Geranylgeranyl pyrophosphate synthase mutant, and method for producing tetraterpene, its precursor, and substance having tetraterpene as precursor using the same

Abstract

The present application relates to a geranylgeranyl pyrophosphate synthase mutant, a polynucleotide encoding the mutant, a vector comprising the polynucleotide, a microorganism comprising at least one of the mutant, the polynucleotide, and the vector, a method for producing a tetraterpene, a precursor of a tetraterpene, or a substance having a precursor of a tetraterpene using the same, a composition for producing a tetraterpene, a precursor of a tetraterpene, or a substance having a precursor of a tetraterpene, and the use of a microorganism comprising at least one of the mutant, the polynucleotide, and the vector for producing tetraterpene, a precursor thereof, or a substance having a precursor of a tetraterpene.

Description

[Technical Field]

[0001] The present application relates to a geranylgeranyl pyrophosphate synthase mutant, a polynucleotide encoding the mutant, a vector containing the polynucleotide, a microorganism containing at least one of the mutant, polynucleotide, and vector, a method for producing tetraterpenes, their precursors, and substances having tetraterpenes as precursors using the same, a composition for producing tetraterpenes, their precursors, and substances having tetraterpenes as precursors, and the use of a microorganism containing at least one of the mutant, polynucleotide, and vector for producing tetraterpenes, their precursors, and substances having tetraterpenes as precursors. [Background technology]

[0002] Terpenoids, a concept that includes carotenoids, are used in a wide range of industrial fields, including food and feed, due to their diverse functions in plants and animals. Terpenoids are known as representative substances that exert medicinal effects in plants and, like carotenoids, are terpenes. In particular, carotenoids such as β-carotene have been reported to have functions such as scavenging free radicals, being the source of vitamin A in animals, enhancing the immune system of vertebrates, and reducing the risk of lung cancer.

[0003] However, despite these advantages, carotenoids, such as β-carotene, are not synthesized in animals, or even if they are synthesized, the amount synthesized is insufficient. Furthermore, although attempts have been made to industrially produce β-carotene using mutated microorganisms (Patent Document 1), it remains difficult to produce β-carotene at high purity due to the by-products.

[0004] Regarding by-products, geranylgeranyl pyrophosphate (GGPP, C20), a key precursor for carotenoid or terpenoid production, is produced by the conjugation of farnesyl pyrophosphate (FPP, C15) and isopentenyl pyrophosphate (IPP, C5) by the enzyme geranylgeranyl pyrophosphate synthase in the isoprenoid biosynthesis pathway. However, squalene (C30) is also produced as a by-product from farnesyl pyrophosphate (FPP, C15) by squalene synthase (ERG9). Therefore, there is a growing need to develop methods to increase the production of GGPP, a key precursor for carotenoid or terpenoid biosynthesis, and reduce the production of squalene, a competing pathway. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] U.S. Patent No. 7,745,170 [Patent Document 2] Korean Patent Publication No. 10-2020-0136813 [Patent Document 3] U.S. Patent No. 7,662,943 [Patent Document 4] U.S. Patent No. 10,584,338 [Patent Document 5] U.S. Patent No. 10,273,491 [Non-patent literature]

[0006] [Non-Patent Document 1] Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453 [Non-licensed document 2] Rice et al., 2000, Trends Genet. 16: 276-277 [Non-licensed document 3] Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

Non-licensed Document 7

Non-licensed literature 9

Non-licensed literature 10

Non-licensed Document 11

[0007] The problem to be solved by the present application is to provide a geranylgeranyl pyrophosphate synthase mutant, and a method for producing a tetraterpene, a precursor thereof, or a substance having a tetraterpene as a precursor using the mutant. [Means for solving the problem]

[0008] The present application aims to provide a geranylgeranyl pyrophosphate synthase mutant 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 substituted with another amino acid.

[0009] Another object of the present application is to provide a polynucleotide encoding the mutant. A further object of the present application is to provide a vector comprising the polynucleotide.

[0010] A further object of the present application is to provide a microorganism comprising at least one of the mutant, polynucleotide, and vector. Furthermore, the present application aims to provide a method for producing tetraterpenes, precursors thereof, and substances having tetraterpenes as precursors, which method comprises the step of culturing the microorganism in a medium.

[0011] Furthermore, the present application aims to provide a composition for producing tetraterpenes, precursors thereof, and substances having tetraterpenes as precursors, which comprises the mutant, vector, microorganism, or culture of the microorganism.

[0012] Furthermore, the present application aims to provide use of at least one of the mutants, polynucleotides, vectors, and microorganisms in the production of tetraterpenes, precursors thereof, and substances having tetraterpene precursors. [Effects of the Invention]

[0013] Microorganisms expressing the geranylgeranyl pyrophosphate synthase variant of the present application can more efficiently produce tetraterpenes, tetraterpene precursors, or substances that have tetraterpene precursors, compared to strains that do not express the variant. DETAILED DESCRIPTION OF THE INVENTION

[0014] The present application will be described in detail below. Note that the description and embodiment of one aspect disclosed in this application also apply to the description and embodiment of other aspects with respect to common matters. In addition, all combinations of various elements disclosed in this application are included in this application. Furthermore, the present application is not limited to the following specific description.

[0015] One aspect of the present application provides a geranylgeranyl pyrophosphate synthase mutant 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 substituted with another amino acid.

[0016] The geranylgeranyl pyrophosphate synthase mutant refers to a polypeptide having geranylgeranyl pyrophosphate synthase activity, or a mutant of geranylgeranyl pyrophosphate synthase in which at least one of the amino acids corresponding to the 29th, 43rd, 90th, 103rd, 122nd, and 141st positions from the N-terminus of SEQ ID NO: 1 has been substituted with another amino acid.

[0017] In the present application, "geranylgeranyl pyrophosphate synthase (GGS1)" refers to an enzyme that catalyzes the synthesis of geranylgeranyl pyrophosphate synthase (GGPP) from farnesyl pyrophosphate (FPP).

[0018] The geranylgeranyl pyrophosphate synthase of the present application may be a geranylgeranyl pyrophosphate synthase that has been modified to produce the geranylgeranyl pyrophosphate synthase variant provided in the present application, or a polypeptide having geranylgeranyl pyrophosphate synthase activity. Specifically, it may be a naturally occurring polypeptide or a wild-type polypeptide, a mature polypeptide thereof, or a mutant or functional fragment thereof, as long as it is a parent of the geranylgeranyl pyrophosphate synthase variant of the present application.

[0019] The geranylgeranyl pyrophosphate synthase in the present application is, but is not limited to, the polypeptide of SEQ ID NO: 1. It may also be a polypeptide having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity with the polypeptide of SEQ ID NO: 1, and any polypeptide having the same or equivalent activity as the polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 is included in the geranylgeranyl pyrophosphate synthase.

[0020] The sequence of the geranylgeranyl pyrophosphate synthase of the present application can be obtained from a publicly known database such as NCBI's GenBank. Specifically, the sequence is a polypeptide encoded by the ggs1 gene, but is not limited thereto.

[0021] In this application, a "variant" refers to a polypeptide that differs from the amino acid sequence of the variant by conservative substitution and / or modification of at least one amino acid, but maintains its functions or properties. Such variants can generally be identified by modifying at least one amino acid in the amino acid sequence of the polypeptide and evaluating the properties of the modified polypeptide. That is, the performance of the variant may be improved, unchanged, or decreased compared to the polypeptide of the variant. Some variants also include variants in which at least one portion, such as an N-terminal leader sequence or a transmembrane domain, has been deleted. Other variants include variants in which a portion has been deleted from the N- and / or C-termini of a mature protein. The term "variant" is often used interchangeably with "mutated type," "modification," "mutated polypeptide," "mutated protein," "mutant," "mutein," "divergent," and the like, but any term meaning a mutation may be used.

[0022] The variant may also include deletions or additions of amino acids that have minimal effect on the properties and secondary structure of the polypeptide. For example, the N-terminus of the variant may be linked to a signal (or leader) sequence involved in co- or post-translational protein translocation. The variant may also be linked to other sequences or linkers to allow identification, purification, or synthesis.

[0023] The mutant of the present application may be a geranylgeranyl pyrophosphate synthase mutant 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 has been substituted with another amino acid. Specifically, the mutant may be one in which at least one, at least two, at least three, at least four, at least five, or at least six of the 29th, 43rd, 90th, 103rd, 122nd, and 141st positions have been substituted, but is not limited thereto.

[0024] In the polypeptide having geranylgeranyl pyrophosphate synthase activity or geranylgeranyl pyrophosphate synthase that is the parent of the mutant, the amino acid corresponding to the 29th position from the N-terminus of the amino acid sequence of SEQ ID NO: 1 is asparagine, the amino acid corresponding to the 43rd position is phenylalanine, the amino acid corresponding to the 90th position is leucine, the amino acid corresponding to the 103rd position is methionine, the amino acid corresponding to the 122nd position is isoleucine, and / or the amino acid corresponding to the 141st position is arginine, but is not limited to these.

[0025] In one example, the variant includes at least one of the following substitutions: substitution of the amino acid corresponding to the 29th position from the N-terminus of the amino acid sequence of SEQ ID NO: 1 with an amino acid other than asparagine; substitution of the amino acid corresponding to the 43rd amino acid with an amino acid other than phenylalanine; substitution of the amino acid corresponding to the 90th amino acid with an amino acid other than leucine; substitution of the amino acid corresponding to the 103rd amino acid with an amino acid other than methionine; substitution of the amino acid corresponding to the 122nd amino acid with an amino acid other than isoleucine; and substitution of the amino acid corresponding to the 141st amino acid with an amino acid other than arginine, but is not limited to these.

[0026] The "other amino acid" may be any amino acid different from the amino acid before substitution. It goes without saying that "a specific amino acid has been substituted" in the present application means that the amino acid has been substituted with an amino acid different from the amino acid before substitution, even if it is not specifically stated that the amino acid has been substituted with another amino acid.

[0027] The substitution with another 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 is selected from the group consisting of glycine, alanine, valine, leucine, isoleucine, methionine, and proline, but is not limited to these. The polar amino acid, which is mixed with a hydrophilic amino acid, is selected from the group consisting of serine, threonine, cysteine, tyrosine, asparagine, and glutamine, and the positively charged (basic) amino acid is selected from the group consisting of arginine, lysine, and histidine, but is not limited to these.

[0028] Specifically, the variants of the present application are variants in which at least one of the amino acids corresponding to positions 29, 43, 90, 103, 122, and 141 of the amino acid sequence of SEQ ID NO: 1, the reference protein, is substituted with a nonpolar amino acid, a polar amino acid, or a positively charged (basic) amino acid different from the original amino acid, but are not limited thereto. The nonpolar amino acid is isoleucine (Ile) or valine (Val), the polar amino acid is threonine (Thr), and the positively charged amino acid is arginine (Arg) or lysine (Lys), but are not limited thereto.

[0029] The other amino acid is any one selected from the group consisting of threonine, isoleucine, arginine, valine, and lysine, but is not limited thereto. As an example, the variant of the present application has at least one amino acid corresponding to positions 29, 43, 90, 103, 122 and 141 of the amino acid sequence of SEQ ID NO: 1 substituted with any amino acid selected from the group consisting of threonine, isoleucine, arginine, valine and lysine, but is not limited thereto.

[0030] As an example, the variant of the present application has a substitution selected from the group consisting of, but not limited to, the substitution of the amino acid corresponding to position 29 of the amino acid sequence of SEQ ID NO: 1 with threonine, the substitution of the amino acid corresponding to position 43 with isoleucine, the substitution of the amino acid corresponding to position 90 with arginine, the substitution of the amino acid corresponding to position 103 with isoleucine, the substitution of the amino acid corresponding to position 122 with valine, the substitution of the amino acid corresponding to position 141 with lysine, and combinations thereof.

[0031] "Corresponding to," as used herein, means the amino acid residue at the recited position in the polypeptide, or an amino acid residue that is similar, identical, or equivalent to the recited residue in the polypeptide. Identifying the amino acid at the corresponding position may also be determining the specific amino acid in a sequence that references a particular sequence. "Corresponding region," as used herein, generally refers to a similar or corresponding position in a related or reference protein.

[0032] For example, when any amino acid sequence is aligned with SEQ ID NO: 1, each amino acid residue in the amino acid sequence can be numbered based on the number and position of the amino acid residue corresponding to the amino acid residue in SEQ ID NO: 1. For example, the sequence alignment algorithm in the present application can identify the amino acid positions or positions where modifications such as substitutions, insertions, deletions, etc. occur when compared with a query sequence (also referred to as a "reference sequence").

[0033] For such alignment, for example, the Needleman-Wunsch algorithm (Non-Patent Document 1) or the Needle program in the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Non-Patent Document 2) can be used, but the present invention is not limited to these. Sequence alignment programs, pairwise sequence comparison algorithms, and the like known in the technical field can also be used as appropriate.

[0034] In one example, the variant of the present application may have about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity with the polypeptide of SEQ ID NO: 1, and at least one of the amino acids corresponding to positions 29, 43, 90, 103, 122, and 141 of SEQ ID NO: 1 may be substituted with another amino acid.

[0035] As an example, a variant of the present application may include an amino acid sequence that has 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.

[0036] Specifically, the variant of the present application may be one having the amino acid sequence shown 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, one comprising the amino acid sequence, one consisting of the amino acid sequence, or one essentially consisting of the amino acid sequence.

[0037] As one example, the variant of the present application may include an amino acid sequence in which at least one of the amino acids corresponding to positions 29, 43, 90, 103, 122, and 141 in the amino acid sequence of SEQ ID NO: 1 is substituted with another amino acid, and which has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.9% or more homology or identity to the amino acid sequence represented by 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.

[0038] As an example, the variants of the present application include a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 3 in which the methionine corresponding to the 103rd position in the amino acid sequence of SEQ ID NO: 1 is substituted with isoleucine; a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 5 in which the phenylalanine corresponding to the 43rd position in the amino acid sequence of SEQ ID NO: 1 is substituted with isoleucine; a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 7 in which the asparagine corresponding to the 29th position in the amino acid sequence of SEQ ID NO: 1 is substituted with threonine and the leucine corresponding to the 90th position is substituted with arginine; a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 9 in which the asparagine corresponding to the 29th position in the amino acid sequence of SEQ ID NO: 1 is substituted with threonine; The polypeptide may be at least one selected from the group consisting of, but not limited to, a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 11 in which the leucine at position 90 in the amino acid sequence is substituted with arginine, a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 13 in which the isoleucine at position 122 in the amino acid sequence of SEQ ID NO: 1 is substituted with valine and the arginine at position 141 in the amino acid sequence of SEQ ID NO: 1 is substituted with lysine, a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 15 in which the isoleucine at position 122 in the amino acid sequence of SEQ ID NO: 1 is substituted with valine, and a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 17 in which the arginine at position 141 in the amino acid sequence of SEQ ID NO: 1 is substituted with lysine.

[0039] Furthermore, it goes without saying that variants having an amino acid sequence in which a portion of the sequence has been deleted, modified, substituted, conservatively substituted, or added are also included in the present application, as long as the variant has such homology or identity and exhibits efficacy equivalent to that of the variant of the present application.

[0040] For example, the variants may have additions or deletions of sequences at the N-terminus, C-terminus and / or internally of the amino acid sequence that do not alter the function of the variants of the present application, naturally occurring mutations, silent mutations or conservative substitutions.

[0041] The term "conservative substitution" refers to the substitution of an amino acid with another amino acid having similar structural and / or chemical properties. Such amino acid substitutions may generally be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues. Typically, conservative substitutions have little or no effect on the activity of a protein or polypeptide.

[0042] In this application, "homology" or "identity" refers to the degree of similarity between two given amino acid or nucleotide sequences, expressed as a percentage. Homology and identity are often used interchangeably.

[0043] Sequence homology or identity of conserved polynucleotides or polypeptides can be determined by standard sequence algorithms, optionally with default gap penalties established by the program used. Substantially homologous or identical sequences will generally hybridize to all or part of the sequence under moderately or highly stringent conditions. Hybridization, of course, also includes hybridization to polynucleotides with common codons or codons that take into account codon degeneracy in the polynucleotide.

[0044] Whether any two polynucleotide or polypeptide sequences have homology, similarity, or identity can be determined using known computer algorithms, such as the "FASTA" program, with default parameters, as described in, for example, Non-Patent Document 3. Alternatively, the Needleman-Wunsch algorithm (Non-Patent Document 1) can be used, as implemented in the Needleman program (EMBOSS: The European Molecular Biology Open Software Suite, Non-Patent Document 2) (version 5.0.0 or later) of the EMBOSS package (which includes the GCG program package (Non-Patent Document 4), BLASTP, BLASTN, and FASTA (Non-Patent Documents 5, 6, and 7)). For example, BLAST or ClustalW from the National Center for Biotechnology Information can be used to determine homology, similarity, or identity.

[0045] Homology, similarity, or identity of polynucleotides or polypeptides can be determined by comparing sequence information using a GAP computer program such as that disclosed in Non-Patent Document 1, as disclosed in, for example, Non-Patent Document 8. Briefly, the GAP program defines a gap as the number of similar sequence symbols (i.e., nucleotides or amino acids) divided by the total number of symbols in the shorter of the two sequences. Default parameters for the GAP program include: (1) a binary comparison matrix (identity takes a value of 1, non-identity a value of 0) and a weighted comparison matrix (or EDNAFULL (the EMBOSS version of NCBINUC4.4) substitution matrix) as disclosed in Non-Patent Document 9; (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap (or a gap open penalty of 10, a gap extension penalty of 0.5); and (3) no penalty for terminal gaps.

[0046] 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 activity that improves the ability to produce tetraterpenes, tetraterpene precursors, or substances derived from tetraterpenes as precursors, compared to wild-type or non-mutated geranylgeranyl pyrophosphate synthase. In one embodiment, the variant of the present application may have activity that reduces the production level of by-products in the production pathway of tetraterpenes, tetraterpene precursors, or substances derived from tetraterpene precursors, compared to wild-type or geranylgeranyl pyrophosphate synthase. In one embodiment, the variant of the present application may have activity that reduces the production level of squalene, compared to wild-type or geranylgeranyl pyrophosphate synthase. In one embodiment, the variant of the present application has improved activity compared to wild-type or non-mutated geranylgeranyl pyrophosphate synthase, but is not limited to this.

[0047] Another aspect of the present application provides polynucleotides encoding the variants of the present application. In this application, the term "polynucleotide" refers to a nucleotide polymer in which nucleotide monomers are linked in a long chain by covalent bonds, and refers to a DNA or RNA chain longer than a certain length, and more specifically refers to a polynucleotide fragment encoding the mutant having geranylgeranyl pyrophosphate activity.

[0048] A polynucleotide encoding a variant of the present application may comprise a nucleotide sequence encoding the amino acid sequence represented by 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. As an example, a polynucleotide of the present application may have or comprise the sequence represented by 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. Furthermore, a polynucleotide of the present application may consist of, or essentially consist of, the sequence represented by 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.

[0049] The polynucleotides of the present application may be modified in various ways in the coding region to the extent that the amino acid sequence of the variants of the present application is not changed, taking into account codon degeneracy or codons preferred in the organism in which the variants of the present application are to be expressed. Specifically, the polynucleotide of the present application has a nucleotide sequence that is 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and less than 100% homologous or identical to the sequence of SEQ ID NO: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 contains the nucleotide sequence; or consists of a nucleotide sequence that is 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and less than 100% homologous or identical to the sequence of SEQ ID NO: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 is essentially composed of the nucleotide sequence, but is not limited to these.

[0050] Here, in the sequence having the homology or identity, the codons encoding the amino acids corresponding to the 29th, 43rd, 90th, 103rd, 122nd and 141st positions 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 may be one of the codons encoding threonine, isoleucine, arginine, valine or lysine.

[0051] Furthermore, the polynucleotide of the present application may be any sequence that hybridizes under stringent conditions with a probe prepared from a known gene sequence, for example, a sequence complementary to all or part of the polynucleotide sequence of the present application. The term "stringent conditions" refers to conditions that allow specific hybridization between polynucleotides. Such conditions are specifically described in the literature (see Non-Patent Documents 11 and 12). For example, conditions include those under which polynucleotides with high homology or identity, such as polynucleotides with a homology or identity of 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, hybridize with each other, but polynucleotides with lower homology or identity do not hybridize with each other; or conditions including washing once, specifically two to three times, at a salt concentration and temperature equivalent to those used in conventional Southern hybridization, namely, 60°C, 1×SSC, 0.1% SDS, specifically, 60°C, 0.1×SSC, 0.1% SDS, more specifically, 68°C, 0.1×SSC, 0.1% SDS.

[0052] Hybridization requires that two nucleic acids have complementary sequences, even if mismatches between bases are possible depending on the stringency of the hybridization. "Complementary" is used to describe the relationship between nucleotide bases that can hybridize to each other. For example, in DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Thus, the polynucleotides of the present application may include not only substantially similar nucleic acid sequences, but also isolated nucleic acid fragments that are complementary to the entire sequence.

[0053] Specifically, polynucleotides having homology or identity to the polynucleotides of the present application can be detected using the hybridization conditions described above, in which the hybridization step is performed at a Tm value of 55° C. The Tm value may be, but is not limited to, 60° C., 63° C., or 65° C., and can be appropriately adjusted by those skilled in the art depending on the purpose.

[0054] The appropriate stringency for hybridizing the polynucleotides depends on the length of the polynucleotides and the degree of complementation, variables known in the art (eg, Non-Patent Document 11).

[0055] Yet another aspect of the present application provides a vector comprising the polynucleotide of the present application, which may be, but is not limited to, an expression vector for expressing the polynucleotide in a host cell.

[0056] The vector of the present application includes a DNA product comprising a base sequence of a polynucleotide encoding a target polypeptide operably linked to a suitable expression control region (or expression control sequence) so as to enable the target polypeptide to be expressed in a suitable host. The expression control region includes a promoter that initiates transcription, an optional operator sequence for regulating the transcription, a sequence encoding a suitable mRNA ribosomal binding site, and a sequence that regulates the termination of transcription and translation. When transformed into a suitable host cell, the vector can replicate and function independently of the host genome and is integrated into the genome itself.

[0057] The vector used in the present application is not particularly limited, and any vector known in the art may be used. Examples of commonly used vectors include naturally occurring or recombinant plasmids, cosmids, viruses, and bacteriophages. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A may be used as phage or cosmid vectors. Examples of plasmid vectors that may be used include pDC, pBR, pUC, pBluescriptII, pGEM, pTZ, pCL, and pET. Specifically, pDC, pDCM2 (Patent Document 2), pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC, and pIMR53 vectors may be used.

[0058] For example, a polynucleotide encoding a target polypeptide can be inserted into a chromosome using a vector for intracellular chromosomal introduction. The polynucleotide can be inserted into a chromosome by any method known in the art, including, but not limited to, homologous recombination. A selection marker for determining whether or not the polynucleotide has been inserted into the chromosome may be further included. The selection marker is used to select cells transformed with the vector, i.e., to determine whether or not the target nucleic acid molecule has been inserted. Markers that confer selectable phenotypes, such as drug resistance, auxotrophy, resistance to cytotoxic agents, and expression of surface polypeptides, are used. In an environment treated with a selective agent, only cells expressing the selection marker survive or exhibit a different phenotype, allowing the selection of transformed cells.

[0059] In the present application, "transformation" refers to introducing a vector containing a polynucleotide encoding a target polypeptide into a host cell or microorganism, thereby expressing the polypeptide encoded by the polynucleotide in the host cell. The transformed polynucleotide may be any polynucleotide that can be expressed in the host cell, regardless of whether it is located intrachromosomally or extrachromosomally. Furthermore, the polynucleotide may comprise DNA and / or RNA encoding the target polypeptide. The polynucleotide may be introduced in any form that can be expressed in the host cell. For example, the polynucleotide may be introduced into the host cell in the form of an expression cassette, which is a genetic construct containing all elements necessary for its own expression. Typically, the expression cassette contains a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal operably linked to the polynucleotide. The expression cassette may be in the form of a self-replicating expression vector. Alternatively, the polynucleotide may be introduced into the host cell in its own form and operably linked to sequences necessary for expression in the host cell, but this is not limited thereto.

[0060] Furthermore, the term "operably linked" means that the polynucleotide sequence is functionally linked to a promoter sequence that initiates and mediates transcription of the polynucleotide encoding the target variant of the present application.

[0061] Yet another aspect of the present application provides a microorganism comprising at least one of a variant of the present application, a polynucleotide encoding said variant, and a vector comprising said polynucleotide.

[0062] The term "microorganism" or "strain" as used herein includes all wild-type microorganisms and naturally or artificially genetically modified microorganisms, and refers to microorganisms in which a specific mechanism has been weakened or strengthened by inserting an exogenous gene or by enhancing or inactivating the activity of an endogenous gene, and which have been genetically modified to produce a desired polypeptide, protein, or product.

[0063] The microorganism of the present application is, but is not limited to, a microorganism comprising at least one of the variants of the present application, the polynucleotides of the present application, and vectors comprising the polynucleotides of the present application; a microorganism modified to express the variants of the present application or the polynucleotides of the present application; a microorganism expressing the variants of the present application or the polynucleotides of the present application; or a microorganism (e.g., a recombinant microorganism) having the activity of the variants of the present application. The microorganism may also be a microorganism modified to further comprise polynucleotides encoding lycopene cyclase / phytoene synthase (crtYB) and phytoene desaturase (crtI) proteins and exhibiting the activity of these proteins, or a microorganism with enhanced activity of these proteins. The lycopene cyclase / phytoene synthase or phytoene desaturase may be, but is not limited to, a protein derived from Xanthophyllomyces dendrorhous. As a 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 coding region of the polynucleotide may be modified in various ways, taking into account codon degeneracy or codons preferred in a microorganism in which the variant of the present application is to be expressed, as long as the amino acid sequence is not changed. Specifically, the polynucleotide has a base sequence that is 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and less than 100% homologous or identical to the sequence of SEQ ID NO: 27 or SEQ ID NO: 28, or contains the base sequence; or consists of a base sequence that is 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and less than 100% homologous or identical to the sequence of SEQ ID NO: 27 or SEQ ID NO: 28, or is essentially composed of the base sequence, but is not limited to these.

[0064] The microorganisms of the present application are those that produce tetraterpenes, precursors of tetraterpenes, or substances that have tetraterpene precursors, those that reduce the production of by-products produced in the tetraterpene production pathway, or those that reduce the production of squalene, but are not limited to these.

[0065] In this application, "tetraterpene" refers to a polymer (C40) of eight isoprene units among terpenes, including tetraterpene-based substances. Terpenes are polymers of isoprene units, and are classified according to the number of isoprene units polymerized, such as a terpene formed by polymerizing two isoprene (C5) units called a monoterpene (C10) and a terpene formed by polymerizing three isoprene units called a sesquiterpene (C15).

[0066] Specifically, the tetraterpene is a carotenoid, but is not limited thereto. In the present application, the precursor of tetraterpene is geranylgeranyl pyrophosphate (GGPP), but is not limited thereto.

[0067] In the present application, the substance having a tetraterpene precursor is at least one selected from the group consisting of retinal and retinol, but is not limited thereto.

[0068] In the present application, the term "tetraterpenoid" refers to a modified tetraterpene. The tetraterpenoid may be a tetraterpene to which a functional group has been attached.

[0069] In this application, the term "carotenoid" refers to tetraterpenes or derivatives thereof that impart colors such as yellow to fruits and vegetables, and is used interchangeably with the terms "tetraterpenes" and "tetraterpenoids."

[0070] Specifically, the carotenoid is at least one selected from the group consisting of xanthophyll, carotene, α-carotene, β-carotene, γ-carotene, lutein, lycopene, zeaxanthin, capsanthin, canthaxanthin, and astaxanthin, but is not limited thereto.

[0071] In the present application, the precursor of carotenoids is geranylgeranyl pyrophosphate (GGPP), but is not limited thereto. In the present application, the substance having a carotenoid precursor is at least one selected from the group consisting of retinal and retinol, but is not limited thereto.

[0072] In the present application, "β-carotene" is a carotenoid substance, and refers to a carotene having β rings at both ends of the molecule. In the present application, the precursor of β-carotene is at least one selected from the group consisting of phytoene, phytofluene, lycopene, and gamma-carotene, but is not limited thereto.

[0073] In the present application, the substance having β-carotene as a precursor is at least one selected from the group consisting of retinal, retinol, and vitamin A, but is not limited thereto.

[0074] The tetraterpene, tetraterpene precursor, or substance having a tetraterpene as a precursor may be a carotenoid, a carotenoid precursor, or a substance having a carotenoid as a precursor, or may be β-carotene, a β-carotene precursor, or a substance having a β-carotene as a precursor, but is not limited to these.

[0075] In the present application, at least one of the mutant, polynucleotide, vector, and microorganism may be for producing a tetraterpene, and the microorganism may be one that produces a tetraterpene, one that produces a precursor of a tetraterpene, or one that produces a substance having a tetraterpene as a precursor.

[0076] In the present application, at least one of the mutant, polynucleotide, vector, and microorganism may be for producing a carotenoid, and the microorganism may be one that produces a carotenoid, one that produces a precursor of a carotenoid, or one that produces a substance whose precursor is a carotenoid.

[0077] In the present application, at least one of the mutant, polynucleotide, vector, and microorganism may be for producing β-carotene, and the microorganism may be one that produces β-carotene, one that produces a precursor of β-carotene, or one that produces a substance whose precursor is β-carotene.

[0078] As one example, since geranylgeranyl pyrophosphate (GGPP) is produced by geranylgeranyl pyrophosphate synthase in the isoprenoid biosynthetic pathway, the microorganism may also produce geranylgeranyl pyrophosphate.

[0079] In this application, by-products produced in the tetraterpene production pathway refer to substances other than tetraterpenes, tetraterpene precursors, and substances derived from tetraterpenes, specifically, but not limited to, squalene. β-Carotene is produced in the carotenoid or isoprenoid production pathway, and in this process, squalene synthase (ERG9) consumes two molecules of farnesyl pyrophosphate (FPP, C15), producing squalene (C30) as a by-product.

[0080] In this application, "squalene" refers to an unsaturated hydrocarbon (C 30 H 50 ) and refers to a substance that is also used in the biosynthesis of steroid hormones, vitamin D, etc. The microorganism of the present application reduces by-products produced in the tetraterpene production pathway, specifically, but not limited to, reducing squalene production.

[0081] The microorganisms or strains of the present application are microorganisms that naturally have the ability to produce geranylgeranyl pyrophosphate synthase or tetraterpene, or are microorganisms into which a mutant of the present application or a polynucleotide encoding it (or a vector containing the polynucleotide) has been introduced into a parent strain that does not have the ability to produce geranylgeranyl pyrophosphate synthase or tetraterpene, and / or which have been conferred the ability to produce tetraterpene, a precursor of a tetraterpene, or a substance whose precursor is a tetraterpene, but are not limited to these.

[0082] For example, the microorganism or strain of the present application is a cell or microorganism that has been transformed with a vector comprising the polynucleotide of the present application or a polynucleotide encoding the variant of the present application and that expresses the variant of the present application. For purposes of the present application, the microorganism or strain of the present application may be any microorganism that includes the variant of the present application and produces a tetraterpene, a tetraterpene precursor, or a substance having a tetraterpene precursor as a precursor. For example, the microorganism or strain of the present application may be a recombinant strain that has been improved in its ability to produce a tetraterpene, a tetraterpene precursor, or a substance having a tetraterpene precursor as a precursor by introducing a polynucleotide encoding the variant of the present application into a naturally occurring wild-type microorganism or a microorganism that produces a tetraterpene, thereby expressing a geranylgeranyl pyrophosphate synthase variant. The recombinant strain is a microorganism that has improved ability to produce tetraterpenes, tetraterpene precursors, or substances with tetraterpene precursors, compared to natural wild-type microorganisms or microorganisms without modified geranylgeranyl pyrophosphate synthase (i.e., microorganisms expressing wild-type geranylgeranyl pyrophosphate synthase), but is not limited to these.

[0083] As an example, the recombinant strain has an improved production capacity of tetraterpenes, tetraterpene precursors, or substances with tetraterpene precursors compared to the parent strain or unmodified microorganism before mutation, specifically an improved production capacity of at least about 1%, specifically about 3%, or about 5% or more. However, any strain that shows an increase in the + value compared to the production capacity of the parent strain or unmodified microorganism before mutation may be used.

[0084] In other examples, the recombinant strain may produce less than about 85%, less than about 80%, less than about 75%, less than about 70%, less than about 65%, less than about 60%, less than about 55%, less than about 50%, less than about 30%, or less than about 10% of by-products generated in the tetraterpene production pathway compared to the parent strain before mutation or an unmodified microorganism, or may not produce any by-products, but this is not limited to these examples.

[0085] The term "about" refers to a range that includes ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and may refer to any numerical value that is equal to or in a similar range to the numerical value following the term "about," but is not limited to these.

[0086] In the present application, the term "unmodified microorganism" does not exclude strains containing mutations that may occur naturally in microorganisms, but refers to a wild-type strain or a naturally occurring strain itself, or a strain before its traits are changed due to genetic mutation caused by natural or artificial factors. For example, the unmodified microorganism refers to a strain into which the geranylgeranyl pyrophosphate synthase mutant of the present application has not been introduced or before it has been introduced. The term "unmodified microorganism" is also used interchangeably with "strain before modification," "microorganism before modification," "non-mutated strain," "non-modified strain," "non-mutated microorganism," or "reference microorganism."

[0087] In one example, the microorganism of the present application is a microorganism of the genus Yarrowia (Yarrowia sp.), specifically, but not limited to, Yarrowia lipolytica.

[0088] In the present application, "enhancing" a polypeptide activity means improving the activity of the polypeptide compared to its endogenous activity. The term "enhancing" is used interchangeably with "activation," "up-regulation," "overexpression," and "increase." Here, activation, enhancement, up-regulation, overexpression, and enhancement all encompass the development of an activity not originally present, as well as an improvement in activity compared to endogenous activity or activity prior to modification. The term "endogenous activity" refers to the activity of a specific polypeptide inherently possessed by a parent strain or unmodified microorganism prior to phenotypic change, when the trait is altered through genetic mutation due to natural or artificial factors. This term is also used interchangeably with "activity prior to modification." "Enhancing," "up-regulating," "overexpressing," or "improving" a polypeptide activity compared to its endogenous activity means an improvement in the activity and / or concentration (expression level) of a specific polypeptide inherently possessed by a parent strain or unmodified microorganism prior to phenotypic change.

[0089] The enhancement may be achieved by introducing a foreign polypeptide, or by enhancing the activity and / or increasing the concentration (expression level) of an endogenous polypeptide. Whether the activity of the polypeptide has been enhanced can be confirmed by an increase in the level of activity of the polypeptide, the expression level, or the amount of a product produced from the polypeptide.

[0090] Various methods well known in the art can be applied to enhance the activity of the polypeptide, and any method can be used as long as it can enhance the activity of the target polypeptide compared to the unmodified microorganism. Specifically, these methods include, but are not limited to, conventional methods in molecular biology that use genetic engineering and / or protein engineering well known to those skilled in the art (e.g., Non-Patent Documents 13 and 14).

[0091] Specifically, the enhancement of a polypeptide of the present application can be achieved by: 1) increasing the intracellular copy number of a polynucleotide encoding the polypeptide; 2) replacing the expression regulatory region of a gene on a chromosome encoding the polypeptide with a sequence with stronger activity; 3) modifying the nucleotide sequence encoding the start codon or 5'UTR region of a gene transcript encoding the polypeptide; 4) modifying the amino acid sequence of the polypeptide so that the activity of the polypeptide is enhanced; 5) modifying the polynucleotide sequence encoding the polypeptide so that the activity of the polypeptide is enhanced (for example, modifying the polynucleotide sequence of the polypeptide gene so that the polypeptide is encoded as a polypeptide modified so that the activity of the polypeptide is enhanced); 6) introducing a foreign polypeptide that exhibits the activity of the polypeptide or a foreign polynucleotide encoding it; 7) optimizing the codons of the polynucleotide encoding the polypeptide; 8) analyzing the tertiary structure of the polypeptide and selectively modifying or chemically modifying exposed portions; or 9) a combination of two or more selected from 1) to 8) above, but is not limited to these.

[0092] More specifically, 1) increasing the intracellular copy number of a polynucleotide encoding a polypeptide is achieved by introducing into a host cell a vector to which a polynucleotide encoding the polypeptide is operably linked, the vector replicating and functioning independently of the host. Alternatively, the polynucleotide encoding the polypeptide may be introduced into a chromosome in the host cell at one or more copies. The introduction into a chromosome is achieved by, but is not limited to, introducing into the host cell a vector capable of inserting the polynucleotide into a chromosome in the host cell. The vector is as described above.

[0093] 2) Replacing the expression regulatory region (or expression regulatory sequence) of a gene on a chromosome encoding a polypeptide with a sequence with stronger activity can be achieved, for example, by generating a mutation in the sequence through deletion, insertion, non-conservative or conservative substitution, or a combination thereof, or by replacing the expression regulatory region with a sequence with higher activity, so as to further enhance the activity of the expression regulatory region. Examples of expression regulatory regions include, but are not limited to, promoters, operator sequences, sequences encoding ribosome binding sites, and sequences regulating the termination of transcription and translation. For example, this can be achieved by replacing the original promoter with a strong promoter, but is not limited to this.

[0094] Examples of known strong promoters include, but are not limited to, the cj1 to cj7 promoters (Patent Document 3), the lac promoter, the trp promoter, the trc promoter, the tac promoter, the lambda phage PR promoter, the PL promoter, the tet promoter, the gapA promoter, the SPL7 promoter, the SPL13 (sm3) promoter (Patent Document 4), the O2 promoter (Patent Document 5), the tkt promoter, and the yccA promoter.

[0095] The nucleotide sequence encoding the start codon or 5'UTR region of the gene transcription product encoding the polypeptide (3) can be modified, for example, by substituting it with a nucleotide sequence encoding another start codon that has a higher polypeptide expression rate than the endogenous start codon, but is not limited to this.

[0096] The modification of the amino acid sequence or polynucleotide sequence in 4) and 5) can be achieved by, but is not limited to, generating a sequence mutation in the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, to enhance the activity of the polypeptide, or by substituting an amino acid sequence or polynucleotide sequence improved to have higher activity or an amino acid sequence or polynucleotide sequence improved to improve activity. Specifically, the substitution can be achieved by, but is not limited to, inserting the polynucleotide into a chromosome by homologous recombination. The vector used here may further include a selection marker for confirming whether or not it has been inserted into the chromosome. The selection marker is as described above.

[0097] 6) Introduction of a foreign polynucleotide that exhibits the activity of a polypeptide is carried out by introducing into a host cell a foreign polynucleotide that encodes a polypeptide that exhibits the same or similar activity as the polypeptide. The foreign polynucleotide may be of any origin or sequence, as long as it exhibits the same or similar activity as the polypeptide. The introduction can be carried out by those skilled in the art using a known transformation method appropriately selected, and the introduced polynucleotide is expressed in the host cell as described above, resulting in the production of the polypeptide and its activity being improved.

[0098] 7) Optimizing the codons of a polynucleotide encoding a polypeptide can be achieved by optimizing the codons of an endogenous polynucleotide so that transcription or translation is increased in a host cell, or by optimizing the codons of an exogenous polynucleotide so that optimized transcription or translation is achieved in a host cell.

[0099] 8) Analyzing the tertiary structure of a polypeptide and selecting and altering or chemically modifying exposed portions can be carried out, for example, by comparing the sequence information of the polypeptide to be analyzed with a database storing sequence information of known proteins, determining candidate template proteins based on the degree of sequence similarity, confirming the structure based on the candidate template proteins, and selecting and altering or modifying exposed portions to be altered or chemically modified.

[0100] Such enhancement of polypeptide activity can be achieved by, but is not limited to, improving the activity, concentration, or expression level of the corresponding polypeptide compared to the activity or concentration of the polypeptide expressed in a wild-type or unmodified microbial strain, or by increasing the amount of product produced from the polypeptide.

[0101] In the microorganisms of the present application, partial or complete modification of a polynucleotide can be induced by, but is not limited to, (a) homologous recombination using a vector for chromosomal introduction into the microorganism, or genome editing using engineered nucleases (e.g., CRISPR-Cas9), and / or (b) light and / or chemical treatment, such as ultraviolet light or radiation. Methods for partially or completely modifying the gene include methods using DNA recombination techniques. For example, partial or complete deletion of a gene can be achieved by introducing a nucleotide sequence or vector containing a nucleotide sequence homologous to the target gene into the microorganism and causing homologous recombination. The introduced nucleotide sequence or vector may include, but is not limited to, a dominant selection marker.

[0102] Yet another aspect of the present application provides a method for producing a tetraterpene, a tetraterpene precursor, or a substance having a tetraterpene precursor, the method comprising culturing in a medium a microorganism containing at least one of a variant of the present application, a polynucleotide encoding the variant, and a vector containing the polynucleotide. The method for producing a tetraterpene, a tetraterpene precursor, or a substance having a tetraterpene precursor is, but is not limited to, a method for reducing squalene production.

[0103] The term "culturing" as used herein means growing the microorganism of the present application under appropriately controlled environmental conditions. The culturing process of the present application can be carried out using a suitable medium and culture conditions known in the art. Those skilled in the art can easily adjust such a culturing process depending on the selected strain. Specifically, the culturing may be batch, continuous, and / or fed-batch culture, but is not limited thereto.

[0104] The term "culture medium" as used herein refers to a mixture of nutrients necessary for culturing the microorganism of the present application as its main components, and provides nutrients such as water essential for survival and growth, growth factors, etc. Specifically, the medium and other culture conditions used to culture the microorganism of the present application may be any medium used to culture ordinary microorganisms, and the microorganism of the present application can be cultured in an ordinary medium containing a suitable carbon source, nitrogen source, phosphorus source, inorganic compounds, amino acids, and / or vitamins under aerobic conditions by adjusting the temperature, pH, etc.

[0105] Examples of carbon sources used in the present application include carbohydrates such as glucose, saccharose, lactose, fructose, sucrose, and maltose; sugar alcohols such as mannitol and sorbitol; organic acids such as pyruvic acid, lactic acid, and citric acid; and amino acids such as glutamic acid, methionine, and lysine. Natural organic nutrient sources such as starch hydrolysates, molasses, blackstrap molasses, rice bran, cassava, bagasse, and corn steeping liquid can also be used. Specifically, carbohydrates such as glucose and sterilized pretreated molasses (i.e., molasses converted into reducing sugars) can be used. Any other carbon source can also be used in an appropriate amount. These carbon sources can be used alone or in combination of two or more, but are not limited to these.

[0106] Examples of the nitrogen source that can be used include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate, and organic nitrogen sources such as amino acids such as glutamic acid, methionine, and glutamine, peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steeping liquid, casein hydrolysate, fish or its degradation products, and defatted soybean cake or its degradation products. These nitrogen sources can be used alone or in combination of two or more, but are not limited to these.

[0107] Examples of the phosphorus source include potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and their corresponding sodium-containing salts. Examples of inorganic compounds that can be used include sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, and calcium carbonate. Other examples include amino acids, vitamins, and / or suitable precursors. These components or precursors can be added to the medium in a batch or continuous manner. However, the present invention is not limited to these.

[0108] Furthermore, during the cultivation of the microorganism of the present application, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid may be added to the medium in a suitable manner to adjust the pH of the medium. Furthermore, during cultivation, foam formation may be suppressed using an antifoaming agent such as a fatty acid polyglycol ester. Furthermore, to maintain an aerobic state in the medium, oxygen or an oxygen-containing gas may be injected into the medium, and to maintain anaerobic and microaerobic states, no gas may be injected, and nitrogen, hydrogen, or carbon dioxide gas may be injected, but this is not limited to these.

[0109] In the culture of the present application, the culture temperature is maintained at 20 to 45°C, specifically 25 to 40°C, 20 to 35°C, or 25 to 35°C, and the culture is carried out for about 10 to 160 hours, but is not limited thereto.

[0110] The tetraterpene, tetraterpene precursor, or substance derived from a tetraterpene precursor produced by the culture of the present application is secreted into the medium or remains intracellularly. The method for producing a tetraterpene, a precursor of a tetraterpene, or a substance having a tetraterpene as a precursor of the present application may further 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.

[0111] The method for producing a tetraterpene, a tetraterpene precursor, or a substance having a tetraterpene precursor of the present application may further include a step of recovering the tetraterpene, the tetraterpene precursor, or the substance having a tetraterpene precursor from the medium used for the culture (the medium in which the culture was performed) or the microorganism of the present application. The recovery step may be further included after the culture step.

[0112] The recovery may involve collecting the target tetraterpene, tetraterpene precursor, or substance having a tetraterpene precursor using a suitable method known in the art depending on the culture method of the microorganism of the present application, such as batch, continuous, or fed-batch culture. For example, centrifugation, filtration, crystallization, treatment with a protein precipitant (salting out), extraction, cell disruption, ultrasonic disruption, ultrafiltration, dialysis, various types of chromatography such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, and affinity chromatography, HPLC, or a combination thereof may be used to recover the target tetraterpene, tetraterpene precursor, or substance having a tetraterpene precursor from the medium or the microorganism using a suitable method known in the art.

[0113] Furthermore, the method for producing a tetraterpene, a tetraterpene precursor, or a substance having a tetraterpene precursor according to the present application may further include a purification step. The purification can be performed by any suitable method known in the art. For example, when the method for producing a tetraterpene, a tetraterpene precursor, or a substance having a tetraterpene precursor according to the present application includes both a recovery step and a purification step, the recovery step and the purification step may be performed at different times (or consecutively) regardless of the order, or may be performed simultaneously or integrated into a single step, but are not limited thereto.

[0114] The method for producing a substance having a tetraterpene as a precursor of the present application may further include a step of converting the tetraterpene into a substance having the tetraterpene as a precursor. The method for producing a substance having a tetraterpene as a precursor of the present application may further include the converting step after the culturing step or the recovering step. The converting step can be carried out by a suitable method known in the art. For example, the conversion can be carried out using β-carotene 15,15'-oxygenase or retinol dehydrogenase, but is not limited to these.

[0115] The mutants, polynucleotides, vectors, microorganisms, tetraterpenes, precursors of tetraterpenes, substances having tetraterpenes as precursors, etc. are as described above. Yet another aspect of the present application provides a composition for producing tetraterpenes, precursors of tetraterpenes, and substances that have tetraterpenes as precursors, comprising any one selected from the group consisting of 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 culture medium in which the same has been cultured, and a combination of at least two of these.

[0116] The compositions of the present application may further comprise any suitable excipient typically used in compositions for producing tetraterpenes, tetraterpene precursors, and tetraterpene precursor substances, including, but not limited to, preservatives, wetting agents, dispersing agents, suspending agents, buffers, stabilizers, isotonicity agents, etc.

[0117] The mutants, polynucleotides, vectors, microorganisms, media, tetraterpenes, precursors of tetraterpenes, substances having tetraterpenes as precursors, etc. are as described above.

[0118] Yet another aspect of the present application provides the use of at least one of the variants, polynucleotides, vectors and microorganisms of the present application in the production of tetraterpenes, precursors of tetraterpenes, and substances that have tetraterpene precursors.

[0119] The mutants, polynucleotides, vectors, microorganisms, tetraterpenes, precursors of tetraterpenes, substances having tetraterpenes as precursors, etc. are as described above. [Example]

[0120] The present application will be described in more detail below with reference to examples. However, these examples and experimental examples are merely illustrative of the present application, and the present application is not limited to these examples and experimental examples.

[0121] [Example 1] Construction of a GGS1 mutant vector library To amplify the GGS1 gene encoding geranylgeranyl pyrophosphate synthase on the Yarrowia lipolytica 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 nucleotide sequence registered in KEGG (Kyoto Encyclopedia of Genes and Genomes). The diversifyPCR random mutagenesis kit (Takara) was used to introduce random mutations by error-prone PCR (ep-PCR). The PCR conditions and method were optimized and performed using buffer condition 1 and buffer condition 2, respectively, as specified in the user manual. Specific error-prone PCR conditions are shown in Table 1.

[0122] [Table 1]

[0123] PCR was performed using the genomic DNA of Yarrowia lipolytica PO1f as a template and primers SEQ ID NO: 19 and SEQ ID NO: 20. PCR conditions consisted of denaturation at 95°C for 1 minute, annealing at 55°C for 1 minute, and polymerization at 68°C for 1 minute, followed by 32 cycles. The GGS1 PCR products amplified by the two PCR conditions were mixed to create a GGS1 mutant expression vector library based on the pIMR53-EXP1p-CYC1t vector.

[0124] 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 primers SEQ ID NO:21 and SEQ ID NO:22. To amplify the CYC1t terminator, PCR was performed using Saccharomyces cerevisiae genomic DNA as a template and primers SEQ ID NO:23 and SEQ ID NO:24. The PCR conditions were denaturation at 95°C for 1 minute, annealing at 55°C for 1 minute, and polymerization at 68°C for 1 minute, followed by 32 cycles. Furthermore, PCR was performed using the pIMR53 vector as a template and primers 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, followed by 32 cycles.

[0125] As a result, a 6,774-bp linearized pIMR53, a 1,050-bp EXP1p, and a 289-bp CYC1t fragment were obtained. The PCR-amplified EXP1p and CYC1t DNA fragments were ligated into the pIMR53 vector using an Infusion Cloning Kit (Invitrogen), transformed into E. coli DH5α, and plated onto LB solid medium containing 30 mg / L ampicillin. Colonies transformed with the plasmid containing the desired EXP1p promoter and CYC1t terminator were selected by PCR, and the plasmid was then isolated using a well-known plasmid extraction method. The resulting plasmid was designated pIMR53-EXP1p-CYC1t.

[0126] [Example 2] Construction of Yarrowia japonica platform strains for β-carotene production Example 2-1. Construction of a strain with crtYB-crtI insertion derived from X. dendrorhous To construct a platform strain for β-carotene production, the lycopene cyclase / phytoene synthase (crtYB) and phytoene desaturase (crtI) genes derived from Xanthophyllomyces dendrorhous were inserted into the genome of the high-fat yeast strain Yarrowia ilipolytica CC08-0125 (accession number KCCM12972P). The polynucleotide sequence of crtYB (SEQ ID NO: 27) was obtained based on the nucleotide sequence registered with the National Center for Biotechnology Information Search database (NCBI) (GenBank: AY177204.1), and the polynucleotide sequence of crtI (SEQ ID NO: 28) was obtained based on the nucleotide sequence registered with NCBI (GenBank: AY177424.1). The polynucleotide sequences of crtYB and crtI were synthesized by Macrogen in the form of TEFINtp-crtYB-CYC1t (SEQ ID NO: 29) and TEFINtp-crtI-CYC1t (SEQ ID NO: 30). A cassette was designed to be inserted into the MHY1 (YALI0B21582g) gene locus using the Y. lipolytica URA3 gene (SEQ ID NO: 31) as a selection marker. PCR was performed using the synthesized crtYB and crtI genes and KCCM12972P genomic DNA as templates, with primers 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 35 cycles of denaturation at 95°C for 1 minute, annealing at 55°C for 1 minute, and polymerization at 72°C for 3 minutes and 30 seconds. The resulting five DNA fragments were combined into one cassette by overlap extension PCR.

[0127] The cassette thus prepared was introduced into the KCCM12972P strain by the heat shock method (Non-Patent Document 15), and colonies were then grown on uracil-free solid medium (YLMM1). Colonies in which the cassette was confirmed to have been inserted into the genome were spotted onto 5-FOA solid medium using primers set forth in SEQ ID NOs: 44 and 45, and cultured at 30°C for 3 days. The URA3 marker was recovered by obtaining colonies grown on 5-FOA solid medium.

[0128] Example 2-2. Construction of HMGR-enhanced strain A cassette was designed to replace the native promoter (SEQ ID NO: 46) of the hydroxymethylglutaryl-CoA reductase (HMGR) gene of the strain containing the crtYB and crtI genes prepared in Example 2-1 with the TEFINt promoter. PCR was performed using the genomic DNA of KCCM12972P as a template and primers SEQ ID NOs: 47 and 48, 49 and 50, 51 and 52, 53 and 54, and 55 and 56. 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. The resulting five DNA fragments were then combined into a single cassette by overlap extension PCR.

[0129] The cassette thus constructed was introduced into the strain constructed in Example 2-1 by the heat shock method, and colonies were then grown on uracil-free solid medium (YLMM1). Colonies in which the cassette was confirmed to have been inserted were spotted onto 5-FOA solid medium using primers set forth in SEQ ID NOs: 57 and 58 and cultured at 30°C for 3 days. The URA3 marker was recovered by obtaining colonies grown on 5-FOA solid medium. The final β-carotene-producing platform strain constructed was designated CC08-1023.

[0130] <Yarrowia lipolytica minimal media1(YLMM1)> Glucose 20g / L, Yeast nitrogen base without amino acids 6.7g / L, Yeast Synthetic Drop-out Medium Supplements without uracil 2g / L, Agar 15g / L 5-Fluoroorotic Acid (5-FOA) Glucose 20g / L, Yeast nitrogen base without amino acids 6.7g / L, Yeast Synthetic Drop-out Medium Supplements without uracil 2g / L, uracil 50μg / mL, 5-Fluoroorotic acid (5-FOA) 1g / L, agar 15g / L [Example 3] Primary selection of GGS1 activity-enhanced strains For primary selection of strains with enhanced GGS1 activity, color-based primary selection was performed.

[0131] First, the Yarrowia ipolytica CC08-1023 strain, which produces a small amount of β-carotene, was transformed by heat shock 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. Then, colonies formed on uracil-free solid medium were isolated.

[0132] 350 μl of uracil-free YLMM2 (Yarrowia lipolytica minimal media 2) liquid medium was dispensed into 25 96-deep-well plates, and the resulting colonies were inoculated and cultured at 30°C for 48 hours. The colonies were then inoculated onto uracil-free YLMM1 solid medium and cultured at 30°C for 3 days. After the culture was completed, 36 strains were initially selected from a total of 2,400 strains, showing a darker orange color than the strain transformed with the positive control vector (CC08-1023 / pIMR53-EXP1p-GGS1-CYC1t). The production method and composition of the aforementioned YLMM2 are as follows:

[0133] <YLMM2(pH7.2)> Glucose 40g / L, yeast nitrogen base without amino acids and ammonium sulfate 1.7g / L, uracil 0.5g / L, ammonium sulfate 2.5g / L dissolved in 0.1M sodium phosphate buffer (pH 7.2) [Example 4] Secondary selection of GGS1 activity-enhanced strains For the secondary selection of strains with enhanced GGS1 activity, flask evaluation was carried out on the 36 strains primarily selected in Example 3.

[0134] The 36 strains and the strain transformed with a PC (positive control) vector were inoculated into a 250 ml corner-baffled flask containing 50 ml of uracil-free YLMM2 (Yarrowia lipolytica minimal media 2) to an initial OD of 1, and cultured at 30°C and 250 rpm for 48 hours with shaking.

[0135] After the cultivation was completed, 1 ml of the culture medium was centrifuged and the supernatant was removed. Next, 0.5 ml of DMSO (dimethyl sulfoxide, Sigma) was added, and the cells were disrupted by shaking at 55°C and 2000 rpm for 10 minutes. Furthermore, 0.5 ml of acetone (Sigma) was added, and the cells were shaken at 45°C and 2000 rpm for 15 minutes to extract β-carotene and squalene. The extracted substances were analyzed for their concentrations using HPLC equipment. The measurement results of OD, β-carotene, and squalene concentrations and contents in the flask evaluation of the selected strains are shown in Table 2.

[0136] [Table 2-1]

[0137] [Table 2-2]

[0138] The concentrations of β-carotene and squalene extracted from the evaluation samples divided by DCW (g / L) were then used as the secondary selection criteria. For the 33 evaluated strains, excluding strains No. 2, No. 4, No. 9, and No. 28, the β-carotene concentration divided by DCW (g / L) (β-carotene (%)) was confirmed to be increased compared to PC (positive control). For all evaluated strains, the squalene concentration divided by DCW (g / L) was confirmed to be decreased compared to PC (positive control).

[0139] Therefore, a standard value was set based on the value obtained from the PC (positive control), and strains that met both conditions were selected, resulting in a total of four candidate strains that simultaneously satisfied both conditions 1 and 2. The standard value for the secondary selection and the four candidates for secondary selection are shown in Table 3.

[0140] [Table 3]

[0141] Plasmids transformed from the four selected strains were extracted and sequenced to confirm random mutations located in the GGS1 ORF. GGS1 plasmids transformed from the selected strains were extracted using Zymoprepyeast Plasmid Miniprep Kit 2 (Zymo Research). To increase the reliability of the sequence analysis, bidirectional sequence analysis was performed using primers with SEQ ID NOs: 59 and 60. The amino acid mutations identified by sequence analysis of the GGS1 ORF are shown in Table 4. As shown in Table 4, the four candidate groups were confirmed to contain one or two mutations.

[0142] [Table 4]

[0143] [Example 5] Activity verification of selected GGS1 mutants The activity of the GGS1 mutants selected in Examples 3 and 4 was verified.

[0144] To this end, flask evaluation was performed on a total of 10 strains prepared by transformation with NC (negative control) and PC (positive control) vectors, and expression vectors for the 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).

[0145] Expression vectors for the four single mutants (N29T, L90R, I122V, and R141K) were constructed as follows. To obtain DNA fragments for the four single mutants (N29T, L90R, I122V, and R141K), PCR was performed using the pIMR53-EXP1p-GGS1-CYC1t vector containing the wild-type GGS1 sequence as a template and primers set forth in SEQ ID NOs: 61 and 62, 63 and 64, 65 and 66, and 67 and 68. PCR conditions included 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 for the double mutants N29T and L90R, or I122V and R141K, were prepared by similar PCR using the N29T expression vector or I122V expression vector prepared as described above as a template and primers of sequence numbers 63 and 64, or primers of sequence numbers 67 and 68.

[0146] The resulting PCR-amplified DNA containing the mutants was transformed into Escherichia coli DH5α using an Infusion Cloning Kit (Invitrogen) and plated onto LB solid medium containing 30 mg / L ampicillin. Colonies transformed with plasmids containing the four GGS1 mutant vectors were selected, and then plasmids were isolated using a well-known plasmid extraction method. The mutant sequences were confirmed using SEQ ID NO: 59 and SEQ ID NO: 60.

[0147] Specifically, the 10 strains were inoculated into a 250 ml corner baffle flask containing 50 ml of uracil-free YLMM2 (Yarrowia lipolytica minimal media 2) to an initial OD of 1, and cultured with shaking at 30°C and 250 rpm for 48 hours.

[0148] After the incubation, 1 ml of the culture medium was centrifuged and the supernatant was removed. Then, 0.5 ml of DMSO (dimethyl sulfoxide, Sigma) was added, and the cells were disrupted by shaking at 55°C and 2000 rpm for 10 minutes. Furthermore, 0.5 ml of acetone (Sigma) was added, and the cells were shaken at 45°C and 2000 rpm for 15 minutes to extract β-carotene and squalene, whose concentrations were analyzed using HPLC equipment. The OD, β-carotene, and squalene concentrations and content measurements are shown in Table 5.

[0149] [Table 5]

[0150] As shown in Table 5, all of the selected GGS1 mutants containing the M103I, F43I, N29T, L90R, I122V, and / or R141K mutations increased β-carotene levels by 143 to 230% and decreased squalene levels by 11 to 51% compared to PC (positive control). Among the six strains containing a single mutation, the F43I mutant showed the greatest effect. The I122V and R141K mutants, when used simultaneously, further increased β-carotene levels and reduced squalene production compared to the two mutants used alone. Furthermore, the N29T and L90R mutants further reduced squalene production compared to the two mutants used alone.

[0151] [Example 6] Construction and evaluation of GGS1 mutant-transduced strains An attempt was made to verify the effect of actually introducing the GGS1F43I mutant, which was determined to be the most excellent in the above-mentioned Examples, into a bacterial strain.

[0152] To this end, we sequentially constructed 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 replaced with isoleucine.

[0153] Example 6-1. Construction of GGS1 promoter-substituted strain ("CC08-1214") To construct a cassette for replacing the GGS1 promoter on the Yarrowia lipolytica chromosome with the TEF1 promoter, we obtained the polynucleotide sequence of SEQ ID NO: 69, which includes the promoter and splicing point of TEF1 (YALI0C09141p) based on the nucleotide sequence registered in KEGG (Kyoto Encyclopedia of Genes and Genomes). To replace the GGS1 promoter, we obtained the polynucleotide sequence of GGS1 (YALI0D17050g) shown in SEQ ID NO: 2. PCR was performed using Yarrowia lipolytica PO1f genomic DNA as a template and primers SEQ ID NOs: 70 and 71, 72 and 73, 74 and 75, and 76 and 77. PCR was also performed using the URA3 auxotrophic marker as a template and primers SEQ ID NOs: 78 and 79. 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.

[0154] The resulting fragments were a 1500-bp 5'UTR arm, a 531-bp 5'UTR_RP, a 1500-bp GGS1 arm, and a 1533-bp URA3 arm. The PCR-amplified DNA fragments were then combined into a single GGS1 promoter-replaced cassette by overlap extension PCR. The cassette design was 5'UTR arm-TEFINt-URA3-TEFINt-GGS1 arm.

[0155] The GGS1 promoter-substituted cassette was transformed into CC08-1023 by heat shock and selected on uracil-free YLMM1 solid medium. A secondary cross was performed with the selected primary strain to generate a strain in which the GGS1 promoter was replaced with the TEFIN promoter. This strain was designated "CC08-1214."

[0156] Example 6-2. Construction of GGS1(F43I) mutant strain ("CC08-1215") To introduce the GGS1 protein F43I mutant expression sequence into the Yarrowia lipolytica chromosome, a cassette was prepared in which thymine at position 127 of the polynucleotide sequence of SEQ ID NO: 2 was replaced with adenine.

[0157] Specifically, PCR was performed using the genomic DNA of Yarrowia PO1f as a template and primers set forth in SEQ ID NOs: 80 and 81, 82 and 83, 84 and 85, 86 and 83, 84 and 87, and 90 and 91. PCR was also performed using the URA3 auxotrophic marker as a template and primers set forth in SEQ ID NOs: 88 and 89. PCR conditions included 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.

[0158] The resulting fragments were a 1500-bp 5'UTR arm, a 661-bp TEFINt-GGS1_1 fragment, a 1390-bp GGS1_2 fragment, a 661-bp RP_1 fragment, a 1390-bp RP_2 fragment, a 1500-bp 3'UTR arm, and a 1533-bp URA3 fragment. The PCR-amplified DNA fragments were then combined into a single GGS1 promoter replacement cassette by overlap extension PCR. The cassette design was 5'UTR arm-TEFINt-GGS1_1-GGS1_2-RP_1-RP_2-3'UTR arm.

[0159] The GGS1(F43I) mutation cassette was transformed into CC08-1214 by heat shock and selected on uracil-free YLMM1 solid medium. A secondary cross was performed with the selected primary strain to generate 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."

[0160] Example 6-3. Evaluation of GGS1 mutant-introduced strains CC08-1023 was used as a negative control and CC08-1214 as a positive control, and flask evaluation was performed together with CC08-1215.

[0161] Specifically, CC08-1215, CC08-1023 (negative control), and CC08-1214 (positive control) were inoculated into a 250 ml corner-baffled flask containing 50 ml of uracil-containing YLMM2 (Yarrowia lipolytica minimal media 2) to an initial OD of 1, and cultured with shaking at 30°C and 250 rpm for 48 hours.

[0162] After the incubation, 1 ml of the culture medium was centrifuged and the supernatant was removed. Then, 0.5 ml of DMSO (dimethyl sulfoxide, Sigma) was added, and the cells were disrupted by shaking at 55°C and 2000 rpm for 10 minutes. Furthermore, 0.5 ml of acetone (Sigma) was added, and the cells were shaken at 45°C and 2000 rpm for 15 minutes to extract β-carotene and squalene, whose concentrations were analyzed using HPLC equipment. The OD, β-carotene, and squalene concentrations and content measurements are shown in Table 6.

[0163] [Table 6]

[0164] As shown in Table 6, in the CC08-1215 strain to which the selected F43I mutation was applied, the β-carotene concentration increased by 51.3% and the squalene concentration decreased by 88.5% compared to CC08-1214 (PC). As a result, it was confirmed that when applied to actual strains, the introduction of the F43I single mutation increased the β-carotene concentration and reduced the squalene production.

[0165] These results confirmed that introduction of the GGS1 mutant of the present invention increases β-carotene production. Furthermore, it was confirmed that introduction of the GGS1 mutant and reduction of squalene synthesis, which competes with β-carotene production, further improves β-carotene production efficiency.

[0166] Furthermore, these results confirmed that, as with β-carotene production, the production efficiency of tetraterpenes involving GGS1 was further improved. From the above description, those skilled in the art to which the present application pertains will understand that the present application can be implemented in other specific forms without changing the technical idea or essential features thereof. It should be understood that the above examples are merely illustrative and not limiting. The present application should be construed as including all modifications and variations derived from the meaning and scope of the claims, rather than the specification, and their equivalent concepts.

[0167] [Table 7]

Claims

1. A geranylgeranyl pyrophosphate synthase mutant having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1 and containing one or more of the amino acid substitutions in which 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 are substituted with threonine (Thr), isoleucine (Ile), arginine (Arg), isoleucine (Ile), valine (Val) and lysine (Lys), respectively, wherein the mutant has geranylgeranyl pyrophosphate synthase activity that improves the ability to produce tetraterpene, a precursor of tetraterpene, or a substance having a tetraterpene precursor, or reduces the squalene production level, compared to a wild-type or non-mutated geranylgeranyl pyrophosphate synthase of SEQ ID NO:

1.

2. The mutant is (i) substitution of the amino acid corresponding to the 29th position from the N-terminus of the amino acid sequence of SEQ ID NO: 1 with threonine (Thr); (ii) substitution of the amino acid corresponding to the 43rd amino acid from the N-terminus of the amino acid sequence of SEQ ID NO: 1 with isoleucine (Ile); (iii) substitution of the amino acid corresponding to the 90th amino acid from the N-terminus of the amino acid sequence of SEQ ID NO: 1 with arginine (Arg); (iv) substitution of the amino acid corresponding to the 103rd amino acid from the N-terminus of the amino acid sequence of SEQ ID NO: 1 with isoleucine (Ile); (v) substitution of the amino acid corresponding to the 122nd amino acid from the N-terminus of the amino acid sequence of SEQ ID NO: 1 with valine (Val); (vi) substitution of the amino acid corresponding to the 141st amino acid from the N-terminus of the amino acid sequence of SEQ ID NO: 1 with lysine (Lys); (vii) substitution of the amino acids corresponding to the 29th and 90th positions from the N-terminus of the amino acid sequence of SEQ ID NO: 1 with threonine (Thr) and arginine (Arg), respectively; and (viii) Substitution of amino acids corresponding to the 122nd and 141st amino acids from the N-terminus of the amino acid sequence of SEQ ID NO: 1 with valine (Val) and lysine (Lys), respectively. The geranylgeranyl pyrophosphate synthase mutant of claim 1, which comprises at least one amino acid substitution selected from the group consisting of:

3. A polynucleotide encoding the geranylgeranyl pyrophosphate synthase mutant of claim 1 or 2.

4. A microorganism comprising at least one of the geranylgeranyl pyrophosphate synthase mutants according to claim 1 or 2 and polynucleotides encoding said mutants.

5. The microorganism according to claim 4, which produces a tetraterpene, a precursor of a tetraterpene, or a substance having a precursor of a tetraterpene.

6. The microorganism of claim 4 , wherein the microorganism is a Yarrowia sp.

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

8. The microorganism according to claim 4, which produces β-carotene, a precursor of β-carotene, or a substance having β-carotene as a precursor.

9. A method for producing a tetraterpene, a precursor of a tetraterpene, or a substance having a tetraterpene as a precursor, comprising the step of culturing the microorganism according to claim 4 in a medium.

10. The method of claim 9, further comprising recovering the tetraterpene, a precursor of the tetraterpene, or a substance having a precursor of the tetraterpene from the culture medium or the microorganism.

11. The method of claim 9, wherein the microorganism is a Yarrowia sp.

12. 10. The method of claim 9, wherein the method reduces squalene production.

13. A composition for producing a tetraterpene, a precursor of a tetraterpene, or a substance having a tetraterpene precursor, comprising the mutant of claim 1, the microorganism of claim 4, or a culture of said microorganism.

14. Use of a microorganism comprising the geranylgeranyl pyrophosphate synthase variant described in claim 1 and at least one polynucleotide encoding said variant in the production of a tetraterpene, a precursor of a tetraterpene, or a substance having a tetraterpene as a precursor.

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