Modified cell

Abstract

To provide a technique for efficiently obtaining non-natural ovalicin compounds (e.g. Demethyl Ovalicin, Demethoxy Ovalicin, Dihydro Ovalicin, Demethyl Chlovalicin).SOLUTION: The present invention provides a modified cell comprising polynucleotide A, which contains the coding sequence of protein A with an amino acid sequence that has at least 70% identity to a specific amino acid sequence and exhibits conversion activity from 5-keto-demethoxyfumagillol to Demethoxy Ovalicin.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to modified cells, etc. [Background technology]

[0002] Amoebiasis is an intestinal protozoal disease that infects approximately 1% of the world's population, and it causes great damage, especially in tropical developing countries. The current drug, metronidazole, has been found to have side effects such as genotoxicity and to cause drug resistance, so the development of safe drugs with a different mode of action is necessary.

[0003] In this context, the natural product Fumagillin has been found to have strong anti-Amebic encephalomyelitis activity, and has been reported to be effective in treating liver abscess in animal models. Fumagillin derivatives have been in clinical development for more than 30 years for the treatment of cancer, obesity, malaria, and other diseases, but have not yet been marketed due to side effects such as neurotoxicity. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] J. Am. Chem. Soc. 2014, 136, 4426-4436. Summary of the Invention [Problem to be solved by the invention]

[0005] The present inventors focused on Ovalicin, which has a structure similar to Fumagillin.

[0006] [ka]

[0007] Derivatization of fumagillin has been performed by converting the sesquiterpene structure at the 5-position to another structure. The present inventors focused on the methoxy at the 6-position of ovalicin and attempted to convert it to another structure. Specifically, if a compound (demethyl ovalicin) in which the methoxy of ovalicin is demethylated and converted to a hydroxyl can be obtained, it is possible to obtain ovalicin derivatives with various structures by using the hydroxyl (for example, by reacting with a carboxyl compound). However, ovalicin contains two epoxides that are easily decomposed, and it is difficult to efficiently obtain demethyl ovalicin by organic synthesis methods.

[0008] Therefore, the present inventors focused on obtaining demethyl ovalicin through biosynthesis (Figure 1). However, the enzyme X (7-hydroxylation enzyme Ova-C7H) that converts 5-keto-demethoxyfumagillol to demethoxy ovalicin, which is essential for the biosynthesis, is still unknown.

[0009] An objective of the present invention is to provide a technique for efficiently obtaining non-natural ovalicins (for example, demethyl ovalicin, demethoxy ovalicin, dihydro ovalicin, demethyl chloricin, etc.). [Means for solving the problem]

[0010] In view of the above problems, the present inventors have conducted intensive research and found that the above problems can be solved by (A) a modified cell comprising polynucleotide A that contains an amino acid sequence having 70% or more identity with the amino acid sequence shown in SEQ ID NO: 1 and that contains a coding sequence for protein A having the activity of converting 5-keto-demethoxyfumagillol to demethoxy ovalicin. Based on this finding, the present inventors have conducted further research and completed the present invention. That is, the present invention encompasses the following aspects.

[0011] Item 1. (A) A modified cell comprising polynucleotide A that contains an amino acid sequence having 70% or more identity to the amino acid sequence shown in SEQ ID NO:1 and a coding sequence for protein A having the activity of converting 5-keto-demethoxyfumagillol to demethoxy ovalicin.

[0012] Item 2. The modified cell according to Item 1, comprising (B) a polynucleotide B comprising a coding sequence for a protein B having an activity of converting demethoxy ovalicin to demethyl ovalicin.

[0013] Item 3. The modified cell according to Item 2, wherein the protein B is (B1) protein B1 comprising an amino acid sequence having 70% or more identity to the amino acid sequence shown in SEQ ID NO:2.

[0014] Item 4. The modified cell according to Item 2, comprising (C) a polynucleotide C including a coding sequence for a protein C having an activity of converting β-trans-bergamotene to 5-keto-demethoxyfumagillol.

[0015] Item 5. The modified cell according to Item 4, wherein the protein C is (C1) protein C1 comprising an amino acid sequence having 70% or more identity to the amino acid sequence shown in SEQ ID NO:3.

[0016] Item 6. The modified cell according to Item 4, comprising (D) a polynucleotide D including a coding sequence for a protein D having an activity of converting farnesyl diphosphate to β-trans-bergamotene.

[0017] Item 7. The modified cell according to Item 6, wherein the protein D is (D1) protein D1 comprising an amino acid sequence having 70% or more identity to the amino acid sequence shown in SEQ ID NO:4.

[0018] Item 8. The polynucleotide A is an exogenous polynucleotide, and / or (EX) The vector does not express protein E having the activity of converting demethyl ovalicin to ovalicin, or is modified so that the expression or function of the protein E is reduced. The modified cell described in item 1.

[0019] Item 9. The modified cell of Item 1, which is a eukaryotic cell.

[0020] Item 10. The modified cell according to Item 9, which is a cell capable of forming mycelium and spores.

[0021] Item 11. A method for producing demethyl ovalicin, comprising culturing the modified cell according to any one of Items 1 to 10 having the ability to produce demethyl ovalicin.

[0022] Item 12. A method for producing ovalicin, comprising culturing the modified cell according to any one of items 1 to 10 having an ability to produce ovalicin.

[0023] Item 13. A method for producing demethoxy ovalicin, comprising culturing the modified cell according to any one of Items 1 to 10 having the ability to produce demethoxy ovalicin.

[0024] Item 14. The production method according to Item 11, wherein a culture medium containing an adsorption resin is used as the culture medium.

[0025] Section 15. Formula (A):

[0026] [ka]

[0027] A compound represented by the formula:

[0028] Section 16. Formula (B):

[0029] [ka]

[0030] A compound represented by the formula: Effect of the Invention

[0031] According to the present invention, it is possible to provide a technique for efficiently obtaining non-natural ovalicin (e.g., demethyl ovalicin, demethoxy ovalicin, dihydro ovalicin, demethyl chloricin, etc.). Specifically, it is possible to provide modified cells containing an enzyme (protein A having the activity of converting 5-keto-demethoxyfumagillol to demethoxy ovalicin) required for this technique, and a method for producing non-natural ovalicin (e.g., demethyl ovalicin, demethoxy ovalicin, dihydro ovalicin, demethyl chloricin, etc.) using the modified cells. [Brief description of the drawings]

[0032] [Figure 1] The biosynthetic pathways of Demethyl Ovalicin and Ovalicin are shown. [Diagram 2] The NMR measurement data of Demethyl Ovalicin is shown below. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0033] 1.Definition In this specification, the expressions "contain" and "comprise" include the concepts of "contain", "include", "consist essentially of" and "consist only of".

[0034] As used herein, the term "identity" of an amino acid sequence refers to the degree of amino acid sequence agreement between two or more comparable amino acid sequences. Thus, the higher the agreement between two amino acid sequences, the higher the identity or similarity between those sequences. The level of identity of an amino acid sequence can be determined, for example, using the sequence analysis tool FASTA with default parameters. Alternatively, it can be determined using the BLAST algorithm by Karlin and Altschul (Karlin S, Altschul SF. "Methods for assessing the statistical significance of molecular sequence features by using general scoring schemes" Proc. Natl. Acad. Sci. USA. 87: 2264-2268 (1990); Karlin S, Altschul SF. "Applications and statistics for multiple high-scoring segments in molecular sequences" Proc. Natl. Acad. Sci. USA. 90: 5873-7 (1993)). A program called BLASTX based on such a BLAST algorithm has been developed. Specific techniques for these analysis methods are known and can be found on the National Center of Biotechnology Information (NCBI) website (http: / / www.ncbi.nlm.nih.gov / ). The "identity" of a base sequence is also defined in the same manner as above.

[0035] In this specification, "conservative substitution" means that an amino acid residue is replaced with an amino acid residue having a similar side chain. For example, substitution between amino acid residues having basic side chains such as lysine, arginine, and histidine is considered to be conservative substitution. In addition, substitution between amino acid residues having acidic side chains such as aspartic acid and glutamic acid; amino acid residues having non-charged polar side chains such as glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine; amino acid residues having non-polar side chains such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; amino acid residues having β-branched side chains such as threonine, valine, and isoleucine; and amino acid residues having aromatic side chains such as tyrosine, phenylalanine, tryptophan, and histidine are also considered to be conservative substitutions.

[0036] In the present specification, polynucleotides such as DNA and RNA may be chemically modified as shown below. To prevent degradation by hydrolases such as nucleases, the phosphoric acid residues (phosphates) of each nucleotide may be replaced with chemically modified phosphoric acid residues such as phosphorothioate (PS), methylphosphonate, and phosphorodithioate. The hydroxyl group at the 2-position of the sugar (ribose) of each ribonucleotide may be replaced with -OR (R represents, for example, CH3(2'-O-Me), CH2CHOCH3(2'-O-MOE), CH2CH2NHC(NH)NH2, CH2CONHCH3, CH2CH2CN, etc.). Furthermore, the base portion (pyrimidine, purine) may be chemically modified, for example, by introducing a methyl group or a cationic functional group into the 5-position of the pyrimidine base, or by replacing the carbonyl group at the 2-position with a thiocarbonyl. Further examples include those in which the phosphate moiety or hydroxyl moiety is modified with, for example, biotin, an amino group, a lower alkylamine group, an acetyl group, etc. In addition, BNA (LNA), in which the conformation of the sugar moiety of the nucleotide is fixed to N-type by bridging the 2' oxygen and 4' carbon of the sugar moiety, can also be preferably used.

[0037] 2. Engineered Cells In one aspect, the present invention relates to a modified cell (also referred to as the "modified cell of the present invention" in this specification) that contains (A) an amino acid sequence having 70% or more identity with the amino acid sequence shown in SEQ ID NO: 1 and a coding sequence of a protein A having an activity of converting 5-keto-demethoxyfumagillol to demethoxy ovalicin. This will be described below.

[0038] SEQ ID NO: 1 is the amino acid sequence of a protein derived from the insect-infectious filamentous fungus Metarhizium robertsii ARSEF23 strain, and is the amino acid sequence of NCBI accession number: XP_007824537.1.

[0039] The identity in (A) above is preferably 80% or more, more preferably 90% or more, even more preferably 95% or more, still more preferably 97% or more, particularly preferably 99% or more, and particularly preferably 100%.

[0040] In the above (A), when the identity is less than 100%, the number of amino acids mutated (substituted, deleted, added, or inserted) relative to the amino acid sequence of SEQ ID NO: 1 is preferably 1 to 10, more preferably 1 to 5, even more preferably 1 to 3, and even more preferably 1 to 2. The amino acid mutation is preferably a substitution, and more preferably a conservative substitution.

[0041] The structures of 5-keto-demethoxyfumagillol and demethoxy ovalicin are shown in FIG.

[0042] The presence or absence of the activity of converting 5-keto-demethoxyfumagillol to demethoxy ovalicin can be confirmed by in vitro or in vivo tests. In the case of an in vivo test, for example, cells that produce 5-keto-demethoxyfumagillol are used as cells or 5-keto-demethoxyfumagillol is introduced into cells, and the cells are cultured with or without expressing a test protein. If demethoxy ovalicin is produced only when the test protein is expressed, it can be determined that the test protein has the activity of converting 5-keto-demethoxyfumagillol to demethoxy ovalicin.

[0043] In addition to the above amino acid sequence, Protein A may contain other sequences such as known protein tags, proteins or peptides such as signal sequences, etc. Examples of protein tags include biotin, His tag, FLAG tag, Halo tag, MBP tag, HA tag, Myc tag, V5 tag, and PA tag.

[0044] In one embodiment, polynucleotide A contains a promoter upstream of the coding sequence for expressing mRNA of protein A. The promoter is not particularly limited and can be appropriately selected depending on the host. Examples of the promoter include the Aspergillus oryzae TAA gene (amyB) promoter, the Aspergillus oryzae GLA gene (glaA) promoter, and the Aspergillus oryzae AGL gene (agdA) promoter. Other examples include the TDH3 promoter, the GAL10 promoter, the CMV promoter, the EF1 promoter, the SV40 promoter, the MSCV promoter, and the CAG promoter.

[0045] Polynucleotide A may contain other elements (e.g., a multicloning site (MCS), a drug resistance gene, a replication origin, etc.) as necessary. For example, in the case where a promoter and a protein coding sequence are arranged in this order from the 5' side in a polynucleotide, an MCS may be arranged between the promoter and the coding sequence (preferably adjacent to either one or both) and on the 3' side of the coding sequence (preferably adjacent to). There are no particular limitations on the MCS, so long as it contains multiple (e.g., 2 to 50, preferably 2 to 20, more preferably 2 to 10) restriction enzyme sites.

[0046] Polynucleotide A may constitute a vector. The type of vector is not particularly limited and is appropriately selected depending on the host. For example, in E. coli, ColE1-based plasmids such as pBR322 derivatives, pACYC-based plasmids having p15A origin, pSC-based plasmids, and mini-F plasmids derived from F factors such as Bac-based plasmids can be mentioned.

[0047] The modified cells of the present invention containing polynucleotide A can be used to prepare cells for producing demethyl ovalicin, and in a preferred embodiment, can be used as cells for producing demethyl ovalicin.

[0048] The modified cell of the present invention preferably further comprises (B) a polynucleotide B comprising a coding sequence for a protein B having an activity of converting demethoxy ovalicin to demethyl ovalicin. This allows the cell to produce demethyl ovalicin under conditions in which 5-keto-demethoxyfumagillol is supplied.

[0049] Protein B having the activity of converting demethoxy ovalicin to demethyl ovalicin is publicly known, or can be easily identified or produced based on homology analysis based on a known amino acid sequence.

[0050] Protein B is preferably (B1) protein B1 comprising an amino acid sequence having 70% or more identity with the amino acid sequence shown in SEQ ID NO:2. SEQ ID NO: 2 is the amino acid sequence of a protein derived from Aspergillus fumigatus strain A1159, and is the amino acid sequence of NCBI accession number: XP_747157.2.

[0051] The identity in (B1) above is preferably 80% or more, more preferably 90% or more, even more preferably 95% or more, still more preferably 97% or more, particularly preferably 99% or more, and particularly preferably 100%.

[0052] In the above (B1), when the identity is less than 100%, the number of amino acids mutated (substituted, deleted, added, or inserted) relative to the amino acid sequence of SEQ ID NO: 2 is preferably 1 to 10, more preferably 1 to 5, even more preferably 1 to 3, and even more preferably 1 to 2. The amino acid mutation is preferably a substitution, and more preferably a conservative substitution.

[0053] The structures of Demethoxy Ovalicin and Demethyl Ovalicin are shown in FIG.

[0054] The presence or absence of the activity of converting demethoxy ovalicin to demethyl ovalicin can be confirmed by in vitro or in vivo tests. In the case of an in vivo test, for example, cells that produce demethoxy ovalicin are used as cells or demethoxy ovalicin is introduced into cells, and the cells are cultured with or without expressing a test protein. If demethyl ovalicin is produced only when the test protein is expressed, it can be determined that the test protein has the activity of converting demethoxy ovalicin to demethyl ovalicin.

[0055] Other sequences contained in protein B and the structure of polynucleotide B are the same as those of protein A and polynucleotide A.

[0056] The modified cell of the present invention further comprising polynucleotide B can be used to prepare a cell for producing demethyl ovalicin, and can be used as a cell for producing demethyl ovalicin.

[0057] The modified cell of the present invention preferably further comprises (C) a polynucleotide C comprising a coding sequence for a protein C having an activity of converting β-trans-bergamotene to 5-keto-demethoxyfumagillol. This allows the cell to produce demethoxy ovalicin under conditions in which β-trans-bergamotene is supplied, and further allows the cell to produce demethyl ovalicin when the cell further comprises polynucleotide B.

[0058] Protein C having the activity of converting β-trans-bergamotene to 5-keto-demethoxyfumagillol is either publicly known or can be easily identified or produced based on homology analysis based on the known amino acid sequence.

[0059] Protein C is preferably (C1) protein C1 comprising an amino acid sequence having 70% or more identity with the amino acid sequence shown in SEQ ID NO:3. SEQ ID NO: 3 is the amino acid sequence of a protein derived from Aspergillus fumigatus strain A1159, and is the amino acid sequence of NCBI accession number: XP_747154.1.

[0060] The identity in (C1) above is preferably 80% or more, more preferably 90% or more, even more preferably 95% or more, still more preferably 97% or more, particularly preferably 99% or more, and particularly preferably 100%.

[0061] In the above (C1), when the identity is less than 100%, the number of amino acids mutated (substituted, deleted, added, or inserted) relative to the amino acid sequence of SEQ ID NO: 3 is preferably 1 to 10, more preferably 1 to 5, even more preferably 1 to 3, and even more preferably 1 to 2. The amino acid mutation is preferably a substitution, and more preferably a conservative substitution.

[0062] The structures of β-trans-bergamotene and 5-keto-demethoxyfumagillol are shown in FIG.

[0063] The presence or absence of the activity of converting β-trans-bergamotene to 5-keto-demethoxyfumagillol can be confirmed by in vitro or in vivo tests. In the case of an in vivo test, for example, cells that produce β-trans-bergamotene are used as cells or β-trans-bergamotene is introduced into cells, and the cells are cultured with or without expressing a test protein. If 5-keto-demethoxyfumagillol is produced only when the test protein is expressed, it can be determined that the test protein has the activity of converting β-trans-bergamotene to 5-keto-demethoxyfumagillol.

[0064] Other sequences contained in protein C and the structure of polynucleotide C are the same as those of protein A and polynucleotide A.

[0065] The modified cell of the present invention further comprising polynucleotide C can be used to prepare a cell for producing demethyl ovalicin, and in a preferred embodiment, can be used as a cell for producing demethyl ovalicin.

[0066] The modified cell of the present invention preferably further comprises (D) a polynucleotide D comprising a coding sequence for protein D having activity of converting farnesyl diphosphate to β-trans-bergamotene. This allows the cell to produce demethoxy ovalicin under conditions in which farnesyl diphosphate is supplied when the cell further comprises polynucleotide C, and allows the cell to produce demethyl ovalicin when the cell further comprises polynucleotide B in addition to polynucleotide C.

[0067] Protein D having the activity of converting farnesyl diphosphate to β-trans-bergamotene is publicly known, or can be easily identified or produced based on homology analysis based on the publicly known amino acid sequence.

[0068] Protein D is preferably (D1) protein D1 comprising an amino acid sequence having 70% or more identity with the amino acid sequence shown in SEQ ID NO: 4. SEQ ID NO: 4 is the amino acid sequence of a protein derived from Aspergillus fumigatus strain A1159, and is the amino acid sequence of NCBI accession number: XP_747153.1.

[0069] The identity in (D1) above is preferably 80% or more, more preferably 90% or more, even more preferably 95% or more, still more preferably 97% or more, particularly preferably 99% or more, and particularly preferably 100%.

[0070] When the identity in (D1) above is less than 100%, the number of amino acids mutated (substituted, deleted, added, or inserted) relative to the amino acid sequence of SEQ ID NO: 4 is preferably 1 to 10, more preferably 1 to 5, even more preferably 1 to 3, and even more preferably 1 to 2. The amino acid mutation is preferably a substitution, and more preferably a conservative substitution.

[0071] The structures of farnesyl diphosphate and β-trans-bergamotene are shown in Figure 1.

[0072] The presence or absence of the activity of converting farnesyl diphosphate to β-trans-bergamotene can be confirmed by in vitro or in vivo tests. In the case of an in vivo test, for example, cells that produce farnesyl diphosphate are used as cells or farnesyl diphosphate is introduced into cells, and the cells are cultured with or without expressing a test protein. If β-trans-bergamotene is produced only when the test protein is expressed, the test protein can be determined to have the activity of converting farnesyl diphosphate to β-trans-bergamotene.

[0073] Other sequences contained in protein D and the structure of polynucleotide D are the same as those of protein A and polynucleotide A.

[0074] The modified cell of the present invention further comprising polynucleotide D can be used to prepare a cell for producing demethyl ovalicin, and in a preferred embodiment, can be used as a cell for producing demethyl ovalicin.

[0075] The modified cell of the present invention can further comprise a polynucleotide E comprising a coding sequence for a protein E having an activity of converting (E) Demethyl Ovalicin to Ovalicin, thereby enabling the production of Ovalicin.

[0076] Protein E having the activity of converting demethyl ovalicin to ovalicin is publicly known, or can be easily identified or produced based on homology analysis based on the publicly known amino acid sequence.

[0077] Protein E is preferably (E1) protein E1 comprising an amino acid sequence having 70% or more identity with the amino acid sequence shown in SEQ ID NO:5. SEQ ID NO:5 is the amino acid sequence of a protein derived from Aspergillus fumigatus strain A1159, and is the amino acid sequence of NCBI accession number: AHL19974.1.

[0078] The identity in (E1) above is preferably 80% or more, more preferably 90% or more, even more preferably 95% or more, still more preferably 97% or more, particularly preferably 99% or more, and particularly preferably 100%.

[0079] In the above (E1), when the identity is less than 100%, the number of amino acids mutated (substituted, deleted, added, or inserted) relative to the amino acid sequence of SEQ ID NO: 5 is preferably 1 to 10, more preferably 1 to 5, even more preferably 1 to 3, and even more preferably 1 to 2. The amino acid mutation is preferably a substitution, and more preferably a conservative substitution.

[0080] The structures of Demethyl Ovalicin and Ovalicin are as shown in FIG.

[0081] The presence or absence of the activity of converting demethyl ovalicin to ovalicin can be confirmed by in vitro or in vivo tests. In the case of an in vivo test, for example, cells that produce demethyl ovalicin are used as cells or demethyl ovalicin is introduced into cells, and the cells are cultured with / without expressing a test protein. If Ovalicin is produced only when the test protein is expressed, the test protein can be determined to have the activity of converting demethyl ovalicin to ovalicin.

[0082] Other sequences contained in protein E and the structure of polynucleotide E are the same as those of protein A and polynucleotide A.

[0083] The modified cell of the present invention can further comprise (F) a polynucleotide F comprising a coding sequence for a protein F having an activity of converting ovalicin to dihydro ovalicin, thereby enabling the production of dihydro ovalicin.

[0084] Protein F having the activity of converting ovalicin to dihydro ovalicin is publicly known, or can be easily identified or produced based on homology analysis based on the publicly known amino acid sequence.

[0085] Protein F is preferably (F1) protein F1 comprising an amino acid sequence having 70% or more identity with the amino acid sequence shown in SEQ ID NO:6. SEQ ID NO: 6 is the amino acid sequence of a protein derived from Aspergillus fumigatus strain A1159, and is the amino acid sequence of NCBI accession number: XP_747156.1.

[0086] The identity in (F1) above is preferably 80% or more, more preferably 90% or more, even more preferably 95% or more, still more preferably 97% or more, particularly preferably 99% or more, and particularly preferably 100%.

[0087] When the identity in (F1) above is less than 100%, the number of amino acids mutated (substituted, deleted, added, or inserted) relative to the amino acid sequence of SEQ ID NO: 6 is preferably 1 to 10, more preferably 1 to 5, even more preferably 1 to 3, and even more preferably 1 to 2. The amino acid mutation is preferably a substitution, and more preferably a conservative substitution.

[0088] The structures of Ovalicin and Dihydro Ovalicin are shown in FIG.

[0089] The presence or absence of the activity of converting ovalicin to dihydro ovalicin can be confirmed by in vitro or in vivo tests. In the case of an in vivo test, for example, cells that produce ovalicin are used as cells or ovalicin is introduced into cells, and the cells are cultured with / without expressing a test protein. If ovalicin is produced only when the test protein is expressed, it can be determined that the test protein has the activity of converting ovalicin to dihydro ovalicin.

[0090] Other sequences contained in protein F and the structure of polynucleotide F are the same as those of protein A and polynucleotide A.

[0091] The above-mentioned polynucleotides can be easily prepared according to known genetic engineering techniques, for example, by using PCR, restriction enzyme digestion, DNA ligation techniques, and the like.

[0092] The modified cells of the present invention are cells obtained by modifying existing cells. The modification is a genetic modification, and includes the introduction of an exogenous polynucleotide, the introduction of a mutation into an endogenous genome, and the like.

[0093] An exogenous polynucleotide is a polynucleotide that contains a base sequence that is not derived from the genomic DNA (particularly chromosomal genomic DNA) of the cell before modification, and is not particularly limited in this respect.

[0094] Examples of mutations in the endogenous genome include gene disruption, mutations in protein coding regions, partial deletions, mutations in splicing regulatory regions, and mutations in expression control regions (e.g., promoters, activators, enhancers, repressor binding elements, etc.).

[0095] The method for genetically modifying cells is not particularly limited, and any method according to or similar to a known method can be used, such as the protoplast-PEG method, electroporation method, lipofection method, particle gun method, CRISPR-Cas method, etc.

[0096] The cell to be modified may be either a eukaryotic cell or a prokaryotic cell. However, when the modified cell of the present invention is used as a cell for producing Demethyl Ovalicin, a eukaryotic cell is preferred. From the viewpoint of efficiency in producing Demethyl Ovalicin, the eukaryotic cell is preferably a fungal cell, and among them, a cell capable of forming mycelium and spores is particularly preferred. The cell capable of forming mycelium and spores is not particularly limited as long as it is a cell that has both a period in which mycelium is formed and a period in which spores are formed in the life cycle, and examples thereof include fungi of the genus Aspergillus, fungi of the genus Penicillium, fungi of the genus Trichoderma, fungi of the genus Cephalosporium, fungi of the genus Acremonium, etc., and particularly preferred is fungi of the genus Aspergillus, and particularly preferred is Aspergillus nidulans.

[0097] When at least one of polynucleotides B to F is included, polynucleotides A to F may all be separate polynucleotide molecules, or two or more of polynucleotides A to F may be the same polynucleotide molecule.

[0098] In one embodiment of the present invention, polynucleotide A is an exogenous polynucleotide. In another embodiment of the present invention, when at least one of polynucleotides B to F is included, two or more (preferably three or more, particularly preferably four) types of polynucleotides including polynucleotide A are exogenous polynucleotides.

[0099] In one embodiment of the present invention, (EX) the strain is modified so as not to express protein E having the activity of converting demethyl ovalicin to ovalicin, or to reduce the expression or function of said protein E. This inhibits the conversion of demethyl ovalicin to ovalicin, and demethyl ovalicin can be obtained more efficiently.

[0100] "Function" refers to the activity of converting dimethyl ovalicin to ovalicin. "Expression" includes both gene mRNA expression and gene protein expression, but is preferably gene protein expression. "Reduction" refers to the activity of the gene protein and / or the expression level of the gene after modification (index value of the function and / or expression of the gene) being, for example, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, 2% or less, 1% or less, 0.5% or less, 0.2% or less, 0.1% or less, 0.05% or less, 0.02% or less, or 0.01% or less, relative to 100% of the activity of the gene protein and / or the expression level of the gene before modification (index value of the function and / or expression when the gene is not modified).

[0101] The modified cells of the present invention have the ability to produce, for example, non-natural ovalicins (e.g., demethyl ovalicin, demethoxy ovalicin, dihydro ovalicin, demethyl chlovalicin, etc.), and preferably demethyl ovalicin. The enzymes and starting materials required for the production of each non-natural ovalicin can be appropriately determined by referring to the scheme in FIG. 1. Specifically, for example, the modified cells of the present invention can produce demethyl ovalicin under the condition of supplying 5-keto-demethoxyfumagillol if they contain polynucleotide B in addition to polynucleotide A. Particularly preferably, the modified cells of the present invention contain all of polynucleotides A to D. This allows demethyl ovalicin to be obtained by utilizing farnesyl diphosphate (which is one of the terpenoids essential for the life support of living organisms and is also a biosynthetic intermediate for various terpenoids such as useful carotenoids, plant hormones, and ubiquinone, and is biosynthesized in many eukaryotic cells).

[0102] 3. Method for producing non-natural ovalicin In one aspect, the present invention relates to a method for producing non-natural ovalicin (e.g., demethyl ovalicin, demethoxy ovalicin, dihydro ovalicin, demethyl chlovalicin, etc.) (sometimes referred to as the "production method of the present invention" in this specification), which comprises culturing a modified cell of the present invention capable of producing non-natural ovalicin. This will be described below.

[0103] The ability to produce non-naturally occurring ovalicin and the modified cells of the present invention are as described above.

[0104] The culture can be carried out according to or in accordance with a method known to those skilled in the art, depending on the species of the modified cells of the present invention.

[0105] The medium (e.g., culture solution) preferably contains a carbon source. Examples of the carbon source include carbohydrates such as monosaccharides, disaccharides, oligosaccharides, and polysaccharides; invert sugar obtained by hydrolyzing sucrose; glycerol; compounds with one carbon atom (hereinafter referred to as C1 compounds) such as methanol, formaldehyde, formate, carbon monoxide, and carbon dioxide; oils such as corn oil, palm oil, and soybean oil; acetate; animal fats and oils; animal oils; fatty acids such as saturated fatty acids and unsaturated fatty acids; lipids; phospholipids; glycerolipids; glycerol fatty acid esters such as monoglycerides, diglycerides, and triglycerides; polypeptides such as microbial proteins and plant proteins; renewable carbon sources such as hydrolyzed biomass carbon sources; yeast extract; and combinations thereof. Examples of the nitrogen source include inorganic ammonium salts such as ammonium sulfate, ammonium chloride, and ammonium phosphate; organic nitrogen such as soybean hydrolysate; ammonia gas; and aqueous ammonia. Examples of the organic trace nutrient source include a required substance such as vitamin B1 and L-homoserine, and yeast extract in an appropriate amount. In addition to these, small amounts of potassium phosphate, magnesium sulfate, iron ions, manganese ions, etc. may be added as necessary. The medium used in the present invention may be either a natural medium or a synthetic medium, so long as it contains a carbon source, a nitrogen source, inorganic ions, and other organic trace components as necessary.

[0106] Examples of monosaccharides include trioses such as ketotriose (dihydroxyacetone) and aldotriose (glyceraldehyde); tetroses such as ketotetrose (erythrulose) and aldotetrose (erythrose, threose); pentoses such as ketopentose (ribulose, xylulose), aldopentose (ribose, arabinose, xylose, lyxose) and deoxysugar (deoxyribose); hexoses such as ketohexose (psicose, fructose, sorbose, tagatose), aldohexose (allose, altrose, glucose, mannose, gulose, idose, galactose, talose) and deoxysugar (fucose, fuculose, rhamnose); and heptoses such as sedoheptulose. Among these, carbohydrates such as C6 sugars such as fructose, mannose, galactose, and glucose; and C5 sugars such as xylose and arabinose are preferred.

[0107] Examples of disaccharides include sucrose, lactose, maltose, trehalose, turanose, cellobiose, and the like, with sucrose and lactose being preferred.

[0108] Examples of oligosaccharides include trisaccharides such as raffinose, melezitose, and maltotriose; tetrasaccharides such as acarbose and stachyose; and other oligosaccharides such as fructooligosaccharides (FOS), galactooligosaccharides (GOS), and mannanoligosaccharides (MOS).

[0109] Examples of polysaccharides include glycogen, starch (amylose, amylopectin), cellulose, dextrin, and glucan (β1,3-glucan). Starch and cellulose are preferred.

[0110] Microbial proteins include polypeptides obtainable from yeast or bacteria.

[0111] Vegetable proteins include polypeptides obtainable from soybean, corn, canola, jatropha, palm, peanut, sunflower, coconut, mustard, cottonseed, palm kernel oil, olive, safflower, sesame, and linseed.

[0112] Lipids include substances that contain one or more saturated or unsaturated fatty acids of C4 or higher.

[0113] The oil is preferably a lipid that contains one or more C4 or higher saturated or unsaturated fatty acids and is liquid at room temperature, and examples of the lipid include lipids that can be obtained from soybean, corn, canola, jatropha, palm, peanut, sunflower, coconut, mustard, cottonseed, palm kernel oil, olive, safflower, sesame, linseed, oleaginous microbial cells, Chinese tallow tree, or a combination of two or more thereof.

[0114] Fatty acids include compounds represented by the formula RCOOH, where "R" represents a hydrocarbon group.

[0115] Unsaturated fatty acids are compounds that have at least one carbon-carbon double bond at "R", and examples include oleic acid, vaccenic acid, linoleic acid, palmitelaidic acid, and arachidonic acid.

[0116] Saturated fatty acids are compounds where "R" is a saturated aliphatic group, and include docosanoic acid, eicosanoic acid, octadecanoic acid, hexadecanoic acid, tetradecanoic acid, dodecanoic acid, and the like.

[0117] Among them, the fatty acid preferably contains one or more C2 to C22 fatty acids, and more preferably contains C12 fatty acids, C14 fatty acids, C16 fatty acids, C18 fatty acids, C20 fatty acids, and C22 fatty acids.

[0118] Further, examples of the carbon source include salts of these fatty acids, derivatives, and salts of the derivatives. Examples of the salts include lithium salts, potassium salts, and sodium salts.

[0119] Further, examples of the carbon source include combinations of lipids, oils, fats, fatty acids, and glycerin fatty acid esters with carbohydrates such as glucose.

[0120] Renewable carbon sources include hydrolyzed biomass carbon sources.

[0121] Biomass carbon sources include cellulosic substrates such as wood, paper, and pulp waste, plant leaves, and fruit pulp; plant parts such as stalks, grains, roots, and tubers.

[0122] Plants that can be used as biomass carbon sources include legumes such as corn, wheat, rye, sorghum, triticale, rice, foxtail millet, barley, cassava, and peas, as well as potatoes, sweet potatoes, bananas, sugarcane, and tapioca.

[0123] When a renewable carbon source such as biomass is added to a medium, it is preferable to pretreat the source by enzymatic pretreatment, chemical pretreatment, or a combination of enzymatic and chemical pretreatment.

[0124] In the production method of the present invention, it is preferable to use a culture solution containing an adsorption resin as the culture solution. This can increase the production efficiency of Demethyl Ovalicin. The adsorption resin is not particularly limited as long as it is a resin material that can adsorb various organic substances by physical interaction between the surface and the adsorbed substance, and typically has a porous structure (preferably a specific surface area of ​​300 to 1200 m2). 2 / dry-g) Examples of the resin include styrene-divinylbenzene, styrene, divinylbenzene, and methacrylic acid ester.

[0125] When an adsorption resin is used, its concentration in the culture medium is preferably 20 to 100 g / L.

[0126] The culture temperature is preferably 20°C to 37°C.

[0127] The culture time is preferably 20 to 100 hours.

[0128] The modified cells of the present invention are preferably cultured under aerobic, anaerobic, or anoxic conditions depending on the nature of the host.

[0129] Examples of methods for culturing the modified cells of the present invention include methods using known fermentation methods such as batch culture, fed-batch culture, and continuous culture.

[0130] After the cultivation is completed, solid-liquid separation such as filtration and centrifugation is performed, and the intracellular components are extracted from the obtained solid sample (for example, organic solvent extraction such as acetone extraction, ultrasonic disruption, etc.), and the obtained soluble portion is purified by chromatography to obtain the target product.

[0131] In one embodiment, the present invention relates to a non-natural ovalicin obtained as described above. For example, in one embodiment, the present invention relates to a non-natural ovalicin represented by the formula (A):

[0132] [ka]

[0133] or a solvate thereof, Formula (B):

[0134] [ka]

[0135] or a solvate thereof, Regarding.

[0136] Examples of the solvent that constitutes the solvate include pharma- ceutically acceptable organic solvents (eg, ethanol, glycerol, acetic acid, etc.). EXAMPLES

[0137] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

[0138] Test Example 1. Biosynthetic design and enzyme screening of Demethyl Ovalicin Based on the biosynthetic pathway of Fumagillin described in Non-Patent Document 1, the biosynthetic pathways of Demethyl Ovalicin and Ovalicin were designed (FIG. 1).

[0139] [ka]

[0140] However, the enzyme X (7-hydroxylation enzyme Ova-C7H) that converts 5-keto-demethoxyfumagillol to demethoxy ovalicin is still unknown.

[0141] Although no biosynthetic gene cluster (BGC) for OVs had been reported, genome mining revealed a candidate BGC in the insect-infecting fungus Metarhizium robertsii ARSEF23. RNA-seq data for this strain under multiple medium conditions have been registered in a public database (SRA), and in silico analysis confirmed high expression of this BGC in Sabouraud medium. M. robertsii was actually cultured under the same conditions and OV production was confirmed. Considering that the predicted BGC contains two copies of alpha-KG-dependent non-heme iron oxidase genes and that the introduction of a hydroxyl group at the C6 position in FM biosynthesis is catalyzed by the same enzyme Fma-C6H, we predicted that the hydroxyl group at the C7 position in OV biosynthesis is also introduced by a similar enzyme. In fact, when four fumagillin biosynthetic genes (fma-TC, fma-P450, fma-C6H, fma-MT) were co-expressed in A. nidulans with the MAA_08348 gene (ova-C7H) cloned from M. robertsii, OV production was confirmed. From these results, we identified the 7-hydroxylation enzyme Ova-C7H responsible for OV production.

[0142] Test Example 2. Production of Demethyl Ovalicin (deMeOV) <Plasmid construction> The following plasmids were constructed by the gap-repair cloning method using Saccharomyces cerevisiae. The fma-TC, fma-P450, fma-C6H, and fma-MT genes were derived from the genomic DNA of Aspergillus fumigatus A1159 strain, while the ova-C7H gene was derived from the genomic DNA of Metarhizium robertsii ARSEF23 strain, and each was prepared by amplifying it by PCR. Plasmids were constructed in the following order: (1) The PCR-amplified fma-TC gene was introduced into the pKW20093 plasmid cut with SwaI to create pKW20517. (2) The PCR-amplified fma-P450 gene was introduced into the pKW10010 plasmid cut with SwaI to create pKW20518. (3) The PCR-amplified fma-C6H gene was introduced into the pKW10012 plasmid cut with SwaI to create pKW20519. (4) The PCR-amplified ova-C7H gene was introduced into the pKW20093 plasmid cut with SwaI to create pKW20522. (5) The fma-TC expression cassette (glaA promoter region and fma-TC gene) was amplified by PCR using pKW20517 (prepared in (1)) as a template, and introduced into the pKW20518 plasmid cut with SwaI to create pKW23502. (6) The PCR-amplified fma-KR gene was introduced into the pKW10010 plasmid cut with SwaI to create pKW20521. (7) The fma-KR expression cassette (gpdA promoter region and fma-KR gene) was amplified by PCR using pKW20521 (prepared in (6)) as a template, and introduced into the pKW23501 plasmid cut with SwaI to produce pKW23503. (8) The PCR-amplified fma-MT gene was introduced into the pKW20093 plasmid cut with SwaI to produce pKW20520. (9) The fma-MT expression cassette (glaA promoter region and fma-MT gene) was amplified by PCR using pKW20520 (prepared in (8)) as a template, and introduced into the pKW23519 plasmid (prepared in (3)) cut with SwaI to produce pKW23501.

[0143]

Table 1

[0144] <Transformation of Aspergillus nidulans (Construction of demethyl ovalicin-producing strain)> The host Aspergillus nidulans ΔPPR strain (ΔpyrG, ΔpyroA, ΔriboB strain) was seeded on CD / PPR agar medium and cultured, and then spores were formed. These spores were suspended in sterilized water, inoculated into CD / PPR liquid medium, and cultured with shaking at 30 °C for 20 hours. Then, Lysing enzyme from Trichoderma harzianum (Sigma-Aldrich) and beta-glucuronidase from Helix pomatia (Sigma-Aldrich) were allowed to act on the collected cells to perform protoplasting. The plasmids (pKW20519, pKW20522, pKW23502) prepared for this protoplast were added, STC solution was added and mixed, and then seeded on CD agar medium. For the obtained colonies, after secondary selective culture, genomic DNA was extracted and transformation was confirmed by PCR method. Through the above procedures, the transformant (Aspergillus nidulans ΔPPR#5) into which the plasmid was correctly introduced was used for subsequent culture experiments.

[0145] <Production of deMeOV> The prepared Aspergillus nidulans ΔPPR#5 strain was inoculated into 30 mL of CDST liquid medium and cultured with shaking at 30 °C. After 72 hours from the start of culture, a part of the culture solution was taken every 24 hours, and the production amount of deMeOV over time was quantified. To 0.5 mL of the aliquoted culture solution, 0.5 mL of ethyl acetate was added and vigorously stirred. Then, centrifugation was performed, and 0.4 mL of the upper ethyl acetate layer was aliquoted into another container, and the solvent was distilled off. To the residue, DMF (100 μL) was added and dissolved, and LC-MS measurement was performed on the supernatant part from which insoluble components were removed by centrifugation.

[0146] <Detection of deMeOV in LC-MS> Analysis of deMeOV by LC-MS was performed using an apparatus from Thermo Fisher Scientific (Dionex U3000 and Orbitrap mass spectrometer Exactive Plus). The column used was Waters AQUITY UPLC HSS C18 (2.1 x 50 mm), and the mobile phase was a gradient mode of acetonitrile and ultrapure water (acetonitrile 5 - 100%, 4 min) at a flow rate of 500 μL / min. Additionally, 0.05% formic acid was added to each solvent. High-precision mass was measured using the ESI method, and the analysis was carried out using Qual browser software.

[0147] <Large-scale culture experiment of deMeOV> A small amount of Aspergillus nidulans ΔPPR#5 strain spores was taken, suspended in 100 μL of 0.1% Tween® 80 aqueous solution, and then the entire amount was inoculated onto CD agar medium and cultured at 30 °C. After confirming spore formation, half of the CD agar medium was cut out and mixed with 10 ml of 0.1% Tween® 80 in a container. The resulting suspension was added in its entirety to a 2 L Erlenmeyer flask containing 400 ml of CDT medium, and pre-culture was initiated. The pre-culture was carried out at 30 °C and 160 rpm for 24 hours. This pre-culture solution was transferred in its entirety to a jar fermenter (Marubishi Bioengineer, bench-top culture apparatus Bioneer) containing 6.0 L of CDST medium (containing adsorption resin Diaion HP-20, 150 g), and main culture was performed. The main culture was carried out at 30 °C, 400 rpm, under an oxygen flow for approximately 48 hours. During the main culture, the culture solution was withdrawn over time, and the deMeOV production amount was confirmed. After confirming sufficient productivity, the culture solution and resin were taken out from the fermenter and filtered to obtain the solids of the cells and resin (HP-20), which were stored frozen. Additionally, using HPLC analysis, it was confirmed that the target component deMeOV was not contained in the culture supernatant (filtrate) part.

[0148] <Purification and structure confirmation of deMeOV> The solid samples obtained from the above culture (6.0 L scale) were pooled together for about 10 times (total of 60 L culture), and extracted three times with 10 L of acetone. Each extraction was carried out for about 2 hours, and the mixture of cells and resin (HP20) was vigorously stirred in acetone every 30 minutes. The insoluble matter was removed by filtration, and the extract was concentrated under reduced pressure, and then separation was carried out three times using 1.0 L of EtOAc. The EtOAc layer was then concentrated under reduced pressure to obtain 27 g of extract. This extract was purified using silica gel column chromatography (BW-820MH 280 g, developing solvent: CHCl3 / MeOH [C / M] = 100 / 0→100 / 2→100 / 1→ 0 / 100). The C / M = 100 / 2 fraction (5.2 g) in which the target component was detected was purified by normal phase flash chromatography (conditions: column = Universal 5 L (silica gel: 300 g), flow rate = 70 ml / min, solvent = n-hexane / EtOAc = 4 / 1, fraction volume = 100 ml / tube). Each fraction obtained was analyzed by TLC, and the fractions containing a large amount of deMeOV were mixed (1.2 g). Finally, normal phase flash chromatography was performed again (conditions: column = Universal 2 L (silica gel: 100 g), flow rate = 30 ml / min, solvent = n-hexane / EtOAc = 17 / 3, fraction volume = 30 ml / tube), and 976 mg of deMeOV was obtained. NMR measurement of the isolated deMeOV in deuterated chloroform was performed, and it was confirmed that various data were consistent with the literature information and that the chemical structure was correct by two-dimensional NMR analysis (Figure 2 and Table 2).

[0149] [ka]

[0150] [Table 2]

[0151] <Aspergillus nidulans Transformation (Construction of Dihydro Ovalicin-Producing Strains)> For the above experiment, the plasmids used were changed to the combination of pKW20522, pKW20503, and pKW20502. Compound production, compound detection, large-scale culture experiments, compound isolation, and chemical structure determination were carried out in the same manner as in the experimental section for demethyl ovalicin.

[0152]

Chemical formula

[0153]

Table 3

[0154] <Aspergillus nidulans Transformation (Construction of Demethoxy Ovalicin-Producing Strains)> For the above experiment, the plasmids used were changed to the combination of pKW20517, pKW20518, and pKW20522. Compound production, compound detection, large-scale culture experiments, compound isolation, and chemical structure determination were carried out in the same manner as in the experimental section for demethyl ovalicin.

[0155]

Chemical formula

[0156]

Table 4

[0157] <Aspergillus nidulans Transformation (Construction of Ovalicin-Producing Strains)> In the above experiments, the plasmids used were changed to a combination of pKW20522, pKW23501, and pKW23502. Compound production, compound detection, mass culture experiments, compound isolation, and chemical structure determination were performed in the same manner as in the experimental section for demethyl ovalicin.

[0158] [ka]

[0159] [Table 5]

Claims

1. (A) A modified cell comprising polynucleotide A including an amino acid sequence having 70% or more identity to the amino acid sequence shown in SEQ ID NO:1 and including a coding sequence for protein A having the activity of converting 5-keto-demethoxyfumagillol to demethoxy ovalicin.

2. The modified cell according to claim 1, further comprising (B) a polynucleotide B comprising a coding sequence for a protein B having an activity of converting demethoxy ovalicin to demethyl ovalicin.

3. The modified cell of claim 2, wherein the protein B is (B1) protein B1 having an amino acid sequence having 70% or more identity to the amino acid sequence shown in SEQ ID NO:

2.

4. The modified cell of claim 2, further comprising (C) a polynucleotide C including a coding sequence for a protein C having an activity of converting β-trans-bergamotene to 5-keto-demethoxyfumagillol.

5. The modified cell of claim 4, wherein the protein C is protein C1 having an amino acid sequence having 70% or more identity to the amino acid sequence shown in SEQ ID NO:3 (C1).

6. The modified cell according to claim 4, further comprising (D) a polynucleotide D comprising a coding sequence for a protein D having an activity of converting farnesyl diphosphate to β-trans-bergamotene.

7. The modified cell of claim 6, wherein the protein D is protein D1 having an amino acid sequence having 70% or more identity to the amino acid sequence shown in SEQ ID NO:4 (D1).

8. said polynucleotide A is an exogenous polynucleotide; and / or (EX) The vector does not express protein E having the activity of converting demethyl ovalicin to ovalicin, or is modified so that the expression or function of the protein E is reduced. The modified cell of claim 1.

9. The modified cell of claim 1 , which is a eukaryotic cell.

10. The modified cell according to claim 9 , which is a cell capable of forming mycelium and spores.

11. A method for producing demethyl ovalicin, comprising culturing the modified cell according to any one of claims 1 to 10 having the ability to produce demethyl ovalicin.

12. A method for producing ovalicin, comprising culturing the modified cell according to any one of claims 1 to 10 having the ability to produce ovalicin.

13. A method for producing demethoxy ovalicin, comprising culturing the modified cell according to any one of claims 1 to 10 having the ability to produce demethoxy ovalicin.

14. The method according to claim 11, wherein a culture medium containing an adsorption resin is used as the culture medium.

15. Formula (A): 【Chemistry 1】 A compound represented by the formula:

16. Formula (B): 【Chemistry 2】 A compound represented by the formula:

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