Retinol-producing microorganisms of the genus Yarrowia with reduced ABC3 protein activity and a method for producing retinol using the same.

Abstract

This application relates to a Yarrowia microorganism having retinol-producing ability in which the activity of the ABC3 protein is reduced compared to its endogenous activity; a method for producing retinol using the same; a method for producing a Yarrowia microorganism having retinol-producing ability; a method for increasing the retinol excretion of microorganisms; and a composition for retinol production.

Description

Technical Field

[0001] This application relates to a Yarrowia microorganism having the ability to produce retinol, in which the activity of the ABC3 protein is reduced compared to the intrinsic activity; a method for producing retinol using the same; a method for producing a Yarrowia microorganism having the ability to produce retinol; a method for increasing the retinol excretion of a microorganism; and a composition for producing retinol.

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, and research is underway to produce retinol based on microbial fermentation. However, different from the technology of stabilizing the retinol compound itself in a composition or product containing retinol (U.S. Patent Publication No. 6858217), the technology of stably producing retinol is in a very limited state of development.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Non-Patent Documents

[0004]

Non-Patent Document 1

Non-Patent Document 2

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

Non-licensed Document 7

Non-licensed literature 9

Non-licensed literature 10

Non-licensed Document 11

[0005] The problems to be solved by the present application are: a Yarrowia sp. microorganism having the ability to produce retinol, in which the activity of the ABC3 protein is decreased compared to the intrinsic activity; a method for producing retinol using the same; a method for producing a Yarrowia sp. microorganism having the ability to produce retinol; a method for increasing the retinol excretion of a microorganism; and a composition for producing retinol.

Means for Solving the Problems

[0006] One aspect of the present application provides a Yarrowia sp. microorganism having the ability to produce retinol, in which the activity of the ABC3 protein is decreased compared to the intrinsic activity.

[0007] Another aspect of the present application provides a method for producing retinol, which includes culturing the microorganism of the present application in a medium.

[0008] Another aspect of the present application provides a method for producing a Yarrowia sp. microorganism having the ability to produce retinol and a method for increasing retinol excretion, which includes decreasing the activity of the ABC3 protein in a Yarrowia sp. microorganism having the ability to produce retinol compared to the intrinsic activity.

[0009] Another aspect of the present application provides a composition for producing retinol, which includes any one or more of the microorganism of the present application and its culture.

[0010] Another aspect of the present application provides the use of the microorganism of the present application or its culture for producing retinol.

Effects of the Invention

[0011] Retinol can be produced using the microorganism of the present application.

Brief Description of the Drawings

[0012] [Figure 1] It is a diagram schematically showing the retinol production ability of strains.

Modes for Carrying Out the Invention

[0013] This can be explained in more detail as follows: On the other hand, each description and embodiment disclosed in this application applies to each other description and embodiment. That is, all combinations of the various elements disclosed in this application fall within the scope of this application. Furthermore, the categories of this application are not limited by the specific descriptions described below. In addition, numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated as references in this specification as a whole, to more clearly explain the level of the technical field to which this application belongs and the content of this application.

[0014] One aspect of this application provides a Yarrowia sp. microorganism capable of producing retinol, in which the activity of the ABC3 (ATP-binding cassette transporter type 3) protein is reduced compared to its endogenous activity.

[0015] The reduced activity of the ABC3 protein can be measured by measuring the amount of the ABC3 protein or the polynucleotide encoding it, or by measuring the retinol production capacity (or yield), but is not limited to these methods. For example, if the retinol production capacity of the microorganism of this application increases compared to the retinol production capacity of a natural wild-type or non-mutant Yarrowia microorganism (e.g., a Yarrowia microorganism expressing a wild-type polypeptide having ABC3 protein activity (e.g., the polypeptide of SEQ ID NO: 1)), the reduced activity of the ABC3 protein can be measured by measuring the increased retinol production capacity, but is not limited to these methods.

[0016] In this application, "ABC3 protein (ATP-binding cassette transporter 3 protein)" refers to type 3 of the ABC protein family. ABC proteins are a type of ATP-binding cassette transporter protein and may also be used interchangeably with ABC transporters. The ABC protein family includes types 1, 2, and 3, of which the type 3 ABC3 protein originates from a precursor having four transmembrane segments (TMS), and may have four, eight, or ten TMS.

[0017] The ABC3 protein of this application may also contain the YALI0B02544 protein. The YALI0B02544 protein may be used interchangeably with YALI0B02544p.

[0018] The ABC3 protein of this application may include any ABC3 protein that increases retinol production and / or efflux capacity. For example, the increase in retinol production capacity may be due to an increase in retinol efflux capacity.

[0019] As one example, the ABC3 protein of this application may have reduced activity in microorganisms of the genus Yarrowia compared to endogenous or wild-type ABC3 proteins, thereby increasing the retinol production and / or efflux capacity of said microorganisms.

[0020] As an example, the ABC3 protein of this application may contain, have, or be composed of an amino acid sequence having 60% or more homology or identity with SEQ ID NO: 1, or substantially consist of said amino acid sequence.

[0021] For example, the amino acid sequence of the ABC3 protein of this application may, but is not limited to, the sequence encoded by the YALI0B02544 gene (i.e., YALI0B02544g). The amino acid sequence may, but is not limited to, the sequence obtained from various databases, such as the NCBI's GenBank, which is a known database.

[0022] As an example, the ABC3 protein of this application may, but is not limited to, be derived from Yarrowia lipolytica.

[0023] Furthermore, although one embodiment of the ABC3 protein of this application is described as a protein containing the sequence of SEQ ID NO: 1, this does not exclude the addition of meaningless sequences before or after the amino acid sequence of SEQ ID NO: 1, or naturally occurring mutations, or silent mutations thereof. It will be obvious to those skilled in the art that any protein having the same or corresponding activity as the protein containing the aforementioned amino acid sequence will qualify as the ABC3 protein of this application.

[0024] For example, the ABC3 protein of this application may include the amino acid sequence of SEQ ID NO: 1, or may include, have, constitute, or substantially consist of an amino acid sequence having at least 60%, 62%, 63%, 64%, 62%, 65%, 70%, 75%, 76%, 77%, 78%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology or identity with the amino acid sequence of SEQ ID NO: 1. Furthermore, it is obvious that any amino acid sequence having the aforementioned homology or identity and exhibiting the efficacy corresponding to the protein is included within the scope of this application, even if some sequences are deleted, modified, substituted, or added.

[0025] As one example, the microorganism of this application may further have increased SNQ2 protein activity compared to its endogenous activity.

[0026] The increased activity of the SNQ2 protein may be measured by measuring the amount of the SNQ2 protein or the polynucleotide encoding it, or by measuring the retinol production capacity (or yield), but is not limited thereto. For example, if the activity of the ABC3 protein of this application decreases, but the activity of the SNQ2 protein does not increase compared to the endogenous activity, or if the retinol production capacity of a microorganism with decreased ABC3 protein activity and increased SNQ2 protein activity increases compared to the retinol production capacity of the Yarrowia microorganism before the increase, the increased activity of the SNQ2 protein can be measured by measuring the increased retinol production capacity, but is not limited thereto.

[0027] The "SNQ2 protein (ATP-binding cassette transporter protein SNQ2)" of this application is a type of ATP-binding cassette transporter protein, and may be a plasma membrane ABC transporter or a multidrug transporter.

[0028] The SNQ2 protein of this application may also contain the YALI0F17996 protein, and the YALI0F17996 protein may be used interchangeably with YALI0F17996p.

[0029] As one example, the SNQ2 protein of this application may increase the activity of endogenous or wild-type SNQ2 proteins in microorganisms of the genus Yarrowia, thereby increasing the retinol production and / or efflux capacity of said microorganisms.

[0030] As an example, the SNQ2 protein of this application may contain, have, or be composed of an amino acid sequence having 60% or more homology or identity with SEQ ID NO: 33, or substantially consist of said amino acid sequence.

[0031] For example, the amino acid sequence of the SNQ2 protein of this application may be encoded by the YALI0F17996 gene (i.e., YALI0F17996g), but is not limited thereto. The amino acid sequence can be obtained from various databases, such as the NCBI's GenBank, which is a known database, but is not limited thereto.

[0032] As an example, the SNQ2 protein of this application may, but is not limited to, be derived from Yarrowia lipolytica.

[0033] Furthermore, although one embodiment of the SNQ2 protein of this application is described as a protein containing SEQ ID NO: 33, this does not exclude the addition of meaningless sequences before or after the amino acid sequence of SEQ ID NO: 33, or naturally occurring mutations, or silent mutations thereof. It will be obvious to those skilled in the art that any protein having the same or corresponding activity as the protein containing the aforementioned amino acid sequence will qualify as the SNQ2 protein of this application.

[0034] For example, the SNQ2 protein of this application may include the amino acid sequence of SEQ ID NO: 33, or may include, have, constitute, or substantially consist of an amino acid sequence having at least 60%, 62%, 63%, 64%, 62%, 65%, 70%, 75%, 76%, 77%, 78%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology or identity with the amino acid sequence of SEQ ID NO: 33. Furthermore, it is obvious that any amino acid sequence having the aforementioned homology or identity and exhibiting the efficacy corresponding to the protein is included within the scope of this application, even if some sequences are deleted, modified, substituted, or added.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] 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).

[0042] 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.

[0043] The polynucleotide sequence encoding the ABC3 protein of this application may be referred to as the YALI0B02544 gene sequence, which may include a polynucleotide sequence or a degenerate sequence thereof encoding the amino acid sequence described in Sequence ID No. 1.

[0044] The polynucleotide sequence encoding the SNQ2 protein of this application may be referred to as the YALI0F17996 gene sequence, which may include a polynucleotide sequence or a degenerate sequence thereof encoding the amino acid sequence described in Sequence ID No. 33.

[0045] The polynucleotide encoding the SNQ2 protein of this application may be subjected to various modifications in the coding region, either due to codon degeneracy or taking into consideration the preferred codons in the organism in which the SNQ2 polypeptide or protein is to be expressed, as long as these modifications do not alter the amino acid sequence of the polypeptide or protein.

[0046] Specifically, the polynucleotide encoding the ABC3 protein may consist of, or substantially consist of, a polynucleotide sequence having 60% or more homology or identity with SEQ ID NO: 2. For example, the polynucleotide encoding the ABC3 protein may consist of, but is not limited to, a base sequence having 60% or more homology or identity with SEQ ID NO: 2, 62% or more, 63% or more, 64% or more, 62% or more, 65% or more, 70% or more, 76% or more, 77% or more, 78% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homology or identity with SEQ ID NO: 2.

[0047] The polynucleotide encoding the SNQ2 protein may consist of, but is not limited to, a polynucleotide sequence having 60% or more homology or identity with SEQ ID NO: 34 or a polynucleotide sequence having 60% or more homology or identity with it. For example, the polynucleotide encoding the SNQ2 protein may consist of, but is not limited to, a base sequence having 60% or more homology or identity with SEQ ID NO: 34, 62% or more, 63% or more, 64% or more, 62% or more, 65% or more, 70% or more, 76% or more, 77% or more, 78% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homology or identity with it.

[0048] 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.

[0049] 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 process may involve hybridizing polynucleotides with high homology or identity, specifically those with 40% or more homology or identity, more specifically 90% or more, more specifically 95% or more, 96% or more, 97% or more, 98% or more, and even more specifically 99% or more homology or identity, with polynucleotides having lower homology or identity than those individuals, and not hybridizing with other polynucleotides. Alternatively, the process may involve washing once, specifically two to three times, at a salt concentration and temperature equivalent to the washing conditions of a typical Southern hybridization: 60°C, 1×SSC, 0.1% SDS, more specifically 60°C, 0.1×SSC, 0.1% SDS, more specifically 68°C, 0.1×SSC, 0.1% SDS.

[0050] 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.

[0051] 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.

[0052] 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.).

[0053] 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.

[0054] 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.

[0055] 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.

[0056] The vector of this application may be, but is not limited to, an insertion vector for inserting a polynucleotide into a chromosome to reduce the activity of the ABC3 protein of this application and / or an insertion vector for inserting a polynucleotide into a chromosome to increase the activity of the SNQ2 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 does not need to contain the origin of replication necessary for replication within the transformed cells.

[0057] 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.

[0058] 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 polynucleotide for reducing the activity of the ABC3 protein or a vector containing a polynucleotide for further increasing the activity of the SNQ2 protein into the host cell. The transformed polynucleotide can be inserted into the chromosome of the host cell or located outside the chromosome. For example, the vector containing the polynucleotide for reducing the activity of the ABC3 protein may be an insertion vector for deleting part or all of the YALI0B02544 gene by being inserted into the chromosome through homologous recombination. The polynucleotide may also include DNA and / or RNA that encode a protein. The polynucleotide can be introduced in a form appropriate to the purpose of introduction. For example, the polynucleotide for increasing the activity of the SNQ2 protein can be introduced into the host cell in the form of an expression cassette, which is a gene structure containing all the elements necessary for its expression. The expression cassette may typically include a promoter operably linked to the coding sequence of the SNQ protein, 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. The polynucleotide may also be introduced into a host cell in its own form and operably linked to the sequence required for expression in the host cell, but is not limited to these.

[0059] 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.

[0060] 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.

[0061] 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 reduced due to causes such as the insertion of external genes or the reduction or inactivation of the activity of endogenous genes, and may be microorganisms that have undergone genetic modification for the production of the target polypeptide, protein, or product. In this application, the terms "microorganism," "strain," "host," and "host cell" are used interchangeably.

[0062] 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.

[0063] For example, the microorganism of this application may be one or more recombinant microorganisms in which the expression of the gene encoding the ABC3 protein is reduced and the activity of the ABC3 protein is reduced compared to its endogenous activity; or recombinant microorganisms in which the expression of the gene encoding the SNQ2 protein is increased and the activity of the SNQ2 protein is increased compared to its endogenous activity.

[0064] The microorganisms of this application may be microorganisms that have the ability to produce retinol. The term "microorganisms that have the ability to produce retinol" may be used interchangeably with "microorganisms that produce retinol."

[0065] 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 have the ability to produce retinol acquire the ability to produce retinol, or so that the retinol production ability of microorganisms that do have the ability to produce retinol is further increased, and which exhibit the 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: 27 or SEQ ID NO: 29, 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, modified, 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 Sequence ID No. 28 or Sequence ID No. 30, 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: 28 or SEQ ID NO: 30.

[0066] 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 microorganisms that do not inherently have the ability to produce retinol have the ability to produce retinol, or so that the retinol production ability of microorganisms that do have the ability to produce retinol is further increased, 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 SEQ ID NO: 31, 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. 32. 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: 32.

[0067] The CC08-2050 microorganism of this application (identical to KCCM13294P) may include a lycopene cyclase / phytoen synthase and / or a polynucleotide encoding it, containing the amino acid sequence of SEQ ID NO: 27, a phytoendesaturase and / or a polynucleotide encoding it, containing the amino acid sequence of SEQ ID NO: 29, and a β-carotene 15,15'-oxygenase and / or a polynucleotide encoding it, containing the amino acid sequence of SEQ ID NO: 31.

[0068] As one example, the microorganism of this application may be a microorganism in which the activity of the ABC3 protein is reduced and the retinol production and / or excretion capacity is increased.

[0069] Microorganisms in which the activity of the ABC3 protein is reduced according to this application may, but are not limited to, those in which the retinol efflux capacity is increased compared to non-mutated Yarrowia microorganisms in which the activity of the ABC3 protein is not reduced.

[0070] As one example of the aforementioned embodiment, the microorganism may further have increased SNQ2 protein activity compared to its endogenous activity, but is not limited to this.

[0071] Microorganisms in which the activity of the ABC3 protein is reduced and the activity of the SNQ2 protein is increased may, but are not limited to, microorganisms in which the activity of the ABC3 protein is reduced and the activity of the SNQ2 protein is not increased, exhibit increased retinol efflux capacity.

[0072] The increase in retinol production in the microorganism of this application may be due to an increase in retinol efflux capacity, but is not limited thereto.

[0073] The microorganisms of this application may, but are not limited to, selectively excreting retinol.

[0074] The microorganisms of this application may selectively excrete retinol from among retinal and retinol.

[0075] The microorganisms of this application may be microorganisms that naturally possess the ability to produce ABC3 protein or retinol, or parent strains that have the ability to produce ABC3 protein or retinol but in which the ABC3 protein of this application has been reduced.

[0076] For example, the microorganisms of this application may include all microorganisms in which the ABC3 protein of this application is reduced and which are capable of producing retinol.

[0077] For example, the microorganisms of this application may be natural wild-type microorganisms, microorganisms capable of producing retinol, and / or recombinant strains of microorganisms containing ABC3 protein in which the activity of ABC3 protein is reduced and retinol production and / or efflux capacity is increased. The recombinant strains with increased retinol production and / or efflux capacity may be, but are not limited to, natural wild-type microorganisms or microorganisms in which the activity of ABC3 protein of this application is not reduced and retinol production and / or efflux capacity is increased.

[0078] As an example, the target strain used to compare whether or not there is an increase in retinol production and / or excretion capacity, in which the activity of the ABC3 protein of this application does not decrease and the activity of the SNQ2 protein does not increase, may be, but is not limited to, CC08-2050 (KCCM13294P, Ref. Park et al., Metabolic engineering 2022;73:26-37). 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.

[0079] For example, recombinant strains and microorganisms with increased retinol production and / or efflux capacity may have increased retinol 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), or in other examples, 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), but are not limited to these, as long as the increase is a positive value compared to the production capacity of the parent strain or non-myxic 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".

[0080] In this application, the term "non-myxoid microorganism (strain)" means a microorganism (strain) that is either wild-type or naturally occurring, or a microorganism (strain) before its traits are altered by genetic mutations due to natural or artificial factors, and does not exclude microorganisms (strains) that include naturally occurring mutations. For example, the non-myxoid microorganism means, but is not limited to, a microorganism (strain) in which the ABC3 protein of this application is not reduced or is before it is reduced. In this application, the term "non-myxoid microorganism (strain)" is used interchangeably with "pre-deformation microorganism (strain)," "non-mutant microorganism (strain)," "parent microorganism," "parent strain," "wild-type microorganism (strain)," or "reference microorganism (strain)."

[0081] The microorganisms of this application may be, but are not limited to, microorganisms of the genus Yarouia.

[0082] As an example, the microorganism of the genus Yarrowia in this application may be, but is not limited to, Yarrowia lipolytica.

[0083] In this application, the term "reduction" of protein (polypeptide) activity encompasses both a decrease in protein (polypeptide) activity compared to its endogenous activity within a host cell (microorganism) or inactivation.

[0084] In other words, the decrease in the activity of the protein (polypeptide) can include a state in which the protein (polypeptide) is not completely inactivated within the host cell (microorganism), and the protein (polypeptide) exhibits reduced activity compared to its endogenous activity or activity before deformation; or it can include the complete inactivation of the protein (polypeptide).

[0085] For example, the aforementioned decrease may include cases where the activity of the protein (polypeptide) is lower or absent compared to the protein (polypeptide) present in the host cell (microorganism) before the phenotype change or in an unmutated host cell (microorganism) due to mutations in the polynucleotide encoding the protein (polypeptide), cases where the overall level of protein (polypeptide) expression within the cell is lower compared to the host cell (microorganism) before the phenotype change or in an unmutated host cell (microorganism) due to inhibition of the expression of the polynucleotide or protein (polypeptide), cases where the expression of the polynucleotide or protein (polypeptide) is not occurring at all, and cases where the activity of the protein (polypeptide) is low or absent even if the protein (polypeptide) is expressed normally.

[0086] The host cell (microorganism) may be a prokaryotic or eukaryotic microorganism.

[0087] A decrease 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 decreased 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).

[0088] Whether or not the activity of the protein (polypeptide) has decreased can be confirmed by an increase in the activity level, expression level, or amount of products resulting from the activity of the protein (polypeptide).

[0089] 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."

[0090] The microorganisms of this application may have improved retinoid production and / or efflux capabilities due to the weakening of the activity of the ABC3 protein.

[0091] The reduction 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 reduce 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., 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.).

[0092] Specifically, the decrease in the activity of the protein (polypeptide) of this application is, 1) Deletion of all or part of the genes that code for proteins (polypeptides); 2) Modification of gene expression regulatory regions on chromosomes that encode proteins (polypeptides) (e.g., introduction of mutations within the regulatory region, replacement with sequences having repressive activity, or insertion of sequences having repressive activity); 3) Modification of the amino acid sequence of a protein (polypeptide) such that its activity is reduced (e.g., deletion / substitution / addition of one or more amino acids in the amino acid sequence); 4) Modification of the polynucleotide sequence encoding the protein (polypeptide) such that the activity of the protein (polypeptide) is reduced (for example, modification of the polynucleotide sequence of the protein (polypeptide) encoding the modified protein (polypeptide) such that the activity of the protein (polypeptide) is reduced); 5) Modifications of the base sequence encoding the start codon or 5'-UTR region of a gene encoding a protein (polypeptide); 6) Introduction of an antisense oligonucleotide (e.g., antisense RNA) that binds complementarily to the transcript of the gene encoding the protein (polypeptide); 7) Addition of a sequence complementary to the Shine-Dalgarno sequence to the Shine-Dalgarno sequence of a protein (polypeptide) encoding gene in order to form a secondary structure, such that ribosome attachment is impossible; 8) Addition of a promoter that is transcribed in the opposite direction to the 3' end of the ORF (open reading frame) of a polynucleotide sequence encoding a protein (polypeptide) (Reverse transcription engineering, RTE); 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.

[0093] for example, The deletion of all or part of the gene encoding the protein (polypeptide) described in 1) above can be carried out by methods utilizing homologous recombination via a chromosome insertion vector in a microorganism, or by using electromagnetic waves such as ultraviolet light, X-rays, gamma rays, or chemical substances, but is not limited thereto.

[0094] Furthermore, the replacement of the gene expression regulatory region on the chromosome encoding the protein (polypeptide) with a sequence having repressive activity may, for example, involve introducing a mutation in the gene expression regulatory region by deletion, insertion, substitution, or a combination thereof, or further replacing it with a sequence having repressive activity, so as to reduce the gene expression induction activity of the gene expression regulatory region. The gene expression regulatory region may include, but is not limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence that regulates the termination of transcription and decoding.

[0095] Furthermore, the modification of the amino acid sequence or polynucleotide sequence of the protein (polypeptide) described in 3) and 4) 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 reduce the activity of the protein (polypeptide), or replacing it with an amino acid sequence or polynucleotide sequence modified to reduce its activity. The modification of the sequence can be, for example, carried out by inserting a polynucleotide of a modified sequence into the chromosome by homologous recombination, but is not limited to these methods. In one specific example, the protein (polypeptide) may be inactivated by introducing a mutation into the polynucleotide sequence encoding the protein (polypeptide) to form a termination codon, but is not limited to this method.

[0096] Furthermore, the modification of the start codon or the base sequence encoding the 5'-UTR of the gene encoding the protein (polypeptide) may, for example, be a substitution with another start codon that results in a lower protein (polypeptide) expression rate compared to the endogenous start codon, or a modification that encodes an RBS sequence that results in a lower protein (polypeptide) expression rate compared to the endogenous RBS (ribosome binding site) sequence, but is not limited to these.

[0097] The introduction of an antisense oligonucleotide (e.g., antisense RNA) that binds complementarily to the transcript of the gene encoding the protein (polypeptide) can be carried out, for example, by referring to the literature [Weintraub, H. et al., Antisense-RNA as a molecular tool for genetic analysis, Reviews - Trends in Genetics, Vol. 1(1) 1986], but is not limited thereto.

[0098] 7) The addition of a sequence complementary to the Shine-Dalgarno sequence before the Shine-Dalgarno sequence of a gene encoding a protein (polypeptide) to form a secondary structure, which may, but is not limited to, making mRNA translation impossible or slowing it down, so that ribosome attachment is impossible.

[0099] 8) The addition of a promoter that is transcribed in the opposite direction to the 3' end of the ORF (open reading frame) of the polynucleotide sequence encoding the protein (polypeptide) (reverse transcription engineering, RTE) may be such that complementary antisense nucleotides are created in the transcript of the gene encoding the polypeptide, thereby suppressing the translation of the protein (polypeptide) and reducing its activity.

[0100] 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.

[0101] Such a decrease in the activity of a protein (polypeptide) may be, but is not limited to, a decrease 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).

[0102] 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.

[0103] As an example, the microorganism of this application may, but is not limited to, having a partial or complete deletion of the gene sequence encoding the ABC3 protein.

[0104] 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.

[0105] The host cell (microorganism) may be a prokaryotic or eukaryotic microorganism.

[0106] The increase in the activity of the protein (polypeptide) may 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.

[0107] 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.

[0108] 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).

[0109] 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).

[0110] 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 genetic 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.).

[0111] 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.

[0112] 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).

[0113] 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.

[0114] Examples of well-known promoters exhibiting potent expression-inducing activity include, but are not limited to, the cj1-cj7 promoter (US Registered Patent US 7662943 B2), the lac promoter, trp promoter, trc promoter, tac promoter, lambda phage PR promoter, PL promoter, tet promoter, gapA promoter, SPL7 promoter, SPL13 (sm3) promoter (US Registered Patent US 10584338 B2), O2 promoter (US Registered Patent US 10273491 B2), tkt promoter, yccA promoter, and TEF promoter.

[0115] 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.

[0116] 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.

[0117] 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.

[0118] 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).

[0119] 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.

[0120] 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.

[0121] 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).

[0122] As one example, the microorganism of this application may have strong SNQ2 protein expression-inducing activity by reducing the activity of the ABC3 protein and further replacing the promoter sequence of the gene encoding the SNQ2 protein with the TEF promoter sequence.

[0123] In this application, “retinol” means the compound known as vitamin A. As an example, retinol can be converted to other retinoid compounds (e.g., retinal, retinoic acid, and retinyl esters, etc.) in ways known in the art.

[0124] In this application, "retinoid" means chemically the vitamin A group or a group of compounds chemically related thereto.

[0125] 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.

[0126] Furthermore, the microorganisms of this application may have or have increased retinoid production ability by further containing a substance (e.g., an enzyme) that converts retinol to other retinoids (e.g., retinoic acid and retinyl esters).

[0127] Another aspect of this application provides a method for producing retinol, comprising the step of culturing a Yarrowia microorganism having retinol-producing ability, wherein the activity of the ABC3 protein is reduced compared to its endogenous activity, in a culture medium.

[0128] The aforementioned microorganisms are as described in other sections.

[0129] As an example, the microorganism may further have increased SNQ2 protein activity compared to its endogenous activity, but is not limited to this.

[0130] 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.

[0131] 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.

[0132] 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.

[0133] 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.

[0134] 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.

[0135] 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.

[0136] 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.

[0137] 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.

[0138] 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.

[0139] The retinol production method of this application may further include recovering retinol from the microorganism or culture medium.

[0140] The target retinol 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, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, and affinity chromatography, as well as HPLC and a combination thereof, can be used, but are not limited to these examples.

[0141] The method may include an additional purification step. The purification step may be performed using a suitable method known in the art.

[0142] As one example, the retinol production method of this application uses microorganisms with reduced ABC3 protein activity that have retinol efflux ability. Therefore, this application can produce retinol without using microbial cell disruption, which is widely used for retinol extraction, or without using dodecane as a solvent, but is not limited thereto.

[0143] The present application provides a method for producing retinol, further comprising the step of converting the retinol expressed by the microorganism of the present application into a retinoid other than retinol. In the retinoid production method of the present application, the conversion step may be further included after the step of culturing the microorganism or the step of recovering the retinol. 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.

[0144] 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.

[0145] Another aspect of this application provides a method for producing retinol-producing microorganisms of the genus Yarrowia, comprising the step of reducing the activity of the ABC3 protein in the retinol-producing Yarrowia microorganism.

[0146] Another aspect of this application provides a method for increasing retinol efflux in a retinol-producing microorganism of the genus Yarrowia, comprising the step of reducing the activity of the ABC3 protein.

[0147] As an example, the method for producing the Yarouia microorganism and the method for increasing retinol excretion may further include, but is not limited to, a step of increasing the activity of the SNQ2 protein in the microorganism.

[0148] The step of reducing the activity of the ABC3 protein may be a step of deforming the Yarouia microorganism so that the activity of the ABC3 protein is reduced compared to its endogenous activity, and the step of increasing the activity of the SNQ2 protein may be a step of deforming the Yarouia microorganism so that the activity of the SNQ2 protein is increased compared to its endogenous activity, as explained in other parts.

[0149] Another aspect of this application provides a composition for retinol production comprising one or more microorganisms of the genus Yarrowia and cultures thereof in which the activity of the ABC3 protein is reduced compared to its endogenous activity.

[0150] As an example, the microorganism may further have increased SNQ2 protein activity compared to its endogenous activity, but is not limited to this.

[0151] The composition of this application may further contain any suitable excipients commonly used in compositions for retinol production, such excipients may include, but are not limited to, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers, or isotonic agents.

[0152] Furthermore, this application can provide a retinoid production composition that further includes a substance that converts retinol to a retinoid other than retinol (for example, an enzyme such as retinol acyltransferase).

[0153] The ABC3 protein, the Yarrowia microorganisms with reduced activity, the SNQ2 protein, the Yarrowia microorganisms with increased activity, retinol, and retinoids are described in other sections.

[0154] Another aspect of this application provides the use of a Yarrowia microorganism or culture thereof, in which the activity of the ABC3 protein of this application is reduced compared to its endogenous activity, for retinol production.

[0155] Another aspect of this application provides the use of a Yarouia microorganism or culture thereof, in which the activity of the ABC3 protein of this application is reduced compared to its endogenous activity, for retinoid production.

[0156] As an example of its use in retinol production and retinoid production, the microorganism may, but is not limited to, have increased SNQ2 protein activity compared to its endogenous activity.

[0157] The ABC3 protein, the Yarrowia microorganisms with reduced activity, the SNQ2 protein, the Yarrowia microorganisms with increased activity, retinol, and retinoids are described in other sections. [Examples]

[0158] 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.

[0159] Example 1. Production of a Yarrowia strain producing retinol with reduced ABC3 activity. In the retinol-producing Yarrowia strain KCCM13294P (CC08-2050, Park et al., Metab Eng. 2022, Jun 6;73:26-37), the activity of the ABC3 protein (YALI0B02544) was reduced.

[0160] To this end, the ORF sequence (SEQ ID NO: 2) of the ABC3 gene (YALI0B02544g) was secured based on the nucleotide sequence registered in KEGG (Kyoto Encyclopedia of Genes and Genomes). Furthermore, an ABC3 gene (YALI0B02544g) deletion cassette was constructed using the Y. lipolytica URA3 gene (SEQ ID NO: 4) as a selection marker. During this process, genomic DNA from CC08-2050 was used as a template, and PCR was performed on the left homologous region, URA3, repeat region, and right homologous region fragments using primers SEQ ID NO: 5 and 6, SEQ ID NO: 7 and 8, SEQ ID NO: 9 and 10, and SEQ ID NO: 11 and 12, 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. Colonies in which cassette insertion into the genome was confirmed using primers SEQ ID NO: 13 and SEQ ID NO: 14 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 resulting strain with reduced activity of the final ABC3 protein was named CC08-2507.

[0161] [Table 1]

[0162] The YLMM1 and 5-FOA media mentioned above were used with the following compositions.

[0163] <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

[0164] <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

[0165] Example 2. Production of a Yarrowia strain producing retinol with increased ABC3 activity. The activity of the ABC3 (YALI0B02544) protein in the Yarrowia strain KCCM13294P, which produces retinol, was increased. For promoter exchange of the ABC3 gene (YALI0B02544g), the ORF sequence (SEQ ID NO: 2) of the ABC3 gene (YALI0B02544g) 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: 4) of Y. lipolytica as a selection marker. The TEF promoter sequence is as shown in SEQ ID NO: 3. At that time, using the genomic DNA of 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 SEQ ID NOs. 15 and 16, SEQ ID NOs. 17 and 18, SEQ ID NOs. 19 and 20, SEQ ID NOs. 21 and 22, and SEQ ID NOs. 23 and 24, 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 constructed as a single cassette via overlap extension PCR. After introducing the thus constructed cassette into the CC08-2050 strain using the heat shock method (D.-C. Chen et al., Appl Microbiol Biotechnol, 1997), colonies formed in uracil-free solid medium (YLMM1) were obtained. Cassette insertion within the genome was confirmed using primers SEQ ID NOs. 25 and 26. 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 ABC3 protein activity obtained through this process was named CC08-2508.

[0166] [Table 2]

[0167] The YLMM1 and 5-FOA media used were those with the same composition as those used in Example 1.

[0168] Example 3. Production of a Yarrowia strain producing retinol with increased SNQ2 activity. The activity of the SNQ2 (YALI0F17996) protein was increased in the Yarrowia strain KCCM13294P (CC08-2050), which produces retinol, and in CC08-2507, which was prepared in Example 1. To do this, the ORF sequence (SEQ ID NO: 34) of the ABC3 gene (YALI0F17996g) was secured based on the nucleotide sequence registered in KEGG (Kyoto Encyclopedia of Genes and Genomes). In addition, a promoter exchange cassette for the SNQ2 gene (YALI0F17996g) was prepared using the URA3 gene (SEQ ID NO: 4) of Y. lipolytica as a selection marker.

[0169] At that time, using the genomic DNA of 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 for SEQ ID NOs. 35 and 36, 37 and 38, 39 and 40, 41 and 42, and 43 and 44, 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 thus assembled 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 uracil-free solid medium (YLMM1) were obtained. Cassette insertion into the genome was confirmed using primers for SEQ ID NOs. 45 and 46. 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 strain obtained through this process, showing increased activity of the final SNQ2 protein of KCCM13294P, was named CC08-2312.

[0170] Furthermore, in the aforementioned CC08-2312 production process, a strain of CC08-2507 was produced using CC08-2507 instead of CC08-2050, and the resulting strain with increased final SNQ2 protein activity was named CC08-2522.

[0171] [Table 3]

[0172] The YLMM1 and 5-FOA media used were those with the same composition as those used in Example 1.

[0173] Example 4. Comparative evaluation of retinol production / excretion capacity of ABC3-decreasing and increasing strains. Flask evaluations were conducted to compare the retinol production and efflux capacity of the strains prepared through Examples 1 to 3 (CC08-2507, CC08-2508, CC08-2312, and CC08-2522) with 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) to an initial OD=2, and cultured for 48 hours at 30°C and 200 rpm. The YPDLU medium used had the following composition.

[0174] <ypdlu> Glucose 60g / L, Bacto peptone 20g / L, Yeast extract 10g / L, Uracil 1g / L, Leucine 1g / L, 1M Phosphate buffer, pH 7.0 100ml / L

[0175] To evaluate the growth rate of each bacterial strain, the OD value was measured at a wavelength of 600 nm using a spectrophotometer. The concentrations of retinol and retinal excreted extracellularly were quantitatively analyzed using an HPLC system after the completion of culture by mixing 0.1 ml of the supernatant (from which bacterial cells were removed) with 0.9 ml of acetone (Sigma) containing 4% BHT.

[0176] The analyzed OD values ​​and retinol and retinal concentrations are shown in Table 4 below, and a diagrammatic representation of these is shown in Figure 1.

[0177] [Table 4]

[0178] Comparing the results of the control group CC08-2050 and the experimental groups CC08-2507 and CC08-2508 in Table 4, the amount of retinol excreted when ABC3 protein activity decreased (CC08-2507) increased 1.37 times compared to the control group (CC08-2050), while the amount of retinal excreted was at a similar level. However, when ABC3 protein activity increased (CC08-2508), the amount of retinol excreted was only 0.83 times that of the control group (CC08-2050), while the amount of retinal excreted was at a similar level.

[0179] Furthermore, comparing the results of the control group CC08-2050 and the experimental groups CC08-2507, CC08-2312, and CC08-2522 in Table 4, the amount of retinol excreted when ABC3 protein activity decreased (CC08-2507) and when SNQ2 protein activity increased (CC08-2312) increased by 1.37 and 1.64 times, respectively, compared to the control group (CC08-2050), while the amount of retinal excreted was at a similar level. On the other hand, the amount of retinol excreted when ABC3 protein activity decreased and SNQ2 protein activity increased simultaneously (CC08-2522) increased by 2.15 times compared to the control group (CC08-2050). This represents a further increase in retinol excretion compared to when ABC3 activity decrease and SNQ2 activity increase were applied individually. Therefore, the simultaneous application of reducing ABC3 activity and increasing SNQ2 activity can increase retinol production / excretion more than applying each individually.

[0180] The results above confirm that ABC3 in *Yarrowia* microorganisms is involved in retinol excretion, and that retinol excretion from microorganisms increases when its activity decreases. Furthermore, a synergistic effect on retinol excretion was confirmed between a decrease in ABC3 activity and an increase in SNQ2 activity.

[0181] 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.

[0182] [Table 5] < / ypdlu>

Claims

1. A Yarrowia sp. microorganism with retinol-producing ability, in which the activity of the ABC3 (ATP-binding cassette transporter type 3) protein is reduced compared to its endogenous activity.

2. The microorganism according to claim 1, wherein the ABC3 protein comprises the YALI0B02544 protein.

3. The microorganism according to claim 1, wherein the ABC3 protein is a protein containing an amino acid sequence of Sequence ID No. 1 or having 60% or more identity therewith.

4. The microorganism according to claim 1, wherein the ABC3 protein is encoded by SEQ ID NO: 2 or a polynucleotide sequence having 60% or more identity therewith.

5. The microorganism according to claim 1, wherein the ABC3 protein is derived from Yarrowia lipolytica.

6. The microorganism according to claim 1, wherein the activity of the SNQ2 protein is further increased compared to its endogenous activity.

7. The microorganism according to claim 6, wherein the SNQ2 protein is a protein containing the amino acid sequence of Sequence ID No. 33 or a sequence having 60% or more identity thereto.

8. The microorganism according to claim 6, wherein the SNQ2 protein is encoded by SEQ ID NO: 34 or a polynucleotide sequence having 60% or more identity therewith.

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

10. The microorganism according to claim 1, wherein the microorganism has increased retinol production or excretion capacity compared to non-mutant Yarrowia microorganisms.

11. A method for producing retinol, comprising the step of culturing a Yarrowia microorganism capable of producing retinol, in which the activity of the ABC3 protein is reduced compared to its endogenous activity, in a culture medium.

12. The method for producing retinol according to claim 11, wherein the microorganism further has increased activity of the SNQ2 protein compared to its endogenous activity.

13. The retinol production method according to claim 11, wherein the production method includes the step of recovering retinol from the cultured medium or microorganism.

14. The retinol production method according to claim 11, wherein the production method does not use microbial cell disruption during retinol extraction, or does not use dodecane as a solvent.

15. A method for producing a retinol-producing microorganism of the genus Yarrowia, comprising the step of reducing the activity of the ABC3 protein in the Yarrowia microorganism having retinol-producing ability.

16. The method for producing microorganisms according to claim 15, further comprising the step of increasing the activity of the SNQ2 protein in the microorganism.

17. A method for increasing retinol excretion in a Yarrowia microorganism capable of producing retinol, comprising the step of reducing the activity of the ABC3 protein.

18. The method for increasing retinol efflux according to claim 17, further comprising the step of increasing the activity of the SNQ2 protein in the microorganism.

19. A composition for retinol production, comprising one or more microorganisms of the genus Yarrowia and their cultures that have retinol-producing ability, wherein the activity of the ABC3 protein is reduced compared to its endogenous activity.

20. The composition according to claim 19, wherein the microorganism further has increased activity of the SNQ2 protein compared to its endogenous activity.

21. Use of Yarrowia microorganisms or their cultures in which ABC3 protein activity is reduced compared to endogenous activity for retinoid production.

22. The use for retinoid production according to claim 21, wherein the microorganism further has increased activity of the SNQ2 protein compared to its endogenous activity.

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