Microorganisms that produce carotenoids or substances derived therefrom, including geranylgeranyl pyrophosphate synthase derived from Haematococcus pluvialis, and a method for producing carotenoids or retinoids using the same.

Abstract

The present application relates to a Yarrowia microorganism that expresses geranylgeranyl pyrophosphate synthase derived from Haematococcus pluvialis and has the ability to produce a carotenoid or a substance whose precursor is a carotenoid, a method for producing a carotenoid or a substance whose precursor is a carotenoid using the same, a composition for producing a carotenoid or a substance whose precursor is a carotenoid, and use of the Yarrowia microorganism or a culture thereof for producing a carotenoid or a substance whose precursor is a carotenoid.

Description

【Technical Field】 【0001】 This application relates to a Yarowia microorganism having the ability to produce a carotenoid or a substance having the carotenoid as a precursor, which expresses geranylgeranyl pyrophosphate synthase derived from Haematococcus pluvialis, a method for producing a carotenoid or a substance having the carotenoid as a precursor using the same, a composition for producing a carotenoid or a substance having the carotenoid as a precursor, and use of the Yarowia microorganism or its culture in the production of a carotenoid or a substance having the carotenoid as a precursor. 【Background Art】 【0002】 Carotenoids and retinoids exhibit various functions in plants and animals, and thus are used in various industrial fields such as food and feed. In particular, carotenoids such as β-carotene are substances reported to have functions such as removal of free radicals, a precursor of vitamin A in animals, enhancement of the immune system of vertebrates, and reduction of the risk of lung cancer, and retinoids are a group of substances chemically related to retinol which is vitamin A, and are also used in cosmetics, skin disease therapeutic agents, and the like. 【0003】 However, despite such advantages, carotenoids (e.g., β-carotene) and retinoids (e.g., retinol) are not synthesized in the animal body or, even if synthesized, the synthesis amount is insufficient. In addition, attempts have been made to industrially produce them using mutant microorganisms (Patent Document 1), but it is still difficult to produce them in high purity. 【0004】 For example, in the process of producing a microorganism that produces a carotenoid or a retinoid, squalene (C30) and the like are produced together as by-products. Therefore, it is essential to find geranylgeranyl pyrophosphate synthase that contributes to efficiently producing a carotenoid or a retinoid in order to increase their production amounts and reduce squalene produced in the competing pathway. [Preliminary Technology Documents] [License] 【0005】 [License 1] U.S. Patent No. 7745170 [License 2] Korean Patent Publication No. 10-2020-0136813 [License 3] U.S. Patent No. 7662943 [License 4] U.S. Patent No. 10584338 [Patent Document 5] U.S. Patent No. 10273491 [Non-licensed literature] 【0006】 [Non-licensed Document 1] Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444 [Non-licensed Document 2] Rice et al., 2000, Trends Genet. 16: 276-277 [Non-licensed Document 3] Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453 [Non-licensed Document 4] Devereux, J., et al, Nucleic Acids Research 12: 387 (1984) [Non-licensed Document 5] Atschul, [S.] [F.,] [ET AL, J MOLEC BIOL 215]: 403 (1990) [Non-licensed Document 6] Guide to Huge Computers, Martin J. Bishop, [ED.,] Academic Press, San Diego, 1994 【Non-licensed Document 7】 [CARILLO ETA / .](1988) SIAM J Applied Math 48: 1073 【Non-licensed Document 8】 Smith and Waterman, Adv. Appl. Math (1981) 2:482 【Non-licensed literature 9】 Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353-358 (1979) 【Non-licensed literature 10】 Gribskov et al(1986) Nucl. Acids Res. 14: 6745 【Non-licensed Document 11】 J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989 【Non-licensed Document 12】 FM Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York 【Non-licensed Document 13】 Sambrook et al., supra, 9.50-9.51, 11.7-11.8 【Non-licensed Document 14】 Sitnicka et al. Functional Analysis of Genes. Advances in Cell Biology. 2010, Vol. 2. 1-16 【Non-licensed Document 15】 Sambrook et al. Molecular Cloning 2012 【Non-Patent Document 16】 D.-C. Chen et al., Appl Microbiol Biotechnol, 1997 【Non-Patent Document 17】 http: / / atgme.org 【Non-Patent Document 18】 Hong et al., Applied Microbiology and Biotechnology, 2019 Jan;103(1):211-223 【Summary of the Invention】 【Problems to be Solved by the Invention】 【0007】 The problem to be solved by the present application is to provide a microorganism that produces a carotenoid or a substance having the carotenoid as a precursor, including geranylgeranyl pyrophosphate synthase derived from Haematococcus pluvialis, and a method and use for producing a carotenoid or a retinoid using the same. 【Means for Solving the Problems】 【0008】 The present application aims to provide a microorganism of the genus Yarrowia that expresses geranylgeranyl pyrophosphate synthase derived from Haematococcus pluvialis and has the ability to produce a carotenoid or a substance having the carotenoid as a precursor. 【0009】 In addition, the present application aims to provide a method for producing a carotenoid or a substance having the carotenoid as a precursor using the microorganism of the genus Yarrowia. 【0010】 Furthermore, the present application aims to provide a composition for producing a carotenoid or a substance having the carotenoid as a precursor, which contains the microorganism of the genus Yarrowia or a culture thereof. 【0011】 Furthermore, the present application aims to provide the use of the microorganism of the genus Yarrowia for the production of a carotenoid or a substance having the carotenoid as a precursor. 【Effects of the Invention】 【0012】 This application demonstrates that the production of carotenoids and their precursors can be effectively increased by introducing the geranylgeranyl pyrophosphate synthase gene derived from Haematococcus pluvialis into microorganisms of the genus Yarowia. [Brief explanation of the drawing] 【0013】 [Figure 1] This figure shows the results of flask evaluations of GGPP synthase gene transfection strains derived from various microorganisms. [Figure 2] This figure shows the results of flask evaluation of Mb.BCO-introduced strains. [Modes for carrying out the invention] 【0014】 These will be explained in detail below. Note that each description and embodiment disclosed in this application applies to other descriptions and embodiments. That is, any combination of the various elements disclosed in this application is included. Furthermore, this application is not limited to the following specific descriptions. In addition, numerous papers and patent documents are referenced throughout this specification, and their citations are indicated. The disclosures of the cited papers and patent documents are incorporated in their entirety as references in this specification, thereby more clearly explaining the level of the art to which this application belongs and the content of this application. 【0015】 One aspect of this application provides a Yarowia microorganism that expresses geranylgeranyl pyrophosphate synthase derived from Haematococcus pluvialis and has the ability to produce carotenoids or substances derived therefrom. 【0016】 In this application, "geranylgeranyl pyrophosphate synthase" refers to an enzyme that catalyzes the synthesis of geranylgeranyl pyrophosphate (GGPP). The substrate of the geranylgeranyl pyrophosphate synthase may be isopentenyl pyrophosphate (IPP) or dimethylallyl pyrophosphate (DMAPP). The geranylgeranyl pyrophosphate synthase is also referred to as "GGS," "GGPPS," "GGPS1," or "a polypeptide having geranylgeranyl pyrophosphate synthase activity." 【0017】 As one example, the microorganism of this application may contain a geranylgeranyl pyrophosphate synthase protein derived from the foreign protein Haematococcus pluvialis, or it may be a Yarowia microorganism that expresses the geranylgeranyl pyrophosphate synthase protein and has the ability to produce carotenoids or substances derived therefrom. 【0018】 The amino acid sequence of the GGPPS protein in this 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 NCBI's GenBank, which are known databases, but is not limited to these. 【0019】 As an example, the GGPPS protein of this application is derived from Haematococcus pluvialis, but any protein having the same sequence or activity may be used. 【0020】 As one embodiment, the GGPPS protein of this application may include an amino acid sequence having 80% or more homology or identity with SEQ ID NO: 103, may have the aforementioned amino acid sequence, may consist of the aforementioned amino acid sequence, or may be substantially composed of the aforementioned amino acid sequence. 【0021】 Furthermore, even if a protein containing SEQ ID NO: 103 is described as one example of the GGPPS protein of this application, this does not exclude meaningless sequence additions before or after the amino acid sequence of SEQ ID NO: 103, naturally occurring mutations, or silent mutations thereof. It will be clear to those skilled in the art that any protein having the same or equivalent activity as the protein containing the aforementioned amino acid sequence is included in the GGPPS protein of this application. 【0022】 Specifically, the GGPPS protein of this application may contain the amino acid sequence of SEQ ID NO: 103, or it may contain 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: 103. Furthermore, it goes without saying that this application also includes amino acid sequences in which some sequences are deleted, modified, substituted, or added, as long as they have such homology or identity and exhibit efficacy equivalent to the aforementioned protein. 【0023】 Even if this application describes a polypeptide or protein containing an amino acid sequence represented by a specific sequence number, a polypeptide or protein consisting of an amino acid sequence represented by a specific sequence number, or a polypeptide or protein having an amino acid sequence represented by a specific sequence number, it goes without saying that any protein having an amino acid sequence in which some sequences are deleted, modified, substituted, conserved substituted, or added may be used in this application, as long as it has the same or equivalent activity as the polypeptide consisting of the amino acid sequence of the said sequence number. Examples include the addition of sequences that do not change the function of the protein at the N-terminus, internal and / or C-terminus of the amino acid sequence, spontaneously occurring mutations, silent mutations, or conserved substitutions. 【0024】 The term "conservative substitution" refers to the substitution of one amino acid with another amino acid having similar structural and / or chemical properties. Such amino acid substitutions can 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 the activity of the polypeptide. 【0025】 In this application, "homology" or "identity" refers to the degree of identical or similarity between two given amino acid sequences or base sequences, expressed as a percentage. Homology and identity are often used interchangeably. 【0026】 The sequence homology or identity of conserved polynucleotides or polypeptides is determined by standard sequence algorithms, and a default gap penalty established by the program used may also be applied. Substantially, homologous or identical sequences generally hybridize with the entire sequence or a portion of at least about 50%, 60%, 70%, 80%, or 90% of the total length under moderate to high stringent conditions. Hybridization also includes hybridization with polynucleotides having common codons or codons considering codon degeneracy. 【0027】 Whether any two polynucleotide or polypeptide sequences are homologous, similar, or identical can be determined, for example, using default parameters as described in Non-Patent Document 1 and known computer algorithms such as the "FASTA" program. Alternatively, it can be determined using the Needleman-Wunsch algorithm (Non-Patent Document 3), as performed 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) (including the GCG program package (Non-Patent Document 4), BLASTP, BLASTN, and FASTA (Non-Patent Documents 5, 6, and 7)). For example, homology, similarity, or identity can be determined using BLAST or Clustal W from the National Center for Biotechnology Information. 【0028】 The homology, similarity, or identity of polynucleotides or polypeptides can be determined by comparing sequence information using a GAP computer program such as Non-Patent Document 3, as disclosed in Non-Patent Document 8, for example. In summary, the GAP program is defined as 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 (with a value of 1 for identity and 0 for non-identity) and a weighted comparison matrix (or EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix) as disclosed in Non-Patent Document 9, as in Non-Patent Document 10; (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 extended penalty of 0.5); and (3) no penalty for terminal gaps. 【0029】 Furthermore, whether any two polynucleotide or polypeptide sequences are homologous, similar, or identical can be confirmed by comparing the sequences in a Southern hybridization experiment under defined stringent conditions, and 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., Non-Patent Documents 11, 12). 【0030】 In this application, protein expression may be performed by introducing a protein-coding gene (polynucleotide) into a microorganism, or by injecting a protein, but is not limited to these methods. 【0031】 As one example, the microorganism of this application may be one into which a geranylgeranyl pyrophosphate synthase gene derived from Haematococcus pluvialis has been introduced. Furthermore, the introduction of the geranylgeranyl pyrophosphate synthase gene may also include further enhancement of its activity after introduction. 【0032】 In this application, the term "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, including the NCBI's GenBank, which is a well-known database, but is not limited to these. 【0034】 As one example, the geranylgeranyl pyrophosphate synthase gene derived from Haematococcus pluvialis may contain the nucleotide sequence of Sequence ID No. 1, may have the aforementioned nucleotide sequence, or may consist of the aforementioned nucleotide sequence, but is not limited to these. 【0035】 As one example, the geranylgeranyl pyrophosphate synthase gene consisting of the nucleotide sequence of Sequence ID No. 1 may have its codons optimized for microorganisms of the genus Yarowia, or more specifically, Yarowia liporitica. 【0036】 In this application, "polynucleotide" refers to a polymer of nucleotides in which nucleotide monomers are covalently linked together in a long chain, and specifically to a DNA chain longer than a predetermined length. 【0037】 The polynucleotide or gene can be modified in various ways in the coding region, either by codon degeneracy or by considering preferred codons in an organism that intends to express the geranylgeranyl pyrophosphate synthase polypeptide, as long as the amino acid sequence of the polypeptide does not change. The polynucleotide or gene may, for example, contain the base sequence of SEQ ID NO: 1, or may consist of a base sequence having 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homology or identity with it, but is not limited to these. 【0038】 Furthermore, the polynucleotide or gene of this application may be any sequence that encodes the amino acid sequence of SEQ ID NO: 103 by hybridizing under stringent conditions with a probe prepared from a known gene sequence, for example, a complementary sequence to all or part of the said base sequence. The “stringent condition” means a condition that enables specific hybridization between polynucleotides. Such conditions are specifically described in the literature (e.g., Non-Patent Document 11). For example, this could involve hybridizing polynucleotides with high homology or identity, specifically 40% or more, more specifically 90% or more, more specifically 95% or more, 96% or more, 97% or more, 98% or more, and even more specifically 99% or more homology or identity, while not hybridizing polynucleotides with lower homology or identity. Alternatively, it could involve washing once, specifically two to three times, at a salt concentration and temperature equivalent to the washing conditions of a typical Southern hybridization: 60°C, 1×SSC, 0.1%SDS, specifically 60°C, 0.1×SSC, 0.1%SDS, or more specifically 68°C, 0.1×SSC, 0.1%SDS. 【0039】 Hybridization requires that the two nucleic acids have complementary sequences, even if mismatches between bases are possible depending on the stringency of the 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. Therefore, the polynucleotides of this application may include not only substantially similar nucleic acid sequences, but also isolated nucleic acid fragments that are complementary to the entire sequence. 【0040】 Specifically, polynucleotides having homology or identity can be detected using hybridization conditions in which the hybridization step is performed at a Tm value of 55°C and the conditions described above. The Tm value may be 60°C, 63°C, or 65°C, but is not limited to these, and can be appropriately adjusted by those skilled in the art depending on the purpose. 【0041】 The appropriate stringency for hybridizing polynucleotides depends on the length and degree of complementarity of the polynucleotides, and these variables are known in the art (see Non-Patent Document 13). 【0042】 As one example, the microorganism of this application may include a vector containing a geranylgeranyl pyrophosphate synthase gene derived from Haematococcus pluvialis, or a polynucleotide encoding geranylgeranyl pyrophosphate synthase derived from Haematococcus pluvialis. 【0043】 The vector of this application comprises a DNA product comprising a polynucleotide sequence encoding a target polypeptide operably linked to a suitable regulatory region (or regulatory sequence) so as to enable expression of the target polypeptide in a suitable host. The regulatory region comprises a promoter for initiating transcription, an optional operator sequence for regulating the transcription, a sequence encoding a suitable mRNA-ribosome binding site, and sequences 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 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, as phage vectors or cosmid vectors, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, Charon21A, etc. can be used, and as plasmid vectors, pDZ series, pBR series, pUC series, pBluescriptII series, pGEM series, pTZ series, pCL series, pET series, etc. can be used. Specifically, pDZ, pDC, pDCM2 (Patent Document 2), pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC, pIMR53 vectors, etc. can be used. 【0045】 For example, a polynucleotide encoding a target polypeptide can be inserted into a chromosome using an intracellular chromosome introduction vector. The insertion of the polynucleotide into the chromosome can be carried out by any method known in the art, such as homologous recombination, but is not limited thereto. The vector may further include a selection marker to confirm whether or not the polynucleotide has been inserted into the chromosome. The selection marker is used to select cells transformed by the vector, that is, to confirm whether or not the target nucleic acid molecule has been inserted, and markers that confer selectable phenotypes such as drug resistance, nutritional requirements, resistance to cytotoxic agents, and expression of surface polypeptides are used. In an environment treated with a selective agent, only cells expressing the selection marker will survive or exhibit different phenotypes, thus allowing for the selection of transformed cells. 【0046】 In this application, "transformation" means introducing a vector containing a polynucleotide encoding a target polypeptide into a host cell or microorganism, thereby causing the polypeptide encoded by the polynucleotide to be expressed in the host cell. The transformed polynucleotide may be any form, regardless of whether it is inserted into or outside the host cell's chromosomes, as long as it is expressed in the host cell. The polynucleotide also contains DNA and / or RNA encoding the target polypeptide. The polynucleotide may be introduced into the host cell in any form, as long as it is introduced and expressed in the host cell. 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. Typically, the expression cassette includes 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. Alternatively, the polynucleotide may be introduced into the host cell in its own form and operably linked to the sequence necessary for expression in the host cell, but is not limited to this. 【0047】 Furthermore, the term "operably linked" means that the polynucleotide sequence is functionally linked to a promoter sequence that initiates and mediates the transcription of the polynucleotide encoding the target polypeptide of this application. 【0048】 As one example, in a Yarowia microorganism expressing GGPPS derived from Haematococcus pluvialis of this application, geranylgeranyl pyrophosphate synthase activity is enhanced compared to Yarowia microorganisms that do not express it, but the invention is not limited to this example. 【0049】 As one example, in a Yarowia microorganism into which the GGPPS gene derived from Haematococcus pluvialis of this application has been introduced, geranylgeranyl pyrophosphate synthase activity was enhanced compared to a Yarowia microorganism in which the GGPPS gene derived from Haematococcus pluvialis has not been introduced, but the invention is not limited to this example. 【0050】 As one example, a Yarrowia microorganism into which geranylgeranyl pyrophosphate synthase encoded by the GGPPS gene derived from Haematococcus pluvialis of this application has been introduced exhibits enhanced geranylgeranyl pyrophosphate synthase activity compared to a Yarrowia microorganism into which geranylgeranyl pyrophosphate synthase encoded by crtE or its mutant gene crtEM1 derived from Xanthophyllomyces dendrorhous, 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 this application, "Yarrowia microorganisms" or "Yarrowia strains" include all wild-type Yarowia microorganisms and Yarowia microorganisms that have been genetically modified naturally or artificially. These include Yarowia microorganisms in which specific mechanisms have been enhanced due to reasons such as the insertion of external genes or the enhancement of endogenous gene activity, and which may contain the GGPPS gene derived from Haematococcus pluvialis for the production of carotenoids or substances that use them as precursors. 【0052】 The microorganism of this application may be a microorganism comprising at least one of the GGPPS protein of this application, the GGPS gene or polynucleotide encoding the GGPPS protein, and a vector comprising the gene or polynucleotide; it may be a microorganism modified to express the GGPPS protein or GGPPS gene derived from Haematococcus pluvialis of this application; it may be a microorganism expressing the GGPPS protein or GGPPS gene derived from Haematococcus pluvialis of this application (e.g., a recombinant strain); or it may be a strain having GGPPS activity derived from Haematococcus pluvialis of this application (e.g., a recombinant strain), but is not limited to these. 【0053】 The strains of this application may be microorganisms that naturally possess the ability to produce geranylgeranyl pyrophosphate synthase, carotenoids, or substances derived therefrom, or they may be microorganisms in which the ability to produce geranylgeranyl pyrophosphate synthase, carotenoids, or substances derived therefrom has been enhanced or conferred by introducing the GGPPS protein, gene, polynucleotide, or vector containing the same derived from Haematococcus pluvialis of this application into a parent strain that does not possess the ability to produce geranylgeranyl pyrophosphate synthase, carotenoids, or substances derived therefrom, but are not limited to these. 【0054】 For example, the strains of this application include all microorganisms that have been transformed with the GGPPS protein, gene, polynucleotide, or vector containing the same derived from Haematococcus pluvialis of this application, thereby enabling them to produce carotenoids or substances derived therefrom, or microorganisms whose production capacity has been improved. For example, the strains of this application may be recombinant strains in which the production capacity of carotenoids or substances derived therefrom has been improved by expressing the GGPPS derived from Haematococcus pluvialis of this application in a naturally occurring wild-type microorganism or a microorganism that produces carotenoids or substances derived therefrom. The recombinant strains exhibiting improved carotenoid production capacity or precursor substances are, but are not limited to, microorganisms exhibiting improved carotenoid production capacity or precursor substances compared to natural wild-type microorganisms or microorganisms without modified geranylgeranyl pyrophosphate synthase (i.e., Yarowia microorganisms containing the wild-type geranylgeranyl pyrophosphate synthase gene (SEQ ID NO: 11), or Yarowia microorganisms in which the geranylgeranyl pyrophosphate synthase gene (SEQ ID NO: 1) derived from Haematococcus pluvialis has not been introduced). 【0055】 For example, the strains of the genus Yarrowia that exhibit improved production capacity for carotenoids or substances derived therefrom, according to this application, are microorganisms that exhibit improved production capacity for carotenoids or substances derived therefrom compared to microorganisms that do not contain GGPPS derived from Haematococcus pluvialis (e.g., SEQ ID NO: 103), or microorganisms of the genus Yarrowia that include CrtE or its variant CrtEM1 derived from Xanthophylromyces dendrosa, BTS1 derived from Saccharomyces cerevisiae, or GGS1 from Yarrowia lipolytica, but are not limited to these. For example, the unmodified microorganism used as a comparison for whether or not the production capacity for the aforementioned carotenoids or substances derived therefrom has improved is strain CC08-1023, but is not limited to this. 【0056】 For example, the recombinant strain with improved production capacity is one in which the β-carotene or retinol production capacity is improved by approximately 0.001% or more, or by 0.01% or more, compared to the β-carotene or retinol production capacity of the parent strain before mutation or the unmodified microorganism. However, any strain that shows a positive increase compared to the production capacity of the parent strain before mutation or the unmodified microorganism is acceptable. The term "approximately" includes a range that encompasses ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc. Any numerical value within a range equivalent to or similar to the numerical value following the term "approximately" is acceptable, but is not limited to these. 【0057】 In this application, "unmodified microorganism" does not exclude strains containing naturally occurring mutations in microorganisms, but rather refers to wild-type strains or natural strains themselves, or strains before they undergo genetic mutation and changes in trait due to natural or artificial factors. For example, the aforementioned unmodified microorganism refers to a strain that does not express GGPPS derived from Haematococcus pluvialis in this application, or a strain before it has been introduced. The aforementioned "unmodified microorganism" may be used interchangeably with "pre-modification strain," "pre-modification microorganism," "non-mutant strain," "unmodified strain," "non-mutant microorganism," or "reference microorganism." 【0058】 The microorganisms of this application belong to the genus Yarrowia, specifically Yarrowia lipolytica, but are not limited to these species. 【0059】 In the microorganisms of this application, modification of part or all of the polynucleotides can be induced by (a) homologous recombination using a chromosome introduction vector in the microorganism, or genome editing using an engineered nuclease (e.g., CRISPR-Cas9), and / or (b) light and / or chemical treatment such as ultraviolet light or radiation. The methods for modifying part or all of the genes include methods using DNA recombination technology. For example, deletion of part or all of the gene can be achieved by introducing a nucleotide sequence or vector containing a nucleotide sequence homologous to the target gene into the microorganism to induce homologous recombination. The introduced nucleotide sequence or vector contains, but is not limited to, a dominant selection marker. 【0060】 The microorganisms of this application may also be Yarowia microorganisms modified to contain polynucleotides encoding lycopene cyclase / phytoene synthase (crtYB), phytoene desaturase (crtI), and beta-carotene 15,15'-oxygenase (BLH) proteins. 【0061】 The microorganisms of this application may be modified to further include polynucleotides encoding lycopene cyclase / phytoene synthase (crtYB) and phytoene desaturase (crtI) proteins, and may exhibit the activity of these proteins, or may be microorganisms with enhanced protein activity. The lycopene cyclase / phytoene synthase or phytoene desaturase is a protein derived from Xanthophyllomyces dendrorhous, but is not limited thereto. As an example, the polynucleotides encoding the lycopene cyclase / phytoene synthase or phytoene desaturase may have nucleotide sequences registered in the NCBI (National Center for Biotechnology Information Search database) (GenBank: AY177204.1 or GenBank: AY177424.1), respectively, or may contain the aforementioned nucleotide sequences. As an example, the polynucleotide encoding the lycopene cyclase / phytoen synthase or phytoendesaturase may have the sequence of SEQ ID NO: 71 or SEQ ID NO: 72, respectively, or may contain the said sequence. The polynucleotide can be modified in various ways in the coding region without changing the amino acid sequence, either by codon degeneracy or by considering the preferred codons in the microorganism intended to express the polypeptide of this application.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% homology or identity with the sequence of SEQ ID NO: 71 or SEQ ID NO: 72, or contains the said 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% homology or identity with the sequence of SEQ ID NO: 71 or SEQ ID NO: 72, or is substantially composed of the said base sequence, but is not limited to these. 【0062】 The microorganisms of this application are modified to further include a polynucleotide encoding the beta-carotene 15,15'-oxygenase (BLH) protein, and exhibit the activity of these proteins, or are microorganisms with enhanced protein activity. The beta-carotene 15,15'-oxygenase is a protein derived from the marine bacterium 66A03 (Uncultured marine bacterium 66A03), but is not limited thereto. As an example, the polynucleotide encoding beta-carotene 15,15'-oxygenase may have the amino acid sequence (Q4PNI0) registered in UniProtKB (UniProt Knowledgebase), or may contain the aforementioned amino acid sequence. As an example, the polynucleotide encoding beta-carotene 15,15'-oxygenase may have the sequence of Sequence ID No. 13, or may contain the aforementioned sequence. The polynucleotide can be modified in various ways in the coding region, within the limits that the amino acid sequence does not change, by codon degeneracy or by considering codons preferred in microorganisms that are to express the polypeptide of this application. 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% homology or identity with the sequence of SEQ ID NO: 13, or contains the said 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% homology or identity with the sequence of SEQ ID NO: 13, or is substantially composed of the said base sequence. 【0063】 In this application, "enhancement" of polypeptide activity means improving the polypeptide activity compared to its endogenous activity. This enhancement is used interchangeably with activation, upregulation, overexpression, and increase. Here, activation, enhancement, upregulation, overexpression, and increase all include exhibiting activity that was not originally present, or improving activity compared to endogenous activity or activity before modification. "Endogenous activity" refers to the activity of a specific polypeptide that was originally present in the parent strain or unmodified microorganism before the trait change, when a trait changes due to genetic mutation caused by natural or artificial factors. This is used interchangeably with "activity before modification." When polypeptide activity is "enhanced," "upregulated," "overexpressed," or "improved" compared to its endogenous activity, it means that the activity and / or concentration (expression level) of the specific polypeptide that was originally present in the parent strain or unmodified microorganism before the trait change is improved. 【0064】 The enhancement may be carried out by introducing an exogenous polypeptide or gene, or by enhancing the activity and / or increasing the concentration (expression level) of an endogenous polypeptide. Whether or not the activity of the polypeptide has been enhanced can be confirmed by an increase in the degree of the polypeptide's activity, its expression level, or the amount of product produced from the polypeptide. 【0065】 Various methods known in the art can be applied to enhance the activity of the polypeptide, and any method that can enhance the activity of the target polypeptide compared to the microorganism before modification is acceptable. Specifically, this includes, but is not limited to, conventional methods in molecular biology, including genetic engineering and / or protein engineering known to those with ordinary skill in the art (see, for example, Non-Patent Documents 14, 15, etc.). 【0066】 Specifically, the enhancement of polypeptide activity in this application is carried out by: 1) increasing the intracellular copy number of the polynucleotide encoding the polypeptide; 2) replacing the expression regulatory region of the gene on the chromosome encoding the polypeptide with a highly active sequence; 3) modifying the start codon or the base sequence encoding the 5'UTR region of the gene transcript encoding the polypeptide; 4) modifying the amino acid sequence of the polypeptide so as to enhance polypeptide activity; 5) modifying the polynucleotide sequence encoding the polypeptide so as to enhance polypeptide activity (for example, modifying the polynucleotide sequence of the polypeptide gene to encode a polypeptide modified to enhance polypeptide activity); 6) introducing an exogenous polypeptide exhibiting polypeptide activity or an exogenous polynucleotide encoding it; 7) optimizing the codon of the polynucleotide encoding the polypeptide; 8) analyzing the tertiary structure of the polypeptide and selectively modifying or chemically modifying exposed portions; or 9) a combination of two or more selected from 1) to 8) above, but is not limited to these methods. 【0067】 More specifically, increasing the intracellular copy number of the polynucleotide encoding the polypeptide (as described in 1) above may be carried out by introducing into a host cell a vector that replicates and functions independently of the host, to which the polynucleotide encoding the polypeptide is operably linked. Alternatively, it may be carried out by introducing one or more copies of the polynucleotide encoding the polypeptide into the chromosomes within the host cell. The introduction into the chromosomes is carried out by introducing into the host cell a vector capable of inserting the polynucleotide into the chromosomes within the host cell, but is not limited to this. The vector is as described above. 【0068】 2) Substitution of a gene expression regulatory region (or expression regulatory sequence) on a chromosome encoding a polypeptide with a more potent sequence is carried out, for example, by causing a sequence mutation through deletion, insertion, non-conservative or conservative substitution, or a combination thereof, or by substituting with a sequence having higher activity, so as to further enhance the activity of the expression regulatory region. The expression regulatory region includes, but is not limited to, promoters, operator sequences, sequences encoding ribosome binding sites, and sequences that regulate the termination of transcription and translation. For example, this is carried out by substituting the original promoter with a potent promoter, but is not limited to this. 【0069】 Examples of known strong promoters include, but are not limited to, the CJ1-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. 【0070】 3) Modifying the start codon or the nucleotide sequence encoding the 5'UTR region of a gene transcript encoding a polypeptide is performed, for example, by substituting it with a nucleotide sequence encoding another start codon that has a higher polypeptide expression rate compared to the endogenous start codon, but is not limited to this. 【0071】 Modifying the amino acid sequence or polynucleotide sequence described in 4) and 5) above is carried out by causing a sequence mutation through deletion, insertion, non-conservative or conservative substitution, or a combination thereof, of the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide, in order to enhance the activity of the polypeptide, or by substituting it with an improved amino acid sequence or polynucleotide sequence that has higher activity, or an improved amino acid sequence or polynucleotide sequence that has improved activity. Specifically, the substitution is carried out by inserting the polynucleotide into the chromosome by homologous recombination, but is not limited to this. The vector used here may further include a selection marker to confirm whether or not it has been inserted into the chromosome. The selection marker is as described above. 【0072】 The introduction of a foreign polynucleotide exhibiting polypeptide activity (6) above may be carried out by introducing a foreign polynucleotide encoding a polypeptide exhibiting identical or similar activity to the polypeptide into the host cell. The foreign polynucleotide may have any origin or sequence, as long as it exhibits identical or similar activity to the polypeptide. The introduction can be carried out by a person skilled in the art using a known transformation method as appropriate, and as described above, the introduction of the polynucleotide into the host cell results in the production of the polypeptide and improvement of its activity. 【0073】 The optimization of codons of polynucleotides encoding polypeptides described in 7) above may be performed by optimizing endogenous polynucleotide codons so as to increase transcription or translation within the host cell, or by optimizing exogenous polynucleotide codons so as to perform optimized transcription or translation within the host cell. 【0074】 The 8) analysis of the tertiary structure of the polypeptide, and the selection and modification or chemical modification of exposed portions may be carried out, for example, by comparing the sequence information of the polypeptide to be analyzed with a database in which sequence information of known proteins is stored, determining candidate template proteins according to the degree of sequence similarity, confirming the structure based on these, and selecting and modifying or chemically modifying exposed portions. 【0075】 Such enhancement of polypeptide activity is achieved by increasing the activity, concentration, or expression level of the corresponding polypeptide compared to the activity or concentration of the polypeptide expressed in the wild-type or pre-modification microbial strain, or by increasing the amount of product produced from the polypeptide, but is not limited to these methods. 【0076】 As one example, the microorganism of this application has had its GGPPS activity enhanced by introducing the GGPPS gene derived from Haematococcus pluvialis, but it is not limited to this. 【0077】 The microorganisms of this application may be capable of producing carotenoids or substances derived therefrom. 【0078】 In this application, "carotenoid" means tetraterpene or its derivatives, which give fruits and vegetables their yellow color. 【0079】 As one example, 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 to these. 【0080】 As one example, the substance having the carotenoid as a precursor is a retinoid, but it is not limited to this. 【0081】 In this application, "retinoid" means either the group of vitamins A chemically or a group of compounds chemically related thereto. 【0082】 As one example, the retinoid is selected from the group consisting of retinol, retinal, retinoic acid, and retinyl ester, but is not limited thereto. 【0083】 As one example, the microorganism described in this application has reduced by-product production capacity, but is not limited to this. 【0084】 In this application, "by-products" refers to any substances other than carotenoids that are produced during the production of carotenoids or substances derived from them. For example, a typical by-product produced during the production of β-carotene is squalene. 【0085】 In this application, "squalene" refers to an unsaturated hydrocarbon (C 30 H 50) refers to substances that are also used in the biosynthesis of steroid hormones, vitamin D, etc. The microorganism of this application reduces by-products generated in the β-carotene production pathway, specifically reducing squalene production, but is not limited to these. 【0086】 Another aspect of this application provides a method for producing carotenoids or substances derived therefrom, comprising the step of culturing the Yarowia microorganism of this application in a culture medium. 【0087】 The aforementioned microorganisms, carotenoids, and substances derived from them are as described above. 【0088】 In this application, "cultivation" means growing the Yarowia microorganisms of this application under appropriately controlled environmental conditions. The cultivation process of this application can be carried out using suitable culture media and cultivation conditions known in the art. Such a cultivation process can be easily adjusted and used by those skilled in the art depending on the selected strain. Specifically, the cultivation is batch, continuous, and / or fed-batch culture, but is not limited to these. 【0089】 The Yarowia microorganisms described in this application are cultured under aerobic conditions in a conventional culture medium containing a suitable carbon source, nitrogen source, phosphorus source, inorganic compounds, amino acids and / or vitamins, with the temperature, pH, etc., adjusted. 【0090】 In the culture described in this application, the culture temperature is maintained at 20-35°C, specifically 25-35°C, and the culture is performed for approximately 10-160 hours, approximately 20-130 hours, approximately 24-120 hours, approximately 36-120 hours, approximately 48-120 hours, approximately 48 hours, approximately 72 hours, or approximately 120 hours, but is not limited to these. 【0091】 The carotenoids or precursors produced by the culture described in this application are either secreted into the culture medium or remain within the microorganisms. 【0092】 The method for producing carotenoids or substances derived therefrom according to this application may further include, for example, the steps of preparing a Yarowia microorganism according to this application, preparing a culture medium for culturing the microorganism, or a combination thereof (in any order), before the culturing step. 【0093】 The method for producing carotenoids or substances derived therefrom according to this application may further include a step of recovering carotenoids or substances derived therefrom from the culture medium used for culturing the Yarowia microorganisms (the culture medium in which the culture was performed) or from the Yarowia microorganisms according to this application. The recovery step may further include a step after the culturing step. 【0094】 The aforementioned recovery may involve collecting the target retinol using a suitable method known in the art, depending on the microorganism culture method of this application, such as batch, continuous, or fed-batch culture. For example, various chromatography methods such as centrifugation, filtration, crystallization, treatment with protein precipitants (salting out), extraction, cell disruption, sonication, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, HPLC, or a combination thereof may be used to recover the target retinol from the culture medium or microorganism using a suitable method known in the art. 【0095】 Furthermore, the method for producing a carotenoid or a substance derived therefrom according to this application may further include a purification step. The purification can be carried out by a preferred method known in the art. For example, if the method for producing a carotenoid or a substance derived therefrom according to this application includes both a recovery step and a purification step, the recovery step and the purification step may be performed separately (or sequentially) in any order, simultaneously, or integrated as a single step, but are not limited to these. 【0096】 The carotenoid production method of this application may further include a step of converting β-carotene produced by the microorganism of this application into a carotenoid other than β-carotene. In the carotenoid production method of this application, the conversion step may further include after the culturing step or the recovery step. The conversion step can be carried out by a preferred method known in the art. For example, the conversion can be carried out chemically or using enzymes, but is not limited to these. 【0097】 The method for producing retinoids according to this application may further include a step of converting retinol produced by the Yarowia microorganisms according to this application into retinoids other than retinol. In the method for producing retinoids according to this application, the conversion step may further include a step after the culturing step or the recovery step. The conversion step can be carried out by a preferred method known in the art. For example, the conversion can be carried out using retinol acyltransferase, but is not limited thereto. 【0098】 As one example, the retinoid other than retinol is selected from the group consisting of retinal, retinoic acid, and retinyl ester, but any retinoid that is included in retinoids is acceptable. 【0099】 Further embodiments of this application provide compositions for producing carotenoids or substances derived therefrom, comprising the Yarowia microorganism of this application or a culture thereof. 【0100】 The aforementioned microorganisms, carotenoids, or substances derived from them are as described above. 【0101】 The compositions of this application may further contain any suitable excipients that are commonly used. Such excipients include, but are not limited to, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers, and isotonic agents. 【0102】 Further embodiments of this application provide the use of the microorganism or culture thereof for the production of carotenoids or substances derived therefrom. 【0103】 The aforementioned microorganisms, carotenoids, or substances derived from them are as described above. [Examples] 【0104】 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 application is not limited thereto. Technical matters not described herein can be fully understood and readily implemented by a skilled technician in the art of this application or a similar art. [Examples] 【0105】 Preparation of platform strains for producing carotenoids or substances derived from them. Example 1-1. Preparation of crtYB-crtI insert strains derived from Xanthophyllomyces dendroas. To create a platform strain for producing carotenoids or substances derived from them, the lycopene cyclase / phytoen synthase (crtYB) and phytoendesaturase (crtI) genes derived from Xanthophyllomyces dendroas were inserted into the genome of the high-fat yeast strain Yarrowia lipolytica CC08-0125 (deposit number KCCM12972P). 【0106】 The polynucleotide sequence for crtYB (SEQ ID NO: 71) was secured based on the nucleotide sequence registered in NCBI (National Center for Biotechnology Information Search database) (GenBank: AY177204.1), and the polynucleotide sequence for crtI (SEQ ID NO: 72) was secured based on the nucleotide sequence registered in NCBI (GenBank: AY177424.1). The polynucleotide sequences of crtYB and crtI were synthesized into the TEFINtp-crtYB-CYC1t (SEQ ID NO: 73) and TEFINtp-crtI-CYC1t (SEQ ID NO: 74) forms by Macrogen. Using the URA3 gene of Yarowia liporitica (SEQ ID NO: 75) as a selection marker, a cassette was designed to be inserted at the MHY1 (YALI0B21582g) gene position. Using the synthesized crtYB and crtI genes and the genomic DNA of KCCM12972P as templates, PCR was performed using primers for SEQ ID NOs. 76 and 77, SEQ ID NOs. 78 and 79, SEQ ID NOs. 80 and 81, SEQ ID NOs. 82 and 83, SEQ ID NOs. 84 and 85, and SEQ ID NOs. 86 and 87. The PCR conditions consisted of denaturation at 95°C for 1 minute, annealing at 55°C for 1 minute, and polymerization at 72°C for 3 minutes, repeated for 35 cycles. The resulting five DNA fragments were assembled into a single cassette by overlap extension PCR. 【0107】 The cassettes prepared in this manner were introduced into the KCCM12972P strain using the heat shock method (Non-Patent Literature 16), and colonies formed in uracil-free solid medium (YLMM1) were then obtained. Colonies in which cassette insertion into the genome was confirmed using primers of SEQ ID NO: 88 and SEQ ID NO: 89 were spotted onto 5-FOA solid medium and cultured at 30°C for 3 days. The URA3 marker was recovered by obtaining colonies grown in 5-FOA solid medium. 【0108】 [Table 1] 【0109】 Examples 1-2. Preparation of HMGR-enhanced strains As described above, a cassette was designed to replace the native promoter (SEQ ID NO: 90) of the hydroxymethylglutaryl-CoA reductase (HMGR) gene of the strain prepared in Example 1-1 with the TEFINt promoter. Using the genomic DNA of KCCM12972P as a template, PCR was performed using primers SEQ ID NOs: 91 and 92, 93 and 94, 95 and 96, 97 and 98, and 99 and 100. The PCR conditions were denaturation at 95°C for 1 minute, annealing at 55°C for 1 minute, and polymerization at 72°C for 1 minute and 30 seconds, repeated for 35 cycles. The resulting five DNA fragments were assembled into a single cassette by overlap extension PCR. 【0110】 The cassette prepared in this manner was introduced into the bacterial strain prepared in Example 1-1 by the heat shock method, and colonies formed on uracil-free solid medium (YLMM1) were then obtained. Colonies in which cassette insertion was confirmed were spotted onto 5-FOA solid medium using primers SEQ ID NO: 101 and SEQ ID NO: 102, and cultured at 30°C for 3 days. The URA3 marker was recovered by obtaining colonies grown on 5-FOA solid medium. The final platform strain 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-Fluororotic acid (5-FOA) 1g / L, Agar 15g / L 【0111】 [Table 2] [Examples] 【0112】 Creation of a strain with a geranylgeranyl pyrophosphate synthase (GGPP synthase) gene insertion from Haematococcus pluvialis. Four different GGPP synthase genes (hereinafter referred to as GGPPS genes) were introduced into the genome of the bacterial strain CC08-1023 prepared in Example 1, as follows. 【0113】 Example 2-1. Preparation of GGPPS insert strains derived from Haematococcus pulvialis To insert the GGPPS1 gene derived from Haematococcus pluvialis (hereinafter referred to as Hp.GGPPS1) onto the chromosome of Yarowia lipopolitica, the codons of Hp.GGPPS1 were optimized to suit Yarowia lipopolitica based on the nucleotide sequence registered in the NCBI (National Center for Biotechnology Information Search database) (GenBank: APX64485.1) according to Non-Patent Literature 17 (Sequence ID 1). The gene was then synthesized in the form of TEFINtp-codon optimized Hp.GGPPS1-CYC1t by Macrogen Corporation (Sequence ID 4). Using the URA3 gene of Yarowia lipopolitica (Sequence ID 5) as a selection marker, a cassette was designed to be inserted at the LIG4 (YALI0D21384g) gene position. Using the synthesized Hp.GGPPS1 gene and KCCM12972P genomic DNA as templates, PCR was performed on the left homologous region, TEFINt promoter, Hp.GGPPS1 ORF, CYC1 terminator, URA3, repeat region, and right homologous region fragments, respectively, using the primers shown in Table 3: SEQ ID NOs. 15 and 16, 17 and 18, 19 and 20, 21 and 22, 23 and 24, 25 and 26, and 27 and 28. The PCR conditions consisted of denaturation at 95°C for 1 minute, annealing at 55°C for 1 minute, and polymerization at 72°C for 2 minutes, repeated for 35 cycles. The resulting DNA fragments were assembled into a single cassette by overlap extension PCR. 【0114】 The cassettes prepared in this manner were introduced into the CC08-1023 strain using the heat shock method, and colonies formed on uracil-free solid medium (YLMM1) were then obtained. Colonies in which cassette insertion into the genome was confirmed using primers of SEQ ID NO: 29 and SEQ ID NO: 30 were streaked onto 5-FOA solid medium and cultured at 30°C for 3 days to obtain colonies formed on 5-FOA solid medium, thereby removing the URA3 marker. 【0115】 [Table 3] 【0116】 Example 2-2. Preparation of a strain with a crtE mutant gene inserted from Xanthophyllomyces dendroas. To insert the crtE mutant gene crtEM1 (SEQ ID NO: 6, Non-Patent Literature 18) derived from Xanthophylromyces dendroas onto the chromosome of Yarowia liporitica, we commissioned Macrogen to synthesize the gene in the form of TEFINtp-crtEM1-TDH3t (SEQ ID NO: 8). Using the URA3 gene of Yarowia liporitica (SEQ ID NO: 5) as a selection marker, we designed a cassette to be inserted at the LIG4 (YALI0D21384g) gene site. 【0117】 Using synthesized crtEM1 DNA and KCCM12972P genomic DNA as templates, PCR was performed on the left homologous region, TEFINt promoter, crtEM1 ORF, TDH3 terminator, URA3, repeat region, and right homologous region fragments, respectively, using the primers shown in Table 4: SEQ ID NOs. 31 and 32, SEQ ID NOs. 33 and 34, SEQ ID NOs. 35 and 36, SEQ ID NOs. 37 and 38, SEQ ID NOs. 39 and 40, SEQ ID NOs. 41 and 42, and SEQ ID NOs. 43 and 44. 【0118】 The PCR conditions consisted of denaturation at 95°C for 1 minute, annealing at 55°C for 1 minute, and polymerization at 72°C for 2 minutes, repeated for 35 cycles. The resulting DNA fragments were then assembled into a single cassette by overlap extension PCR. 【0119】 The cassettes prepared in this manner were introduced into the CC08-1023 strain using the heat shock method, and colonies formed on uracil-free solid medium (YLMM1) were then obtained. Colonies in which cassette insertion into the genome was confirmed using primers of SEQ ID NO: 45 and SEQ ID NO: 46 were streaked onto 5-FOA solid medium and cultured at 30°C for 3 days to obtain colonies formed on 5-FOA solid medium, thereby removing the URA3 marker. 【0120】 [Table 4] 【0121】 Examples 2-3. Preparation of BTS1 insert strains derived from Saccharomyces cerevisiae To insert the BTS1 gene derived from Saccharomyces cerevisiae (hereinafter referred to as Sc.BTS1) onto the chromosome of Yarowia lipopolitica, the polynucleotide of sequence number 9 was secured based on the nucleotide sequence (YPL069C) registered in KEGG (Kyoto Encyclopedia of Genes and Genomes). Using the aforementioned polynucleotide of BTS1, the gene was synthesized in the form of TEFINtp-Sc.BTS1-TDH3t (sequence number 10). Using the URA3 gene of Yarowia lipopolitica (sequence number 5) as a selection marker, a cassette was designed to be inserted at the LIG4 (YALI0D21384g) gene position. 【0122】 Using synthesized Sc.BTS1 DNA and KCCM12972P genomic DNA as templates, PCR was performed on the left homologous region, TEFINt promoter, Sc.BTS1 ORF, TDH3 terminator, URA3, repeat region, and right homologous region fragments, respectively, using the primers shown in Table 5: SEQ ID NOs. 31 and 32, 33 and 47, 48 and 49, 50 and 38, 39 and 40, 41 and 42, and 43 and 44. The PCR conditions consisted of denaturation at 95°C for 1 minute, annealing at 55°C for 1 minute, and polymerization at 72°C for 2 minutes, repeated for 35 cycles. The resulting DNA fragments were assembled into a single cassette by overlap extension PCR. 【0123】 The cassettes prepared in this manner were introduced into the CC08-1023 strain using the heat shock method, and colonies formed on uracil-free solid medium (YLMM1) were then obtained. Colonies in which cassette insertion into the genome was confirmed using primers of SEQ ID NO: 45 and SEQ ID NO: 46 were streaked onto 5-FOA solid medium and cultured at 30°C for 3 days to obtain colonies formed on 5-FOA solid medium, thereby removing the URA3 marker. 【0124】 [Table 5] 【0125】 Example 2-4. Preparation of GGS1 insert strains derived from Yarowia liporitica To insert the GGS1 gene derived from Yarowia lipopolitica (hereinafter referred to as Yl.GGS1) onto the chromosome of Yarowia lipopolitica, the polynucleotide of SEQ ID NO: 11 was secured based on the nucleotide sequence (YALI0D17050g) registered in KEGG (Kyoto Encyclopedia of Genes and Genomes). Using the aforementioned Yl.GGS1 polynucleotide, the gene was synthesized in the form of TEFINtp-Yl.GGS1-TDH3t (SEQ ID NO: 12). Using the URA3 gene of Yarowia lipopolitica (SEQ ID NO: 5) as a selection marker, a cassette was designed to be inserted at the LIG4 (YALI0D21384g) gene position. 【0126】 Using the synthesized Yl.GGS1 gene and KCCM12972P genomic DNA as templates, PCR was performed on the left homologous region, TEFINt promoter, Yl.GGS1 ORF, TDH3 terminator, URA3, repeat region, and right homologous region fragments, respectively, using the primers shown in Table 6: SEQ ID NOs. 31 and 32, SEQ ID NOs. 33 and 51, SEQ ID NOs. 52 and 53, SEQ ID NOs. 54 and 38, SEQ ID NOs. 39 and 40, SEQ ID NOs. 41 and 42, and SEQ ID NOs. 43 and 44. The PCR conditions consisted of denaturation at 95°C for 1 minute, annealing at 55°C for 1 minute, and polymerization at 72°C for 2 minutes, repeated for 35 cycles. The resulting DNA fragments were then assembled into a single cassette by overlap extension PCR. 【0127】 The cassettes prepared in this manner were introduced into the CC08-1023 strain using the heat shock method, and colonies formed on uracil-free solid medium (YLMM1) were then obtained. Colonies in which cassette insertion into the genome was confirmed using primers of SEQ ID NO: 45 and SEQ ID NO: 46 were streaked onto 5-FOA solid medium and cultured at 30°C for 3 days to obtain colonies formed on 5-FOA solid medium, thereby removing the URA3 marker. 【0128】 [Table 6] [Examples] 【0129】 Comparative evaluation of β-carotene production capacity based on GGPP synthase-introduced strains. Flask evaluation was performed on all five strains: the bacterial strains secured in Examples 2-1 to 2-4 and the parent strain CC08-1023 secured in Example 1. The bacterial strains were inoculated into 250 ml corner baffle flasks containing 20 ml of YPD (Yeast extract-Peptone-Dextrose) medium to achieve an initial OD of 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 and the supernatant was removed. The composition of the YPD medium was as follows. <YPD liquid media> Dissolved in 0.1M sodium phosphate buffer (pH 7.0) in a ratio of 4% glucose, 1% yeast extract, and 2% peptone. 【0130】 Next, 0.5 ml of DMSO (Dimethyl sulfoxide, Sigma, CAS number 67-68-5) was added, and the cells were disrupted by agitation at 55°C for 10 minutes (2,000 rpm). Furthermore, 0.5 ml of acetone (Sigma, CAS number 67-64-1) was added, and β-carotene and squalene were extracted by agitation at 45°C for 15 minutes (2,000 rpm), and the concentrations were analyzed using an HPLC system. The results of the β-carotene and squalene concentration measurements are shown in Figure 1. 【0131】 As a result, as shown in Figure 1, the β-carotene concentrations measured in CC08-1023 (parent strain), Hp.GGPPS1-introduced strain, crtEM1-introduced strain, Sc.BTS1-introduced strain, and Yl.GGS1-introduced strain were 5.49 mg / L, 58.73 mg / L, 40.58 mg / L, 5.21 mg / L, and 49.22 mg / L, respectively. In particular, the introduction of Hp.GGPPS1 increased β-carotene by 53.24 mg / L compared to the parent strain, confirming that it had the best effect in increasing β-carotene. 【0132】 Furthermore, the measured squalene concentrations were 313.24 mg / L, 200.31 mg / L, 235.27 mg / L, 253.28 mg / L, and 221.22 mg / L, respectively. Similarly, when Hp.GGPPS1 was introduced, the squalene concentration decreased by 112.93 mg / L compared to the CC08-1023 strain, confirming that it had the best effect in reducing squalene production. 【0133】 These results confirmed that Hp.GGPPS1 exhibited the most superior GGPP synthase activity among microorganisms of the genus Yarovia. Surprisingly, while the effects were minimal when geranylgeranyl pyrophosphate synthase from closely related Saccharomyces cerevisiae, Yarovia lipopolitica, and Xanthophylromyces dendroas were introduced, the effects were significant when geranylgeranyl pyrophosphate synthase from the relatively unrelated Haematococcus pluvialis was introduced. [Examples] 【0134】 Creation of a β-carotene 15,15' oxygenase (BCO) gene-transformed strain To insert the β-carotene 15,15' oxygenase (hereinafter referred to as Mb.BCO) gene derived from the marine bacterium 66A03 (Uncultured marine bacterium 66A03) onto the chromosome of Yarowia lipopolitica, a polynucleotide sequence (SEQ ID NO: 13) was secured by optimizing the codons of Mb.BCO to suit Yarowia lipopolitica based on the amino acid sequence (Q4PNI0) registered in UniProtKB (UniProt Knowledgebase) according to Non-Patent Literature 17. Using the aforementioned polynucleotide of Mb.BCO, the gene was synthesized in the form of TEFINtp-codon optimized Mb.BCO-CYC1t (SEQ ID NO: 14). Using the URA3 gene (SEQ ID NO: 5) of Yarowia lipopolitica as a selection marker, a cassette was designed to be inserted at the KU70 (YALI0C08701g) gene position. Using synthesized Mb.BCO and KCCM12972P genomic DNA as templates, PCR was performed on the left homologous region, TEFINt promoter, Mb.BCO ORF, CYC1 terminator, URA3, repeat region, and right homologous region, respectively, using the primers shown in Table 7: SEQ ID NOs. 55 and 56, 57 and 58, 59 and 60, 61 and 62, 63 and 64, 65 and 66, and 67 and 68. The PCR conditions consisted of denaturation at 95°C for 1 minute, annealing at 55°C for 1 minute, and polymerization at 72°C for 2 minutes, repeated 35 times. The resulting DNA fragments were assembled into a single cassette by overlap extension PCR. 【0135】 The cassettes prepared in this manner were introduced into the bacterial strains prepared in Examples 2-1 to 2-4 using the heat shock method, and colonies formed on uracil-free solid medium (YLMM1) were then obtained. Colonies in which cassette insertion into the genome was confirmed using primers of SEQ ID NO: 69 and SEQ ID NO: 70 were streaked onto 5-FOA solid medium and cultured at 30°C for 3 days to obtain colonies formed on 5-FOA solid medium, thereby removing the URA3 marker. 【0136】 [Table 7] [Examples] 【0137】 Comparative evaluation of retinol production capacity of β-carotene 15,15' oxygenase (BCO) gene-transformed strains. Flask evaluation was performed on all five strains, including the strains secured in Example 4 and the parent strain CC08-1023 secured in Example 1. The strains were inoculated into 250 ml corner baffle flasks containing 20 ml of YPD (Yeast extract-Peptone-Dextrose) medium and 0.05% butlated hydroxytoluene, so that the initial OD was 2, and the flasks were cultured with shaking at 30°C and 200 rpm for 48 hours. After the culture period, 1 ml of the culture medium was centrifuged and the supernatant was removed. 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 retinol, retinal, β-carotene, and squalene were extracted by shaking (agitation, 2,000 rpm) at 45°C for 15 minutes, and their concentrations were analyzed using an HPLC system. Figure 2 shows the concentration measurements of retinol, retinal, β-carotene, and squalene that were analyzed. 【0138】 As a result, as shown in Figure 2, no retinol was detected in the strain in which Mb.BCO was introduced into the CC08-1023 strain. In contrast, in four strains in which Mb.BCO was introduced after introducing Hp.GGPPS1, crtEM1, Sc.BTS1, and Yl.GGS1, respectively, into the CC08-1023 base, retinol concentrations of 8.44 mg / L, 2.78 mg / L, 0 mg / L, and 4.35 mg / L were measured, respectively. 【0139】 The β-carotene concentrations in the five strains were 3.68 mg / L, 0.35 mg / L, 2.47 mg / L, 3.58 mg / L, and 0.98 mg / L, respectively, confirming that β-carotene was converted to retinol, leading to a decrease in β-carotene concentration. In addition, the measured squalene concentrations in the five strains were 309.88 mg / L, 202.18 mg / L, 282.19 mg / L, 306.34 mg / L, and 269.18 mg / L, respectively. 【0140】 These results confirm that enhancing GGPP biosynthesis has a positive effect on improving retinol production capacity. 【0141】 Furthermore, these results confirmed that Hp.GGPPS1 has excellent effects on β-carotene production, squalene reduction, and retinol production. 【0142】 From the above explanation, 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. It should be understood that the above embodiments are merely illustrative and not limiting. This application should be interpreted as including all modified or altered forms derived from the meaning and scope of the claims and their equivalent concepts, rather than the specification. 【0143】 Table 8 shows the sequence of sequence numbers for this application. 【0144】 [Table 8-1] 【0145】 Table 8-2 【0146】 Table 8-3 【0147】 Table 8-4 【0148】 Table 8-5 【0149】 Table 8-6 【0150】 Table 8-7 【0151】 Table 8-8 【0152】 Table 8-9 【0153】 Table 8-10 【0154】 Table 8-11 【0155】 Table 8-12 【0156】 Table 8-13 【0157】 Table 8-14 【0158】 Table 8-15 【0159】 Table 8-16 【0160】 Table 8-17 【0161】 Table 8-18 【0162】 Table 8-19 【0163】 Table 8-20 【0164】 Table 8-21 【0165】 Table 8-22 【0166】 Table 8-23 【0167】 Table 8-24 【0168】 Table 8-25 【0169】 JPEG0007872854000033.jpg211145

Claims

[Claim 1] A Yarowia microorganism expressing geranylgeranyl pyrophosphate synthase derived from Haematococcus pluvialis, having the ability to produce carotenoids or substances derived therefrom, wherein the geranylgeranyl pyrophosphate synthase contains the amino acid sequence of SEQ ID NO: 103 or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO:

103. [Claim 2] The Yarowia microorganism according to claim 1, wherein the geranylgeranyl pyrophosphate synthase consists of the amino acid sequence of SEQ ID NO:

103. [Claim 3] The Yarowia microorganism according to claim 1, wherein the geranylgeranyl pyrophosphate synthase is encoded by a polynucleotide consisting of the base sequence of Sequence ID No.

1. [Claim 4] The Yarowia microorganism according to claim 1, wherein the Yarowia microorganism is Yarowia liporitica. [Claim 5] The Yarowia microorganism according to claim 1, wherein the substance having the carotenoid as a precursor is a retinoid. [Claim 6] The Yarowia microorganism according to claim 1, wherein the carotenoid is β-carotene. [Claim 7] The Yarowia microorganism according to claim 5, wherein the retinoid is retinol. [Claim 8] The Yarowia microorganism according to claim 1, wherein the Yarowia microorganism has reduced by-product production capacity. [Claim 9] The Yarowia microorganism according to claim 8, wherein the by-product is squalene. [Claim 10] A step of culturing a Yarowia microorganism according to any one of claims 1 to 9 in a culture medium, A method for producing carotenoids or substances derived from carotenoids, comprising the step of recovering carotenoids or substances derived from carotenoids or substances derived from carotenoids or substances derived from carotenoids from the Yarowia microorganism or culture medium. [Claim 11] The steps include converting the β-carotene produced by the Yarowia microorganism into carotenoids other than β-carotene, or A method for producing a carotenoid or a substance derived therefrom, according to claim 10, further comprising the step of converting the retinol produced by the Yarowia microorganism into a retinoid other than retinol. [Claim 12] A composition for producing a carotenoid or a substance derived therefrom, comprising a microorganism of the genus Yarowia as described in any one of claims 1 to 9. [Claim 13] Use of a Yarowia microorganism capable of producing carotenoids or carotenoid precursors, which expresses geranylgeranyl pyrophosphate synthase derived from Haematococcus pluvialis, for the production of carotenoids or carotenoid precursors, wherein the geranylgeranyl pyrophosphate synthase comprises the amino acid sequence of SEQ ID NO: 103 or an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 103.

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