WO2022260455A1 - 제라닐제라닐 피로포스페이트 신타아제 변이체 및 이를 이용한 테트라테르펜, 이의 전구체, 및 테트라테르펜을 전구체로 하는 물질의 생산방법 - Google Patents
제라닐제라닐 피로포스페이트 신타아제 변이체 및 이를 이용한 테트라테르펜, 이의 전구체, 및 테트라테르펜을 전구체로 하는 물질의 생산방법 Download PDFInfo
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- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1085—Transferases (2.) transferring alkyl or aryl groups other than methyl groups (2.5)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P5/00—Preparation of hydrocarbons or halogenated hydrocarbons
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P5/00—Preparation of hydrocarbons or halogenated hydrocarbons
- C12P5/007—Preparation of hydrocarbons or halogenated hydrocarbons containing one or more isoprene units, i.e. terpenes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y205/00—Transferases transferring alkyl or aryl groups, other than methyl groups (2.5)
- C12Y205/01—Transferases transferring alkyl or aryl groups, other than methyl groups (2.5) transferring alkyl or aryl groups, other than methyl groups (2.5.1)
- C12Y205/01001—Dimethylallyltranstransferase (2.5.1.1)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y205/00—Transferases transferring alkyl or aryl groups, other than methyl groups (2.5)
- C12Y205/01—Transferases transferring alkyl or aryl groups, other than methyl groups (2.5) transferring alkyl or aryl groups, other than methyl groups (2.5.1)
- C12Y205/01029—Geranylgeranyl diphosphate synthase (2.5.1.29)
Definitions
- the present application relates to geranylgeranyl pyrophosphate synthase (Geranylgeranyl pyrophosphate synthase) variants; a polynucleotide encoding the variant; a vector containing the polynucleotide; a microorganism comprising any one or more of the above mutants, polynucleotides, and vectors; a method for producing a tetraterpene using the same, a precursor thereof, and a material using the tetraterpene as a precursor; tetraterpenes, precursors thereof, and compositions for producing substances containing tetraterpenes as precursors; and a tetraterpene of a microorganism including at least one of the variant, polynucleotide, and vector, a precursor thereof, and a use for producing a material using the tetraterpene as a precursor.
- Terpenoid is a concept including carotenoid, and as it exerts various functions in plants and animals, it is used in various industrial fields such as food and feed.
- Terpenoids are known as representative substances that produce medicinal effects of plants, and are terpenes like carotenoids.
- carotenoids such as beta-carotene are reported to have functions such as removing free radicals, being the mother of vitamin A in animals, enhancing the immune system of vertebrates, and reducing the risk of lung cancer.
- carotenoids such as beta-carotene
- beta-carotene are not synthesized or synthesized in insufficient amounts in the body of animals.
- mutated microorganisms US Patent Registration No. 7745170
- geranylgeranyl pyrophosphate (GGPP, C20), a key precursor for the production of carotenoids or terpenoids, activates geranylgeranyl pyrophosphate synthase (isoprenoid) in the biosynthetic pathway. It is produced by combining farnesyl pyrophosphate (FPP, C15) and isopentenyl pyrophosphate (IPP, C5) by the enzyme geranylgeranyl pyrophosphate synthase.
- squalene (C30) may be produced as a by-product from farnesyl pyrophosphate (FPP, C15) by squalene synthase (ERG9). Accordingly, there is a need to develop a method for increasing the production of GGPP, a key precursor for carotenoid or terpenoid biosynthesis, and reducing the production of squalene, which is a competitive pathway.
- An object to be solved by the present application is to provide a geranylgeranyl pyrophosphate synthase variant, a tetraterpene using the same, a precursor thereof, and a method for producing a material using the tetraterpene as a precursor.
- One object of the present application is to convert any one or more 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 to another amino acid. It is to provide a substituted, geranylgeranyl pyrophosphate synthase (Geranylgeranyl pyrophosphate synthase) mutant.
- Another object of the present application is to provide a polynucleotide encoding the variant.
- Another object of the present application is to provide a vector containing the polynucleotide.
- Another object of the present application is to provide a microorganism comprising any one or more of the above mutants, polynucleotides, and vectors.
- Another object of the present application is to provide a tetraterpene (tetraterpene), a precursor thereof, and a method for producing a material using the tetraterpene as a precursor, including the step of culturing the microorganism in a medium.
- tetraterpene tetraterpene
- a method for producing a material using the tetraterpene as a precursor including the step of culturing the microorganism in a medium.
- Another object of the present application is to provide a tetraterpene, a precursor thereof, and a composition for producing a material containing the tetraterpene as a precursor, including the mutant, the vector, the microorganism, or the culture of the microorganism.
- Another object of the present application is to provide a tetraterpene of any one or more of the above mutants, polynucleotides, vectors, and microorganisms, a precursor thereof, and a use for producing a material using the tetraterpene as a precursor.
- Microorganisms expressing the geranylgeranyl pyrophosphate synthase variant of the present application can effectively produce tetraterpenes, precursors of tetraterpenes, or materials containing tetraterpenes as precursors, compared to strains that do not express them.
- any one or more 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
- a variant of geranylgeranyl pyrophosphate synthase is substituted with another amino acid.
- the geranylgeranyl pyrophosphate synthase variant is a polypeptide having geranylgeranyl pyrophosphate synthase activity or geranylgeranyl pyrophosphate synthase at positions 29, 43, and 90 from the N-terminus of SEQ ID NO: 1.
- 103, 122, and 141 means any one or more of the amino acids corresponding to the amino acid is substituted with another amino acid variant.
- Geranylgeranyl pyrophosphate synthase (GGS1) can catalyze the synthesis of geranylgeranyl pyrophosphate synthase (GGPP) from farnesylpyrophosphate (FPP). is an enzyme in
- the geranylgeranyl pyrophosphate synthase of the present application is a geranylgeranyl pyrophosphate synthase or geranylgeranyl pyrophosphate synthase modified to prepare a geranylgeranyl pyrophosphate synthase variant provided in the present application. It may be a polypeptide having an ase activity. Specifically, it may be a naturally occurring polypeptide or wild-type polypeptide, may be a mature polypeptide thereof, may include a variant or functional fragment thereof, but the parent of the geranylgeranyl pyrophosphate synthase variant of the present application Included without limitation where possible.
- the geranylgeranyl pyrophosphate synthase is not limited thereto, but may be a polypeptide of SEQ ID NO: 1. Also, about 80% with the polypeptide of SEQ ID NO: 1. It may be a polypeptide having a sequence identity of 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more, and may have the same or equivalent activity as the polypeptide consisting of the amino acid sequence of SEQ ID NO: 1. included in the scope of geranylgeranyl pyrophosphate synthase.
- 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.
- a “variant” is a polypeptide that differs from the amino acid sequence prior to the mutation by conservative substitution and/or modification of one or more amino acids, but retains functions or properties. refers to 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 reduced compared to the polypeptide before the mutation.
- some variants may include modifications in which one or more portions are removed, such as an N-terminal leader sequence or a transmembrane domain.
- Other variants may include modifications in which portions are removed from the N- and/or C-terminus of the mature protein.
- variant may be used interchangeably with terms such as variant, variant, variant polypeptide, mutated protein, mutation and variant (in English, modification, modified polypeptide, modified protein, mutant, mutein, divergent, etc.) And, if the term is used in a mutated sense, it is not limited thereto.
- Variants may also include deletions or additions of amino acids that have minimal impact on the secondary structure and properties of the polypeptide.
- a signal (or leader) sequence involved in protein translocation may be conjugated to the N-terminus of the variant, either co-translationally or post-translationally.
- the variant may be conjugated with other sequences or linkers to enable identification, purification, or synthesis.
- the variant of the present application is a substitution of any one or more amino acids among 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 with another amino acid. , geranylgeranyl pyrophosphate synthase variants. Specifically, among the positions 29, 43, 90, 103, 122, and 141, one or more, two or more, three or more, four or more, five or more, or six or more amino acids may be substituted. However, it is not limited thereto.
- the amino acid corresponding to position 29 from the N-terminus of the amino acid sequence of SEQ ID NO: 1 is asparagine. ;
- the amino acid corresponding to position 43 is phenylalanine;
- the amino acid corresponding to position 90 is leucine;
- the amino acid corresponding to position 103 is methionine;
- the amino acid corresponding to position 122 is isoleucine;
- the amino acid corresponding to position 141 may be arginine, but is not limited thereto.
- the amino acid corresponding to position 29 from the N-terminus of the amino acid sequence of SEQ ID NO: 1 is substituted with an amino acid other than asparagine; the amino acid corresponding to amino acid 43 is substituted with an amino acid other than phenylalanine; an amino acid corresponding to amino acid 90 is substituted with an amino acid other than leucine; an amino acid corresponding to amino acid number 103 is substituted with an amino acid other than methionine; an amino acid corresponding to amino acid 122 is substituted with an amino acid other than isoleucine; and substitution of an amino acid other than arginine for the amino acid corresponding to amino acid 141, but is not limited thereto.
- the "other amino acid” is not limited as long as it is an amino acid different from the amino acid before substitution and the amino acid before substitution.
- it is expressed as 'a specific amino acid is substituted' in the present application, it is obvious that it is substituted with an amino acid different from the amino acid before substitution, even if it is not separately indicated that the amino acid is substituted with another amino acid.
- 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.
- 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 is arginine , lysine, and may be selected from the group consisting of histidine, but is not limited thereto.
- the variant of the present application has any one or more amino acids among 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.
- non-polar amino acids polar amino acids, or positively charged (basic) amino acids
- it may be a variant substituted with an amino acid different from the amino acid before substitution, 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)
- the positively charged amino acid may be arginine (Arg) or lysine (Lys). Not limited.
- 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.
- the variant of the present application is 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 threonine, isoleucine, It may be substituted with any one selected from the group consisting of arginine, valine, and lysine, but is not limited thereto.
- 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 it may be selected and substituted from the group consisting of combinations thereof, but is not limited thereto.
- corresponding to refers to an amino acid residue at a recited position in a polypeptide, or an amino acid residue that is similar, identical, or homologous to a recited residue in a polypeptide. Identification of the amino acid at the corresponding position may be determining the specific amino acid in the sequence that references the specific sequence.
- corresponding region generally refers to a similar or corresponding position in a related or reference protein.
- 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 with reference to the numerical position of the amino acid residue corresponding to the amino acid residue of SEQ ID NO: 1.
- sequence alignment algorithms such as those described herein, can identify the location of amino acids, or locations where modifications such as substitutions, insertions, or deletions occur, compared to a query sequence (also referred to as a “reference sequence”).
- Such alignments include, for example, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453), the Needle program in the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al. , 2000), Trends Genet. 16: 276-277) may be used, but it is not limited thereto, and a sequence alignment program known in the art, a pairwise sequence comparison algorithm, and the like may be appropriately used.
- the variant of the present application is about 80% of the polypeptide of SEQ ID NO: 1. Positions 29, 43, 90, 103, 122, and 141 of SEQ ID NO: 1, having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity Any one or more of the amino acids corresponding to may be substituted with other amino acids.
- the variant of the present application is at least 80% identical 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, It may comprise amino acid sequences having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% homology or identity.
- the variant of the present application has the amino acid sequence described 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, It may comprise, consist of, or consist essentially of the amino acid sequence.
- the variant of the present application differs in any one or more of the amino acids corresponding to positions 29, 43, 90, 103, 122, and 141 based on the amino acid sequence of SEQ ID NO: 1. substituted with, and at least 80%, 85%, 90% of the amino acid sequence described 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 %, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% homology or identity.
- the variant of the present application is a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 3, in which methionine, an amino acid corresponding to position 103 of the amino acid sequence of SEQ ID NO: 1, is substituted with isoleucine; a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 5, in which phenylalanine, an amino acid corresponding to position 43 of the amino acid sequence of SEQ ID NO: 1, is substituted with isoleucine; A polypeptide comprising the amino acid sequence shown in SEQ ID NO: 7 in which asparagine, an amino acid corresponding to position 29 of the amino acid sequence of SEQ ID NO: 1, is substituted with threonine, and leucine, an amino acid corresponding to position 90, is substituted with arginine; a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 9, in which asparagine, which is an amino acid corresponding to position 29 of the amino acid sequence of SEQ ID NO: 1, is substituted
- sequence additions or deletions naturally occurring mutations, silent mutations or conservations to the amino acid sequence N-terminus, C-terminus and/or within that do not alter the function of the variants of the present application. This is the case with redundant substitution.
- the "conservative substitution” refers to the substitution of one amino acid with another amino acid having similar structural and/or chemical properties. Such amino acid substitutions can generally occur based on similarities in 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 the protein or polypeptide.
- the term 'homology' or 'identity' refers to the degree of similarity between two given amino acid sequences or base sequences and can be expressed as a percentage.
- the terms homology and identity are often used interchangeably.
- Sequence homology or identity of conserved polynucleotides or polypeptides can be determined by standard alignment algorithms, together with default gap penalties established by the program used. Substantially homologous or identical sequences are generally capable of hybridizing with all or part of the sequence under moderate or high stringent conditions. It is obvious that hybridization also includes hybridization with polynucleotides containing common codons or codons in consideration of codon degeneracy in polynucleotides.
- GAP program can define 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).
- the default parameters for the GAP program are (1) a binary comparison matrix (containing values of 1 for identity and 0 for non-identity) and Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation , pp. 353-358 (1979), Gribskov et al (1986) Nucl. Acids Res. 14: weighted comparison matrix of 6745 (or EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix); (2) a penalty of 3.0 for each gap and an additional penalty of 0.10 for each symbol in each gap (or 10 gap opening penalty, 0.5 gap extension penalty); and (3) no penalty for end gaps.
- 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 ability to produce a tetraterpene, a precursor of a tetraterpene, or a material using a tetraterpene as a precursor, compared to wild-type or non-mutated geranylgeranyl pyrophosphate synthase. .
- the variant of the present application has an activity to reduce the production level of a by-product of a tetraterpene, a precursor of a tetraterpene, or a material production pathway using a tetraterpene as a precursor, compared to wild-type or geranylgeranyl pyrophosphate synthase.
- the variant of the present application may have an activity that reduces the level of squalene production compared to wild-type or geranylgeranyl pyrophosphate synthase.
- the variant of the present application may have enhanced activity compared to wild-type or non-mutated geranylgeranyl pyrophosphate synthase, but is not limited thereto.
- Another aspect of the present application provides a polynucleotide encoding a variant of the present application.
- polynucleotide is a polymer of nucleotides in which nucleotide monomers are connected in a long chain shape by covalent bonds, and is a DNA or RNA strand of a certain length or more, more specifically, geranylgeranylpyramidal. It refers to a polynucleotide fragment encoding the variant having phosphate activity.
- the polynucleotide encoding the variant of the present application is a base encoding 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 sequence may be included.
- the polynucleotide of the present application may have or include 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 have.
- polynucleotide of the present application may consist of or consist essentially of 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 have.
- polynucleotides of the present application are various in the coding region within the range that does not change the amino acid sequence of the variants of the present application in consideration of codon degeneracy or preferred codons in organisms intended to express the variants of the present application. Transformations can be made.
- the polynucleotide of the present application has 80% 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 has or comprises at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, and less than 100% of the sequence of SEQ ID NO: 4, SEQ ID NO: 6, or 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 homology or identity to the sequence of 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% Or more, 98% or more, and may consist of or essentially consist of a nucleotide sequence of less than 100%, but is not limited thereto.
- the polynucleotide of the present application is not limited as long as it is a probe that can be prepared from a known gene sequence, for example, a sequence that can hybridize under stringent conditions with a sequence complementary to all or part of the polynucleotide sequence of the present application.
- the "stringent condition” means a condition that allows specific hybridization between polynucleotides. These conditions are described in 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).
- polynucleotides with high homology or identity 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 a condition in which polynucleotides having 99% or more homology or identity hybridize and polynucleotides having lower homology or identity do not hybridize, or 60 ° C., which is a washing condition for normal southern hybridization, 1 ⁇ SSC, 0.1% SDS, specifically at 60°C, 0.1 ⁇ SSC, 0.1% SDS, more specifically at a salt concentration and temperature equivalent to 68°C, 0.1 ⁇ SSC, 0.1% SDS, washed once, specifically 2 to 3 times conditions can be enumerated.
- Hybridization requires that two nucleic acids have complementary sequences, although mismatches between bases are possible depending on the stringency of hybridization.
- complementary is used to describe the relationship between nucleotide bases that are capable of hybridizing to each other. For example, with respect to DNA, adenine is complementary to thymine and cytosine is complementary to guanine.
- the polynucleotides of the present application may also include substantially similar nucleic acid sequences as well as isolated nucleic acid fragments complementary to the entire sequence.
- a polynucleotide having homology or identity to 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 above-described conditions.
- the Tm value may be 60 °C, 63 °C, or 65 °C, but is not limited thereto and may be appropriately adjusted by those skilled in the art according to the purpose.
- Appropriate stringency for hybridizing the polynucleotides depends on the length of the polynucleotides and the degree of complementarity, parameters well known in the art (e.g., J. Sambrook et al., supra).
- the vector may be an expression vector for expressing the polynucleotide in a host cell, but is not limited thereto.
- the vector of the present application may include a DNA product containing the nucleotide sequence of a polynucleotide encoding the target polypeptide operably linked to a suitable expression control region (or expression control sequence) so as to express the target polypeptide in a suitable host.
- the expression control region may include a promoter capable of initiating transcription, an arbitrary operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence regulating termination of transcription and translation.
- the vector After transformation into a suitable host cell, the vector can replicate or function independently of the host genome and can integrate into the genome itself.
- Vectors used in the present application are not particularly limited, and any vectors known in the art may be used.
- Examples of commonly used vectors include natural or recombinant plasmids, cosmids, viruses and bacteriophages.
- pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A may be used as phage vectors or cosmid vectors
- pDC-based, pBR-based, and pUC-based plasmid vectors may be used.
- pBluescriptII-based, pGEM-based, pTZ-based, pCL-based, pET-based, etc. can be used.
- pDC pDC
- pDCM2 Public of Korea Patent Publication No. 10-2020-0136813
- pACYC177 pACYC184
- pCL pECCG117
- pUC19 pUC19
- pBR322 pMW118
- pCC1BAC pIMR53 vectors and the like
- a polynucleotide encoding a target polypeptide may be inserted into a chromosome through a vector for chromosomal insertion into a cell. Insertion of the polynucleotide into the chromosome may be performed by any method known in the art, for example, homologous recombination, but is not limited thereto.
- a selection marker for determining whether the chromosome is inserted may be further included.
- the selectable marker is used to select cells transformed with a vector, that is, to determine whether a target nucleic acid molecule has been inserted, and can exhibit selectable phenotypes such as drug resistance, auxotrophy, resistance to cytotoxic agents, or surface polypeptide expression. markers may be used. In an environment treated with a selective agent, only cells expressing the selectable marker survive or exhibit other expression traits, so transformed cells can be selected.
- the term "transformation” means 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 can be expressed in the host cell, it may be inserted into and located in the chromosome of the host cell or located outside the chromosome.
- the polynucleotide includes DNA and/or RNA encoding a polypeptide of interest.
- the polynucleotide may be introduced in any form as long as it can be introduced and expressed into a host cell.
- the polynucleotide may be introduced into a host cell in the form of an expression cassette, which is a genetic construct containing all elements required for self-expression.
- the expression cassette may include a promoter operably linked to the polynucleotide, a transcription termination signal, a ribosome binding site, and a translation termination signal.
- the expression cassette may be in the form of an expression vector capable of self-replication.
- 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.
- operably linked means that the polynucleotide sequence is functionally linked to a promoter sequence that initiates and mediates the transcription of the polynucleotide encoding the target variant of the present application.
- Another aspect of the present application is a variant of the present application; a polynucleotide encoding the variant; And it provides a microorganism comprising any one or more of the vectors containing the polynucleotide.
- microorganism or “strain” includes both wild-type microorganisms and naturally or artificially genetically modified microorganisms, and is caused by insertion of foreign genes or enhancement or inactivation of endogenous gene activity.
- a microorganism whose specific mechanism is attenuated or enhanced due to this, it may be a microorganism including genetic modification for the production of a desired polypeptide, protein or product.
- the microorganism of the present application includes a microorganism comprising any one or more of a vector comprising a variant of the present application, a polynucleotide of the present application, and a polynucleotide of the present application; microorganisms modified to express a variant of the present application or a polynucleotide of the present application; microorganisms expressing the variants of the present application, or the polynucleotides of the present application; Or it may be a microorganism (eg, recombinant microorganism) having the mutant activity of the present application, but is not limited thereto.
- a microorganism comprising any one or more of a vector comprising a variant of the present application, a polynucleotide of the present application, and a polynucleotide of the present application
- microorganisms modified to express a variant of the present application or a polynucleotide of the present application microorganis
- the microorganism is modified to further include polynucleotides encoding lycopene cyclase / phytoene synthase (crtYB) and phytoene desaturase (crtI) proteins, and these protein activities It may be a microorganism that expresses or a microorganism for which these protein activities are enhanced.
- the lycopene cyclase/phytoene synthase or phytoene desaturase may be a protein derived from Xanthophyllomyces dendrohous , but is not limited thereto.
- 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.
- various modifications may be made to the coding region within a range that does not change the amino acid sequence in consideration of codon degeneracy or codons preferred in microorganisms to express the variant of the present application.
- the polynucleotide has 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97% homology or identity to the sequence of SEQ ID NO: 27 or SEQ ID NO: 28, having or comprising a nucleotide sequence of less than 100%; Or more, 98% or more, and may consist of or essentially consist of a nucleotide sequence of less than 100%, but is not limited thereto.
- the microorganism of the present application may be one that produces tetraterpene, a precursor of tetraterpene, or a material using tetraterpene as a precursor, and the production of by-products generated in the tetraterpene production pathway may be reduced, and the production of squalene may be reduced.
- tetraterpene a precursor of tetraterpene
- a material using tetraterpene as a precursor a material using tetraterpene as a precursor
- the production of by-products generated in the tetraterpene production pathway may be reduced, and the production of squalene may be reduced.
- tetraterpene means a polymer (C40) in which 8 isoprene is polymerized among terpenes, and may include a tetraterpene-based material.
- the terpene is a polymer of isoprene units, a terpene obtained by polymerizing two isoprene (C5) units is a monoterpene (C10), and a terpene obtained by polymerizing three isoprene units is a sesquiterpene (C15).
- terpenes can be classified according to the number of polymerized isoprene units.
- the tetraterpene may be a carotenoid, but is not limited thereto.
- the tetraterpene precursor may be geranylgeranyl pyrophosphate (GGPP), but is not limited thereto.
- GGPP geranylgeranyl pyrophosphate
- the material using tetraterpene as a precursor may be at least one selected from the group consisting of retinal and retinol, but is not limited thereto.
- a "tetraterpenoid" in this application is a modified tetraterpene.
- the tetraterpenoid may be a functional group-linked tetraterpene.
- carotenoid is a tetraterpene or a derivative thereof that gives colors such as yellow in fruits and vegetables, and may be used interchangeably with the tetraterpene and tetraterpenoid.
- the carotenoids are xanthophyll, carotene, alpha-carotene, beta-carotene, gamma-carotene, lutein, and lycopene. ), zeaxanthin, capsanthin, canthaxanthin, and astaxanthin, but may be any one or more selected from the group consisting of, but is not limited thereto.
- the precursor of carotenoid may be geranylgeranyl pyrophosphate (GGPP), but is not limited thereto.
- GGPP geranylgeranyl pyrophosphate
- the material using carotenoid as a precursor may be at least one selected from the group consisting of retinal and retinol, but is not limited thereto.
- beta-carotene is one of carotenoid-based substances, and has beta rings at both ends of the molecule in carotene.
- the beta-carotene precursor may be at least one selected from the group consisting of phytoene, phytofluene, lycopene, and gamma-carotene, but is not limited thereto. don't
- the material containing beta-carotene as a precursor may be any one or more selected from the group consisting of retinal, retinol, and vitamin A, but is not limited thereto.
- the tetraterpene, a precursor of tetraterpene, or a material using tetraterpene as a precursor may be a carotenoid, a precursor of carotenoid, or a material using a carotenoid as a precursor, and may be beta-carotene, a precursor of beta-carotene, or a material using beta-carotene as a precursor. It may, but is not limited thereto.
- any one or more of the variant, polynucleotide, vector, and microorganism may be for producing tetraterpene, and the microorganism may be for producing tetraterpene, and may produce a precursor of tetraterpene or a precursor of tetraterpene. It may be to produce a substance to be.
- any one or more of the variants, polynucleotides, vectors, and microorganisms may be for producing carotenoids, and the microorganisms may be for producing carotenoids, and may produce precursors of carotenoids or materials containing carotenoids as precursors. may be producing
- any one or more of the variant, polynucleotide, vector, and microorganism may be for beta-carotene production, and the microorganism may be to produce beta-carotene, produce a beta-carotene precursor or beta-carotene as a precursor It may be to produce a substance to be.
- GGPP geranylgeranyl pyrophosphate
- the microorganism can also produce geranylgeranyl pyrophosphate. may be producing
- by-products produced in the tetraterpene production pathway refer to tetraterpenes, precursors of tetraterpenes, and materials other than materials using tetraterpenes as precursors, and may specifically be squalene, but are not limited thereto.
- don't Beta-carotene can be produced in the carotenoid or isoprenoid production pathway, and in the process, squalene synthase (ERG9) consumes 2 molecules of farnesyl pyrophosphate (FPP, C15), C30) can be produced together as a by-product.
- squalene is an unsaturated hydrocarbon (C 30 H 50 ), which is also used for biosynthesis of steroid hormones and vitamin D.
- the microorganism of the present application may be one in which by-products produced in the tetraterpene production pathway are reduced, and may specifically be one in which squalene production is reduced, but is not limited thereto.
- the microorganism or strain of the present application is a microorganism naturally having the ability to produce geranylgeranyl pyrophosphate synthase or tetraterpene, or a variant or A polynucleotide encoding it (or a vector containing the polynucleotide) may be introduced and/or a microorganism endowed with the ability to produce a tetraterpene, a precursor of a tetraterpene, or a material using a tetraterpene as a precursor, but is not limited thereto.
- the microorganism or strain of the present application is a cell or microorganism that is transformed with a vector containing a polynucleotide of the present application or a polynucleotide encoding a variant of the present application and expresses the variant of the present application.
- the microorganisms or strains of the present application may include all microorganisms capable of producing tetraterpenes, precursors of tetraterpenes, or substances containing tetraterpenes as precursors, including the variants of the present application.
- a polynucleotide encoding the variant of the present application is introduced into a natural wild-type microorganism or a microorganism that produces a tetraterpene, so that a geranylgeranyl pyrophosphate synthase variant is expressed, and the tetraterpene , It may be a recombinant strain with increased production ability of a precursor of tetraterpene or a material using tetraterpene as a precursor.
- the recombinant strain is a tetraterpene, a precursor of tetraterpene, or a tetraterpene compared to a natural wild-type microorganism or a non-modified geranylgeranyl pyrophosphate synthase microorganism (ie, a microorganism expressing wild-type geranylgeranyl pyrophosphate synthase). It may be a microorganism with increased production ability of a material using a precursor, but is not limited thereto.
- the recombinant strain is about 1% or more, specifically, about 3%, about 5%, compared to the production ability of the parent strain or the unmodified microorganism before the mutation, the tetraterpene, the precursor of the tetraterpene, or the material using the tetraterpene as a precursor. It may be higher than or higher, but is not limited thereto as long as it has an increase of + value compared to the production capacity of the parent strain or unmodified microorganism before mutation.
- the recombinant strain has about 85% or less, about 80% or less, about 75% or less, about 70% or less, about 65% or less, about 85% or less, about 80% or less, about 65% or less, % or less, about 60% or less, about 55% or less, about 50% or less, about 30% or less, or about 10% or less, or by-products may not be produced, but is not limited thereto.
- the term "unmodified microorganism” does not exclude strains containing mutations that may occur naturally in microorganisms, and are wild-type strains or wild-type strains themselves, or are genetically modified by natural or artificial factors. It may mean a strain before change.
- the unmodified microorganism may mean a strain before or without introduction of the geranylgeranyl pyrophosphate synthase variant of the present application.
- the "unmodified microorganism” may be used interchangeably with "strain before transformation", “microorganism before transformation”, “non-mutated strain”, “unmodified strain”, "non-mutated microorganism” or "reference microorganism".
- the microorganism of the present application may be a microorganism of the genus Yarrowia sp ., specifically Yarrowia lipolytica , but may be, but is not limited thereto.
- the term "enhancement" of polypeptide activity means that the activity of the polypeptide is increased relative to the intrinsic activity.
- the enhancement may be used interchangeably with terms such as activation, up-regulation, overexpression, and increase.
- activation, enhancement, upregulation, overexpression, and increase may include those that exhibit an activity that was not originally possessed, or those that exhibit enhanced activity compared to intrinsic activity or activity before modification.
- intrinsic activity refers to the activity of a specific polypeptide originally possessed by the parent strain or non-transformed microorganism before transformation when the character changes due to genetic mutation caused by natural or artificial factors.
- activity before transformation and “Enhancement”, “upregulation”, “overexpression” or “increase” of the activity of a polypeptide compared to its intrinsic activity means the activity of a specific polypeptide originally possessed by the parent strain or non-transformed microorganism before transformation. And / or improved compared to the concentration (expression amount).
- the enhancement can be achieved by introducing a foreign polypeptide or by enhancing the activity and/or concentration (expression level) of an endogenous polypeptide. Whether or not the activity of the polypeptide is enhanced can be confirmed from an increase in the activity level, expression level, or amount of a product released from the corresponding polypeptide.
- Enhancement of the activity of the polypeptide can be applied by 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 the microorganism before transformation. Specifically, it may be using genetic engineering and / or protein engineering, which is well known to those skilled in the art, which is a routine method of molecular biology, 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.).
- modification of the polynucleotide sequence encoding the polypeptide to enhance the activity of the polypeptide eg, modification of the polynucleotide sequence of the polypeptide gene to encode the modified polypeptide to enhance the activity of the polypeptide
- It may be a combination of two or more selected from 1) to 8), but is not particularly limited thereto.
- the increase in intracellular copy number of the polynucleotide encoding the polypeptide is achieved by introducing into the host cell a vector capable of replicating and functioning independently of the host, to which the polynucleotide encoding the corresponding polypeptide is operably linked. it may be Alternatively, it may be achieved by introducing one copy or two or more copies of a polynucleotide encoding the corresponding polypeptide into a chromosome of a host cell.
- the introduction into the chromosome may be performed by introducing a vector capable of inserting the polynucleotide into the chromosome of the host cell into the host cell, but is not limited thereto.
- the vector is as described above.
- the expression control region may include a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence regulating termination of transcription and translation.
- the original promoter may be replaced with a strong promoter, but is not limited thereto.
- Examples of known strong promoters include cj1 to cj7 promoter (US Patent US 7662943 B2), lac promoter, trp promoter, trc promoter, tac promoter, lambda phage PR promoter, PL promoter, tet promoter, gapA promoter, SPL7 promoter, SPL13 (sm3) promoter (US Patent US 10584338 B2), O2 promoter (US Patent US 10273491 B2), tkt promoter, yccA promoter, etc., but are not limited thereto.
- Modification of the nucleotide sequence encoding the initiation codon or 5'-UTR region of the gene transcript encoding the polypeptide is, for example, a nucleotide sequence encoding another initiation codon with a higher polypeptide expression rate than the endogenous initiation codon. It may be substituted, but is not limited thereto.
- Modification of the amino acid sequence or polynucleotide sequence of 4) and 5) above may include deletion, insertion, non-conservative or conservative substitution of the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide to enhance the activity of the polypeptide.
- the combination thereof may be a sequence mutation, 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 increase activity, but is not limited thereto.
- 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 further include a selection marker for checking whether the chromosome is inserted.
- the selectable marker is as described above.
- Introduction of a foreign polynucleotide exhibiting the activity of the polypeptide may be introduction of a foreign polynucleotide encoding a polypeptide exhibiting the same/similar activity as the polypeptide into a host cell.
- the foreign polynucleotide is not limited in origin or sequence as long as it exhibits the same/similar activity as the polypeptide.
- the method used for the introduction can be performed by appropriately selecting a known transformation method by a person skilled in the art, and expression of the introduced polynucleotide in a host cell can generate a polypeptide and increase its activity.
- the codon optimization of the polynucleotide encoding the polypeptide is codon optimization of the endogenous polynucleotide to increase transcription or translation in the host cell, or optimization of the transcription or translation of the foreign polynucleotide in the host cell. It may be that the codons of this have been optimized.
- Analyzing the tertiary structure of the polypeptide to select and modify or chemically modify the exposed site for example, by comparing the sequence information of the polypeptide to be analyzed with a database in which sequence information of known proteins is stored, depending on the degree of sequence similarity. It may be to determine a template protein candidate according to the method, confirm the structure based on this, and modify or modify an exposed portion to be chemically modified to be modified or modified.
- the enhancement of such a polypeptide activity is an increase in the activity or concentration of the corresponding polypeptide based on the activity or concentration of the polypeptide expressed in the wild-type or pre-modified microbial strain, or an increase in the amount of the product produced from the corresponding polypeptide. It may be, but is not limited thereto.
- Modification of some or all of the polynucleotides in the microorganism of the present application is (a) genome editing using homologous recombination or genetic scissors (engineered nuclease, e.g., CRISPR-Cas9) using a vector for chromosomal insertion into the microorganism and / or (b) It may be induced by light and/or chemical treatment, such as ultraviolet light and radiation, but is not limited thereto.
- a method of modifying part or all of the gene may include a method using DNA recombination technology.
- a part or all of a gene may be deleted by injecting a nucleotide sequence or vector containing a nucleotide sequence homologous to a target gene into the microorganism to cause homologous recombination.
- the injected nucleotide sequence or vector may include a dominant selection marker, but is not limited thereto.
- Another aspect of the present application is a variant of the present application; a polynucleotide encoding the variant; and culturing a microorganism comprising any one or more of the vectors containing the polynucleotide in a medium, and a method for producing a tetraterpene, a precursor of the tetraterpene, or a material using the tetraterpene as a precursor.
- the method for producing the tetraterpene, the precursor of the tetraterpene, or a material using the tetraterpene as a precursor may be a method for reducing squalene production, but is not limited thereto.
- the term "culture” means growing the microorganism of the present application under appropriately controlled environmental conditions.
- the culture process of the present application may be performed according to suitable media and culture conditions known in the art. This culturing process can be easily adjusted and used by those skilled in the art according to the selected strain.
- the culture may be batch, continuous and/or fed-batch, but is not limited thereto.
- the term "medium” refers to a material in which nutrients necessary for culturing the microorganisms of the present application are mixed as main components, and supplies nutrients and growth factors, including water essential for survival and growth.
- the medium and other culture conditions used for culturing the microorganisms of the present application can be any medium without particular limitation as long as it is a medium used for culturing ordinary microorganisms, but the microorganisms of the present application are suitable as carbon sources, nitrogen sources, personnel, and inorganic materials. It can be cultured while controlling temperature, pH, etc. under aerobic conditions in a conventional medium containing compounds, amino acids, and/or vitamins.
- Examples of the carbon source 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, citric acid and the like; Amino acids such as glutamic acid, methionine, lysine, and the like may be included.
- natural organic nutrients such as starch hydrolysate, molasses, blackstrap molasses, rice winter, cassava, sugarcane pomace and corn steep liquor can be used, specifically glucose and sterilized pretreated molasses (i.e. converted to reducing sugar).
- Carbohydrates such as molasses
- various other carbon sources in appropriate amounts may be used without limitation. These carbon sources may be used alone or in combination of two or more, but are not limited thereto.
- nitrogen source examples include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate; Amino acids such as glutamic acid, methionine, glutamine, etc., organic nitrogen sources such as peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolysate, fish or degradation products thereof, defatted soybean cake or degradation products thereof, etc. can be used These nitrogen sources may be used alone or in combination of two or more, but are not limited thereto.
- inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate
- Amino acids such as glutamic acid, methionine, glutamine, etc.
- organic nitrogen sources such as peptone, NZ-amine,
- the number of persons may include monopotassium phosphate, dipotassium phosphate, or a sodium-containing salt corresponding thereto.
- the inorganic compound sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, etc. may be used, and amino acids, vitamins, and/or appropriate precursors may be included. These components or precursors may be added to the medium either batchwise or continuously. However, it is not limited thereto.
- the pH of the medium can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid and the like to the medium in an appropriate manner during the cultivation of the microorganism of the present application.
- an antifoaming agent such as a fatty acid polyglycol ester.
- oxygen or oxygen-containing gas may be injected into the medium, or nitrogen, hydrogen or carbon dioxide gas may be injected without gas injection or nitrogen, hydrogen or carbon dioxide gas may be injected to maintain the anaerobic and non-aerobic state. It is not.
- the culture temperature may be maintained at 20 to 45 ° C, specifically 25 to 40 ° C, 20 to 35 ° C, or 25 to 35 ° C, and may be cultured for about 10 to 160 hours, but is not limited thereto not.
- Tetraterpenes produced by the culture of the present application may be secreted into the medium or remain in cells.
- the method for producing a tetraterpene, a precursor of tetraterpene, or a material using tetraterpene as a precursor of the present application includes preparing a microorganism of the present application, preparing a medium for culturing the strain, or a combination thereof (in the order Regardless, in any order), for example, prior to the culturing step, may be further included.
- the tetraterpene of the present application is a tetraterpene, a precursor of tetraterpene, or a tetraterpene from the culture medium (culture medium) or the microorganism of the present application.
- a step of recovering a material using as a precursor may be further included.
- the recovering step may be further included after the culturing step.
- the recovery is carried out using a suitable method known in the art according to the culture method of the microorganism of the present application, for example, batch, continuous or fed-batch culture method, etc. It may be to collect a substance to be. For example, centrifugation, filtration, treatment with a precipitating agent for crystallized proteins (salting out), extraction, cell disruption, sonication, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography It may be used in combination with various chromatography such as chromatography, affinity chromatography, HPLC, or these methods, and the desired tetraterpene, precursor of tetraterpene, or tetraterpene is obtained from a medium or microorganism using a suitable method known in the art. A material using as a precursor can be recovered.
- a suitable method known in the art can be recovered.
- the method for producing a tetraterpene, a precursor of tetraterpene, or a material using tetraterpene as a precursor of the present application may additionally include a purification step.
- the purification may be performed using suitable methods known in the art.
- the recovery step and the purification step are temporary regardless of the order ( or continuously), or may be performed simultaneously or integrated into one step, but is not limited thereto.
- the method for producing a material using tetraterpene as a precursor of the present application may further include converting the tetraterpene into a material using tetraterpene as a precursor.
- the converting step may be further 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.
- mutants, polynucleotides, vectors, microorganisms, tetraterpenes, precursors of tetraterpenes, and materials containing tetraterpenes as precursors are as described above.
- Another aspect of the present application is a variant of the present application; vectors of the present application; The variant of the present application, the polynucleotide encoding the variant, the vector comprising the polynucleotide, or the microorganism comprising the polynucleotide of the present application; medium in which it was cultured; Or to provide a tetraterpene containing a combination of two or more of them, a precursor of the tetraterpene, and a composition for producing a material using the tetraterpene as a precursor.
- composition of the present application may further include any suitable excipient commonly used in compositions for producing tetraterpenes, precursors of tetraterpenes, and substances containing tetraterpenes as precursors, and these excipients include, for example, preservatives, wetting agents, It may be a dispersing agent, a suspending agent, a buffering agent, a stabilizing agent or an isotonic agent, etc., but is not limited thereto.
- mutants, polynucleotides, vectors, microorganisms, media, tetraterpenes, precursors of tetraterpenes, materials containing tetraterpenes as precursors, and the like are as described above.
- Another aspect of the present application provides a use for producing a tetraterpene of any one or more of the variant, polynucleotide, vector, and microorganism of the present application, a precursor of the tetraterpene, and a material using the tetraterpene as a precursor.
- mutants, polynucleotides, vectors, microorganisms, tetraterpenes, precursors of tetraterpenes, and materials containing tetraterpenes as precursors are as described above.
- GGS1 In order to amplify the GGS1 gene encoding geranylgeranyl pyrophosphate synthase on the Yarrowia lioplytica chromosome, GGS1 (YALI0D17050 ) The amino acid sequence of SEQ ID NO: 1 and the polynucleotide sequence of SEQ ID NO: 2 were obtained.
- a diversify PCR random mutagenesis kit (Takara Co.) was used to induce random mutations through error-prone PCR (ep-PCR). PCR conditions and methods were optimized through buffer condition 1 and buffer condition 2 of the user manual, respectively. Specific Error-prone PCR conditions are shown in Table 1 below.
- Buffer condition 1 Buffer condition 2 PCR grade water 40 ⁇ l 39 ⁇ l 10X Titanium Taq buffer 5 ⁇ l 5 ⁇ l MnSO4 0 ⁇ l 1 ⁇ l dGTP 1 ⁇ l 1 ⁇ l 50X Diversify dNTP Mix 1 ⁇ l 1 ⁇ l Primer mix 1 ⁇ l 1 ⁇ l Template DNA 1 ⁇ l 1 ⁇ l Titanium Taq polymerase 1 ⁇ l 1 ⁇ l
- PCR was performed using Yarrowia lipolytica PO1f genomic DNA as a template and primers of SEQ ID NO: 19 and SEQ ID NO: 20. PCR conditions, denaturation 95 °C, 1 min; Anneal 55°C, 1 min; And the polymerization reaction was repeated 32 times at 68°C for 1 minute.
- a GGS1 mutant expression vector library based on the pIMR53-EXP1p-CYC1t vector was constructed by mixing the GGS1 PCR products of the two conditions amplified by PCR.
- 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 of SEQ ID NO: 21 and SEQ ID NO: 22, and Saccharomyces cerevisiae to amplify the CYC1t terminator. PCR was performed using Saccharomyce cerevisiae genomic DNA as a template and primers of SEQ ID NO: 23 and SEQ ID NO: 24. PCR conditions, denaturation 95 °C, 1 min; Anneal 55°C, 1 min; And the polymerization reaction was repeated 32 times at 68°C for 1 minute.
- PCR was performed using the pIMR53 vector as a template and using the primers of SEQ ID NO: 25 and SEQ ID NO: 26. PCR conditions, denaturation 95 °C, 1 min; Anneal 55°C, 1 min; And the polymerization reaction was repeated 32 times at 68 ° C for 5 minutes.
- Colonies transformed with the plasmid into which the desired EXP1p promoter and CYC1t terminator were inserted were selected by PCR, and then the plasmid was obtained using a commonly known plasmid extraction method, and this plasmid was named pIMR53-EXP1p-CYC1t.
- Example 2-1 X. dendrorhous Production of derived crtYB-crtI insert strain
- crtYB lycopene cyclase/phytoene synthase
- crtI phytoene desaturase
- crtYB secured the polynucleotide of SEQ ID NO: 27 based on the base sequence registered in NCBI (National Center for Biotechnology Information Search database) (GenBank: AY177204.1), and crtI obtained the base sequence registered in NCBI (GenBank: Based on AY177424.1), the polynucleotide of SEQ ID NO: 28 was obtained.
- 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), and URA3 of Y. lipolytica was used as a selection marker.
- a cassette inserted into the MHY1 (YALI0B21582g) gene locus was designed using the gene (SEQ ID NO: 31).
- Each PCR was performed using the primers of SEQ ID NO: 41, SEQ ID NO: 42, and SEQ ID NO: 43.
- PCR conditions were denaturing 95 °C for 1 min; Anneal 55°C, 1 min; And the polymerization reaction was repeated 35 times at 72°C for 3 minutes and 30 seconds. Five DNA fragments obtained as a result were produced as one cassette through overlap extension PCR.
- the prepared cassette was introduced into KCCM12972P strain by heat shock method ( D.-C. Chen et al., Appl Microbiol Biotechnol, 1997 ), and then colonies formed on uracil-free solid medium (YLMM1) were obtained.
- YLMM1 uracil-free solid medium
- SEQ ID NO: 44 and SEQ ID NO: 45 colonies in which cassette insertion was confirmed in the genome were spotted on 5-FOA solid medium, cultured at 30 ° C for 3 days, and colonies grown on 5-FOA solid medium were obtained to obtain URA3 Markers were recovered.
- Example 2-2 HMGR fortified wine production
- a cassette was designed to replace the site with the TEFINt promoter, using KCCM12972P genomic DNA as a template and 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, SEQ ID NO: 55, and SEQ ID NO: 56, respectively, PCR was performed.
- PCR conditions were denaturing 95 °C for 1 min; Anneal 55°C, 1 min; And the polymerization reaction was repeated 35 times at 72°C for 1 minute and 30 seconds. Five DNA fragments obtained as a result were produced as one cassette through overlap extension PCR.
- 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
- the first color-based selection was performed.
- YLMM2 Yarrowia lipolytica minimal media 2
- YLMM1 Yarrowia lipolytica minimal media 2
- the positive control CC08-1023/pIMR53-EXP1p-GGS1-CYC1t
- 36 strains of dark orange color compared to strains were first screened.
- the preparation method and composition of the above-described YLMM2 are as follows.
- Glucose 40 g/L Yeast nitrogen base without amino acids and ammonium sulfate 1.7 g/L, Uracil 0.5 g/L, Ammonium sulfate 2.5 g/L in 0.1 M phosphate buffer (sodium phosphate buffer) (pH 7.2).
- a reference value was set based on the value calculated from PC (positive control), strains corresponding to both conditions were selected, and a total of four candidate groups corresponding to conditions 1 and 2 were selected at the same time.
- the secondary selection criteria values and four secondary selection candidates are shown in Table 3 below.
- Transformed plasmids were extracted from the selected four strains, and sequence analysis was performed to identify random mutations located in the GGS1 ORF.
- the transformed GGS1 plasmid was extracted from the selected strain using Zymoprep yeast plasmid miniprep kit 2 (Zymo research), and sequencing was performed in both directions using primers of SEQ ID NO: 59 and SEQ ID NO: 60 to increase the reliability of sequencing. proceeded.
- Amino acid mutations identified as a result of the GGS1 ORF sequence analysis are shown in Table 4 below, and four candidate groups were confirmed to contain one or two mutations in Table 4 below.
- NC (negative control) and PC (positive control) vectors and 4 variants selected in Example 4 (M103I variant, F43I variant, N29T and L90R variant, and I122V and R141K variant), and 4 related single variants (N29T mutant, L90R mutant, I122V mutant, and R141K mutant) flask evaluation of a total of 10 strains constructed by transforming expression vectors was performed.
- Expression vectors for each of the four single variants are prepared as follows. To secure the DNA fragments of four single variants (N29T variant, L90R variant, I122V variant, and R141K variant), the pIMR53-EXP1p-GGS1-CYC1t vector containing the wild-type GGS1 sequence was used as a template with SEQ ID NO: 61 and SEQ ID NO: 62; PCR was performed using primers of 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.
- Expression vectors of the double mutants, N29T and L90R variants, or I122V and R141K variants, respectively, use the above-constructed N29T expression vector or I122V expression vector as a template, and primers of SEQ ID NO: 63 and SEQ ID NO: 64 or SEQ ID NO: 67 and SEQ ID NO: 68 It was prepared by performing PCR in the same way using the primers of.
- the PCR-amplified DNA into which the variant was introduced was transformed into E. coli DH5 ⁇ using an Infusion cloning kit (Invitrogen), and plated on LB solid medium containing 30 mg/L ampicillin. After screening colonies transformed with the plasmid into which the 4 transformed GGS1 variant vectors were inserted, plasmids were obtained using a commonly known plasmid extraction method, and the variant sequences were identified using SEQ ID NO: 59 and SEQ ID NO: 60. .
- Genotype OD ⁇ -carotene (mg/L) Squalene (mg/L) ⁇ -carotene (%) Squalene (%) NC 62 5 208 0.03 1.34 PC 56 30 133 0.21 0.95 M103I 62 59 91 0.38 0.59 F43I 63 69 71 0.44 0.45 N29T/L90R 64 67 89 0.42 0.56 N29T 61 55 88 0.36 0.58 L90R 64 58 90 0.36 0.56 I122V/R141K 62 62 65 0.40 0.42 I122V 57 43 118 0.30 0.83 R141K 59 44 111 0.30 0.75
- the beta-carotene concentration increased by 143 to 230% in all of the GGS1 variants to which M103I, F43I, N29T, L90R, I122V, and/or R141K mutations were selected compared to PC (positive control), and the squalene concentration was A decrease of 11 to 51% was confirmed.
- the strain introduced with the F43I variant had the best effect, and in the case of the I122V and R141K variants, beta-carotene concentration increased and squalene increased when applied simultaneously compared to the application of the two variants alone. It was confirmed that the production reduction effect was further increased.
- the N29T and L90R variants it was confirmed that the effect of reducing squalene production was further increased when applied simultaneously compared to the application of the two variants alone.
- a strain (CC08-1214) in which the promoter of the GGS1 gene was replaced (CC08-1214) and a strain expressing a mutant in which the 43rd phenylalanine of GGS1 was substituted with isoleucine (CC08-1215) were sequentially prepared.
- Promoter of TEF1 (YALI0C09141p) based on the nucleotide sequence registered in KEGG (Kyoto Encyclopedia of Genes and Genomes) for the production of a cassette capable of replacing the promoter of GGS1 with the TEFINt promoter on the Yarrowia lipolytica chromosome And a polynucleotide sequence of SEQ ID NO: 69 including a splicing point was obtained.
- the polynucleotide sequence of SEQ ID NO: 2 of GGS1 (YALI0D17050g) was secured for promoter substitution of GGS1.
- PCR was performed using Yarrowia lipolytica PO1f 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. did Then, PCR was performed using the primers of SEQ ID NO: 78 and SEQ ID NO: 79 using the URA3 auxotrophic marker as a template. PCR conditions were denaturing 95° C. for 1 min; Annealing 55° C., 1 min; And the polymerization reaction was repeated 35 times at 72° C. for 1 minute and 30 seconds.
- the GGS1 promoter replacement cassette was transformed into CC08-1023 by heat-shock and selected on YLMM1 solid medium without uracil. The selected primary strains were subjected to secondary crossover, and finally, strains in which the promoter of the GGS1 gene was replaced with the TEFINt promoter were produced. The strain thus produced was named "CC08-1214".
- PCR was performed using the primers of SEQ ID NO: 90 and SEQ ID NO: 91, respectively.
- PCR was performed using the primers of SEQ ID NO: 88 and SEQ ID NO: 89 using the URA3 auxotrophic marker as a template. PCR conditions were denaturing 95 °C for 1 min; Anneal 55°C, 1 min; And the polymerization reaction was repeated 35 times at 72°C for 1 minute and 30 seconds.
- the GGS1 (F43I) mutant replacement cassette was transformed into CC08-1214 by heat-shock, and selected on YLMM1 solid medium without uracil. The selected primary strain was again subjected to secondary crossover, and finally a strain expressing the F43I mutant in which the 43rd amino acid residue of GGS1 was replaced with isoleucine was prepared. The strain thus produced was named "CC08-1215".
- Flask evaluation was performed with CC08-1023 as negative control and CC08-1214 as positive control along with CC08-1215.
- CC08-1215, CC08-1023 (negative control), and CC08-1214 (positive control) were added to a 250 ml corner-baffle flask containing 50 ml of YLMM2 (Yarrowia lipolytica minimal media 2) containing uracil. Inoculated to an initial OD of 1, and cultured at 30° C. for 48 hours with shaking at 250 rpm.
- Genotype OD ⁇ -carotene (mg/L) Squalene (mg/L) ⁇ -carotene (%) Squalene (%) CC08-1023(NC) 46 5 41 0.04 0.36 CC08-1214 (PC) 50 32 17 0.26 0.14 CC08-1215 48 49 2 0.41 0.02
- beta-carotene production can be increased when introducing the GGS1 variant of the present application.
- the beta-carotene production efficiency can be further increased by introducing the GGS1 variant to reduce beta-carotene production and squalene synthesis, which is a competitive pathway.
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Abstract
Description
Buffer 조건 1 | Buffer 조건 2 | |
PCR grade water | 40 ㎕ | 39 ㎕ |
10X Titanium Taq buffer | 5 ㎕ | 5 ㎕ |
MnSO4 | 0 ㎕ | 1 ㎕ |
dGTP | 1 ㎕ | 1 ㎕ |
50X Diversify dNTP Mix | 1 ㎕ | 1 ㎕ |
Primer mix | 1 ㎕ | 1 ㎕ |
Template DNA | 1 ㎕ | 1 ㎕ |
Titanium Taq polymerase | 1 ㎕ | 1 ㎕ |
Numb. | OD | β-carotene(mg/L) | Squalene(mg/L) | β-carotene(%) | Squalene(%) |
PC | 60 | 38 | 248 | 0.25 | 1.66 |
1 | 59 | 54 | 154 | 0.36 | 1.04 |
2 | 61 | 35 | 214 | 0.23 | 1.41 |
3 | 65 | 62 | 212 | 0.38 | 1.32 |
4 | 50 | 30 | 102 | 0.24 | 0.82 |
5 | 62 | 61 | 194 | 0.39 | 1.24 |
6 | 62 | 55 | 282 | 0.36 | 1.83 |
7 | 55 | 46 | 173 | 0.33 | 1.27 |
8 | 48 | 40 | 180 | 0.34 | 1.52 |
9 | 56 | 34 | 132 | 0.25 | 0.95 |
10 | 50 | 46 | 185 | 0.36 | 1.48 |
11 | 51 | 41 | 120 | 0.33 | 0.94 |
12 | 62 | 52 | 218 | 0.34 | 1.42 |
13 | 63 | 57 | 210 | 0.37 | 1.34 |
14 | 59 | 53 | 230 | 0.36 | 1.57 |
15 | 58 | 57 | 185 | 0.39 | 1.29 |
16 | 53 | 40 | 219 | 0.31 | 1.66 |
17 | 60 | 60 | 121 | 0.40 | 0.81 |
18 | 59 | 53 | 206 | 0.36 | 1.40 |
19 | 62 | 50 | 207 | 0.32 | 1.33 |
20 | 62 | 59 | 189 | 0.38 | 1.22 |
21 | 60 | 69 | 108 | 0.46 | 0.72 |
22 | 59 | 60 | 148 | 0.41 | 1.00 |
23 | 62 | 58 | 134 | 0.37 | 0.86 |
24 | 60 | 57 | 198 | 0.38 | 1.33 |
25 | 64 | 48 | 214 | 0.30 | 1.33 |
26 | 67 | 56 | 228 | 0.34 | 1.37 |
27 | 59 | 47 | 214 | 0.32 | 1.45 |
28 | 61 | 35 | 183 | 0.23 | 1.20 |
29 | 60 | 51 | 219 | 0.34 | 1.45 |
30 | 59 | 48 | 201 | 0.33 | 1.38 |
31 | 64 | 61 | 198 | 0.38 | 1.23 |
32 | 65 | 66 | 169 | 0.40 | 1.04 |
33 | 59 | 47 | 206 | 0.32 | 1.40 |
34 | 62 | 51 | 238 | 0.32 | 1.53 |
35 | 53 | 37 | 189 | 0.28 | 1.42 |
36 | 61 | 53 | 225 | 0.35 | 1.47 |
조건 | 기준 | 선별 결과(플라스크 번호) | 비고 |
#1 | 베타카로틴 함량(%) ≥ 0.38 | 3, 5, 15, 17, 20, 21, 22, 24, 31, 32 | GGS1(PC)=0.25 |
#2 | 스쿠알렌 함량(%) < 1.05 | 2, 4, 9, 11, 17, 21, 22, 23, 32 | GGS1(PC)=1.66 |
조건 1 + 조건 2 | 17, 21, 22, 32 |
변이주 플라스크 번호. | Genotype |
17 | GGS1(I122V, R141K) |
21 | GGS1(N29T, L90R) |
22 | GGS1(M103I) |
32 | GGS1(F43I) |
Genotype | OD | β-carotene (mg/L) |
Squalene (mg/L) |
β-carotene (%) |
Squalene (%) |
NC | 62 | 5 | 208 | 0.03 | 1.34 |
PC | 56 | 30 | 133 | 0.21 | 0.95 |
M103I | 62 | 59 | 91 | 0.38 | 0.59 |
F43I | 63 | 69 | 71 | 0.44 | 0.45 |
N29T/L90R | 64 | 67 | 89 | 0.42 | 0.56 |
N29T | 61 | 55 | 88 | 0.36 | 0.58 |
L90R | 64 | 58 | 90 | 0.36 | 0.56 |
I122V/R141K | 62 | 62 | 65 | 0.40 | 0.42 |
I122V | 57 | 43 | 118 | 0.30 | 0.83 |
R141K | 59 | 44 | 111 | 0.30 | 0.75 |
Genotype | OD | β-carotene (mg/L) |
Squalene (mg/L) |
β-carotene (%) |
Squalene (%) |
CC08-1023(NC) | 46 | 5 | 41 | 0.04 | 0.36 |
CC08-1214(PC) | 50 | 32 | 17 | 0.26 | 0.14 |
CC08-1215 | 48 | 49 | 2 | 0.41 | 0.02 |
Claims (16)
- 서열번호 1의 아미노산 서열의 N-말단으로부터 29번, 43번, 90번, 103번, 122번, 및 141번 위치에 상응하는 아미노산 중 어느 하나 이상의 아미노산이 다른 아미노산으로 치환된, 제라닐제라닐 피로포스페이트 신타아제(Geranylgeranyl pyrophosphate synthase) 변이체.
- 제1항에 있어서, 상기 변이체는 서열번호 1의 아미노산 서열의 N-말단으로부터 29번 위치에 상응하는 아미노산이 아스파라긴 외의 아미노산으로 치환; 43번 아미노산에 상응하는 아미노산이 페닐알라닌 외의 아미노산으로 치환; 90번 아미노산에 상응하는 아미노산이 류신 외의 아미노산으로 치환; 103번 아미노산에 상응하는 아미노산이 메티오닌 외의 아미노산으로 치환; 122번 아미노산에 상응하는 아미노산이 이소류신 외의 아미노산으로 치환; 및 141번 아미노산에 상응하는 아미노산이 아르기닌 외의 아미노산으로 치환 중 하나 이상의 치환을 포함하는, 제라닐제라닐 피로포스페이트 신타아제 변이체.
- 제1항에 있어서, 다른 아미노산으로의 치환은 비극성(nonpolar) 아미노산, 극성(polar) 아미노산, 또는 양으로 하전된(염기성) 아미노산으로의 치환인 것인, 제라닐제라닐 피로포스페이트 신타아제 변이체.
- 제1항에 있어서, 상기 다른 아미노산은 트레오닌(Thr), 이소류신(Ile), 아르기닌(Arg), 발린(Val), 및 라이신(Lys)으로 구성되는 군에서 선택되는 어느 하나인 것인, 제라닐제라닐 피로포스페이트 신타아제 변이체.
- 제1항 내지 제4항 중 어느 한 항의 제라닐제라닐 피로포스페이트 신타아제 변이체를 코딩하는 폴리뉴클레오티드.
- 제1항 내지 제4항 중 어느 한 항의 제라닐제라닐 피로포스페이트 신타아제 변이체 및 상기 변이체를 코딩하는 폴리뉴클레오티드 중 어느 하나 이상을 포함하는, 미생물.
- 제6항에 있어서, 상기 미생물은 테트라테르펜, 테트라테르펜의 전구체, 또는 테트라테르펜을 전구체로 하는 물질을 생산하는 것인, 미생물.
- 제6항에 있어서, 상기 미생물은 야로위아 속(Yarrowia sp.)인, 미생물.
- 제6항에 있어서. 상기 미생물은 야로위아 리폴리티카 (Yarrowia lipolytica) 인, 미생물.
- 제6항에 있어서, 상기 미생물은 베타카로틴, 베타카로틴의 전구체, 또는 베타카로틴을 전구체로 하는 물질을 생산하는 것인, 미생물.
- 제6항의 미생물을 배지에서 배양하는 단계를 포함하는, 테트라테르펜, 테트라테르펜의 전구체, 또는 테트라테르펜을 전구체로 하는 물질 생산 방법.
- 제11항에 있어서, 상기 방법은 상기 배지 또는 미생물로부터 테트라테르펜, 테트라테르펜의 전구체, 또는 테트라테르펜을 전구체로 하는 물질을 회수하는 단계를 추가로 포함하는, 생산 방법.
- 제11항에 있어서, 상기 미생물은 야로위아 속(Yarrowia sp.)인, 생산 방법.
- 제11항에 있어서, 상기 방법은 스쿠알렌 생산을 감소시키는 것인, 생산 방법.
- 제1항의 변이체, 제6항의 미생물, 또는 상기 미생물의 배양물을 포함하는, 테트라테르펜, 테트라테르펜의 전구체, 또는 테트라테르펜을 전구체로 하는 물질 생산용 조성물.
- 서열번호 1의 아미노산 서열의 N-말단으로부터 29번, 43번, 90번, 103번, 122번, 및 141번 위치에 상응하는 아미노산 중 어느 하나 이상의 아미노산이 다른 아미노산으로 치환된, 제라닐제라닐 피로포스페이트 신타아제 변이체; 및 상기 변이체를 코딩하는 폴리뉴클레오티드 중 어느 하나 이상을 포함하는, 미생물의 테트라테르펜, 테트라테르펜의 전구체, 또는 테트라테르펜을 전구체로 하는 물질 생산 용도.
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