Microorganism of genus Yarrowia having reduced ABC3 protein activity for producing retinol, and method for producing retinol using same

Abstract

The present application relates to: a Yarrowia genus microorganism having a retinol-producing ability, in which the activity of an ABC3 protein is reduced as compared to the endogenous activity thereof; a retinol production method using the same; a method for producing a microorganism of the genus Yarrowia having a retinol-producing ability; the invention discloses a method for enhancing retinol release of microorganisms. And a composition for producing retinol.

Description

Technical Field

[0001] This disclosure relates to a Yarrowia sp. microorganism capable of producing retinol, wherein the activity of the ABC3 protein is reduced compared to its endogenous activity; a method for producing retinol using the microorganism; a method for preparing a Yarrowia sp. microorganism capable of producing retinol; a method for increasing microbial retinol secretion; and a composition for producing retinol. Background Technology

[0002] Retinol is a fat-soluble vitamin, an essential vitamin related to eye health, and is used to improve night blindness, enhance immunity, and promote skin health. Currently, it is mainly produced and marketed by leading global companies through chemical synthesis, and research is underway on the production of retinol based on microbial fermentation. However, unlike the technology for stabilizing the retinol compound itself in compositions or products containing retinol (US 6858217 B2), there is limited development in technologies for the stable production of retinol. Summary of the Invention

[0003] [Technical Issues]

[0004] The problem to be solved by this disclosure is to provide a *Yersinia* microorganism with retinol production capability, wherein the activity of the ABC3 protein is reduced compared with its endogenous activity; a method for producing retinol using the microorganism; a method for preparing a *Yersinia* microorganism with retinol production capability; a method for increasing microbial retinol secretion; and a composition for producing retinol.

[0005] [Technical Solution]

[0006] One aspect of this disclosure provides a Yersinia spp. microorganism capable of producing retinol, wherein the activity of the ABC3 protein is reduced compared to its endogenous activity.

[0007] Another aspect of this disclosure provides a method for producing retinol, which includes culturing the microorganisms of this disclosure in a culture medium.

[0008] Another aspect of this disclosure provides a method for preparing a *Yersinia* microorganism capable of retinol production, comprising reducing the activity of the ABC3 protein in the *Yersinia* microorganism capable of retinol production (compared to its endogenous activity) and increasing retinol secretion.

[0009] Another aspect of this disclosure provides a composition for the production of retinol comprising one or more of the microorganisms of this disclosure and their cultures.

[0010] Another aspect of this disclosure provides the use of the microorganisms of this disclosure or their cultures for the production of retinol.

[0011] [Beneficial Effects]

[0012] The microorganisms disclosed herein can be used to produce retinol. Attached Figure Description

[0013] Figure 1 This is a graph illustrating the retinol production capacity of the strain. Detailed Implementation

[0014] This disclosure will be described in detail below. Furthermore, each description and embodiment described herein can be applied to other descriptions and embodiments. That is, all combinations of the various elements described herein fall within the scope of this disclosure. Moreover, the scope of this disclosure is not limited by the specific descriptions below. Additionally, this specification references numerous academic papers and patent documents throughout. The contents of the cited academic papers and patent documents are incorporated herein by reference in their entirety to more clearly describe the level of the technical field to which this disclosure pertains and the content of this disclosure.

[0015] One aspect of this disclosure provides a Yersinia spp. microorganism capable of producing retinol, wherein the activity of the ABC3 (ATP-binding cassette transporter type 3) protein is reduced compared to its endogenous activity.

[0016] The reduced ABC3 protein activity can be measured by measuring the amount of ABC3 protein or the polynucleotide encoding it, or by measuring retinol production capacity (or yield), but the measurement is not limited thereto. For example, in the case where the retinol production capacity of the microorganisms of this disclosure is increased compared to the retinol production capacity of natural wild-type Yersinia spp. or unmodified Yersinia spp. (e.g., Yersinia spp. expressing a wild-type polypeptide (e.g., the polypeptide of SEQ ID NO: 1) with wild-type ABC3 protein activity), the increased ABC3 protein activity can be measured by measuring the increased retinol production capacity, but the measurement is not limited thereto.

[0017] The "ABC3 protein (ATP-binding cassette transporter 3 protein)" disclosed herein refers to type 3 of the ABC protein family. ABC protein is an ATP-binding cassette transporter and is used interchangeably with the term ABC transporter. The ABC protein family includes type 1, type 2, and type 3 proteins. Type 3 protein, namely ABC3 protein, is derived from a precursor having four transmembrane segments (TMS), and may have 4, 8, or 10 TMS.

[0018] The ABC3 protein disclosed herein may include the YALI0B02544 protein. The YALI0B02544 protein is interchangeable with YALI0B02544p.

[0019] The ABC3 protein disclosed herein may include any ABC3 protein capable of increasing retinol production and / or retinol secretion. In one instance, increased retinol production may be a result of increased retinol secretion.

[0020] In one embodiment, the ABC3 protein of this disclosure may have reduced activity compared to endogenous or wild-type ABC3 proteins in Yersinia spp. microorganisms, thereby increasing the retinol production and / or retinol secretion capacity of the microorganisms.

[0021] In one embodiment, the ABC3 protein of this disclosure may comprise, have an amino acid sequence having 60% or more homology or identity with the amino acid sequence of SEQ ID NO: 1, or consist of, or substantially consist of, an amino acid sequence having 60% or more homology or identity with the amino acid sequence of SEQ ID NO: 1.

[0022] For example, the amino acid sequence of the ABC3 protein disclosed herein may be encoded by the YALI0B02544 gene (i.e., YALI0B02544g), but is not limited thereto. The amino acid sequence of the ABC3 protein can be obtained from various databases, such as NCBI's GenBank, but is not limited thereto.

[0023] In one embodiment, the ABC3 protein of this disclosure may be derived from Yarrowia lipolytica, but is not limited thereto.

[0024] Furthermore, although embodiments of the ABC3 protein of this disclosure are described as proteins comprising the amino acid sequence of SEQ ID NO: 1, this does not preclude the addition of non-functional sequences, naturally occurring mutations, or silent mutations thereof to the upstream or downstream regions of the amino acid sequence of SEQ ID NO: 1. It will be apparent to those skilled in the art that any protein exhibiting the same or corresponding activity as a protein comprising that amino acid sequence falls within the scope of the ABC3 protein of this disclosure.

[0025] For example, the ABC3 protein of this disclosure may comprise the amino acid sequence of SEQ ID NO: 1, or comprise, have, or substantially have, an amino acid sequence having at least 60% or more, 62% or more, 63% or more, 64% or more, 62% or more, 65% or more, 70% or more, 75% or more, 76% or more, 77% or more, 78% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homology or identity with the amino acid sequence of SEQ ID NO: 1, or be composed of, or substantially composed of, an amino acid sequence having, or substantially composed of, an amino acid sequence having, at least 60% or more, 62% or more, 63% or more, 96% or more, 97% or more, 98% or more, or 99% or more homology or identity with the amino acid sequence of SEQ ID NO: 1, or be composed of, or substantially composed of, an amino acid sequence having, at least 60% or more, 62% or more, 63% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homology or identity with, the amino acid sequence of SEQ ID NO: 1, or be composed of, or substantially composed of, an amino acid sequence having, at least 60% or more, 62% or more, 63% or more, 94% or more, 95% or more, 96% or more, 97% or more The amino acid sequence of 1 has an amino acid sequence composition of at least 60% or higher, 62% or higher, 63% or higher, 64% or higher, 62% or higher, 65% or higher, 70% or higher, 75% or higher, 76% or higher, 77% or higher, 78% or higher, 80% or higher, 85% or higher, 86% or higher, 87% or higher, 88% or higher, 89% or higher, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher. Furthermore, it is apparent that as long as the amino acid sequence has the aforementioned homology or identity and exhibits efficacy corresponding to the efficacy of the protein, even if some sequences are deleted, modified, substituted, or added, it falls within the scope of this disclosure.

[0026] In one embodiment, the microorganisms of this disclosure may additionally exhibit increased SNQ2 protein activity compared to their endogenous activity.

[0027] Increased SNQ2 protein activity can be measured by measuring the amount of SNQ2 protein or the polynucleotide encoding it, or by measuring retinol production capacity (or yield), but is not limited to these methods. For example, when comparing the retinol production capacity of a *Yarrowia* microorganism in which the activity of the ABC3 protein of this disclosure is reduced compared to its endogenous activity but the activity of the SNQ2 protein is not increased or has not yet increased compared to its endogenous activity, the increased retinol production capacity of a microorganism in which the activity of the ABC3 protein of this disclosure is reduced and the activity of the SNQ2 protein is increased can be measured to measure the increased SNQ2 protein activity, but the measurement of increased SNQ2 protein activity is not limited to these methods.

[0028] The term "SNQ2 protein (ATP-binding cassette transporter SNQ2)" in this disclosure refers to a type of ATP-binding cassette transporter. The SNQ2 protein of this disclosure can be a plasma membrane ABC transporter and can be a multidrug transporter.

[0029] The SNQ2 protein disclosed herein may include the YALI0F17996 protein, and the YALI0F17996 protein may be used interchangeably with YALI0F17996p.

[0030] In one embodiment, the SNQ2 protein of this disclosure may have increased activity compared to endogenous or wild-type SNQ2 proteins in Yersinia spp. microorganisms, thereby increasing the retinol production and / or retinol secretion capacity of the microorganisms.

[0031] In one embodiment, the SNQ2 protein of this disclosure may comprise, have an amino acid sequence having 60% or more homology or identity with the amino acid sequence of SEQ ID NO: 33, or consist of, or substantially consist of, an amino acid sequence having 60% or more homology or identity with the amino acid sequence of SEQ ID NO: 33.

[0032] For example, the amino acid sequence of the SNQ2 protein disclosed herein may be encoded by the YALI0F17996 gene (i.e., YALI0F17996g), but is not limited thereto. The amino acid sequence of the SNQ2 protein can be obtained from various databases, such as NCBI's GenBank, but is not limited thereto.

[0033] In one embodiment, the SNQ2 protein of this disclosure may be derived from Yersinia lipolytica, but is not limited thereto.

[0034] Furthermore, although embodiments of the SNQ2 protein of this disclosure are described as proteins comprising the sequence of SEQ ID NO: 33, this does not preclude the addition of non-functional sequences, naturally occurring mutations, or silent mutations thereof upstream or downstream of the amino acid sequence of SEQ ID NO: 33. It will be apparent to those skilled in the art that any protein exhibiting the same or corresponding activity as a protein comprising that amino acid sequence falls within the scope of the SNQ2 protein of this disclosure.

[0035] For example, the SNQ2 protein of this disclosure may comprise the amino acid sequence of SEQ ID NO: 33, or comprise, having, or substantially comprise, an amino acid sequence having at least 60% or higher, 62% or higher, 63% or higher, 64% or higher, 62% or higher, 65% or higher, 70% or higher, 75% or higher, 76% or higher, 77% or higher, 78% or higher, 80% or higher, 85% or higher, 86% or higher, 87% or higher, 88% or higher, 89% or higher, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher homology or identity with the amino acid sequence of SEQ ID NO: 33, or composed of, or substantially composed of, an amino acid sequence having, or substantially composed of, an amino acid sequence having, at least 60% or higher, 62% or higher, 63% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher homology or identity with, the amino acid sequence of SEQ ID NO: 33, or composed of, or substantially composed of, an amino acid sequence having, at least 60% or higher, 62% or higher, 63% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher homology or identity with, the amino acid sequence of SEQ ID NO: 33, or composed of, an ... The amino acid sequence of 33 has an amino acid sequence composition of at least 60% or higher, 62% or higher, 63% or higher, 64% or higher, 62% or higher, 65% or higher, 70% or higher, 75% or higher, 76% or higher, 77% or higher, 78% or higher, 80% or higher, 85% or higher, 86% or higher, 87% or higher, 88% or higher, 89% or higher, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher. Furthermore, it is apparent that as long as the amino acid sequence has the aforementioned homology or identity and exhibits efficacy corresponding to the efficacy of the protein, even if some sequences are deleted, modified, substituted, or added, it falls within the scope of this disclosure.

[0036] Even though this disclosure describes "a polypeptide (or protein) comprising an amino acid sequence represented by a specific SEQ ID NO", "a polypeptide (or protein) composed of an amino acid sequence represented by a specific SEQ ID NO", or "a polypeptide (or protein) having an amino acid sequence represented by a specific SEQ ID NO", it is clear that proteins with missing, modified, substituted, or added amino acid sequences in a portion of the sequence can also be used in this invention, as long as they have the same or corresponding activity as the polypeptide or protein composed of the amino acid sequence corresponding to the SEQ ID NO. For example, this includes the addition of sequences that do not alter protein function to the N-terminus and / or C-terminus of the amino acid sequence, naturally occurring mutations, their silent mutations, or conserved substitutions.

[0037] The term "conservative substitution" refers to the replacement of an amino acid with a different amino acid that has similar structure and / or chemical properties. Such amino acid substitutions typically occur based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphiphilicity of the residues. Generally, conservative substitutions have little or no effect on protein activity.

[0038] As used herein, the term “identity” or “homology” refers to the degree of similarity between two given amino acid sequences or nucleotide sequences, and may be expressed as a percentage. The terms “homology” and “identity” are often used interchangeably in this disclosure.

[0039] Sequence homology or identity of conserved polynucleotides or polypeptides (including proteins) is determined by standard alignment algorithms and can be used together with a default gap penalty established by the procedure used. Essentially, under moderately or highly stringent conditions, homologous or identical sequences are generally capable of hybridizing with the entire sequence or a portion corresponding to at least approximately 50%, 60%, 70%, 80%, or 90% of the full length. Clearly, hybridization also includes hybridization with polynucleotides containing universal codons or codons that account for codon degeneracy in polynucleotides.

[0040] Whether any two polynucleotide or polypeptide (including protein) sequences are homologous, similar, or identical can be determined, for example, by known computer algorithms, such as the “FASTA” program with default parameters as described in Pearson et al. (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444. Alternatively, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) can be used to determine this, as in the Needleman program (version 5.0.0 or later) of the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277) (including the GCG 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.,] AcademicPress, San This is performed as described in Diego, 1994 and [CARILLO ET AL.] (1988) SIAM J Applied Math 48: 1073. For example, homology, similarity, or identity can be determined by using BLAST or ClustalW from the National Center for Biotechnology Information.

[0041] Homology, similarity, or identity between polynucleotides or polypeptides (including proteins) can be determined by comparing sequence information, for example, using a GAP computer program (such as Needleman et al. (1970), J Mol Biol. 48:443), as described, for example, in Smith and Waterman, Adv. Appl. Math (1981) 2:482. In summary, the GAP program can be defined as a value obtained by dividing the number of similarly arranged symbols (i.e., nucleotides or amino acids) by the total number of symbols in the shorter sequence of the two sequences. The default parameters of the GAP procedure may include: (1) a binary comparison matrix (containing a value of 1 for identity and a value of 0 for non-identity) and a weighted comparison matrix as disclosed in Schwartz and Dayhoff, Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353–358 (1979) by Gribskov et al. (1986) Nucl. Acids Res. 14: 6745 (or an EDNAFULL (EMBOSS version of NCBI NUC4.4) replacement matrix); (2) a penalty of 3.0 for each vacancy and an additional penalty of 0.10 for each symbol in each vacancy (or a penalty of 10 for vacancy opening and 0.5 for vacancy extension); and (3) no penalty for terminal vacancy.

[0042] Furthermore, whether any two polynucleotide or polypeptide (including protein) sequences are homologous, similar, or identical can be confirmed by comparing the sequences under suitable hybridization conditions, which are within the scope of this art and can be determined by methods well known to those skilled in the art (such as 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), but not limited thereto.

[0043] As used herein, the term "polynucleotide" is a polymer in which nucleotide monomers are covalently linked together to form a long chain of nucleotides, which is a DNA chain of a certain length or longer.

[0044] The polynucleotide sequence encoding the ABC3 protein disclosed herein may be referred to as the YALI0B02544 gene sequence, and may include a polynucleotide sequence or a degenerate sequence thereof encoding the amino acid sequence represented by SEQ ID NO: 1.

[0045] The polynucleotide sequence encoding the SNQ2 protein disclosed herein may be referred to as the YALI0F17996 gene sequence, and may include a polynucleotide sequence or a degenerate sequence thereof encoding the amino acid sequence represented by SEQ ID NO: 33.

[0046] In the polynucleotide encoding the SNQ2 protein disclosed herein, various modifications can be made to the coding region, taking into account codon degeneracy or preferred codons in an organism intended to express the SNQ2 polypeptide or protein, as long as the amino acid sequence of the polypeptide or protein remains unchanged.

[0047] Specifically, the polynucleotide encoding the ABC3 protein may comprise, or consist of, or substantially consist of, the polynucleotide sequence of SEQ ID NO: 2 or having 60% or higher homology or identity with it, but is not limited thereto. For example, the polynucleotide encoding the ABC3 protein may consist of a base sequence having 60% or higher homology or identity with the sequence of SEQ ID NO: 2, 62% or higher, 63% or higher, 64% or higher, 62% or higher, 65% or higher, 70% or higher, 76% or higher, 77% or higher, or 78% or higher, 80% or higher, 85% or higher, 86% or higher, 87% or higher, 88% or higher, 89% or higher, 90% or higher, 91% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher, but is not limited thereto.

[0048] The polynucleotide encoding the SNQ2 protein may comprise, but is not limited to, the polynucleotide sequence of SEQ ID NO: 34 or having 60% or more homology or identity with it. For example, the polynucleotide encoding the SNQ2 protein may consist of a base sequence having 60% or more, 62% or more, 63% or more, 64% or more, 62% or more, 65% or more, 70% or more, 76% or more, 77% or more, or 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 the sequence of SEQ ID NO: 34.

[0049] Furthermore, the polynucleotides disclosed herein may contain probes, for example, without limitation, as long as they are sequences capable of hybridizing with all or part of the complementary sequence of the polynucleotide base sequence under stringent conditions.

[0050] As used herein, the term “strict conditions” refers to conditions that enable specific hybridization between polynucleotides. These conditions are specifically described in the literature (e.g., J. Sambrook et al., see above). Examples of strict conditions may be conditions in which polynucleotides with high homology or identity hybridize with each other at 40% or higher, specifically 90% or higher, more specifically 95% or higher, 96% or higher, 97% or higher, or 98% or higher, or even more specifically 99% or higher, but polynucleotides with low homology or identity do not hybridize with each other; or washing conditions typical of Southern hybridization, i.e., washing once, or specifically two to three times, at salt concentrations and temperatures corresponding to 1×SSC, 0.1% SDS at 60°C, specifically 0.1×SSC, 0.1% SDS at 60°C, or more specifically 0.1×SSC, 0.1% SDS at 68°C.

[0051] Hybridization can occur between nucleotides with complementary base sequences, but depending on the stringency of the hybridization, hybridized polynucleotides may include some base mismatches. The term "complementary" is used to describe the relationship between nucleotide bases that can hybridize with each other. For example, in DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Therefore, the polynucleotides of this disclosure may also include isolated nucleic acid fragments complementary to the whole sequence as well as nucleic acid sequences substantially similar to them.

[0052] Specifically, homologous or identical polynucleotides can be detected using hybridization conditions, which include hybridization at a Tm value of 55°C and the conditions described above. Furthermore, the Tm value can be 60°C, 63°C, or 65°C, but is not limited to these, and can be appropriately adjusted by those skilled in the art.

[0053] The appropriate stringency of hybrid polynucleotides depends on the length and complementarity of the polynucleotides, and the variables are well known in the relevant technical fields (see J. Sambrook et al., above).

[0054] For example, homologous or identical polynucleotide sequences can typically hybridize at least about 50%, 60%, 70%, 80%, or 90% along the entire sequence or full length under stringent conditions.

[0055] The term "vector" in this disclosure refers to a DNA construct for delivering a desired polynucleotide to a suitable host or host cell. In one instance, it may contain a base sequence of a polynucleotide encoding a desired polypeptide or protein, which is operatively linked to a suitable expression regulatory region (or expression control sequence) such that the desired polypeptide or protein can be expressed in a suitable host, but is not limited thereto.

[0056] The expression regulatory sequence may include a promoter capable of initiating transcription, any operon sequence for regulating transcription, a sequence encoding a suitable mRNA ribosome binding site, and a termination sequence for regulating transcription and translation. After being transformed into a suitable host cell (microorganism), the vector may replicate or function independently of the host genome, or it may integrate into the genome itself to replicate or function.

[0057] The vectors disclosed herein may be insert vectors for inserting polynucleotides for reducing the activity of the ABC3 protein of this disclosure into chromosomes and / or insert vectors for inserting polynucleotides for increasing the activity of the SNQ2 protein of this disclosure into chromosomes, but are not limited thereto. The polynucleotides may be inserted into chromosomes by any method known in the art, such as homologous recombination, but are not limited thereto. The vectors may also contain selection markers to confirm transformation into host cells, or further, insertion into the chromosome of host cells. The selection markers are used to select cells transformed with the vector, or to confirm the chromosome insertion of the desired polynucleotide, and may use markers conferring selectable phenotypes such as drug resistance, auxotrophic phenotype, cytotoxic agent resistance, or surface polypeptide or protein expression. In an environment treated with a selection agent, only cells expressing the selection marker survive or exhibit different phenotypic traits, thereby enabling the selection of transformed cells. The insert vector may not contain the origin of replication required for replication in the transformed cells.

[0058] There are no particular limitations on the vectors used in this disclosure, and any vector known in the art may be used. Examples of commonly used vectors include plasmids, granules, viruses, and phages in their native or recombinant states. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A can be used as phage vectors or granule vectors; and those based on pDZ, pBR, pUC, pBluescriptII, pGEM, pTZ, pCL, pET, etc., can be used as plasmid vectors. Specifically, pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, and pCC1BAC vectors may be used.

[0059] As used herein, the term "transformation" refers to the introduction of a desired polynucleotide and / or a vector containing that polynucleotide into a host cell (microorganism), thereby altering the genetic traits of the host cell (microorganism). In this disclosure, transformation can refer to the introduction of a vector containing a polynucleotide for reducing ABC3 protein activity, or further containing a polynucleotide for increasing SNQ2 protein activity, into a host cell, thereby altering the genetic traits of the host cell. The transformed polynucleotide can be inserted into the chromosome of the host cell or located extrachromosomally. For example, a vector containing a polynucleotide for reducing ABC3 protein activity can be an insertion vector for inserting into the chromosome via homologous recombination to delete part or all of the YALI0B02544 gene. Furthermore, the polynucleotide can contain DNA and / or RNA encoding the protein. Depending on the purpose of the introduction, the polynucleotide can be introduced in a suitable form. Additionally, for example, the polynucleotide for increasing SNQ2 protein activity can be introduced into the host cell in the form of an expression cassette, which is a gene construct containing all the elements necessary for self-expression. The expression cassette may include a promoter, transcription termination signal, ribosome binding site, and translation termination signal operably linked to the coding sequence of the SNQ protein. The expression cassette may be in the form of a self-replicating expression vector. Furthermore, the polynucleotide is introduced into a host cell in its native form and operably linked to the desired sequence for expression in the host cell, but is not limited thereto.

[0060] As used herein, the term "operably linked" refers to a composition in which a regulatory sequence is positioned appropriately to control the expression of a coding sequence. Therefore, "operably linked" involves attaching or linking a regulatory region with a functional domain having known or desired activity, such as a promoter, terminator, signaling sequence, or enhancer region, to a target (gene or polypeptide) to regulate its expression, secretion, or function according to known or desired activity. For example, it might mean the functional linking of a polynucleotide sequence encoding a polypeptide to a promoter sequence that initiates and mediates polynucleotide transcription.

[0061] Methods for transforming the vectors of this disclosure include any method of introducing nucleic acids into cells and can be performed by selecting suitable standard techniques known in the art, depending on the host cell. For example, methods such as electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) methods, DEAE-dextran methods, cationic liposome methods, lithium acetate-DMSO methods can be used, but the methods are not limited thereto.

[0062] As used herein, the term "microorganism (or strain)" includes wild-type microorganisms and naturally or artificially genetically modified prokaryotic or eukaryotic microorganisms. It may be a genetically modified microorganism for the production of a target polypeptide, protein, or product, in which a specific mechanism is weakened or reduced due to factors such as the insertion of a foreign gene or the reduction or inactivation of an endogenous gene. In this disclosure, the terms "microorganism," "strain," "host," and "host cell" are used interchangeably.

[0063] As used herein, the term "recombinant microorganism" refers to a microorganism that has been genetically modified to exhibit a genotype and / or phenotype different from that of naturally occurring microorganisms (e.g., when the genetic modification affects the coding nucleic acid sequence of the microorganism), and may include all or potential offspring of that microorganism. In this disclosure, the terms "recombinant microorganism," "genetically modified microorganism," "recombinant host cell," "recombinant cell," and "recombinant strain" are used interchangeably. For example, a recombinant microorganism may express genes not found in its natural (non-recombinant) form, not express genes expressed in its natural form, or express natural genes in a manner different from those expressed in its natural form.

[0064] For example, the microorganism disclosed herein may be any one or more of the following: recombinant microorganisms, wherein the ABC3 protein activity is reduced compared to its endogenous activity due to reduced expression of the gene encoding the ABC3 protein; and recombinant microorganisms, wherein the SNQ2 protein activity is additionally increased compared to its endogenous activity due to increased expression of the gene encoding the SNQ2 protein, but not limited thereto.

[0065] The microorganisms disclosed herein may be microorganisms capable of producing retinol. The term "microorganism capable of producing retinol" may be used interchangeably with "microorganism producing retinol".

[0066] The microorganisms disclosed herein may be microorganisms in which polynucleotides encoding lycopene cyclase / hydrophobic lycopene synthase (crtYB) and phobic lycopene desaturase (crtI) proteins have been introduced, to enable microorganisms inherently lacking retinol production capacity to produce retinol, or to further increase the retinol production capacity of microorganisms already possessing retinol production capacity, thereby exhibiting or having increased activity of these proteins. The lycopene cyclase / hydrophobic lycopene synthase or phobic lycopene desaturase may be proteins derived from *Xanthophyllomyces dendrorhous*, but are not limited thereto, as long as they are proteins exhibiting the same or similar activities. In one specific embodiment, the lycopene cyclase / hydrophobic lycopene synthase or phobic lycopene desaturase may consist of, or contain, the amino acid sequences of SEQ ID NO: 27 or SEQ ID NO: 29, respectively. The amino acid sequence of 29 may also consist of an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology or identity with the above-mentioned amino acid sequence, while exhibiting activities corresponding to lycopene cyclase / hydrophobic lycopene synthase or hydrophobic lycopene desaturase, or contain an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology or identity with the above-mentioned amino acid sequence, while exhibiting activities corresponding to lycopene cyclase / hydrophobic lycopene synthase or hydrophobic lycopene desaturase. Furthermore, it is evident that the lycopene cyclase / hydrolycopene synthase or hydrolycopene desaturase also includes proteins with deletions, modifications, substitutions, or additions in a portion of their sequence, provided that the protein possesses the aforementioned homology or identity and exhibits activity corresponding to the lycopene cyclase / hydrolycopene synthase or hydrolycopene desaturase. In addition, in one specific embodiment, the polynucleotide encoding the lycopene cyclase / hydrolycopene synthase or hydrolycopene desaturase may consist of or contain the sequences of SEQ ID NO: 28 or SEQ ID NO: 30, respectively. Within the polynucleotide, various modifications can be made to the coding region, taking into account codon degeneracy or preferred codons in the microorganisms disclosed herein, provided the amino acid sequence remains unchanged.Specifically, the polynucleotide may comprise 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, or may consist of 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, but is not limited thereto.

[0067] Furthermore, the microorganisms disclosed herein may be microorganisms in which a polynucleotide encoding a β-carotene 15,15'-oxygenase (BLH) protein has been introduced, so as to enable microorganisms that are inherently lacking in retinol production capacity to produce retinol, or to further increase the retinol production capacity of microorganisms that already possess retinol production capacity, thereby exhibiting β-carotene 15,15'-oxygenase activity or having increased β-carotene 15,15'-oxygenase activity. β-carotene 15,15'-oxygenase may be a protein derived from unculturable marine bacteria 66A03, but is not limited thereto, as long as it is a protein exhibiting the same or similar activity. In one specific embodiment, β-carotene 15,15'-oxygenase may consist of or contain the amino acid sequence of SEQ ID NO: 31, but may also consist of an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology or identity with the amino acid sequence of SEQ ID NO: 31, while exhibiting activity corresponding to β-carotene 15,15'-oxygenase activity, or may contain an amino acid sequence that is homologous to or contains the amino acid sequence of SEQ ID NO: 31. The amino acid sequence of SEQ ID NO: 31 has at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology or identity, and exhibits an amino acid sequence corresponding to β-carotene 15,15'-oxygenase activity. Furthermore, it is apparent that β-carotene 15,15'-oxygenase also includes proteins with deletions, modifications, substitutions, or additions in portions of their sequence, provided that such proteins have the aforementioned homology or identity and exhibit β-carotene 15,15'-oxygenase activity. In addition, in one specific embodiment, the polynucleotide encoding β-carotene 15,15'-oxygenase may have or contain the sequence of SEQ ID NO: 32. In the polynucleotide, various modifications can be made to the coding region, taking into account codon degeneracy or preferred codons in the microorganisms of this disclosure, as long as the amino acid sequence remains unchanged.Specifically, the polynucleotide may consist of 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, or may contain 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, but is not limited thereto.

[0068] The CC08-2050 microorganism disclosed herein (identical to KCCM13294P) may comprise: a lycopene cyclase / hydrophobic lycopene synthase comprising the amino acid sequence of SEQ ID NO: 27 and / or a polynucleotide encoding thereof; a hydrophobic lycopene desaturase comprising the amino acid sequence of SEQ ID NO: 29 and / or a polynucleotide encoding thereof; and a β-carotene 15,15'-oxygenase comprising the amino acid sequence of SEQ ID NO: 31 and / or a polynucleotide encoding thereof.

[0069] In one embodiment, the microorganisms disclosed herein may be microorganisms that increase retinol production and / or retinol secretion capacity due to reduced ABC3 protein activity.

[0070] Compared to unmodified Yersinia spp. microorganisms in which the activity of ABC3 protein is not reduced, the microorganisms of this disclosure in which the activity of ABC3 protein is reduced may exhibit increased retinol secretion capacity, but are not limited thereto.

[0071] In one embodiment of the foregoing implementation scheme, the microorganism may additionally exhibit increased SNQ2 protein activity compared to its endogenous activity, but is not limited thereto.

[0072] Compared to microorganisms in which the activity of SNQ2 protein is not increased but the activity of ABC3 protein is reduced, the microorganisms of this disclosure in which the activity of ABC3 protein is reduced and the activity of SNQ2 protein is additionally increased may exhibit an increased retinol secretion capacity, but are not limited thereto.

[0073] The increased ability of the microorganisms disclosed herein to produce retinol may be a result of increased retinol secretion capacity, but is not limited thereto.

[0074] The microorganisms disclosed herein may selectively secrete retinol, but are not limited thereto.

[0075] The microorganisms disclosed herein can selectively secrete retinol from retinaldehyde and retinol, but are not limited thereto.

[0076] The microorganisms disclosed herein may be naturally occurring microorganisms capable of producing ABC3 protein or retinol, or parental strains of which the ABC3 protein of this disclosure is reduced but still capable of producing ABC3 protein or retinol.

[0077] In one instance, the microorganisms of this disclosure may include all microorganisms in which the activity of the ABC3 protein of this disclosure is reduced and which are capable of producing retinol.

[0078] For example, the microorganisms disclosed herein may be recombinant strains in which retinol production and / or retinol secretion capacity is increased due to reduced ABC3 protein activity in naturally wild-type microorganisms, microorganisms with retinol production capacity, and / or microorganisms containing ABC3 protein. The recombinant strains with increased retinol production and / or retinol secretion capacity may be microorganisms with increased retinol production and / or retinol secretion capacity compared to naturally wild-type microorganisms, or microorganisms in which the activity of the ABC3 protein of this disclosure is not reduced, but are not limited thereto.

[0079] For example, a microorganism in which the activity of the ABC3 protein of this disclosure is not reduced and the activity of the SNQ2 protein is not increased, used as a reference strain for comparing whether retinol production capacity and / or retinol secretion capacity are increased, could be CC08-2050 (KCCM13294P, reference Park et al., Metabolic Engineering 2022;73:26-37), but is not limited thereto. The depositary strain name of the CJ2050 strain disclosed in the cited reference (Park et al., Metabolic Engineering 2022;73:26-37) is the CC08-2050 strain of this disclosure; therefore, the CC08-2050 strain and the CJ2050 strain of this disclosure are the same strain.

[0080] In one instance, the retinol production capacity of recombinant strains and microorganisms with increased retinol production capacity and / or retinol secretion capacity may be enhanced by about 1% or more, about 2% or more, about 5% or more, about 10% or more, about 20% or more, or about 30% or more (with no particular upper limit, for example, it may be about 200% or less) compared to the retinol production capacity of the parental strain before modification or the unmodified microorganism, and in another instance, it may be enhanced by about 1.01 times or more, about 1.02 times or more, about 1.05 times or more, about 1.07 times or more, about 1.1 times or more, about 1.2 times or more, or about 1.3 times or more (with no particular upper limit, for example, it may be about 10 times or less), but is not limited thereto, as long as there is a positive increase compared to the production capacity of the parental strain before modification or the unmodified microorganism. The term “about” refers to a range including ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., which includes all values ​​equal to or similar to the value following the term “about”, but is not limited to this range.

[0081] As used herein, the term "unmodified microorganism (strain)" does not exclude naturally occurring mutant microorganisms (strains) and may refer to wild-type microorganisms (strains) or the natural microorganisms (strains) themselves, or microorganisms (strains) before their traits are altered due to genetic variation caused by natural or artificial factors. For example, an unmodified microorganism may refer to, but is not limited to, a microorganism (strain) in which the ABC3 protein of this disclosure is not reduced or has not yet been reduced. In this disclosure, the term "unmodified microorganism (strain)" may be used interchangeably with "pre-modified microorganism (strain)," "unmutated microorganism (strain)," "parental strain," "parental microorganism," "wild-type microorganism (strain)," or "reference microorganism (strain)."

[0082] The microorganisms disclosed herein may be microorganisms of the genus Yersinia, but are not limited thereto.

[0083] In one embodiment, the Yersinia species disclosed herein may be Yersinia lipophila, but is not limited thereto.

[0084] As used herein, the term “reduction” in protein (peptide) activity is a concept that includes a decrease and inactivation of protein (peptide) activity in the host cell (microbe) compared to its endogenous activity.

[0085] In other words, a reduction in protein (peptide) activity can include a state in which the protein (peptide) in the host cell (microorganism) is not completely inactivated and therefore exhibits reduced protein (peptide) activity compared to its endogenous or pre-modified activity; or a state in which the protein (peptide) activity is completely inactivated.

[0086] For example, the reduction may also include the following situations: the protein (peptide) has low activity or is absent compared to the protein (peptide) originally possessed by the host cell (microorganism) before transformation or the unmodified host cell (microorganism) due to mutations in polynucleotides encoding the protein (peptide); the overall intracellular expression level of the protein (peptide) is reduced compared to the host cell (microorganism) before transformation or the unmodified host cell (microorganism) due to inhibition of polynucleotide or protein (peptide) expression; the polynucleotide or protein (peptide) is not expressed at all; and even if the protein (peptide) is normally expressed, the protein (peptide) has low activity or is absent.

[0087] The host cell (microorganism) can be a prokaryotic or eukaryotic microorganism.

[0088] The reduction in protein (peptide) activity compared to its endogenous activity refers to a decrease in the activity and / or concentration (expression level) of the protein (peptide) in the host cell (microorganism) compared to the activity and / or concentration (expression level) of the protein (peptide) originally present in the unmodified host cell (microorganism) or the unmodified host cell (microorganism).

[0089] Whether the activity of a protein (peptide) is reduced can be confirmed by the degree of protein (peptide) activity, its expression level, or the amount of product produced by the activity of the protein (peptide).

[0090] "Endogenous activity" refers to the activity of a specific protein (peptide) that was originally present in the host cell (microorganism) before transformation or in the unmodified host cell (microorganism) when the trait is altered due to genetic variation caused by natural or artificial factors. This term is interchangeable with "pre-modification activity".

[0091] The microorganisms disclosed herein can exhibit enhanced retinoid production and / or retinoid secretion capacity due to reduced activity of the ABC3 protein.

[0092] The reduction of protein (peptide) activity can be achieved by a variety of methods well known in the art, and is not limited thereto, as long as the desired protein (peptide) activity can be reduced compared to the activity of the host cell (microbe) before modification. Specifically, genetic engineering and / or protein engineering, which are conventional molecular biology techniques well known to those skilled in the art, can be used, but are not limited thereto (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.).

[0093] Specifically, the reduction in protein (peptide) activity disclosed herein may be due to:

[0094] 1) The gene encoding the protein (peptide) is partially or completely missing;

[0095] 2) Modify the gene expression regulatory region on the chromosome encoding the protein (peptide) (e.g., introduce mutations into the expression regulatory region, replace with a sequence that exhibits expression repression activity, or insert a sequence that exhibits expression repression activity).

[0096] 3) Modify the amino acid sequence of the protein (peptide) (e.g., delete / replace / add one or more amino acids in the amino acid sequence) to reduce the activity of the protein (peptide);

[0097] 4) Modifying the polynucleotide sequence encoding the protein (peptide) (e.g., modifying the polynucleotide sequence of a gene encoding a protein (peptide) such that the gene encodes a protein (peptide) modified to have reduced protein (peptide) activity) reduces the activity of the protein (peptide).

[0098] 5) Modify the start codon or 5'-UTR of the gene encoding the protein (polypeptide);

[0099] 6) Introduce an antisense oligonucleotide (e.g., antisense RNA) that binds complementary to the transcript of the gene encoding the protein (peptide).

[0100] 7) Add a sequence complementary to the SD sequence upstream of the Shine-Dalgarno (SD) sequence of the gene encoding the protein (peptide) to form a secondary structure that prevents ribosome attachment.

[0101] 8) Reverse transcription engineering (RTE) involves adding a promoter to be reverse transcribed to the 3' end of the open reading frame (ORF) encoding the polynucleotide sequence of the protein (peptide);

[0102] 9) Regulating the cellular localization of the protein (peptide); or

[0103] 10) Selected from two or more of the above 1) to 9), but not particularly limited thereto.

[0104] For example,

[0105] 1) The deletion of part or all of the gene encoding the protein (peptide) can be achieved by methods involving homologous recombination using a chromosome insertion vector within a microorganism, and / or by exposure to electromagnetic waves such as ultraviolet light or radiation, or by treatment with chemical reagents, but is not limited thereto.

[0106] 2) Modifying the gene expression regulatory region on the chromosome encoding the protein (peptide) can be achieved, for example, by introducing modifications into the gene expression regulatory region through deletion, insertion, substitution, or a combination thereof to further reduce the expression-inducing activity of the expression regulatory region, or by replacing the sequence with a sequence having further expression-inhibiting activity. The expression regulatory region may include, but is not particularly limited to, promoters, operon sequences, sequences encoding ribosome binding sites, sequences controlling transcription and translation termination, etc.

[0107] 3) and 4) Modification of the amino acid sequence or polynucleotide sequence of the protein (peptide) may include introducing mutations in the sequence by deletion, insertion, substitution, or a combination thereof in the amino acid sequence of the protein (peptide) or the polynucleotide sequence encoding the protein (peptide) to reduce the activity of the protein (peptide), or by replacing the amino acid sequence or polynucleotide sequence with a modified sequence to reduce activity, but is not limited thereto. Sequence modification may be achieved, for example, by inserting a polynucleotide having the modified sequence into a chromosome through homologous recombination, but is not limited thereto. In one specific embodiment, the protein (peptide) may be inactivated by introducing a mutation in the polynucleotide sequence encoding the protein (peptide) to form a stop codon, but the modification is not limited thereto.

[0108] 5) Modifying the start codon or 5'-UTR of a gene encoding the protein (peptide) may include, for example, replacing it with another start codon that has a lower protein (peptide) expression rate compared to the endogenous start codon, or modifying it to encode an RBS sequence that has a lower protein (peptide) expression rate compared to the endogenous ribosome binding site (RBS) sequence, but is not limited thereto.

[0109] 6) The introduction of an antisense oligonucleotide (e.g., antisense RNA) that binds complementary to the transcript of the gene encoding the protein (peptide) can be achieved, but is not limited to, the reference [Weintraub, H. et al., Antisense-RNA as a molecular tool for genetic analysis, Reviews - Trends in Genetics, Vol. 1(1) 1986].

[0110] 7) Adding a sequence complementary to the SD sequence upstream of the Shine-Dalgarno (SD) sequence of the gene encoding the protein (peptide) to form a secondary structure that prevents ribosome attachment, which may prevent or slow down mRNA translation, but is not limited thereto.

[0111] In addition, 8) reverse transcription engineering (RTE), which adds a promoter to be reverse transcribed to the 3' end of the open reading frame (ORF) of the polynucleotide sequence encoding the protein (peptide), can be achieved by generating an antisense nucleotide complementary to the transcript of the gene encoding the peptide to inhibit the translation of the protein (peptide) and reduce its activity.

[0112] 9) A method for regulating the cellular localization of the protein (peptide) may be to target the protein (peptide) to a specific organelle or a specific space within the cell. For example, it may be by adding or removing a leader sequence that plays a role in protein targeting, to target the protein (peptide) to the periplasm or cytoplasm, but is not limited thereto.

[0113] This reduction in the activity of a protein (peptide) compared to the activity or concentration of a protein (peptide) expressed in a wild-type host cell (microorganism) or a host cell (microorganism) before modification may lead to a reduction in the activity or concentration of the corresponding protein (peptide), but is not limited thereto.

[0114] The modification of some or all of the polynucleotides in the host cell (microorganism) disclosed herein can be induced by: (a) using homologous recombination or genome editing methods, homologous recombination using a vector for chromosome insertion, genome editing using an engineered nuclease (e.g., CRISPR-Cas9), and / or (b) treatment with light (e.g., ultraviolet light and radiation) and / or chemicals, but not limited thereto.

[0115] In one embodiment, the microorganism disclosed herein may lack part or all of the gene sequence encoding the ABC3 protein, but is not limited thereto.

[0116] As used herein, “increased” protein (peptide) activity refers to an increase in the activity of a protein (peptide) compared to its endogenous activity in the host cell (microbe). This increase may be used interchangeably with terms such as “activation,” “upregulation,” “overexpression,” or “enhancement.”

[0117] The host cell (microorganism) can be a prokaryotic or eukaryotic microorganism.

[0118] Increased protein (peptide) activity may include the host cell (microbe) exhibiting protein (peptide) activity that it does not possess itself, or exhibiting improved protein (peptide) activity compared to its endogenous activity or pre-modified activity.

[0119] For example, "exhibiting protein (peptide) activity that it does not possess on its own" or "exhibiting improved protein (peptide) activity" may be due to "the introduction of a protein (peptide)," but is not limited to this. The introduction of a protein (peptide) may be due to the introduction of a gene encoding the protein (peptide) into a host cell (microorganism). For example, a polynucleotide encoding a specific protein (peptide) may be introduced into the chromosome of a host cell (microorganism), or a vector containing a polynucleotide encoding a specific protein (peptide) may be introduced into a host cell (microorganism), thereby exhibiting or enhancing its activity.

[0120] An increase in protein (peptide) activity compared to its endogenous activity refers to an enhanced activity and / or concentration (expression level) of the protein (peptide) in the host cell (microorganism) compared to the activity and / or concentration (expression level) of the protein (peptide) originally present in the unmodified host cell (microorganism) or the unmodified host cell (microorganism).

[0121] Increased protein (peptide) activity can be achieved by introducing exogenous proteins (peptides) or increasing the activity of endogenous proteins (peptides). Whether protein (peptide) activity has increased can be confirmed by the degree of protein (peptide) activity, its expression level, or the increase in the amount of product generated by the protein (peptide) activity.

[0122] Increased protein (peptide) activity can be achieved by a variety of methods well known in the art, and is not limited thereto, as long as the desired protein (peptide) activity can be increased compared to the activity of the host cell (microbe) before modification. Specifically, genetic engineering and / or protein engineering, which are conventional methods in molecular biology and well known to those skilled in the art, can be used, but the methods are not limited thereto (e.g., Sitnicka et al., Functional Analysis of Genes. Advances in Cell Biology. 2010, Vol. 2, pp. 1-16; Sambrook et al., Molecular Cloning 2012, etc.).

[0123] Specifically, the increase in protein (peptide) activity disclosed herein may be due to:

[0124] 1) Increase the intracellular copy number of the polynucleotide encoding the protein (peptide);

[0125] 2) Modify the gene expression regulatory region on the chromosome that encodes the protein (peptide) (e.g., introduce mutations into the expression regulatory region, replace it with a sequence that exhibits strong expression induction activity, or insert a sequence that exhibits strong expression induction activity).

[0126] 3) Modify the start codon or 5'-UTR base sequence of the gene transcript (which encodes the protein (polypeptide));

[0127] 4) Modify the amino acid sequence of the protein (peptide) to increase its activity;

[0128] 5) Modify the polynucleotide sequence encoding the protein (peptide) to increase the activity of the protein (peptide) (e.g., modify the polynucleotide sequence of the gene encoding the protein (peptide) so that the gene encodes a protein (peptide) modified to have increased activity).

[0129] 6) Introduce an exogenous protein (peptide) exhibiting the activity of the stated protein (peptide), or an exogenous polynucleotide encoding the protein (peptide);

[0130] 7) Codon optimization of the polynucleotides encoding the protein (peptide);

[0131] 8) By analyzing the tertiary structure of the protein (peptide), select and modify or chemically modify the exposed regions of the protein (peptide);

[0132] 9) Regulating the cellular localization of the protein (peptide); or

[0133] 10) Selected from two or more of the above 1) to 9), but not particularly limited thereto.

[0134] For example,

[0135] 1) Increasing the intracellular copy number of the polynucleotide encoding the protein (peptide) may include introducing the polynucleotide encoding the protein (peptide) into a host cell (microorganism), the polynucleotide being operatively linked to a suitable regulatory sequence in the form of a vector containing the polynucleotide. Alternatively, the method may be achieved by introducing one or two or more copies of the polynucleotide encoding the protein (peptide) into the chromosome of the host cell (microorganism), the polynucleotide being operatively linked to a suitable regulatory sequence. Chromosomal introduction may be performed by introducing a vector capable of inserting the polynucleotide into the host cell (microorganism) chromosome, but is not limited thereto. The vector is as described above. Regarding the polynucleotide sequence encoding the protein, the regulatory sequence may be a natural sequence (of the same origin) or a foreign sequence (derived from a different gene), a variant thereof, or another artificial sequence, and may induce the expression of the polynucleotide in the host cell (microorganism).

[0136] 2) Replacing the gene expression regulatory region (or expression control sequence) on the chromosome encoding the protein (peptide) with a sequence exhibiting strong expression-inducing activity may include, for example, introducing mutations into the sequence through deletion, insertion, substitution, or a combination thereof, thereby further enhancing the expression-inducing activity of the expression regulatory region, or replacing it with a sequence exhibiting stronger expression-inducing activity. The expression regulatory region may include, but is not particularly limited to, promoters, operon sequences, sequences encoding ribosome binding sites, and sequences regulating transcription and translation termination. In one instance, it may include replacing the original promoter with a promoter exhibiting strong expression-inducing activity, but is not limited thereto.

[0137] Examples of known promoters exhibiting strong expression-inducing activity include, but are not limited to, the cj1 to cj7 promoters (US 7662943B2), the lac promoter, the trp promoter, the trc promoter, the tac promoter, the λ 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 (US10273491 B2), the tkt promoter, the yccA promoter, and the TEF promoter.

[0138] 3) Modifying the start codon or 5'-UTR of a gene encoding the protein (peptide) may include, for example, substitution with another start codon that has a higher protein (peptide) expression rate compared to the endogenous start codon, or a modification that results in an RBS sequence that encodes a protein (peptide) expression rate that has a higher protein (peptide) expression rate compared to the endogenous ribosome binding site (RBS) sequence, but is not limited thereto.

[0139] 4) and 5) Modification of the amino acid sequence or polynucleotide sequence of the protein (peptide) may include introducing mutations in the sequence through deletions, insertions, substitutions, or combinations thereof in the amino acid sequence of the protein (peptide) or the polynucleotide sequence encoding the protein (peptide), or by replacing them with modified amino acid sequences or polynucleotide sequences to exhibit increased activity, but is not limited thereto. Sequence modification may be achieved, for example, by inserting a polynucleotide having the modified sequence into the chromosome through homologous recombination, but is not limited thereto.

[0140] 6) The method for introducing exogenous polynucleotides exhibiting the activity of the stated protein (peptide) can be achieved by introducing exogenous polynucleotides encoding a protein (peptide) exhibiting the same / similar activity as the stated protein (peptide) into a host cell (microorganism). The exogenous polynucleotide is not limited by its source or sequence, as long as it exhibits the same / similar activity as the stated protein (peptide). Introduction can be carried out using transformation methods known in the art, appropriately selected by those skilled in the art, and the expression of the introduced polynucleotide in the host cell can produce the stated protein (peptide), thereby increasing its activity.

[0141] 7) Codon optimization can be performed on the polynucleotides encoding the protein (peptide) to increase the transcription or translation of endogenous polynucleotides in the host cell (microorganism) or to optimize the transcription or translation of exogenous polynucleotides in the host cell (microorganism).

[0142] 8) Selecting and modifying or chemically modifying the exposed regions of a protein (peptide) by analyzing its tertiary structure may include, for example, comparing the sequence information of the protein (peptide) to be analyzed with a database storing the sequence information of known proteins, determining candidate template proteins based on sequence similarity, confirming the structure based on this, selecting the exposed sites to be modified or chemically modified, and modifying or chemically modifying them.

[0143] 9) Regulation of the cellular localization of the protein (peptide) may involve targeting the protein (peptide) to specific organelles or specific intracellular spaces. For example, it may include, but is not limited to, targeting the protein (peptide) to the periplasm or cytoplasm by adding or removing a leader sequence that plays a role in the targeting of the protein (peptide).

[0144] This increase in protein (peptide) activity can lead to, but is not limited to, an increase in the activity or concentration of the corresponding protein (peptide) compared to the activity or concentration of the protein (peptide) expressed in wild-type host cells (microorganisms) or unmodified host cells (microorganisms).

[0145] In one embodiment, by reducing the activity of the ABC3 protein and additionally exchanging the promoter sequence of the gene encoding the SNQ2 protein with a TEF promoter, the microorganism of this disclosure can exhibit strong SNQ2 protein expression induction activity.

[0146] In this disclosure, "retinol" refers to a compound known as vitamin A. In one embodiment, retinol can be converted into other retinol compounds (e.g., retinal, retinoic acid, and retinyl esters) by methods known in the art.

[0147] In this disclosure, "retinoids" refers to a group of compounds that are chemically related to or belong to the vitamin A family.

[0148] In one embodiment, the retinoid may be any one selected from the group consisting of retinol, retinaldehyde, retinoic acid, and retinyl esters, but is not limited thereto.

[0149] Additionally, by further including substances (e.g., enzymes) that convert retinol into other retinoids (e.g., retinoic acid and retinyl esters), the microorganisms of this disclosure may have, or already have, an increased capacity to produce retinoids.

[0150] Another aspect of this disclosure provides a method for producing retinol, comprising culturing a Yersinia spp. microorganism with retinol-producing capacity in a culture medium, wherein the activity of the ABC3 protein is reduced compared to its endogenous activity.

[0151] Microorganisms, as described in another aspect.

[0152] In one embodiment, the microorganism may additionally exhibit increased SNQ2 protein activity compared to its endogenous activity, but is not limited thereto.

[0153] As used herein, the term "culture" refers to the cultivation of the microorganisms of this disclosure under appropriately controlled environmental conditions. The cultivation process of this disclosure can be carried out using suitable culture media and cultivation conditions known in the art. Those skilled in the art can readily adapt and use such a cultivation process according to the selected strain. Specifically, the cultivation can be batch, continuous, and / or fed-batch, but is not limited thereto.

[0154] The microorganisms disclosed herein can be cultured under aerobic conditions in a universal culture medium containing appropriate carbon sources, nitrogen sources, phosphorus sources, inorganic compounds, amino acids and / or vitamins, while controlling temperature, pH and other conditions.

[0155] In this disclosure, the carbon source may include carbohydrates such as glucose, saccharose, lactose, fructose, sucrose, maltose, etc.; sugar alcohols such as mannitol, sorbitol, etc.; organic acids such as pyruvic acid, lactic acid, citric acid, etc.; and amino acids such as glutamic acid, methionine, lysine, etc. In addition, natural organic nutrient sources such as starch hydrolysates, molasses, molasses, rice bran, cassava, sugarcane residue, and corn steep liquor may be used. Specifically, carbohydrates such as glucose and sterilized pretreated molasses (i.e., molasses converted to reducing sugars) may be used, and other carbon sources may be used in unlimited quantities and in various ways. These carbon sources may be used alone or in combination of two or more, but are not limited thereto.

[0156] As for the nitrogen source, inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate can be used; amino acids such as glutamic acid, methionine, and glutamine; and organic nitrogen sources such as peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolysate, fish or its decomposition products, and defatted soybean meal or its degradation products can be used. These nitrogen sources can be used alone or in combination of two or more, but are not limited to these.

[0157] The phosphorus source may include potassium dihydrogen phosphate, dipotassium hydrogen phosphate, or their corresponding sodium-containing salts. As for inorganic compounds, sodium chloride, calcium chloride, ferric chloride, magnesium sulfate, ferric sulfate, manganese sulfate, calcium carbonate, etc., may be used. In addition, amino acids, vitamins, and / or suitable precursors may be included. These components or precursors may be added to the culture medium in batches or continuously, but are not limited thereto. However, the culture medium is not limited thereto.

[0158] During the cultivation of the microorganisms disclosed herein, the pH of the culture medium can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid in a suitable manner. Furthermore, foaming can be suppressed during cultivation by using antifoaming agents such as polyethylene glycol fatty acids. Additionally, oxygen or oxygen-containing gases can be injected into the culture medium to maintain an aerobic state, or no gas can be injected, or nitrogen, hydrogen, or carbon dioxide gas can be injected to maintain an anaerobic or micro-aerobic state, but these methods are not limited to these.

[0159] In addition, the culture medium may contain metal salts necessary for growth, such as magnesium sulfate or ferric sulfate. Finally, besides the above-mentioned substances, essential growth substances such as amino acids and vitamins may also be used. Furthermore, suitable precursors may be used in the culture medium. The above-mentioned raw materials may be added to the culture in batches or continuously during the culture process using suitable methods, but are not limited thereto.

[0160] During the cultivation of the microorganisms disclosed herein, the pH of the culture can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid to the culture in a suitable manner. Furthermore, foaming can be suppressed during cultivation by using antifoaming agents such as polyethylene glycol fatty acids. Additionally, oxygen or oxygen-containing gases can be injected into the culture to maintain an aerobic state, or no gas can be injected, or nitrogen, hydrogen, or carbon dioxide gas can be injected to maintain an anaerobic or micro-aerobic state, but these methods are not limited to these.

[0161] In the cultivation process disclosed herein, the cultivation temperature can be maintained between 20°C and 35°C, specifically between 25°C and 35°C. The cultivation time can continue until an amount of useful substance is obtained, and can be approximately 10 hours to 160 hours, approximately 20 hours to 130 hours, approximately 24 hours to 120 hours, approximately 36 hours to 120 hours, approximately 48 hours to 120 hours, approximately 48 hours or longer, or approximately 48 hours, approximately 72 hours or approximately 120 hours, but is not limited thereto.

[0162] The method for producing retinol disclosed herein may further include recovering retinol from the microorganism or the culture medium.

[0163] According to the microbial culture methods disclosed herein, such as batch, continuous, or fed-batch culture methods, the desired retinol can be recovered from the culture medium by using appropriate methods known in the art. For example, centrifugation, filtration, treatment with a crystalline protein precipitant (salting out), extraction, ultrafiltration, dialysis, various types of chromatography (such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, etc.), HPLC, and combinations of these methods can be used, but are not limited thereto.

[0164] Methods for producing retinol may also include a purification process. The purification process can be carried out using suitable methods known in the art.

[0165] In one embodiment, the method for producing retinol disclosed herein uses microorganisms with reduced ABC3 protein activity and retinol secretion capacity. Therefore, the present disclosure can produce retinol without using cell disruption of microorganisms widely used for retinol extraction, or without using dodecane as a solvent, but is not limited thereto.

[0166] The method for producing retinol disclosed herein may further include converting retinol expressed by the microorganisms of this disclosure into retinoids other than retinol; therefore, this disclosure can provide a method for producing retinoids. The method for producing retinoids of this disclosure may further include conversion after culturing the microorganisms or recovering the retinol. Conversion can be carried out using suitable methods known in the art. For example, conversion can be carried out using retinyl acyltransferases, but is not limited thereto.

[0167] In one embodiment, the retinoid other than retinol can be any one selected from the group consisting of retinaldehyde, retinoic acid and retinyl esters, but is not limited thereto, as long as it is included in the retinoid group.

[0168] Another aspect of this disclosure provides a method for preparing a *Yersinia* microorganism capable of retinol production, comprising reducing the activity of the ABC3 protein in the *Yersinia* microorganism capable of retinol production.

[0169] Another aspect of this disclosure provides a method for increasing retinol secretion, which includes reducing the activity of the ABC3 protein in Yersinia spp. microorganisms capable of retinol production.

[0170] In one embodiment, the method for preparing Yersinia spp. microorganisms and the method for increasing retinol secretion may further include increasing the activity of SNQ2 protein in the microorganisms, but are not limited thereto.

[0171] Steps to reduce ABC3 protein activity may include modifying Yersinia spp. microorganisms such that the activity of ABC3 protein is reduced compared to its endogenous activity, while steps to increase SNQ2 protein activity may include modifying Yersinia spp. microorganisms such that the activity of SNQ2 protein is increased compared to its endogenous activity, as described elsewhere.

[0172] Another aspect of this disclosure provides a composition for producing retinol, comprising one or more of a Yersinia spp. microorganism and its cultures in which the activity of the ABC3 protein is reduced compared to its endogenous activity and is capable of producing retinol.

[0173] In one embodiment, the microorganism may additionally exhibit increased SNQ2 protein activity compared to its endogenous activity, but is not limited thereto.

[0174] The compositions disclosed herein may also contain any suitable excipients commonly used in compositions for the production of retinol. Such excipients may be, for example, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers, or isotonic agents, but are not limited thereto.

[0175] Additionally, this disclosure may provide a composition for producing retinoids by further comprising a substance (e.g., an enzyme such as retinyl acyltransferase) for converting retinol into retinoids other than retinol.

[0176] ABC3 protein, Yersinia species with reduced ABC3 protein activity, SNQ2 protein, Yersinia species with increased SNQ2 protein activity, retinol and retinoids, as described elsewhere.

[0177] Another aspect of this disclosure provides the use of a Yersinia spp. microorganism or a culture thereof for retinol production, wherein the activity of the ABC3 protein in the microorganism is reduced compared to its endogenous activity.

[0178] Another aspect of this disclosure provides the use of a Yersinia genus microorganism in which the activity of the ABC3 protein is reduced compared to its endogenous activity.

[0179] In one embodiment, the microorganism may additionally exhibit increased SNQ2 protein activity, but is not limited thereto, in relation to the use of retinol production and the use of retinoid production.

[0180] ABC3 protein, Yersinia species with reduced ABC3 protein activity, SNQ2 protein, Yersinia species with increased SNQ2 protein activity, retinol and retinoids, as described elsewhere.

[0181] [Modes for Implementing the Invention]

[0182] This disclosure will be described in detail by way of examples. However, these examples are for illustrative purposes only, and the scope of the invention is not limited to these examples. Furthermore, technical matters not described in this specification can be fully understood and readily implemented by those skilled in the art or similar fields.

[0183] Example 1. Construction of a Yarrowia spp. strain with reduced ABC3 activity for retinol production.

[0184] In the retinol-producing Yersinia strain KCCM13294P (CC08-2050, Park et al., MetabEng.2022, Jun 6;73:26-37), the protein activity of ABC3 (YALI0B02544) was reduced.

[0185] To this end, the ORF sequence (SEQ ID NO: 2) of the ABC gene (YALI0B02544g) was obtained based on the nucleotide sequence registered in KEGG (Kyoto Encyclopedia of Genes and Genomes). Furthermore, a deletion cassette for the ABC3 gene (YALI0B02544g) was constructed using the URA3 gene (SEQ ID NO: 4) of *Yarrowia lipolytica* as a selection marker. Genomic DNA from CC08-2050 was then 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 SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, and SEQ ID NO: 11 and SEQ ID NO: 12, respectively. The PCR conditions consisted of the following: denaturation at 95°C for 1 minute, annealing at 55°C for 1 minute, and polymerization at 72°C for 2 minutes, repeated for 35 cycles. The obtained DNA fragments were assembled into individual cassettes by overlap extension PCR. The cassettes thus constructed were introduced into strain CC08-2050 using a heat shock method (D.-C. Chen et al., Appl Microbiol Biotechnol, 1997), and colonies were obtained on uracil-deficient solid medium (YLMM1). Using primers of SEQ ID NO: 13 and SEQ ID NO: 14, colonies were cultured on 5-FOA solid medium at 30°C for 3 days to confirm that the cassettes were inserted into the genome, and the URA3 marker was recovered by obtaining colonies growing on 5-FOA solid medium. The final strain thus obtained with reduced ABC3 protein activity was named CC08-2507.

[0186] [Table 1]

[0187]

[0188] The YLMM1 and 5-FOA culture media used above have the following composition:

[0189] <Yarrowia lipolyticis basal medium 1 (YLMM1)>

[0190] Glucose 20 g / L, amino acid-free yeast nitrogen base 6.7 g / L, uracil-free yeast selective auxotrophic medium (Synthetic Drop-out Medium) supplement 2 g / L, agar 15 g / L

[0191] <5-Fluororhodic acid (5-FOA) medium>

[0192] Glucose 20 g / L, amino acid-free yeast nitrogen base 6.7 g / L, uracil-free yeast selective auxotrophic medium supplement 2 g / L, uracil 50 μg / mL, 5-fluoroorotic acid (5-FOA) 1 g / L, agar 15 g / L

[0193] Example 2. Construction of a Yersinia spp. strain with increased ABC3 activity for retinol production.

[0194] The activity of the ABC3 (YALI0B02544) protein in the retinol-producing *Yarrowia* strain KCCM13294P was increased. To replace the promoter of the ABC3 gene (YALI0B02544g), the ORF sequence (SEQ ID NO: 2) of the ABC gene (YALI0B02544g) was obtained based on the nucleotide sequence registered in KEGG (Kyoto Encyclopedia of Genetics and Genomes). Furthermore, a promoter replacement cassette was constructed using the *Yarrowia lipolytica* URA3 gene (SEQ ID NO: 4) as a selection marker. The TEF promoter sequence corresponds to SEQ ID NO: 3. At this point, genomic DNA from CC08-2050 was used as a template, and PCR was performed on the left homologous region, TEF promoter, URA3, repeat region, and right homologous region fragments using primers of SEQ ID NO: 15 and SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22, and SEQ ID NO: 23 and SEQ ID NO: 24, respectively. The PCR conditions consisted of denaturation at 95°C for 1 minute, annealing at 55°C for 1 minute, and polymerization at 72°C for 2 minutes, repeated for 35 cycles. The obtained DNA fragments were assembled into individual cassettes by overlap extension PCR. The constructed cassette was introduced into strain CC08-2050 using a heat shock method (D.-C. Chen et al., Appl Microbiol Biotechnol, 1997), and colonies were obtained on uracil-deficient solid medium (YLMM1). The insertion of the cassette into the genome was confirmed using primers of SEQ ID NO: 25 and SEQ ID NO: 26. The resulting colonies were incubated at 30°C for 3 days on 5-FOA solid medium, and the URA3 marker was recovered by obtaining colonies growing on 5-FOA solid medium. The final strain thus obtained with increased ABC3 protein activity was named CC08-2508.

[0195] [Table 2]

[0196]

[0197] The YLMM1 and 5-FOA culture media used above have the same composition as those described in Example 1.

[0198] Example 3. Construction of a Yersinia spp. strain with increased SNQ2 activity for retinol production.

[0199] The activity of the SNQ2 (YALI0F17996) protein from the retinol-producing *Yarrowia* strain KCCM13294P (CC08-2050) and the CC08-2507 constructed in Example 1 was increased. Therefore, the ORF sequence (SEQ ID NO: 34) of the ABC gene (YALI0F17996g) was obtained based on the nucleotide sequence registered in KEGG (Kyoto Encyclopedia of Genetics and Genomes). Furthermore, the promoter replacement cassette of the SNQ2 gene (YALI0F17996g) was constructed using the *Yarrowia lipolytica* URA3 gene (SEQ ID NO: 4) as a selection marker.

[0200] At this point, genomic DNA from CC08-2050 was used as a template, and PCR was performed on the left homologous region, TEF promoter, URA3, repeat region, and right homologous region fragments using primers of SEQ ID NO: 35 and SEQ ID NO: 36, SEQ ID NO: 37 and SEQ ID NO: 38, SEQ ID NO: 39 and SEQ ID NO: 40, SEQ ID NO: 41 and SEQ ID NO: 42, and SEQ ID NO: 43 and SEQ ID NO: 44, respectively. The PCR conditions consisted of denaturation at 95°C for 1 minute, annealing at 55°C for 1 minute, and polymerization at 72°C for 2 minutes, repeated for 35 cycles. The obtained DNA fragments were assembled into individual cassettes by overlap extension PCR. The constructed cassette was introduced into strain CC08-2050 using a heat shock method (D.-C. Chen et al., Appl Microbiol Biotechnol, 1997), and colonies were obtained on uracil-deficient solid medium (YLMM1). The insertion of the cassette into the genome was confirmed using primers of SEQ ID NO: 45 and SEQ ID NO: 46. The obtained colonies were incubated on 5-FOA solid medium at 30°C for 3 days, and the URA3 marker was recovered by obtaining colonies growing on 5-FOA solid medium. The resulting final KCCM13294P strain with increased SNQ2 protein activity was named CC08-2312.

[0201] In addition, CC08-2050 in the aforementioned process of constructing CC08-2312 was replaced with CC08-2507 to construct the final CC08-2507 strain with increased SNQ2 protein activity, and the final strain was named CC08-2522.

[0202] [Table 3]

[0203]

[0204] The YLMM1 and 5-FOA culture media used above have the same composition as those described in Example 1.

[0205] Example 4. Comparative evaluation of retinol production / secretion capacity of strains with reduced ABC3 and strains with increased ABC3.

[0206] To compare the retinol production and secretion capabilities of the strains (CC08-2507, CC08-2508, CC08-2312, and CC08-2522) constructed in Examples 1-3 with those of the control strain (CC08-2050), a shake-flask evaluation was performed. Each strain was inoculated into 250 mL corner-baffle flasks to an initial OD of 2, containing 25 mL of YPDLU medium supplemented with 0.05% BHT (3,5-di-tert-4-butylhydroxytoluene), and cultured at 30°C and 200 rpm for 48 hours. The YPDLU medium used had the following composition:

[0207] <ypdlu>

[0208] Glucose 60 g / L, Bacto peptone 20 g / L, yeast extract 10 g / L, uracil 1 g / L, leucine 1 g / L, 1 M phosphate buffer (pH 7.0) 100 ml / L

[0209] To evaluate the growth level of each strain, OD values ​​were measured at a wavelength of 600 nm using a spectrophotometer. The concentrations of extracellularly secreted retinol and retinal were quantified by mixing 0.1 mL of the supernatant (in which microbial cells were removed from the culture medium) after culture was completed with 0.9 mL of acetone containing 4% BHT (Sigma) and then analyzing the mixture using HPLC.

[0210] The analyzed OD values ​​and concentrations of retinol and retinaldehyde are shown in Table 4 below. Figure 1 The illustration is shown in the image.

[0211] [Table 4]

[0212]

[0213] Comparing the values ​​of CC08-2507 and CC08-2508 (experimental strains) with those of CC08-2050 (control strain) in Table 4, when ABC3 protein activity decreased, the retinol secreted by CC08-2507 increased by 1.37 times compared to the control (CC08-2050), while the retinaldehyde secretion remained at the same level. However, when ABC3 protein activity increased, the retinol secreted by CC08-2508 was only 0.83 times that of the control (CC08-2050), while the retinaldehyde secretion remained at the same level.

[0214] Additionally, comparing the values ​​of CC08-2507, CC08-2312, and CC08-2522 (experimental strains) with those of CC08-2050 (control strain) in Table 4, the retinol secreted when ABC3 protein activity was reduced (CC08-2507) and when SNQ2 protein activity was increased (CC08-2312) increased by 1.37-fold and 1.66-fold, respectively, compared to the control (CC08-2050), while the secreted retinaldehyde remained at the same level. Simultaneously, when both ABC3 and SNQ2 protein activities were reduced (CC08-2522), the retinol secreted increased by 2.15-fold compared to the control (CC08-2050). Retinol secretion was further increased compared to either reducing ABC3 activity alone or increasing SNQ2 activity alone. Therefore, the simultaneous application of reduced ABC3 activity and increased SNQ2 activity can further increase retinol production / secretion compared to applying each alone.

[0215] Based on the above results, it was confirmed that ABC3 in *Yersinia* microorganisms plays a role in retinol secretion, and that reducing ABC3 activity increases retinol secretion in microorganisms. Furthermore, the synergistic effect of reduced ABC3 activity and increased SNQ2 activity on retinol secretion was demonstrated.

[0216] As described above, those skilled in the art will understand that the present invention can be implemented in other specific forms without departing from the technical spirit or essential characteristics of the invention. Therefore, the above embodiments should be interpreted as exemplary and not as limiting the scope of this disclosure. It should be understood that all changes or modifications derived from the definitions and scope of the claims and their equivalents fall within the scope of this disclosure.

[0217] PCT / RO / 134 form < / ypdlu>

Claims

1. A Yersinia species capable of producing retinol, wherein 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 comprises the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having 60% or more identity with it.

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

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

6. The microorganism of claim 1, wherein the microorganism additionally exhibits increased SNQ2 protein activity compared to its endogenous activity.

7. The microorganism according to claim 6, wherein the SNQ2 protein comprises the amino acid sequence of SEQ ID NO: 33 or an amino acid sequence having 60% or more identity with it.

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

9. The microorganism according to claim 1, wherein the Yersinia genus microorganism is Yersinia lipophila.

10. The microorganism according to claim 1, wherein the microorganism has increased retinol production or retinol secretion capacity compared with unmodified Yersinia spp. microorganisms.

11. A method for producing retinol, comprising culturing in a culture medium a Yersinia spp. microorganism with retinol-producing capacity in which the activity of ABC3 protein is reduced compared to its endogenous activity.

12. The method of claim 11, wherein the microorganism additionally exhibits increased SNQ2 protein activity compared to its endogenous activity.

13. The method of claim 11, further comprising recovering retinol from the culture medium or microorganisms.

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

15. A method for preparing a *Yersinia* microorganism capable of retinol production, comprising reducing the activity of ABC3 protein in the *Yersinia* microorganism capable of retinol production.

16. The method of claim 15, further comprising increasing the activity of the SNQ2 protein in the microorganism.

17. A method for increasing retinol secretion, comprising reducing the activity of ABC3 protein in Yersinia spp. microorganisms capable of retinol production.

18. The method of claim 17, further comprising increasing the activity of the SNQ2 protein in the microorganism.

19. A composition for producing retinol comprising one or more of a Yersinia spp. microorganism with retinol-producing capacity, wherein the activity of the ABC3 protein is reduced compared to its endogenous activity.

20. The composition of claim 19, wherein the microorganism additionally exhibits increased SNQ2 protein activity compared to its endogenous activity.

21. Use of a Yersinia spp. microorganism or a culture thereof for the production of retinol, wherein the activity of the ABC3 protein in the microorganism is reduced compared to its endogenous activity.

22. The use according to claim 21, wherein the microorganism additionally exhibits increased SNQ2 protein activity compared to its endogenous activity.

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