 15,15'-oxygenase may be a protein derived from the marine bacterium 66A03 (Uncultured marine bacterium 66A03), but is not limited to this as long as it is a protein that exhibits the same or similar activity. As a specific example, the β-carotene 15,15'-oxygenase may consist of or contain the amino acid sequence of Sequence ID No. 49, but it may also consist of or contain an amino acid sequence that exhibits activity corresponding to the β-carotene 15,15'-oxygenase while 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 aforementioned amino acid sequence. Furthermore, it is obvious that proteins in which some sequences are deleted, modified, substituted, or added are also included in the β-carotene 15,15'-oxygenase, as long as they exhibit activity corresponding to the β-carotene 15,15'-oxygenase while having the aforementioned homology or identity. Furthermore, as a specific example, the polynucleotide encoding β-carotene 15,15'-oxygenase may have or contain the sequence of Sequence ID No. 50. The polynucleotide may be subjected to various modifications in the coding region, within the limits that do not alter the amino acid sequence, taking into consideration the degeneracy of the codon or the codon preferred in the microorganism of this application.Specifically, the polynucleotide may consist of, but is not limited to, a base sequence having 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 99% or more homology or identity with the sequence of SEQ ID NO: 50.

[0056] The CC08-2050 microorganism of this application (identical to KCCM13294P) may include a lycopene cyclase / phytoen synthase and / or a polynucleotide encoding it, a phytoendesaturase and / or a polynucleotide encoding it, and a β-carotene 15,15'-oxygenase and / or a polynucleotide encoding it, each containing the amino acid sequence of SEQ ID NO: 45.

[0057] As one example, the microorganism of this application may be a microorganism in which the activity of the PHO84 protein has increased and the retinoid production capacity has increased.

[0058] The microorganisms of this application may be microorganisms that naturally possess the ability to produce PHO84 protein or retinoids, or parent strains that possess the ability to produce PHO84 protein or retinoids, with further increases in the PHO84 protein of this application.

[0059] For example, the microorganisms of this application may include all microorganisms that increase the PHO84 protein of this application and are capable of producing retinoids.

[0060] For example, the microorganisms of this application may be natural wild-type microorganisms, microorganisms capable of producing retinoids, and / or recombinant strains of microorganisms containing the PHO84 protein in which the activity of the PHO84 protein of this application has increased, thereby increasing retinoid production capacity. The recombinant strains with increased retinoid production capacity may be, but are not limited to, natural wild-type microorganisms or microorganisms in which the activity of the PHO84 protein of this application has not increased.

[0061] For example, the microorganism whose PHO84 protein has not increased, which is the target strain for comparing the presence or absence of the increase in retinoid production capacity in this application, may be CC08-2050 (KCCM13294P, Ref. Park et al., Metabolic engineering 2022;73:26-37), but is not limited to this. The deposited strain name of the CJ2050 strain described in the aforementioned reference (Park et al., Metabolic engineering 2022;73:26-37) is the CC08-2050 strain of this application, and the CC08-2050 strain of this application and the CJ2050 strain are the same strain.

[0062] For example, recombinant strains and microorganisms with increased retinoid production capacity may have increased retinoid production capacity by approximately 1% or more, approximately 2% or more, approximately 5% or more, approximately 10% or more, approximately 20% or more, or approximately 30% or more (there is no special limit on the upper limit, and for example, it may be approximately 200% or less) compared to the retinoid production capacity of the parent strain or non-myxoid microorganism before mutation. In other examples, the increase may be approximately 1.01 times or more, approximately 1.02 times or more, approximately 1.05 times or more, approximately 1.07 times or more, approximately 1.1 times or more, approximately 1.2 times or more, or approximately 1.3 times or more (there is no special limit on the upper limit, and for example, it may be approximately 10 times or less). However, the increase is not limited to these, as long as it is a positive value increase compared to the production capacity of the parent strain or non-myxoid microorganism before mutation. The term "about" includes all ranges such as ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, and includes, but is not limited to, all numerical values ​​within a range equivalent to or similar to the numerical value following the term "about".

[0063] In this application, the term "non-myxoid microorganism (strain)" does not exclude microorganisms (strains) that include naturally occurring mutations, but rather means a wild-type microorganism (strain) or a naturally occurring microorganism (strain) itself, or a microorganism (strain) before its traits are altered by genetic mutations due to natural or artificial factors. For example, the non-myxoid microorganism refers to a strain in which the activity of the PHO84 protein of this application has not increased, or before it increases. The term "non-myxoid microorganism" is used interchangeably with "pre-deformation microorganism (strain)," "non-mutant microorganism (strain)," "parent microorganism," "parent strain," "wild-type microorganism (strain)," or "reference microorganism (strain)."

[0064] The microorganisms of this application may be, but are not limited to, microorganisms of the genus Yarouia.

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

[0066] In this application, the term "increase" of protein (polypeptide) activity means that the activity of a protein (polypeptide) within a host cell (microorganism) increases compared to its endogenous activity. This increase may be used interchangeably with terms such as activation, upregulation, overexpression, and enhancement.

[0067] The host cell (microorganism) may be a prokaryotic or eukaryotic microorganism.

[0068] The increase in the activity of the aforementioned protein (polypeptide) can include exhibiting protein (polypeptide) activity that was not inherently present in the host cell (microorganism), or exhibiting protein (polypeptide) activity that is improved compared to its intrinsic activity or activity before deformation.

[0069] For example, "showing activity of a protein (polypeptide) that was not intrinsically present" or "showing improved activity of a protein (polypeptide)" may be achieved by "introduction of a protein (polypeptide)," but is not limited to this. The introduction of the protein (polypeptide) may be achieved by introducing a gene encoding the protein (polypeptide) into the host cell (microorganism). For example, a polynucleotide encoding a specific protein (polypeptide) may be introduced into the chromosome of the host cell (microorganism), or a vector containing a polynucleotide encoding a specific protein (polypeptide) may be introduced into the host cell (microorganism) to show or improve its activity.

[0070] The aforementioned "intrinsic activity" refers to the activity of a specific protein (polypeptide) that was originally present in the host cell (microorganism) before transformation or in the non-transformed host cell (microorganism) when a trait has changed due to genetic mutation caused by natural or artificial factors. This can be confused with "activity before transformation."

[0071] An increase in protein (polypeptide) activity compared to its endogenous activity means that the activity and / or concentration (expression level) of the protein (polypeptide) in the host cell (microorganism) has improved compared to the activity and / or concentration (expression level) of the said protein (polypeptide) that was originally present in the host cell (microorganism) before transformation or in the non-transformed host cell (microorganism).

[0072] For example, the increase may indicate that the corresponding protein (polypeptide) was inactive, or that its activity or concentration was increased by approximately 1%, 10%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400%, or 500%, up to approximately 1000% or 2000%, relative to the activity or concentration in the host cell (microorganism) before transformation or in the non-transformed host cell (microorganism).

[0073] An increase in the activity of the aforementioned protein (polypeptide) can be achieved by introducing an exogenous protein (polypeptide) or by increasing the activity of an endogenous protein (polypeptide). Whether or not there has been an increase in the activity of the aforementioned protein (polypeptide) can be confirmed by an increase in the activity level, expression level, or the amount of products resulting from the activity of the aforementioned protein (polypeptide).

[0074] The increase in the activity of the aforementioned protein (polypeptide) can be achieved by various methods well known in the field, and is not limited as long as it can increase the activity of the target protein (polypeptide) compared to that of the host cell (microorganism) before deformation. Specifically, this may, but is not limited to, methods of gene engineering and / or protein engineering that are routine methods of molecular biology and are well known to ordinary technicians in the field (e.g., Sitnicka et al. Functional Analysis of Genes. Advances in Cell Biology. 2010, Vol. 2. 1-16, Sambrook et al. Molecular Cloning 2012, etc.).

[0075] Specifically, the increase in the activity of the protein (polypeptide) of this application is, 1) Increase in the intracellular copy number of polynucleotides that encode proteins (polypeptides); 2) Modification of gene expression regulatory regions on chromosomes that encode proteins (polypeptides) (e.g., introduction of mutations within the expression regulatory region, replacement with sequences having strong expression-inducing activity, or insertion of sequences having strong expression-inducing activity); 3) Modifications of the base sequence encoding the start codon or 5'-UTR of a gene transcription that codes for a protein (polypeptide); 4) Modification of the amino acid sequence of the protein (polypeptide) so as to increase the activity of the protein (polypeptide); 5) Modification of the polynucleotide sequence encoding the protein (polypeptide) so as to increase the activity of the protein (polypeptide) (for example, modification of the polynucleotide sequence of the protein (polypeptide) encoding gene so as to encode a protein (polypeptide) that has been modified to increase the activity of the protein (polypeptide); 6) Introduction of a foreign protein (polypeptide) or a foreign polynucleotide encoding a protein (polypeptide) that exhibits protein (polypeptide) activity; 7) Codon optimization of polynucleotides that code for proteins (polypeptides); 8) Analyze the tertiary structure of the protein (polypeptide), select exposed sites, and deform or chemically modify them; 9) Regulation of the intracellular localization of proteins (polypeptides); or 10) A combination of two or more selected from items 1) to 9) above is also acceptable, but is not particularly limited thereto.

[0076] for example, The increase in the intracellular copy number of the polynucleotide encoding the protein (polypeptide) described in 1) above may be achieved by introducing the polynucleotide encoding the protein (polypeptide), operably linked to an appropriate regulatory sequence, into a host cell (microorganism) in the form of a vector containing it. Alternatively, one or more copies of the polynucleotide encoding the protein (polypeptide), operably linked to an appropriate regulatory sequence, may be introduced into the chromosomes of the host cell (microorganism). The introduction into the chromosomes can be achieved, but is not limited to, by introducing into the host cell (microorganism) a vector that can insert the polynucleotide into the chromosomes of the host cell (microorganism). The vector is as described above. The regulatory sequence may be native to the encoding polynucleotide sequence (of the same origin), or a foreign (derived from another gene) sequence, or a variant thereof, or another artificial sequence, and may induce the expression of the polynucleotide in the host cell (microorganism).

[0077] 2) The replacement of a gene expression regulatory region (or expression regulatory sequence) on a chromosome encoding a protein (polypeptide) with a sequence having strong expression-inducing activity may, for example, involve introducing a mutation in the sequence by deletion, insertion, substitution, or a combination thereof, or replacing it with a sequence having stronger expression-inducing activity, so as to further increase the expression-inducing activity of the expression regulatory region. The expression regulatory region may include, but is not limited to, promoters, operator sequences, sequences encoding ribosome binding sites, and sequences that regulate transcription and decoding termination. As an example, the original promoter may be replaced with a promoter that exhibits strong expression-inducing activity, but is not limited to this.

[0078] Examples of known promoters exhibiting potent gene expression-inducing activity include, but are not limited to, the cj1-cj7 promoters (US 7662943 B2), the lac promoter, the trp promoter, the trc promoter, the tac promoter, the lambda phage PR promoter, the PL promoter, the tet promoter, the gapA promoter, the SPL7 promoter, the SPL13 (sm3) promoter (US 10584338 B2), the O2 promoter (US 10273491 B2), the tkt promoter, the yccA promoter, and the TEF promoter. As one example, the microorganism of this application may have the promoter of the gene encoding the PHO84 protein (e.g., YALI0A15125, YALIOA21307, and / or YALIOB19008 protein) replaced with the TEF promoter, but is not limited to this.

[0079] The modification of the start codon or the base sequence encoding the 5'-UTR of the gene encoding the protein (polypeptide) described in 3) above may, but is not limited to, substitution with another start codon that has a higher protein (polypeptide) expression rate than the endogenous start codon, or modification that encodes an RBS (ribosome binding site) sequence that has a higher protein (polypeptide) expression rate than the endogenous RBS (ribosome binding site) sequence.

[0080] The modification of the amino acid sequence or polynucleotide sequence of the protein (polypeptide) described in 4) and 5) above may be, but is not limited to, introducing a sequence mutation such as deletion, insertion, substitution, or combination thereof into the amino acid sequence or polynucleotide sequence encoding the protein (polypeptide) so as to increase the activity of the protein (polypeptide), or replacing it with an amino acid sequence or polynucleotide sequence modified to increase activity. The modification of the sequence may be, for example, carried out by inserting a polynucleotide of the modified sequence into the chromosome by homologous recombination, but is not limited to these methods.

[0081] The introduction of a foreign polynucleotide exhibiting protein (polypeptide) activity as described in 6) above may also be the introduction of a foreign polynucleotide encoding a protein (polypeptide) exhibiting the same or similar activity as the aforementioned protein (polypeptide) into a host cell (microorganism). The foreign polynucleotide is not restricted in its origin or sequence, as long as it exhibits the same or similar activity as the aforementioned protein (polypeptide). The method used for the introduction can be appropriately selected by those skilled in the art from known transformation methods, and the introduction of the polynucleotide in the host cell can generate a protein (polypeptide) and increase its activity.

[0082] The codon optimization of polynucleotides encoding proteins (polypeptides) described in 7) above can be used to increase the transcription or translation of endogenous polynucleotides within the host cell (microorganism), or to optimize the transcription and translation of exogenous polynucleotides within the host cell (microorganism).

[0083] 8) Analyzing the tertiary structure of a protein (polypeptide) and selecting exposed sites to deform or chemically modify may, for example, involve comparing the sequence information of the protein (polypeptide) to be analyzed with a database containing sequence information of known proteins to determine candidate template proteins according to the degree of sequence similarity, confirming the structure based on that, and selecting exposed sites to deform or chemically modify.

[0084] The intracellular positional regulation of a protein (polypeptide) described in 9) above may involve targeting the protein (polypeptide) to a specific organelle or specific intracellular space within the cell. For example, it may involve targeting the periplasm or cytoplasm through the addition or removal of a leader sequence that functions to target the protein (polypeptide), but is not limited thereto.

[0085] Such an increase in the activity of a protein (polypeptide) may be, but is not limited to, an increase in the activity or concentration of the corresponding protein (polypeptide) relative to the activity or concentration of the protein (polypeptide) expressed in the wild-type or pre-deformation host cell (microorganism).

[0086] Modification of some or all of the polynucleotides in the host cells (microorganisms) of this application may be induced by (a) homologous recombination using a chromosome insertion vector or genome editing using an engineered nuclease (e.g., CRISPR-Cas9) and / or (b) treatment with light such as ultraviolet light and radiation and / or chemical substances, but is not limited to these.

[0087] In this application, "retinoid" means chemically the vitamin A group or a group of compounds chemically related thereto.

[0088] As an example, the retinoid may be any one selected from the group consisting of retinol, retinal, retinoic acid, and retinyl esters, but is not limited thereto.

[0089] As an example, retinol can be converted to other retinoid compounds (e.g., retinal, retinoic acid, and retinyl esters) in ways known in the art.

[0090] Another aspect of this application provides a method for producing retinoids, comprising the step of culturing a Yarrowia microorganism having retinoid-producing ability in a culture medium, wherein the activity of the PHO84 protein is increased compared to its endogenous activity.

[0091] The aforementioned microorganisms are as described in other sections.

[0092] In this application, the term "culture" means growing the microorganisms of this application under appropriately controlled environmental conditions. In this application, the culture process can be carried out according to suitable culture media and culture conditions known in the art. Such a culture process can be easily adapted and used by those skilled in the art depending on the selected microorganisms. Specifically, the culture may be batch, continuous, and / or fed-batch.

[0093] The microorganisms of this application can be cultured in a conventional culture medium containing a suitable carbon source, nitrogen source, phosphorus source, inorganic compounds, amino acids and / or vitamins, under aerobic conditions while adjusting the temperature, pH, etc.

[0094] In this application, the carbon source may include carbohydrates such as glucose, sucrose, lactose, fructose, 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. In addition, natural organic nutrient sources such as starch hydrolysates, molasses, blackstrap molasses, rice bran, cassava, bagasse, and corn maceration can be used. Specifically, carbohydrates such as glucose and sterilized pre-treated molasses (i.e., molasses converted to reducing sugars) can be used, and other appropriate amounts of carbon sources can be used in a variety of ways without limitation. These carbon sources may be used alone or in combination of two or more, and are not limited to these uses.

[0095] The nitrogen sources may 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 like glutamic acid, methionine, and glutamine, peptone, NZ-amine, meat extracts, yeast extracts, malt extracts, corn maceration, casein hydrolysates, fish or their decomposition products, defatted soy cake or its decomposition products. These nitrogen sources may be used individually or in combination of two or more, and are not limited to these uses.

[0096] The phosphorus source may include monopotassium phosphate, dipotassium phosphate, or their corresponding sodium-containing salts. Inorganic compounds that can be used include sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, and calcium carbonate, and may also include amino acids, vitamins, and / or suitable precursors. These components or precursors may be added to the culture medium in batches or continuously, but are not limited to these methods.

[0097] During the cultivation of the microorganisms described in this application, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid can be added to the culture medium in an appropriate manner to adjust the pH of the culture medium. Furthermore, during cultivation, antifoaming agents such as fatty acid polyglycol esters can be used to suppress bubble formation. In addition, oxygen or oxygen-containing gas can be injected into the culture medium to maintain an aerobic state, or nitrogen, hydrogen, or carbon dioxide gas can be injected without gas injection to maintain an anaerobic and microaerobic state, but are not limited to these.

[0098] Furthermore, the culture medium may contain metal salts such as magnesium sulfate or iron sulfate necessary for growth. Finally, essential growth substances such as amino acids and vitamins are used in addition to the aforementioned substances. Appropriate precursors are also used in the culture medium. The raw materials may, but are not limited to, be added to the culture in a batch or continuous manner in an appropriate manner during the culture process.

[0099] This application allows for the adjustment of the culture's pH by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid to the culture in an appropriate manner during microbial cultivation. Furthermore, during cultivation, the formation of bubbles can be suppressed using an antifoaming agent such as fatty acid polyglycol ester. In addition, oxygen or oxygen-containing gas can be injected into the culture to maintain an aerobic state, or, to maintain anaerobic and microaerobic states, no gas can be injected, or nitrogen, hydrogen, or carbon dioxide gas can be injected, but this is not limited to these methods.

[0100] In the culture described in this application, the culture temperature can be maintained at 20-35°C, specifically 25-35°C, and the culture period can be continued until a useful amount of substance is obtained, and can be, but is not limited to, approximately 10-160 hours, approximately 20-130 hours, approximately 24-120 hours, approximately 36-120 hours, approximately 48-120 hours, approximately 48 hours or more, or approximately 48 hours, approximately 72 hours, or approximately 120 hours.

[0101] The retinoid production method of this application may further include recovering retinoids from the microorganism or culture medium.

[0102] The target retinoid can be recovered from the culture medium using appropriate methods known in the art, such as batch, continuous, or fed-batch culture methods, as described in this application. For example, various chromatography methods such as centrifugation, filtration, treatment with crystallizing protein precipitants (salting-out method), extraction, sonication, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, HPLC, and combinations thereof can be used, but are not limited to these examples.

[0103] The method may include an additional purification step. The purification step may be performed using a suitable method known in the art.

[0104] The method for producing retinoids according to this application may further include a step of converting retinol expressed by the microorganism of this application into a retinoid other than retinol. In the method for producing retinoids according to this application, the conversion step may further be included after the culturing step or the recovery step. The conversion step can be carried out using a suitable method known in the art. For example, the conversion can be carried out using retinol acyltransferase, but is not limited thereto.

[0105] As an example, the retinoid other than retinol may be any one selected from the group consisting of retinal, retinoic acid, and retinyl esters, but is not limited to these as long as it is included in the category of retinoids.

[0106] Another aspect of this application provides a method for producing retinoid-producing Yarouia microorganisms, comprising the step of increasing the activity of the PHO84 protein in the Yarouia microorganisms having retinoid-producing ability.

[0107] Another aspect of this application provides a method for increasing retinoid production in a Yarouia microorganism capable of producing retinoids, comprising the step of increasing the activity of the PHO84 protein.

[0108] The step of increasing the activity of the PHO84 protein may also be a step of deforming the Yaroia microorganism so that the activity of the PHO84 protein is increased compared to its endogenous activity, as described in other sections.

[0109] Another aspect of this application provides a composition for retinoid production comprising one or more Yarouia microorganisms and cultures thereof in which the activity of the PHO84 protein is increased compared to its endogenous activity.

[0110] The composition of this application may further contain any suitable excipients commonly used in compositions for retinoid production, such excipients may include, but are not limited to, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers, or isotonic agents.

[0111] The PHO84 protein, the Yarouia microorganisms with increased activity, and retinoids are described in other sections.

[0112] Another aspect of this application provides the use of a Yarouia microorganism or culture thereof, in which the activity of the PHO84 protein of this application is increased compared to its endogenous activity, for retinoid production.

[0113] The PHO84 protein, the Yarouia microorganisms with increased activity, and retinoids are described in other sections. [Examples]

[0114] The present application will be described in more detail below with reference to experimental examples. However, the following embodiments are merely preferred embodiments for illustrative purposes of the present application and are not intended to limit the scope of the rights of this application. On the other hand, technical matters not described herein can be fully understood and easily implemented by a person of ordinary skill who is skilled in the art of this application or a similar art.

[0115] Example 1. Production of a PHO84 (YALI0A15125) activity-enhanced strain of Yarrowia species that produces retinoids. In the Yarrowia strain KCCM13294P (CC08-2050, Park et al., Metab Eng. 2022, Jun 6;73:26-37), which produces retinoids, three PHO84 genes were identified: YALI0A15125, YALI0A21307, and YALI0B19008. To increase the expression of the YALI0A15125 gene, the promoter of the YALI0A15125 gene was replaced with the TEF promoter, which is an enhanced promoter. The sequence of the TEF promoter is as shown in Sequence ID No. 7.

[0116] To this end, the ORF sequence (SEQ ID NO: 2) of the YALI0A15125 gene was secured based on the nucleotide sequence registered in KEGG (Kyoto Encyclopedia of Genes and Genomes). Furthermore, a promoter exchange cassette was constructed using the URA3 gene (SEQ ID NO: 8) of Y. lipolytica as a selection marker. Using genomic DNA from CC08-2050 as a template, PCR was performed on the left homologous region, TEF promoter, URA3, repeat region, and right homologous region fragments using primers as shown in Table 1: SEQ ID NOs. 9 and 10, SEQ ID NOs. 11 and 12, SEQ ID NOs. 13 and 14, SEQ ID NOs. 15 and 16, and SEQ ID NOs. 17 and 18, 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, repeated 35 times. The resulting DNA fragments were then assembled into a single cassette via overlap extension PCR. The cassettes prepared in this manner were introduced into the CC08-2050 strain using the heat shock method (D.-C. Chen et al., Appl Microbiol Biotechnol, 1997), and colonies formed in uracil-free solid medium (YLMM1) were obtained. Cassette insertion within the genome was confirmed using primers of SEQ ID NO: 19 and SEQ ID NO: 20. The secured colonies were cultured in 5-FOA solid medium at 30°C for 3 days, and the URA3 marker was recovered by obtaining colonies grown in 5-FOA solid medium. The final strain with increased YALI0A15125 activity obtained through this process was named CC08-2483.

[0117] [Table 1]

[0118] The YLMM1 and 5-FOA media mentioned above were used with the following compositions.

[0119] <Yarrowia lipolytica 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

[0120] <5-Fluoroorotic Acid (5-FOA) Medium> 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-Fluororotic acid (5-FOA) 1 g / L, Agar 15 g / L

[0121] Example 2. Production of a retinoid-producing strain of the genus Yarrowia with increased PHO84 (YALI0A21307) activity. Among the three PHO84 genes identified in the retinoid-producing Yarouia strain KCCM13294P—YALI0A15125, YALI0A21307, and YALI0B19008—we decided to replace the promoter of the YALI0A21307 gene with the TEF promoter, an enhanced promoter, in order to increase the expression of the YALI0A21307 gene.

[0122] To this end, the ORF sequence (SEQ ID NO: 4) of the YALI0A21307 gene was secured based on the nucleotide sequence registered in KEGG (Kyoto Encyclopedia of Genes and Genomes). Furthermore, a promoter exchange cassette was constructed using the URA3 gene (SEQ ID NO: 8) of Y. lipolytica as a selection marker. Using genomic DNA from CC08-2050 as a template, PCR was performed on the left homologous region, TEF promoter, URA3, repeat region, and right homologous region fragments using primers as shown in Table 2: SEQ ID NOs. 21 and 22, 23 and 24, 25 and 26, 27 and 28, and 29 and 30, 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, repeated 35 times. The resulting DNA fragments were then assembled into a single cassette via overlap extension PCR. The cassettes prepared in this manner were introduced into the CC08-2050 strain using the heat shock method (D.-C. Chen et al., Appl Microbiol Biotechnol, 1997), and colonies formed in uracil-free solid medium (YLMM1) were obtained. Cassette insertion into the genome was confirmed using primers of SEQ ID NO: 31 and SEQ ID NO: 32. The secured colonies were cultured in 5-FOA solid medium at 30°C for 3 days, and the URA3 marker was recovered by obtaining colonies grown in 5-FOA solid medium. The final strain with increased YALI0A21307 activity obtained through this process was named CC08-2484.

[0123] [Table 2]

[0124] The YLMM1 and 5-FOA media used were those with the same composition as those used in Example 1.

[0125] Example 3. Production of a PHO84 (YALI0B19008) activity-enhanced strain of Yarrowia species that produces retinoids. In the retinoid-producing Yarouia strain KCCM13294P, three PHO84 genes were identified: YALI0A15125, YALI0A21307, and YALI0B19008. To increase the activity of the YALI0B19008 protein compared to its endogenous activity, the promoter of the YALI0B19008 gene was replaced with the TEF promoter, which is an enhanced promoter.

[0126] To this end, the ORF sequence (SEQ ID NO: 6) of the YALI0B19008 gene was secured based on the nucleotide sequence registered in KEGG (Kyoto Encyclopedia of Genes and Genomes). Furthermore, a promoter exchange cassette was constructed using the URA3 gene (SEQ ID NO: 8) of Y. lipolytica as a selection marker. Using genomic DNA from CC08-2050 as a template, PCR was performed on the left homologous region, TEF promoter, URA3, repeat region, and right homologous region fragments using primers as shown in Table 3: SEQ ID NOs: 33 and 34, 35 and 36, 37 and 38, 39 and 40, and 41 and 42, 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, repeated 35 times. The resulting DNA fragments were then assembled into a single cassette via overlap extension PCR. The cassette prepared in this manner was introduced into the CC08-2050 strain using the heat shock method (D.-C. Chen et al., Appl Microbiol Biotechnol, 1997), and colonies formed in a solid medium (YLMM1) without uracil were obtained. Cassette insertion into the genome was confirmed using primers of sequence number 43 and sequence number 44. The secured colonies were cultured in 5-FOA solid medium at 30°C for 3 days, and the URA3 marker was recovered by obtaining colonies grown in 5-FOA solid medium. The final strain with increased YALI0B19008 activity obtained through this process was named CC08-2506.

[0127] [Table 3]

[0128] The YLMM1 and 5-FOA media used were those with the same composition as those used in Example 1.

[0129] Example 4. Comparative evaluation of retinoid production capacity of strains with increased expression levels of three PHO84 genes. Flask evaluations were performed to compare the retinoid production capacity of the strains prepared through Examples 1-3 (CC08-2483, CC08-2484, and CC08-2506) with that of the control strain (CC08-2050). Each strain was inoculated into a 250 ml corner baffle flask containing 25 ml of YPDLU medium with 0.05% BHT (3,5-Di-tert-4-butylhydroxytoluene) and 5% Tween 20 to an initial OD=2, and incubated at 30°C and 200 rpm for 48 hours. The YPDLU medium used had the following composition.

[0130] <ypdlu> Glucose 40g / L, Bacto peptone 20g / L, Yeast extract 10g / L, Uracil 1g / L, Leucine 1g / L, 1M Phosphate buffer, pH 7.0 100ml / L

[0131] To evaluate the growth rate of each strain, the OD value was measured at a wavelength of 600 nm using a spectrophotometer. Retinol and retinal concentrations were quantitatively analyzed using HPLC after mixing 0.1 ml of culture medium with 0.9 ml of acetone (Sigma) containing 4% BHT following the completion of cultivation.

[0132] The analyzed OD values ​​and retinoid concentrations are shown in Table 4 below, and when visualized, they are shown in Figure 1.

[0133] [Table 4]

[0134] Comparing the results of the control group CC08-2050 and the experimental groups CC08-2483, CC08-2484, and CC08-2506 in Table 4, it was found that increased expression of the three PHO84 genes (CC08-2483, CC08-2484, CC08-2506) resulted in increased retinol and retinal concentrations in all groups compared to the control group (CC08-2050).

[0135] CC08-2483, which increased the activity of the YALI0A15125 protein, showed a 1.34-fold increase in retinol concentration and a 1.32-fold increase in retinal concentration compared to the control group. CC08-2484, which increased the activity of the YALI0A21307 protein, showed a 2.09-fold increase in retinol concentration and a 1.72-fold increase in retinal concentration compared to the control group. CC08-2506, which increased the activity of the YALI0B19008 protein, showed a 1.73-fold increase in retinol concentration and a 1.69-fold increase in retinal concentration compared to the control group.

[0136] The results above confirm that retinoid production capacity can be increased in microorganisms of the genus Yarrowia through increased activity of the PHO84 protein.

[0137] From the above description, a person skilled in the art to which this application pertains will understand that this application can be implemented in other specific forms without altering its technical idea or essential features. In this regard, it should be understood that the embodiments described above are merely illustrative and not limiting. The scope of this application should be interpreted as encompassing all modified or altered forms derived from the meaning and scope of the claims, as described below, and their equivalent concepts, rather than from the above detailed description.

[0138] [Table 5] < / ypdlu>

Claims

1. A Yarrowia sp. microorganism with retinoid production ability, exhibiting increased activity of the PHO84 (high-affinity inorganic phosphate transporter PHO84) protein compared to its endogenous activity.

2. The microorganism according to claim 1, wherein the PHO84 protein comprises one or more proteins selected from YALI0A15125, YALI0A21307, and YALI0B19008.

3. The microorganism according to claim 1, wherein the PHO84 protein contains one or more of the sequences of Sequence ID No. 1, Sequence ID No. 3, and Sequence ID No. 5, or an amino acid sequence having 80% or more identity therewith.

4. The microorganism according to claim 1, wherein the PHO84 protein is derived from Yarrowia lipolytica.

5. The microorganism according to claim 1, wherein the PHO84 protein is encoded by one or more of SEQ ID NOs: 2, SEQ ID NOs: 4, and SEQ ID NOs: 6, or by a polynucleotide sequence having 80% or more identity thereto.

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

7. The microorganism according to claim 1, wherein the microorganism has increased retinoid production capacity compared to non-mutated Yarrowia microorganisms.

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

9. A method for producing retinoids, comprising the step of culturing a Yarrowia microorganism having retinoid-producing ability in which the activity of the PHO84 protein is increased compared to its endogenous activity, in a culture medium.

10. The retinoid production method according to claim 9, comprising the step of recovering a retinoid from the cultured medium or microorganism.

11. The method for producing a retinoid according to claim 9, wherein the retinoid comprises one or more selected from the group consisting of retinol, retinal, retinoic acid, and retinyl esters.

12. A method for producing a retinoid-producing microorganism of the genus Yarrowia, comprising the step of increasing the activity of the PHO84 protein in the Yarrowia microorganism having retinoid-producing ability.

13. A composition for retinoid production comprising one or more Yarrowia sp. microorganisms and their cultures that have retinoid-producing ability, wherein the activity of the PHO84 protein is increased compared to its endogenous activity.

14. Use of Yarrowia sp. microorganisms or cultures thereof, in which the activity of the PHO84 protein is increased compared to its endogenous activity, for retinoid production.

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