Geranylgeranyl pyrophosphate synthase variant and method of producing tetraterpene, precursor thereof, and material having tetraterpene as precursor using the same

Abstract

The present disclosure relates to a geranylgeranyl pyrophosphate synthase variant; a polynucleotide encoding the variant; a vector including the polynucleotide; a microorganism including any one or more of the variant, the polynucleotide, and the vector; a method of producing tetraterpene, a precursor thereof, or a material having tetraterpene as a precursor using the same; a composition for producing tetraterpene, a precursor thereof, or a material having tetraterpene as a precursor; and use of the microorganism including any one or more of the variant, the polynucleotide, and the vector, for producing tetraterpene, a precursor thereof, and a material having tetraterpene as a precursor.

Description

Geranylgeranyl pyrophosphate synthase variant and method for producing tetraterpene, precursor thereof, and substance using tetraterpene as precursor using the same

[0001] The present application relates to a geranylgeranyl pyrophosphate synthase variant; a polynucleotide encoding the variant; a vector comprising the polynucleotide; a microorganism comprising at least one of the variant, the polynucleotide, and the vector; a method for producing tetraterpene, a precursor thereof, and a substance using the variant; a composition for producing tetraterpene, a precursor thereof, and a substance using tetraterpene as a precursor; and a use of a microorganism comprising at least one of the variant, the polynucleotide, and the vector for producing tetraterpene, a precursor thereof, and a substance using tetraterpene as a precursor.

[0002]

[0003] Terpenoids, a concept encompassing carotenoids, exhibit diverse functions in plants and animals and are utilized in a wide range of industrial fields, including food and feed. Terpenoids are known as representative substances responsible for the medicinal properties of plants and, like carotenoids, are terpenes. Among them, carotenoids, such as beta-carotene, have been reported to have functions such as scavenging free radicals, being the precursor to vitamin A in animals, enhancing the immune system in vertebrates, and reducing the risk of lung cancer.

[0004] However, despite these advantages, carotenoids, such as beta-carotene, are not synthesized in animals, or are synthesized in insufficient amounts. Furthermore, even when industrial production is attempted using mutated microorganisms (U.S. Patent No. 7,745,170), high-purity beta-carotene production remains difficult due to byproducts.

[0005] With regard to by-products, geranylgeranyl pyrophosphate (GGPP, C20), a key precursor for carotenoid or terpenoid production, is produced by the isoprenoid biosynthetic pathway from the conjugation of farnesyl pyrophosphate (FPP, C15) and isopentenyl pyrophosphate (IPP, C5) by the enzyme geranylgeranyl pyrophosphate synthase. However, squalene (C30) can also be produced as a by-product from farnesyl pyrophosphate (FPP, C15) by squalene synthase (ERG9). Accordingly, there is an emerging need to develop a method to increase the production of GGPP, a key precursor for carotenoid or terpenoid biosynthesis, and to reduce the production of squalene, a competing pathway.

[0006]

[0007] The problem to be solved by the present application is to provide a geranylgeranyl pyrophosphate synthase variant and a method for producing tetraterpene, a precursor thereof, and a material using tetraterpene as a precursor.

[0008]

[0009] One object of the present application is to provide a geranylgeranyl pyrophosphate synthase variant, in which at least one amino acid corresponding to positions 29, 43, 90, 103, 122, and 141 from the N-terminus of the amino acid sequence of SEQ ID NO: 1 is replaced with another amino acid.

[0010] Another object of the present application is to provide a polynucleotide encoding the above variant.

[0011] Another object of the present application is to provide a vector comprising the polynucleotide.

[0012] Another object of the present application is to provide a microorganism comprising at least one of the above mutant, polynucleotide, and vector.

[0013] Another object of the present application is to provide a method for producing tetraterpene, a precursor thereof, and a substance using tetraterpene as a precursor, which comprises a step of culturing the microorganism in a medium.

[0014] Another object of the present application is to provide a composition for producing a tetraterpene, a precursor thereof, and a substance using a tetraterpene as a precursor, comprising the mutant, vector, microorganism or a culture of the microorganism.

[0015] Another object of the present application is to provide a use for producing a tetraterpene, a precursor thereof, and a material using a tetraterpene as a precursor, by using any one or more of the above mutants, polynucleotides, vectors, and microorganisms.

[0016]

[0017] A microorganism expressing the geranylgeranyl pyrophosphate synthase variant of the present invention can effectively produce tetraterpene, a precursor of tetraterpene, or a substance containing tetraterpene as a precursor, compared to a strain that does not express the geranylgeranyl pyrophosphate synthase variant.

[0018]

[0019] Hereinafter, the contents of this application will be described in detail. The description and embodiments of one aspect disclosed in this application may also be applied to other aspects regarding common elements. Furthermore, all combinations of the various elements disclosed in this application fall within the scope of this application. Furthermore, the scope of this application is not limited by the specific descriptions described below.

[0020]

[0021] One aspect of the present application provides a geranylgeranyl pyrophosphate synthase variant, wherein at least one amino acid corresponding to positions 29, 43, 90, 103, 122, and 141 from the N-terminus of the amino acid sequence of SEQ ID NO: 1 is substituted with another amino acid.

[0022] The above geranylgeranyl pyrophosphate synthase variant refers to a polypeptide having geranylgeranyl pyrophosphate synthase activity or a variant in which at least one amino acid among the amino acids corresponding to positions 29, 43, 90, 103, 122, and 141 from the N-terminus of SEQ ID NO: 1 in geranylgeranyl pyrophosphate synthase is substituted with another amino acid.

[0023]

[0024] In the present application, “Geranylgeranyl pyrophosphate synthase (GGS1)” is an enzyme capable of catalyzing the synthesis of geranylgeranyl pyrophosphate synthase (GGPP) from farneylpyrophosphate (FPP).

[0025] The geranylgeranyl pyrophosphate synthase of the present application may be a geranylgeranyl pyrophosphate synthase or a polypeptide having geranylgeranyl pyrophosphate synthase activity that is modified to produce a geranylgeranyl pyrophosphate synthase variant provided in the present application. Specifically, it may be a naturally occurring polypeptide or a wild-type polypeptide, and may include a mature polypeptide thereof, a variant thereof, or a functional fragment thereof, but is included without limitation as long as it can be a parent of the geranylgeranyl pyrophosphate synthase variant of the present application.

[0026] In the present application, the geranylgeranyl pyrophosphate synthase is not limited thereto, but may be a polypeptide of SEQ ID NO: 1. In addition, it may be a polypeptide having a sequence identity of about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more with the polypeptide of SEQ ID NO: 1, and if it has the same or corresponding activity as the polypeptide consisting of the amino acid sequence of SEQ ID NO: 1, it is included in the scope of geranylgeranyl pyrophosphate synthase without limitation.

[0027] The sequence of the geranylgeranyl pyrophosphate synthase of the present application can be obtained from a known database such as NCBI's GenBank. Specifically, it may be a polypeptide encoded by the ggs1 gene, but is not limited thereto.

[0028]

[0029] In this application, the term "variant" refers to a polypeptide in which one or more amino acids are conservatively substituted and / or modified, thereby differing from the amino acid sequence of the variant before the mutation, but retaining functions or properties. Such variants can generally be identified by modifying one or more amino acids in the amino acid sequence of the polypeptide and evaluating the properties of the modified polypeptide. That is, the ability of the variant may be increased, unchanged, or decreased compared to the polypeptide before the mutation. Furthermore, some variants may include modifications in which one or more portions, such as the N-terminal leader sequence or the transmembrane domain, are deleted. Other variants may include modifications in which portions are deleted from the N- and / or C-termini of the mature protein. The above term "variant" may be used interchangeably with terms such as variant, modification, variant polypeptide, variant protein, variant and variant (in English, modification, modified polypeptide, modified protein, mutant, mutein, divergent, etc.), and is not limited thereto if the term is used in the meaning of variant.

[0030] Additionally, variants may include deletions or additions of amino acids that have minimal impact on the properties and secondary structure of the polypeptide. For example, the N-terminus of the variant may be conjugated with a signal (or leader) sequence involved in co-translational or post-translational protein translocation. Furthermore, the variant may be conjugated to other sequences or linkers to facilitate identification, purification, or synthesis.

[0031]

[0032] The variant of the present application may be a geranylgeranyl pyrophosphate synthase variant in which any one or more amino acids corresponding to positions 29, 43, 90, 103, 122, and 141 from the N-terminus of the amino acid sequence of SEQ ID NO: 1 are substituted with other amino acids. Specifically, the variant may be one in which amino acids at positions 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, or 6 or more among positions 29, 43, 90, 103, 122, and 141 are substituted, but is not limited thereto.

[0033] In the polypeptide having geranylgeranyl pyrophosphate synthase activity or geranylgeranyl pyrophosphate synthase that is the parent of the above mutant, the amino acid corresponding to position 29 from the N-terminus of the amino acid sequence of SEQ ID NO: 1 may be asparagine; the amino acid corresponding to position 43 may be phenylalanine; the amino acid corresponding to position 90 may be leucine; the amino acid corresponding to position 103 may be methionine; the amino acid corresponding to position 122 may be isoleucine; and / or the amino acid corresponding to position 141 may be arginine, but is not limited thereto.

[0034] In one embodiment, the variant may include, but is not limited to, one or more substitutions among: a substitution of an amino acid corresponding to position 29 from the N-terminus of the amino acid sequence of SEQ ID NO: 1 with an amino acid other than asparagine; a substitution of an amino acid corresponding to amino acid 43 with an amino acid other than phenylalanine; a substitution of an amino acid corresponding to amino acid 90 with an amino acid other than leucine; a substitution of an amino acid corresponding to amino acid 103 with an amino acid other than methionine; a substitution of an amino acid corresponding to amino acid 122 with an amino acid other than isoleucine; and a substitution of an amino acid corresponding to amino acid 141 with an amino acid other than arginine.

[0035] The above "other amino acid" is not limited to an amino acid that is different from the amino acid before substitution. Furthermore, when the present application states that "a specific amino acid has been substituted," it is self-evident that the amino acid has been substituted with an amino acid that is different from the amino acid before substitution, even if it is not specifically stated that it has been substituted with another amino acid.

[0036] The substitution with the above other amino acid may be a substitution with a nonpolar amino acid, a polar amino acid, or a positively charged (basic) amino acid. Specifically, the nonpolar amino acid may be selected from the group consisting of glycine, alanine, valine, leucine, isoleucine, methionine, and proline, but is not limited thereto. The polar amino acid may be mixed with a hydrophilic amino acid, and may be selected from the group consisting of serine, threonine, cysteine, tyrosine, asparagine, and glutamine, and the positively charged (basic) amino acid may be selected from the group consisting of arginine, lysine, and histidine, but is not limited thereto.

[0037] Specifically, the variant of the present application may be a variant in which any one or more of the amino acids corresponding to positions 29, 43, 90, 103, 122, and 141 of the amino acid sequence of SEQ ID NO: 1, which is a reference protein, is substituted with an amino acid different from the amino acid before substitution among non-polar amino acids, polar amino acids, or positively charged (basic) amino acids, but is not limited thereto. The non-polar amino acid may be isoleucine (Ile) or valine (Val), the polar amino acid may be threonine (Thr), and the positively charged amino acid may be arginine (Arg) or lysine (Lys), but is not limited thereto.

[0038] Additionally, the other amino acid may be any one selected from the group consisting of threonine, isoleucine, arginine, valine, and lysine, but is not limited thereto.

[0039] In one embodiment, the variant of the present application may be one in which any one or more of the amino acids corresponding to positions 29, 43, 90, 103, 122, and 141 of the amino acid sequence of SEQ ID NO: 1 is substituted with any one selected from the group consisting of threonine, isoleucine, arginine, valine, and lysine, but is not limited thereto.

[0040] As an example of implementation, the variant of the present application may be selected from the group consisting of, but is not limited to, a substitution in which the amino acid corresponding to position 29 of the amino acid sequence of SEQ ID NO: 1 is substituted with threonine; the amino acid corresponding to position 43 is substituted with isoleucine; the amino acid corresponding to position 90 is substituted with arginine; the amino acid corresponding to position 103 is substituted with isoleucine; the amino acid corresponding to position 122 is substituted with valine; the amino acid corresponding to position 141 is substituted with lysine; and combinations thereof.

[0041]

[0042] As used herein, the term "corresponding to" refers to an amino acid residue at a position listed in a polypeptide, or an amino acid residue that is similar, identical, or homologous to the residue listed in the polypeptide. Identifying an amino acid at a corresponding position may be determining a specific amino acid in a sequence that references a particular sequence. As used herein, "corresponding region" generally refers to a similar or corresponding position in a related or reference protein.

[0043] For example, any amino acid sequence can be aligned with SEQ ID NO: 1, and based on this, each amino acid residue of the amino acid sequence can be numbered by referring to the numerical position of the amino acid residue corresponding to the amino acid residue in SEQ ID NO: 1. For example, a sequence alignment algorithm such as that described in the present application can identify the position of an amino acid, or the position at which a modification such as a substitution, insertion, or deletion occurs, by comparing it to a query sequence (also referred to as a “reference sequence”).

[0044] For such alignment, for example, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453), the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000), Trends Genet. 16: 276-277) can be used, but is not limited thereto, and any sequence alignment program known in the art, pairwise sequence comparison algorithm, etc. can be appropriately used.

[0045]

[0046] In one embodiment, the variant of the present application may have a sequence identity of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% with the polypeptide of SEQ ID NO: 1, wherein any one or more of the amino acids corresponding to positions 29, 43, 90, 103, 122, and 141 of SEQ ID NO: 1 is substituted with another amino acid.

[0047] In one embodiment, the variant of the present application may comprise an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% homology or identity to the amino acid sequence set forth in SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15 or SEQ ID NO: 17.

[0048] Specifically, the variant of the present application may have, comprise, consist of, or consist essentially of an amino acid sequence set forth in SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, or SEQ ID NO: 17.

[0049] In one embodiment, the variant of the present application may include an amino acid sequence in which any one or more amino acids corresponding to positions 29, 43, 90, 103, 122, and 141 of the amino acid sequence of SEQ ID NO: 1 are replaced with another amino acid, and have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.9% homology or identity with the amino acid sequence set forth in SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, or SEQ ID NO: 17.

[0050] In one embodiment, the variant of the present application comprises a polypeptide comprising an amino acid sequence as set forth in SEQ ID NO: 3, wherein methionine, which is an amino acid corresponding to position 103 of the amino acid sequence of SEQ ID NO: 1, is substituted with isoleucine; a polypeptide comprising an amino acid sequence as set forth in SEQ ID NO: 5, wherein phenylalanine, which is an amino acid corresponding to position 43 of the amino acid sequence of SEQ ID NO: 1, is substituted with isoleucine; a polypeptide comprising an amino acid sequence as set forth in SEQ ID NO: 7, wherein asparagine, which is an amino acid corresponding to position 29 of the amino acid sequence of SEQ ID NO: 1, is substituted with threonine and leucine, which is an amino acid corresponding to position 90 of the amino acid sequence of SEQ ID NO: 1, is substituted with arginine; a polypeptide comprising an amino acid sequence as set forth in SEQ ID NO: 9, wherein asparagine, which is an amino acid corresponding to position 29 of the amino acid sequence of SEQ ID NO: 1, is substituted with threonine; a polypeptide comprising an amino acid sequence as set forth in SEQ ID NO: 11, wherein leucine, which is an amino acid corresponding to position 90 of the amino acid sequence of SEQ ID NO: 1, is substituted with arginine; The polypeptide may be at least one selected from, but is not limited to, a polypeptide comprising an amino acid sequence as set forth in SEQ ID NO: 13, wherein isoleucine, an amino acid corresponding to position 122 of the amino acid sequence of SEQ ID NO: 1, is substituted with valine and arginine, an amino acid corresponding to position 141 of the amino acid sequence of SEQ ID NO: 1, is substituted with lysine; a polypeptide comprising an amino acid sequence as set forth in SEQ ID NO: 15, wherein isoleucine, an amino acid corresponding to position 122 of the amino acid sequence of SEQ ID NO: 1, is substituted with valine; and a polypeptide comprising an amino acid sequence as set forth in SEQ ID NO: 17, wherein arginine, an amino acid corresponding to position 141 of the amino acid sequence of SEQ ID NO: 1, is substituted with lysine.

[0051]

[0052]

[0053] *In addition, it is obvious that a variant having an amino acid sequence in which some sequences are deleted, modified, substituted, conservatively substituted or added is also included within the scope of the present application, provided that the amino acid sequence has such homology or identity and exhibits an effect corresponding to that of the variant of the present application.

[0054] For example, if the amino acid sequence has sequence additions or deletions, naturally occurring mutations, silent mutations or conservative substitutions that do not alter the function of the variant of the present application at the N-terminus, C-terminus and / or within the amino acid sequence.

[0055] The term "conservative substitution" refers to the replacement of one amino acid with another amino acid having similar structural and / or chemical properties. Such amino acid substitutions may generally be based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues. Typically, conservative substitutions may have little or no effect on the activity of a protein or polypeptide.

[0056]

[0057] In this application, the terms "homology" or "identity" refer to the degree of similarity between two given amino acid sequences or base sequences, which may be expressed as a percentage. The terms homology and identity are often used interchangeably.

[0058] Sequence homology or identity of conserved polynucleotides or polypeptides is determined by standard alignment algorithms, and may be combined with default gap penalties established by the program being used. In practice, homologous or identical sequences can generally hybridize with all or part of the sequence under moderate or high stringency conditions. It should be appreciated that hybridization also includes hybridization with polynucleotides containing common codons or codons that take codon degeneracy into account.

[0059] Whether any two polynucleotide or polypeptide sequences are homologous, similar or identical can be determined using known computer algorithms such as the "FASTA" program using default parameters, for example as in Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444. Alternatively, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277) (version 5.0.0 or later) can be determined using 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 of the National Center for Biotechnology Information Database.

[0060] Homology, similarity, or identity of polynucleotides or polypeptides can be determined by comparing sequence information, for example, using a GAP computer program such as that of Needleman et al. (1970), J Mol Biol. 48:443, as disclosed, for example, in Smith and Waterman, Adv. Appl. Math (1981) 2:482. In brief, the GAP program can be defined as the total number of symbols in the shorter of the two sequences divided by the number of similarly arranged symbols (i.e., nucleotides or amino acids). Default parameters for the GAP program include (1) a binary comparison matrix (containing values ​​of 1 for identity and 0 for non-identity) and (2) a comparison matrix as disclosed by Gribskov et al. (1986) Nucl. Acids Res. 48:443, as disclosed by Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353-358 (1979). 14: 6745 weighted comparison matrix (or EDNAFULL (EMBOSS version of NCBI NUC4.4) permutation matrix); (2) a penalty of 3.0 for each gap and an additional penalty of 0.10 for each symbol in each gap (or a gap opening penalty of 10, a gap extension penalty of 0.5); and (3) no penalty for terminal gaps.

[0061]

[0062] In one embodiment, the variant of the present application may have geranylgeranyl pyrophosphate synthase activity. In one embodiment, the variant of the present application may have an activity that increases the production of a tetraterpene, a tetraterpene precursor, or a substance that uses a tetraterpene as a precursor, compared to a wild-type or unmutated geranylgeranyl pyrophosphate synthase. In one embodiment, the variant of the present application may have an activity that reduces the production level of a byproduct of a tetraterpene, a tetraterpene precursor, or a substance that uses a tetraterpene as a precursor, compared to a wild-type or unmutated geranylgeranyl pyrophosphate synthase. In one embodiment, the variant of the present application may have an activity that reduces the production level of squalene, compared to a wild-type or geranylgeranyl pyrophosphate synthase. In one embodiment, the variant of the present application may have, but is not limited to, enhanced activity compared to wild-type or unmutated geranylgeranyl pyrophosphate synthase.

[0063]

[0064] Another aspect of the present application provides a polynucleotide encoding a variant of the present application.

[0065] In this application, the term "polynucleotide" means a polymer of nucleotides in which nucleotide units are covalently bonded to form a long chain, a DNA or RNA strand of a certain length or longer, and more specifically, a polynucleotide fragment encoding the variant having geranylgeranyl pyrophosphate activity.

[0066] A polynucleotide encoding a variant of the present application may include a base sequence encoding an amino acid sequence set forth in SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, or SEQ ID NO: 17. In one embodiment, the polynucleotide of the present application may have or include a sequence of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, or SEQ ID NO: 18. In addition, the polynucleotide of the present application may consist of, or consist essentially of, a sequence of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, or SEQ ID NO: 18.

[0067] The polynucleotide of the present application may have various modifications made to the coding region within a range that does not change the amino acid sequence of the variant of the present application, taking into account the degeneracy of the codon or the codon preferred in the organism that is intended to express the variant of the present application. Specifically, the polynucleotide of the present application may have or include a base sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, and less than 100% homology or identity with the sequence of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, or SEQ ID NO: 18, or may consist of or consist essentially of a base sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, and less than 100% homology or identity with the sequence of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, or SEQ ID NO: 18, but is not limited thereto.

[0068] At this time, the codon encoding the amino acid corresponding to positions 29, 43, 90, 103, 122, and 141 of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, or SEQ ID NO: 18 of the sequence having the above homology or identity may be one of the codons encoding threonine, isoleucine, arginine, valine, or lysine.

[0069] In addition, the polynucleotide of the present application may include, without limitation, a probe that can be prepared from a known genetic sequence, for example, a sequence that can hybridize under stringent conditions with a complementary sequence to all or part of the polynucleotide sequence of the present application. The term "stringent conditions" refers to conditions that enable specific hybridization between polynucleotides. Such conditions are specifically described in the literature (see J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989; F. M. Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, 9.50-9.51, 11.7-11.8). For example, conditions in which polynucleotides having high homology or identity hybridize with each other, polynucleotides having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homology or identity hybridize with each other, and polynucleotides having lower homology or identity do not hybridize with each other, or conditions in which washing is performed once, specifically twice or three times, at a salt concentration and temperature equivalent to 60°C, 1ХSSC, 0.1% SDS, specifically 60°C, 0.1ХSSC, 0.1% SDS, and more specifically 68°C, 0.1ХSSC, 0.1% SDS, which are washing conditions of typical southern hybridization, are performed.

[0070] Hybridization requires that two nucleic acids have complementary sequences, although mismatches between bases are possible depending on the stringency of hybridization. The term "complementary" is used to describe the relationship between nucleotide bases that can hybridize with each other. For example, in DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Therefore, the polynucleotides of the present application may also include isolated nucleic acid fragments that are complementary in their entirety, as well as substantially similar nucleic acid sequences.

[0071] Specifically, a polynucleotide having homology or identity with the polynucleotide of the present application can be detected using hybridization conditions including a hybridization step at a Tm value of 55°C and using the conditions described above. In addition, the Tm value may be, but is not limited to, 60°C, 63°C, or 65°C, and can be appropriately adjusted by a person skilled in the art depending on the purpose.

[0072] The appropriate stringency for hybridizing the polynucleotides depends on the length and degree of complementarity of the polynucleotides, variables which are well known in the art (e.g., J. Sambrook et al., supra).

[0073]

[0074] Another aspect of the present application provides a vector comprising the polynucleotide of the present application. The vector may be, but is not limited to, an expression vector for expressing the polynucleotide in a host cell.

[0075] The vector of the present application may comprise a DNA construct comprising a base sequence of a polynucleotide encoding a target polypeptide operably linked to a suitable expression control region (or expression control sequence) so as to enable expression of the target polypeptide in a suitable host. The expression control region may comprise a promoter capable of initiating transcription, an optional operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosome binding site, and sequences regulating the termination of transcription and translation. The vector may be capable of replicating or functioning independently of the host genome after being transformed into a suitable host cell, or may be integrated into the genome itself.

[0076] The vector used in the present application is not particularly limited, and any vector known in the art can be used. Examples of commonly used vectors include plasmids, cosmids, viruses, and bacteriophages in a natural or recombinant state. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A can be used as phage vectors or cosmid vectors, and pDC series, pBR series, pUC series, pBluescriptII series, pGEM series, pTZ series, pCL series, and pET series can be used as plasmid vectors. Specifically, pDC, pDCM2 (Korean Patent Publication No. 10-2020-0136813), pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC, pIMR53 vectors, etc. can be used.

[0077] For example, a polynucleotide encoding a target polypeptide can be inserted into a chromosome via a vector for intracellular chromosomal insertion. The insertion of the polynucleotide into the chromosome can be achieved by any method known in the art, for example, homologous recombination, but is not limited thereto. A selection marker for confirming the chromosomal insertion can be additionally included. The selection marker is used to select cells transformed with the vector, i.e., to confirm the insertion of the target nucleic acid molecule. Markers that confer a selectable phenotype, such as drug resistance, nutrient requirement, cytotoxic agent resistance, or expression of a surface polypeptide, can be used. In an environment treated with a selective agent, only cells expressing the selection marker survive or exhibit other phenotypic traits, so that transformed cells can be selected.

[0078] The term "transformation" in this application refers to introducing 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. The transformed polynucleotide may be located within the chromosome of the host cell or located extrachromosomally, as long as it can be expressed in the host cell. In addition, the polynucleotide includes DNA and / or RNA encoding the target polypeptide. The polynucleotide may be introduced in any form as long as it can be introduced into the host cell and expressed. For example, the polynucleotide may be introduced into the host cell in the form of an expression cassette, which is a genetic construct containing all elements necessary for autonomous expression. The expression cassette may typically include a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal, all of which are operably linked to the polynucleotide. The above expression cassette may be in the form of a self-replicating expression vector. Furthermore, the polynucleotide may be introduced into a host cell in its own form and operably linked to a sequence necessary for expression in the host cell, but is not limited thereto.

[0079] Additionally, the term "operably linked" as used herein means that the polynucleotide sequence is functionally linked to a promoter sequence that initiates and mediates transcription of the polynucleotide encoding the target variant of the present application.

[0080]

[0081] Another aspect of the present application provides a microorganism comprising at least one of a variant of the present application; a polynucleotide encoding the variant; and a vector comprising the polynucleotide.

[0082] In this application, the term "microorganism" or "strain" includes both wild-type microorganisms and microorganisms that have undergone genetic modification naturally or artificially, and may be a microorganism that includes genetic modification for the production of a desired polypeptide, protein or product, as a microorganism whose specific mechanism has been weakened or strengthened due to causes such as the insertion of an external gene or the enhancement or inactivation of the activity of an endogenous gene.

[0083] The microorganism of the present application may be, but is not limited to, a microorganism comprising at least one of a variant of the present application, a polynucleotide of the present application, and a vector comprising a polynucleotide of the present application; a microorganism modified to express the variant of the present application or the polynucleotide of the present application; a microorganism expressing the variant of the present application or the polynucleotide of the present application; or a microorganism (e.g., a recombinant microorganism) having the activity of the variant of the present application. The microorganism may be modified to further include a polynucleotide encoding lycopene cyclase / phytoene synthase (crtYB) and phytoene desaturase (crtI) proteins, thereby exhibiting these protein activities or a microorganism having enhanced activity of these proteins. The above lycopene cyclase / phytoene synthase or phytoene desaturase may be a protein derived from Xanthophyllomyces dendrorhous, but is not limited thereto. In one specific example, the polynucleotide encoding the lycopene cyclase / phytoene synthase or phytoene desaturase may have or include the sequence of SEQ ID NO: 27 or SEQ ID NO: 28, respectively. The polynucleotide may have various modifications in the coding region within a range that does not change the amino acid sequence, taking into account the degeneracy of the codon or the codon preferred in the microorganism to which the variant of the present application is to be expressed.Specifically, the polynucleotide may have or include a base sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, and less than 100% homology or identity with the sequence of SEQ ID NO: 27 or SEQ ID NO: 28, or may consist of or consist essentially of a base sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, and less than 100% homology or identity with the sequence of SEQ ID NO: 27 or SEQ ID NO: 28, but is not limited thereto.

[0084]

[0085] The microorganism of the present application may produce a tetraterpene, a precursor of a tetraterpene, or a substance using a tetraterpene as a precursor, may have reduced production of by-products generated in the tetraterpene production pathway, and may have reduced production of squalene, but is not limited thereto.

[0086]

[0087] In this application, "tetraterpene" means a polymer (C40) in which eight isoprene units are polymerized among terpenes, and may include tetraterpene-based substances. The terpene is a polymer of isoprene units, and a terpene in which two isoprene (C5) units are polymerized is a monoterpene (C10), and a terpene in which three isoprene units are polymerized is a sesquiterpene (C15). In this way, terpenes can be classified according to the number of polymerized isoprene units.

[0088] Specifically, the tetraterpene may be, but is not limited to, a carotenoid.

[0089] In the present application, the precursor of the tetraterpene may be, but is not limited to, geranylgeranyl pyrophosphate (GGPP).

[0090] In the present application, the substance using tetraterpene as a precursor may be at least one selected from the group consisting of retinal and retinol, but is not limited thereto.

[0091] In this application, "tetraterpenoid" refers to a modified tetraterpene. The tetraterpenoid may be a tetraterpene having a functional group attached thereto.

[0092] In this application, “carotenoid” refers to a tetraterpene or a derivative thereof that imparts a color such as yellow to fruits and vegetables, and may be used interchangeably with the tetraterpene and tetraterpenoid.

[0093] Specifically, the carotenoid may be at least one selected from the group consisting of xanthophyll, carotene, alpha-carotene, beta-carotene, gamma-carotene, lutein, lycopene, zeaxanthin, capsanthin, canthaxanthin, and astaxanthin, but is not limited thereto.

[0094] In the present application, the precursor of the carotenoid may be, but is not limited to, geranylgeranyl pyrophosphate (GGPP).

[0095] In the present application, the substance using carotenoid as a precursor may be at least one selected from the group consisting of retinal and retinol, but is not limited thereto.

[0096] In this application, “beta-carotene” is one of the carotenoid substances, which has beta rings at both ends of the molecule among carotenes.

[0097] In the present application, the precursor of beta-carotene may be at least one selected from the group consisting of phytoene, phytofluene, lycopene, and gamma-carotene, but is not limited thereto.

[0098] In the present application, the substance using beta-carotene as a precursor may be at least one selected from the group consisting of retinal, retinol, and vitamin A, but is not limited thereto.

[0099] The above tetraterpene, precursor of tetraterpene or substance having tetraterpene as a precursor may be a carotenoid, a precursor of carotenoid or a substance having carotenoid as a precursor, and may be beta-carotene, a precursor of beta-carotene or a substance having beta-carotene as a precursor, but is not limited thereto.

[0100] In the present application, any one or more of the mutant, polynucleotide, vector, and microorganism may be for producing tetraterpene, and the microorganism may be one that produces tetraterpene, one that produces a precursor of tetraterpene, or one that produces a substance using tetraterpene as a precursor.

[0101] In the present application, any one or more of the variant, polynucleotide, vector, and microorganism may be for producing carotenoids, and the microorganism may be one that produces carotenoids, one that produces precursors of carotenoids, or one that produces substances using carotenoids as precursors.

[0102] In the present application, any one or more of the mutant, polynucleotide, vector, and microorganism may be for producing beta-carotene, and the microorganism may be one that produces beta-carotene, or one that produces a precursor of beta-carotene or one that produces a substance using beta-carotene as a precursor.

[0103] In one embodiment, geranylgeranyl pyrophosphate (GGPP) is produced by geranylgeranyl pyrophosphate synthase in the isoprenoid biosynthetic pathway, so the microorganism may also produce geranylgeranyl pyrophosphate.

[0104]

[0105] *In this application, the by-product generated in the tetraterpene production pathway refers to a substance other than tetraterpene, a precursor of tetraterpene, and a substance that uses tetraterpene as a precursor, and specifically may be squalene, but is not limited thereto. Beta-carotene can be produced in a carotenoid or isoprenoid production pathway, and in the process, squalene (C30) can be produced together as a by-product by consuming two molecules of farnesyl pyrophosphate (FPP, C15) by squalene synthase (ERG9).

[0106] In this application, “squalene” means an unsaturated hydrocarbon (C 30 H 50 ) is a substance used in the biosynthesis of steroid hormones, vitamin D, etc. The microorganism of the present application may be one that reduces byproducts produced in the tetraterpene production pathway, and may specifically reduce squalene production, but is not limited thereto.

[0107]

[0108] The microorganism or strain of the present application may be, but is not limited to, a microorganism that naturally has geranylgeranyl pyrophosphate synthase or tetraterpene production ability, or a parent strain that does not have geranylgeranyl pyrophosphate synthase or tetraterpene production ability, into which a variant of the present application or a polynucleotide encoding the same (or a vector including the polynucleotide) is introduced and / or a microorganism that is endowed with the production ability of tetraterpene, a precursor of tetraterpene, or a substance using tetraterpene as a precursor.

[0109] For example, the microorganism or strain of the present application is a cell or microorganism that is transformed with a vector including a polynucleotide encoding the polynucleotide of the present application or a variant of the present application, and expresses the variant of the present application. For the purpose of the present application, the microorganism or strain of the present application may include all microorganisms capable of producing tetraterpene, a precursor of tetraterpene, or a substance using tetraterpene as a precursor, including the variant of the present application. For example, the microorganism or strain of the present application may be a recombinant strain in which a polynucleotide encoding the variant of the present application is introduced into a natural wild-type microorganism or a microorganism producing tetraterpene, thereby expressing a geranylgeranyl pyrophosphate synthase variant, and thus having an increased ability to produce tetraterpene, a precursor of tetraterpene, or a substance using tetraterpene as a precursor. The above recombinant strain may be a microorganism having increased production capacity of tetraterpene, a precursor of tetraterpene, or a substance using tetraterpene as a precursor compared to a natural wild-type microorganism or a non-modified microorganism expressing wild-type geranylgeranyl pyrophosphate synthase (i.e., a microorganism expressing wild-type geranylgeranyl pyrophosphate synthase), but is not limited thereto.

[0110]

[0111] For example, the recombinant strain may have an increase of about 1% or more, specifically about 3% or about 5% or more, in the production of tetraterpene, a precursor of tetraterpene, or a substance using tetraterpene as a precursor compared to the production ability of the parent strain or the non-transformed microorganism before mutation, but is not limited thereto as long as it has a positive increase compared to the production ability of the parent strain or the non-transformed microorganism before mutation.

[0112] As another example, the recombinant strain may have a production amount of a by-product generated in a tetraterpene production pathway reduced by about 85% or less, about 80% or less, about 75% or less, about 70% or less, about 65% or less, about 60% or less, about 55% or less, about 50% or less, about 30% or less, or about 10% or less compared to the parent strain or unmodified microorganism before mutation, or may not produce any by-product, but is not limited thereto.

[0113] The term "about" above includes, but is not limited to, a range including ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and includes all numerical values ​​in a range equal to or similar to the numerical value following the term "about."

[0114]

[0115] In this application, the term "unmodified microorganism" does not exclude a strain that contains a mutation that can occur naturally in a microorganism, and may refer to a wild-type strain or a natural strain itself, or a strain before its characteristics are changed by genetic mutation due to natural or artificial factors. For example, the unmodified microorganism may refer to a strain into which the geranylgeranyl pyrophosphate synthase variant of the present application has not been introduced or before it has been introduced. The "unmodified microorganism" may be used interchangeably with "pre-modified strain," "pre-modified microorganism," "unmutated strain," "unmodified microorganism," or "reference microorganism."

[0116] In one embodiment, the microorganism of the present application may be a microorganism of the genus Yarrowia sp., specifically, Yarrowia lipolytica, but is not limited thereto.

[0117]

[0118] In this application, the term "enhancement" of polypeptide activity means that the activity of the polypeptide is increased compared to the intrinsic activity. The enhancement may be used interchangeably with terms such as activation, up-regulation, overexpression, and increase. Here, activation, enhancement, up-regulation, overexpression, and increase may all include exhibiting an activity that was not originally present, or exhibiting an activity that is enhanced compared to the intrinsic activity or the activity before modification. The "intrinsic activity" refers to the activity of a specific polypeptide that a parent strain or an unmodified microorganism originally had before the trait change when the trait is changed due to genetic mutation caused by natural or artificial factors. This may be used interchangeably with "activity before modification." "Enhanced," "upregulated," "overexpressed," or "increased" the activity of a polypeptide relative to its intrinsic activity means that the activity and / or concentration (expression amount) of the specific polypeptide is improved compared to the activity and / or concentration (expression amount) that the parent strain or unmodified microorganism originally had prior to the transformation.

[0119] The above enhancement can be achieved by introducing an exogenous polypeptide, or by enhancing the activity and / or concentration (expression level) of an endogenous polypeptide. Whether the activity of the polypeptide is enhanced can be determined by an increase in the level of activity, expression level, or amount of product excreted from the polypeptide.

[0120] Enhancement of the activity of the above polypeptide can be achieved by applying various methods well known in the art, and is not limited as long as the activity of the target polypeptide can be enhanced compared to that of the microorganism before modification. Specifically, it may be achieved by using genetic engineering and / or protein engineering, which are routine methods of molecular biology and are well known to those skilled in the art, but 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.).

[0121] Specifically, the enhancement of the polypeptide of the present application is

[0122] 1) Increase in the intracellular copy number of a polynucleotide encoding a polypeptide;

[0123] 2) Replacing the gene expression control region on the chromosome encoding the polypeptide with a highly active sequence;

[0124] 3) Modification of the base sequence encoding the initiation codon or 5'-UTR region of a gene transcript encoding a polypeptide;

[0125] 4) Modification of the amino acid sequence of the polypeptide so as to enhance polypeptide activity;

[0126] 5) Modification of the polynucleotide sequence encoding the polypeptide so as to enhance the activity of the polypeptide (e.g., modification of the polynucleotide sequence of the polypeptide gene so as to encode a polypeptide modified so as to enhance the activity of the polypeptide);

[0127] 6) Introduction of a foreign polypeptide exhibiting the activity of the polypeptide or a foreign polynucleotide encoding the same;

[0128] 7) Codon optimization of a polynucleotide encoding a polypeptide;

[0129] 8) Analyzing the tertiary structure of the polypeptide and selecting the exposed portion to modify or chemically modify; or

[0130] 9) It may be a combination of two or more of the above 1) to 8), but is not particularly limited thereto.

[0131] More specifically,

[0132] The increase in the intracellular copy number of the polynucleotide encoding the polypeptide described above may be achieved by introducing into the host cell a vector capable of replicating and functioning independently of the host, to which the polynucleotide encoding the polypeptide is operably linked. Alternatively, the polynucleotide encoding the polypeptide may be achieved by introducing one copy or two or more copies into the chromosome of the host cell. The introduction into the chromosome may be performed by introducing into the host cell a vector capable of inserting the polynucleotide into the chromosome of the host cell, but is not limited thereto. The vector is as described above.

[0133] 2) Replacing the gene expression control region (or expression control sequence) on the chromosome encoding the polypeptide with a sequence having strong activity may be, for example, a mutation in the sequence such as deletion, insertion, non-conservative or conservative substitution, or a combination thereof to further enhance the activity of the expression control region, or replacement with a sequence having stronger activity. The expression control region may include, but is not particularly limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence regulating the termination of transcription and translation. As an example, it may be, but is not limited to, replacing the original promoter with a strong promoter.

[0134] Examples of known strong promoters include, but are not limited to, the cj1 to cj7 promoters (US Patent No. US 7662943 B2), 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 (US Patent No. US 10584338 B2), the O2 promoter (US Patent No. US 10273491 B2), the tkt promoter, and the yccA promoter.

[0135] The above 3) modification of the base sequence encoding the initiation codon or 5'-UTR region of the gene transcript encoding the polypeptide may be, for example, a substitution with a base sequence encoding another initiation codon having a higher polypeptide expression rate than the endogenous initiation codon, but is not limited thereto.

[0136] The modification of the amino acid sequence or polynucleotide sequence of the above 4) and 5) may be, but is not limited to, a mutation in the sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof in the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide to enhance the activity of the polypeptide, or replacement with an amino acid sequence or polynucleotide sequence improved to have stronger activity, or an amino acid sequence or polynucleotide sequence improved to have increased activity. The replacement may be specifically performed by inserting the polynucleotide into a chromosome by homologous recombination, but is not limited thereto. The vector used at this time may additionally include a selection marker to confirm whether or not the chromosome has been inserted. The selection marker is as described above.

[0137] The introduction of the foreign polynucleotide exhibiting the activity of the polypeptide as described above 6) may be the introduction into the host cell of a foreign polynucleotide encoding a polypeptide exhibiting the same / similar activity as the polypeptide. The foreign polynucleotide is not limited in its origin or sequence as long as it exhibits the same / similar activity as the polypeptide. The method used for the introduction may be performed by a person skilled in the art by appropriately selecting a known transformation method, and the polypeptide may be produced by expressing the introduced polynucleotide in the host cell, thereby increasing its activity.

[0138] The above 7) codon optimization of a polynucleotide encoding a polypeptide may be codon optimization of an endogenous polynucleotide to increase transcription or translation within a host cell, or codon optimization of a foreign polynucleotide to achieve optimized transcription or translation within a host cell.

[0139] The above 8) analyzing the tertiary structure of a polypeptide and selecting an exposed portion to modify or chemically modify may be done by, for example, comparing the sequence information of the polypeptide to be analyzed with a database storing the sequence information of known proteins, determining a template protein candidate based on the degree of sequence similarity, confirming the structure based on this, and selecting an exposed portion to modify or chemically modify, and modifying or modifying it.

[0140] Such enhancement of polypeptide activity may be, but is not limited to, an increase in the activity or concentration of the corresponding polypeptide relative to the activity or concentration of the polypeptide expressed in the wild type or pre-transformed microbial strain, or an increase in the amount of a product produced from the polypeptide.

[0141] In the microorganism of the present application, modification of part or all of the polynucleotide may be induced by, but is not limited to, (a) homologous recombination using a vector for chromosome insertion into the microorganism or genome editing using engineered nucleases (e.g., CRISPR-Cas9) and / or (b) treatment with light and / or chemicals such as ultraviolet rays and radiation. The method for modifying part or all of the gene may include a method using DNA recombination technology. For example, a nucleotide sequence or vector containing a nucleotide sequence homologous to a target gene may be injected into the microorganism to cause homologous recombination, thereby causing deletion of part or all of the gene. The injected nucleotide sequence or vector may include, but is not limited to, a dominant selection marker.

[0142]

[0143] Another aspect of the present application provides a method for producing a tetraterpene, a precursor of a tetraterpene, or a substance using a tetraterpene as a precursor, the method comprising the step of culturing a microorganism in a medium, the microorganism comprising at least one of a variant of the present application; a polynucleotide encoding the variant; and a vector including the polynucleotide. The method for producing a tetraterpene, a precursor of a tetraterpene, or a substance using a tetraterpene as a precursor may be, but is not limited to, a method for reducing squalene production.

[0144] In this application, the term "cultivation" refers to growing the microorganism of this application under appropriately controlled environmental conditions. The culturing process of this application can be performed using any suitable medium and culture conditions known in the art. This culturing process can be easily adjusted and used by those skilled in the art depending on the selected strain. Specifically, the culturing process may be batch, continuous, and / or fed-batch, but is not limited thereto.

[0145] In this application, the term "medium" refers to a material containing nutrients as a main component necessary for culturing the microorganism of this application, and supplies nutrients and growth factors, including water, which is essential for survival and growth. Specifically, the medium and other culture conditions used for culturing the microorganism of this application may be any medium used for culturing general microorganisms without particular limitation, but the microorganism of this application may be cultured under aerobic conditions while controlling temperature, pH, etc. in a general medium containing an appropriate carbon source, nitrogen source, phosphorus source, inorganic compound, amino acid, and / or vitamin.

[0146] In the present application, the carbon source may include carbohydrates such as glucose, saccharose, lactose, fructose, sucrose, maltose, etc.; sugar alcohols such as mannitol, sorbitol, etc.; organic acids such as pyruvic acid, lactic acid, citric acid, etc.; amino acids such as glutamic acid, methionine, lysine, etc. In addition, natural organic nutrients such as starch hydrolysate, molasses, blackstrap molasses, rice winter, cassava, sugarcane bagasse, and corn steep liquor may be used, and specifically, carbohydrates such as glucose and sterilized pretreated molasses (i.e., molasses converted to reducing sugar) may be used, and other appropriate amounts of carbon sources may be used in various ways without limitation. These carbon sources may be used alone or in combination of two or more, but are not limited thereto.

[0147] The nitrogen source may include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, ammonium nitrate, etc.; organic nitrogen sources such as amino acids such as glutamic acid, methionine, glutamine, etc.; peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolysate, fish or its decomposition product, defatted soybean cake or its decomposition product, etc. These nitrogen sources may be used alone or in combination of two or more, but are not limited thereto.

[0148] The above-mentioned components may include potassium phosphate monobasic, potassium phosphate dibasic, or their corresponding sodium-containing salts. Inorganic compounds may include sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, etc. In addition, amino acids, vitamins, and / or suitable precursors may be included. These components or precursors may be added to the medium in batch or continuous manner, but are not limited thereto.

[0149] In addition, during the cultivation of the microorganism of the present application, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid, etc. may be added to the medium in an appropriate manner to adjust the pH of the medium. In addition, during the cultivation, foaming may be suppressed by using an antifoaming agent such as fatty acid polyglycol ester. In addition, in order to maintain the aerobic state of the medium, oxygen or an oxygen-containing gas may be injected into the medium, or in order to maintain the anaerobic and microaerobic state, nitrogen, hydrogen, or carbon dioxide gas may be injected without gas injection, but is not limited thereto.

[0150] In the culture of the present application, the culture temperature can be maintained at 20 to 45°C, specifically 25 to 40°C, 20 to 35°C, or 25 to 35°C, and the culture can be performed for about 10 to 160 hours, but is not limited thereto.

[0151] The tetraterpene, tetraterpene precursor, or substance using tetraterpene as a precursor produced by the culture of the present application may be secreted into the medium or remain within the cell.

[0152]

[0153] The method for producing a tetraterpene, a precursor of a tetraterpene, or a substance using a tetraterpene as a precursor of the present application may additionally include a step of preparing a microorganism of the present application, a step of preparing a medium for culturing the strain, or a combination thereof (in any order), for example, before the culturing step.

[0154] The method for producing a tetraterpene, a precursor of a tetraterpene, or a substance using a tetraterpene as a precursor of the present application may further include a step of recovering the tetraterpene, a precursor of a tetraterpene, or a substance using a tetraterpene as a precursor from the culture medium (the medium in which the culture is performed) or the microorganism of the present application. The recovering step may be additionally included after the culturing step.

[0155] The above recovery may be performed by collecting the target tetraterpene, the precursor of the tetraterpene, or the substance containing the tetraterpene as a precursor using a suitable method known in the art according to the culture method of the microorganism of the present application, for example, a batch, continuous, or fed-batch culture method. For example, various chromatographies such as centrifugation, filtration, treatment with a crystallizing protein precipitant (salting out method), extraction, cell disruption, ultrasonic disruption, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, HPLC, or a combination thereof may be used, and the target tetraterpene, the precursor of the tetraterpene, or the substance containing the tetraterpene as a precursor can be recovered from the medium or the microorganism using a suitable method known in the art.

[0156] In addition, the method for producing a tetraterpene, a precursor of a tetraterpene, or a substance using a tetraterpene as a precursor of the present application may additionally include a purification step. The purification may be performed using a suitable method known in the art. In one example, when the method for producing a tetraterpene, a precursor of a tetraterpene, or a substance using a tetraterpene as a precursor of the present application includes both a recovery step and a purification step, the recovery step and the purification step may be performed sequentially (or consecutively) regardless of the order, or may be performed simultaneously or integrated into one step, but is not limited thereto.

[0157] The method for producing a substance using a tetraterpene as a precursor of the present application may additionally include a step of converting a tetraterpene into a substance using a tetraterpene as a precursor. In the method for producing a substance using a tetraterpene as a precursor of the present application, the conversion step may be additionally included after the culturing step or the recovering step. The conversion step may be performed using a suitable method known in the art. For example, the conversion may be performed using beta-carotene 15,15'-oxygenase or retinol dehydrogenase, but is not limited thereto.

[0158] The above mutants, polynucleotides, vectors, microorganisms, tetraterpenes, precursors of tetraterpenes, and substances containing tetraterpenes as precursors are as described above.

[0159]

[0160] Another aspect of the present application provides a composition for producing tetraterpene, a precursor of tetraterpene, and a substance using tetraterpene as a precursor, comprising a variant of the present application; a vector of the present application; a variant of the present application, a polynucleotide encoding the variant, a vector comprising the polynucleotide, or a microorganism comprising the polynucleotide of the present application; a medium for culturing the same; or a combination of two or more thereof.

[0161] The composition of the present application may further comprise any suitable excipients commonly used in compositions for producing tetraterpenes, precursors of tetraterpenes and materials using tetraterpenes as precursors, and such excipients may be, for example, but are not limited to, preservatives, wetting agents, dispersing agents, suspending agents, buffers, stabilizers or isotonic agents.

[0162] The above mutants, polynucleotides, vectors, microorganisms, media, tetraterpenes, precursors of tetraterpenes, and substances containing tetraterpenes as precursors are as described above.

[0163]

[0164] Another aspect of the present application provides a use of any one or more of the variants, polynucleotides, vectors, and microorganisms of the present application for producing tetraterpenes, precursors of tetraterpenes, and materials using tetraterpenes as precursors.

[0165] The above mutants, polynucleotides, vectors, microorganisms, tetraterpenes, precursors of tetraterpenes, and substances containing tetraterpenes as precursors are as described above.

[0166]

[0167] Hereinafter, this application will be described in more detail through examples. However, these examples and experimental examples are intended to exemplify this application and the scope of this application is not limited to these examples and experimental examples.

[0168]

[0169] Example 1. Construction of a GGS1 mutant vector library

[0170] In order to amplify the GGS1 gene encoding geranylgeranyl pyrophosphate synthase on the Yarrowia lioplytica chromosome, the amino acid sequence of SEQ ID NO. 1 and the polynucleotide sequence of SEQ ID NO. 2 of GGS1 (YALI0D17050) were obtained based on the base sequence registered in the Kyoto Encyclopedia of Genes and Genomes (KEGG). A diversify PCR random mutagenesis kit (Takara) was used to induce random mutations through error-prone PCR (ep-PCR). The PCR conditions and methods were optimized and performed under buffer conditions 1 and 2 of the user manual, respectively. The specific error-prone PCR conditions are shown in Table 1 below.

[0171]

[0172] Buffer conditions 1Buffer conditions 2 PCR grade water40 ㎕39 ㎕10X Titanium Taq buffer5 ㎕5 ㎕MnSO40 ㎕1 ㎕dGTP1 ㎕1 ㎕50X Diversify dNTP Mix1 ㎕1 ㎕Primer mix1 ㎕1 ㎕Template DNA1 ㎕1 ㎕Titanium Taq polymerase1 ㎕1 ㎕

[0173] PCR was performed using Yarrowia lipolytica PO1f genomic DNA as a template and primers of SEQ ID NO: 19 and SEQ ID NO: 20. The PCR conditions were 32 cycles of denaturation at 95°C for 1 min; annealing at 55°C for 1 min; and polymerization at 68°C for 1 min. The GGS1 PCR products amplified under two conditions were mixed to construct a pIMR53-EXP1p-CYC1t vector-based GGS1 mutant expression vector library.

[0174] The pIMR53-EXP1p-CYC1t vector was constructed as follows. To amplify EXP1p, PCR was performed using Yarrowia lipolytica PO1f genomic DNA as a template and the primers of SEQ ID NO: 21 and SEQ ID NO: 22. To amplify the CYC1t terminator, PCR was performed using Saccharomyce cerevisiae genomic DNA as a template and the primers of SEQ ID NO: 23 and SEQ ID NO: 24. The PCR conditions were 32 cycles of denaturation at 95°C for 1 minute; annealing at 55°C for 1 minute; and polymerization at 68°C for 1 minute. Additionally, PCR was performed using the pIMR53 vector as a template and the primers of SEQ ID NO: 25 and SEQ ID NO: 26. The PCR conditions were denaturation at 95°C for 1 minute; Annealing at 55°C for 1 minute and polymerization at 68°C for 5 minutes were repeated 32 times.

[0175] As a result, 6,774 bp linearized pIMR53, 1,050 bp EXP1p, and 289 bp CYC1t were obtained. The EXP1p and CYC1t DNA fragments amplified by PCR were ligated into the pIMR53 vector using an Infusion cloning kit (Invitrogen), transformed into E. coli DH5α, and plated on LB solid medium containing 30 mg / L ampicillin. Colonies transformed with the plasmid into which the desired EXP1p promoter and CYC1t terminator were inserted through PCR were selected, and the plasmid was obtained using a conventionally known plasmid extraction method, and this plasmid was named pIMR53-EXP1p-CYC1t.

[0176]

[0177] Example 2. Production of a Yarrowia lipolytica platform strain for beta-carotene production.

[0178] Example 2-1. Production of crtYB-crtI insertion strain derived from X. dendrorhous

[0179] To construct a platform strain for beta-carotene production, the lycopene cyclase / phytoene synthase (crtYB) and phytoene desaturase (crtI) genes from Xanthophyllomyces dendrorhous were inserted into the genome of the high-fat yeast strain Yarrowia lipolytica CC08-0125 (accession number KCCM12972P). The polynucleotide of sequence number 27 for crtYB was obtained based on the nucleotide sequence registered in the National Center for Biotechnology Information Search database (NCBI) (GenBank: AY177204.1), and the polynucleotide of sequence number 28 for crtI was obtained based on the nucleotide sequence registered in the NCBI (GenBank: AY177424.1). The polynucleotide sequences of crtYB and crtI were synthesized as TEFINtp-crtYB-CYC1t (SEQ ID NO: 29) and TEFINtp-crtI-CYC1t (SEQ ID NO: 30) genes through Macrogen. As a selection marker, a cassette was designed to be inserted into the MHY1 (YALI0B21582g) gene position using the URA3 gene of Y. lipolytica (SEQ ID NO: 31). The synthesized crtYB, crtI genes and KCCM12972P genomic DNA were used as templates, and each PCR was performed using the primers of SEQ ID NO: 32 and SEQ ID NO: 33, SEQ ID NO: 34 and SEQ ID NO: 35, SEQ ID NO: 36 and SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 39, SEQ ID NO: 40 and SEQ ID NO: 41, and SEQ ID NO: 42 and SEQ ID NO: 43. The PCR conditions were denaturation 95°C, 1 min; Annealing at 55°C for 1 minute and polymerization at 72°C for 3 minutes and 30 seconds were repeated 35 times. The resulting five DNA fragments were then combined into a single cassette through overlap extension PCR.

[0180] The cassette thus constructed was introduced into the KCCM12972P strain by the heat shock method (D.-C. Chen et al., Appl Microbiol Biotechnol, 1997), and colonies formed on solid medium (YLMM1) without uracil were obtained. Colonies in which the cassette insertion into the genome was confirmed using primers of SEQ ID NO: 44 and SEQ ID NO: 45 were spotted on 5-FOA solid medium and cultured at 30°C for 3 days, and the URA3 marker was recovered by obtaining colonies grown on the 5-FOA solid medium.

[0181]

[0182] Example 2-2. Production of HMGR reinforced liquor

[0183] A cassette was designed to replace the native promoter (SEQ ID NO: 46) of the hydroxymethylglutaryl reductase (3-hydroxy-3-methylglutaryl-CoA reductase, HMGR) gene of the strain into which the crtYB and crtI genes produced in Example 2-1 were inserted with the TEFINt promoter, and each PCR was performed using the KCCM12972P genomic DNA as a template and the primers of SEQ ID NO: 47 and SEQ ID NO: 48, SEQ ID NO: 49 and SEQ ID NO: 50, SEQ ID NO: 51 and SEQ ID NO: 52, SEQ ID NO: 53 and SEQ ID NO: 54, and SEQ ID NO: 55 and SEQ ID NO: 56. 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 35 times. The five resulting DNA fragments were produced into one cassette through overlap extension PCR.

[0184] The cassette thus constructed was introduced into the strain constructed in Example 2-1 by the heat shock method, and colonies formed on solid medium (YLMM1) not containing uracil were obtained. Colonies in which cassette insertion was confirmed using primers of SEQ ID NO: 57 and SEQ ID NO: 58 were spotted on 5-FOA solid medium and cultured at 30°C for 3 days, and the URA3 marker was recovered by obtaining colonies grown on the 5-FOA solid medium. The finally constructed beta-carotene production platform strain was named CC08-1023.

[0185]

[0186] < Yarrowia lipolytica minimal media1 (YLMM1)>

[0187] Glucose 20 g / L, Yeast nitrogen base without amino acids 6.7 g / L, Yeast Synthetic Drop-out Medium Supplements without uracil 2 g / L, Agar 15 g / L

[0188]

[0189] <5-Fluoroorotic Acid (5-FOA)>

[0190] Glucose 20 g / L, Yeast nitrogen base without amino acids 6.7 g / L, Yeast Synthetic Drop-out Medium Supplements without uracil 2 g / L, Uracil 50 μg / mL, 5-Fluoroorotic acid (5-FOA) 1 g / L, Agar 15 g / L

[0191]

[0192] Example 3. First screening of GGS1 activity-enhanced strains

[0193] For the primary screening of GGS1 activity-enhanced strains, color-based primary screening was conducted.

[0194] First, the Yarrowia lipolytica CC08-1023 strain, which produces small amounts of beta-carotene, was transformed with pIMR53 (negative control; NC), pIMR53-EXP1p-GGS1 (wild type, SEQ ID NO: 2)-CYC1t vector (positive control; PC), and the GGS1 random mutant expression vector library constructed in Example 1 by heat shock. Thereafter, colonies formed on a solid medium that did not contain uracil were obtained.

[0195] 350 ㎕ of YLMM2 (Yarrowia lipolytica minimal media 2) liquid medium without uracil was dispensed into 25 96 deep-well plates, and the obtained colonies were inoculated and cultured at 30℃ for 48 hours. Afterwards, they were inoculated into YLMM1 solid medium without uracil and cultured at 30℃ for 3 days. After the culture was completed, 36 strains with a dark orange color compared to the strain transformed with the positive control (CC08-1023 / pIMR53-EXP1p-GGS1-CYC1t) vector were first selected from a total of 2,400 strains. The manufacturing method and composition of the above-mentioned YLMM2 are as follows.

[0196]

[0197] <YLMM2 (pH 7.2)>

[0198] Glucose 40 g / L, yeast nitrogen base without amino acids and ammonium sulfate 1.7 g / L, uracil 0.5 g / L, and ammonium sulfate 2.5 g / L were dissolved in 0.1 M sodium phosphate buffer (pH 7.2).

[0199]

[0200] Example 4. Secondary screening of GGS1 activity-enhanced strains

[0201] For the secondary screening of GGS1 activity-enhanced strains, flask evaluation was conducted on the 36 strains selected in the first stage in Example 3.

[0202] The 36 strains and the strain transformed with the PC (positive control) vector were each inoculated into a 250 ml corner-baffle flask containing 50 ml of YLMM2 (Yarrowia lipolytica minimal media 2) without uracil to an initial OD of 1, and cultured with shaking at 250 rpm at 30°C for 48 hours.

[0203] After the culture was completed, 1 ml of the culture medium was centrifuged to remove the supernatant. Next, 0.5 ml of DMSO (Dimethyl sulfoxide, Sigma) was added and the cells were disrupted by shaking at 2000 rpm for 10 minutes at 55°C. Additionally, 0.5 ml of acetone (Sigma) was added and shaken at 2000 rpm for 15 minutes at 45°C to extract beta-carotene and squalene. The extracted substances were analyzed for concentration using HPLC equipment. The results of the selection flask evaluation, OD, concentration and content of beta-carotene and squalene are shown in Table 2 below.

[0204]

[0205] Numb.ODβ-carotene(mg / L)Squalene(mg / L)β-carotene(%)Squalene(%)PC60382480.251.661595415 40.361.04261352140.231.41365622120.381.32450301020.240.82562611940.391.24662552820.36 1.83755461730.331.27848401800.341.52956341320.250.951050461850.361.481151411200.330.941262522180.341.421363572100.371.341459532300.361.571558571850.391.291653402190.311.6 61760601210.400.811859532060.361.401962502070.321.332062591890.381.222160691080.460.722259601480.411.002362581340.370.862460571980.381.332564482140.301.332667562280.341.3 72759472140.321.452861351830.231.202960512190.341.453059482010.331.383164611980.381.233265661690.401.043359472060.321.403462512380.321.533553371890.281.423661532250.351.47

[0206] Afterwards, the value of the beta-carotene and squalene concentrations extracted from the evaluation samples divided by DCW (g / L) was used as the standard for the secondary selection. In 33 evaluated strains, excluding strains No. 2, No. 4, No. 9, and No. 28, the value of the beta-carotene concentration divided by DCW (g / L) (β-carotene (%)) was confirmed to increase compared to the PC (positive control). The value of the squalene concentration divided by DCW (g / L) was confirmed to decrease compared to the PC (positive control) in all evaluated strains.

[0207] Therefore, a reference value was set based on the value calculated from the PC (positive control), and strains that met both conditions were selected, and a total of four candidates that met both conditions 1 and 2 were selected. The secondary selection reference value and the four secondary selection candidates are shown in Table 3 below.

[0208]

[0209] Conditional criteria selection results (flask number) Note #1 Beta-carotene content (%) ≥ 0.383, 5, 15, 17, 20, 21, 22, 24, 31, 32 GGS1 (PC) = 0.25 #2 Squalene content (%) < 1.052, 4, 9, 11, 17, 21, 22, 23, 32 GGS1 (PC) = 1.66 Condition 1 + Condition 2 17, 21, 22, 32

[0210] Transformed plasmids were extracted from the four selected strains and sequenced to identify random mutations located in the GGS1 ORF. Using the Zymoprep yeast plasmid miniprep kit 2 (Zymo research), the transformed GGS1 plasmids were extracted from the selected strains, and to increase the reliability of the sequence analysis, bidirectional sequence analysis was performed using primers of SEQ ID NO: 59 and SEQ ID NO: 60. The amino acid mutations identified as a result of the GGS1 ORF sequence analysis are shown in Table 4 below, and the four candidates were confirmed to contain one or two mutations among Table 4 below.

[0211]

[0212] Mutant flask number. Genotype 17GGS1 (I122V, R141K) 21GGS1 (N29T, L90R) 22GGS1 (M103I) 32GGS1 (F43I)

[0213] Example 5. Verification of activity of GGS1 selected variants

[0214] An activity verification experiment was conducted on the GGS1 variants selected in Examples 3 and 4.

[0215] To this end, a flask evaluation was conducted on a total of 10 strains produced by transforming NC (negative control) and PC (positive control) vectors and expression vectors of four mutants selected in Example 4 (M103I mutant, F43I mutant, N29T and L90R mutants, and I122V and R141K mutants), and four related single mutants (N29T mutant, L90R mutant, I122V mutant, and R141K mutant).

[0216] The method for constructing expression vectors for each of the four single mutants (N29T mutant, L90R mutant, I122V mutant, and R141K mutant) is as follows. To obtain DNA fragments for the four single mutants (N29T mutant, L90R mutant, I122V mutant, and R141K mutant), PCR was performed using the pIMR53-EXP1p-GGS1-CYC1t vector containing the wild-type GGS1 sequence as a template and the primers of 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, respectively. The PCR conditions were 32 cycles of denaturation at 95°C for 1 minute; annealing at 55°C for 1 minute; and polymerization at 68°C for 5 minutes. Expression vectors of the double mutants N29T and L90R mutants, or I122V and R141K mutants, were constructed by performing PCR in the same manner using the N29T expression vector or I122V expression vector constructed above as a template, and the primers of SEQ ID NO: 63 and SEQ ID NO: 64, or the primers of SEQ ID NO: 67 and SEQ ID NO: 68.

[0217] As a result, the DNA containing the above-mentioned mutants amplified by PCR was transformed into E. coli DH5α using an Infusion cloning kit (Invitrogen) and plated on LB solid medium containing 30 mg / L of ampicillin. After selecting colonies transformed with plasmids inserted with the four types of transformed GGS1 mutant vectors, plasmids were obtained using a commonly known plasmid extraction method, and the mutant sequences were confirmed using SEQ ID NOs: 59 and 60.

[0218] Specifically, the 10 strains were inoculated into a 250 ml corner-baffle flask containing 50 ml of YLMM2 (Yarrowia lipolytica minimal media 2) without uracil to an initial OD of 1, and cultured at 30°C for 48 hours with shaking at 250 rpm.

[0219] After the culture was completed, 1 ml of the culture medium was centrifuged to remove the supernatant. Thereafter, 0.5 ml of DMSO (Dimethyl sulfoxide, Sigma) was added and the cells were disrupted by shaking at 2000 rpm for 10 minutes at 55°C. Additionally, 0.5 ml of acetone (Sigma) was added and the cells were disrupted by shaking at 2000 rpm for 15 minutes at 45°C. Beta-carotene and squalene were extracted and their concentrations were analyzed using HPLC equipment. The results of measuring the OD, concentration, and content of beta-carotene and squalene are shown in Table 5 below.

[0220]

[0221] GenotypeODβ-carotene(mg / L)Squalene(mg / L)β-carotene(%)Squalene(%)NC6252080.031.34PC56301330.210.95M103I6259910.380.59F43I6369710.4 40.45N29T / L90R6467890.420.56N29T6155880.360.58L90R6458900.360.56 I122V / R141K6262650.400.42I122V57431180.300.83R141K59441110.300.75

[0222] As shown in Table 5 above, it was confirmed that the beta-carotene concentration increased by 143 to 230% and the squalene concentration decreased by 11 to 51% in all GGS1 mutants applied with the selected M103I, F43I, N29T, L90R, I122V, and / or R141K mutations compared to the PC (positive control). As a result, among the six strains introduced with a single mutant, the strain introduced with the F43I mutant had the best effect, and in the case of the I122V and R141K mutants, the effects of increasing the beta-carotene concentration and decreasing the squalene production were further increased when applied simultaneously compared to when the two mutants were applied alone. In addition, in the case of the N29T and L90R mutants, the effects of decreasing the squalene production were further increased when applied simultaneously compared to when the two mutants were applied alone.

[0223]

[0224] Example 6. Production and evaluation of GGS1 mutant strains

[0225] We aimed to verify the effect of the GGS1 F43I variant, which was judged to be the best through the previous examples, when introduced into an actual strain.

[0226] To this end, a strain (CC08-1214) in which the promoter of the GGS1 gene was replaced based on CC08-1023 and a strain (CC08-1215) expressing a mutant in which the 43rd phenylalanine of GGS1 was substituted with isoleucine were sequentially produced.

[0227]

[0228] Example 6-1. Production of GGS1 promoter substitution strain ("CC08-1214")

[0229] In order to construct a cassette capable of replacing the promoter of GGS1 with the TEFINt promoter on the Yarrowia lipolytica chromosome, a polynucleotide sequence of SEQ ID NO: 69 including the promoter and splicing point of TEF1 (YALI0C09141p) was secured based on the base sequence registered in KEGG (Kyoto Encyclopedia of Genes and Genomes). In addition, a polynucleotide sequence of SEQ ID NO: 2 of GGS1 (YALI0D17050g) was secured for promoter replacement of GGS1. PCR was performed using Yarrowia lipolyticaPO1f genomic DNA as a template and primers of SEQ ID NO: 70 and SEQ ID NO: 71, SEQ ID NO: 72 and SEQ ID NO: 73, SEQ ID NO: 74 and SEQ ID NO: 75, and SEQ ID NO: 76 and SEQ ID NO: 77, respectively. Then, PCR was performed using the URA3 auxotrophic marker as a template and primers of SEQ ID NO: 78 and SEQ ID NO: 79. 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 35 times.

[0230] As a result, 1500 bp 5'UTR arm, 531 bp 5'UTR_RP, 1500 bp GGS1 arm, and 1533 bp URA3 were obtained. The PCR-amplified DNA fragment was constructed into a single GGS1 promoter replacement cassette through overlap extension PCR, and the cassette design was performed in the following order: 5'UTR arm-TEFINt-URA3-TEFINt-GGS1 arm.

[0231] The GGS1 promoter replacement cassette was transformed into CC08-1023 by heat shock and selected on YLMM1 solid medium without uracil. The selected primary strain was then subjected to a second round of crossing, ultimately generating a strain in which the GGS1 gene promoter was replaced with the TEFINt promoter. This generated strain was named "CC08-1214."

[0232]

[0233] Example 6-2. Production of GGS1 (F43I) mutant strain ("CC08-1215")

[0234] To introduce a GGS1 protein F43I mutant expression sequence onto the Yarrowia lipolytica chromosome, a cassette was constructed in which thymine at position 127 of the polynucleotide sequence of SEQ ID NO: 2 was substituted with adenine.

[0235] Specifically, PCR was performed using the Yarrowia lipolyticaPO1f genomic DNA as a template and the primers 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, SEQ ID NO: 86 and SEQ ID NO: 83, SEQ ID NO: 84 and SEQ ID NO: 87, and SEQ ID NO: 90 and SEQ ID NO: 91, respectively. In addition, PCR was performed using the URA3 auxotrophic marker as a template and the primers SEQ ID NO: 88 and SEQ ID NO: 89. The PCR conditions were 35 cycles of 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.

[0236] As a result, 1500 bp 5'UTR arm, 661 bp TEFINt-GGS1_1, 1390 bp GGS1_2, 661 bp RP_1, 1390 bp RP_2, 1500 bp 3'UTR arm, and 1533 bp URA3 were obtained. The PCR-amplified DNA fragments were constructed into a single GGS1 promoter replacement cassette through overlap extension PCR, and the cassette design was as follows: 5'UTR arm- TEFINt-GGS1_1- GGS1_2- RP_1- RP_2-3'UTR arm.

[0237] The GGS1 (F43I) mutation replacement cassette was transformed into CC08-1214 by heat shock and selected on YLMM1 solid medium without uracil. The selected primary strain underwent a second round of crossover, ultimately generating a strain expressing the F43I mutant in which the 43rd amino acid residue of GGS1 was replaced with isoleucine. This strain was designated "CC08-1215."

[0238]

[0239] Example 6-3. Evaluation of GGS1 mutant strains introduced

[0240] Flask evaluation was conducted with CC08-1023 as a negative control and CC08-1214 as a positive control, along with CC08-1215.

[0241] Specifically, CC08-1215, CC08-1023 (negative control), and CC08-1214 (positive control) were inoculated into a 250 ml corner-baffle flask containing 50 ml of YLMM2 (Yarrowia lipolytica minimal media 2) containing uracil to an initial OD of 1, and cultured at 30°C for 48 hours with shaking at 250 rpm.

[0242] After the culture was completed, 1 ml of the culture medium was centrifuged to remove the supernatant. Thereafter, 0.5 ml of DMSO (Dimethyl sulfoxide, Sigma) was added and the cells were disrupted by shaking at 2000 rpm for 10 minutes at 55°C. Additionally, 0.5 ml of acetone (Sigma) was added and the cells were shaken at 2000 rpm for 15 minutes at 45°C to extract beta-carotene and squalene, and their concentrations were analyzed using HPLC equipment. The results of measuring the OD, concentrations, and contents of beta-carotene and squalene are shown in Table 6 below.

[0243]

[0244] GenotypeODβ-carotene(mg / L)Squalene(mg / L)β-carotene(%)Squalene(%)CC08-1023(NC)465410.040.36CC08-1214(PC)5032170.260.14CC08-1215484920.410.02

[0245] As shown in Table 6 above, the beta-carotene concentration was confirmed to increase by 51.3% and the squalene concentration was confirmed to decrease by 88.5% in the CC08-1215 strain to which the selected F43I mutation was applied compared to CC08-1214 (PC). Consequently, it was confirmed that the effect of introducing the F43I single mutant was effective in increasing beta-carotene concentration and decreasing squalene production even when applied to an actual strain.

[0246]

[0247] From the above results, it was confirmed that introduction of the GGS1 variant of the present invention can increase beta-carotene production. Furthermore, it was confirmed that introduction of the GGS1 variant can further increase beta-carotene production efficiency by reducing squalene synthesis, a competitive pathway with beta-carotene production.

[0248] In addition, from the above results, it was confirmed that, similar to beta-carotene production, the efficiency of tetraterpene production involving GGS1 can be further increased.

[0249]

[0250] From the above description, those skilled in the art will understand that the present application can be implemented in other specific forms without altering its technical concept or essential characteristics. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of this application should be interpreted to include all changes or modifications derived from the meaning and scope of the following claims and their equivalents, rather than the detailed description above.

[0251]

Claims

1. A geranylgeranyl pyrophosphate synthase mutant in which at least one amino acid corresponding to positions 29, 43, 90, 103, 122, and 141 from the N-terminus of the amino acid sequence of sequence number 1 is replaced with another amino acid.

2. A geranylgeranyl pyrophosphate synthase variant according to claim 1, wherein the variant comprises one or more of the following substitutions: a substitution of an amino acid corresponding to position 29 from the N-terminus of the amino acid sequence of SEQ ID NO: 1 with an amino acid other than asparagine; a substitution of an amino acid corresponding to amino acid 43 with an amino acid other than phenylalanine; a substitution of an amino acid corresponding to amino acid 90 with an amino acid other than leucine; a substitution of an amino acid corresponding to amino acid 103 with an amino acid other than methionine; a substitution of an amino acid corresponding to amino acid 122 with an amino acid other than isoleucine; and a substitution of an amino acid corresponding to amino acid 141 with an amino acid other than arginine.

3. A geranylgeranyl pyrophosphate synthase variant, wherein the substitution with another amino acid in paragraph 1 is a substitution with a nonpolar amino acid, a polar amino acid, or a positively charged (basic) amino acid.

4. A geranylgeranyl pyrophosphate synthase variant, wherein the other amino acid in paragraph 1 is any one selected from the group consisting of threonine (Thr), isoleucine (Ile), arginine (Arg), valine (Val), and lysine (Lys).

5. A polynucleotide encoding a geranylgeranyl pyrophosphate synthase variant of any one of claims 1 to 4.

6. A microorganism comprising at least one of the geranylgeranyl pyrophosphate synthase variants of any one of claims 1 to 4 and a polynucleotide encoding the variant.

7. A microorganism according to claim 6, wherein the microorganism produces a tetraterpene, a precursor of a tetraterpene, or a substance using a tetraterpene as a precursor.

8. In the 6th paragraph, the microorganism is a microorganism of the genus Yarrowia sp.

9. In paragraph 6, the microorganism is Yarrowia lipolytica.

10. A microorganism in paragraph 6, wherein the microorganism produces beta-carotene, a precursor of beta-carotene, or a substance using beta-carotene as a precursor.

11. A method for producing tetraterpene, a precursor of tetraterpene, or a substance using tetraterpene as a precursor, comprising a step of culturing the microorganism of clause 6 in a medium.

12. A production method according to claim 11, wherein the method further comprises a step of recovering a tetraterpene, a precursor of a tetraterpene, or a substance containing a tetraterpene as a precursor from the medium or microorganism.

13. A production method according to claim 11, wherein the microorganism is Yarrowia sp.

14. A production method according to claim 11, wherein the method reduces squalene production.

15. A composition for producing a tetraterpene, a precursor of a tetraterpene, or a substance using a tetraterpene as a precursor, comprising a mutant of paragraph 1, a microorganism of paragraph 6, or a culture of the microorganism.

16. A use for producing a tetraterpene, a precursor of a tetraterpene, or a substance using a tetraterpene as a precursor of a microorganism, comprising a geranylgeranyl pyrophosphate synthase variant in which at least one of the amino acids corresponding to positions 29, 43, 90, 103, 122, and 141 from the N-terminus of the amino acid sequence of SEQ ID NO: 1 is replaced with another amino acid; and at least one polynucleotide encoding the variant.