Microorganisms with enhanced SNQ2 protein activity and retinoid-producing ability, and a method for producing retinoids using the same.

Abstract

This application relates to a Yarowia liporitica microorganism with enhanced SNQ2 protein activity and retinoid-producing ability, a method for producing retinoids using the same, a composition for retinoid production, its use in retinoid production, and a method for producing the said microorganism.

Description

Technical Field

[0001] This application relates to a Yarrowia lipolytica microorganism having retinoid-producing ability with enhanced activity of the SNQ2 protein, a method for producing retinoids using the same, a composition for producing retinoids, use in retinoid production, and a method for producing the microorganism.

Background Art

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

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

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Non-Patent Documents

[0005]

Non-Patent Document 1

Non-licensed Document 2

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

Non-licensed Document 7

Non-licensed Document 8

Non-licensed literature 9

Non-licensed literature 10

Non-licensed Document 11

[0006] The problem that this application aims to solve is to provide a Yarowia liporitica microorganism with enhanced SNQ2 protein activity and retinoid-producing ability, a method for producing retinoids using the same, a composition for retinoid production, its use in retinoid production, and a method for producing the said microorganism. [Means for solving the problem]

[0007] This application provides a Yarrowia lipolytica microorganism having the ability to produce retinoids, with enhanced activity of the SNQ2 protein.

[0008] This application provides a method for producing retinoids, which includes the step of culturing the microorganism in a medium.

[0009] This application provides a method for producing the microorganism.

[0010] This application provides a method for increasing the excretion of retinoids in the microorganism.

[0011] This application provides a composition for producing retinoids, which includes any one or more of the microorganism and its culture.

[0012] This application provides the use of the microorganism and / or its culture for producing retinoids.

Advantages of the Invention

[0013] Retinoids can be produced using the microorganism of this application.

Brief Description of the Drawings

[0014] [Figure 1] It is a diagram showing the retinoid concentration measured in the flask culture evaluation of the microorganism.

Modes for Carrying Out the Invention

[0015] This can be explained in more detail as follows: On the other hand, each description and embodiment disclosed in this application applies to each other description and embodiment. That is, all combinations of the various elements disclosed in this application fall within the scope of this application. Furthermore, the categories of this application are not limited by the specific descriptions described below. In addition, numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated as references in this specification as a whole, to more clearly explain the level of the technical field to which this application belongs and the content of this application.

[0016] One aspect of this application provides a Yarrowia lipolytica microorganism with enhanced SNQ2 protein activity and retinoid-producing ability.

[0017] The "SNQ2 protein" of this application is a type of ATP-binding cassette transporter protein, and ATP-binding cassette transporters can be used interchangeably with ABC transporters.

[0018] As an example, the SNQ2 protein of this application may have ABC transporter activity.

[0019] In this application, "ABC transporter" refers to a protein that is part of the transport system superfamily, commonly possesses an ATP-binding cassette domain, and uses the energy obtained from the hydrolysis of ATP to perform biological functions (for example, transporting various substrates across membranes).

[0020] In this application, the SNQ2 protein may enhance retinoid production and / or efflux capacity. For example, the increase in retinoid production capacity may be due to an increase in retinoid efflux capacity, but is not limited thereto.

[0021] As one example, the SNQ2 protein of this application may be enhanced in activity in the microorganism Yarowia liporitica, increasing the retinoid production and / or efflux capacity of said microorganism.

[0022] The SNQ2 protein in this application refers to the endogenous SNQ2 protein of the microorganism Yarowia liporitica, the SNQ2 protein derived from Yarowia liporitica, SEQ ID NO: 1, or a protein containing an amino acid sequence having 70% or more homology thereto.

[0023] As an example, the SNQ2 protein of this application may contain, have, or consist of an amino acid sequence having 90% or more homology or identity with SEQ ID NO: 1, or substantially consist of said amino acid sequence.

[0024] Specifically, the amino acid sequence of the SNQ2 protein of this application may be a protein sequence having the activity of the ABC transporter encoded by the SNQ2 gene. The amino acid sequence can be obtained from various databases, such as the NCBI's GenBank, which is a known database, but is not limited thereto.

[0025] As one example, the SNQ2 protein of this application may be derived from Yarrowia lipolytica.

[0026] Furthermore, although one embodiment of the SNQ2 protein of this application is described as a protein containing SEQ ID NO: 1, this does not exclude the addition of meaningless sequences before or after the amino acid sequence of SEQ ID NO: 1, or naturally occurring mutations, or silent mutations thereof. It is obvious to those skilled in the art that any protein having the same or corresponding activity as the protein containing the aforementioned amino acid sequence is considered to be the SNQ2 protein of this application.

[0027] Specifically, the SNQ2 protein of this application may include the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence having at least 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: 1. Furthermore, it is obvious that any amino acid sequence having the aforementioned homology or identity and exhibiting the efficacy corresponding to the protein is included within the scope of this application, even if some sequences are deleted, modified, substituted, or added.

[0028] Even if this application describes a polypeptide (including a protein) containing an amino acid sequence described by a specific sequence number, a polypeptide (including a protein) consisting of an amino acid sequence described by a specific sequence number, or a polypeptide (including a protein) having an amino acid sequence described by a specific sequence number, it is obvious that proteins having amino acid sequences in which some sequences are deleted, modified, substituted, conserved substituted, or added may also be used in this application, as long as they have the same or corresponding activity as the polypeptide or protein consisting of the amino acid sequence of said sequence number. For example, this includes cases where the N-terminus and / or C-terminus of the amino acid sequence have an added sequence that does not alter the function of the protein, a naturally occurring mutation, a silent mutation, or a conserved substitution.

[0029] The term "conservative substitution" refers to the replacement of one amino acid with another amino acid having similar structural and / or chemical properties. Such amino acid substitutions generally occur based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues. Typically, conservative substitutions have little to no effect on protein activity.

[0030] In this application, the terms "homology" and "identity" refer to the degree of identical or similarity between two given amino acid sequences or base sequences, and can be expressed as a percentage. The terms homology and identity are often used interchangeably.

[0031] The homology or identity of sequences of conserved polynucleotides or polypeptides (including proteins) is determined by standard sequencing algorithms, which may also be used in conjunction with a default gap penalty established by the program used. Substantially homologous or identical sequences can generally be hybridized under moderate to high stringent conditions with the entire sequence or with portions representing at least about 50%, 60%, 70%, 80%, or 90% of the total length. It is obvious that hybridization also includes hybridization with polynucleotides containing codons in general or codons considering codon degeneracy in polynucleotides.

[0032] Whether any two polynucleotide or polypeptide (including protein) sequences are homologous, similar, or identical can be determined using known computer algorithms such as the "FASTA" program with default parameters, for example, as described in Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]:2444. Alternatively, it can be determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453), as performed in the Needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277) (version 5.0.0 or later) (GCG program package (Devereux, J., et al, Nucleic Acids Research 12:387 (1984)), BLASTP, BLASTN, FASTA (Atschul, [S.] [F.] [ET AL, J MOLEC BIOL 215]:403 (1990); Guide to Huge Computers, Martin J. Bishop, [ED.] Academic Press, San (Including Diego, 1994, and [CARILLO ETA / .](1988) SIAM J Applied Math 48:1073). For example, homology, similarity, or identity can be determined using BLAST or ClustalW from the National Center for Biotechnology Information Databases.

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

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

[0035] The Yarowia lipopolitica microorganism in which the activity of the SNQ2 protein of this application is enhanced may be a microorganism comprising one or more of the following: an SNQ2 protein derived from Yarowia lipopolitica and / or an SNQ2 protein having an amino acid sequence having 70% or more homology with SEQ ID NO: 1; a polynucleotide encoding the same; and a vector containing the polynucleotide.

[0036] In this application, the term "polynucleotide" refers to a polymer of nucleotides in which nucleotide monomers are covalently linked together in a long chain, and is a DNA chain of a certain length or longer.

[0037] The polynucleotide sequence encoding the SNQ2 protein of this application may be referred to as the "SNQ2 gene," and this may include the polynucleotide sequence encoding the amino acid sequence described in Sequence ID No. 1.

[0038] The polynucleotide may undergo various modifications to its coding region, within the limits that do not alter the amino acid sequence of the polypeptide or protein, taking into consideration the degeneracy of the codons or the preferred codons in the organism that intends to express the polypeptide or protein. Specifically, the polynucleotide may consist of, or substantially consist of, SEQ ID NO: 2 or a polynucleotide sequence having 70% or more homology or identity thereto, but is not limited thereto. For example, the polynucleotide may consist of, but is not limited thereto, a base sequence having 70% or more homology or identity with SEQ ID NO: 2, specifically 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0039] Furthermore, the polynucleotides of this application may include, without limitation, any probes, such as sequences that can hybridize under stringent conditions with a complementary sequence to all or part of the polynucleotide base sequence. “Stringent conditions” means conditions that enable specific hybridization between polynucleotides. Such conditions are specifically described in the literature (e.g., J. Sambrook et al., ibid.). For example, the conditions may be such that polynucleotides with high homology or identity hybridize with each other, with 40% or more homology or identity, specifically 90% or more, more specifically 95% or more, 96% or more, 97% or more, 98% or more, and more specifically 99% or more, and do not hybridize with polynucleotides with lower homology or identity, or that the washing conditions are the same as those for normal Southern hybridization, with a salt concentration and temperature equivalent to 60°C, 1×SSC, 0.1% SDS, specifically 60°C, 0.1×SSC, 0.1% SDS, more specifically 68°C, 0.1×SSC, 0.1% SDS, and the washing is performed once, specifically two to three times.

[0040] Hybridization requires that two nucleic acids have complementary sequences, even if mismatches between bases are possible depending on the stringency of the hybridization. The term “complementary” is used to describe the relationships between nucleotide bases that can hybridize with one another. For example, with respect to DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Thus, the polynucleotides of this application may also include not only substantially similar nucleic acid sequences, but also isolated nucleic acid fragments that are complementary throughout the entire sequence.

[0041] Specifically, polynucleotides having homology or identity can be detected using hybridization conditions that include a hybridization step at a Tm value of 55°C, and under the conditions described above. The Tm value may be 60°C, 63°C, or 65°C, but is not limited thereto and can be appropriately adjusted by those skilled in the art depending on the purpose.

[0042] The appropriate stringency for hybridizing polynucleotides depends on the length and degree of complementarity of the polynucleotides, and these variables are well known in the art (J. Sambrook et al., ibid.).

[0043] In this application, “vector” may include a DNA product for inserting a polynucleotide encoding the SNQ2 protein of this application into a host chromosome, or a DNA product comprising a nucleotide sequence of the polynucleotide encoding the SNQ2 protein operably linked to a suitable regulatory region (or regulatory sequence) so as to enable expression of the target polypeptide or protein in a suitable host. The regulatory region may include a promoter capable of initiating transcription, an optional operator sequence for regulating such transcription, a sequence encoding a suitable mRNA-ribosome binding site, and sequences regulating the termination of transcription and decoding. The vector, after being transformed into a suitable host cell, can replicate or function independently of the host genome and integrate into the genome itself.

[0044] The vector of this application may, but is not limited to, be an expression vector for expressing the SNQ2 protein of this application in host cells (including microorganisms).

[0045] Furthermore, the vector of this application may, but is not limited to, an insertion vector for inserting a polynucleotide encoding the SNQ2 protein of this application into a chromosome. The insertion of the polynucleotide into the chromosome may, but is not limited to, any method known in the art, such as homologous recombination. The vector may further include a selection marker for confirming the presence or absence of the chromosomal insertion. The selection marker is for selecting cells transformed with the vector, i.e., confirming the presence or absence of insertion into the target nucleic acid molecule, and may be a marker that confers a selectable phenotype, such as drug resistance, nutritional requirements, resistance to cytotoxic agents, or expression of a surface polypeptide or protein. Transformed cells can be selected so that only cells expressing the selection marker survive or exhibit other phenotypes in an environment treated with the selective agent. The insertion vector may not contain origins of replication necessary for replication in the transformed cells.

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

[0047] In this application, the term "transformation" means introducing a vector containing a polynucleotide encoding the SNQ2 protein of this application into a host cell so that the protein encoded by the polynucleotide can be expressed in the host cell. The transformed polynucleotide may be inserted into or extrachromosomal regions of the host cell, as long as it can be expressed in the host cell. The polynucleotide also includes DNA and / or RNA encoding the protein. The polynucleotide may be introduced into the host cell in any form that allows for expression. For example, the polynucleotide may be introduced into the host cell in the form of an expression cassette, which is a gene structure containing all the elements necessary for its expression. The expression cassette may typically include a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal operably linked to the polynucleotide. The expression cassette may also be in the form of a self-replicating expression vector. The polynucleotide may also be introduced into the host cell in its own form and operably linked to the sequences necessary for expression in the host cell, but is not limited to these forms.

[0048] Furthermore, the term "operably linked" in this application means that the gene sequence is functionally linked to a promoter sequence that initiates and mediates the transcription of the polynucleotide encoding the SNQ2 protein of this application.

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

[0050] In this application, the terms "microorganism" or "strain" include all wild-type microorganisms and microorganisms that have undergone natural or artificial genetic modification, and are microorganisms in which a particular mechanism has been weakened or strengthened due to causes such as the insertion of external genes or the enhancement or inactivation of the activity of endogenous genes, and may include microorganisms that undergo genetic modification for the SNQ2 enhancement or product production of this application.

[0051] The microorganism of this application is a Yarowia lipopolitica microorganism capable of producing retinoids. The term "Yarowia lipopolitica microorganism capable of producing retinoids" may be used interchangeably with "Yarowia lipopolitica microorganism that produces retinoids."

[0052] The microorganism described in this application can produce retinoids without using microbial cell disruption, which is widely used for retinoid extraction, or without using dodecane as a solvent.

[0053] The microorganism of this application may be a Yarowia liporitica microorganism in which the activity of the SNQ2 protein is enhanced and the retinoid production and / or efflux capacity is increased.

[0054] In the microorganisms of this application, the increase in retinoid production capacity may be due to an increase in retinoid efflux capacity, but is not limited thereto.

[0055] The microorganisms of this application may, but are not limited to, selectively excreting retinoids.

[0056] As one example, the microorganism of this application may selectively excrete retinoids among β-carotenes and retinoids.

[0057] The microorganisms of this application may be those with enhanced endogenous SNQ2 protein activity, microorganisms naturally possessing the ability to produce SNQ2 protein or retinoids, parent strains lacking the ability to produce SNQ2 protein or retinoids into which the SNQ2 protein of this application, which has enhanced activity compared to the endogenous SNQ2 protein, has been introduced, or parent strains possessing the ability to produce SNQ2 protein or retinoids into which the SNQ2 protein of this application has been further introduced.

[0058] For example, the microorganisms of this application are cells or microorganisms transformed with a polynucleotide encoding the SNQ2 protein of this application, thereby enhancing the activity of the SNQ2 protein. For the purposes of this application, the microorganisms of this application may include all microorganisms that can produce retinoids containing the SNQ2 protein of this application.

[0059] The microorganisms of this application may have increased retinoid efflux capacity compared to Yarowia liporitica microorganisms that have retinoid production capacity but do not have enhanced SNQ2 protein activity.

[0060] For example, the microorganisms of this application may be naturally occurring wild-type microorganisms, microorganisms capable of producing retinoids, and / or recombinant strains in which the activity of the SNQ2 protein of this application is enhanced and retinoid production and / or efflux capacity is increased by further introduction of the polynucleotide encoding the SNQ2 protein of this application into a microorganism containing the SNQ2 protein. The recombinant strains with increased retinoid production and / or efflux capacity may be, but are not limited to, naturally occurring wild-type microorganisms or microorganisms in which the activity of the SNQ2 protein of this application is not enhanced or which do not overexpress the SNQ2 protein.

[0061] For example, the microorganism not enhanced with the SNQ2 protein of this application, which is used as a target strain for comparing the presence or absence of increased retinoid production and excretion capacity, may be, but is not limited to, CC08-2050 (KCCM13294P, Ref. Park et al., Metabolic engineering 2022;73:26-37). The deposited strain name of the CJ2050 strain described in the aforementioned reference (Park et al., Metabolic engineering 2022;73:26-37) is the CC08-2050 strain of this application, and the CC08-2050 strain of this application and the CJ2050 strain are identical to each other.

[0062] As an example, a recombinant strain with increased retinoid production and / or efflux capacity may have an improvement of approximately 0.001% or more, or 0.01% or more, compared to the retinoid production and / or efflux capacity of the parent strain or non-myxoid microorganism before mutation, but is not limited to these values ​​as long as it has a positive increase compared to the production and / or efflux capacity of the parent strain or non-myxoid microorganism before mutation. The term "about" includes, but is not limited to, all values ​​within a range equivalent to or similar to the value following the term "about".

[0063] In this application, the term "non-myxoid microorganism" does not exclude strains containing naturally occurring mutations in microorganisms, but rather means the wild-type or native strain itself, or a strain before its characteristics are altered by genetic mutations due to natural or artificial factors. For example, the non-myxoid microorganism means a strain in which the SNQ2 protein of this application is not enhanced, or before it is enhanced. The term "non-myxoid microorganism" may be used interchangeably with "pre-deformation strain," "pre-deformation microorganism," "non-mutant strain," "non-myxoid strain," "non-mutant microorganism," or "reference microorganism."

[0064] The microorganisms having retinoid production ability according to this application may be microorganisms in which polynucleotides encoding lycopene cyclase / phytoene synthase (crtYB) and phytoene desaturase (crtI) proteins have been introduced, so that microorganisms that do not inherently have retinoid production ability become retinoid production ability, or so that the retinoid production ability of microorganisms that already have retinoid production ability is further enhanced, and which exhibit the activity of these proteins or whose activity of these proteins is enhanced. The lycopene cyclase / phytoene synthase or phytoene desaturase may be proteins derived from Xanthophyllomyces dendrorhous, but are not limited to these as long as they are proteins that exhibit the same or similar activity. As a specific example, the lycopene cyclase / phytoen synthase or phytoendesaturase may consist of or contain the amino acid sequence of SEQ ID NO: 11 or SEQ ID NO: 13, respectively, but may also consist of or contain an amino acid sequence that exhibits activity corresponding to the lycopene cyclase / phytoen synthase or phytoendesaturase while having at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology or identity with the aforementioned amino acid sequence. Furthermore, it is obvious that proteins with partial sequence deletions, alterations, substitutions, or additions are also included in lycopene cyclase / phytoen synthase or phytoendesaturase, as long as they possess the aforementioned homology or identity and exhibit activity corresponding to the lycopene cyclase / phytoen synthase or phytoendesaturase. As a specific example, the polynucleotide encoding the lycopene cyclase / phytoen synthase or phytoendesaturase may consist of or contain the sequence of Sequence ID No. 12 or Sequence ID No. 14, respectively.The polynucleotide may be subjected to various modifications in the coding region, taking into consideration the degeneracy of the codon or the codon preferred in the microorganism of this application, within the limits that the amino acid sequence is not altered. Specifically, the polynucleotide may consist of, but is not limited to, a base sequence having homology or identity with the sequence of SEQ ID NO: 12 or SEQ ID NO: 14 by 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and less than 100%.

[0065] The microorganisms of this application may be microorganisms in which a polynucleotide encoding a geranylgeranyl pyrophosphate synthase (GGPPS) protein has been introduced into the microorganism so that a microorganism that does not inherently have the ability to produce retinoids becomes capable of producing retinoids, or so that a microorganism that has the ability to produce retinoids is further enhanced, thereby exhibiting geranylgeranyl pyrophosphate synthase activity, or microorganisms in which geranylgeranyl pyrophosphate synthase activity has been enhanced. The geranylgeranyl pyrophosphate synthase may be a protein derived from Haematococcus pluvialis, but is not limited to this as long as it is a protein that exhibits the same or similar activity. As a specific example, the geranylgeranyl pyrophosphate synthase may consist of or contain the amino acid sequence of SEQ ID NO: 15, but may also consist of or contain an amino acid sequence that exhibits activity corresponding to the geranylgeranyl pyrophosphate synthase while having at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology or identity with the aforementioned amino acid sequence. Furthermore, it is obvious that proteins in which some sequences are deleted, modified, substituted, or added are also included in the geranylgeranyl pyrophosphate synthase, as long as they exhibit activity corresponding to the geranylgeranyl pyrophosphate synthase while having the aforementioned homology or identity. Furthermore, as a specific example, the polynucleotide encoding geranylgeranyl pyrophosphate synthase may have or contain the sequence of Sequence ID No. 16. The polynucleotide may be subjected to various modifications in the coding region, within the limits that do not alter the amino acid sequence, taking into consideration the degeneracy of the codon or the codon preferred in the microorganism of this application.Specifically, the polynucleotide may consist of, but is not limited to, a base sequence having homology or identity with the sequence of Sequence ID No. 16 of 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and less than 100%.

[0066] Furthermore, the microorganisms of this application may be microorganisms in which a polynucleotide encoding a β-carotene 15,15'-oxygenase (BLH) protein has been introduced into the microorganism so that microorganisms that do not inherently have the ability to produce retinoids have the ability to produce retinoids, or so that the retinoid production ability of microorganisms that do have the ability to produce retinoids is further enhanced, thereby exhibiting β-carotene 15,15'-oxygenase activity, or microorganisms in which β-carotene 15,15'-oxygenase activity has been enhanced. The β-carotene 15,15'-oxygenase may be a protein derived from the marine bacterium 66A03 (Uncultured marine bacterium 66A03), but is not limited thereto as long as it is a protein that exhibits the same or similar activity. As a specific example, the β-carotene 15,15'-oxygenase may consist of or contain the amino acid sequence of Sequence ID No. 17, but it may also consist of or contain an amino acid sequence that exhibits activity corresponding to the β-carotene 15,15'-oxygenase while having at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology or identity with the aforementioned amino acid sequence. Furthermore, it is obvious that proteins in which some sequences are deleted, modified, substituted, or added are also included in the β-carotene 15,15'-oxygenase, as long as they exhibit activity corresponding to the β-carotene 15,15'-oxygenase while having the aforementioned homology or identity. Furthermore, as a specific example, the polynucleotide encoding β-carotene 15,15'-oxygenase may have or contain the sequence of Sequence ID No. 18. The polynucleotide may be subjected to various modifications in the coding region, within the limits that do not alter the amino acid sequence, taking into consideration the degeneracy of the codon or the codon preferred in the microorganism of this application.Specifically, the polynucleotide may consist of, but is not limited to, a base sequence having homology or identity with the sequence of Sequence ID No. 18 of 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and less than 100%.

[0067] As one example, CC08-2050 (KCCM13294P), one of the microorganisms capable of producing retinoids in this application, contains a lycopene cyclase / phytoen synthase containing the amino acid sequence of SEQ ID NO: 11; a phytoendesaturase containing the amino acid sequence of SEQ ID NO: 13; a geranylgeranyl pyrophosphate synthase containing the amino acid sequence of SEQ ID NO: 15; and a β-carotene 15,15'-oxygenase containing the amino acid sequence of SEQ ID NO: 17.

[0068] In this application, the term "enhancement" of protein activity means that the activity of a protein increases compared to its endogenous activity. This enhancement may be used interchangeably with terms such as activation, upregulation, overexpression, and increase.

[0069] Here, activation, enhancement, upregulation, overexpression, and increase can all include exhibiting activity that was not originally present, or exhibiting improved activity compared to endogenous activity or activity before modification.

[0070] Therefore, microorganisms expressing the SNQ2 protein or microorganisms into which the SNQ2 protein has been introduced can also be described as microorganisms with enhanced activity compared to endogenous SNQ2 proteins.

[0071] The term "intrinsic activity" refers to the activity of a specific protein that was originally present in the parent strain or non-myxoid microorganism before the trait change, when the trait has been altered due to genetic mutation caused by natural or artificial factors. This can be used interchangeably with "activity before the trait change." When a protein's activity is described as "enhanced," "upregulated," "overexpressed," or "increased" compared to its intrinsic activity, it means that the activity and / or concentration (expression level) of the specific protein that was originally present in the parent strain or non-myxoid microorganism before the trait change has improved.

[0072] The aforementioned enhancement can be achieved by introducing an exogenous protein (however, in the case of enhancing the activity of the SNQ2 protein, the introduction means the introduction of a protein derived from Yarowia liporitica that exhibits SNQ2 activity; a protein containing the amino acid sequence of Sequence ID No. 1 or a sequence having 70% or more homology thereto; and / or the polynucleotide encoding it), or by enhancing the activity and / or concentration (expression level) of an endogenous protein. Whether or not the activity of the protein has been enhanced can be confirmed by an increase in the activity level, expression level, or amount of product excreted from the protein.

[0073] The enhancement of the activity of the aforementioned protein can be achieved by applying a variety of methods well known in the field, and is not limited as long as it enhances the activity of the target protein compared to the microorganism before deformation. Specifically, it may, but is not limited to, methods of gene engineering and / or protein engineering that are routine methods of molecular biology and are well known to ordinary technicians in this field (e.g., Sitnicka et al. Functional Analysis of Genes. Advances in Cell Biology. 2010, Vol. 2. 1-16, Sambrook et al. Molecular Cloning 2012, etc.).

[0074] Specifically, the enhancement of the protein activity of this application is 1) Increase in the intracellular copy number of polynucleotides that code for proteins; 2) Replacement of gene expression regulatory regions on protein-coding chromosomes with highly active sequences; 3) Modifications of the nucleotide sequence encoding the start codon or 5'-UTR region of a protein-coding gene transcript; 4) Modification of the amino acid sequence of the protein so as to enhance its activity; 5) Modification of the polynucleotide sequence encoding the protein so as to enhance the protein's activity (for example, modification of the polynucleotide sequence of the protein gene so as to encode a protein modified to enhance its activity); 6) Introduction of a foreign protein exhibiting protein activity or a foreign polynucleotide encoding it (however, in the case of enhancing the activity of the SNQ2 protein, the introduction means the introduction of a protein derived from Yarowia liporitica exhibiting that activity; a protein containing the amino acid sequence of Sequence ID No. 1 or having 70% or more homology thereto; and / or a polynucleotide encoding it); 7) Codon optimization of polynucleotides that code for proteins; 8) Analyze the tertiary structure of the protein, select exposed sites, and deform or chemically modify them; 9) Regulation of the intracellular localization of proteins (polypeptides); or 10) A combination of two or more selected from items 1) to 9) above is also acceptable, but is not particularly limited thereto.

[0075] for example, The increase in the intracellular copy number of the polynucleotide encoding the protein (polypeptide) described in 1) above may be achieved by introducing a vector containing the polynucleotide encoding the protein (polypeptide) operably linked to an appropriate regulatory sequence into a host cell (microorganism). Alternatively, one or more copies of the polynucleotide encoding the protein (polypeptide) operably linked to an appropriate regulatory sequence may be introduced into the chromosomes of the host cell (microorganism). The introduction into the chromosomes can be achieved by introducing a vector into the host cell (microorganism) that can insert the polynucleotide into the chromosomes of the host cell (microorganism), but is not limited to this. The vector is as described above. The regulatory sequence may be native to the encoding polynucleotide sequence (of the same origin), or a foreign (derived from another gene) sequence, or a variant thereof, or another artificial sequence, and may induce the expression of the polynucleotide in the host cell (microorganism).

[0076] As one example, in the enhancement of SNQ2 protein activity according to this application, the regulatory sequence of the gene encoding the SNQ2 protein may be, but is not limited to, a TEF promoter.

[0077] 2) The replacement of a gene expression regulatory region (or expression regulatory sequence) on a chromosome encoding a protein (polypeptide) with a more potent sequence may, for example, involve introducing a mutation in the sequence by deletion, insertion, substitution, or a combination thereof, or replacing it with a sequence having stronger activity, in order to further increase the activity of the expression regulatory region. The expression regulatory region may include, but is not limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence that regulates the termination of transcription and decoding. For example, the original promoter may be replaced with a potent promoter, but is not limited to this.

[0078] Examples of well-known strong promoters include, but are not limited to, the cj1-cj7 promoter (US Registered Patent US 7662943 B2), lac promoter, trp promoter, trc promoter, tac promoter, lambda phage PR promoter, PL promoter, tet promoter, gapA promoter, SPL7 promoter, SPL13 (sm3) promoter (US Registered Patent US 10584338 B2), O2 promoter (US Registered Patent US 10273491 B2), tkt promoter, yccA promoter, and TEF promoter.

[0079] As one example, in the Yarowia liporitica microorganism of this application, the existing promoter (native promoter) of the SNQ2 coding gene may be replaced with the TEF promoter, but this is not limited to this example.

[0080] The above 3) Modification of the base sequence of the start codon or the region encoding the 5'-UTR of a gene encoding a protein (polypeptide) may be, for example, a modification that encodes another start codon with a higher protein (polypeptide) expression rate than the endogenous start codon, or a modification that encodes an RBS sequence with a higher protein (polypeptide) expression rate than the endogenous RBS (ribosome binding site) sequence, but is not limited thereto.

[0081] The modification of the amino acid sequence or polynucleotide sequence of the protein (polypeptide) described in 4) and 5) above may be, but is not limited to, introducing a sequence mutation by deletion, insertion, substitution, or combination thereof into the amino acid sequence of the protein (polypeptide) or the polynucleotide sequence encoding the protein (polypeptide), or replacing it with an amino acid sequence or polynucleotide sequence modified to increase its activity. Such replacement can be carried out, for example, by inserting a polynucleotide into the chromosome by homologous recombination, but is not limited to these methods.

[0082] The introduction of a foreign polynucleotide exhibiting protein (polypeptide) activity as described in 6) above may also be the introduction of a foreign polynucleotide encoding a protein (polypeptide) exhibiting the same or similar activity as the aforementioned protein (polypeptide) into a host cell (microorganism). The foreign polynucleotide is not restricted in its origin or sequence, as long as it exhibits the same or similar activity as the aforementioned protein (polypeptide). The method used for the introduction can be appropriately selected by those skilled in the art from known transformation methods, and the introduction of the polynucleotide in the host cell can generate a protein (polypeptide) and increase its activity.

[0083] The codon optimization of the polynucleotide encoding the protein (polypeptide) described in 7) above may be codon optimization of the endogenous polynucleotide so that transcription or translation is increased within the host cell (microorganism), or the codon of the exogenous polynucleotide may be optimized so that optimized transcription or translation occurs within the host cell (microorganism).

[0084] 8) Analyzing the tertiary structure of a protein (polypeptide) and selecting exposed sites to deform or chemically modify may, for example, involve comparing the sequence information of the protein (polypeptide) to be analyzed with a database containing sequence information of known proteins to determine candidate template proteins according to the degree of sequence similarity, confirming the structure based on that, and selecting exposed sites to deform or chemically modify.

[0085] The intracellular positional regulation of a protein (polypeptide) described in 9) above may involve targeting the protein (polypeptide) to a specific organelle or specific intracellular space within the cell. For example, it may involve targeting the periplasm or cytoplasm through the addition or removal of a leader sequence that functions to target the protein (polypeptide), but is not limited thereto.

[0086] Such an increase in the activity of a protein (polypeptide) may be due to an increase in the activity or concentration of the corresponding protein (polypeptide) relative to the activity or concentration of the protein (polypeptide) expressed in the wild-type or pre-deformation host cell (microorganism), or it may be due to an increase in the amount of product resulting from the activity of the protein (polypeptide).

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

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

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

[0090] Another aspect of this application provides a method for producing retinoids, comprising the step of culturing the microorganism of this application in a culture medium.

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

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

[0093] The Yarowia liporitica microorganism described in this application can be cultured in a conventional culture medium containing a suitable carbon source, nitrogen source, phosphorus source, inorganic compounds, amino acids and / or vitamins, under aerobic conditions with controlled temperature, pH, etc.

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

[0095] The nitrogen sources may include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate; and organic nitrogen sources such as amino acids like glutamic acid, methionine, and glutamine, peptone, NZ-amine, meat extracts, yeast extracts, malt extracts, corn maceration, casein hydrolysates, fish or their decomposition products, defatted soy cake or its decomposition products. These nitrogen sources may be used individually or in combination of two or more, and are not limited to these uses.

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

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

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

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

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

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

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

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

[0104] As one example, the retinoid production method of this application uses microorganisms with enhanced SNQ2 protein activity and retinoid efflux ability. Therefore, this application can produce retinoids without using microbial cell disruption, which is widely used for retinoid extraction, or without using dodecane as a solvent, but is not limited thereto.

[0105] The retinoid production method of this application may further include a step of converting the retinol expressed by the microorganism of this application into a retinoid other than retinol. In the retinoid production method of this application, the conversion step may further be included after the culturing step or the recovery step. The conversion step can be carried out using a suitable method known in the art. For example, the conversion can be carried out using retinol acyltransferase, but is not limited thereto.

[0106] As an example, the retinoid may be any one selected from the group consisting of retinal, retinoic acid, and retinyl ester, but is not limited to these as long as it is included in the category of retinoids.

[0107] Another aspect of this application provides a method for producing retinoid-producing Yarowia lipopolitica microorganisms, comprising the step of enhancing the activity of the SNQ2 protein in the retinoid-producing Yarowia lipopolitica microorganism.

[0108] Another aspect of this application provides a method for increasing retinoid efflux, comprising the step of enhancing the activity of the SNQ2 protein in a Yarowia liporitica microorganism capable of producing retinoids.

[0109] The method for increasing the excretion of retinoids may also be a method for increasing the excretion of retinoids in Yarowia liporitica microorganisms that have the ability to produce retinoids.

[0110] The step of enhancing the activity of the SNQ2 protein may also be a step of deforming the Yarowia liporitica microorganism to express the SNQ2 protein, as described in other sections.

[0111] The SNQ2 protein, its activity enhancement, microorganisms, and retinoids mentioned above are as described in other sections.

[0112] Another aspect of this application provides a composition for retinoid production comprising a Yarowia liporitica microorganism or culture thereof in which the activity of the SNQ2 protein is enhanced.

[0113] The composition of this application may further include any buffer and / or suitable excipients commonly used in retinoid production compositions, such excipients may include, but are not limited to, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers, or isotonic agents.

[0114] The SNQ2 protein, its activity enhancement, microorganisms, and retinoids mentioned above have been described in other sections.

[0115] Another aspect of this application provides the use of the SNQ2 protein of this application for increasing retinoid production.

[0116] Another aspect of this application provides the use of the Yarowia liporitica microorganism, in which the activity of the SNQ2 protein of this application is enhanced, for retinoid production.

[0117] The SNQ2 protein, its activity enhancement, microorganisms, and retinoids mentioned above have been described in other sections. [Examples]

[0118] The present application will be described in more detail below with reference to experimental examples. However, the following embodiments are merely preferred embodiments for illustrative purposes of the present application and are not intended to limit the scope of the rights of this application. On the other hand, technical matters not described herein can be fully understood and easily implemented by a person of ordinary skill who is skilled in the art of this application or a similar art.

[0119] Example 1. DPP1 deficiency in a retinoid-producing Y. lipolytica strain. In the retinoid-producing Yarovia lipolytica strain KCCM13294P, the DPP1 (YALI0C11297g) gene was deleted from the genome to secure a site for the insertion of a foreign gene. For this purpose, the ORF sequence (SEQ ID NO: 19) of DPP1 (YALI0C11297g) was secured based on the nucleotide sequence registered in KEGG (Kyoto Encyclopedia of Genes and Genomes). Furthermore, a DPP1 (YALI0C11297g) deletion cassette was constructed using the Yarovia lipolytica URA3 gene (SEQ ID NO: 22) as a selection marker.

[0120] To this end, using the genomic DNA of KCCM13294P as a template, PCR was performed on the left homologous region, URA3, repeat region, and right homologous region fragments using primers for SEQ ID NOs. 24 and 25, 26 and 27, 28 and 29, and 30 and 31, respectively, as shown in Table 1. The PCR conditions were denaturation at 95°C for 1 minute; annealing at 55°C for 1 minute; and polymerization at 72°C for 2 minutes, repeated 35 times. The resulting DNA fragments were then prepared as a single cassette via overlap extension PCR. The cassette thus prepared was introduced into the KCCM13294P strain using the heat shock method (D.-C. Chen et al., Appl Microbiol Biotechnol, 1997), and colonies formed on uracil-free solid medium (YLMM1) were obtained. Colonies in which cassette insertion into the genome was confirmed using primers for SEQ ID NO: 32 and SEQ ID NO: 33 were cultured in 5-FOA solid medium at 30°C for 3 days, and the URA3 marker was recovered by obtaining colonies grown in 5-FOA solid medium.

[0121] The final strain lacking DPP1 obtained through this process was named CC08-2373.

[0122] [Table 1]

[0123] The YLMM1 medium and 5-FOA medium used, as mentioned above, had the following compositions.

[0124] < Yarrowia lipolytica minimal media 1 (YLMM1)> Glucose 20g / L, Yeast nitrogen base without amino acids 6.7g / L, Yeast Synthetic Drop-out Medium Supplements without uracil 2g / L, Agar 15g / L

[0125] <5-Fluoroorotic Acid (5-FOA) Medium> Glucose 20 g / L, Yeast nitrogen base without amino acids 6.7 g / L, Yeast Synthetic Drop-out Medium Supplements without uracil 2 g / L, Uracil 50 μg / mL, 5-Fluororotic acid (5-FOA) 1 g / L, Agar 15 g / L

[0126] Example 2. SNQ2 enhancement of Y. lipolytica strains producing retinoids. To enhance the expression of endogenous SNQ2 based on the CC08-2373 strain prepared in Example 1, the existing SNQ2 promoter (native promoter) was replaced with a TEF promoter. For this purpose, the ORF sequence (SEQ ID NO: 2) and promoter region sequence (SEQ ID NO: 20) of SNQ2 (YALI0F17996g) were secured based on the nucleotide sequence registered in KEGG (Kyoto Encyclopedia of Genes and Genomes). Furthermore, an SNQ2-enhanced cassette was prepared using the TEF promoter (SEQ ID NO: 21) and the URA3 gene of Yarowia liporitica (SEQ ID NO: 22), which is used as a selection marker.

[0127] For this purpose, using genomic DNA from CC08-2373 as a template, PCR was performed on the left homologous region, TEF promoter, URA3, repeat region, and right homologous region fragments using primers from Table 2: SEQ ID NOs. 34 and 35, 36 and 37, 38 and 39, 40 and 41, and 42 and 43, respectively. The PCR conditions were denaturation at 95°C for 1 minute; annealing at 55°C for 1 minute; and polymerization at 72°C for 2 minutes, repeated 35 times. The resulting DNA fragments were then assembled into a single cassette via overlap extension PCR.

[0128] [Table 2]

[0129] The cassettes prepared in this manner were introduced into the CC08-2373 strain using the heat shock method (D.-C. Chen et al., Appl Microbiol Biotechnol, 1997), and colonies formed in a uracil-free solid medium (YLMM1; identical composition to the YLMM1 medium in Example 1) were obtained. Colonies in which cassette insertion into the genome was confirmed using primers of SEQ ID NO: 44 and SEQ ID NO: 45 were cultured in 5-FOA solid medium at 30°C for 3 days, and the URA3 marker was recovered by obtaining colonies grown in 5-FOA solid medium (identical composition to the 5-FOA medium in Example 1). The final strain obtained through this process, with enhanced SNQ2, was named CC08-2374.

[0130] Example 3. Enhancement of PDR10 derived from S. cerevisiae with a Y. lipolytica strain producing retinoids. We decided to enhance PDR10 (hereinafter, Sc.PDR10) based on the CC08-2373 strain prepared in Example 1. To this end, we obtained the polynucleotide sequence of PDR10 derived from S. cerevisiae based on the sequence registered in NCBI (National Center for Biotechnology Information Search database) (GenBank: KAG2512237.1), and performed codon optimization to suit Y. lipolytica via http: / / genscript.com. Furthermore, we synthesized this gene (SEQ ID NO: 4) through Macrogen, and designed a cassette to be inserted at the position of the DPP1 gene using the TEF promoter (SEQ ID NO: 21), the TDH3 terminator (SEQ ID NO: 23), and the URA3 gene (SEQ ID NO: 22) as a selection marker for its expression.

[0131] To this end, using the synthesized Sc.PDR10 gene and CC08-2373 genomic DNA as templates, PCR was performed on the left homologous region, TEF promoter, Sc.PDR10 ORF, TDH3 terminator, URA3, repeat region, and right homologous region fragments, respectively, using primers for SEQ ID NOs. 46 and 47, 48 and 49, 50 and 51, 52 and 53, 54 and 55, 56 and 57, and 58 and 59, as shown in Table 3. The PCR conditions were denaturation at 95°C for 1 minute; annealing at 55°C for 1 minute; and polymerization at 72°C for 2 minutes, repeated 35 times. The resulting DNA fragments were then assembled into a single cassette via overlap extension PCR.

[0132] [Table 3]

[0133] The cassettes prepared in this manner were introduced into the CC08-2373 strain using the heat shock method, and colonies formed on a uracil-free solid medium (YLMM1; identical in composition to the YLMM1 medium in Example 1) were obtained. Colonies in which cassette insertion into the genome was confirmed using primers of SEQ ID NO: 32 and SEQ ID NO: 33 were streaked onto 5-FOA solid medium (identical in composition to the 5-FOA medium in Example 1) and cultured at 30°C for 3 days to obtain colonies formed on the 5-FOA solid medium, thereby recovering the URA3 marker. The final strain expressing Sc.PDR10 obtained through this process was named CC08-2488.

[0134] Example 4. Expression of SNQ2 derived from S. cerevisiae in a Y. lipolytica strain that produces retinoids. Based on the CC08-2373 strain prepared in Example 1, we decided to express SNQ2 derived from S. cerevisiae (hereinafter, Sc.SNQ2). To this end, we obtained the polynucleotide sequence of S. cerevisiae-derived SNQ2 based on the sequence registered in the NCBI (National Center for Biotechnology Information Search database) (NCBI Reference Sequence: NP_010294.1) and performed codon optimization to suit Y. lipolytica via http: / / genscript.com. Furthermore, we synthesized the gene through Macrogen (SEQ ID NO: 6), and for expression, we designed a cassette to be inserted at the position of the DPP1 gene using the TEF promoter (SEQ ID NO: 21), the TDH3 terminator (SEQ ID NO: 23), and the URA3 gene (SEQ ID NO: 22) as a selection marker.

[0135] For this purpose, the synthesized Sc.SNQ2 gene and CC08-2373 genomic DNA were used as templates, and PCR was performed on the left homologous region, TEF promoter, Sc.SNQ2 ORF, TDH3 terminator, URA3, repeat region, and right homologous region fragments using primers as shown in Table 4: SEQ ID NOs. 46 and 47, SEQ ID NOs. 48 and 60, SEQ ID NOs. 61 and 62, SEQ ID NOs. 63 and 53, SEQ ID NOs. 54 and 55, SEQ ID NOs. 56 and 57, and SEQ ID NOs. 58 and 59, respectively. The PCR conditions were denaturation at 95°C for 1 minute; annealing at 55°C for 1 minute; and polymerization at 72°C for 2 minutes, repeated 35 times. The resulting DNA fragments were then assembled into a single cassette via overlap extension PCR.

[0136] [Table 4]

[0137] The cassettes prepared in this manner were introduced into the CC08-2373 strain by heat shock, and colonies formed on a uracil-free solid medium (YLMM1; identical in composition to the YLMM1 medium in Example 1) were obtained. Colonies in which cassette insertion into the genome was confirmed using primers SEQ ID NO: 32 and SEQ ID NO: 33 were streaked onto 5-FOA solid medium (identical in composition to the 5-FOA medium in Example 1) and cultured at 30°C for 3 days. The URA3 marker was recovered by obtaining colonies formed on the 5-FOA solid medium. The final strain expressing Sc.SNQ2 obtained through this process was named CC08-2492.

[0138] Example 5. Expression of Escherichia coli-derived MsbA in a Y. lipolytica strain producing retinoids. Based on the CC08-2373 strain prepared in Example 1, we decided to express MsbA (hereinafter referred to as Ec.MsbA) derived from Escherichia coli str. K-12 substr. MG1655. To this end, we obtained the polynucleotide sequence of E. coli-derived MsbA based on the sequence registered in NCBI (National Center for Biotechnology Information Search database) (GenBank: AAC74000.1) and performed codon optimization to suit Y. lipolytica via http: / / genscript.com. Furthermore, we synthesized this gene through Macrogen (SEQ ID NO: 8), and for expression, we designed a cassette to be inserted at the position of the DPP1 gene using the TEF promoter (SEQ ID NO: 21), the TDH3 terminator (SEQ ID NO: 23), and the URA3 gene (SEQ ID NO: 22) as a selection marker.

[0139] For this purpose, the synthesized Ec.MsbA gene and CC08-2373 genomic DNA were used as templates, and PCR was performed on the left homologous region, TEF promoter, Ec.MsbA ORF, TDH3 terminator, URA3, repeat region, and right homologous region fragments using primers as shown in Table 5: SEQ ID NOs. 46 and SEQ ID NOs. 47, SEQ ID NOs. 48 and SEQ ID NOs. 64, SEQ ID NOs. 65 and SEQ ID NOs. 65 and SEQ ID NOs. 67 and SEQ ID NOs. 53, SEQ ID NOs. 54 and SEQ ID NOs. 55 and SEQ ID NOs. 58 and SEQ ID NOs. The PCR conditions were denaturation at 95°C for 1 minute; annealing at 55°C for 1 minute; and polymerization at 72°C for 2 minutes, repeated 35 times. The resulting DNA fragments were then assembled into a single cassette via overlap extension PCR.

[0140] [Table 5]

[0141] The cassettes prepared in this manner were introduced into the CC08-2373 strain by heat shock, and colonies formed on a uracil-free solid medium (YLMM1; identical in composition to the YLMM1 medium in Example 1) were obtained. Colonies in which cassette insertion into the genome was confirmed using primers SEQ ID NO: 32 and SEQ ID NO: 33 were streaked onto 5-FOA solid medium (identical in composition to the 5-FOA medium in Example 1) and cultured at 30°C for 3 days to obtain colonies formed on the 5-FOA solid medium, thereby recovering the URA3 marker. The final strain expressing Ec.MsbA obtained through this process was named CC08-2376.

[0142] Example 6. Expression of Salmonella enterica-derived MsbA in a Y. lipolytica strain producing retinoids. Based on the CC08-2373 strain prepared in Example 1, we decided to express MsbA (hereinafter, Se.MsbA) derived from Salmonella enterica serovar. Typhimurium. To this end, we obtained the polynucleotide sequence of S. enterica-derived MsbA based on the sequence registered in the NCBI (National Center for Biotechnology Information Search database) (NCBI Reference Sequence: WP_000551246.1) and performed codon optimization to suit Y. lipolytica via http: / / genscript.com. Furthermore, we synthesized this gene through Macrogen (SEQ ID NO: 10), and for expression, we designed a cassette to be inserted at the position of the DPP1 gene using the TEF promoter (SEQ ID NO: 21), the TDH3 terminator (SEQ ID NO: 23), and the URA3 gene (SEQ ID NO: 22) as a selection marker.

[0143] For this purpose, the synthesized Se.MsbA gene and CC08-2373 genomic DNA were used as templates, and PCR was performed on the left homologous region, TEF promoter, Se.MsbA ORF, TDH3 terminator, URA3, repeat region, and right homologous region fragments using primers as shown in Table 6: SEQ ID NOs. 46 and 47, SEQ ID NOs. 48 and 68, SEQ ID NOs. 69 and 70, SEQ ID NOs. 71 and 53, SEQ ID NOs. 54 and 55, SEQ ID NOs. 56 and 57, and SEQ ID NOs. 58 and 59, respectively. The PCR conditions were denaturation at 95°C for 1 minute; annealing at 55°C for 1 minute; and polymerization at 72°C for 2 minutes, repeated 35 times. The resulting DNA fragments were then assembled into a single cassette via overlap extension PCR.

[0144] [Table 6]

[0145] The cassettes prepared in this manner were introduced into the CC08-2373 strain by heat shock, and colonies formed in a uracil-free solid medium (YLMM1; identical in composition to the YLMM1 medium in Example 1) were obtained. Colonies in which cassette insertion into the genome was confirmed using primers of SEQ ID NO: 32 and SEQ ID NO: 33 were streaked onto 5-FOA solid medium and cultured at 30°C for 3 days. The URA3 marker was recovered by obtaining colonies formed in 5-FOA solid medium (identical in composition to the 5-FOA medium in Example 1). The final strain expressing Se.MsbA obtained through this process was named CC08-2377.

[0146] Example 7. SNQ2 deficiency in Y. lipolytica strains producing retinoids. Based on the CC08-2373 strain prepared in Example 1, the ORF sequence was removed to inactivate endogenous SNQ2. To prepare an SNQ2-deficient cassette, genomic DNA from CC08-2373 was used as a template, and PCR was performed on the left homologous region, URA3, repeat region, and right homologous region fragments using primers SEQ ID NOs. 72 and 73, 74 and 75, 76 and 77, and 78 and 79, respectively, as shown in Table 7. The PCR conditions were denaturation at 95°C for 1 minute; annealing at 55°C for 1 minute; and polymerization at 72°C for 2 minutes, repeated 35 times. The resulting DNA fragments were then assembled into a single cassette via overlap extension PCR.

[0147] [Table 7]

[0148] The SNQ2-deficient cassette prepared in this manner was introduced into the CC08-2373 strain by heat shock, and colonies formed in a uracil-free solid medium (YLMM1; identical in composition to the YLMM1 medium in Example 1) were obtained. Colonies in which cassette insertion into the genome was confirmed using primers SEQ ID NO: 80 and SEQ ID NO: 81 were cultured in 5-FOA solid medium (identical in composition to the 5-FOA medium in Example 1) at 30°C for 3 days, and the URA3 marker was recovered by obtaining colonies grown in 5-FOA solid medium. The final strain obtained through this process, which lacked SNQ2, was named CC08-2378.

[0149] Example 8. Comparative evaluation of retinoid efflux capacity of endogenous SNQ2 and exogenous transporter-enhanced strains. Flask evaluations were performed to compare the retinoid production and excretion capabilities of the strains produced through Examples 1-7. The following strains were evaluated at 0.05% each: a retinoid-producing Yarovia liporitica strain (CJ2050; KCCM13294P; control group), a DPP1-deficient strain produced in Example 1 (CC08-2373), an SNQ2-enhanced strain produced in Example 2 (CC08-2374), a Sc.PDR10-expressing strain produced in Example 3 (CC08-2488), a Sc.SNQ2-expressing strain produced in Example 4 (CC08-2492), an Ec.MsbA-expressing strain produced in Example 5 (CC08-2376), a Se.MsbA-expressing strain produced in Example 6 (CC08-2377), and an SNQ2-deficient strain produced in Example 7 (CC08-2378). A 250 ml corner baffled flask containing 25 ml of YPDLU medium containing BHT (3,5-Di-tert-4-butylhydroxytoluene) was inoculated to an initial OD of 2, and incubated at 30°C and 200 rpm for 48 hours. The YPDLU medium used had the following composition.

[0150] <ypdlu> Glucose 40g / L, Bacto peptone 20g / L, Yeast extract 10g / L, Uracil 1g / L, Leucine 1g / L, 1M Phosphate buffer, pH 7.0 100ml / L

[0151] The growth rate of each bacterial strain was measured by measuring the OD value at a wavelength of 600 nm using a spectrophotometer. The concentrations of retinol, retinal, and β-carotene excreted extracellularly from each strain were quantitatively analyzed using an HPLC system after mixing 0.1 ml of the supernatant obtained by removing bacterial cells from the culture medium after the end of cultivation with 0.9 ml of acetone (Sigma) containing 4% BHT.

[0152] The analyzed OD values ​​and concentrations of retinoids and β-carotene are shown in Table 8, and the retinoid concentration is plotted in Figure 1.

[0153] [Table 8]

[0154] A comparison of the values ​​for CC08-2050 (control group) and CC08-2373 (DPP1-deficient strain) in Table 8 confirmed that the DPP1 deficiency due to the insertion of an exogenous gene did not affect retinoid production. Therefore, the CC08-2373 strain was used as a base for evaluation of endogenous SNQ2-enhanced strains of Yarovia liporitica (CC08-2374) and Yarovia liporitica strains with ABC transporters derived from other microorganisms (S. cerevisiae, E. coli, and S. enteria) (CC08-2488, CC08-2492, CC08-2376, CC08-2377).

[0155] As a result, only when the endogenous SNQ2 of Yarowia liporitica was enhanced (CC08-2374; SNQ2-enhanced strain) did the amount of retinol and retinal excreted from the microbial cells increase 2.7 times and 1.4 times, respectively, compared to the control group. In contrast, when SNQ2 was deficient (CC08-2378), the amount of retinol and retinal excreted from the microorganisms decreased to 0.1 times the level of the control group.

[0156] The results suggest that in Yarowia liporitica, the enhancement of its own SNQ2 plays a key role in retinoid efflux. Furthermore, since β-carotene was not analyzed in the culture medium, this suggests that the SNQ2 protein in Yarowia liporitica selectively effluxes retinoids.

[0157] From the above description, a person skilled in the art to which this application pertains will understand that this application can be implemented in other specific forms without altering its technical idea or essential features. In this regard, it should be understood that the embodiments described above are merely illustrative and not limiting. The scope of this application should be interpreted as encompassing all modified or altered forms derived from the meaning and scope of the claims, as described below, and their equivalent concepts, rather than from the above detailed description.

[0158] [Table 9] < / ypdlu>

Claims

1. A Yarrowia lipolytica microorganism with enhanced SNQ2 protein activity and retinoid production capabilities.

2. The microorganism according to claim 1, wherein the SNQ2 protein is a protein containing an amino acid sequence of Sequence ID No. 1 or a sequence having 90% or more homology thereto.

3. The microorganism according to claim 1, wherein the SNQ2 protein is derived from Yarrowia lipolytica.

4. The microorganism according to claim 1, wherein the protein is encoded by the SNQ2 gene of Sequence ID No. 2 or a polynucleotide sequence having 90% or more homology thereto.

5. The microorganism according to claim 1, wherein the microorganism has increased retinoid efflux capacity compared to Yarowia liporitica microorganisms having retinoid production capacity without enhanced SNQ2 protein activity.

6. The microorganism according to claim 1, further comprising a lycopene cyclase / phytoen synthase containing the amino acid sequence of Sequence ID No. 11 or having 90% or more homology thereto.

7. The microorganism according to claim 1, further comprising a phytoendesaturase containing the amino acid sequence of Sequence ID No. 13 or having 90% or more homology thereto.

8. The microorganism according to claim 1, further comprising geranylgeranyl pyrophosphate synthase containing the amino acid sequence of Sequence ID No. 15 or having 90% or more homology thereto.

9. The microorganism according to claim 8, wherein the geranylgeranyl pyrophosphate synthase is derived from Haematococcus pluvialis.

10. The microorganism according to claim 1, further comprising β-carotene 15,15'-oxygenase containing the amino acid sequence of Sequence ID No. 17 or having 90% or more homology thereto.

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

12. A method for producing retinoids, comprising the step of culturing a microorganism according to any one of claims 1 to 11 in a culture medium.

13. The retinoid production method according to claim 12, further comprising the step of recovering retinoids from the cultured medium or microorganisms.

14. The retinoid production method according to claim 12, wherein the production method does not use microbial cell disruption during retinoid extraction, or does not use dodecane as a solvent.

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

16. A method for producing Yarowia lipopolitica microorganisms capable of producing retinoids, comprising the step of enhancing the activity of the SNQ2 protein in the Yarowia lipopolitica microorganisms capable of producing retinoids.

17. A method for increasing retinoid excretion in the Yarowia liporitica microorganism capable of producing retinoids, comprising the step of enhancing the activity of the SNQ2 protein.

18. A composition for retinoid production comprising one or more Yarrowia lipolytica microorganisms and their cultures, which have enhanced SNQ2 protein activity and retinoid production capabilities.

19. Use of Yarowia liporitica microorganisms with enhanced SNQ2 protein activity for retinoid production.

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