Microorganisms producing carotenoids or substances having precursors thereof, containing geranylgeranyl pyrophosphate synthase derived from Dunaliella salina, and a method for producing carotenoids or retinoids using the same

Abstract

The present application relates to a Yarrowia microorganism that expresses Dunaliella salina-derived geranylgeranyl pyrophosphate synthase and has the ability to produce carotenoids or substances whose precursors are carotenoids, a method for producing carotenoids or substances whose precursors are carotenoids using the same, a composition for producing carotenoids or substances whose precursors are carotenoids, and use of the Yarrowia microorganism or a culture thereof for producing carotenoids or substances whose precursors are carotenoids.

Description

[Technical field]

[0001] The present application relates to a Yarrowia microorganism that expresses Dunaliella salina-derived geranylgeranyl pyrophosphate synthase and has the ability to produce carotenoids or substances whose precursors are carotenoids, a method for producing carotenoids or substances whose precursors are carotenoids using the same, a composition for producing carotenoids or substances whose precursors are carotenoids, and use of the Yarrowia microorganism or a culture thereof for producing carotenoids or substances whose precursors are carotenoids. [Background technology]

[0002] Carotenoids and retinoids have various functions in plants and animals, and are therefore used in many industrial fields such as food and feed. In particular, carotenoids such as β-carotene are substances that have been reported to have functions such as removing free radicals, acting as the mother of vitamin A in animals, enhancing the immune system of vertebrates, and reducing the risk of lung cancer. Retinoids are a group of substances chemically related to retinol, which is vitamin A, and are also used in cosmetics and skin disease treatments.

[0003] However, despite these advantages, carotenoids (e.g., β-carotene) and retinoids (e.g., retinol) are not synthesized in the animal body, or even if they are synthesized, the amount synthesized is insufficient. In addition, although attempts have been made to industrially produce them using mutated microorganisms (Patent Document 1), it remains difficult to produce them in high purity.

[0004] For example, in the process of producing a microorganism that produces carotenoids or retinoids, squalene (C30) and other by-products are also produced. Therefore, finding a geranylgeranyl pyrophosphate synthase that contributes to the efficient production of carotenoids or retinoids is essential for increasing their production and reducing squalene produced in competing pathways.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Patent Document 5

Non-Patent Documents

[0006]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Non-licensed literature 9

Non-licensed literature 10

Non-licensed Document 11

Non-licensed Document 12

Non-licensed Document 13

Non-licensed Document 14

Non-licensed Document 15

Non-licensed Document 16

[0007] The problem to be solved by this application is to provide a microorganism that produces carotenoids or substances that have precursors thereof, which contains geranylgeranyl pyrophosphate synthase derived from Dunaliella salina, as well as a method and use for producing carotenoids or retinoids using the same. [Means for solving the problem]

[0008] The present application aims to provide a Yarrowia microorganism that expresses geranylgeranyl pyrophosphate synthase derived from Dunaliella salina and has the ability to produce carotenoids or substances that have precursors thereof.

[0009] Another object of the present application is to provide a method for producing a carotenoid or a substance having a precursor thereof using the Yarrowia microorganism.

[0010] Furthermore, the present application has an object to provide a composition for producing a carotenoid or a substance having a precursor thereof, comprising the Yarrowia microorganism or a culture thereof.

[0011] A further object of the present application is to provide use of the Yarrowia microorganism for producing a carotenoid or a substance having a precursor thereof. Effect of the Invention

[0012] The present application discloses that the production of carotenoids and substances derived from their precursors can be effectively increased by introducing the geranylgeranyl pyrophosphate synthase gene derived from Dunaliella salina into a Yarrowia microorganism. [Brief description of the drawings]

[0013] [Figure 1] FIG. 1 shows the results of flask evaluation of strains into which GGPP synthase genes derived from various microorganisms have been introduced. [Diagram 2] FIG. 1 shows the results of flask evaluation of Mb.BCO-introduced strains. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] These will be described in detail below. Each description and embodiment disclosed in this application is also applicable to other descriptions and embodiments. That is, all combinations of various elements disclosed in this application are included in this application. In addition, this application is not limited to the following specific description. Furthermore, many papers and patent documents are referenced throughout this specification, and citations are provided. The disclosure contents of the cited papers and patent documents are incorporated in their entirety into this specification by reference, thereby more clearly explaining the state of the art to which this application pertains and the contents of this application.

[0015] One aspect of the present application provides a Yarrowia microorganism that expresses geranylgeranyl pyrophosphate synthase derived from Dunaliella salina and has the ability to produce carotenoids or substances containing precursors thereof.

[0016] In the present application, "geranylgeranyl pyrophosphate synthase" refers to an enzyme that catalyzes the synthesis of geranylgeranyl pyrophosphate (GGPP). The substrates of the geranylgeranyl pyrophosphate synthase may be isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP). The geranylgeranyl pyrophosphate synthase is also referred to as "GGS", "GGPPS", "GGPS" or "polypeptide having geranylgeranyl pyrophosphate synthase activity".

[0017] As one example, the microorganism of the present application may be a Yarrowia microorganism that contains a foreign protein, a geranylgeranyl pyrophosphate synthase protein derived from Dunaliella salina, or expresses the geranylgeranyl pyrophosphate synthase protein, and has the ability to produce carotenoids or substances that have carotenoids as precursors.

[0018] The amino acid sequence of the GGPPS protein of the present application may be a protein sequence having geranylgeranyl pyrophosphate synthase activity encoded by the GGPPS gene. The amino acid sequence can be obtained from various databases, such as GenBank of NCBI, which is a publicly known database, but is not limited thereto.

[0019] As an example, the GGPPS protein of the present application is derived from Dunaliella salina, and may be any protein having the same sequence or activity.

[0020] As one example, the GGPPS protein of the present application may comprise, have, consist of, or be essentially composed of the amino acid sequence set forth in SEQ ID NO:91 or an amino acid sequence having 80% or more homology or identity thereto.

[0021] Furthermore, even if a protein containing SEQ ID NO: 91 is described as an example of the GGPPS protein of the present application, this does not exclude meaningless addition of sequences before or after the amino acid sequence of SEQ ID NO: 91, naturally occurring mutations, or silent mutations thereof, and it is clear to a person skilled in the art that any protein having the same or equivalent activity as a protein containing the amino acid sequence is included in the GGPPS protein of the present application.

[0022] Specifically, the GGPPS protein of the present application may comprise the amino acid sequence of SEQ ID NO: 91, or may comprise an amino acid sequence having at least 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: 91. It goes without saying that the present application also includes amino acid sequences in which a portion of the sequence has been deleted, modified, substituted or added, so long as the amino acid sequence has such homology or identity and exhibits an effect equivalent to that of the protein.

[0023] Even if the present application describes "a polypeptide or protein comprising an amino acid sequence represented by a specific SEQ ID NO," "a polypeptide or protein consisting 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 goes without saying that any protein having an amino acid sequence in which a part of the sequence has been deleted, modified, substituted, conservatively substituted, or added is also used in the present application, so long as it has the same or corresponding activity as the polypeptide consisting of the amino acid sequence of the SEQ ID NO. For example, there are those having an addition of a sequence that does not change the function of the protein at the N-terminus, internal, and / or C-terminus of the amino acid sequence, naturally occurring mutations, silent mutations, or conservative substitutions thereof.

[0024] The term "conservative substitution" refers to the replacement of an amino acid with another amino acid having similar structural and / or chemical properties. Such amino acid substitutions may generally be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues. Typically, conservative substitutions have little or no effect on the activity of a polypeptide.

[0025] In this application, "homology" or "identity" means the degree of identity or similarity between two given amino acid or nucleotide sequences, expressed as a percentage. Homology and identity are often used interchangeably.

[0026] Sequence homology or identity of conserved polynucleotides or polypeptides may be determined by standard sequence algorithms, with default gap penalties established by the program used. Substantially homologous or identical sequences will hybridize under moderate or high stringent conditions, generally with the entire sequence or a portion thereof representing at least about 50%, 60%, 70%, 80% or 90% of the entire length. Hybridization, of course, also includes hybridization with polynucleotides having common codons or codons that take into account codon degeneracy in the polynucleotide.

[0027] Whether any two polynucleotide or polypeptide sequences have homology, similarity or identity can be determined using known computer algorithms such as the "FASTA" program with default parameters as in, for example, Non-Patent Document 1. Alternatively, it can be determined using the Needleman-Wunsch algorithm (Non-Patent Document 3), as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Non-Patent Document 2) (version 5.0.0 or later), which includes the GCG program package (Non-Patent Document 4), BLASTP, BLASTN, FASTA (Non-Patent Documents 5, 6 and 7). For example, BLAST or Clustal W from the National Center for Biotechnology Information can be used to determine homology, similarity or identity.

[0028] Homology, similarity or identity of polynucleotides or polypeptides can be determined by comparing sequence information using a GAP computer program such as that disclosed in Non-Patent Document 3, as disclosed in Non-Patent Document 8. In summary, the GAP program defines the number of similar sequence symbols (i.e., nucleotides or amino acids) divided by the total number of symbols in the shorter of the two sequences. Default parameters for the GAP program include (1) a binary comparison matrix (identity takes a value of 1 and non-identity takes a value of 0) and a weighted comparison matrix of Non-Patent Document 10 (or an EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix) as disclosed in Non-Patent Document 9, (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 open penalty of 10 and a gap extension penalty of 0.5), and (3) no penalty for terminal gaps.

[0029] Furthermore, whether any two polynucleotide or polypeptide sequences have homology, similarity or identity can be ascertained by comparing the sequences in a Southern hybridization experiment under defined stringent conditions, and appropriate defined hybridization conditions are within the skill of the art and can be determined by methods well known to those skilled in the art (e.g., Non-Patent Documents 11 and 12).

[0030] In the present application, expression of a protein may be achieved by introducing a gene (polynucleotide) encoding the protein into a microorganism, or by injecting the protein, but is not limited thereto.

[0031] As an example, the microorganism of the present application may be one into which a geranylgeranyl pyrophosphate synthase gene derived from Dunaliella salina has been introduced. Introduction of the geranylgeranyl pyrophosphate synthase gene also includes further enhancing the activity of the gene after the introduction.

[0032] In the present application, "geranylgeranyl pyrophosphate synthase gene" is used interchangeably with "ggs", "ggpps", "ggps", "GGS gene", "GGPPS gene", "GGPS gene", "gene encoding geranylgeranyl pyrophosphate synthase", "polynucleotide encoding geranylgeranyl pyrophosphate synthase" or "polynucleotide encoding a polypeptide having geranylgeranyl pyrophosphate synthase activity".

[0033] The sequence of the geranylgeranyl pyrophosphate synthase gene can be obtained from various databases, such as GenBank of NCBI, which is a publicly known database, but is not limited thereto.

[0034] As one example, the geranylgeranyl pyrophosphate synthase gene derived from Dunaliella salina may include, have, or consist of the base sequence of SEQ ID NO: 1, but is not limited to these.

[0035] As one example, the geranylgeranyl pyrophosphate synthase gene consisting of the base sequence of SEQ ID NO: 1 may be codon-optimized for Yarrowia microorganisms, or more specifically, for Yarrowia lipolytica.

[0036] In the present application, the term "polynucleotide" refers to a nucleotide polymer in which nucleotide monomers are linked in a long chain by covalent bonds, and refers to a DNA chain longer than a certain length.

[0037] The polynucleotide or gene may be modified in various ways in the coding region by codon degeneracy or by taking into consideration the codons preferred in the organism in which the geranylgeranyl pyrophosphate synthase polypeptide is to be expressed, as long as the amino acid sequence of the polypeptide is not changed. The polynucleotide or gene may, for example, include the nucleotide sequence of SEQ ID NO: 1, or may be composed of a nucleotide sequence having a homology or identity thereto of 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, but is not limited thereto.

[0038] Furthermore, the polynucleotide or gene of the present application may be any sequence that encodes the amino acid sequence of SEQ ID NO: 1 by hybridizing under stringent conditions with a probe prepared from a known gene sequence, for example, a complementary sequence to all or a part of the base sequence. The term "stringent conditions" refers to conditions that allow specific hybridization between polynucleotides. Such conditions are specifically described in the literature (e.g., Non-Patent Document 11). For example, the conditions include conditions under which polynucleotides having high homology or identity, such as 40% or more, specifically 90% or more, more specifically 95% or more, 96% or more, 97% or more, 98% or more, and even more specifically 99% or more, hybridize with each other, while polynucleotides having lower homology or identity than that do not hybridize with each other, or conditions under which washing is performed once, specifically 2 to 3 times, at a salt concentration and temperature equivalent to those used in normal Southern hybridization, specifically 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.

[0039] Hybridization requires that two nucleic acids have complementary sequences, even though mismatches between bases are possible depending on the stringency of hybridization. "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. Thus, the polynucleotides of the present application may include isolated nucleic acid fragments that are complementary to the entire sequence, as well as substantially similar nucleic acid sequences.

[0040] Specifically, polynucleotides having homology or identity can be detected using the hybridization conditions described above, in which the hybridization step is performed at a Tm value of 55° C. The Tm value may be, but is not limited to, 60° C., 63° C., or 65° C., and may be appropriately adjusted by those skilled in the art depending on the purpose.

[0041] The appropriate stringency for hybridizing polynucleotides depends on the length of the polynucleotides and the degree of complementation, and variables are known in the art (see Non-Patent Document 13).

[0042] As an example, the microorganism of the present application may contain the Dunaliella salina-derived geranylgeranyl pyrophosphate synthase gene of the present application, or a vector containing a polynucleotide encoding the Dunaliella salina-derived geranylgeranyl pyrophosphate synthase.

[0043] The vector of the present application includes a DNA product comprising a base sequence of a polynucleotide encoding a target polypeptide operably linked to a suitable expression control region (or expression control sequence) so that the target polypeptide can be expressed in a suitable host. The expression control region includes a promoter for initiating transcription, an optional operator sequence for regulating the transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence for regulating the termination of transcription and translation. When transformed into a suitable host cell, the vector can replicate and function independently of the host genome, and is integrated into the genome itself.

[0044] The vector used in the present application is 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 a natural or recombinant state. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, Charon21A, etc. may be used as phage or cosmid vectors, and pDZ, pBR, pUC, pBluescriptII, pGEM, pTZ, pCL, pET, etc. may be used as plasmid vectors. Specifically, pDZ, pDC, pDCM2 (Patent Document 2), pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC, pIMR53 vectors, etc. may be used.

[0045] For example, a polynucleotide encoding a target polypeptide can be inserted into a chromosome by a vector for intracellular chromosome introduction. The polynucleotide can be inserted into a chromosome by any method known in the art, such as, but not limited to, homologous recombination. A selection marker for confirming whether or not it has been inserted into the chromosome may be further included. The selection marker is for selecting cells transformed with the vector, i.e., for confirming whether or not the target nucleic acid molecule has been inserted, and a marker that confers a selectable phenotype such as drug resistance, auxotrophy, resistance to cytotoxic agents, or expression of a surface polypeptide is used. In an environment treated with a selection agent, only cells expressing the selection marker survive or show a different phenotype, so that transformed cells can be selected.

[0046] In the present application, "transformation" means introducing a vector containing a polynucleotide encoding a target polypeptide into a host cell or a microorganism, thereby expressing the polypeptide encoded by the polynucleotide in the host cell. The transformed polynucleotide may be any polynucleotide that is expressed in the host cell, regardless of whether it is inserted into the chromosome of the host cell or located extrachromosomally. The polynucleotide may include DNA and / or RNA that encodes the target polypeptide. The polynucleotide may be introduced in any form as long as it is introduced into the host cell and expressed. For example, the polynucleotide is introduced into the host cell in the form of an expression cassette, which is a genetic structure that contains all elements necessary for its own expression. Typically, the expression cassette contains a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal operably linked to the polynucleotide. The expression cassette may be in the form of a self-replicating expression vector. The polynucleotide may be introduced into the host cell in its own form and operably linked to a sequence necessary for expression in the host cell, but is not limited thereto.

[0047] Additionally, the term "operably linked" means that the polynucleotide sequence is functionally linked to a promoter sequence that initiates and mediates transcription of the polynucleotide encoding the target polypeptide of the present application.

[0048] As an example, a Yarrowia microorganism expressing the Dunaliella salina-derived GGPPS of the present application has enhanced geranylgeranyl pyrophosphate synthase activity compared to a Yarrowia microorganism not expressing the GGPPS, but the present invention is not limited to this example.

[0049] As one example, in a Yarrowia microorganism into which the Dunaliella salina-derived GGPPS gene of the present application has been introduced, geranylgeranyl pyrophosphate synthase activity is enhanced compared to a Yarrowia microorganism into which the Dunaliella salina-derived GGPPS gene has not been introduced, but the present invention is not limited to this example.

[0050] As an example, a Yarrowia microorganism into which geranylgeranyl pyrophosphate synthase encoded by the GGPPS gene derived from Dunaliella salina of the present application has been introduced has enhanced geranylgeranyl pyrophosphate synthase activity compared to a Yarrowia microorganism into which geranylgeranyl pyrophosphate synthase encoded by crtE derived from Xanthophyllomyces dendrorhous or its mutant gene crtEM1, the BTS1 gene derived from Saccharomyces cerevisiae, or the GGS1 gene derived from Yarrowia lipolytica has been introduced, but is not limited to this example.

[0051] In the present application, a "Yarrowia strain" or a "Yarrowia microorganism" includes all wild-type Yarrowia microorganisms and Yarrowia microorganisms that have been genetically modified naturally or artificially, and may be a Yarrowia microorganism in which a specific mechanism has been enhanced by, for example, inserting an exogenous gene or enhancing the activity of an endogenous gene, and may be a Yarrowia microorganism that contains the GGPPS gene derived from Dunaliella salina for producing carotenoids or substances having carotenoids as precursors.

[0052] The microorganism of the present application may be a microorganism containing at least one of the GGPPS protein of the present application, the GGPS gene or polynucleotide encoding the GGPPS protein, and a vector containing the gene or polynucleotide, may be a microorganism modified to express the GGPPS protein or GGPPS gene derived from Dunaliella salina of the present application, may be a microorganism (e.g., recombinant strain) that expresses the GGPPS protein or GGPPS gene derived from Dunaliella salina of the present application, or may be a strain (e.g., recombinant strain) having GGPPS activity derived from Dunaliella salina of the present application, but is not limited thereto.

[0053] The strain of the present application may be a microorganism that naturally has the ability to produce geranylgeranyl pyrophosphate synthase, carotenoids, or substances having precursors thereof, or a microorganism in which the ability to produce geranylgeranyl pyrophosphate synthase, carotenoids, or substances having precursors thereof has been enhanced or imparted by introducing the Dunaliella salina-derived GGPPS protein, gene, polynucleotide, or vector containing them of the present application into a parent strain that does not have the ability to produce geranylgeranyl pyrophosphate synthase, carotenoids, or substances having precursors thereof, but is not limited to these.

[0054] For example, the strain of the present application includes all microorganisms that have been transformed with the Dunaliella salina-derived GGPPS protein, gene, polynucleotide, or vectors containing them to produce carotenoids or substances having precursors thereof, or microorganisms with improved production ability. For example, the strain of the present application may be a recombinant strain in which the production ability of carotenoids or substances having precursors thereof is improved by expressing the Dunaliella salina-derived GGPPS of the present application in a natural wild-type microorganism or a microorganism that produces carotenoids or substances having precursors thereof. The recombinant strain having improved production ability of carotenoids or substances having precursors thereof is a microorganism having improved production ability of carotenoids or substances having precursors thereof compared to a natural wild-type microorganism or a microorganism having unmodified geranylgeranyl pyrophosphate synthase (i.e., a Yarrowia microorganism having a wild-type geranylgeranyl pyrophosphate synthase gene (SEQ ID NO: 10), or a Yarrowia microorganism having no Dunaliella salina-derived geranylgeranyl pyrophosphate synthase gene (SEQ ID NO: 1) introduced therein), but is not limited thereto.

[0055] For example, the strain of the present application having improved ability to produce carotenoids or substances having precursors thereof is a microorganism having improved ability to produce carotenoids or substances having precursors thereof compared to a microorganism not containing GGPPS (e.g., SEQ ID NO: 91) derived from Dunaliella salina, or a Yarrowia microorganism containing CrtE or its mutant CrtEM1 derived from Xanthophyllomyces dendroas, BTS1 derived from Saccharomyces cerevisiae, or GGS1 from Yarrowia lipolytica, but is not limited thereto. For example, the unmodified microorganism that is the subject strain for comparing whether the ability to produce the carotenoids or substances having precursors thereof is improved is the CC08-1023 strain, but is not limited thereto.

[0056] For example, the recombinant strain with improved production ability has an improved β-carotene or retinol production ability of about 0.001% or more, or 0.01% or more, compared to the β-carotene or retinol production ability of the parent strain or unmodified microorganism before mutation, but may be any value that shows an increase in + value compared to the production ability of the parent strain or unmodified microorganism before mutation. The term "about" refers to a range that includes ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and may be any value that is equal to or in a similar range to the value following the term "about," but is not limited thereto.

[0057] In the present application, the term "unmodified microorganism" does not exclude strains containing naturally occurring mutations in microorganisms, but refers to a wild-type strain or a natural strain itself, or a strain before its characteristics are changed by genetic mutation due to natural or artificial factors. For example, the unmodified microorganism refers to a strain before the Dunaliella salina-derived GGPPS of the present application is expressed or introduced. The term "unmodified microorganism" is used interchangeably with "strain before modification", "microorganism before modification", "non-mutated strain", "non-modified strain", "non-mutated microorganism" or "reference microorganism".

[0058] The microorganism of the present application is of the genus Yarrowia, specifically, but not limited to, Yarrowia lipolytica.

[0059] In the microorganism of the present application, modification of a part or the whole of a polynucleotide can be induced by (a) homologous recombination using a vector for chromosomal introduction in the microorganism, or genome editing using engineered nuclease (e.g., CRISPR-Cas9), and / or (b) light such as ultraviolet light or radiation and / or chemical treatment, but is not limited thereto. Methods for modifying a part or the whole of the gene include methods using DNA recombination techniques. For example, a nucleotide sequence or vector containing a nucleotide sequence homologous to a target gene is introduced into the microorganism to cause homologous recombination, thereby deleting a part or the whole of the gene. The introduced nucleotide sequence or vector contains a dominant selection marker, but is not limited thereto.

[0060] The microorganism of the present application may be a microorganism modified to further contain a polynucleotide encoding a lycopene cyclase / phytoene synthase (crtYB) or a phytoene desaturase (crtI) protein, and exhibit the activity of the protein, or may be a microorganism in which the activity of the protein is enhanced. The lycopene cyclase / phytoene synthase or phytoene desaturase is a protein derived from Xanthophyllomyces dendrorhous, but is not limited thereto. In one embodiment, the polynucleotide encoding the lycopene cyclase / phytoene synthase or phytoene desaturase may have or include a base sequence registered in the National Center for Biotechnology Information Search database (NCBI) (GenBank: AY177204.1 or GenBank: AY177424.1), respectively. As an example, the polynucleotide encoding the lycopene cyclase / phytoene synthase or phytoene desaturase may have or include the sequence of SEQ ID NO: 59 or SEQ ID NO: 60, respectively. The polynucleotide may have various modifications in the coding region, without altering the amino acid sequence, due to codon degeneracy or taking into account the codons preferred in the microorganism in which the polypeptide of the present application is to be expressed.Specifically, the polynucleotide has a base sequence that is 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% homologous or identical to the sequence of SEQ ID NO:59 or SEQ ID NO:60, or contains the base sequence, or consists of a base sequence that is 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% homologous or identical to the sequence of SEQ ID NO:59 or SEQ ID NO:60, or is substantially composed of the base sequence, but is not limited to these.

[0061] The microorganism of the present application is a microorganism that has been modified to further contain a polynucleotide encoding a β-carotene 15,15'-oxygenase (BLH) protein and exhibits the protein activity or has enhanced protein activity, but is not limited thereto. β-carotene 15,15'-oxygenase is a protein derived from uncultured marine bacterium 66A03, but is not limited thereto. As an example, the polynucleotide encoding β-carotene 15,15'-oxygenase may have an amino acid sequence (Q4PNI0) registered in UniProtKB (UniProt Knowledgebase), or may include said amino acid sequence. As an example, the polynucleotide encoding β-carotene 15,15'-oxygenase may have a sequence of SEQ ID NO: 12, or may include said sequence. The polynucleotide may be modified in various ways in the coding region to the extent that the amino acid sequence is not changed, taking into consideration the codon degeneracy or the codons preferred in the microorganism in which the polypeptide of the present application is to be expressed. Specifically, the polynucleotide may have a base sequence having 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% homology or identity to the sequence of SEQ ID NO: 12, or may contain the base sequence, or may consist of a base sequence having 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% homology or identity to the sequence of SEQ ID NO: 12, or may essentially consist of the base sequence, but is not limited thereto.

[0062] In the present application, "enhancement" of a polypeptide activity means improving the activity of the polypeptide compared to its endogenous activity. The enhancement is used interchangeably with activation, up-regulation, overexpression, increase, etc. Here, activation, enhancement, up-regulation, overexpression, and increase all include the display of an activity that was not originally present, and the improvement of activity compared to the endogenous activity or activity before modification. The "endogenous activity" means the activity of a specific polypeptide originally possessed by a parent strain or an unmodified microorganism before the trait change when the trait is changed due to genetic mutation caused by natural or artificial factors. This is used interchangeably with "activity before modification." The "enhancement," "up-regulation," "overexpression," or "improvement" of a polypeptide activity compared to its endogenous activity means the improvement of the activity and / or concentration (expression level) of a specific polypeptide originally possessed by a parent strain or an unmodified microorganism before the trait change.

[0063] The enhancement may be achieved by introducing a foreign polypeptide or gene, or by enhancing the activity and / or increasing the concentration (expression level) of an endogenous polypeptide. Whether the activity of the polypeptide has been enhanced can be confirmed by an increase in the level of activity of the polypeptide, the expression level, or the amount of a product produced from the polypeptide.

[0064] To enhance the activity of the polypeptide, various methods well known in the art can be applied, and any method can be used as long as it can enhance the activity of the target polypeptide compared to the unmodified microorganism. Specifically, the method is a conventional method in molecular biology that uses genetic engineering and / or protein engineering well known to those skilled in the art, but is not limited to these (e.g., Non-Patent Documents 14, 15, etc.).

[0065] Specifically, the enhancement of the polypeptide activity of the present application is carried out by 1) increasing the intracellular copy number of a polynucleotide encoding the polypeptide, 2) replacing the expression regulatory region of a gene on a chromosome encoding the polypeptide with a sequence having a stronger activity, 3) modifying the base sequence encoding the start codon or 5'UTR region of a gene transcript encoding the polypeptide, 4) modifying the amino acid sequence of the polypeptide so as to enhance the polypeptide activity, 5) modifying the polynucleotide sequence encoding the polypeptide so as to enhance the polypeptide activity (e.g., modifying the polynucleotide sequence of the polypeptide gene so as to encode a polypeptide modified so as to enhance the polypeptide activity), 6) introducing a foreign polypeptide exhibiting the activity of the polypeptide or a foreign polynucleotide encoding the same, 7) optimizing the codons of the polynucleotide encoding the polypeptide, 8) analyzing the tertiary structure of the polypeptide and selecting and modifying or chemically modifying exposed portions, or 9) a combination of two or more selected from 1) to 8) above, but is not particularly limited thereto.

[0066] More specifically, the above 1) increasing the intracellular copy number of the polynucleotide encoding the polypeptide may be carried out by introducing into the host cell a vector to which the polynucleotide encoding the polypeptide is operably linked, the vector replicating and functioning independently of the host. Alternatively, the above may be carried out by introducing one or more copies of the polynucleotide encoding the polypeptide into a chromosome in the host cell. The introduction into the chromosome is carried out by introducing into the host cell a vector capable of inserting the polynucleotide into a chromosome in the host cell, but is not limited thereto. The vector is as described above.

[0067] The expression regulatory region (or expression regulatory sequence) of a gene on a chromosome encoding a polypeptide is replaced with a sequence having a stronger activity, for example, by generating a mutation in the sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, so that the activity of the expression regulatory region is further enhanced, or by replacing the expression regulatory region with a sequence having a higher activity. The expression regulatory region includes, but is not limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, a sequence regulating the termination of transcription and translation, and the like. For example, the expression regulatory region is replaced with a strong promoter, but is not limited to this.

[0068] Examples of known strong promoters include, but are not limited to, the CJ1 to CJ7 promoters (Patent Document 3), the lac promoter, the trp promoter, the trc promoter, the tac promoter, the lambda phage PR promoter, the PL promoter, the tet promoter, the gapA promoter, the SPL7 promoter, the SPL13 (sm3) promoter (Patent Document 4), the O2 promoter (Patent Document 5), the tkt promoter, the yccA promoter, and the TEFINt promoter.

[0069] The nucleotide sequence encoding the start codon or 5'UTR region of the gene transcription product encoding the polypeptide (3) can be modified, for example, by substituting it with a nucleotide sequence encoding another start codon that has a higher expression rate of the polypeptide compared to the endogenous start codon, but is not limited to this.

[0070] The modification of the amino acid sequence or polynucleotide sequence in 4) and 5) above can be performed by, but is not limited to, generating a mutation in the sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, in the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide, so as to enhance the activity of the polypeptide, or by replacing the sequence with an improved amino acid sequence or polynucleotide sequence having a higher activity, or an improved amino acid sequence or polynucleotide sequence having an improved activity. Specifically, the replacement can be performed by, but is not limited to, inserting the polynucleotide into a chromosome by homologous recombination. Here, the vector used may further include a selection marker for checking whether or not it has been inserted into a chromosome. The selection marker is as described above.

[0071] The above 6) introduction of a foreign polynucleotide exhibiting the activity of the polypeptide may be carried out by introducing into a host cell a foreign polynucleotide encoding a polypeptide exhibiting the same / similar activity as the polypeptide. The foreign polynucleotide may be of any origin or sequence as long as it exhibits the same / similar activity as the polypeptide. The introduction can be carried out by a person skilled in the art by appropriately selecting a known transformation method, and the introduced polynucleotide is expressed in the host cell as described above, thereby producing a polypeptide and improving its activity.

[0072] 7) Optimizing the codons of a polynucleotide encoding a polypeptide may be carried out by optimizing the codons of an endogenous polynucleotide so as to increase transcription or translation in a host cell, or by optimizing the codons of an exogenous polynucleotide so as to perform optimized transcription or translation in a host cell.

[0073] 8) Analyzing the tertiary structure of a polypeptide and selecting and altering or chemically modifying an exposed portion may be carried out, for example, by comparing sequence information of the polypeptide to be analyzed with a database in which sequence information of known proteins is stored, determining candidates for template proteins depending on the degree of sequence similarity, confirming the structure based on the candidates, and selecting and altering or modifying the exposed portion to be altered or chemically modified.

[0074] Such enhancement of polypeptide activity can be achieved by, but is not limited to, improving the activity, concentration, or expression level of the corresponding polypeptide compared to the activity or concentration of the polypeptide expressed in a wild-type or unmodified microbial strain, or by increasing the amount of a product produced from the polypeptide.

[0075] As an example, the microorganism of the present application has GGPPS activity enhanced by introducing a GGPPS gene derived from Dunaliella salina, but is not limited thereto.

[0076] The microorganism of the present application may be capable of producing a carotenoid or a substance having a precursor thereof.

[0077] By "carotenoid" in this application is meant tetraterpenes or derivatives thereof that impart color, such as yellow, in fruits and vegetables.

[0078] In one embodiment, the carotenoid is at least one selected from the group consisting of xanthophyll, carotene, alpha-carotene, beta-carotene, gamma-carotene, phytoene, phytofluene, neurosporene, lutein, lycopene, zeaxanthin, capsanthin, canthaxanthin, and astaxanthin, but is not limited thereto.

[0079] In one embodiment, the carotenoid precursor is a retinoid, but is not limited thereto.

[0080] By "retinoid" in this application is meant chemically the vitamin A group or a group of compounds chemically related thereto.

[0081] In one embodiment, the retinoid is any one selected from the group consisting of retinol, retinal, retinoic acid, and retinyl esters, but is not limited thereto.

[0082] As an example, but not limited to, the microorganism of the present application has a reduced ability to produce by-products.

[0083] In this application, by-product means any substance that is generated other than carotenoids during the production of carotenoids or substances that have carotenoids as precursors. For example, a typical by-product generated during the production of β-carotene is squalene.

[0084] In this application, "squalene" refers to an unsaturated hydrocarbon (C 30 H 50) and is also used in the biosynthesis of steroid hormones, vitamin D, etc. The microorganism of the present application reduces by-products produced in the β-carotene production pathway, specifically, but not limited to, reducing squalene production.

[0085] Another aspect of the present application provides a method for producing a carotenoid or a precursor thereof, comprising the step of culturing a Yarrowia microorganism of the present application in a medium.

[0086] The microorganisms, carotenoids, and substances having the carotenoids as precursors are as described above.

[0087] In the present application, "culturing" refers to growing the Yarrowia microorganism of the present application under appropriately adjusted environmental conditions. The culturing process of the present application can be carried out in a suitable medium and under suitable culture conditions known in the art. Such a culturing process can be easily adjusted and used by a person skilled in the art depending on the strain selected. Specifically, the culturing can be batch, continuous and / or fed-batch culture, but is not limited thereto.

[0088] The Yarrowia microorganism of the present application is cultured in a conventional medium containing suitable carbon sources, nitrogen sources, phosphorus sources, inorganic compounds, amino acids and / or vitamins, etc., under aerobic conditions with controlled temperature, pH, etc.

[0089] In the culture of the present application, the culture temperature is maintained at 20 to 35°C, specifically 25 to 35°C, and the culture is performed for about 10 to 160 hours, about 20 to 130 hours, about 24 to 120 hours, about 36 to 120 hours, about 48 to 120 hours, about 48 hours, about 72 hours, or about 120 hours, but is not limited to these.

[0090] The carotenoids or precursors thereof produced by the culture of the present application are secreted into the medium or remain within the microorganisms.

[0091] The method for producing a carotenoid or a substance having a precursor thereof of the present application may further include a step of preparing a Yarrowia microorganism of the present application, a step of preparing a medium for culturing the microorganism, or a combination thereof (in any order), for example, prior to the culturing step.

[0092] The method for producing a carotenoid or a substance having a precursor thereof according to the present application may further include a step of recovering the carotenoid or a substance having a precursor thereof from a medium used for culturing the Yarrowia microorganism (a medium in which the culture was performed) or from the Yarrowia microorganism according to the present application. The recovery step may be further included after the culturing step.

[0093] The recovery may involve collecting the target retinol using a suitable method known in the art depending on the culture method of the microorganism of the present application, such as batch, continuous, or fed-batch culture. For example, centrifugation, filtration, crystallization, treatment with a protein precipitant (salting out), extraction, cell disruption, ultrasonic disruption, ultrafiltration, dialysis, various chromatographies such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, and affinity chromatography, HPLC, or a combination thereof may be used, and the target retinol can be collected from the medium or the microorganism using a suitable method known in the art.

[0094] In addition, the method for producing a carotenoid or a substance having a precursor thereof of the present application may further include a purification step. The purification may be performed by a suitable method known in the art. For example, when the method for producing a carotenoid or a substance having a precursor thereof of the present application includes both a recovery step and a purification step, the recovery step and the purification step may be performed at different times (or consecutively) regardless of the order, or may be performed simultaneously or integrated into one step, but is not limited thereto.

[0095] The method for producing carotenoids of the present application may further include a step of converting β-carotene produced by the Yarrowia microorganism of the present application into a carotenoid other than β-carotene. In the method for producing carotenoids of the present application, the converting step may be further included after the culturing step or the recovering step. The converting step may be performed by any suitable method known in the art. For example, the conversion may be performed chemically or using an enzyme, but is not limited thereto.

[0096] The method for producing a retinoid of the present application may further include a step of converting the retinol produced by the microorganism of the present application into a retinoid other than retinol. In the method for producing a retinoid of the present application, the converting step may further be included after the culturing step or the recovering step. The converting step may be carried out by any suitable method known in the art. For example, the conversion may be carried out using retinol acyltransferase, but is not limited thereto.

[0097] In one embodiment, the retinoid other than retinol is any one selected from the group consisting of retinal, retinoic acid, and retinyl ester, but may be any one included in the retinoids.

[0098] Yet another aspect of the present application provides a composition for producing a carotenoid or a substance having a precursor thereof, comprising the Yarrowia microorganism of the present application or a culture thereof.

[0099] The microorganisms, carotenoids and substances having precursors thereof are as described above.

[0100] The compositions of the present application may further contain any suitable excipients that are commonly used, such as, but not limited to, preservatives, wetting agents, dispersing agents, suspending agents, buffers, stabilizers, isotonicity agents, etc.

[0101] Yet another aspect of the present application provides the use of a microorganism or a culture thereof of the present application for producing a carotenoid or a precursor thereof.

[0102] The microorganisms, carotenoids and substances having precursors thereof are as described above. EXAMPLES

[0103] The present application will be described in more detail below with reference to examples. However, these examples are merely preferred embodiments illustrating the present application, and the present application is not limited thereto. Note that technical matters not described in this specification are fully understood and easily implemented by a skilled engineer in the technical field of the present application or a similar technical field. EXAMPLES

[0104] Construction of platform strains for producing carotenoids or their precursors Example 1-1. Construction of a strain with crtYB-crtI insertion derived from Xanthophyllomyces dendroas To construct a platform strain for producing carotenoids or their precursors, the lycopene cyclase / phytoene synthase (crtYB) and phytoene desaturase (crtI) genes from Xanthophyllomyces dendroas were inserted into the genome of the high-fat yeast Yarrowia lipolytica CC08-0125 (accession number KCCM12972P).

[0105] The polynucleotide sequence of crtYB was obtained as SEQ ID NO: 59 based on the nucleotide sequence (GenBank: AY177204.1) registered in the National Center for Biotechnology Information Search database (NCBI), and the polynucleotide sequence of crtI was obtained as SEQ ID NO: 60 based on the nucleotide sequence (GenBank: AY177424.1) registered in the NCBI. The polynucleotide sequences of crtYB and crtI were synthesized by Macrogen in the form of TEFINtp-crtYB-CYC1t (SEQ ID NO: 61) and TEFINtp-crtI-CYC1t (SEQ ID NO: 62). A cassette was designed to be inserted into the MHY1 (YALI0B21582g) gene position using the URA3 gene (SEQ ID NO: 63) of Yarrowia lipolytica as a selection marker. Using the synthesized crtYB and crtI genes and the genomic DNA of KCCM12972P as templates, PCR was performed using primers of SEQ ID NO:64 and SEQ ID NO:65, SEQ ID NO:66 and SEQ ID NO:67, SEQ ID NO:68 and SEQ ID NO:69, SEQ ID NO:70 and SEQ ID NO:71, SEQ ID NO:72 and SEQ ID NO:73, and SEQ ID NO:74 and SEQ ID NO:75. The PCR conditions were 35 cycles of denaturation at 95°C for 1 minute, annealing at 55°C for 1 minute, and polymerization reaction at 72°C for 3 minutes. The five resulting DNA fragments were prepared into one cassette by overlap extension PCR.

[0106] The cassette thus prepared was introduced into the KCCM12972P strain by the heat shock method (Non-Patent Document 16), and then colonies formed on a uracil-free solid medium (YLMM1) were obtained. Colonies in which the cassette was confirmed to have been inserted into the genome were spotted on a 5-FOA solid medium using primers of SEQ ID NO: 76 and SEQ ID NO: 77, and cultured at 30°C for 3 days to obtain colonies grown on the 5-FOA solid medium, thereby recovering the URA3 marker.

[0107] [Table 1]

[0108] Example 1-2. Preparation of HMGR-enhanced strain As described above, a cassette was designed to replace the native promoter (SEQ ID NO: 78) site of the hydroxymethylglutaryl-CoA reductase (HMGR) gene of the strain prepared in Example 1-1 with the TEFINt promoter, and PCR was performed using the genomic DNA of KCCM12972P as a template and primers SEQ ID NO: 79 and SEQ ID NO: 80, SEQ ID NO: 81 and SEQ ID NO: 82, SEQ ID NO: 83 and SEQ ID NO: 84, SEQ ID NO: 85 and SEQ ID NO: 86, and SEQ ID NO: 87 and SEQ ID NO: 88. The PCR conditions were 35 cycles of denaturation at 95°C for 1 minute, annealing at 55°C for 1 minute, and polymerization reaction at 72°C for 1 minute and 30 seconds. The five resulting DNA fragments were prepared into one cassette by overlap extension PCR.

[0109] The cassette thus prepared was introduced into the strain prepared in Example 1-1 by the heat shock method, and then colonies formed on a uracil-free solid medium (YLMM1) were obtained. Colonies in which the cassette insertion was confirmed were spotted on a 5-FOA solid medium using primers of SEQ ID NO: 89 and SEQ ID NO: 90, and cultured at 30°C for 3 days to obtain colonies grown on the 5-FOA solid medium, thereby recovering the URA3 marker. The platform strain finally prepared was named CC08-1023. <Yarrowia lipolytica minimal media1(YLMM1)> Glucose 20g / L, Yeast nitrogen base without amino acids 6.7g / L, Yeast Synthetic Drop-out Medium Supplements without uracil 2g / L, Agar 15g / L <5-Fluoroorotic Acid (5-FOA)> Glucose 20g / L, Yeast nitrogen base without amino acids 6.7g / L, Yeast Synthetic Drop-out Medium Supplements without uracil 2g / L, Uracil 50μg / mL, 5-fluoroorotic acid (5-FOA) 1g / L, agar 15g / L

[0110] [Table 2] EXAMPLES

[0111] Construction of a strain with the geranylgeranyl pyrophosphate synthase (GGPP synthase) gene inserted from Dunaliella salina Four types of GGPP synthase genes (hereinafter, GGPPS genes) having different origins were introduced into the genome of the strain CC08-1023 prepared in Example 1 as follows.

[0112] Example 2-1. Construction of a Dunaliella salina-derived GGPPS insertion strain To insert the Dunaliella salina-derived GGPPS gene (hereinafter referred to as Ds.GGPPS) into the chromosome of Yarrowia lipolytica, Ds.GGPPS was codon-optimized according to Non-Patent Document 17 based on the base sequence (GenBank: APW83741.1) registered in the NCBI (National Center for Biotechnology Information Search database) to match Yarrowia lipolytica (SEQ ID NO: 1), and the gene was synthesized in the form of TEFINtp-codon-optimized GGPPS-TDH3t (SEQ ID NO: 4) by requesting Macrogen. Using the URA3 gene of Yarrowia lipolytica (SEQ ID NO: 5) as a selection marker, a cassette to be inserted at the LIG4 (YALI0D21384g) gene position was designed.

[0113] Using the synthesized Ds.GGPPS gene and the genomic DNA of KCCM12972P as templates, PCR was performed on the left homologous region, TEFINt promoter, Ds.GGPPS ORF, TDH3 terminator, URA3, repeat region, and right homologous region fragments using primers 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, SEQ ID NO:23 and SEQ ID NO:24, SEQ ID NO:25 and SEQ ID NO:26, and SEQ ID NO:27 and SEQ ID NO:28 shown in Table 3. The PCR conditions were 35 cycles of denaturation at 95°C for 1 minute, annealing at 55°C for 1 minute, and polymerization reaction at 72°C for 2 minutes. The resulting DNA fragments were prepared into one cassette by overlap extension PCR.

[0114] The cassette thus prepared was introduced into the CC08-1023 strain by the heat shock method, and then colonies formed on a uracil-free solid medium (YLMM1) were obtained. Colonies in which the cassette was confirmed to have been inserted into the genome were smeared on a 5-FOA solid medium using primers of SEQ ID NO:29 and SEQ ID NO:30, and cultured at 30°C for 3 days to obtain colonies formed on the 5-FOA solid medium, thereby removing the URA3 marker.

[0115] [Table 3]

[0116] Example 2-2. Construction of a strain containing the crtE mutant gene from Xanthophyllomyces dendroas To insert the crtE mutant gene crtEM1 (SEQ ID NO: 6, Non-Patent Document 18) derived from Xanthophyllomyces dendroas into the chromosome of Yarrowia lipolytica, we requested Macrogen to synthesize the gene in the form of TEFINtp-crtEM1-TDH3t (SEQ ID NO: 7). Using the URA3 gene (SEQ ID NO: 5) of Yarrowia lipolytica as a selection marker, we designed a cassette to be inserted into the LIG4 (YALI0D21384g) gene position.

[0117] Using the synthesized crtEM1 DNA and KCCM12972P genomic DNA as templates, PCR was performed for the left homologous region, TEFINt promoter, crtEM1 ORF, TDH3 terminator, URA3, repeat region, and right homologous region fragments using primers of sequence numbers 15 and 16, 17 and 31, 32 and 33, 34 and 22, 23 and 24, 25 and 26, and 27 and 28 shown in Table 4.

[0118] The PCR conditions were 35 cycles of denaturation at 95° C. for 1 min, annealing at 55° C. for 1 min, and polymerization at 72° C. for 2 min. The resulting DNA fragments were generated into one cassette by overlap extension PCR.

[0119] The cassette thus prepared was introduced into the CC08-1023 strain by the heat shock method, and then colonies formed on a uracil-free solid medium (YLMM1) were obtained. Colonies in which the cassette was confirmed to have been inserted into the genome were smeared on a 5-FOA solid medium using primers of SEQ ID NO:29 and SEQ ID NO:30, and cultured at 30°C for 3 days to obtain colonies formed on the 5-FOA solid medium, thereby removing the URA3 marker.

[0120] [Table 4]

[0121] Example 2-3. Construction of a Saccharomyces cerevisiae-derived BTS1 insertion strain To insert the BTS1 gene (hereinafter referred to as Sc.BTS1) derived from Saccharomyces cerevisiae into the chromosome of Yarrowia lipolytica, a polynucleotide of SEQ ID NO: 8 was obtained based on the base sequence (YPL069C) registered in KEGG (Kyoto Encyclopedia of Genes and Genomes) for BTS1. The gene was synthesized in the form of TEFINtp-Sc.BTS1-TDH3t (SEQ ID NO: 9) using the BTS1 polynucleotide. A cassette to be inserted into the LIG4 (YALI0D21384g) gene position was designed using the URA3 gene (SEQ ID NO: 5) of Yarrowia lipolytica as a selection marker.

[0122] The synthesized DNA of Sc.BTS1 and the genomic DNA of KCCM12972P were used as templates, and the primers shown in Table 5, SEQ ID NO: 15 and SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 35, SEQ ID NO: 36 and SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26, and SEQ ID NO: 27 and SEQ ID NO: 28, were used to perform PCR of the left homologous region, TEFINt promoter, Sc.BTS1 ORF, TDH3 terminator, URA3, repeat region, and right homologous region fragments. The PCR conditions were 35 cycles of denaturation at 95°C for 1 minute, annealing at 55°C for 1 minute, and polymerization reaction at 72°C for 2 minutes. The resulting DNA fragments were prepared into one cassette by overlap extension PCR.

[0123] The cassette thus prepared was introduced into the CC08-1023 strain by the heat shock method, and then colonies formed on a uracil-free solid medium (YLMM1) were obtained. Colonies in which the cassette was confirmed to have been inserted into the genome were smeared on a 5-FOA solid medium using primers of SEQ ID NO:29 and SEQ ID NO:30, and cultured at 30°C for 3 days to obtain colonies formed on the 5-FOA solid medium, thereby removing the URA3 marker.

[0124] [Table 5]

[0125] Example 2-4. Construction of a Yarrowia lipolytica-derived GGS1 insertion strain To insert the GGS1 gene (hereinafter referred to as Yl.GGS1) derived from Y. lipolytica onto the chromosome of Y. lipolytica, a polynucleotide of SEQ ID NO: 10 was obtained based on the base sequence (YALI0D17050g) registered in KEGG (Kyoto Encyclopedia of Genes and Genomes) for GGS1. The Yl.GGS1 polynucleotide was used to synthesize a gene in the form of TEFINtp-Yl.GGS1-TDH3t (SEQ ID NO: 11). A cassette to be inserted into the LIG4 (YALI0D21384g) gene position was designed using the URA3 gene (SEQ ID NO: 5) of Y. lipolytica as a selection marker.

[0126] Using the synthesized Yl.GGS1 gene and the genomic DNA of KCCM12972P as templates, PCR was performed for the left homologous region, TEFINt promoter, Yl.GGS1 ORF, TDH3 terminator, URA3, repeat region, and right homologous region fragments using primers SEQ ID NO:15 and SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:39, SEQ ID NO:40 and SEQ ID NO:41, SEQ ID NO:42 and SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24, SEQ ID NO:25 and SEQ ID NO:26, and SEQ ID NO:27 and SEQ ID NO:28 shown in Table 6. The PCR conditions were 35 cycles of denaturation at 95°C for 1 minute, annealing at 55°C for 1 minute, and polymerization reaction at 72°C for 2 minutes. The resulting DNA fragments were prepared into one cassette by overlap extension PCR.

[0127] The cassette thus prepared was introduced into the CC08-1023 strain by the heat shock method, and then colonies formed on a uracil-free solid medium (YLMM1) were obtained. Colonies in which the cassette was confirmed to have been inserted into the genome were smeared on a 5-FOA solid medium using primers of SEQ ID NO:29 and SEQ ID NO:30, and cultured at 30°C for 3 days to obtain colonies formed on the 5-FOA solid medium, thereby removing the URA3 marker.

[0128] [Table 6] EXAMPLES

[0129] Comparative evaluation of β-carotene production based on GGPP synthase-transduced strains Flask evaluation was performed on all five strains obtained in Examples 2-1 to 2-4 and the parent strain CC08-1023 obtained in Example 1. The strains were inoculated into a 250 ml corner baffle flask containing 20 ml of YPD (Yeast extract-Peptone-Dextrose) medium so that the initial OD was 2, and cultured with shaking at 30°C and 200 rpm for 48 hours. After the culture was completed, 1 ml of the culture solution was centrifuged to remove the supernatant. The composition of the YPD medium was as follows: <YPD liquid media> 4% glucose, 1% yeast extract, 2% peptone dissolved in 0.1M sodium phosphate buffer (pH 7.0)

[0130] Next, 0.5 ml of DMSO (Dimethyl sulfoxide, Sigma, CAS number 67-68-5) was added, and the cells were disrupted by shaking (agitation, 2,000 rpm) at 55°C for 10 minutes. Furthermore, 0.5 ml of acetone (Sigma, CAS number 67-64-1) was added, and the cells were extracted by shaking (agitation, 2,000 rpm) at 45°C for 15 minutes, and the concentrations of β-carotene and squalene were analyzed using HPLC equipment. The results of the concentration measurements of β-carotene and squalene are shown in Figure 1.

[0131] As a result, as shown in Figure 1, the β-carotene concentrations measured in CC08-1023 (parent strain), the Ds.GGPPS-introduced strain, the crtEM1-introduced strain, the Sc.BTS1-introduced strain, and the Yl.GGS1-introduced strain were 5.49 mg / L, 54.74 mg / L, 40.58 mg / L, 5.21 mg / L, and 49.22 mg / L, respectively. In particular, when Ds.GGPPS was introduced, β-carotene increased by 49.25 mg / L compared to the parent strain, confirming that the β-carotene-increasing effect was the most excellent.

[0132] Furthermore, the measured squalene concentrations were 313.24 mg / L, 211.86 mg / L, 235.27 mg / L, 253.28 mg / L, and 221.22 mg / L, respectively. As above, when Ds.GGPPS was introduced, the squalene concentration decreased by 101.38 mg / L compared to the CC08-1023 strain, confirming that it had the most excellent effect of reducing squalene production.

[0133] These results confirmed that Ds.GGPPS is the most effective GGPP synthase among Yarrowia spp. Surprisingly, the effect was minimal when geranylgeranyl pyrophosphate synthases from closely related species Saccharomyces cerevisiae, Yarrowia lipolytica, and Xanthophyllomyces dendroas were introduced, but the effect was remarkable when geranylgeranyl pyrophosphate synthase from the relatively unrelated Dunaliella salina was introduced. EXAMPLES

[0134] Construction of β-carotene 15,15'oxygenase (BCO) gene-transfected strain To insert the β-carotene 15,15' oxygenase (hereinafter referred to as Mb.BCO) gene derived from marine bacterium 66A03 (Uncultured marine bacterium 66A03) into the chromosome of Yarrowia lipolytica, a polynucleotide sequence (SEQ ID NO: 12) was obtained in which the codons were optimized for Yarrowia lipolytica according to Non-Patent Document 17 based on the amino acid sequence (Q4PNI0) registered in UniProtKB (UniProt Knowledgebase). The gene was synthesized in the form of TEFINtp-codon-optimized Mb.BCO-CYC1t (SEQ ID NO: 13) using the Mb.BCO polynucleotide. A cassette was designed to be inserted into the KU70 (YALI0C08701g) gene position using the URA3 gene (SEQ ID NO: 5) of Yarrowia lipolytica as a selection marker.

[0135] Using the synthesized genomic DNA of Mb.BCO and KCCM12972P as templates, PCR was performed on the left homologous region, TEFINt promoter, Mb.BCO ORF, CYC1 terminator, URA3, repeat region, and right homologous region using primers SEQ ID NO: 43 and SEQ ID NO: 44, SEQ ID NO: 45 and SEQ ID NO: 46, SEQ ID NO: 47 and SEQ ID NO: 48, SEQ ID NO: 49 and SEQ ID NO: 50, SEQ ID NO: 51 and SEQ ID NO: 52, SEQ ID NO: 53 and SEQ ID NO: 54, and SEQ ID NO: 55 and SEQ ID NO: 56 shown in Table 7. The PCR conditions were 35 cycles of denaturation at 95°C for 1 minute, annealing at 55°C for 1 minute, and polymerization reaction at 72°C for 2 minutes. The resulting DNA fragments were prepared into one cassette by overlap extension PCR.

[0136] The cassettes thus prepared were introduced into the strains prepared in Examples 2-1 to 2-4 by the heat shock method, and then colonies formed on a uracil-free solid medium (YLMM1) were obtained. Colonies in which the cassette insertion into the genome was confirmed were smeared on a 5-FOA solid medium using primers of SEQ ID NO: 57 and SEQ ID NO: 58, and cultured at 30°C for 3 days to obtain colonies formed on the 5-FOA solid medium, thereby removing the URA3 marker.

[0137] [Table 7] EXAMPLES

[0138] Comparative evaluation of retinol production in β-carotene 15,15'-oxygenase (BCO) gene-transfected strains Flask evaluation was performed on the five strains obtained in Example 4 and the parent strain CC08-1023 obtained in Example 1. The strains were inoculated into a 250 ml corner baffle flask containing 20 ml of YPD (Yeast extract-Peptone-Dextrose) medium and 0.05% butylated hydroxytoluene so that the initial OD was 2, and cultured with shaking at 30°C and 200 rpm for 48 hours. After the culture was completed, 1 ml of the culture solution was centrifuged to remove the supernatant. Next, 0.5 ml of DMSO (Dimethyl sulfoxide, Sigma) was added, and the cells were disrupted by shaking (agitation; 2,000 rpm) at 55°C for 10 minutes. Furthermore, 0.5 ml of acetone (Sigma) was added, and the cells were extracted by shaking (agitation, 2,000 rpm) at 45°C for 15 minutes to extract retinol, retinal, β-carotene, and squalene, and their concentrations were analyzed using HPLC equipment. The results of the measurement of the concentrations of retinol, retinal, β-carotene, and squalene are shown in FIG.

[0139] As a result, as shown in Figure 2, retinol was not measured in the strains in which Mb.BCO was introduced into the CC08-1023 strain. In contrast, retinol concentrations of 5.88 mg / L, 2.78 mg / L, 0 mg / L, and 4.35 mg / L were measured in the four strains in which Mb.BCO was introduced after Ds.GGPPS, crtEM1, Sc.BTS1, and Yl.GGS1 were introduced into the CC08-1023 strain, respectively.

[0140] The β-carotene concentrations were 3.68 mg / L, 0.63 mg / L, 2.47 mg / L, 3.58 mg / L, and 0.98 mg / L in the five strains, respectively, confirming that β-carotene was converted to retinol and the β-carotene concentration decreased.The squalene concentrations measured in the five strains were 309.88 mg / L, 233.52 mg / L, 282.19 mg / L, 306.34 mg / L, and 269.18 mg / L, respectively.

[0141] These results confirmed that enhancing GGPP biosynthesis had a positive effect on improving retinol production capacity.

[0142] These results also verified that Ds.GGPPS has excellent effects on β-carotene production, squalene reduction, and retinol production.

[0143] From the above description, a person skilled in the art to which the present application pertains will understand that the present application can be implemented in other specific forms without changing its technical ideas or essential features. It should be understood that the above examples are merely illustrative and not limiting. This application should be interpreted as including all modifications or alterations derived from the meaning and scope of the claims and their equivalent concepts, rather than the specification.

[0144] The sequences of the SEQ ID NOs of the present application are shown in Table 8.

[0145] [Table 8-1]

[0146]

Table 8-2

[0147]

Table 8-3

[0148]

Table 8-4

[0149]

Table 8-5

[0150]

Table 8-6

[0151]

Table 8-7

[0152]

Table 8-8

[0153]

Table 8-9

[0154]

Table 8-10

[0155]

Table 8-11

[0156]

Table 8-12

[0157]

Table 8-13

[0158]

Table 8-14

[0159]

Table 8-15

[0160]

Table 8-16

[0161]

Table 8-17

[0162]

Table 8-18

[0163]

Table 8-19

[0164]

Table 8-20

[0165]

Table 8-21

[0166]

Table 8-22

[0167]

Table 8-23

[0168]

Table 8-24

[0169]

Table 8-25

[0170] JPEG2025510210000034.jpg196134

Claims

1. A Yarrowia microorganism that expresses geranylgeranyl pyrophosphate synthase derived from Dunaliella salina and has the ability to produce carotenoids or substances that have precursors thereof.

2. The Yarrowia microorganism according to claim 1, wherein the geranylgeranyl pyrophosphate synthase consists of the amino acid sequence of SEQ ID NO:

91.

3. The Yarrowia microorganism according to claim 1 , wherein the geranylgeranyl pyrophosphate synthase is encoded by a polynucleotide consisting of the base sequence of SEQ ID NO:

1.

4. The Yarrowia microorganism of claim 1 , wherein the Yarrowia microorganism is Yarrowia lipolytica.

5. The Yarrowia microorganism according to claim 1 , wherein the substance having a carotenoid as a precursor is a retinoid.

6. The Yarrowia microorganism according to claim 1, wherein the carotenoid is β-carotene.

7. The Yarrowia microorganism of claim 5 , wherein the retinoid is retinol.

8. The Yarrowia microorganism according to claim 1 , wherein the Yarrowia microorganism has a reduced ability to produce by-products.

9. The Yarrowia microorganism of claim 8 , wherein the by-product is squalene.

10. Cultivating the Yarrowia microorganism according to any one of claims 1 to 9 in a medium; and recovering the carotenoid or a substance having a precursor thereof from the Yarrowia microorganism or the culture medium.

11. A step of converting the β-carotene produced by the Yarrowia microorganism into a carotenoid other than β-carotene; or The method for producing a carotenoid or a substance having a precursor thereof according to claim 10, further comprising a step of converting the retinol produced by the Yarrowia microorganism into a retinoid other than retinol.

12. A composition for producing a carotenoid or a substance having a precursor thereof, comprising the Yarrowia microorganism according to any one of claims 1 to 9 or a culture thereof.

13. 2. Use of a Yarrowia microorganism that expresses Dunaliella salina-derived geranylgeranyl pyrophosphate synthase and has the ability to produce a carotenoid or a substance having a precursor thereof for the production of a carotenoid or a substance having a precursor thereof.

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