Microorganism for producing carotenoid or substance having carotenoid as precursor, comprising haematococcus pluvialis geranylgeranyl pyrophosphate synthase, and method for producing carotenoid or retinoid using same

Abstract

The present application relates to: a microorganism of the genus Yarrowia, which has the ability to produce a carotenoid or a substance having a carotenoid as a precursor, and which expresses a geranylgeranyl pyrophosphate synthase from Haematococcus pluvialis; a method for producing a carotenoid or a substance having a carotenoid as a precursor using the microorganism; a composition for producing a carotenoid or a substance using a carotenoid as a precursor; and a use of the microorganism of the genus Yarrowia or a culture thereof in the production of carotenoid or a substance taking carotenoid as a precursor.

Description

Technical Field

[0001] The present disclosure relates to a Yarrowia microorganism having the ability to produce carotenoids or substances with carotenoids as precursors, wherein the microorganism expresses geranylgeranyl pyrophosphate synthase from Haematococcus pluvialis; a method for producing carotenoids or substances with carotenoids as precursors using the microorganism; a composition for producing carotenoids or substances with carotenoids as precursors; and use of a Yarrowia microorganism or a culture thereof in producing carotenoids or substances with carotenoids as precursors. Background Art

[0002] Since carotenoids and retinoids exert various functions in plants and animals, they are used in various industrial fields such as food, feed, etc. Among them, carotenoids such as β-carotene are substances reported to have functions such as eliminating free radicals, serving as a precursor of vitamin A in animals, enhancing the immune system of vertebrates, and reducing the risk of lung cancer, and retinoids are a class of substances chemically related to retinol (i.e., vitamin A) and are also used in cosmetics, skin disease therapeutic agents, etc.

[0003] However, despite these advantages, carotenoids (such as β-carotene) and retinoids (such as retinol) cannot be synthesized in animals or are synthesized in insufficient amounts. In addition, even if attempts are made to use mutant microorganisms (U.S. Patent No. 7745170) for industrial production, it is still difficult to produce them with high purity.

[0004] For example, squalene (C30) may also be produced as a byproduct in the production process of microorganisms producing carotenoids or retinoids. Therefore, the discovery of geranylgeranyl pyrophosphate synthase that contributes to the efficient production of carotenoids or retinoids is crucial to increase their production and reduce squalene produced in competing pathways. Summary of the invention

[0005] [Technical issues]

[0006] The problem to be solved in the present disclosure is to provide a microorganism for producing carotenoids or substances with carotenoids as precursors, the microorganism comprising geranylgeranyl pyrophosphate synthase from Haematococcus pluvialis; a method for producing carotenoids or retinoids using the microorganism; and uses thereof.

[0007] [Technical solution]

[0008] An object of the present disclosure is to provide a microorganism of the genus Yarrowia having the ability to produce carotenoids or substances with carotenoids as precursors, the microorganism expressing geranylgeranyl pyrophosphate synthase from Haematococcus pluvialis.

[0009] Another object of the present disclosure is to provide a method for producing carotenoids or substances using carotenoids as precursors using a microorganism of the genus Yarrowia.

[0010] Another object of the present disclosure is to provide a composition for producing carotenoids or substances with carotenoids as precursors, the composition comprising a microorganism of the genus Yarrowia or a culture thereof.

[0011] Another object of the present disclosure is to provide use of a microorganism of the genus Yarrowia in producing carotenoids or substances using carotenoids as precursors.

[0012] [Beneficial Effects]

[0013] The present invention can effectively increase the production of carotenoids and substances using carotenoids as precursors by introducing a geranylgeranyl pyrophosphate synthase gene from Haematococcus pluvialis into a microorganism of the genus Yarrowia. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The results of shake flask experiments on strains into which GGPP synthase genes from various microorganisms were introduced are shown; and

[0015] Figure 2 The results of the flask test of the strain into which Mb.BCO was introduced are shown. DETAILED DESCRIPTION

[0016] The present disclosure will be described in detail below. At the same time, each description and embodiment disclosed in the present disclosure can also be applied to other descriptions and embodiments. That is to say, all combinations of the various elements disclosed in the present disclosure fall within the scope of the present disclosure. In addition, the scope of the present disclosure is not limited by the specific description described below. In addition, many papers and patent documents are referenced and cited throughout the specification. The disclosures of the cited papers and patent documents are incorporated herein by reference as a whole to further illustrate the level and scope of the subject matter to which the present disclosure belongs.

[0017] One aspect of the present disclosure provides a Yarrowia microorganism having the ability to produce carotenoids or substances with carotenoids as precursors, the microorganism expressing geranylgeranyl pyrophosphate synthase from Haematococcus pluvialis.

[0018] As used herein, "geranylgeranyl pyrophosphate synthase" is an enzyme that can catalyze the synthesis of geranylgeranyl pyrophosphate (GGPP). The substrates of geranylgeranyl pyrophosphate synthase can be isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP). Geranylgeranyl pyrophosphate synthase can also be named "GGS", "GGPPS", "GGPS", "GGPPS1" or "a polypeptide having geranylgeranyl pyrophosphate synthase activity".

[0019] In one embodiment, the microorganism of the present disclosure may be a Yarrowia microorganism that contains or expresses a geranylgeranyl pyrophosphate synthase protein from Haematococcus pluvialis, which is an exogenous protein, and may have the ability to produce carotenoids or substances with carotenoids as precursors.

[0020] The amino acid sequence of the GGPPS protein disclosed herein may be a protein sequence having geranylgeranyl pyrophosphate synthase activity, which is encoded by a GGPPS gene. The amino acid sequence may be obtained in various databases, such as NCBI GenBank, etc. (known databases), but is not limited thereto.

[0021] In one embodiment, the GGPPS protein of the present disclosure may be from Haematococcus pluvialis, and any protein may be included in the present disclosure as long as it has the same sequence or activity as that thereof.

[0022] In one embodiment, the GGPPS protein of the present disclosure may comprise, have, or consist of SEQ ID NO: 103, or an amino acid sequence having 80% or more homology or identity thereto, or may consist essentially of the amino acid sequence.

[0023] In addition, although one embodiment of the GGPPS protein of the present disclosure is described as a protein comprising SEQ ID NO: 103, such expression does not exclude mutations that may occur by adding a nonsense sequence upstream or downstream of the amino acid sequence of SEQ ID NO: 103, or naturally occurring mutations therein, or silent mutations thereof, and it is obvious to those skilled in the art that any protein may belong to the GGPPS protein of the present disclosure as long as it has the same or corresponding activity as the protein comprising the amino acid sequence.

[0024] Specifically, the GGPPS protein of the present disclosure may comprise the amino acid sequence of SEQ ID NO: 103 or 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. In addition, it is obvious that proteins having amino acid sequences in which some sequences are deleted, modified, substituted or added are also included in the scope of the present disclosure, as long as the amino acid sequence has such homology or identity and exhibits the efficacy corresponding to the above-mentioned protein.

[0025] Although described in the present disclosure as "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 is apparent that a protein having an amino acid sequence with deletion, modification, substitution, conservative substitution or addition of certain sequences can also be used in the present disclosure, as long as it has the same or corresponding activity as a polypeptide consisting of an amino acid sequence of the corresponding SEQ ID NO. Examples thereof include those having a sequence added to the N-terminus, interior and / or C-terminus of the amino acid sequence that does not change the function of the protein, a naturally occurring mutation, a silent mutation or a conservative substitution thereof.

[0026] "Conservative substitution" refers to the replacement of one amino acid with another amino acid of similar structure and / or chemical properties. Such amino acid substitutions generally occur based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathicity of the residues. Typically, conservative substitutions may have little or no effect on the activity of the polypeptide.

[0027] As used herein, the term "homology" or "identity" refers to the degree of similarity between two given amino acid sequences or nucleotide sequences, which can be expressed as a percentage. The terms "homology" and "identity" are often used interchangeably.

[0028] The sequence homology or identity of conservative polynucleotide or polypeptide can be determined by standard comparison algorithm, and can use the default gap penalty set up by the program to be used together.Basically, under moderate or highly stringent conditions, homologous or identical sequences can hybridize with each other along at least about 50%, 60%, 70%, 80% or 90% of the entire sequence or entire length usually.Obviously, the hybridization also comprises the hybridization of the polynucleotide containing universal codon or the codon taking into account the degeneracy of codons in the polynucleotide.

[0029] Whether any two polynucleotide or polypeptide sequences have homology, similarity or identity can be determined using known computer algorithms, such as the "FASTA" program using default parameters, for example, as described in Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]:2444. Alternatively, they can be determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453), such as the Needleman program in the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277) (version 5.0.0 or later) (including the GCG program package (Devereux, J., et al, Nucleic Acids Research 12:387 (1984)), BLASTP, BLASTN, FASTA (Atschul, [S.] [F.,] [ET AL, J MOLEC BIOL 215]:403 (1990); Guide to Huge Computers, Martin J. Bishop, [ED.,] Academic Press, San Diego, 1994 and [CARILLO ETA / .] (1988) SIAM J Applied Math 48: 1073). For example, homology, similarity or identity can be determined using BLAST or ClustalW from the National Center for Biotechnology.

[0030] Homology, similarity or identity of polynucleotides or polypeptides can be determined by comparing sequence information using the GAP computer program (e.g., Needleman et al. (1970), J Mol Biol. 48:443), for example, as disclosed in Smith and Waterman, Adv. Appl. Math (1981) 2:482. Briefly, the GAP program defines similarity as the number of aligned symbols (i.e., nucleotides or amino acids) that are similar divided by the total number of symbols in the shorter of the two sequences. The default parameters of the GAP program can include: (1) the binary comparison matrix of Gribskov et al (1986) Nucl. Acids Res. 14:6745 (including a value of 1 for identity and a value of 0 for non-identity) and a weighted comparison matrix (or the EDNAFULL (EMBOSS version of NCBINUC4.4) substitution matrix), as disclosed in Atlas Of Protein Sequence And Structure, edited by Schwartz and Dayhoff, National Biomedical Research Foundation, pp. 353-358 (1979); (2) a penalty of 3.0 for each gap and an additional penalty of 0.10 for each symbol in each gap (or a gap open penalty of 10 and a gap extension penalty of 0.5); and (3) no penalty for end gaps.

[0031] In addition, whether any two polynucleotide or polypeptide sequences have homology, similarity or identity can be determined by comparing these sequences through Southern hybridization experiments under defined stringent conditions. The appropriate hybridization conditions to be determined can be within the scope of the technology and can be determined by methods well known to those of ordinary skill in the art (e.g., J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989; FM Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York).

[0032] In the present disclosure, the expression of a protein may be achieved by introducing a gene (polynucleotide) encoding the protein into a microorganism or injecting the protein therein, but is not limited thereto.

[0033] In one embodiment, the microorganism of the present disclosure may be introduced with a geranylgeranyl pyrophosphate synthase gene from Haematococcus pluvialis. In addition, the introduction of the geranylgeranyl pyrophosphate synthase gene may further include enhancing its activity after the introduction.

[0034] As used herein, "geranylgeranyl pyrophosphate synthase gene" can be 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".

[0035] The sequence of the geranylgeranyl pyrophosphate synthase gene can be obtained in various databases, such as NCBI GenBank and the like (known databases), but is not limited thereto.

[0036] In one embodiment, the geranylgeranyl pyrophosphate synthase gene from Haematococcus pluvialis may include, have or consist of the nucleotide sequence of SEQ ID NO: 1, but is not limited thereto.

[0037] In one embodiment, the geranylgeranyl pyrophosphate synthase gene consisting of the nucleotide sequence of SEQ ID NO: 1 may be codon-optimized for a microorganism of the genus Yarrowia, or more specifically, Yarrowia lipolytica.

[0038] As used herein, the term "polynucleotide", ie, a nucleotide polymer in which nucleotide monomers are covalently bonded in a long chain, refers to a DNA chain having a predetermined length or longer.

[0039] In addition, due to codon degeneracy or considering preferred codons for expressing geranylgeranyl pyrophosphate synthase polypeptide in an organism, the polynucleotide or gene may have various modifications in the coding region within the range of not changing the amino acid sequence of the polypeptide. The polynucleotide or gene may include, for example, the nucleotide sequence of SEQ ID NO: 1, and may be composed of a nucleotide 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 thereto, but is not limited thereto.

[0040] In addition, the polynucleotides or genes disclosed herein may include probes that can be prepared from known gene sequences, for example, any sequence, without limitation, as long as it is a sequence that hybridizes with all or part of the complementary sequence of the nucleotide sequence under stringent conditions to encode the amino acid sequence of SEQ ID NO: 103. "Stringent conditions" refer to conditions that enable specific hybridization between polynucleotides. These conditions are specifically described in the literature (e.g., J. Sambrook et al., supra). For example, stringent conditions may include conditions under which polynucleotides having a high degree of homology or identity (e.g., 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 homology or identity) can hybridize to each other, while polynucleotides having a homology or identity lower than the above-mentioned homology or identity cannot hybridize to each other; or ordinary washing conditions for Southern hybridization, wherein washing is performed once, specifically two to three times using a salt concentration and temperature equivalent to 60°C, 1X SSC, 0.1% SDS, specifically 60°C, 0.1X SSC, 0.1% SDS, more specifically 68°C, 0.1X SSC, 0.1% SDS.

[0041] Hybridization requires that the two nucleic acids have complementary sequences, although mismatches between nucleotides are possible, depending on the stringency of the hybridization. The term "complementary" is used to describe the relationship between nucleotides that can hybridize with each other. For example, with respect to DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Therefore, the polynucleotides of the present disclosure may also include isolated nucleic acid fragments that are complementary to the complete sequence, as well as nucleic acid sequences substantially similar thereto.

[0042] Specifically, polynucleotides having homology or identity can be detected using hybridization conditions, which include a Tm value of 55° C. and a hybridization step under the above conditions. In addition, the Tm value may be 60° C., 63° C., or 65° C., but is not limited thereto, and can be appropriately adjusted by those skilled in the art according to the purpose.

[0043] The appropriate stringency for hybridization of polynucleotides depends on the length of the polynucleotides and the degree of complementation, and variables are well known in the art (see Sambrook et al., supra, 9.50-9.51, 11.7-11.8).

[0044] In one embodiment, the microorganism of the present disclosure may comprise a vector comprising the geranylgeranyl pyrophosphate synthase gene from Haematococcus pluvialis of the present disclosure or a polynucleotide encoding the geranylgeranyl pyrophosphate synthase from Haematococcus pluvialis.

[0045] The vector of the present disclosure may comprise a DNA construct comprising a nucleotide sequence of a polynucleotide encoding a target polypeptide, which is operably linked to a suitable expression regulatory region (or expression control sequence) so that the target polypeptide can be expressed in a suitable host. The expression regulatory region may comprise a promoter capable of initiating transcription, any operon sequence for controlling transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence controlling transcription and translation termination. The vector may be transformed into a suitable host cell and then replicated or functioned independently of the host genome, or may be integrated into the genome itself.

[0046] The vector used in the present disclosure is not particularly limited, but any vector known in the art can be used. Examples of commonly used vectors include natural or recombinant plasmids, cosmids, viruses and bacteriophages. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, Charon21A, etc. can be used as phage vectors or cosmid vectors, and pDC systems, pBR systems, pUC systems, pBluescript II systems, pGEM systems, pTZ systems, pCL systems, pET systems, etc. can be used as plasmid vectors. Specifically, pDZ, pDC, pDCM2 (Korean Patent Publication No. 10-2020-0136813), pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC, pIMR53 vectors, etc. can be used.

[0047] For example, the polynucleotide encoding the target polypeptide can be inserted into the chromosome by a vector for intracellular chromosome insertion. The polynucleotide can be inserted into the chromosome by any method known in the art, such as homologous recombination, but is not limited thereto. The vector can also include a selection marker for confirming chromosome insertion. The selection marker is used to select cells transformed with the vector, i.e., to confirm the insertion of the target nucleic acid molecule, and can use markers that confer selectable phenotypes such as drug resistance, auxotrophy, cytotoxic agent resistance or surface polypeptide expression. In an environment treated with a selection agent, only cells expressing the selection marker survive or show other phenotypic traits, so the transformed cells can be selected.

[0048] As used herein, the term "transformation" refers to the introduction of a vector containing a polynucleotide encoding a target polypeptide into a host cell or microorganism so that the polypeptide encoded by the polynucleotide can be expressed in the host cell. Regardless of the position, the converted polynucleotide can be located by inserting into the chromosome of the host cell or outside the chromosome, as long as it can be expressed in the host cell. In addition, the polynucleotide comprises DNA and / or RNA encoding the target polypeptide. The polynucleotide can be introduced in any form, as long as it can be introduced into the host cell and expressed. For example, the polynucleotide can be introduced into the host cell in the form of an expression cassette, which is a gene construct containing all the elements required for self-expression. The expression cassette can generally include a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal operably connected to the polynucleotide. The expression cassette can be in the form of an expression vector capable of self-replication. In addition, the polynucleotide can be introduced into the host cell in its own form and operably connected to a sequence required for expression in the host cell, but is not limited thereto.

[0049] Furthermore, the term "operably linked" means that a polynucleotide sequence is functionally linked to a promoter sequence, which initiates and mediates the transcription of the polynucleotide encoding the desired polypeptide of the present disclosure.

[0050] In one embodiment, a Yarrowia microorganism expressing the GGPPS from Haematococcus pluvialis of the present disclosure may have enhanced geranylgeranyl pyrophosphate synthase activity compared to a Yarrowia microorganism not expressing the GGPPS from Haematococcus pluvialis of the present disclosure, but is not limited thereto.

[0051] In one embodiment, a microorganism of the genus Yarrowia into which the GGPPS gene from Haematococcus pluvialis of the present disclosure is introduced may have enhanced geranylgeranyl pyrophosphate synthase activity compared to a microorganism of the genus Yarrowia into which the GGPPS gene from Haematococcus pluvialis is not introduced, but is not limited thereto.

[0052] In one embodiment, a Yarrowia microorganism introduced with the geranyl pyrophosphate synthase of the present disclosure encoded by the GGPPS gene from Haematococcus pluvialis can have enhanced geranyl pyrophosphate synthase activity compared to a Yarrowia microorganism introduced with the geranyl pyrophosphate synthase encoded by crtE or its variant gene crtEM1 from Xanthophyllomyces dendrorhous, the BTS1 gene from Saccharomyces cerevisiae, or the GGS1 gene from Yarrowia lipolytica, but is not limited thereto.

[0053] As used herein, the term "microorganism of the genus Yarrowia" or "strain of the genus Yarrowia" includes all wild-type microorganisms of the genus Yarrowia or naturally or artificially genetically modified microorganisms of the genus Yarrowia, and it can be a microorganism of the genus Yarrowia in which a specific mechanism is enhanced due to the insertion of foreign genes or the enhancement of the activity of endogenous genes, and it can be a microorganism of the genus Yarrowia comprising the GGPPS gene from Haematococcus pluvialis for producing carotenoids or substances with carotenoids as precursors.

[0054] The microorganism disclosed herein may be a microorganism comprising any one or more of the GGPPS proteins disclosed herein, a GGPS gene or polynucleotide encoding a GGPPS protein, and a vector comprising the gene or polynucleotide; a microorganism modified to express the GGPPS protein or GGPPS gene from Haematococcus pluvialis disclosed herein; a microorganism (e.g., a recombinant strain) expressing the GGPPS protein or GGPPS gene from Haematococcus pluvialis disclosed herein; or a strain having the activity of the GGPPS from Haematococcus pluvialis disclosed herein (e.g., a recombinant strain), but is not limited thereto.

[0055] The strain disclosed herein may be a microorganism that naturally possesses geranylgeranyl pyrophosphate synthase or has the ability to produce carotenoids or substances with carotenoids as precursors; or a microorganism in which geranylgeranyl pyrophosphate synthase or the ability to produce carotenoids or substances with carotenoids as precursors is enhanced or provided by introducing the GGPPS protein, gene, polynucleotide or vector containing them from Haematococcus pluvialis disclosed herein into a parent strain that does not possess geranylgeranyl pyrophosphate synthase or does not have the ability to produce carotenoids or substances with carotenoids as precursors, but is not limited thereto.

[0056] For example, the strains disclosed herein may include all microorganisms transformed with the GGPPS protein, gene, polynucleotide or vector comprising the GGPPS protein from Haematococcus pluvialis disclosed herein, to produce carotenoids or substances with carotenoids as precursors or to have enhanced production capacity. For example, the strains disclosed herein may be recombinant strains that have enhanced production capacity of carotenoids or substances with carotenoids as precursors by expressing the GGPPS from Haematococcus pluvialis disclosed herein in natural wild-type microorganisms or microorganisms that produce carotenoids or substances with carotenoids as precursors. The recombinant strain having an enhanced ability to produce carotenoids or substances with carotenoids as precursors may be a microorganism having an enhanced ability to produce carotenoids or substances with carotenoids as precursors, compared to a natural wild-type microorganism or a microorganism in which geranylgeranyl pyrophosphate synthase is not modified (i.e., a Yarrowia microorganism comprising a wild-type geranylgeranyl pyrophosphate synthase gene (SEQ ID NO: 11) or a Yarrowia microorganism into which the geranylgeranyl pyrophosphate synthase gene (SEQ ID NO: 1) from Haematococcus pluvialis has not been introduced), but is not limited thereto.

[0057] For example, compared to a Yarrowia microorganism that does not contain GGPPS from Haematococcus pluvialis (e.g., SEQ ID NO: 103); or a Yarrowia microorganism that contains CrtE or its variant CrtEM1 derived from Phaffia rhodozyma, BTS1 derived from Saccharomyces cerevisiae, or Yarrowia lipolytica GGS1, the strain with enhanced ability to produce carotenoids or substances with carotenoids as precursors disclosed herein may be a microorganism with enhanced ability to produce carotenoids or substances with carotenoids as precursors, but is not limited thereto. For example, an unmodified microorganism may be strain CC08-1023, which is a target strain for comparing whether the ability to produce carotenoids or substances with carotenoids as precursors is enhanced, but is not limited thereto.

[0058] For example, compared to the parent strain before modification or unmodified microorganism, the recombinant strain with enhanced production capacity can have an enhanced production capacity of beta-carotene or retinol of about 0.001% or more, or 0.01% or more. However, as long as the microorganism has an increased + value capacity compared to the parent strain before modification or unmodified microorganism, it is not limited thereto. The term "about" refers to a range including all ± 0.5, ± 0.4, ± 0.3, ± 0.2, ± 0.1, etc., and includes all values ​​that are equal or similar to the value after the term "about", but the scope is not limited thereto.

[0059] As used herein, the term "unmodified microorganism" does not exclude the bacterial strain comprising a mutation that may occur naturally in the microorganism, and can be a wild-type strain or a natural strain itself, or can be a strain before the character is changed due to natural or artificial factors by genetic variation. For example, an unmodified microorganism can be a bacterial strain that does not express the GGPPS from Haematococcus pluvialis, or a bacterial strain that has not yet imported the GGPPS from Haematococcus pluvialis. The term "unmodified microorganism" can be used interchangeably with "strain before modification", "microorganism before modification", "unvaried strain", "unmodified strain", "unvaried microorganism" or "reference microorganism".

[0060] The microorganism of the present disclosure may be a microorganism of the genus Yarrowia, specifically, Yarrowia lipolytica, but is not limited thereto.

[0061] In the microorganisms disclosed herein, some or all of the modification of the polynucleotides may be induced by (a) homologous recombination for chromosome insertion in the microorganism using a vector or genome editing using an engineered nuclease (e.g., CRISPR-Cas9) and / or (b) treatment with light (e.g., ultraviolet light and radiation) and / or chemicals, but is not limited thereto. Methods for modifying some or all of the genes may include methods using DNA recombination technology. For example, by introducing a nucleotide sequence or vector containing a nucleotide sequence homologous to the target gene into the microorganism to cause homologous recombination, some or all of the genes may be deleted. The nucleotide sequence or vector to be introduced may include a dominant selection marker, but is not limited thereto.

[0062] The microorganism of the present disclosure may be a microorganism of the genus Yarrowia that is modified to contain polynucleotides encoding lycopene cyclase / phytoene synthase (crtYB), phytoene desaturase (crtI), and β-carotene 15,15′-oxygenase (BLH) proteins.

[0063] The microorganism of the present disclosure may be a microorganism modified to further include polynucleotides encoding lycopene cyclase / phytoene synthase (crtYB) and phytoene desaturase (crtI) proteins, thereby exhibiting the activity of the protein, or a microorganism in which the activity of the protein is enhanced. Lycopene cyclase / phytoene synthase or phytoene desaturase may be a protein derived from Phaffia rhodozyma, but is not limited thereto. In one embodiment, the polynucleotide encoding lycopene cyclase / phytoene synthase or phytoene desaturase may have or may include a sequence based on a nucleotide sequence registered in the National Center for Biotechnology Information Search Database (NCBI) (GenBank: AY177204.1 or GenBank: AY177424.1), respectively. In one embodiment, the polynucleotide encoding lycopene cyclase / phytoene synthase or phytoene desaturase may have or include SEQ ID NO: 71 or SEQ ID NO: 72, respectively. In the polynucleotide, various modifications may be made in the coding region, taking into account codon degeneracy or preferred codons in the microorganisms in which the polypeptide of the present disclosure is intended to be expressed, as long as the amino acid sequence is not changed. Specifically, the polynucleotide may have or may include a nucleotide 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 with the sequence of SEQ ID NO: 71 or SEQ ID NO: 72, or may consist of or may consist essentially of a nucleotide 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 with the sequence of SEQ ID NO: 71 or SEQ ID NO: 72, but is not limited thereto.

[0064] The microorganism of the present disclosure may be a microorganism modified to further include a polynucleotide encoding a β-carotene 15,15'-oxygenase (BLH) protein, thereby exhibiting the activity of the protein, or a microorganism in which the activity of the protein is enhanced, but is not limited thereto. The β-carotene 15,15'-oxygenase may be a protein from an uncultured marine bacterium 66A03, but is not limited thereto. In one embodiment, the polynucleotide encoding the β-carotene 15,15'-oxygenase may have or include an amino acid sequence based on Q4PNI0 registered in the UniProt Knowledge Base (UniProtKB). In one embodiment, the polynucleotide encoding the β-carotene 15,15'-oxygenase may have or include a sequence of SEQ ID NO: 13. Taking into account codon degeneracy or preferred codons in a microorganism intended to express a polypeptide of the present disclosure, the polynucleotide may undergo various modifications in the coding region within the range of not changing the amino acid sequence. Specifically, the polynucleotide may have or comprise a nucleotide 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 with the sequence of SEQ ID NO: 13, or may consist of or consist essentially of a nucleotide 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 with the sequence of SEQ ID NO: 13, but is not limited thereto.

[0065] As used herein, the term "enhancing" of polypeptide activity refers to that the activity of polypeptide increases compared with endogenous activity.Enhancing can be used interchangeably with terms such as activation, up-regulation, over-expression and increase.Here, activation, enhancement, up-regulation, over-expression and increase can include showing an activity that is not initially present and showing an activity that is improved compared with the activity before endogenous activity or modification."Endogenous activity" refers to the activity of a specific polypeptide originally possessed by the parent strain or unmodified microorganism before the property changes when the genetic variation caused by natural or artificial factors changes.This can be used interchangeably with "activity before modification".Compared with endogenous activity, the fact that the activity of polypeptide is "enhanced", "up-regulated", "over-expressed" or "increased" means that, compared with the activity and / or concentration (expression level) of a specific polypeptide originally possessed by the parent strain or unmodified microorganism before the property changes, the activity of polypeptide is improved.

[0066] This enhancement can be achieved by introducing exogenous polypeptides or genes or enhancing the endogenous activity and / or concentration (expression level) of the polypeptide. The enhancement of polypeptide activity can be confirmed by an increase in the activity degree and expression level of the corresponding polypeptide or an increase in the amount of product produced by the corresponding polypeptide.

[0067] In order to enhance the activity of polypeptide, various methods well known in the art can be applied, and the method is not limited, as long as the activity of the target polypeptide can be enhanced compared with the microorganism before modification. Specifically, genetic engineering and / or protein engineering well known to those skilled in the art can be used, which are conventional methods of molecular biology, but the method is not limited thereto (e.g., Sitnicka et al. Functional Analysis of Genes. Advances in Cell Biology. 2010, Vol. 2.1-16, Sambrook et al. Molecular Cloning 2012, etc.).

[0068] Specifically, the enhancement of the polypeptide in the present disclosure can be:

[0069] 1) increasing the intracellular copy number of a polynucleotide encoding a polypeptide;

[0070] 2) replacing the gene expression regulatory region on the chromosome encoding the polypeptide with a sequence showing strong activity;

[0071] 3) modifying the nucleotide sequence of the start codon or 5′-UTR region of the gene transcript encoding the polypeptide;

[0072] 4) Modify the amino acid sequence of a polypeptide to enhance the activity of the polypeptide;

[0073] 5) modifying a polynucleotide sequence encoding a polypeptide to enhance the activity of the polypeptide (e.g., modifying a polynucleotide sequence of a polypeptide gene to encode a polypeptide that has been modified to enhance the activity of the polypeptide);

[0074] 6) introducing an exogenous polypeptide exhibiting polypeptide activity or an exogenous polynucleotide encoding the polypeptide;

[0075] 7) Codon optimization of polynucleotides encoding polypeptides;

[0076] 8) analyzing the tertiary structure of the polypeptide to select and modify or chemically modify exposed sites; or

[0077] 9) A combination of two or more selected from 1) to 8), but not limited thereto.

[0078] More specifically,

[0079] 1) The increase in the number of intracellular copies of a polynucleotide encoding a polypeptide can be achieved by introducing a vector into a host cell, the vector can replicate and function independently of the host, and the polynucleotide encoding the corresponding polypeptide is operably linked to the vector. Alternatively, the increase can be achieved by introducing one copy or two or more copies of the polynucleotide encoding the corresponding polypeptide into the chromosome of the host cell. The introduction of the chromosome can be carried out by introducing a vector capable of inserting the polynucleotide into the chromosome of the host cell, but is not limited to this. The vector is as described above.

[0080] 2) Replacing the gene expression regulatory region (or expression control sequence) on the chromosome encoding the polypeptide with a sequence showing strong activity may be, for example, a variation in the sequence due to deletion, insertion, non-conservative or conservative substitution or a combination thereof, or replacing it with a sequence showing stronger activity to further enhance the activity of the expression regulatory region. The expression regulatory region may include but is not particularly limited to a promoter, an operator sequence, a sequence encoding a ribosome binding site, a sequence controlling transcription and translation termination, etc. For example, the replacement may be replacing the original promoter with a strong promoter, but is not limited thereto.

[0081] Examples of known strong promoters include CJ1 to CJ7 promoters (U.S. Patent No. US 7662943 B2), lac promoter, trp promoter, trc promoter, tac promoter, lambda phage PR promoter, PL promoter, tet promoter, gapA promoter, SPL7 promoter, SPL13 (sm3) promoter (U.S. Patent No. US10584338B2), O2 promoter (U.S. Patent No. US10273491B2), tkt promoter, yccA promoter, TEFINt promoter, etc., but are not limited to these.

[0082] 3) Modification of the nucleotide sequence of the start codon or 5'-UTR region of the gene transcript encoding the polypeptide may be, for example, replacement with a nucleotide sequence encoding another start codon having a higher polypeptide expression rate than the endogenous start codon, but is not limited thereto.

[0083] 4) and 5) The modification of the amino acid sequence or polynucleotide sequence may be due to the 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, or the amino acid sequence or polynucleotide sequence modified to show stronger activity, or the amino acid sequence or polynucleotide sequence modified to be more active, thereby enhancing the activity of the polypeptide, but not limited thereto. The polynucleotide may be specifically replaced by inserting it into the chromosome by homologous recombination, but not limited thereto. The vector used here may further include a selection marker for confirming chromosome insertion. The selection marker is as described above.

[0084] 6) The introduction of an exogenous polynucleotide that exhibits polypeptide activity can be the introduction of an exogenous polynucleotide encoding a polypeptide that exhibits the same or similar activity as the polypeptide into a host cell. There is no limitation on its source or sequence, as long as the exogenous polynucleotide exhibits the same or similar activity as the polypeptide. The method used in the introduction process can be appropriately selected by a person skilled in the art by a known transformation method. When the introduced polynucleotide is expressed in a host cell, a polypeptide can be produced, and its activity can be increased.

[0085] 7) Codon optimization of a polynucleotide encoding a polypeptide can be codon optimization of an endogenous polynucleotide to increase transcription or translation in a host cell, or codon optimization of an exogenous polynucleotide to optimize transcription and translation in a host cell.

[0086] 8) Analyzing the tertiary structure of a polypeptide to select and modify or chemically modify an exposed site can be, for example, by comparing the sequence information of the polypeptide to be analyzed with a database storing known protein sequence information, determining a template protein candidate according to the degree of sequence similarity, confirming the structure based on this, and modifying or chemically modifying the exposed site to be modified or chemically modified.

[0087] Such enhancement of polypeptide activity may be an increase in the activity or concentration expression level of the corresponding polypeptide based on the activity or concentration of the polypeptide expressed in the wild-type or pre-modified microbial strain, or an increase in the amount of the product produced by the corresponding polypeptide, but is not limited thereto.

[0088] In one embodiment, the microorganism of the present disclosure may have enhanced GGPPS activity by introducing a GGPPS gene from Haematococcus pluvialis, but is not limited thereto.

[0089] The microorganism of the present disclosure may have the ability to produce carotenoids or substances with carotenoids as precursors.

[0090] As used herein, the term "carotenoid" refers to tetraterpenes or their derivatives that produce color (eg, yellow) in fruits and vegetables.

[0091] In one embodiment, the carotenoid may be any one or more selected from the group consisting of lutein, beta carotene, α-carotene, β-carotene, γ-carotene, phytoene, phytoene, neurosporene, lutein, lycopene, zeaxanthin, capsanthin, canthaxanthin and astaxanthin, but is not limited thereto.

[0092] In one embodiment, the substance having carotenoid as a precursor may be retinoid, but is not limited thereto.

[0093] As used herein, the term "retinoid" refers chemically to the Vitamin A family or a group of compounds chemically related thereto.

[0094] In one embodiment, the retinoid may be any one selected from the group consisting of retinol, retinal, retinoic acid, and retinyl esters, but is not limited thereto.

[0095] In one embodiment, the microorganisms of the present disclosure may have reduced ability to produce byproducts, but are not limited thereto.

[0096] In the present disclosure, by-products may refer to any substance other than carotenoids or substances that use carotenoids as precursors in their production process. For example, a representative by-product produced during the production of β-carotene may be squalene.

[0097] As used herein, "squalene" is an unsaturated hydrocarbon (C 30 H 50 ), and is also a substance used for the biosynthesis of steroid hormones, vitamin D, etc. The microorganism of the present disclosure may have reduced byproducts produced in the β-carotene production pathway, specifically, may have reduced squalene production, but is not limited thereto.

[0098] Another aspect of the present disclosure provides a method for producing carotenoids or substances with carotenoids as precursors, the method comprising the step of culturing the Yarrowia microorganism of the present disclosure in a culture medium.

[0099] The microorganisms, carotenoids and substances with carotenoids as precursors are as described in other aspects.

[0100] As used herein, the term "culture" refers to growing the Yarrowia microorganism of the present disclosure under appropriately adjusted environmental conditions. In the present disclosure, the culture procedure can be carried out according to suitable culture media or culture conditions known in the art. Those skilled in the art can easily adjust this culture procedure according to the selected microorganism. Specifically, the culture can be batch type, continuous type and / or fed-batch type, but is not limited thereto.

[0101] The Yarrowia microorganism of the present disclosure can be cultured under aerobic conditions in a common medium containing a suitable carbon source, nitrogen source, phosphorus source, inorganic compound, amino acid and / or vitamin while controlling temperature, pH and the like.

[0102] In the culture of the present disclosure, the culture temperature can be maintained at 20°C to 35°C, specifically, at 25°C to 35°C, and the culture can be performed for about 10 hours to about 160 hours, about 20 hours to about 130 hours, about 24 hours to about 120 hours, about 36 hours to about 120 hours, about 48 hours to about 120 hours, about 48 hours, about 72 hours, or about 120 hours, but is not limited thereto.

[0103] The carotenoid or the substance with carotenoid as a precursor produced by the culture of the present disclosure may be released into the culture medium or may be retained in the microorganism.

[0104] The method for producing carotenoids or substances with carotenoids as precursors disclosed herein may further include a step of preparing the Yarrowia microorganism disclosed herein, a step of preparing a culture medium for culturing the microorganism, or a combination of these steps (regardless of the order, in any order), for example, before or after the culturing step.

[0105] The method for producing carotenoids or substances with carotenoids as precursors disclosed herein may further include a step of recovering carotenoids or substances with carotenoids as precursors from a culture medium obtained by culturing a microorganism of the genus Yarrowia (a culture medium in which culturing has been performed) or from a microorganism of the genus Yarrowia disclosed herein. A recovery step may be further included after the culturing step.

[0106] Recovery can be a method of culturing a microorganism according to the present disclosure, such as batch, continuous or fed-batch type culture, by using suitable methods known in the art to collect the desired retinol. For example, centrifugation, filtration, treatment with a crystalline protein precipitant (salting out), extraction, cell disruption, ultrasonic treatment, ultrafiltration, dialysis, various types of chromatography (such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography and affinity chromatography, etc.), HPLC and combinations of these methods can be used, and retinol can be recovered from the culture medium or microorganism by using suitable methods known in the art.

[0107] In addition, the method for producing carotenoids or substances with carotenoids as precursors disclosed herein may further include a purification step. Purification may be performed using a suitable method known in the art. In an exemplary embodiment, when the method for producing carotenoids or substances with carotenoids as precursors disclosed herein includes both a recovery step and a purification step, the recovery step and the purification step may be performed discontinuously (or continuously) without regard to order, or may be performed simultaneously or integrated into one step, but is not limited thereto.

[0108] The method for producing carotenoids disclosed herein may further include a step of converting the beta-carotene produced by the microorganism disclosed herein into a carotenoid other than beta-carotene. In the method for producing carotenoids disclosed herein, a conversion step may also be included after the culturing step or the recovery step. The conversion step may be performed using a suitable method known in the art. For example, the conversion may be performed by a chemical method or using an enzyme, but is not limited thereto.

[0109] The method for producing retinoids disclosed herein may further include a step of converting the retinol produced by the Yarrowia microorganism disclosed herein into a retinoid other than retinol. In the method for producing retinoids disclosed herein, a conversion step may also be included after the culturing step or the recovery step. The conversion step may be performed using a suitable method known in the art. For example, retinol acyltransferase may be used for conversion, but is not limited thereto.

[0110] In one embodiment, the retinoid other than retinol may be any one selected from the group consisting of retinal, retinoic acid, and retinyl ester, but is not limited thereto as long as it is included in the retinoid.

[0111] Another aspect of the present disclosure provides a composition for producing carotenoids or substances with carotenoids as precursors, the composition comprising the Yarrowia microorganism of the present disclosure or a culture thereof.

[0112] The microorganism, carotenoid or substance with carotenoid as precursor is as described in other aspects.

[0113] The composition of the present disclosure may further include any commonly used suitable excipients, and the excipients may include, for example, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers, isotonic agents, etc., but are not limited thereto.

[0114] Yet another aspect of the present disclosure provides use of the microorganism or culture thereof in producing carotenoids or substances with carotenoids as precursors.

[0115] The microorganism, carotenoid or carotenoid-based precursor are as described in other aspects.

[0116] Example

[0117] Hereinafter, the present disclosure will be described in more detail by way of exemplary embodiments. However, the following exemplary embodiments are only used to illustrate preferred embodiments of the present disclosure, and are therefore not intended to limit the scope of the present disclosure thereto. At the same time, technical matters not described in this specification can be fully understood and easily implemented by those skilled in the art of the present disclosure or similar technical fields.

[0118] Example 1. Preparation of a platform strain for producing carotenoids or substances with carotenoids as precursors

[0119] Example 1-1. Preparation of crtYB-crtI Insertion Strain from Phaffia rhodozyma

[0120] In order to prepare a platform strain for producing carotenoids or substances with carotenoids as precursors, the lycopene cyclase / phytoene synthase (crtYB) and phytoene desaturase (crtI) genes derived from Phaffia rhodozyma were inserted into the genome of the high-fat yeast Yarrowia lipolytica CC08-0125 (Accession No. KCCM12972P).

[0121] Regarding crtYB, the polynucleotide of SEQ ID NO: 71 was obtained based on the nucleotide sequence (GenBank: AY177204.1) registered in the National Center for Biotechnology Information Search Database (NCBI), and regarding crtI, the polynucleotide of SEQ ID NO: 72 was obtained based on the nucleotide sequence (GenBank: AY177424.1) registered in NCBI. The polynucleotide sequences of crtYB and crtI were synthesized by Macrogen in the form of TEFINtp-crtYB-CYC1t (SEQ ID NO: 73) and TEFINtp-crtI-CYC1t (SEQ ID NO: 74), respectively. Using the URA3 gene (SEQ ID NO: 75) of Yarrowia lipolytica as a selection marker, a cassette was designed to be inserted into the MHY1 (YALI0B21582g) gene site. Each PCR was performed using synthetic crtYB and crtI genes and KCCM12972P genomic DNA as templates, and primers SEQ ID NO: 76 and SEQ ID NO: 77, SEQ ID NO: 78 and SEQ ID NO: 79, SEQ ID NO: 80 and SEQ ID NO: 81, SEQ ID NO: 82 and SEQ ID NO: 83, SEQ ID NO: 84 and SEQ ID NO: 85, and SEQ ID NO: 86 and SEQ ID NO: 87. PCR was performed by 35 cycles, including denaturation at 95° C. for 1 minute; annealing at 55° C. for 1 minute; and polymerization at 72° C. for 3 minutes. The resulting DNA fragments were prepared into a single cassette by overlap extension PCR.

[0122] The cassette thus prepared was introduced into the KCCM12972P strain by the heat shock method (D.-C. Chen et al., Appl Microbiol Biotechnol, 1997), and then colonies were obtained, which were formed on a solid medium (YLMM1) without uracil. Colonies whose cassette insertion into the genome was confirmed using primers of SEQ ID NO: 88 and SEQ ID NO: 89 were plated on a 5-FOA solid medium and cultured at 30° C. for 3 days to obtain colonies grown on a 5-FOA solid medium to recover the URA3 marker.

[0123] [Table 1]

[0124]

[0125] Example 1-2. Preparation of HMGR enhanced strains

[0126] A cassette for replacing the natural promoter (SEQ ID NO: 90) region of the 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) gene of the strain prepared by Example 1-1 with the TEFINt promoter was designed, and each PCR was performed using the genomic DNA of KCCM12972P as a template, and primers SEQ ID NO: 91 and SEQ ID NO: 92, SEQ ID NO: 93 and SEQ ID NO: 94, SEQ ID NO: 95 and SEQ ID NO: 96, SEQ ID NO: 97 and SEQ ID NO: 98, and SEQ ID NO: 99 and SEQ ID NO: 100. PCR was performed by 35 cycles, including 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. The resulting five DNA fragments were prepared into a single cassette by overlap extension PCR.

[0127] The cassette thus prepared was introduced into the strain prepared in Example 1-1 by the heat shock method, and then a colony was obtained, which was formed on a solid medium (YLMM1) without uracil. The colonies in which the cassette insertion was confirmed using primers of SEQ ID NO: 101 and SEQ ID NO: 102 were plated on a 5-FOA solid medium and cultured at 30° C. for 3 days to obtain colonies grown on a 5-FOA solid medium to recover the URA3 marker. Therefore, the platform strain finally prepared was named CC08-1023.

[0128] <Yarrowia lipolytica minimal medium 1 (YLMM1)>

[0129] 20g / L glucose, 6.7g / L yeast nitrogen base without amino acids, 2g / L yeast synthetic dropout medium supplement without uracil, 15g / L agar.

[0130] <5-Fluoroorotic acid (5-FOA)>

[0131] 20g / L glucose, 6.7g / L yeast nitrogen base without amino acids, 2g / L yeast synthetic dropout medium supplement without uracil, 50μg / mL uracil, 1g / L 5-fluoroorotic acid (5-FOA), 15g / L agar

[0132] [Table 2]

[0133]

[0134] Example 2. Preparation of a strain into which the geranylgeranyl pyrophosphate synthase (GGPP synthase) gene from Haematococcus pluvialis is inserted

[0135] Four types of GGPP synthase genes derived from different sources (hereinafter referred to as GGPPS genes) were introduced into the genome of the strain CC08-1023 prepared in Example 1 as follows.

[0136] Example 2-1. Preparation of a strain containing GGPPS from Haematococcus pluvialis

[0137] In order to insert the GGPPS1 gene from Haematococcus pluvialis (hereinafter referred to as Hp.GGPPS1) into the chromosome of Yarrowia lipolytica, Hp.GGPPS1 was codon-optimized (SEQ ID NO: 1) through http: / / atgme.org based on the nucleotide sequence registered in the National Center for Biotechnology Information Search Database (NCBI) (GenBank: APX64485.1) to make it suitable for Yarrowia lipolytica, and the gene (SEQ ID NO: 4) was synthesized by Macrogen in the form of TEFINtp-codon-optimized Hp.GGPPS1-CYC1t. Using the URA3 gene (SEQ ID NO: 5) of Yarrowia lipolytica as a selection marker, a cassette was designed to insert into the LIG4 (YALI0D21384g) gene site.

[0138] Using the synthetic Hp.GGPPS1 gene and the genomic DNA of KCCM12972P 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 with primers of SEQ ID NO: 15 and SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22, 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, respectively. PCR was performed by 35 cycles, including denaturation at 95°C for 1 minute; annealing at 55°C for 1 minute; and polymerization at 72°C for 2 minutes. The resulting DNA fragments were prepared into a single cassette by overlap extension PCR.

[0139] The cassette thus prepared was introduced into the CC08-1023 strain by the heat shock method, and then colonies were obtained, which were formed on a solid medium (YLMM1) without uracil. The colonies whose cassette insertion into the genome was confirmed using primers of SEQ ID NO: 29 and SEQ ID NO: 30 were plated on a 5-FOA solid medium and cultured at 30° C. for 3 days to obtain colonies grown on a 5-FOA solid medium to remove the URA3 marker.

[0140] [Table 3]

[0141]

[0142]

[0143] Example 2-2. Preparation of a strain into which a crtE variant gene from Phaffia rhodozyma is inserted

[0144] In order to insert the crtE variant gene crtEM1 (SEQ ID NO: 6, Hong et al., Applied Microbiology and Biotechnology, 2019 Jan; 103 (1): 211-223) from Phaffia rhodozyma into the chromosome of Yarrowia lipolytica, the gene was synthesized by Macrogen in the form of TEFINtp-crtEM1-TDH3t (SEQ ID NO: 8). The URA3 gene (SEQ ID NO: 5) of Yarrowia lipolytica was used as a selection marker to design a cassette for insertion into the LIG4 (YALI0D21384g) gene site.

[0145] Synthetic crtEM1 DNA and genomic DNA of KCCM12972P were used as templates, and PCR was performed on the left homologous region, TEFINt promoter, crtEM1 ORF, TDH3 terminator, URA3, repeat region and right homologous region fragments using primers of SEQ ID NO:31 and SEQ ID NO:32, SEQ ID NO:33 and SEQ ID NO:34, SEQ ID NO:35 and SEQ ID NO:36, SEQ ID NO:37 and SEQ ID NO:38, SEQ ID NO:39 and SEQ ID NO:40, SEQ ID NO:41 and SEQ ID NO:42, and SEQ ID NO:43 and SEQ ID NO:44 shown in Table 4, respectively.

[0146] PCR was performed by 35 cycles, including denaturation at 95° C. for 1 minute, annealing at 55° C. for 1 minute, and polymerization at 72° C. for 2 minutes. The resulting DNA fragments were prepared into a single cassette by overlap extension PCR.

[0147] The cassette thus prepared was introduced into the CC08-1023 strain by the heat shock method, and then colonies were obtained, which were formed on a solid medium (YLMM1) without uracil. The colonies whose cassette insertion into the genome was confirmed using primers of SEQ ID NO: 45 and SEQ ID NO: 46 were plated on a 5-FOA solid medium and cultured at 30° C. for 3 days to obtain colonies grown on a 5-FOA solid medium to remove the URA3 marker.

[0148] [Table 4]

[0149]

[0150]

[0151] Example 2-3. Preparation of a strain containing BTS1 from Saccharomyces cerevisiae

[0152] In order to insert the BTS1 gene from Saccharomyces cerevisiae (hereinafter referred to as Sc.BTS1) into the chromosome of Yarrowia lipolytica, the polynucleotide of SEQ ID NO: 9 of BTS1 was obtained based on the nucleotide sequence (YPL069C) registered in Kyoto Encyclopedia of Genes and Genomes (KEGG). The gene was synthesized by Macrogen in the form of TEFINtp-Sc.BTS1-TDH3t (SEQ ID NO: 10) using the polynucleotide of BTS1. Using the URA3 gene (SEQ ID NO: 5) of Yarrowia lipolytica as a selection marker, a cassette inserted into the LIG4 (YALI0D21384g) gene site was designed.

[0153] Synthetic Sc.BTS1 DNA and genomic DNA of KCCM12972P were used as templates, and PCR was performed on the left homologous region, TEFINt promoter, Sc.BTS1 ORF, TDH3 terminator, URA3, repeat region and right homologous region fragments with primers of SEQ ID NO:31 and SEQ ID NO:32, SEQ ID NO:33 and SEQ ID NO:47, SEQ ID NO:48 and SEQ ID NO:49, SEQ ID NO:50 and SEQ ID NO:38, SEQ ID NO:39 and SEQ ID NO:40, SEQ ID NO:41 and SEQ ID NO:42, and SEQ ID NO:43 and SEQ ID NO:44 shown in Table 5, respectively. PCR was performed by 35 cycles, including denaturation at 95°C for 1 minute; annealing at 55°C for 1 minute; and polymerization at 72°C for 2 minutes. The resulting DNA fragments were prepared into a single cassette by overlap extension PCR.

[0154] The cassette thus prepared was introduced into the CC08-1023 strain by the heat shock method, and then colonies were obtained, which were formed on a solid medium (YLMM1) without uracil. The colonies whose cassette insertion into the genome was confirmed using primers of SEQ ID NO: 45 and SEQ ID NO: 46 were plated on a 5-FOA solid medium and cultured at 30° C. for 3 days to obtain colonies grown on a 5-FOA solid medium to remove the URA3 marker.

[0155] [Table 5]

[0156]

[0157] Example 2-4. Preparation of a strain containing GGS1 from Yarrowia lipolytica

[0158] In order to insert the GGS1 gene from Yarrowia lipolytica (hereinafter referred to as Y1.GGS1) into the chromosome of Yarrowia lipolytica, the polynucleotide of SEQ ID NO: 11 of GGS1 was obtained based on the nucleotide sequence (YALI0D17050g) registered in Kyoto Encyclopedia of Genes and Genomes (KEGG). The gene was synthesized in the form of TEFINtp-Y1.GGS1-TDH3t (SEQ ID NO: 12) using the polynucleotide of Y1.GGS1. Using the URA3 gene (SEQ ID NO: 5) of Yarrowia lipolytica as a selection marker, a cassette for insertion into the LIG4 (YALI0D21384g) gene site was designed.

[0159] Using the synthetic Yl.GGS1 gene and the genomic DNA of KCCM12972P as templates, and using the primer pairs shown in SEQ ID NO:31 and SEQ ID NO:32, SEQ ID NO:33 and SEQ ID NO:51, SEQ ID NO:52 and SEQ ID NO:53, SEQ ID NO:54 and SEQ ID NO:38, SEQ ID NO:39 and SEQ ID NO:40, SEQ ID NO:41 and SEQ ID NO:42, and SEQ ID NO:43 and SEQ ID NO:44 in Table 6 respectively, the left homologous region, TEFINt promoter, Yl.GGS1 ORF, TDH3 terminator, URA3, repeat region, and right homologous region fragments were subjected to PCR. The PCR was carried out through 35 cycles, including denaturation at 95 °C for 1 minute; annealing at 55 °C for 1 minute; and polymerization at 72 °C for 2 minutes. The obtained DNA fragments were prepared into a single cassette by overlap extension PCR.

[0160] The cassette thus prepared was introduced into the CC08-1023 strain by heat shock method, and then colonies were obtained, which were formed on a solid medium (YLMM1) without uracil. The colonies in which the cassette was confirmed to be inserted into the genome using the primers of SEQ ID NO:45 and SEQ ID NO:46 were spread on a 5-FOA solid medium and cultured at 30 °C for 3 days to obtain colonies growing on the 5-FOA solid medium to remove the URA3 marker.

[0161] [Table 6]

[0162]

[0163] Example 3. Comparative evaluation of β-carotene production capacity of strains based on the introduction of GGPP synthase

[0164] A shake flask test was carried out on a total of 5 species, including the strains obtained in Examples 2-1 to 2-4 and the parental strain CC08-1023 obtained in Example 1. Each strain was inoculated with an initial OD of 2 into a 250 ml Erlenmeyer flask with baffles containing 20 ml of yeast extract-peptone-dextrose (YPD) medium and cultured with stirring at 200 rpm at 30 °C for 48 hours. After the culture was completed, 1 ml of the culture broth was centrifuged and the supernatant was removed. The composition of the YPD medium is as follows.

[0165] <YPD liquid medium>

[0166] 4% glucose, 1% yeast extract, and 2% peptone dissolved in 0.1 M phosphate buffer (sodium phosphate buffer) (pH 7.0).

[0167] Next, 0.5 ml of dimethyl sulfoxide (DMSO, Sigma, CAS No. 67-68-5) was added, and the cells were disrupted by stirring at 55°C (2000 rpm) for 10 minutes. In addition, 0.5 ml of acetone (Sigma, CAS No. 67-64-1) was added, and stirred at 45°C (2000 rpm) for 15 minutes to extract β-carotene and squalene, and their concentrations were analyzed using an HPLC device. The results of the measured β-carotene and squalene concentrations are shown in Figure 1 middle.

[0168] The results are as follows Figure 1 As shown, the concentrations of β-carotene in CC08-1023 (parent strain), strain introduced with Hp.GGPPS1, strain introduced with crtEM1, strain introduced with Sc.BTS1, and strain introduced with Yl.GGS1 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, when Hp.GGPPS1 was introduced, β-carotene increased by 53.24 mg / L compared to the parent strain, indicating that the effect of increasing β-carotene was the best.

[0169] In addition, the concentrations of squalene were 313.24 mg / L, 200.31 mg / L, 235.27 mg / L, 253.28 mg / L and 221.22 mg / L, respectively. Similarly, compared with the CC08-1023 strain, when Hp.GGPPS1 was introduced, squalene was reduced by 112.93 mg / L, indicating that the effect of reducing squalene was the best.

[0170] Based on these results, Hp.GGPPS1 was confirmed to be the most efficient GGPP synthase in Yarrowia microorganisms. Surprisingly, when geranylgeranyl pyrophosphate synthases from the closely related Saccharomyces cerevisiae, Yarrowia lipolytica and Phaffia rhodozyma were introduced, the effect was not significant, while when geranylgeranyl pyrophosphate synthases from the relatively unrelated Haematococcus pluvialis were introduced, the effect was significant.

[0171] Example 4. Preparation of a strain into which the β-carotene 15,15' oxygenase (BCO) gene is introduced

[0172] In order to introduce the β-carotene 15,15' oxygenase (hereinafter referred to as Mb.BCO) gene from the uncultured marine bacterium 66A03 into the chromosome of Yarrowia lipolytica, a polynucleotide sequence (SEQ ID NO: 13) was obtained by codon optimization of Mb.BCO through http: / / atgme.org based on the amino acid sequence (Q4PNI0) registered in the UniProt Knowledge Base (UniProtKB) to be suitable for Yarrowia lipolytica. The gene was synthesized in the form of TEFINtp codon-optimized Mb.BCO-CYC1t (SEQ ID NO: 14) using the polynucleotide of Mb.BCO. Using the URA3 gene (SEQ ID NO: 5) of Yarrowia lipolytica as a selection marker, a cassette inserted into the KU70 (YALI0C08701g) gene site was designed.

[0173] Using the synthetic genomic DNA of Mb.BCO and KCCM12972P as a template, PCR was performed on the left homologous region, TEFINt promoter, Mb.BCO ORF, CYC1 terminator, URA3, repeat region and right homologous region fragments with primers of SEQ ID NO: 55 and SEQ ID NO: 56, SEQ ID NO: 57 and SEQ ID NO: 58, SEQ ID NO: 59 and SEQ ID NO: 60, SEQ ID NO: 61 and SEQ ID NO: 62, SEQ ID NO: 63 and SEQ ID NO: 64, SEQ ID NO: 65 and SEQ ID NO: 66, and SEQ ID NO: 67 and SEQ ID NO: 68 shown in Table 7, respectively. PCR was performed by 35 cycles, including denaturation at 95°C for 1 minute; annealing at 55°C for 1 minute; and polymerization at 72°C for 2 minutes. The resulting DNA fragments were prepared into a single cassette by overlap extension PCR.

[0174] The cassette thus prepared was introduced into each strain prepared in Examples 2-1 to 2-4 by a heat shock method, and then colonies were obtained, which were formed on a solid medium (YLMM1) without uracil. The colonies in which the cassette was inserted into the genome using primers of SEQ ID NO: 69 and SEQ ID NO: 70 were plated on a 5-FOA solid medium and cultured at 30° C. for 3 days to obtain colonies grown on a 5-FOA solid medium to remove the URA3 marker.

[0175] [Table 7]

[0176]

[0177] Example 5. Comparative evaluation of the ability of strains introduced with the β-carotene 15,15' oxygenase (BCO) gene to produce retinol

[0178] A shake flask test was performed on a total of 5 species, including the strain obtained in Example 4 and the parent strain CC08-1023 obtained in Example 1. Each strain was inoculated into a 250 ml baffled flask containing 20 ml yeast extract-peptone-glucose (YPD) medium and 0.05% butylated hydroxytoluene at an initial OD of 2, and cultured at 30°C with stirring at 200 rpm for 48 hours. After the culture was completed, 1 ml of the culture medium was centrifuged and the supernatant was removed. Next, 0.5 ml of dimethyl sulfoxide (DMSO, Sigma) was added, and the cells were broken by stirring at 55°C (2000 rpm) for 10 minutes. In addition, 0.5 ml of acetone (Sigma) was added and stirred at 45°C (2000 rpm) for 15 minutes to extract retinol, retinal, β-carotene and squalene, and their concentrations were analyzed using HPLC equipment. The measurement results of the analyzed retinol, retinal, β-carotene and squalene concentrations are as shown in Figure 2 shown.

[0179] The results are as follows Figure 2 As shown, no retinol was detected in the strain prepared by introducing Mb.BCO into the CC08-1023 strain. In contrast, after introducing Hp.GGPPS1, crtEM1, Sc.BTS1 and Yl.GGS1 into CC08-1023, Mb.BCO was introduced into four types of strains, and the resulting retinol concentrations were 8.44 mg / L, 2.78 mg / L, 0 mg / L and 4.35 mg / L, respectively.

[0180] The concentrations of β-carotene in the five types of strains were 3.68 mg / L, 0.35 mg / L, 2.47 mg / L, 3.58 mg / L and 0.98 mg / L, respectively, indicating that β-carotene was converted into retinol, resulting in low β-carotene concentrations. In addition, the concentrations of squalene in the five types of strains were 309.88 mg / L, 202.18 mg / L, 282.19 mg / L, 306.34 mg / L and 269.18 mg / L, respectively.

[0181] These results confirm that enhancement of GGPP biosynthesis has a positive effect on increasing retinol productivity.

[0182] The above results also verified that Hp.GGPPS1 had excellent effects on β-carotene production, squalene reduction and retinol production.

[0183] Based on the above description, it will be appreciated by those skilled in the art that the present disclosure can be implemented in different specific forms without changing its technical spirit or basic features. In this regard, it should be understood that the above embodiments are not restrictive, but illustrative in all respects. The scope of the present disclosure is limited by the appended claims rather than by the description before them, so all changes and modifications falling within the boundaries and scope of the claims, or the equivalents of these boundaries and scopes, are included by the claims.

[0184] Each sequence of SEQ ID NO. according to the present disclosure is shown in Table 8 below.

[0185] [Table 8]

[0186]

[0187]

[0188]

[0189]

[0190]

[0191]

[0192]

[0193]

[0194]

[0195]

[0196]

[0197]

[0198]

[0199]

[0200]

[0201]

[0202]

[0203]

[0204]

[0205]

[0206]

[0207]

[0208]

[0209]

[0210]

[0211]

[0212]

[0213]

[0214]

[0215]

Claims

1. A Yarrowia microorganism having the ability to produce carotenoids or substances with carotenoids as precursors, wherein the microorganism expresses geranylgeranyl pyrophosphate synthase from Haematococcus pluvialis.

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

103.

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

1. The microorganism according to claim 1 , wherein the microorganism of the genus Yarrowia is Yarrowia lipolytica. The microorganism according to claim 1 , wherein the substance having carotenoid as a precursor is retinoid. The microorganism according to claim 1 , wherein the carotenoid is β-carotene.

7. The microorganism according to claim 5, wherein the retinoid is retinol.

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

9. The microorganism according to claim 8, wherein the by-product is squalene.

10. A method for producing carotenoids or substances with carotenoids as precursors, the method comprising the following steps: Cultivating the Yarrowia microorganism according to any one of claims 1 to 9 in a culture medium; and The carotenoid or a substance with the carotenoid as a precursor is recovered from the Yarrowia microorganism or the culture medium.

11. The method according to claim 10, further comprising the steps of: converting the β-carotene produced by the Yarrowia microorganism into carotenoids other than β-carotene; or The retinol produced by the Yarrowia microorganism is converted into a retinoid other than retinol.

12. A composition for producing carotenoids or substances with carotenoids as precursors, the composition comprising the microorganism of the genus Yarrowia according to any one of claims 1 to 9 or a culture thereof.

13. Use of a microorganism of the genus Yarrowia in producing carotenoids or substances with carotenoids as precursors, wherein the microorganism has the ability to produce carotenoids or substances with carotenoids as precursors by expressing geranylgeranyl pyrophosphate synthase from Haematococcus pluvialis.

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