WO2018128140A1 - 環状ペプチド化合物合成関連遺伝子、これを用いた環状ペプチド化合物の製造方法及びこれを有する形質転換体 - Google Patents
環状ペプチド化合物合成関連遺伝子、これを用いた環状ペプチド化合物の製造方法及びこれを有する形質転換体 Download PDFInfo
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- C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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Definitions
- the present invention relates to a novel cyclic peptide compound synthesis-related gene involved in the synthesis of a cyclic peptide compound produced by Curvularia fungi and having a bactericidal action against fungi, and the production of the cyclic peptide compound using the cyclic peptide compound synthesis-related gene
- the present invention relates to a method and a transformant having a gene related to synthesis of the cyclic peptide compound.
- KK-1 A specific cyclic peptide compound (hereinafter referred to as CAS143380-71-6, produced by Curvularia filamentous fungi is known to exhibit a strong bactericidal action against phytopathogenic fungi, especially fungi.
- Patent Document 1 A specific cyclic peptide compound (hereinafter referred to as CAS143380-71-6), produced by Curvularia filamentous fungi is known to exhibit a strong bactericidal action against phytopathogenic fungi, especially fungi.
- Patent Document 1 A specific cyclic peptide compound (hereinafter referred to as KK-1), known as CAS143380-71-6, produced by Curvularia filamentous fungi is known to exhibit a strong bactericidal action against phytopathogenic fungi, especially fungi.
- Patent Document 1 A specific cyclic peptide compound (hereinafter referred to as KK-1), known as CAS143380-71-6, produced by Curvularia filamentous fungi is known to exhibit a strong bacterici
- Non-patent Document 1 a biosynthetic gene cluster involved in biosynthesis of metabolites exists.
- Non-patent Document 3 a sufficient amount of secondary metabolism can be achieved by activating the biosynthetic gene cluster (Non-patent Document 3) or expressing it in an appropriate heterologous species such as budding yeast. So-called synthetic biology techniques that try to synthesize products have been attempted.
- KK-1 produced by Curvularia spp. May be produced by using synthetic biology techniques, similar to the secondary metabolites described above. However, the genome of Curvularia spp. Has hardly been elucidated, and the gene cluster involved in the production of KK-1 has not been elucidated.
- the present invention aims to identify a gene cluster involved in biosynthesis of KK-1 produced by Curvularia spp. And provide a system for synthesizing KK-1.
- NRPS nonribosomal peptide synthase
- the present invention includes the following.
- a first adenylation domain consisting of the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence having 70% or more identity to the amino acid sequence shown in SEQ ID NO: 1, and the amino acid sequence shown in SEQ ID NO: 2,
- a second adenylation domain consisting of an amino acid sequence having 70% or more identity to the amino acid sequence, and 70% or more identity to the amino acid sequence shown in SEQ ID NO: 5 or the amino acid sequence shown in SEQ ID NO: 5
- A a protein comprising the amino acid sequence shown in SEQ ID NO: 37
- B an amino acid sequence having 70% or more identity to the amino acid sequence shown in SEQ ID NO: 37
- C a cyclic peptide compound that is encoded by a polynucleotide that hybridizes under stringent conditions to a complementary strand of the base sequence shown in SEQ ID NO: 36 and that is produced by Curvularia spp.
- the cyclic peptide compound synthesis-related gene according to (1) which is derived from a Curvularia genus filamentous fungus.
- A a protein consisting of the amino acid sequence shown in SEQ ID NO: 39
- b a protein consisting of an amino acid sequence having 70% or more identity to the amino acid sequence shown in SEQ ID NO: 39 and having transcription factor activity
- c SEQ ID NO: A protein encoded by a polynucleotide that hybridizes under stringent conditions to the complementary strand of the base sequence shown in No. 38 and having a transcription factor activity (6) characterized by being derived from Curvularia spp. (5) The cyclic peptide compound synthesis-related gene as described.
- Amino acid sequence or sequence shown in SEQ ID NO: 43 A gene encoding a protein comprising an amino acid sequence having 70% or more identity to the amino acid sequence shown in No.
- Amino acid sequence or sequence shown in SEQ ID NO: 43 A gene encoding a protein comprising an amino acid sequence having 70% or more identity to the amino acid sequence shown in No.
- Curvularia spp. Having the cyclic peptide compound synthesis-related gene according to any one of (1) to (4) above.
- Curvularia spp. Described in (14), which is Curvularia clavata.
- a gene encoding a non-ribosomal peptide synthase involved in the synthesis of a cyclic peptide compound produced by Curvularia spp. And a gene group involved in the synthesis of the other cyclic peptide compound are provided.
- a system for synthesizing a cyclic peptide compound produced by Curvularia spp. Can be constructed, and the cyclic peptide compound can be efficiently produced.
- FIG. 3 is a characteristic diagram showing the results of examining the production amount of KK-1 separately for the wild type and the high expression factor of the transcription factor separately on the outside and inside of the cells at 3 and 7 days after the main culture. It is a characteristic figure which shows the result of having investigated the production amount of KK-1 when a wild strain and a transcription factor high expression strain are cultured by "culture 3", and the mode of solid culture. It is a figure which shows typically the construction of the plasmid for CcpyrG gene destruction. It is a figure which shows typically the construct for destroying a transcription factor gene (TF068-005 gene).
- the cyclic peptide compound synthesis-related gene means individual genes included in a gene group (gene cluster) involved in the synthesis of a cyclic peptide compound produced by Curvularia spp.
- the cyclic peptide compound is represented by the following formula as disclosed in JP-T-8-504165 (or WO93 / 12659).
- each amino acid residue and lactate residue can independently take L-form or D-form.
- the compound name of the cyclic peptide compound (hereinafter sometimes referred to as KK-1) is Tyrosine, N- [N- [N- [N- [N- [N- [N- [N- [N-[[1 -(2-hydroxy-1-oxopropyl) -2-piperidinyl] carbonyl] -N-methylvalyl] valyl] -N-methyl-a-aspartyl] -N-methylvalyl] -Nmethylisoleucyl] glycyl] -N-methylvalyl] -O -methyl-, d2-lactone (9CI).
- Curvularia genus fungus that produces this cyclic peptide compound can typically include Curvularia clavata, but also C. affinis, C. brachyspora, C. caricae-papayae, C. eragrostidis (Cochliobolus eragrostidis), C. fallax, C. geniculata (Cochliobolus geniculatus), C. harveyi, C. lunata (Cochliobolus lunatus), C. ovoidea, C. pallescens, C. penniseti, C. prasadii, C. protuberata, C. senegalensis, C.
- Curvularia clavata includes Curvularia clavata BAUA-2787 stock sold by Akita Imano Co., Ltd.
- Curvularia clavata BAUA-2787 dated December 28, 2016, the National Institute of Technology and Evaluation (NPMD) (2-5-Kazusa Kamashi, Kisarazu City, Chiba Prefecture 292-0818, Japan) No. 8 122) was deposited under the accession number NITE BP-02399.
- the gene group involved in the synthesis of the present cyclic peptide compound can be defined as a gene group containing 10 types of genes, preferably 9 types of genes, as shown in the Examples described later.
- These 10 genes encode O-methyltransferase gene, non-ribosomal peptide synthase gene (NRPS gene), amidase gene, genes with unknown function (2 types), transcription factor gene, and leptomycin B resistance protein.
- pmd1 gene pyrroline-5-carboxylic acid reductase-like gene and ⁇ / ⁇ hydrolase gene.
- O-methyltransferase gene, non-ribosomal peptide synthase gene (NRPS gene), amidase gene, gene with unknown function (1 type) are particularly involved in the synthesis of cyclic peptide compounds.
- Transcription factor gene encoding leptomycin B resistance protein, pyrroline-5-carboxylate reductase-like gene and ⁇ / ⁇ hydrolase gene are defined as genes involved in the synthesis of this cyclic peptide compound You can also
- the NRPS gene encodes NRPS having a function of forming the basic skeleton of the present cyclic peptide compound. That is, this NRPS is [alanine (Ala) -pipecolic acid (Pip) -valine (Val) -valine-aspartic acid (Asp) -valine-isoleucine (Ile) -glycine (Gly) -valine-tyrosine (Tyr)] A peptide backbone consisting of 10 amino acids is formed.
- this NRPS forms a peptide bond between the carboxyl group of alanine and the amino group of pipecolic acid, forms a peptide bond between the carboxyl group of pipecolic acid and the amino group of valine, and A peptide bond is formed between the carboxyl group and the amino group of valine, a peptide bond is formed between the carboxyl group of the valine and the amino group of aspartic acid, and the carboxyl group of the aspartic acid and the amino group of valine A peptide bond is formed between the carboxyl group of the valine and the amino group of isoleucine, a peptide bond is formed between the carboxyl group of the isoleucine and the amino group of glycine, A peptide bond is formed between the carboxyl group of glycine and the amino group of valine.
- This NRPS also methylates the peptide bond between pipecolic acid and valine, methylates the peptide bond between valine and aspartic acid, methylates the peptide bond between aspartic acid and valine, It has an activity of methylating a peptide bond between isoleucine and methylating a peptide bond between glycine and valine.
- This NRPS has 10 modules corresponding to 10 amino acids constituting the basic peptide skeleton described above [alanine-pipecolic acid-valine-valine-aspartic acid-valine-isoleucine-glycine-valine-tyrosine]. ing. Each module has an A domain (adenylation domain) that takes in an amino acid of interest and synthesizes an aminoacyl AMP by binding AMP (adenosine monophosphate) to the amino acid. Each module has a phosphopantethein and a PCP domain (peptidyl carrier protein domain) that binds the aminoacyl AMP by a thioester formed between the serine site of phosphopantethein and aminoacyl AMP. Have.
- each module has a C domain (condensation domain) that forms a peptide bond between aminoacylAMPs bound to adjacent PCP domains. Furthermore, some modules have an nMT domain (N-methyltransferase domain) that methylates the formed peptide bond.
- NRPS having the activity described above is composed of first to ten modules as shown in FIG.
- the position of each module of NRPS coincides with the position of the amino acid constituting the peptide skeleton to be synthesized.
- the position of the module having the nMT domain coincides with the position of the N-methylated peptide bond.
- the first module has a first A domain consisting of the amino acid sequence shown in SEQ ID NO: 1 and a first PCP domain consisting of the amino acid sequence shown in SEQ ID NO: 2 in this order from the N-terminal side.
- the first A domain and the first PCP domain in the first module are not limited to the amino acid sequences shown in SEQ ID NOs: 1 and 2, respectively, so long as they function as the A domain and the PCP domain, respectively.
- Amino acid sequences having the same identity may be used.
- the second module consists of a first C domain consisting of the amino acid sequence shown in SEQ ID NO: 3, a second A domain consisting of the amino acid sequence shown in SEQ ID NO: 4, and a second PCP consisting of the amino acid sequence shown in SEQ ID NO: 5. Domains in this order from the N-terminal side. However, the first C domain, the second A domain, and the second PCP domain in the second module are not limited to the amino acid sequences shown in SEQ ID NOs: 3, 4, and 5, respectively.
- identity preferably 80% or more identity, more preferably 90% identity or more, more preferably, to the amino acid sequences shown in SEQ ID NOs: 3, 4 and 5 It may be an amino acid sequence having 95% or more identity, most preferably 97% or more identity.
- the third module includes a second C domain consisting of the amino acid sequence shown in SEQ ID NO: 6, a third A domain consisting of the amino acid sequence shown in SEQ ID NO: 7, and a first nMT consisting of the amino acid sequence shown in SEQ ID NO: 8. It has a domain and a third PCP domain consisting of the amino acid sequence shown in SEQ ID NO: 9 in this order from the N-terminal side.
- the second C domain, the third A domain, the first nMT domain, and the third PCP domain in the third module are not limited to the amino acid sequences shown in SEQ ID NOs: 6, 7, 8, and 9, respectively.
- identity 70% or more identity, preferably 80% or more identity to the amino acid sequences shown in SEQ ID NOs: 6, 7, 8 and 9, if functioning as C domain, A domain, nMT domain and PCP domain, respectively More preferably, it may be an amino acid sequence having 90% or more identity, more preferably 95% or more identity, and most preferably 97% or more identity.
- the fourth module includes a third C domain consisting of the amino acid sequence shown in SEQ ID NO: 10, a fourth A domain consisting of SEQ ID NO: 11, and a fourth PCP domain consisting of the amino acid sequence shown in SEQ ID NO: 12. It has in this order from the terminal side.
- the third C domain, the fourth A domain, and the fourth PCP domain in the fourth module are not limited to the amino acid sequences shown in SEQ ID NOs: 10, 11, and 12, respectively, and the C domain, A domain, and PCP, respectively.
- identity preferably 80% or more identity, more preferably 90% or more identity to the amino acid sequences shown in SEQ ID NOs: 10, 11, and 12, more preferably It may be an amino acid sequence having 95% or more identity, most preferably 97% or more identity.
- the fifth module includes a fourth C domain consisting of the amino acid sequence shown in SEQ ID NO: 13, a fifth A domain consisting of the amino acid sequence shown in SEQ ID NO: 14, and a second nMT consisting of the amino acid sequence shown in SEQ ID NO: 15. It has a domain and a fifth PCP domain consisting of the amino acid sequence shown in SEQ ID NO: 16 in this order from the N-terminal side.
- the fourth C domain, the fifth A domain, the second nMT domain and the fifth PCP domain in the fifth module are not limited to the amino acid sequences shown in SEQ ID NOs: 13, 14, 15 and 16, respectively.
- identity 70% or more identity, preferably 80% or more identity to the amino acid sequences shown in SEQ ID NOs: 13, 14, 15 and 16 if they function as C domain, A domain, nMT domain and PCP domain, respectively. More preferably, it may be an amino acid sequence having 90% or more identity, more preferably 95% or more identity, and most preferably 97% or more identity.
- the sixth module includes a fifth C domain consisting of the amino acid sequence shown in SEQ ID NO: 17, a sixth A domain consisting of the amino acid sequence shown in SEQ ID NO: 18, and a third nMT consisting of the amino acid sequence shown in SEQ ID NO: 19. It has a domain and a sixth PCP domain consisting of the amino acid sequence shown in SEQ ID NO: 20 in this order from the N-terminal side.
- the fifth C domain, the sixth A domain, the third nMT domain, and the sixth PCP domain in the sixth module are not limited to the amino acid sequences shown in SEQ ID NOs: 17, 18, 19, and 20, respectively.
- identity 70% or more identity, preferably 80% or more identity to the amino acid sequence shown in SEQ ID NOs: 17, 18, 19 and 20 if functioning as C domain, A domain, nMT domain and PCP domain, respectively More preferably, it may be an amino acid sequence having 90% or more identity, more preferably 95% or more identity, and most preferably 97% or more identity.
- the seventh module includes a sixth C domain consisting of the amino acid sequence shown in SEQ ID NO: 21, a seventh A domain consisting of the amino acid sequence shown in SEQ ID NO: 22, and a fourth nMT consisting of the amino acid sequence shown in SEQ ID NO: 23. It has a domain and a seventh PCP domain consisting of the amino acid sequence shown in SEQ ID NO: 24 in this order from the N-terminal side.
- the sixth C domain, the seventh A domain, the fourth nMT domain, and the seventh PCP domain in the seventh module are not limited to the amino acid sequences shown in SEQ ID NOs: 21, 22, 23, and 24, respectively.
- identity 70% or more identity, preferably 80% or more identity to the amino acid sequences shown in SEQ ID NOs: 21, 22, 23 and 24, if they function as C domain, A domain, nMT domain and PCP domain, respectively. More preferably, it may be an amino acid sequence having 90% or more identity, more preferably 95% or more identity, and most preferably 97% or more identity.
- the eighth module includes a seventh C domain consisting of the amino acid sequence shown in SEQ ID NO: 25, an eighth A domain consisting of SEQ ID NO: 26, and an eighth PCP domain consisting of the amino acid sequence shown in SEQ ID NO: 27. It has in this order from the terminal side.
- the seventh C domain, the eighth A domain, and the eighth PCP domain in the eighth module are not limited to the amino acid sequences shown in SEQ ID NOs: 25, 26, and 27, respectively. If functioning as a domain, 70% or more identity, preferably 80% or more identity, more preferably 90% identity or more, more preferably, to the amino acid sequences shown in SEQ ID NOs: 25, 26 and 27 It may be an amino acid sequence having 95% or more identity, most preferably 97% or more identity.
- the ninth module includes an eighth C domain consisting of the amino acid sequence shown in SEQ ID NO: 28, a ninth A domain consisting of the amino acid sequence shown in SEQ ID NO: 29, and a fifth nMT consisting of the amino acid sequence shown in SEQ ID NO: 30. It has a domain and a ninth PCP domain consisting of the amino acid sequence shown in SEQ ID NO: 31 in this order from the N-terminal side.
- the eighth C domain, the ninth A domain, the fifth nMT domain, and the ninth PCP domain in the ninth module are not limited to the amino acid sequences shown in SEQ ID NOs: 28, 29, 30, and 31, respectively.
- identity 70% or more identity, preferably 80% or more identity to the amino acid sequences shown in SEQ ID NOs: 28, 29, 30 and 31 if functioning as C domain, A domain, nMT domain and PCP domain respectively. More preferably, it may be an amino acid sequence having 90% or more identity, more preferably 95% or more identity, and most preferably 97% or more identity.
- the tenth module includes a ninth C domain consisting of the amino acid sequence shown in SEQ ID NO: 32, a tenth A domain consisting of the SEQ ID NO: 33, a tenth PCP domain consisting of the amino acid sequence shown in SEQ ID NO: 34, and a sequence It has a 10th C domain consisting of the amino acid sequence shown in No. 35 in this order from the N-terminal side.
- the ninth C domain, the tenth A domain, the tenth PCP domain, and the tenth C domain in the tenth module are not limited to the amino acid sequences shown in SEQ ID NOs: 32, 33, 34, and 35, respectively.
- identity 70% or more identity, preferably 80% or more identity to the amino acid sequences shown in SEQ ID NOs: 32, 33, 34 and 35, if functioning as C domain, A domain, PCP domain and C domain respectively. More preferably, it may be an amino acid sequence having 90% or more identity, more preferably 95% or more identity, and most preferably 97% or more identity.
- a mutant NRPS gene is designed to encode the first mutant A domain designed to be different from the amino acid sequence of SEQ ID NO: 1.
- This mutant NRPS gene is expressed in an appropriate host, and it is confirmed whether a compound having the basic peptide skeleton of the cyclic peptide compound is synthesized in the metabolite in the host and in the culture supernatant.
- the designed first mutant A domain functions as an A domain corresponding to alanine. it can.
- the second to tenth A domains are different from the amino acid sequences of SEQ ID NOs: 4, 7, 11, 14, 18, 22, 26, 29, and 33, it is similarly evaluated whether they can function as A domains. Can do.
- first PCP domain can function as a PCP domain when it differs from the amino acid sequence of SEQ ID NO: 2
- a mutant NRPS gene is designed to encode the first mutant PCP domain designed to be different from the amino acid sequence of SEQ ID NO: 2.
- This mutant NRPS gene is expressed in an appropriate host, and it is confirmed whether a compound having the basic peptide skeleton of the cyclic peptide compound is synthesized in the metabolite in the host and in the culture supernatant.
- a compound having a basic peptide skeleton of the above cyclic peptide compound is synthesized in a metabolite, it can be evaluated that the designed first mutant PCP domain functions as a PCP domain.
- even when the second to tenth PCP domains are different from the amino acid sequences of SEQ ID NOs: 5, 9, 12, 16, 20, 24, 27, 31 and 34, it is evaluated whether they can function as PCP domains in the same manner. Can do.
- first C domain is different from the amino acid sequence of SEQ ID NO: 3
- whether it can function as the C domain can be evaluated as follows.
- a mutant NRPS gene is designed to encode a first mutant C domain designed to be different from the amino acid sequence of SEQ ID NO: 3.
- This mutant NRPS gene is expressed in an appropriate host, and it is confirmed whether a compound having the basic peptide skeleton of the cyclic peptide compound is synthesized in the metabolite in the host and in the culture supernatant.
- a compound having a basic peptide skeleton of the above cyclic peptide compound is synthesized in a metabolite, it can be evaluated that the designed first mutant C domain functions as a C domain.
- even when the 2nd to 10th C domains are different from the amino acid sequences of SEQ ID NOs: 6, 10, 13, 17, 21, 25, 28, 32 and 35, it is evaluated whether they can function as C domains in the same manner. Can do.
- first nMT domain is different from the amino acid sequence of SEQ ID NO: 8
- whether it can function as an nMT domain can be evaluated as follows.
- a mutant NRPS gene is designed to encode a first mutant nMT domain designed to be different from the amino acid sequence of SEQ ID NO: 8. This mutant NRPS gene is expressed in an appropriate host, and it is confirmed whether a compound having the basic peptide skeleton of the cyclic peptide compound is synthesized in the metabolite in the host and in the culture supernatant.
- nMT domain When a compound having a basic peptide skeleton of the above cyclic peptide compound is synthesized in a metabolite, it can be evaluated that the designed first mutant nMT domain functions as an nMT domain. Even when the second to fifth nMT domains are different from the amino acid sequences of SEQ ID NOs: 15, 19, 23 and 30, it can be similarly evaluated whether they can function as nMT domains.
- the NRPS for synthesizing the basic peptide skeleton of the cyclic peptide compound can be defined by the first module to the tenth module.
- the amino acid sequence of NRPS derived from Curvularia clavata and having the synthetic activity of the basic peptide skeleton in the cyclic peptide compound is shown in SEQ ID NO: 37, and the base sequence of the coding region corresponding to the amino acid sequence shown in SEQ ID NO: 37 This is shown in SEQ ID NO: 36.
- the NRPS gene according to the present invention comprises the first module to the tenth module defined by the amino acid sequences shown in SEQ ID NOs: 1 to 35, and is 70% or more with respect to the amino acid sequence shown in SEQ ID NO: 37.
- Amino acid sequences having the same identity preferably more than 80% identity, more preferably more than 90% identity, still more preferably more than 95% identity, most preferably more than 97% identity, It may be a gene encoding a protein having a synthetic activity of the basic peptide skeleton in the cyclic peptide compound.
- the value of identity between amino acid sequences can be calculated by a BLASTN or BLASTX program that implements the BLAST algorithm (default setting). The identity value is calculated as a ratio of all amino acid residues compared by calculating the amino acid residues that completely match when a pair of amino acid sequences are subjected to pairwise alignment analysis.
- the NRPS gene according to the present invention comprises the first module to the tenth module defined by the amino acid sequences shown in SEQ ID NOs: 1 to 35 described above, and is one or several relative to the amino acid sequence of SEQ ID NO: 37. It may have an amino acid sequence in which one amino acid is substituted, deleted, inserted or added, and encodes a protein having a synthetic activity of a basic peptide skeleton in the cyclic peptide compound.
- the term “several” means, for example, 2 to 1300, preferably 2 to 1000, more preferably 2 to 700, still more preferably 2 to 500, still more preferably 2 to 250, still more preferably 2. ⁇ 100, more preferably 2-50.
- the NRPS gene according to the present invention comprises the first module to the tenth module defined by the amino acid sequences shown in SEQ ID NOs: 1 to 35 described above, and all the complementary strands of DNA consisting of the base sequence of SEQ ID NO: 36 Alternatively, it may be a protein that hybridizes to a part under stringent conditions and encodes a protein having a synthetic activity of the basic peptide skeleton in the cyclic peptide compound.
- stringent conditions as used herein means the conditions under which a so-called specific hybrid is formed and a non-specific hybrid is not formed, and is appropriately determined with reference to, for example, Molecular Cloning: A Laboratory Manual (Third Edition) can do.
- the stringency can be set according to the temperature at the time of Southern hybridization and the salt concentration contained in the solution, and the temperature at the time of the washing step of Southern hybridization and the salt concentration contained in the solution. More specifically, as stringent conditions, for example, the sodium concentration is 25 to 500 mM, preferably 25 to 300 mM, and the temperature is 42 to 68 ° C., preferably 42 to 65 ° C. More specifically, it is 5 ⁇ SSC (83 mM NaCl, 83 mM sodium citrate), and the temperature is 42 ° C.
- the NRPS gene according to the present invention is not limited to the one encoding a protein comprising the first module to the tenth module defined by the amino acid sequences shown in SEQ ID NOs: 1 to 35 described above.
- the amino acid sequence of NRPS derived from Curvularia clavata and having the synthetic activity of the basic peptide skeleton in the cyclic peptide compound is shown in SEQ ID NO: 37
- the base sequence of the coding region corresponding to the amino acid sequence is shown in SEQ ID NO:
- the NRPS gene according to the present invention can also be defined by these SEQ ID NOs: 36 and 37.
- the NRPS gene according to the present invention can be a gene encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 37.
- the NRPS gene according to the present invention has 70% or more identity, preferably 80% or more identity, more preferably 90% or more identity, more preferably 95%, to the amino acid sequence shown in SEQ ID NO: 37. It may be a gene encoding a protein having an amino acid sequence having at least% identity, most preferably at least 97% identity, and having a basic peptide backbone synthesis activity in the cyclic peptide compound.
- the identity value between amino acid sequences can be calculated by the BLASTN or BLASTX program in which the BLAST algorithm is implemented, as described above (default setting). Similar to the above, the identity value is calculated as a percentage of the total amino acid residues that are calculated by calculating amino acid residues that completely match when a pair of amino acid sequences are subjected to pairwise alignment analysis.
- the NRPS gene according to the present invention has a high coverage, a low E-value, and a high identity value with respect to the base sequence shown in SEQ ID NO: 36 using a known database storing base sequence information. It is possible to identify genes that satisfy such conditions.
- the coverage can be 90% or more, preferably 95% or more, more preferably 99% or more.
- E-value can be 1.0e-5 or less, preferably 1.0e-15 or less, more preferably 0.0.
- the value of identity can be 70% or more, preferably 75% or more, more preferably 80% or more.
- a gene identified as satisfying these conditions has a very high probability of being a homologous gene of the NRPS gene consisting of the base sequence of SEQ ID NO: 36, and the cyclic peptide as in the case of the NRPS gene consisting of the base sequence of SEQ ID NO: 36. It can be identified as a gene encoding a protein having a synthetic activity of the basic peptide backbone in the compound.
- a microorganism having the gene may be obtained, and the ability of the cyclic peptide compound to be synthesized in the microorganism may be verified.
- the ability of the cyclic peptide compound in the obtained microorganism can be verified by culturing the microorganism and confirming whether the cyclic peptide compound is contained in the cultured microbial cells or in the culture supernatant.
- the nucleotide sequence of the NRPS gene according to the present invention is determined, it is prepared by chemical synthesis, by PCR using genomic DNA as a template, or by hybridizing a DNA fragment having the nucleotide sequence as a probe. can do. Furthermore, a gene consisting of a base sequence different from SEQ ID NO: 36 or a gene encoding an amino acid sequence different from SEQ ID NO: 37 is synthesized by site-directed mutagenesis or the like with respect to a polynucleotide consisting of the base sequence shown in SEQ ID NO: 36.
- a known method such as the Kunkel method or the Gapped-duplex method or a method equivalent thereto can be employed.
- a mutagenesis kit for example, Mutant-K (manufactured by Takara Bio Inc.) or Mutant-G (manufactured by Takara Bio Inc.)
- Site-directed mutagenesis or LA-PCR by Takara Bio Inc. Mutation is introduced using in vitro Mutagenesis series kit.
- the NRPS gene according to the present invention can be isolated from microorganisms known to produce the above cyclic peptide compounds.
- an NRPS gene (an NRPS gene encoding the amino acid sequence shown in SEQ ID NO: 37) can be isolated from Curvularia clavata.
- the NRPS gene according to the present invention has a high probability of being isolated from Curvularia spp. Other than Curvularia clavata using the nucleotide sequence shown in SEQ ID NO: 36. That is, by using a hybridization reaction using as a probe a polynucleotide comprising a part of a continuous base sequence selected from the base sequence shown in SEQ ID NO: 36, derived from the genome or transcript of Curvularia spp. Other than Curvularia clavata From this cDNA, the NRPS gene according to the present invention can be isolated.
- Curvularia genus filamentous fungi other than Curvularia clavata may not produce the cyclic peptide compound, or may produce the cyclic peptide compound. This is because even Curvularia spp. That do not produce the cyclic peptide compound may have the NRPS gene according to the present invention.
- Curvularia sp. Other than Curvularia clavata include, for example, C. affinis, C. brachyspora, C. caricae-papayae, C. eragrostidis (Cochliobolus eragrostidis (Teleomorph)), C. fallax, C. geniculata (Cochliobolus geniculatus (Cochliobolus geniculatus )), C. harveyi, C. lunata (Cochliobolus lunatus (Teleomorph)), C. ovoidea, C. pallescens, C. penniseti, C. prasadii, C. protuberata, C.
- Curvularia spp. The Curvularia genus fungus according to the present invention is characterized by having the NRPS gene described above. As described above, the NRPS gene according to the present invention is highly likely to be isolated from Curvularia spp. Other than Curvularia clavata. That is, the Curvularia genus fungus according to the present invention is not limited to Curvularia clavata, and C. affinis, C. brachyspora, C. caricae-papayae, C.
- eragrostidis (Cochliobolus eragrostidis), C. fallax, which produce cyclic peptide compounds.
- C. geniculata (Cochliobolus geniculatus), C. harveyi, C. lunata (Cochliobolus lunatus), C. ovoidea, C. pallescens, C. penniseti, C. prasadii, C. protuberata, C. senegalensis, C. trifolii, C tuberculata (Cochliobolus tuberculatus) and the like.
- Curvularia genus fungus according to the present invention is preferably Curvularia clavata.
- Curvularia genus filamentous fungi according to the present invention include Curvularia clavata BAUA-2787 strain sold by Akita Imano Co., Ltd.
- NPMD Patent Microorganisms Deposit Center
- the transcription factor gene contained in the gene group involved in the synthesis of the cyclic peptide compound described above encodes a protein having a function of positively controlling the expression of each gene contained in the gene group at the transcription level.
- the transcription factor gene according to the present invention is derived from Curvularia clavata as an example, and the amino acid sequence of a protein having transcription promoting activity for genes contained in a group of genes involved in the synthesis of cyclic peptide compounds is shown in SEQ ID NO: 39, The base sequence of the coding region corresponding to the amino acid sequence is shown in SEQ ID NO: 38.
- the transcription factor gene according to the present invention can be defined by these SEQ ID NOs: 38 and 39.
- the transcription factor gene according to the present invention can be a gene encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 39.
- the transcription factor gene according to the present invention has 70% or more identity, preferably 80% or more identity, more preferably 90% or more identity, and still more preferably, to the amino acid sequence shown in SEQ ID NO: 39. It may be an amino acid sequence having 95% or more identity, most preferably 97% or more identity, and may be a gene encoding a protein having the above transcription promoting activity.
- the identity value between amino acid sequences can be calculated by the BLASTN or BLASTX program in which the BLAST algorithm is implemented, as described above (default setting). Similar to the above, the identity value is calculated as a percentage of the total amino acid residues that are calculated by calculating amino acid residues that completely match when a pair of amino acid sequences are subjected to pairwise alignment analysis.
- the transcription factor gene according to the present invention has an amino acid sequence in which one or several amino acids are substituted, deleted, inserted or added to the amino acid sequence of SEQ ID NO: 39, and has the above-mentioned transcription promoting activity. It may be one that encodes a protein.
- the term “several” refers to, for example, 2 to 40, preferably 2 to 30, more preferably 2 to 20, further preferably 2 to 10, and further preferably 2 to 5, as described above. It is.
- the protein that hybridizes under stringent conditions with the transcription factor gene according to the present invention all or part of the complementary strand of DNA consisting of the nucleotide sequence of SEQ ID NO: 38, and has the above-mentioned transcription promoting activity.
- stringent condition means a condition in which a so-called specific hybrid is formed and a non-specific hybrid is not formed, for example, Molecular Cloning: A Laboratory Manual (Third Edition) Can be determined as appropriate.
- the stringency is set according to the temperature at the time of Southern hybridization and the salt concentration contained in the solution, and the temperature at the time of the washing step of Southern hybridization and the salt concentration contained in the solution.
- the sodium concentration is 25 to 500 mM, preferably 25 to 300 mM, and the temperature is 42 to 68 ° C., preferably 42 to 65 ° C. More specifically, it is 5 ⁇ SSC (83 mM NaCl, 83 mM sodium citrate), and the temperature is 42 ° C.
- a gene having a nucleotide sequence different from SEQ ID NO: 38 or a gene encoding an amino acid sequence different from SEQ ID NO: 39 encodes a protein having transcription promoting activity is determined by determining whether the gene is It can be confirmed by introducing into a host producing a cyclic peptide compound (for example, Curvularia clavata as an example) so that it can be expressed, and verifying the expression of genes involved in the synthesis of the cyclic peptide compound in the host at the transcription level.
- a cyclic peptide compound for example, Curvularia clavata as an example
- nucleotide sequence of the transcription factor gene according to the present invention is determined, by chemical synthesis, by PCR using genomic DNA as a template, or by hybridizing a DNA fragment having the nucleotide sequence as a probe. Can be produced.
- a gene consisting of a base sequence different from SEQ ID NO: 38 or a gene encoding an amino acid sequence different from SEQ ID NO: 39 is synthesized by site-directed mutagenesis or the like with respect to a polynucleotide consisting of the base sequence shown in SEQ ID NO: 38
- Mutant-K manufactured by Takara Bio Inc.
- Mutant-G manufactured by Takara Bio Inc.
- LA-PCR LA-PCR
- the transcription factor gene according to the present invention can be isolated from microorganisms known to produce the above-mentioned cyclic peptide compounds.
- a transcription factor gene (a transcription factor gene encoding the amino acid sequence shown in SEQ ID NO: 39) can be isolated from Curvularia clavata.
- the transcription factor gene according to the present invention has a high probability of being isolated from Curvularia spp. Other than Curvularia clavata using the base sequence shown in SEQ ID NO: 38. That is, by using a hybridization reaction using as a probe a polynucleotide comprising a part of a continuous base sequence selected from the base sequence shown in SEQ ID NO: 38, derived from the genome or transcript of Curvularia spp. Other than Curvularia clavata
- the transcription factor gene according to the present invention can be isolated from the cDNA.
- Curvularia genus filamentous fungi other than Curvularia clavata may not produce the cyclic peptide compound, or may produce the cyclic peptide compound. This is because even Curvularia spp. That do not produce the cyclic peptide compound may have the NRPS gene according to the present invention.
- Curvularia sp. Other than Curvularia clavata include, for example, C. affinis, C. brachyspora, C. caricae-papayae, C. eragrostidis (Cochliobolus eragrostidis (Teleomorph)), C. fallax, C. geniculata (Cochliobolus geniculatus (Cochliobolus geniculatus )), C. harveyi, C. lunata (Cochliobolus lunatus (Teleomorph)), C. ovoidea, C. pallescens, C. penniseti, C. prasadii, C. protuberata, C.
- a transformant having the ability to synthesize a compound having a basic peptide skeleton in the cyclic peptide compound is prepared by introducing the NRPS gene into a host so that it can be expressed.
- a transformant having the ability to synthesize a cyclic peptide compound can be prepared by introducing a cyclic peptide compound synthetic gene other than the NRPS gene into a host so that it can be expressed together with the NRPS gene.
- the transcription factor gene described above may or may not be introduced as a cyclic peptide compound synthesis-related gene other than the NRPS gene.
- the NRPS gene and other genes may be introduced into the host in a state where they are arranged downstream of a constitutive expression promoter that can function in the host, the expression of these NRPS genes and other genes can be constantly induced. In this case, even if the transcription factor gene described above is not introduced, the cyclic peptide compound synthesis-related gene can be expressed to produce the cyclic peptide compound.
- the host is not particularly limited, and any organism, particularly any microorganism, can be used as the host.
- Microorganisms that can be used as a host are not particularly limited, and for example, Escherichia such as Escherichia coli, Corynebacterium such as Corynebacterium glutamicum, and Bacillus such as Bacillus subtilis. Examples include bacteria belonging to the genus, Pseudomonas putida, Pseudomonas genus, Rhizobium meliloti, Rhizobium genus, Saccharomyces cerevisiae, Schicharocyces pom Examples include yeasts such as Pichia pastoris, and fungi including filamentous fungi.
- the expression vector is preferably composed of a promoter, a ribosome binding sequence, the above-described gene, and a transcription termination sequence as well as being capable of autonomous replication in the bacterium.
- the expression vector may contain a gene that controls promoter activity.
- Any promoter may be used as long as it can be expressed in a host such as E. coli.
- those derived from Escherichia coli such as trp promoter, lac promoter, PL promoter, PR promoter, and those derived from phage such as T7 promoter are used.
- artificially designed and modified promoters such as the tac promoter may be used.
- the method for introducing the expression vector is not particularly limited as long as it is a method for introducing DNA into bacteria.
- a method using calcium ions [Cohen, S.N., et al .: Proc. Natl. Acad. Sci., USA, 69: 2110-2114 (1972)], electroporation method and the like can be mentioned.
- yeasts that can be used as a host are not particularly limited, but Candida genus yeasts such as Candida Shehatae, Pichia genus yeasts such as Pichia stipitis, Pachysolen genus yeasts such as Pachysolen tannophilus, and Saccharomyces genus yeasts such as Saccharomyces cerevisiae. And yeast belonging to the genus Schizosaccharomyces such as Schizosaccharomyces pombe, and Saccharomyces cerevisiae is particularly preferred.
- an appropriate promoter having high transcription activity is used.
- Such promoters are not particularly limited, but include, for example, the glyceraldehyde 3-phosphate dehydrogenase gene (TDH3) promoter, the 3-phosphoglycerate kinase gene (PGK1) promoter, and the hyperosmotic response 7 gene (HOR7). Promoters can be used. Of these, the pyruvate decarboxylase gene (PDC1) promoter is preferred because of its high ability to highly express a downstream target gene.
- downstream genes can be strongly expressed by using gal1 promoter, gal10 promoter, heat shock protein promoter, MF ⁇ 1 promoter, PHO5 promoter, GAP promoter, ADH promoter, AOX1 promoter and the like.
- the filamentous fungus that can be used for the host is not particularly limited. Rhizomucor genus fungi such as pusillus and Rhizomucor miehei, Penicillium atumnotatum, Penicillium genus fungi such as Penicillium Rchrysogenum, Rhizopus genus fungi such as Rhizopus oryzae, Acremonium cellulolyticus, Humicola grisea, Thermocusus aurant
- the host is preferably Aspergillus sp.
- ⁇ -amylase gene (amyB) promoter When expressing the above-mentioned NRPS gene and other genes in filamentous fungi, ⁇ -amylase gene (amyB) promoter, ⁇ -glucosidase gene (agdA) promoter, glucoamylase gene promoter (glaA), tryptophan biosynthesis Gene (trpC) promoter, alcohol dehydrogenase gene (alcA) promoter, translation elongation factor gene (tef1) promoter, triose phosphate isomerase gene (tpiA) promoter, glyceraldehyde 3-phosphate dehydrogenase (gpdA) gene A promoter, an enolase (enoA) promoter, a pyruvate carboxylase (pdcA) promoter, a cellobiohydrase gene (cbh1) promoter, and the like can be used.
- ⁇ -amylase gene (amyB) promoter
- any conventionally known technique known as a method for transforming yeast and filamentous fungi can be applied. Specifically, for example, transformation method, transfection method, conjugation method, protoplast method, spheroplast method, electroporation method, lipofection method, lithium acetate method and the like can be used. (Production of cyclic peptide compound) By using the transformant described above, a target cyclic peptide compound can be produced.
- a compound having a basic peptide skeleton in the above-mentioned cyclic peptide compound is produced by using a transformant in which the NRPS gene is introduced so that it can be expressed.
- the cyclic peptide compound can be produced from the obtained compound by a chemical synthesis reaction.
- the said cyclic peptide compound can be manufactured by utilizing the transformant which introduce
- the cyclic peptide compound synthesized with the transformant and the compound having the basic peptide skeleton are separated from the culture supernatant with a centrifuge, Miracloth, etc., and then an organic solvent such as ethyl acetate is added. Can be extracted. Also, it is released from the cells by physical destruction methods (homogenizer, glass bead crushing, freeze thawing, etc.) and chemical destruction methods (solvent treatment, acid, base treatment, osmotic pressure treatment, enzyme treatment, etc.). Then, an organic solvent such as ethyl acetate can be added for extraction. Purification of the extracted cyclic peptide compound and the compound having the basic peptide skeleton can be performed by an existing purification method (column chromatography, salt precipitation, etc.). These methods can be implemented in appropriate combination as required.
- the cyclic peptide compound produced as described above can be used as a bactericidal agent having a bactericidal action against, for example, phytopathogenic fungi, particularly fungi. More specifically, when the above cyclic peptide compound is used as a bactericidal agent, it may be used as it is, but usually mixed with an appropriate solid carrier, liquid carrier, etc., a surfactant and other formulation adjuvants, an emulsion, EW, liquid, suspension, wettable powder, granule wettable powder, powder, DL powder, fine powder, fine powder F, granule, tablet, oil, aerosol, flowable, dry flowable, microcapsule, etc. Any dosage form can be used.
- solid carriers include animal and vegetable powders such as starch, activated carbon, soybean flour, wheat flour, wood flour, fish flour, and milk powder, talc, kaolin, bentonite, calcium carbonate, zeolite, diatomaceous earth, white carbon, clay, alumina, ammonium sulfate, and urea.
- animal and vegetable powders such as starch, activated carbon, soybean flour, wheat flour, wood flour, fish flour, and milk powder, talc, kaolin, bentonite, calcium carbonate, zeolite, diatomaceous earth, white carbon, clay, alumina, ammonium sulfate, and urea.
- Inorganic powders such as
- liquid carrier examples include water; alcohols such as isopropyl alcohol and ethylene glycol; ketones such as cyclohexanone and methyl ethyl ketone; ethers such as dioxane and tetrahydrofuran; aliphatic hydrocarbons such as kerosene and light oil; xylene, trimethylbenzene, Aromatic hydrocarbons such as tetramethylbenzene, methylnaphthalene and solvent naphtha; Halogenated hydrocarbons such as chlorobenzene; Acid amides such as dimethylacetamide; Esters such as glycerin esters of fatty acids; Nitriles such as acetonitrile; And sulfur-containing compounds such as sulfoxide.
- alcohols such as isopropyl alcohol and ethylene glycol
- ketones such as cyclohexanone and methyl ethyl ketone
- ethers such as dioxane and tetrahydrofuran
- surfactant examples include alkylbenzene sulfonic acid metal salt, dinaphthylmethane disulfonic acid metal salt, alcohol sulfate ester salt, alkylaryl sulfonate, lignin sulfonate, polyoxyethylene glycol ether, polyoxyethylene alkylaryl ether, Examples include polyoxyethylene sorbitan monoalkylate.
- adjuvants include, for example, carboxymethyl cellulose, gum arabic, sodium alginate, guar gum, tragacanth gum, polyvinyl alcohol and other sticking agents or thickeners, metal soap and other antifoaming agents, fatty acids, alkyl phosphates, silicones, paraffins And the like, and physical property improvers, colorants and the like can be used.
- Application of various preparations of a bactericidal agent or dilutions thereof is generally performed by a commonly used application method, that is, spraying (for example, spraying, misting, atomizing, dusting, dusting, water surface application, box application, etc.) , Soil application (for example, mixing, irrigation, etc.), surface application (for example, application, powder coating, coating, etc.), immersion, poison bait, smoke application, etc. It can also be applied by the so-called ultra-high concentration and small quantity spraying method. In this method, it is possible to contain 100% of the active ingredient.
- the fungicide containing the cyclic peptide compound as an active ingredient is sufficiently effective as an active ingredient when the cyclic peptide compound alone is used.
- other fertilizers, agricultural chemicals such as insecticides are used.
- Can be used in combination with or in combination with acaricides, nematicides, other fungicides, antiviral agents, attractants, herbicides, plant growth regulators, etc. is there.
- KK-1 itself exhibits control effects
- gray mold fungus Botrytis cinerea
- powdery mildew fungus Blumeria graminis
- blast fungus Magnnaporthe oryzae
- blight fungus Tehanatephorus cucumeris (Frank) Donk
- Example 1 [Genome analysis of Curvularia clavata] Conidia of C. clavata BAUA-2787 strain distributed from Akita Imano Co., Ltd. were inoculated into a 200 ml CM liquid medium (500 ml Erlenmeyer flask) and cultured at 26 ° C. and 130 rpm for 48 hours. After collecting the cultured cells with Miracloth, the cells were dehydrated by pressing with a spatula, put into a mortar that had been cooled to -20 ° C in advance, and then poured into liquid nitrogen and frozen. After rapidly crushing with a pestle until powdery, genomic DNA was extracted using DNeasy Plant Maxi Kit.
- Genome analysis was performed using two types of next-generation sequencers (5500xl SOLiD (life technologies) and MiSeq (illumina)). Libraries were created from the prepared genomic DNA of C. clavata using 5500 SOLiD Mate-Paired Library Kit (for 5500xl SOLiD) and Nextera DNA Sample Prep Kit (for MiSeq), and genome analysis was performed using a next-generation sequencer.
- the cyclic peptide (hereinafter referred to as KK-1) produced by the C. clavata BAUA-2787 strain is a cyclic peptide consisting of 10 amino acids as shown in FIG. 2, characterized by 5 out of 9 peptide bonds.
- NRPS nonribosomal peptide synthetase
- the NRPS gene that biosynthesizes the basic skeleton of the peptide was estimated based on the genomic sequence information of the C. clavata BAUA-2787 strain.
- NRPS is an enzyme that synthesizes a peptide by linking amino acids without using a ribosome, and has a module structure that matches the number of amino acid residues constituting the product peptide in order. Therefore, it can be presumed that an NRPS having a module and domain structure that matches the structural characteristics of a compound is an NRPS that biosynthesizes the peptide skeleton of the compound.
- filamentous fungi have about 10 NRPS (12 NRPS for C. heterostrophus and 14 NRPS for Asperillus fumigatus), the top 20 genes that were hit for each of the queried genes were extracted. As a result, it was narrowed down to the following 24 genes.
- TRAF01000140000154 TRAF01000135000001, TRAF01000070000001, TRAF01000068000001, TRAF01000108000067, TRAF01000130000847, TRAF01000117000049, TRAF01000117000050, TRAF01000099000028, TRAF01000088000002, TRAF01000082000001, TRAF01000081000001, TRAF01000117000368, TRAF01000142000376, TRAF01000109000032, TRAF01000142000383, TRAF01000136000233, TRAF01000100000101, TRAF01000061000021, TRAF01000108000142, TRAF01000139000099, TRAF01000140000122, TRAF01000117000201, TRAF01000136000219
- CDS gene sequence
- clavata is predicted by a dedicated program based on the genomic DNA sequence analyzed by the next-generation sequencer.
- this CDS prediction is often wrong, and in particular due to the presence of introns, the 5'-side and 3'-side sequences of the CDS may be deleted and predicted to be shorter than the actual CDS. Many.
- a blastx search was performed on the GenBank database using a genomic DNA sequence from 3000 bp upstream of the estimated start codon to 3000 bp downstream of the estimated stop codon of each of the 24 selected genes as a query.
- TRAF01000117000049 and TRAF01000117000050 were estimated as different genes, it became clear that they were actually one gene (referred to as TRAF01000117000049-50). Also, because the genomic DNA sequence surrounding the gene used for the search cannot be obtained sufficiently, the 5'-side is deleted (start codon cannot be found) or the 3'-side is deleted (stop codon cannot be found). Some genes were also present. These genes are shown below.
- TRAF01000135000001 5 'deletion
- TRAF01000070000001 Bottom 5 'and 3' are deleted
- TRAF01000068000001 (3 'deletion)
- TRAF01000088000002 (3 'deletion)
- TRAF01000082000001 5 'deletion
- TRAF01000081000001 3 'deletion
- TRAF01000117000368 5 'deletion
- Further analysis of these sequences revealed 2285 bp identical sequences at the 3′-end of TRAF01000068000001, the 5′- and 3′-ends of TRAF01000070000001 and the 5′-end of TRAF01000135000001. That is, it was estimated that these three genes were actually one gene (TRAF01000135000001_J3G) in which TRAF01000068000001, TRAF01000070000001 and TRAF01000135000001 were connected in this order.
- clavata BAUA-2787 strain has 12 NRPS. It was suggested to have a gene.
- TRAF01000142000383 was found only in the A domain and C domain by analysis with antiSMASH, but it was estimated to be NRPS from the PCP domain-like sequence found on the N-terminal side from the analysis by InterProScan. did.
- TRAF01000109000032 was thought to be a PKS-NRPS hybrid because it had a typical PKS (polyketide synthase) domain on the N-terminal side. [Estimation of NRPS involved in KK-1 biosynthesis] Since the C.
- clavata BAUA-2787 strain was considered to have 12 NRPS genes, a gene that biosynthesizes the basic peptide skeleton of KK-1 was searched for.
- KK-1 uses a cyclic peptide consisting of 10 amino acids as a basic skeleton.
- the peptide bond between Tyr and Ala is not a peptide bond but an ester bond.
- a peptide biosynthesized by NRPS has a feature that the number of amino acids constituting it matches the number of biosynthetic NRPS modules. Therefore, NRPS that biosynthesizes the basic peptide backbone of KK-1 consisting of 10 amino acids is considered to have 10 modules, that is, 10 A domains. Therefore, when the number of A domain of 12 putative NRPS genes of C. clavata was examined, only TRAF01000135000001_J3G had 10 A domains, so this gene is involved in the biosynthesis of KK-1. (Fig. 3-1).
- TRAF01000135000001_J3G there are five N-methy transferase domain (nMT domain) that N-methylates peptide bonds. Located in the module, the seventh module and the ninth module. The position of each module of NRPS coincides with the position of the amino acid constituting the biosynthesized peptide, and similarly, the position of the N-methylated peptide bond coincides with the module having the nMT domain. Assuming that the first module of TRAF01000135000001_J3G corresponds to the Ala residue of KK-1, the position of the N-methylated peptide bond is exactly the same as the module with the nMT domain. This strongly suggested that TRAF01000135000001_J3G is an NRPS that biosynthesizes the basic peptide skeleton of KK-1.
- TRAF01000135000001_J3G also has a C domain at the C terminus of the 10th module, and this C domain may be cyclized.
- KK-1 is biosynthesized by modification with various enzymes.
- the following table summarizes the domains included in the first to tenth modules constituting the estimated NRPS, the sequence numbers related to the amino acid sequences, and the like.
- the numerical range described in the column of “amino acid sequence” indicates the position of the amino acid residue in the entire amino acid sequence (SEQ ID NO: 37) of the estimated NRPS.
- TRAF01000135000002 is annotated as O-methyltransferase, which is thought to be responsible for O-methylation of tyrosine (Tyr) residues in the KK-1 molecule. It was. TRAF01000068000006, which is annotated as pmd1 encoding a leptomycin B resistant protein, is an ABC transporter as a protein function, and may be involved in the export of KK-1 to the outside of the cell. In addition, a transcription factor gene (TRAF01000068000005) was present in the cluster.
- MIDDAS-M detects gene clusters based on gene expression information.
- comprehensive expression information of production host genes in each production and non-production conditions of the substance is compared, and a group of genes that move in conjunction with the production conditions Is detected as a cluster.
- KK-1 production in CM liquid culture of C. clavata BAUA-2786 strain is not confirmed when the culture temperature is raised to 37 °C, so the normal culture temperature is 26 °C, production condition is 37 °C and non-production condition is 37 °C It was.
- the gene expression of C. clavata under these conditions was comprehensively analyzed by RNA-seq using a next-generation DNA sequencer, and cluster detection by MIDDAS-M was performed. As shown in FIG. The same gene group as the gene cluster estimated in (2) was detected (a and b in FIG. 6). Two peaks are detected because the gene cluster was fragmented in the original genome sequence data as described above.
- Example 2 the function of the transcription factor gene was analyzed from the gene group included in the KK-1 biosynthetic gene cluster estimated in Example 1.
- the gene encoding the transcription factor designated TRAF01000068000005 in Example 1 is designated TF068-005.
- Figure 7 A construct that highly expresses the TF068-005 gene is schematically shown in FIG. In this construct, 1000 bp upstream from the start codon of Ccnmt1 (TRAF01000124000183) gene of C.
- clavata BAUA-2787 strain is used as a promoter
- 355 bp downstream of Ccnmt1 gene is a terminator
- the selection marker is an aureobasidin A (AurA) resistance gene and did.
- the promoter and terminator of the Ccnmt1 gene are PCR using C. clavata genomic DNA as a template
- the aureobasidin A (AurA) resistance gene is PCR using the pAUR316 plasmid (TaKaRa) as a template
- the TF068-005 gene is C. clavata.
- Amplification was performed by PCR using cDNA as a template.
- the target plasmid (pUC-Pnmt1-TF-Tnmt1-aurA) was prepared by performing an In-Fusion reaction with linear pUC19 of In-Fusion® HD® Cloning® Kit (Clontech). The used primers and reaction conditions are shown below.
- nmt1-pro_In-Fus_FW1 5'-cggtacccggggatcTAGTCTGTTGATTACTCG-3 '(SEQ ID NO: 56)
- nmt1-pro_In-Fus_RV1 5'-ctcgacaaggtcatTTTGACTTTGAATACCGGTG-3 '(SEQ ID NO: 57)
- nmt1-ter_FW1 5'-GCAGTTGCCGTTGGACCAGAGG-3'
- nmt1-ter_In-Fus_RV2 5'-atagtcataacaagcCGCGACACTGTAATATTAAAGC-3 '(SEQ ID NO: 59)
- TF-CDS_FW1 5'-ATGACCTTTGTCGAGACTGTAGCC-3 '(SEQ ID NO: 60)
- TF-CDS_In-Fus_RV1 5'-TCCAACGGCAACTGCCTATGATATACTCAT
- initial denaturation 30 ° C. at 98 ° C.
- denaturation 10 ° C. at 98 ° C.
- annealing 30 ° C. at 60 ° C.
- elongation 30 sec at 72 ° C. for 30 cycles
- final elongation 7 min at 72 ° C. did.
- AnaurA-mark_In-Fus_FW1 5'-cgactctagaggatcCTGATGGTCAGATGGATCTG-3 '(SEQ ID NO: 62)
- AnaurA-mark_RV1 5'-GCTTGTTATGACTATGTATACATATGCG-3 '(SEQ ID NO: 63)
- Phusion Hot Start II High-Fidelity DNA Polymerase (Thermo Fisher Scientific) was used.
- initial denaturation 30 ° C. at 98 ° C.
- denaturation 10 ° C. at 98 ° C.
- annealing 30 ° C.
- CM + 1.2M sucrose + 10 ⁇ g / ml AbA Place 0.2ml of protoplast suspension on 5 selective plates, 6-7ml (per 90mm ⁇ petri dish) CM + 1.2M sucrose + 10 ⁇ g / ml AbA soft agar (1%) A selective medium was added, the layers were quickly overlaid so that the protoplast was uniform, and cultured at 26 ° C. for 6 days.
- “Culture 1” The conidia of the wild strain and the high expression strain of TF068-005 were inoculated into 100 ml CM medium (500 mL Erlenmeyer flask) and cultured with shaking at 26 ° C., 160 rpm, 72 h.
- "Culture 2” Conidia of wild strain and TF068-005 high-expressing strain were inoculated into 30ml K1 medium (conical flask with 100mL baffle), pre-cultured at 26 ° C, 200rpm, 72h, and 500 ⁇ l of culture solution with glucose concentration of 5% CM The medium was transferred to a medium (conical flask with 500 mL baffle) and main culture was performed at 26 ° C.
- RNA-seq analysis of TF068-005 highly expressing strains The cells cultured in liquid were frozen in liquid nitrogen, ground with a mortar and pestle, and total RNA was prepared using ISOGEN (Nippon Gene).
- RNA-Seq RNA-Seq library was prepared using the Truseq RNA Sample Prep Kit v2 and subjected to a next-generation sequencer (MiSeq) (Paired-End, Read Length 75). The obtained sequence data was mapped to the genomic sequence of C. clavata using the TopHat program. 5) Extraction and quantification of KK-1 In liquid culture, 15 ml of ethyl acetate was directly added to a 30 ml culture system, shaken at 130 rpm for 1 hour, and then centrifuged at 4,700 xg for 15 minutes. The supernatant was collected and concentrated by centrifugation, and this was used as the extracellular fraction.
- clavata using this plasmid was carried out in two ways: using the plasmid in a circular form and using the plasmid in a linear form after cutting at one place.
- RNA-seq analysis and KK-1 productivity were confirmed for one strain (ox_TF_1) introduced with a linear plasmid and one strain (ox_TF_2) introduced with a circular chain.
- FIG. 9 shows the results of RNA-seq analysis of gene transcription on the 2nd and 4th days after the main culture.
- the transcription of the KK-1 biosynthetic gene cluster constituent gene in the TF068-005 high expression strain was significantly higher than that in the wild strain.
- the difference from the expression level in the wild strain was larger after 4 days than after 2 days.
- the production amount of KK-1 was examined separately on the outside and inside of the cells at 3 and 7 days after the main culture.
- the results are shown in FIG.
- the total amount of KK-1 in the culture system increased in the TF068-005 highly expressing strain on both day 3 and day 7, In eyes, it was about twice that of the wild strain. From the results shown in FIG. 10, it was considered that about 20 to 30% of the total amount of KK-1 was accumulated in the microbial cells.
- FIG. 11 shows a photograph of the state in which the TF068-005 high expression strain is cultured in solid. As shown in FIG. 11, the productivity of KK-1 in the TF068-005 high expression strain was about 6 times that of the wild strain even in solid culture.
- the pyrG gene-disrupted strain cannot grow 5-fluoroorotic acid (5-FOA) into 5-fluorouridine phosphate (an inhibitor of thymine biosynthetic enzyme) and can therefore grow in a medium containing 5-FOA. .
- 5-fluoroorotic acid 5-FOA
- 5-fluorouridine phosphate an inhibitor of thymine biosynthetic enzyme
- PCR was performed with the following primer set using the genomic DNA of C. clavata BAUA-2787 as a template to amplify the region from 2005 bp upstream of the start codon of CcpyrG gene to 1261 bp downstream of the stop codon. .
- CcPyrG-del_FW3 5'-GACAGACTCTTCGTCGACGTC-3 '(SEQ ID NO: 64)
- CcPyrG-del_RV3 5'-GTTGTGGTTGGTGTTCCTGAGG-3 '(SEQ ID NO: 65)
- Phusion Hot Start II High-Fidelity DNA Polymerase (Thermo Fisher Scientific) was used. Temperature conditions were initial denaturation: 98 ° C for 3 min, denaturation: 98 ° C for 10 sec, annealing: 60 ° C for 30 sec, extension: 72 ° C for 2.5 min for 30 cycles, final extension: 72 ° C for 7 min. did.
- the end of the amplified DNA fragment (4463 bp) containing the CcpyrG gene was phosphorylated with T4 Polynucleotide Kinase (TOYOBO).
- pUC18 was digested with Sma I, dephosphorylated with E. coli Alkaline Phosphatase (TOYOBO), and ligated with the phosphorylated DNA fragment containing the CcpyrG gene.
- the pUC18 side including the upstream and downstream regions of the CcpyrG gene was PCR amplified with the following primer set to delete the region containing the CcpyrG gene.
- CcPyrG-del_FW2 5'-CACTCGATCTACCAAATCGACG-3 '(SEQ ID NO: 66)
- CcPyrG-del_RV2 5'-CCTATCCGGATATGCAGTCAC-3 '(SEQ ID NO: 67)
- Temperature conditions were initial denaturation: 98 ° C for 3 min, denaturation: 98 ° C for 10 sec, annealing: 60 ° C for 30 sec, elongation: 72 ° C for 3 min for 30 cycles, and final elongation: 72 ° C for 7 min. .
- PCR fragment was phosphorylated with T4 Polynucleotide Kinase and self-ligated to construct a target CcpyrG gene disruption construct (pUC-CcPyrG-del_C1).
- CcpyrG gene disruption transformation of C. clavata BAUA-2787 strain A fragment obtained by PCR amplification with pUC-CcPyrG-del_C1 as a template and a primer set of CcPyrG-del_FW3 / CcPyrG-del_RV3 was used for CcpyrG gene disruption.
- Phusion Hot Start II High-Fidelity DNA Polymerase was used for PCR. Temperature conditions were initial denaturation: 98 ° C. for 30 sec, denaturation: 98 ° C. for 10 sec, annealing: 63 ° C. for 30 sec, extension: 72 ° C. for 30 cycles for 35 cycles, and final extension: 72 ° C. for 5 min. .
- Transformation of the C. clavata BAUA-2787 strain was basically performed according to the protocol described in [Analysis using a Transcription Factor High-Expression Strain]. However, the selection medium was CM + 1 mg / ml 5-FOA + 0.2% Uridine + 0.02% Uracil. 3) Construction of TF068-005 gene disruption construct (Figure 13) A scheme for constructing a construct for disrupting the TF068-005 gene, which was found to encode a transcription factor as described above, is shown in FIG.
- primers TF068-005_L-arm_FW1 and TF068-005_L-arm_RV1 complementary to the upstream region of the TF068-005 gene were used. (1147 bp) was amplified. Similarly, right arm (1205 bp) was amplified using primers TF068-005_R-arm_FW1 and TF068-005_R-arm_RV1 complementary to the downstream region of the TF068-005 gene.
- selection marker gene pyrG (2231 bp) was amplified using primers CcPyrG-mark_FW1 and CcPyrG-mark_RV1. An electrophoresis photograph of each amplified fragment is shown in FIG.
- the left arm, pyrG marker and right arm that were amplified by the PCR were inserted in this order into the linear pUC19 plasmid vector attached to the kit.
- the obtained vector was introduced into Escherichia coli JM109 strain, and a plasmid was prepared from 3 clones of the transformant and sequenced.
- TF068-005_L-arm_FW1 5'-cggtacccggggatcCTCTGAAGCGGTCAAGGATAACG-3 '(SEQ ID NO: 68)
- TF068-005_L-arm_RV1 5'-atgaagcagagcggcGAGCCTAAGATATGCCAGGAGG-3 '(SEQ ID NO: 69)
- TF068-005_R-arm_FW1 5'-ctagcaaccgtcatgCCATAGACGTGGCACTCGAACG-3 '(SEQ ID NO: 70)
- TF068-005_R-arm_RV1 5'-cgactctagaggatcCGTCTTAAGGATGGTTCAGCTGC-3 '(SEQ ID NO: 71)
- CcPyrG-mark_FW1 5'-CATGACGGTTGCTAGGGTCG-3 '(SEQ ID NO: 72
- FIG. 15 schematically shows a method for transforming the CcpyrG gene disrupted strain of C. clavata BAUA-2787 strain prepared in 2) above with the TF068-005 gene disrupted construct.
- the TF068-005 gene disruption construct was linearized by digestion with restriction enzyme EcoR I (TaKaRa) and purified using Ethachinimate (Nippon Gene). Subsequently, the linearized construct was introduced into a C. clavata BAUA-2787 pyrG gene disruption strain and transformed. Transformation was basically performed according to the protocol described in [Analysis using a Transcription Factor High-Expression Strain].
- CM + 5 mM uridine + 5 mM uracil medium is used for culturing C. clavata BAUA-2787 pyrG gene disruption strain, and MM agar medium [1% glucose, 0.6% NaNO 3 , 0.052% KCl, 0.052% MgSO 4 .7H 2 O, 0.152% KH 2 PO 4 , and Hutner's trace elements (pH 6.5)] were used.
- MM agar medium 1% glucose, 0.6% NaNO 3 , 0.052% KCl, 0.052% MgSO 4 .7H 2 O, 0.152% KH 2 PO 4 , and Hutner's trace elements (pH 6.5)] were used.
- MM agar medium 1% glucose, 0.6% NaNO 3 , 0.052% KCl, 0.052% MgSO 4 .7H 2 O, 0.152% KH 2 PO 4 , and Hutner's trace elements (pH 6.5)] were used.
- the culture solution was filtered through Miracloth to remove the bacterial cells, sterilized through a 0.22 ⁇ m filter, and then impregnated into a paper disk.
- the paper disk and the mycelium of Botrytis cinerea cut out together with the agar medium were placed about 2.5 cm apart on the PDA medium, and cultured at 26 ° C. for 3 days.
- the positive control was a culture solution of the wild strain C. clavata BAUA-2787, and the negative control was a CM medium in which the cells were not cultured.
- RNA of the transcription factor gene (TF068-005) disrupted strain Inoculate the TF068-005 gene disrupted strain and wild-type conidial suspension in 30 ml of CM medium and shake at 26 ° C and 130 rpm for 72 hours. Cultured. Subsequently, the culture solution was filtered using Miracloth to collect the cells. 0.8 g of cells were weighed and frozen with liquid nitrogen, and then ground with a mortar and pestle. The cells were suspended in 10 ml of ISOGEN (Nippon Gene) and allowed to stand for 10 minutes. After 2 ml of chloroform was added and vortexed, the mixture was centrifuged at 4,700 ⁇ g for 10 minutes.
- ISOGEN Natural Gene
- RNA pellet was dissolved in 200 ⁇ l of Nucrease-free water.
- the obtained RNA solution was purified again using RNeasy Plant Mini Kit (QIAGEN).
- RNA-seq analysis of the transcription factor gene (TF068-005) disrupted strain Prepare an RNA-Seq library from total RNA prepared as described above using Truseq RNA Sample Prep Kit v2 (illumina). Used for generation sequencer (MiSeq) (Paired-End, Read Length 75). The obtained sequence data was mapped to the genomic sequence of C. clavata using the TopHat program. 8) Extraction and quantification of KK-1 in transcription factor gene disruption strain and wild strain TF068-005 gene-disrupted strain and wild-type conidial suspension were inoculated into 30 ml of CM medium and cultured with shaking at 26 ° C. and 130 rpm for 10 days.
- FIG. 16 shows the antibacterial activity test results for three of the five TF068-005 gene disrupted strains obtained.
- the positive control wild-type culture broth
- the TF068-005 gene-disrupted culture broth showed inhibitory activity.
- the negative control culture medium only
- RNA-seq comprehensive gene expression analysis
- TK-1 production were analyzed for the TF068-005 gene disrupted strain.
- TF068-005 gene disruption strains (2 strains) were analyzed using the wild strain as a comparative control.
- the results are shown in FIG. In FIG. 17, “del_TF” indicates the result of the TF068-005 gene disrupted strain.
- “RPKM” on the vertical axis in the graph shown in FIG. 17 is reads per kilobase of exon per million mapped sequence ⁇ reads, and the number of mapped sequences (reads) is the total number of reads and the sequence length of transcripts. It is a normalized value.
- the expression level of the gene contained in the estimated biosynthetic cluster is significantly lower than that in the wild strain, whereas it is contained in this cluster. It was clarified that the expression level of no surrounding gene group was equivalent to that of the wild strain except for the TRAF01000068000011 gene.
- the function of the TRAF01000068000011 gene is presumed from the annotation information as Nucleotide-sugar transporter involved in transport of sugar nucleotides synthesized in the cytosol or nucleus to the endoplasmic reticulum or Golgi apparatus. Since there was no sugar nucleotide in the structure of KK-1, this gene was thought to be less involved in biosynthesis.
- Example 3 gene disruption strains were prepared for each gene contained in the KK-1 biosynthesis gene cluster estimated in Example 1, and the function of each gene was analyzed.
- each cluster gene disruption construct ( Figure 19) Except for TRAF01000068000005 (transcription factor gene) verified in Example 2, for each gene contained in the KK-1 biosynthetic gene cluster, an upstream region of about 1,000 bp is L-arm, and a downstream region of about 1,000 bp is R-arm was obtained, and both gene fragments were obtained by PCR using the genomic DNA of C. clavata BAUA-2787 strain as a template. In addition, the pyrG gene serving as a selection marker for transformants was PCR amplified.
- the production of KK-1 is completely lost, and this gene is essential for the biosynthesis of KK-1. It has been suggested.
- the KK-1 production amount of the 5 types of genes (TRAF01000135000002, TRAF01000068000002, TRAF01000068000003, TRAF01000068000007 and TRAF01000068000008) disrupted strains in the gene cluster included in the gene cluster is significantly larger than that of the wild strain. It had fallen to. This suggests that these genes are deeply involved in the production of KK-1 at the stage of modification to the cyclic peptide skeleton biosynthesized by NRPS.
- the KK-1 production amount of the three types of genes (TRAF01000068000004, TRAF01000068000006 and TRAF01000068000009) disrupted strains is smaller than that of the above five genes, although the fluctuation range is small. Was falling. This suggests that these three genes are also involved in KK-1 production.
- the protein encoded by TRAF01000068000006 is presumed to be an ABC transporter, and is thought to be involved in the excretion of KK-1 produced in the cells outside the cell.
- TRAF01000068000009 which is annotated as ⁇ / ⁇ hydrolase, is a thioesterase that hydrolyzes an erroneously incorporated substrate within the polyketide antibiotic lankamycin biosynthetic cluster produced by Streptomyces rochei 7434AN4. Since the gene coding for is included, it is considered that this gene may have the same function. Since TRAF01000068000004 has no similar protein as small as 8.1 kDa, there was a possibility that TRAF01000068000004 does not have a specific function.
- Example 4 the KK-1 biosynthetic gene cluster whose function was analyzed in Examples 1 to 3 was introduced into Neisseria gonorrhoeae, and heterologous production of KK-1 in Neisseria gonorrhoeae was examined.
- A. oryzae strain and growth media NS4 ⁇ adeA strain (sC-, niaD-, ⁇ ligD :: sC, ⁇ adeA :: ptrA) was used as a parent strain into which the KK-1 biosynthetic gene cluster was introduced in A. oryzae.
- Czapek-dox (CD) minimal medium that meets the auxotrophy of the strain was used [0.6% NaNO 3 , 0.052% KCl, 0.152% KH 2 PO 4 , 0.0001% FeSO 4 ⁇ 7H 2 O, 0.00088% ZnSO 4 ⁇ 7H 2 O, 0.00004% CuSO 4 ⁇ 5H 2 O, 0.000015% MnSO 4 ⁇ 4H 2 O, 0.00001% Na 2 B 4 O 7 ⁇ 10H 2 O, 0.000005% (NH 4 ) 6 Mo 7 O 24 ⁇ 4H 2 O, 0.059% MgSO 4 ⁇ 7H 2 O and 2% glucose].
- CD Czapek-dox
- FIG. 22 schematically shows a construction scheme of a vector used for introducing the KK-1 biosynthetic gene cluster. As shown in FIG.
- the NRPS gene was divided into two and introduced into koji molds. That is, the first half (about 20 kb) and the second half (about 20 kb) of the 39-kb full-length gene were separately subcloned into a vector plasmid and introduced into koji molds. Thereafter, transformants in which both fragments were ligated in the koji mold were selected.
- pAAG-Cre capable of marker recycling by endogenous Cre recombinase expression was used.
- PCR was performed using primers NRPS-fh-F and NRPS-fh-R to amplify the gene in the first half.
- genomic DNA of C. clavata BAUA-2787 strain was used.
- the enzyme used for PCR was PrimeSTAR GXL DNA Polymerase (Takara), and PCR was performed according to the product instructions.
- the obtained PCR product was once ligated to the Eco RV cleavage site of pZErO-2 (Invitrogen).
- the first half of the NRPS gene was excised by NotI cleavage and ligated with the same site of pAAG-Cre.
- a plasmid in which the gonococcal promoter PamyB and the NRPS gene were ligated in the correct orientation was selected and used as the NRPS gene first half introduction vector pAAG-Cre / KK1-F.
- Each fragment and NotA-I digested pAAG-Cre were ligated by the In-Fusion cloning method, and the correctly ligated plasmid was designated as the NRPS gene latter half introduction vector pAAG-Cre / NRPSrh.
- In-Fusion cloning was performed using In-Fusion HD Cloning kit (Clontech) according to the method described in the attached product manual. The gene sequences of both the NRPS gene first half transfer vector and the second half transfer vector were confirmed, and it was confirmed that no mutation occurred in NRPS.
- NRPS-fh-F TCGACAAGCTTGCGGCCGCCACGTGACTAGTATGGCCAGCGACATCAATACTCATCCAG
- NRPS-fh-R ACTAGTCACGTGGCGGCCGCGCGGCGCGCCAAGATCGTCTTGCTGTACG
- NRPS-rh-IF-Fa GATGCGCTAGCGGCCGCGAAGTGGTCCTTGTCGCTGGTGAC
- NRPS-rh-IF-Ra TGCCGTTCGCATTCATAGGCATCTCGTC (SEQ ID NO: 113)
- NRPS-rh-IF-Fb TGAATGCGAACGGCAAGGTTGACAG
- NRPS-rh-IF-Rb CTTGGTTGCTGGCTTCGTCGTTGTC
- NRPS-rh-IF-Fc AAGCCAGCAACCA
- TRAF01000068000004 is not essential for the biosynthesis of KK-1 among the genes contained in the KK-1 biosynthesis gene cluster.
- TRAF01000068000005 is a transcription factor that controls the expression of cluster genes, it is expected that it is not necessary when all genes are controlled by a promoter for Neisseria gonorrhoeae.
- a gene introduction vector carrying these genes was constructed as shown in FIG.
- This plasmid is also equipped with a Cre-loxP marker recycling system using Cre recombinase and loxP sequences, and is a vector suitable for multi-step gene transfer.
- PCR was performed using the cDNA of C. clavata BAUA-2787 strain as a template to amplify each gene.
- a set of primers TR02-SpeI-F and TR02-SpeI-R was used for amplification of the TRAF01000068000002 gene, and a set of primers TR03-NotI-F and TR03-NotI-R was used for amplification of the TRAF01000068000003 gene.
- the obtained amplified fragments were once ligated to the Eco RV cleavage site of pZErO-2 (named pZTR02 and pZTR03, respectively).
- TRAF01000068000002 gene was excised from pZTR02 by SpeI digestion
- TRAF01000068000003 gene was excised from pZTR03 by NotI digestion.
- the excised genes were introduced into the SpeI and NotI sites of pA3AXPC in the order TRAF01000068000002 (SpeI) and TRAF01000068000003 (NotI).
- a plasmid with the correct insertion direction of both genes was selected and designated as pATR0203.
- a set of primers TR06-NheI-F and TR06-NheI-R is used for amplification of the TRAF01000068000006 gene
- a set of primers TR07-NotI-F and TR07-NotI-R is used for amplification of the TRAF01000068000007 gene
- a set of TRAF01000068000009 gene is used.
- the obtained PCR product was once ligated to the Eco RV cleavage site of pZErO-2 (named pZTR06, pZTR07 and pZTR08, respectively).
- TRAF01000068000006 gene was excised from pZTR06 by NheI digestion, TRAF01000068000007 gene from pZTR07 by NotI digestion, and TRAF01000068000008 gene from pZTR08 by SpeI digestion.
- the excised genes were introduced into the Nhe I, Spe I and Not I sites of pA3AXPC in the order of TRAF01000068000006 (Nhe I), TRAF01000068000008 (Spe I) and TRAF01000068000007 (Not I), respectively.
- a plasmid with the correct insertion direction of all genes was selected and designated pATR678.
- a set of primers TR09-NheI-F and TR09-NheI-R was used for amplification of the TRAF01000068000009 gene, and a set of primers OMT-NotI-F and OMT-NotI-R was used for the TRAF01000135000002 gene.
- the obtained PCR product was once ligated to the Eco RV cleavage site of pZErO-2 (named pZTR09 and pZOMT, respectively). Thereafter, the TRAF01000068000009 gene was excised from pZTR09 by NheI digestion and the TRAF01000135000001 gene was excised from pZOMT by NotI digestion.
- the excised genes were introduced into the Nhe I and Not I sites of pA3AXPC in the order TRAF01000068000009 (Nhe I) and TRAF01000135000001OMT (Not I).
- a plasmid with the correct insertion direction of both genes was selected and designated as pATR09OMT.
- TR02-SpeI-F GGACTAGTATGACTGAACCCACATGGAAG
- TR02-SpeI-R GGACTAGTTTAATAATCTACTTCAAGCAC
- TR03-NotI-F ATAAGAATGCGGCCGCATGGCGTTGCAAGAGCG
- TR03-NotI-R ATAAGAATGCGGCCGCTCAAGATGGGAAAGCCGCTG
- TR06-NheI-F CTAGCTAGCATGAGTGCTATCGAGCTGC
- TR06-NheI-R CTAGCTAGCTCAGCGATTGAGGGCCTGG
- TR07-NotI-F ATAAGAATGCGGCCGCATGAAGCTCACCGTTTTCAG
- TR07-NotI-R TR07-NotI-R: ATAAGAATGCGGCCGCTC
- Protoplasts were prepared by digesting the cell wall by shaking at 30 ° C. and 83 rpm for 3 hours. After the reaction, undigested cells were filtered with sterilized Miracloth, and the filtrate was centrifuged at 2,500 xg for 5 minutes at 4 ° C to recover protoplasts.
- the collected protoplasts were washed with 10 ml of 0.8 M NaCl, and centrifuged again at 4 ° C. and 2,500 ⁇ g for 5 minutes to precipitate and collect the protoplasts.
- Sol.I [0.8 M NaCl, 10 mM CaCl 2 , 10 mM Tris-HCl (pH 8.0)] was added so that the protoplast would be 2 ⁇ 10 8 cells / ml, and after suspension, 1/5 volume of Sol. II [40% (w / v) PEG4000, 50 mM CaCl 2 , 50 mM Tris-HCl (pH 8.0)] was added and mixed well.
- Cre recombinase by placing an adeA selection marker and a Cre recombinase between mutant loxP sequences (lox66 and lox71), expressing the Cre recombinase, inducing a loop-out between the loxP sequences, and extracting the adeA selection marker is there.
- the expression of Cre recombinase can be induced by an Xylose-inducible promoter. That is, a strain in which Cre recombinase is expressed and an adeA selection marker is lost can be obtained by inoculating a strain incorporating the above system into a medium containing Xylose as a carbon source.
- FIG. 23 schematically shows a scheme for introducing the first half and the second half of the NRPS gene in two stages using the Cre-loxP system.
- pAAG-Cre / NRPSfh carrying the first half of the NRPS gene was first introduced when introducing the vector into Neisseria gonorrhoeae.
- transduction of the vector was confirmed by PCR.
- Cre-loxP Cre-loxP
- the cells were grown in a CDE medium containing Xylose and supplemented with adenine, that is, a CDEAX medium. From the grown cells, a reddish colony characteristic of deletion of the marker adeA gene was selected. These strains were subjected to nuclear purification, and it was confirmed that they could not grow on a medium without addition of adenine.
- the strain was cultured with shaking in a YPM medium (containing 2% maltose as a promoter-inducing substrate) for 24 hours, and then the culture was filtered through Miracloth to recover the cells.
- the cells were snap frozen with liquid nitrogen and quickly crushed with a mortar and pestle under liquid nitrogen.
- the disrupted cells were transferred to a 1.5 ml Eppendorf tube, and RNA was extracted according to the protocol of RNeasy Plant Mini Kit (QIAGEN). In order to avoid DNA contamination, DNase treatment was also performed on-column according to the attached protocol. Finally, total RNA was obtained by elution twice with 50 ⁇ l of RNase free water.
- cDNA was synthesized from the obtained total RNA.
- High-Capacity cDNA-Reverse Transcription Kit (Applied Biosystem) was used for cDNA synthesis, and the method was performed according to the attached protocol.
- CDNA was synthesized from mRNA using the obtained total RNA equivalent of 4 ⁇ g in a 40 ⁇ l reaction system.
- the reverse transcription reaction conditions were preincubation at 25 ° C. for 10 minutes and then reaction at 37 ° C. for 120 minutes. The reaction was stopped by heating at 85 ° C. for 5 seconds.
- the obtained cDNA was stored at ⁇ 20 ° C. until use.
- qRT-PCR quantitative real-time PCR
- TOYOBO THUNDERBIRD SYBR qRCR Mix
- This mixed solution contains 400 ng equivalent of cDNA synthesized by reverse transcription.
- Three measurements were performed per sample.
- Mini Opticon real-time PCR analysis system BioRad
- BioRad CFX Manager 2.1 was used.
- the relative expression intensity was calculated as the expression ratio of each gene to the expression level of the internal standard gene (histone H2B) measured under the same conditions.
- O-MT means TRAF01000135000002 gene
- NRPS-1 to NRPS-3 mean TRAF01000135000001 gene (NRPS gene of Example 1)
- TR02 means TRAF01000068000002 gene
- TR03 means TRAF01000068000003 gene
- TR06 means the TRAF01000068000006 gene
- TR07 means the TRAF01000068000007 gene
- TR08 means the TRAF01000068000008 gene
- TR09 means the TRAF01000068000009 gene.
- all the introduced genes showed the same level of expression as histone, and all the genes required for KK-1 biosynthesis were highly expressed in the koji mold into which the cluster gene was introduced. It became clear that 9) Evaluation of KK-1 productivity Further, KK-1 productivity was evaluated for the transformed koji molds into which the KK-1 biosynthetic cluster gene prepared as described above was introduced.
- the conidia suspension of the transformed koji mold prepared in this example was inoculated into 100 ml of YPM (maltose 2%) or CM (maltose 2%) and cultured with shaking at 26 ° C. and 140 rpm for 5 days. Subsequently, the cultured cells and the culture solution were extracted with acetone / ethyl acetate and concentrated to dryness. This was dissolved in acetonitrile and subjected to LC / MS analysis.
- the conditions for LC / MS analysis were the same as those for evaluating KK-1 production in Curvularia sp.
- the antibacterial property of the said extract was evaluated by the growth inhibitory effect with respect to the gray mold which is the object of antibacterial evaluation.
- the extract was soaked in a paper disc (thin, ⁇ 6 mm), and this paper disc and the mycelium of Botrytis cinerea cut out together with the agar medium were placed on a CM agar medium and subjected to counterculture. Subsequently, the antibacterial activity was evaluated based on the extent of colony growth up to the periphery of the paper disc of gray mold.
- FIG. 26A shows the LC / MS analysis result
- B shows the antibacterial activity test result.
- FIG. 26A in the extract derived from the transformed koji mold prepared in this example, a peak completely matching the retention time and molecular weight of the KK-1 preparation was detected. Furthermore, as shown in B of FIG. 26, the mycelial elongation inhibitory activity of the gray mold fungus was observed in this extract. From these results, the transformed gonococcus prepared in this example was introduced in a form that functions the KK-1 biosynthetic gene cluster identified in C. clavata, and can produce KK-1 heterogeneously. It was proved that.
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Abstract
Description
配列番号3に示すアミノ酸配列又は配列番号3に示すアミノ酸配列に対して70%以上の同一性を有するアミノ酸配列からなる第1の縮合ドメインと、配列番号4に示すアミノ酸配列又は配列番号4に示すアミノ酸配列に対して70%以上の同一性を有するアミノ酸配列からなる第2のアデニレーションドメインと、配列番号5に示すアミノ酸配列又は配列番号5に示すアミノ酸配列に対して70%以上の同一性を有するアミノ酸配列からなる第2のペプチジルキャリアタンパク質ドメインとをN末端側からこの順で有する第2モジュールと、
配列番号6に示すアミノ酸配列又は配列番号6に示すアミノ酸配列に対して70%以上の同一性を有するアミノ酸配列からなる第2の縮合ドメインと、配列番号7に示すアミノ酸配列又は配列番号7に示すアミノ酸配列に対して70%以上の同一性を有するアミノ酸配列からなる第3のアデニレーションドメインと、配列番号8に示すアミノ酸配列又は配列番号8に示すアミノ酸配列に対して70%以上の同一性を有するアミノ酸配列からなる第1のN-メチルトランスフェラーゼドメインと、配列番号9に示すアミノ酸配列又は配列番号9に示すアミノ酸配列に対して70%以上の同一性を有するアミノ酸配列からなる第3のペプチジルキャリアタンパク質ドメインとをN末端側からこの順で有する第3モジュールと、
配列番号10に示すアミノ酸配列又は配列番号10に示すアミノ酸配列に対して70%以上の同一性を有するアミノ酸配列からなる第3の縮合ドメインと、配列番号11に示すアミノ酸配列又は配列番号11に示すアミノ酸配列に対して70%以上の同一性を有するアミノ酸配列からなる第4のアデニレーションドメインと、配列番号12に示すアミノ酸配列又は配列番号12に示すアミノ酸配列に対して70%以上の同一性を有するアミノ酸配列からなる第4のペプチジルキャリアタンパク質ドメインとをN末端側からこの順で有する第4モジュールと、
配列番号13に示すアミノ酸配列又は配列番号13に示すアミノ酸配列に対して70%以上の同一性を有するアミノ酸配列からなる第4の縮合ドメインと、配列番号14に示すアミノ酸配列又は配列番号14に示すアミノ酸配列に対して70%以上の同一性を有するアミノ酸配列からなる第5のアデニレーションドメインと、配列番号15に示すアミノ酸配列又は配列番号15に示すアミノ酸配列に対して70%以上の同一性を有するアミノ酸配列からなる第2のN-メチルトランスフェラーゼドメインと、配列番号16に示すアミノ酸配列又は配列番号16に示すアミノ酸配列に対して70%以上の同一性を有するアミノ酸配列からなる第5のペプチジルキャリアタンパク質ドメインとをN末端側からこの順で有する第5モジュールと、
配列番号17に示すアミノ酸配列又は配列番号17に示すアミノ酸配列に対して70%以上の同一性を有するアミノ酸配列からなる第5の縮合ドメインと、配列番号18に示すアミノ酸配列又は配列番号18に示すアミノ酸配列に対して70%以上の同一性を有するアミノ酸配列からなる第6のアデニレーションドメインと、配列番号19に示すアミノ酸配列又は配列番号19に示すアミノ酸配列に対して70%以上の同一性を有するアミノ酸配列からなる第3のN-メチルトランスフェラーゼドメインと、配列番号20に示すアミノ酸配列又は配列番号20に示すアミノ酸配列に対して70%以上の同一性を有するアミノ酸配列からなる第6のペプチジルキャリアタンパク質ドメインとをN末端側からこの順で有する第6モジュールと、
配列番号21に示すアミノ酸配列又は配列番号21に示すアミノ酸配列に対して70%以上の同一性を有するアミノ酸配列からなる第6の縮合ドメインと、配列番号22に示すアミノ酸配列又は配列番号22に示すアミノ酸配列に対して70%以上の同一性を有するアミノ酸配列からなる第7のアデニレーションドメインと、配列番号23に示すアミノ酸配列又は配列番号23に示すアミノ酸配列に対して70%以上の同一性を有するアミノ酸配列からなる第4のN-メチルトランスフェラーゼドメインと、配列番号24に示すアミノ酸配列又は配列番号24に示すアミノ酸配列に対して70%以上の同一性を有するアミノ酸配列からなる第7のペプチジルキャリアタンパク質ドメインとをN末端側からこの順で有する第7モジュールと、
配列番号25に示すアミノ酸配列又は配列番号25に示すアミノ酸配列に対して70%以上の同一性を有するアミノ酸配列からなる第7の縮合ドメインと、配列番号26に示すアミノ酸配列又は配列番号26に示すアミノ酸配列に対して70%以上の同一性を有するアミノ酸配列からなる第8のアデニレーションドメインと、配列番号27に示すアミノ酸配列又は配列番号27に示すアミノ酸配列に対して70%以上の同一性を有するアミノ酸配列からなる第8のペプチジルキャリアタンパク質ドメインとをN末端側からこの順で有する第8モジュールと、
配列番号28に示すアミノ酸配列又は配列番号28に示すアミノ酸配列に対して70%以上の同一性を有するアミノ酸配列からなる第8の縮合ドメインと、配列番号29に示すアミノ酸配列又は配列番号29に示すアミノ酸配列に対して70%以上の同一性を有するアミノ酸配列からなる第9のアデニレーションドメインと、配列番号30に示すアミノ酸配列又は配列番号30に示すアミノ酸配列に対して70%以上の同一性を有するアミノ酸配列からなる第5のN-メチルトランスフェラーゼドメインと、配列番号31に示すアミノ酸配列又は配列番号31に示すアミノ酸配列に対して70%以上の同一性を有するアミノ酸配列からなる第9のペプチジルキャリアタンパク質ドメインとをN末端側からこの順で有する第9モジュールと、
配列番号32に示すアミノ酸配列又は配列番号32に示すアミノ酸配列に対して70%以上の同一性を有するアミノ酸配列からなる第9の縮合ドメインと、配列番号33に示すアミノ酸配列又は配列番号33に示すアミノ酸配列に対して70%以上の同一性を有するアミノ酸配列からなる第10のアデニレーションドメインと、配列番号34に示すアミノ酸配列又は配列番号34に示すアミノ酸配列に対して70%以上の同一性を有するアミノ酸配列からなる第10のペプチジルキャリアタンパク質ドメインと、配列番号35に示すアミノ酸配列又は配列番号35に示すアミノ酸配列に対して70%以上の同一性を有するアミノ酸配列からなる第10の縮合ドメインとをN末端側からこの順で有する第10モジュールと、
をN末端側からこの順で有し、Curvularia属糸状菌が生産する環状ペプチド化合物の非リボソーム型ペプチド合成活性を有するタンパク質をコードする環状ペプチド化合物合成関連遺伝子。
(b)配列番号37に示すアミノ酸配列に対して70%以上の同一性を有するアミノ酸配列からなり、Curvularia属糸状菌が生産する環状ペプチド化合物の非リボソーム型ペプチド合成活性を有するタンパク質
(c)配列番号36に示す塩基配列の相補鎖に対してストリンジェントな条件下でハイブリダイズするポリヌクレオチドによりコードされ、Curvularia属糸状菌が生産する環状ペプチド化合物の非リボソーム型ペプチド合成活性を有するタンパク質
(3)Curvularia属糸状菌由来であることを特徴とする(1)記載の環状ペプチド化合物合成関連遺伝子。
(b)配列番号39に示すアミノ酸配列に対して70%以上の同一性を有するアミノ酸配列からなり、転写因子活性を有するタンパク質
(c)配列番号38に示す塩基配列の相補鎖に対してストリンジェントな条件下でハイブリダイズするポリヌクレオチドによりコードされ、転写因子活性を有するタンパク質
(6)Curvularia属糸状菌由来であることを特徴とする(5)記載の環状ペプチド化合物合成関連遺伝子。
培養した形質転換体及び/又は培養液から上記環状ペプチド化合物を回収する工程とを含む、
Curvularia属糸状菌が生産する環状ペプチド化合物の製造方法。
[2]配列番号43に示すアミノ酸配列又は配列番号43に示すアミノ酸配列に対して70%以上の同一性を有するアミノ酸配列からなるタンパク質をコードする遺伝子
[3]配列番号45に示すアミノ酸配列又は配列番号45に示すアミノ酸配列に対して70%以上の同一性を有するアミノ酸配列からなるタンパク質をコードする遺伝子
[4]配列番号47に示すアミノ酸配列又は配列番号47に示すアミノ酸配列に対して70%以上の同一性を有するアミノ酸配列からなるタンパク質をコードする遺伝子
[5]配列番号49に示すアミノ酸配列又は配列番号49に示すアミノ酸配列に対して70%以上の同一性を有するアミノ酸配列からなるタンパク質をコードする遺伝子
[6]配列番号51に示すアミノ酸配列又は配列番号51に示すアミノ酸配列に対して70%以上の同一性を有するアミノ酸配列からなるタンパク質をコードする遺伝子
[7]配列番号53に示すアミノ酸配列又は配列番号53に示すアミノ酸配列に対して70%以上の同一性を有するアミノ酸配列からなるタンパク質をコードする遺伝子
(10)上記形質転換体は、麹菌(Aspergillus oryzae)を宿主とすることを特徴とする(8)記載の環状ペプチド化合物の製造方法。
[2]配列番号43に示すアミノ酸配列又は配列番号43に示すアミノ酸配列に対して70%以上の同一性を有するアミノ酸配列からなるタンパク質をコードする遺伝子
[3]配列番号45に示すアミノ酸配列又は配列番号45に示すアミノ酸配列に対して70%以上の同一性を有するアミノ酸配列からなるタンパク質をコードする遺伝子
[4]配列番号47に示すアミノ酸配列又は配列番号47に示すアミノ酸配列に対して70%以上の同一性を有するアミノ酸配列からなるタンパク質をコードする遺伝子
[5]配列番号49に示すアミノ酸配列又は配列番号49に示すアミノ酸配列に対して70%以上の同一性を有するアミノ酸配列からなるタンパク質をコードする遺伝子
[6]配列番号51に示すアミノ酸配列又は配列番号51に示すアミノ酸配列に対して70%以上の同一性を有するアミノ酸配列からなるタンパク質をコードする遺伝子
[7]配列番号53に示すアミノ酸配列又は配列番号53に示すアミノ酸配列に対して70%以上の同一性を有するアミノ酸配列からなるタンパク質をコードする遺伝子
(13)麹菌(Aspergillus oryzae)を宿主とすることを特徴とする(11)記載の形質転換体。
なお、環状ペプチド化合物(以下、KK-1と称する場合もある)の化合物名は、Tyrosine,N-[N-[N-[N-[N-[N-[N-[N-[[1-(2-hydroxy-1-oxopropyl)-2-piperidinyl]carbonyl]-N-methylvalyl]valyl]-N-methyl-a-aspartyl]-N-methylvalyl]-Nmethylisoleucyl]glycyl]-N-methylvalyl]-O-methyl-, d2-lactone (9CI)である。
これら遺伝子群のうちNRPS遺伝子は、本環状ペプチド化合物の基本骨格を形成する機能を有するNRPSをコードしている。すなわち、本NRPSは、[アラニン(Ala)-ピペコリン酸(Pip)-バリン(Val)-バリン-アスパラギン酸(Asp)-バリン-イソロイシン(Ile)-グリシン(Gly)-バリン-チロシン(Tyr)]という10個のアミノ酸からなるペプチド骨格を形成する。すなわち、本NRPSは、アラニンのカルボシキル基とピペコリン酸のアミノ基との間にペプチド結合を形成し、当該ピペコリン酸のカルボキシル基とバリンのアミノ基との間にペプチド結合を形成し、当該バリンのカルボキシル基とバリンのアミノ基との間にペプチド結合を形成し、当該バリンのカルボキシル基とアスパラギン酸のアミノ基との間にペプチド結合を形成し、当該アスパラギン酸のカルボキシル基とバリンのアミノ基との間にペプチド結合を形成し、当該バリンのカルボキシル基とイソロイシンのアミノ基との間にペプチド結合を形成し、当該イソロイシンのカルボキシル基とグリシンのアミノ基との間にペプチド結合を形成し、当該グリシンのカルボキシル基とバリンのアミノ基との間にペプチド結合を形成し、当該バリンのカルボキシル基とチロシンのアミノ基との間にペプチド結合を形成し、当該チロシンのカルボキシル基と上記アラニンのアミノ基との間にペプチド結合を形成する活性を有している。また、本NRPSは、ピペコリン酸とバリンとの間のペプチド結合をメチル化し、バリンとアスパラギン酸との間のペプチド結合をメチル化し、アスパラギン酸とバリンとの間のペプチド結合をメチル化し、バリンとイソロイシンとの間のペプチド結合をメチル化し、グリシンとバリンとの間のペプチド結合をメチル化する活性を有している。
〔Curvularia属糸状菌〕
本発明に係るCurvularia属糸状菌は、上述したNRPS遺伝子を有するという特徴を有する。上述したように、本発明に係るNRPS遺伝子は、Curvularia clavata以外のCurvularia属糸状菌からも単離できる蓋然性が高い。すなわち、本発明に係るCurvularia属糸状菌は、Curvularia clavataに限定されず、環状ペプチド化合物を生産するC. affinis、C. brachyspora、C. caricae-papayae、C. eragrostidis (Cochliobolus eragrostidis) 、C. fallax、C. geniculata (Cochliobolus geniculatus)、C. harveyi、C. lunata (Cochliobolus lunatus)、C. ovoidea、C. pallescens、C. penniseti、C. prasadii、C. protuberata、C. senegalensis、C. trifolii、C. tuberculata (Cochliobolus tuberculatus)等を挙げることができる。
〔転写因子遺伝子〕
上述した、環状ペプチド化合物の合成に関与する遺伝子群に含まれる転写因子遺伝子は、当該遺伝子群に含まれる各遺伝子の発現を転写レベルで正に制御する機能を有するタンパク質をコードする。本発明に係る転写因子遺伝子は、一例としてCurvularia clavata由来であって、環状ペプチド化合物の合成に関与する遺伝子群に含まれる遺伝子に対する転写促進活性を有するタンパク質のアミノ酸配列を配列番号39に示し、当該アミノ酸配列に対応するコーディング領域の塩基配列を配列番号38に示す。本発明に係る転写因子遺伝子は、これら配列番号38及び39により規定することができる。
〔形質転換体〕
上述した本発明に係る環状ペプチド化合物合成関連遺伝子のうち、NRPS遺伝子を発現可能に宿主に導入することによって、上記環状ペプチド化合物におおける基本ペプチド骨格を有する化合物合成能を有する形質転換体を作製することができ、また、NRPS遺伝子とともに、NRPS遺伝子以外の環状ペプチド化合物合成遺伝子を発現可能に宿主に導入することによって上記環状ペプチド化合物合成能を有する形質転換体を作製することができる。
〔環状ペプチド化合物の製造〕
上述した形質転換体を利用することで、目的とする環状ペプチド化合物を製造することができる。
〔実施例1〕
[Curvularia clavataのゲノム解析]
株式会社秋田今野商店から分譲されたC. clavata BAUA-2787株の分生子を200ml CM液体培地(500ml 三角フラスコ)に植菌し、26℃、130rpmで48時間培養した。培養菌体をミラクロスで集菌した後、スパーテルで菌体をプレスして脱水し、予め-20℃に冷却しておいた乳鉢に菌体を入れ、液体窒素を注いで凍結させた。粉状になるまで乳棒で素早く破砕した後、DNeasy Plant Maxi Kitを用いてゲノムDNAを抽出した。
[C. clavataが有するNRPS遺伝子の探索]
C. clavata BAUA-2787株が産生する環状ペプチド(以下KK-1)は、図2に示すように、10個のアミノ酸からなる環状ペプチドで、特徴としては、9つあるペプチド結合のうち、5つがN-メチル化されている点と、分子内のチロシン(Tyr)残基がO-メチル化されている点が挙げられる。本実施例では、本KK-1の基本骨格を合成する非リボソーム型ペプチド合成酵素(nonribosomal peptide synthetase(NRPS))遺伝子を探索した。
TRAF01000140000154, TRAF01000135000001, TRAF01000070000001, TRAF01000068000001, TRAF01000108000067, TRAF01000130000847, TRAF01000117000049, TRAF01000117000050, TRAF01000099000028, TRAF01000088000002, TRAF01000082000001, TRAF01000081000001, TRAF01000117000368, TRAF01000142000376, TRAF01000109000032, TRAF01000142000383, TRAF01000136000233, TRAF01000100000101, TRAF01000061000021, TRAF01000108000142, TRAF01000139000099, TRAF01000140000122, TRAF01000117000201, TRAF01000136000219
C. clavataの遺伝子配列(CDS)は、次世代シークエンサーによって解析されたゲノムDNA配列を基に、専用プログラムによって予測されている。しかしながら、このCDS予測は間違っていることも少なくはなく、特にイントロンの存在などにより、CDSの5'-側及び3'-側の配列が削られ、実際のCDSよりも短く予測されることが多い。より正確なCDSを得るためには、遺伝子が位置するゲノム領域周辺の配列情報などを利用して、ひとつひとつ詳細に検討する必要がある。そこでまず、絞り込んだ24の遺伝子それぞれの推定開始コドンの上流3000bpから推定終止コドンの下流3000bpまでのゲノムDNA配列をクエリーとして、GenBankのデータベースに対してblastx検索をかけた。この検索により、既知のタンパク質配列と相同性を示す領域が明らかになったことから、遺伝子の開始および終止コドンを推定した。また、同じく既知のタンパク質配列との相同性からイントロンが存在する部位が示されたので、GU-AG ruleに従いイントロン部位を予測し、より正確なCDSを推定した。
TRAF01000135000001 (5' 欠失)
TRAF01000070000001 (5', 3' ともに欠失)
TRAF01000068000001 (3' 欠失)
TRAF01000088000002 (3' 欠失)
TRAF01000082000001 (5' 欠失)
TRAF01000081000001 (3' 欠失)
TRAF01000117000368 (5' 欠失)
これらの配列をさらに詳細に解析したところ、TRAF01000068000001の3'-末端、TRAF01000070000001の5'-及び3'-末端、TRAF01000135000001の5'-末端に2285bpの全く同一の配列が見出された。すなわち、これら3つの遺伝子は、実際には、TRAF01000068000001、TRAF01000070000001及びTRAF01000135000001がこの順につながった1つの遺伝子 (TRAF01000135000001_J3Gとする) であると推定された。
1) TRAF01000088000002 - TRAF01000082000001
2) TRAF01000088000002 - TRAF01000117000368
3) TRAF01000081000001 - TRAF01000082000001
4) TRAF01000081000001 - TRAF01000117000368
の4通りの組み合わせでつながる可能性があるが、配列情報からでは正しい組み合わせは決定できなかった。
[KK-1の生合成に関与するNRPSの推定]
C. clavata BAUA-2787株が12個のNRPS遺伝子を有していると考えられたことから、この中から、KK-1の基本ペプチド骨格を生合成している遺伝子の探索を行った。KK-1は図2に示したように、10個のアミノ酸からなる環状のペプチドを基本骨格としている。TyrとAlaの間はペプチド結合でなく、エステル結合となっているが、何らかの修飾を受けてこのような形になっていると考えられる。NRPSにより生合成されるペプチドは、構成するアミノ酸の数が生合成NRPSのモジュールの数と一致するという特徴を持つ。ゆえに、10個のアミノ酸からなるKK-1の基本ペプチド骨格を生合成するNRPSは10個のモジュール、すなわち10個のAドメインを有すると考えられる。そこで、C. clavataの12個の推定NRPS遺伝子のAドメインの数を調べたところ、TRAF01000135000001_J3Gのみが10個のAドメインを有していたことから、本遺伝子がKK-1の生合成に関わるNRPSであると考えられた (図3-1)。
[KK-1の生合成遺伝子クラスターの推定]
以上のように、KK-1の基本ペプチド骨格を形成するNRPS遺伝子としてTRAF01000135000001_J3Gを推定したことから、このNRPS(TRAF01000135000001_J3G)を含む領域にKK-1生合成遺伝子クラスターを構成する遺伝子群が存在していると考えられた。そこで、当該NRPS周辺に位置する遺伝子に関して、コードするタンパク質のアミノ酸配列と、アミノ酸配列に基づいて推定される機能とを基に、生合成遺伝子クラスターを構成する遺伝子群の推定を試みた。なお、TRAF01000135000001_J3Gは、ゲノムシークエンスおよび遺伝子予測の際に分断されていた3つの配列を結合したものであることから、以下の遺伝子クラスターの予測には結合後の配列を用いた。
[遺伝子発現情報を基にしたKK-1の生合成遺伝子クラスターの推定]
KK-1生合成遺伝子クラスターの推定を行うにあたり、MIDDAS-M(Umemura M. et. al.,Plos one, 8(5), e63673. (2013))アルゴリズムを用いたバイオインフォマティクスの手法によるクラスター検出も行った。
本実施例では、実施例1で推定したKK-1生合成遺伝子クラスターに含まれる遺伝子群のうち転写因子遺伝子について機能を解析した。なお、本実施例では、実施例1において、TRAF01000068000005と表記した転写因子をコードする遺伝子をTF068-005と表記する。
[転写因子高発現株を用いた解析]
1)TF068-005高発現コンストラクトの構築(図7)
TF068-005遺伝子を高発現するコンストラクトを図7に模式的に示した。このコンストラクトにおいては、C. clavata BAUA-2787株のCcnmt1(TRAF01000124000183)遺伝子の開始コドンより上流1000bpをプロモーターとし、Ccnmt1遺伝子の下流355bpをターミネーターとし、選抜マーカーをオーレオバシジンA(AurA)耐性遺伝子とした。なお、Ccnmt1遺伝子のプロモーター及びターミネーターはC. clavataゲノムDNAを鋳型としたPCR、オーレオバシジンA(AurA)耐性遺伝子はpAUR316プラスミド(TaKaRa)を鋳型としたPCR、TF068-005遺伝子はC. clavataのcDNAを鋳型としたPCRにより増幅した。
nmt1-pro_In-Fus_RV1: 5'-ctcgacaaaggtcatTTTGACTTTGAATACCGGTG-3'(配列番号57) nmt1-ter_FW1: 5'-GCAGTTGCCGTTGGACCAGAGG-3'(配列番号58)
nmt1-ter_In-Fus_RV2: 5'-atagtcataacaagcCGCGACACTGTAATATTAAAGC-3'(配列番号59)
TF-CDS_FW1: 5'-ATGACCTTTGTCGAGACTGTAGCC-3'(配列番号60)
TF-CDS_In-Fus_RV1: 5'-TCCAACGGCAACTGCCTATGATATACTCATGTTCTCGTC-3'(配列番号61)
PCRには、Phusion Hot Start II High-Fidelity DNA Polymerase(Thermo Fisher Scientific)を使用した。温度条件としては、初期変性:98℃で30secとし、変性:98℃で10sec、アニーリング:60℃で30sec、伸長:72℃で30secを1サイクルとして30サイクル行い、最終伸長:72℃で7minとした。
AnaurA-mark_RV1: 5'-GCTTGTTATGACTATGTATACATATGCG-3'(配列番号63)
PCRには、Phusion Hot Start II High-Fidelity DNA Polymerase(Thermo Fisher Scientific)を使用した。温度条件としては、初期変性:98℃で30secとし、変性:98℃で10sec、アニーリング:60℃で30sec及び伸長:72℃で2minを1サイクルとして30サイクル行い、最終伸長:72℃で7minとした。
2)C. clavata BAUA-2787株の形質転換
100mlのCM培地(300 ml三角フラスコ)にC. clavata BAUA-2787株の胞子懸濁液を植菌し、30℃で40時間振盪培養した後、ガラスろ過器(11G1)を用いたろ過により菌糸を集め、滅菌水で洗浄後、スパーテル等で押さえて十分に水分を除いた。菌体を10mlのprotoplast化溶液(YL組成)に加え懸濁し、30℃で3時間ゆるやかに振盪しprotoplast化を行った。ミラクロスでろ過し、ろ液を1,500xgで5分間遠心し、protoplastを集め、0.8M NaClで2回洗浄した。protoplastを2×108/mlとなるようにSolution 1 [0.8M NaCl、10mM CaCl2、10mM Tris-HCl(pH8.0)]に懸濁し、0.2容量のSolution 2[40%(w/v) PEG4000、50mM CaCl2、50mM Tris-HCl(pH8.0)]を加えて緩やかに懸濁し、0.2mlのprotoplast懸濁液にpUC-Pnmt1-TF-Tnmt1-aurA(7.8μg/20μl)を加え、氷中で10分間静置した。1mlのSolution 2を加え、緩やかに懸濁し、室温で15分間静置した。10mlのSolution 1を加えて緩やかに懸濁し、遠心でprotoplastを集め、上清をできるだけ除き、protoplastを1 mlのSolution 1に懸濁した。CM+1.2M sucrose+10μg/ml AbA 選択プレート5枚にprotoplast懸濁液を0.2mlずつのせ、6~7ml(90mmφシャーレ当たり)のCM+1.2M sucrose+10μg/ml AbA軟寒天(1%)選択培地を加え、protoplastが均一になるようにすばやく重層し、26℃で6日間培養した。
3) TF068-005高発現株の培養条件
TF068-005高発現株は以下の3通りの培養を行い、RNAの調製ならびにKK-1の生産を調べた。
「培養1」
野生株及びTF068-005高発現株の分生子を100ml CM培地(500mL 三角フラスコ)に植菌し、26℃、160rpm、72h振盪培養した。
「培養2」
野生株及びTF068-005高発現株の分生子を30ml K1培地(100mL バッフル付三角フラスコ)に植菌し、26℃、200rpm、72h前培養し、培養液500μlをglucose濃度を5%としたCM培地(500mL バッフル付三角フラスコ)に移し、26℃、130rpmで本培養を行った。
「培養3」
50 mlファルコンチューブに玄米2.5gと水2mlを入れ、オートクレーブした。これに野生株及びTF068-005高発現株の分生子を植菌し、26℃で8日間静置培養した。
4) TF068-005高発現株のRNA-seq解析
液体培養した菌体を液体窒素で凍結させ、乳鉢と乳棒で摩砕し、ISOGEN(ニッポンジーン)を用いてtotal RNAを調製した。このtotal RNAからTruseq RNA Sample Prep Kit v2を用いて、RNA-Seq用ライブラリーを調製し、次世代シークエンサー(MiSeq)に供した(Paired-End, Read Length 75)。得られた配列データはTopHatプログラムを用いてC. clavataのゲノム配列にマッピングした。
5) KK-1の抽出と定量
液体培養においては、30mlの培養系に直接15ml酢酸エチル添加し、130rpmで1時間振盪した後、4,700xgで15分間遠心した。上清を回収・遠心濃縮し、これを菌体外画分とした。続いて、酢酸エチル回収後の水層に15mlアセトンを添加し、ボルテックスにて撹拌後、4,700xgで15分間遠心した。デカントで上清を回収し、遠心濃縮でアセトンを除いた。これに酢酸エチルを15ml添加し、4,700xgで15分間遠心した。酢酸エチル層を50mlチューブに回収し、遠心濃縮したものを菌体内画分とした。
KK-1の定量はUPLCによって行った。条件を以下に示す。
・ 装置:ACQUITY UPLC I-Class システム(Waters)
・ カラム: Acquity UPLC BEH C18 2.1x100mm
・ Solvent: Gradient 50%-98% Acetonitrile+0.1% Fomic Acid(3min)
・ 流速: 0.6ml/min
・ 検出波長: 273nm
<結果と考察>
本実施例では、C. clavataにおいて高発現している遺伝子として見出したnmt-1遺伝子ホモログ(TRAF01000124000183;Ccnmt1)のプロモーターとターミネーターを用いて転写因子を高発現させるコンストラクト(pUC-Pnmt1-TF-Tnmt1-aurA)を作製した(図7)。このプラスミドを用いたC. clavataの形質転換は、プラスミドを環状のまま用いるものと、1ヵ所切断し直鎖状で用いるものの2通りの方法で行った。その結果得られた、プラスミドを直鎖導入した1株(ox_TF_1)と環状導入した1株(ox_TF_2)についてRNA-seq解析ならびに、KK-1の生産性の確認を行った。
[転写因子破壊株を用いた解析]
1)CcpyrG遺伝子破壊用プラスミドの構築(図12)
C. clavata BAUA-2787株におけるpyrG遺伝子(CcpyrG遺伝子)を破壊するためのプラスミド構築のスキームを図12に示した。なお、pyrG遺伝子破壊株は、5-フルオロオロチン酸(5-FOA)を5-フルオロウリジンリン酸(チミン生合成酵素の阻害剤)へ変換できないため5-FOAを含む培地でも生育することができる。
CcPyrG-del_RV3: 5'-GTTGTGGTTGGTGTTCCTGAGG-3'(配列番号65)
PCRはPhusion Hot Start II High-Fidelity DNA Polymerase(Thermo Fisher Scientific)を使用した。温度条件は、初期変性:98℃で3minとし、変性:98℃で10sec、アニーリング:60℃で30sec、伸長:72℃で2.5minを1サイクルとして30サイクル行い、最終伸長:72℃で7minとした。
CcPyrG-del_RV2: 5'-CCTATCCGGATATGCAGTCAC-3'(配列番号67)
PCRはPhusion Hot Start II High-Fidelity DNA Polymeraseを使用した。温度条件は、初期変性:98℃で3minとし、変性:98℃で10sec、アニーリング:60℃で30sec、伸長:72℃で3minを1サイクルとして30サイクル行い、最終伸長:72℃で7minとした。
2)C. clavata BAUA-2787株のCcpyrG遺伝子破壊形質転換
pUC-CcPyrG-del_C1を鋳型にCcPyrG-del_FW3/CcPyrG-del_RV3のプライマーセットでPCR増幅した断片をCcpyrG遺伝子破壊に用いた。PCRはPhusion Hot Start II High-Fidelity DNA Polymeraseを使用した。温度条件は、初期変性:98℃で30secとし、変性:98℃で10sec、アニーリング:63℃で30sec、伸長:72℃で30secを1サイクルとして35サイクル行い、最終伸長:72℃で5minとした。
3)TF068-005遺伝子破壊コンストラクトの構築(図13)
上述のように転写因子をコードすることが判明したTF068-005遺伝子を破壊するためのコンストラクトを構築するスキームを図13に示した。
TF068-005_L-arm_FW1:5’-cggtacccggggatcCTCTGAAGCGGTCAAGGATAACG-3’(配列番号68)
TF068-005_L-arm_RV1:5’-atgaagcagagcggcGAGCCTAAGATATGCCAGGAGG-3’(配列番号69)
TF068-005_R-arm_FW1:5’-ctagcaaccgtcatgCCATAGACGTGGCACTCGAACG-3’(配列番号70)
TF068-005_R-arm_RV1:5’-cgactctagaggatcCGTCTTAAGGATGGTTCAGCTGC-3’(配列番号71)
CcPyrG-mark_FW1:5’-CATGACGGTTGCTAGGGTCG-3’(配列番号72)
CcPyrG-mark_RV1:5’-GCCGCTCTGCTTCATTGCTG-3’(配列番号73)
(小文字はIn-Fusion反応のための15 bpのオーバーラップ配列)
4)TF068-005遺伝子破壊コンストラクトでの形質転換(図15)
図15に、上記2)で作製したC. clavata BAUA-2787株のCcpyrG遺伝子破壊株をTF068-005遺伝子破壊コンストラクトでの形質転換する方法を模式的に示した。先ず、TF068-005遺伝子破壊コンストラクトを制限酵素EcoR I(TaKaRa)消化により直鎖化し、Ethachinimate(ニッポン・ジーン)を用いて精製した。続いて、直鎖化したコンストラクトをC. clavata BAUA-2787 pyrG遺伝子破壊株に導入し形質転換した。形質転換は、基本的に[転写因子高発現株を用いた解析]で説明したプロトコルに従って行った。ただし、C. clavata BAUA-2787 pyrG遺伝子破壊株の培養にはCM+5mM uridine+5mM uracil培地を、形質転換体の選抜にはMM寒天培地[1% glucose、0.6% NaNO3、0.052% KCl、0.052% MgSO4・7H2O、0.152% KH2PO4、and Hutner’s trace elements(pH6.5)]を用いた。
5) TF068-005遺伝子破壊株の抗菌活性試験
以上のように作製されたTF068-005遺伝子破壊株の分生子懸濁液を100mlのCM培地に植菌し、26℃、130rpmで72時間培養した。ミラクロスにより培養液を濾過して菌体を除き、0.22μmのフィルターに通して滅菌した後、ペーパーディスクに染み込ませた。このペーパーディスクと、寒天培地ごと切り抜いた灰色かび病菌(Botrytis cinerea)の菌糸をPDA培地上に2.5cm程度離して置き、26℃で3日間対峙培養した。ポジティブコントロールは野生株であるC. clavata BAUA-2787株の培養液、ネガティブコントロールは菌体を培養していないCM培地とした。
6) 転写因子遺伝子(TF068-005)破壊株のtotal RNAの調整
TF068-005遺伝子破壊株及び野生株の分生子懸濁液を30mlのCM培地に植菌し、26℃、130rpmで72時間振盪培養した。続いて、ミラクロスを用いて培養液濾過し、菌体を回収した。菌体0.8gを秤量して液体窒素で凍結させた後、乳鉢と乳棒で摩砕した。その菌体を10mlのISOGEN(ニッポンジーン)に懸濁して10分間静置し、クロロホルムを2ml添加してボルテックスで撹拌した後、4,700xgで10分間遠心した。水層を回収し、5mlのイソプロパノールを添加してボルテックスで撹拌した後、4,700xgで10分間遠心した。上清を捨て、5mlの75%エタノールを加えて洗浄し4,700xgで10分間遠心した後再度上清を捨て、RNAのペレットを200μlのNucrease-free waterに溶解した。得られたRNA溶液はRNeasy Plant Mini Kit (QIAGEN)を用いて再度精製した。
7) 転写因子遺伝子(TF068-005)破壊株のRNA-seq解析
以上のように調製したtotal RNAから、Truseq RNA Sample Prep Kit v2 (illumina)を用いてRNA-Seq用ライブラリーを調製し、次世代シーケンサー(MiSeq)に供した (Paired-End, Read Length 75)。得られた配列データはTopHatプログラムを用いてC. clavataのゲノム配列にマッピングした。
8) 転写因子遺伝子破壊株及び野生株におけるKK-1の抽出と定量
TF068-005遺伝子破壊株及び野生株の分生子懸濁液を30mlのCM培地に植菌し、26℃、130rpmで10日間振盪培養した。培養液に15mlの酢酸エチルを添加して1時間振盪した後、4,700xgで15分間遠心した。酢酸エチル層を回収し、乾固させて菌体外画分とした。続いて、酢酸エチル層回収後の水層にアセトンを10ml添加し、ボルテックスにて撹拌後、遠心濃縮でアセトンを除いた。これに酢酸エチルを15ml添加し、ボルテックスにて撹拌した後、4,700xgで10分間遠心した。酢酸エチル層を回収して遠心濃縮し、これを菌体内画分とした。得られた抽出物は再度酢酸エチルに溶解した後、LC/MS分析に供した。
<LC>
装置:ACQUITY UPLC I-Classシステム (Waters)
カラム: Acquity UPLC BEH C18 2.1 x 100 mm
移動相: DW+0.1%Formic Acid/アセトニトリル+0.1% Formic Acid=
50/50 (0.5min)→2/98(3.4min)
Gradient 50%-98% Acetonitrile + 0.1% Formic Acid (0.5-3.4 min)
流速: 0.6 ml/min
検出波長: 273 nm
<MS>
装置:Xevo G2 QTof (Waters)
イオン化条件: Negative
<結果と考察>
本実施例では、TF068-005遺伝子の破壊及び核の純化が確認された株が5株得られたため、これらの株の培養上清における抗菌活性を調べた。上述した抗菌活性試験の結果を図16に示した。図16には、取得した5株のTF068-005遺伝子破壊株のうち3株についての抗菌活性試験の結果を掲載した。図16に示すように、ポジティブコントロール(野生株の培養液)では灰色かび病菌のペーパーディスク側に伸びる菌糸伸長の阻害が見られたのに対し、TF068-005遺伝子破壊株の培養液では阻害活性は検出されず、ネガティブコントロール(培養液のみ)と同様の結果が得られた。このことから、TF068-005遺伝子破壊株においてKK-1生産能が著しく低下していると考えられ、TF068-005遺伝子がKK-1の生合成に深く関与していることが強く示唆された。
〔実施例3〕
本実施例では、実施例1で推定したKK-1生合成遺伝子クラスターに含まれる各遺伝子について遺伝子破壊株を作製し、各遺伝子の機能を解析した。
1) 各クラスター遺伝子破壊コンストラクトの構築(図19)
実施例2で検証したTRAF01000068000005(転写因子遺伝子)を除いて、KK-1生合成遺伝子クラスターに含まれる各遺伝子について、上流側約1,000bpの領域をL-arm、下流側約1,000bpの領域をR-armとし、両遺伝子断片をC. clavata BAUA-2787株のゲノムDNAを鋳型にしたPCRにより得た。また、形質転換体の選抜マーカーとなるpyrG遺伝子をPCR増幅した。続いてIn-Fusion Cloning Kit (Clontech)を用いて、上記PCRで増幅したL-arm、pyrG遺伝子、R-armをこの順に連結させた遺伝子断片をpUC19に挿入し、各遺伝子破壊コンストラクトを作製した(図19)。
・pyrG選抜マーカー増幅用(実施例2で使用したプライマー)
CcPyrG-mark_FW1:5’-CATGACGGTTGCTAGGGTCG-3’(配列番号72)
CcPyrG-mark_RV1:5’-GCCGCTCTGCTTCATTGCTG-3’(配列番号73)
・TRAF01000135000002破壊コンストラクトL-arm(982bp)増幅用
TRAF135-002_del_L-arm_FW:5’-cggtacccggggatcGACCCATTGCAGCTTGTG-3’ (配列番号74)
TRAF135-002_del_L-arm_RV:5’-atgaagcagagcggcGTGCAGTATGGTGTCTAAAACG-3’ (配列番号75)
・TRAF01000135000002破壊コンストラクトR-arm(950bp)増幅用
TRAF135-002_del_R-arm_FW:5’-ctagcaaccgtcatgGATGAATGAGCACCCTGTTAG-3’ (配列番号76)
TRAF135-002_del_R-arm_RV:5’-cgactctagaggatcGTACATTACAAAAACCTGTTGCAG-3’ (配列番号77)
・TRAF01000135000001破壊コンストラクトL-arm(1,000bp)増幅用
TRAF135-001_del_L-arm_FW:5’-cggtacccggggatcGTCCCACGTGCAGCTTCAAC-3’ (配列番号78)
TRAF135-001_del_L-arm_RV:5’-atgaagcagagcggcCGTGGAGTATCCCAGGATGG-3’ (配列番号79)
・TRAF01000135000001破壊コンストラクトR-arm(982bp)増幅用
TRAF135-001_del_R-arm_FW:5’-ctagcaaccgtcatgCCAGCCAAAGGGTATCATGG-3’ (配列番号80)
TRAF135-001_del_R-arm_RV:5’-cgactctagaggatcTGAGGGCAGCGTAGCCTG-3’ (配列番号81)
・TRAF01000068000002破壊コンストラクトL-arm(992bp)増幅用
TRAF068-002_del_L-arm_FW:5’-cggtacccggggatcGTGGATAAATTCGTACCCTTTG-3’ (配列番号82)
TRAF068-002_del_L-arm_RV:5’-atgaagcagagcggcCTGATCTTTGTTGTGGTCGTG-3’ (配列番号83)
・TRAF01000068000002破壊コンストラクトR-arm(1,014bp)増幅用
TRAF068-002_del_R-arm_FW:5’-ctagcaaccgtcatgCAGTTTGGCACTTGAGCATC-3’ (配列番号84)
TRAF068-002_del_R-arm_RV:5’-cgactctagaggatcCACGGAAAGGAACTCCTACAG-3’ (配列番号85)
・TRAF01000068000003破壊コンストラクトL-arm(912bp)増幅用
TRAF068-003_del_L-arm_FW:5’-cggtacccggggatcCTCTGGGAAAAGCGGTTAG-3’ (配列番号86)
TRAF068-003_del_L-arm_RV:5’-atgaagcagagcggcGAAGAACCGAGAGCGAGAG-3’ (配列番号87)
・TRAF01000068000003破壊コンストラクトR-arm(995bp)増幅用
TRAF068-003_del_R-arm_FW:5’-ctagcaaccgtcatgCTTGCATCTACCTAGATATTTCACG-3’ (配列番号88)
TRAF068-003_del_R-arm_RV:5’-cgactctagaggatcCAGAGAATCAGCAGAGACACC-3’ (配列番号89)
・TRAF01000068000004破壊コンストラクトL-arm(991bp)増幅用
TRAF068-004_del_L-arm_FW:5’-cggtacccggggatcCCCTGGTAGTTCAGTGGAAGTAAG-3’ (配列番号90)
TRAF068-004_del_L-arm_RV:5’-atgaagcagagcggcTGATAGAGGTACGGGGGTG-3’ (配列番号91)
・TRAF01000068000004破壊コンストラクトR-arm(1,003bp)増幅用
TRAF068-004_del_R-arm_FW:5’-ctagcaaccgtcatgTGCTTGGCTGCTTCAAATC-3’ (配列番号92)
TRAF068-004_del_R-arm_RV:5’-CGACTCTAGAGGATCCTAATACTTGTCGTCCCACTGATG-3’ (配列番号93)
・TRAF01000068000006破壊コンストラクトL-arm(993bp)増幅用
TRAF068-006_del_L-arm_FW:5’-cggtacccggggatcGCAGTACATCGTCAGGGTC-3’ (配列番号94)
TRAF068-006_del_L-arm_RV:5’-atgaagcagagcggcGATGAATAAGGCGAAGGAAAG-3’ (配列番号95)
・TRAF01000068000006破壊コンストラクトR-arm (579 bp)増幅用
TRAF068-006_del_R-arm_FW:5’-ctagcaaccgtcatgCCCTCTTTTTTCTTGCTGTCTC-3’ (配列番号96)
TRAF068-006_del_R-arm_RV:5’-cgactctagaggatcGAAGGAAGGACGGATACTGG-3’ (配列番号97)
・TRAF01000068000007破壊コンストラクトL-arm(769bp)増幅用
TRAF068-007_del_L-arm_FW:5’-cggtacccggggatcGATGAGCGTAGAATTCGTAAAAAG-3’ (配列番号98)
TRAF068-007_del_L-arm_RV:5’-atgaagcagagcggcGCGAACGGGCGTTTTTC-3’ (配列番号99)
・TRAF01000068000007破壊コンストラクトR-arm(579bp)増幅用
TRAF068-007_del_R-arm_FW:5’-ctagcaaccgtcatgGAAGGAAGGACGGATACTGG-3’ (配列番号100)
TRAF068-007_del_R-arm_RV:5’-cgactctagaggatcCCCTCTTTTTTCTTGCTGTCTC-3’ (配列番号101)
・TRAF01000068000008破壊コンストラクトL-arm(716bp)増幅用
TRAF068-008_del_L-arm_FW:5’-cggtacccggggatcCTCCTTATTTTGCAACTTCTGATAC-3’ (配列番号102)
TRAF068-008_del_L-arm_RV:5’-atgaagcagagcggcCGTGTTGATTTTGGTAATTTTG-3’ (配列番号103)
・TRAF01000068000008破壊コンストラクトR-arm(769bp)増幅用
TRAF068-008_del_R-arm_FW:5’-ctagcaaccgtcatgGATGAGCGTAGAATTCGTAAAAAG-3’ (配列番号104)
TRAF068-008_del_R-arm_RV:5’-cgactctagaggatcGCGAACGGGCGTTTTTC-3’ (配列番号105)
・TRAF01000068000009破壊コンストラクトL-arm(716bp)増幅用
TRAF068-009_del_L-arm_FW:5’-cggtacccggggatcCGTGTTGATTTTGGTAATTTTG-3’ (配列番号106)
TRAF068-009_del_L-arm_RV:5’-atgaagcagagcggcCTCCTTATTTTGCAACTTCTGATAC-3’ (配列番号107)
・TRAF01000068000009破壊コンストラクトR-arm(989bp)増幅用
TRAF068-009_del_R-arm_FW:5’-ctagcaaccgtcatgCTAGCAGCCATAAGAGACGTAACC-3’ (配列番号108)
TRAF068-009_del_R-arm_RV:5’- cgactctagaggatcGTTTTCATTGCATGCTCCG-3’ (配列番号109)
・PCR反応条件
PCRには、Phusion High-Fidelity DNA Polymerase(Thermo Fisher Scientific)を使用した。温度条件は、初期変性:98℃で30secとし、変性:98℃で10sec、アニーリング:60℃で30sec及び伸長:72℃で45secを1サイクルとして30サイクル行い、最終伸長:72℃で5min行った。
2) C. clavata pyrG破壊株の形質転換
図20のAに、実施例2の2)で作製したC. clavata BAUA-2787株のCcpyrG遺伝子破壊株を各遺伝子破壊コンストラクトで形質転換する方法を模式的に示した。構築した各遺伝子破壊コンストラクトを所定の制限酵素(図20のBに示した)により直鎖化した。続いて、実施例2の[転写因子高発現株を用いた解析]で説明したプロトコルに従って、直鎖化したコンストラクトをC. clavata BAUA-2787 pyrG遺伝子破壊株に導入した。宿主のC. clavata BAUA-2787 pyrG遺伝子破壊株を培養する際には、5mMのuridine及びuracilを添加した液体CM培地を用い、形質転換体の選抜はMM寒天培地で行った。
3) クラスター遺伝子破壊株の培養
上記2)で得られた各遺伝子破壊株の分生子懸濁液を、KM培地30ml(バッフル付き100ml三角フラスコ)に植菌し、26℃、130rpmで3日間振盪培養したものを前培養液とした。続いて、CM培地30ml(バッフル付き100ml三角フラスコ)に前培養液を300μl植菌し、26℃、130rpmで7日間振盪培養した。
4) 培養系内の代謝物の抽出および分析
上記3)で得られた培養液に15mlの酢酸エチルを添加して130rpmで1時間振盪した後、4,200xgで15分間遠心した。酢酸エチル層を回収し、遠心濃縮したものを菌体外画分とした。続いて、酢酸エチル層回収後の水層にアセトンを10ml添加し、ボルテックスにて撹拌後、遠心濃縮でアセトンを除いた。これに酢酸エチルを15ml添加し、ボルテックスにて撹拌した後、4,200xgで10分間遠心した。酢酸エチル層を回収して遠心濃縮し、これを菌体内画分とした。得られた抽出物は、菌体内外の画分をまとめて500μlの酢酸エチルに溶解した後、1μlをLC/MS分析に供した。
・LC/MS分析条件
<LC>
装置: ACQUITY UPLC I-Classシステム(Waters)
カラム: Acquity UPLC BEH C18 2.1x100mm
移動相: DW/MeCN=50/50(0.5min)→2/98(3.4min) (各溶媒0.1%ギ酸を含む)
流速: 0.6ml/min
検出波長: 273nm
<MS>
装置: Xevo G2 QTof(Waters)
イオン化条件: Negative
<結果と考察>
本実施例で作製した各遺伝子破壊株及び実施例2で作製した転写因子遺伝子破壊株について、KK-1の生産性を調べた。その結果を図21に示した。図21においては、各株のKK-1生産量(Wild株に対する相対量)を平均±標準偏差(n=2)で示した。図21に示すように、基本骨格の環状ペプチドを生合成するNRPS(TRAF01000135000001)遺伝子を破壊した株ではKK-1の生産が完全に消失し、本遺伝子がKK-1の生合成に必須であることが示唆された。
〔実施例4〕
本実施例では、実施例1~3で機能解析されたKK-1生合成遺伝子クラスターを麹菌に導入し、麹菌におけるKK-1の異種生産を検討した。
1)麹菌の菌株、培地(A. oryzae strain and growth media)
麹菌(A. oryzae)においてKK-1生合成遺伝子クラスターを導入する親株としては、NS4 ΔadeA株(sC-、niaD-、ΔligD::sC、ΔadeA::ptrA)を使用した。通常の生育および分生子の形成には、菌株の栄養要求性を満たしたCzapek-dox (CD)最小培地を使用した [0.6% NaNO3、0.052% KCl、0.152% KH2PO4、0.0001% FeSO4・7H2O、0.00088% ZnSO4・7H2O、0.00004% CuSO4・5H2O、0.000015% MnSO4・4H2O、0.00001% Na2B4O7・10H2O、0.000005% (NH4)6Mo7O24・4H2O、0.059% MgSO4・7H2O及び2% glucose]。すなわち、窒素源としてNaNO3の替わりに70mMのグルタミン酸ナトリウムを添加した培地(CDE)あるいはCDEに0.01%アデニンを添加した培地(CDEA)を使用した。麹菌 amyB プロモーターで導入した遺伝子の発現を誘導する際には、YPM 培地(1% yeast extract、2% polypeptone、2% maltose)を使用し、KK-1の生産性を評価する際も同様にYPM 培地を使用した。
2) NRPS遺伝子分割導入ベクターの構築(図22)
図22に、KK-1生合成遺伝子クラスターを導入する際に使用するベクターの構築スキームを模式的に示した。図22に示すように、KK-1生合成遺伝子クラスターに含まれる遺伝子のうち、NRPS遺伝子については2分割して麹菌に導入した。すなわち、全長39kbの遺伝子を前半部分(約20kb)と後半部分(約20kb)とを別々にベクタープラスミドへサブクローニングし、麹菌に導入した。その後、麹菌内で両断片が連結した形質転換体を選抜した。ベクタープラスミドとして、内在性Creリコンビナーゼ発現によりマーカーリサイクルが可能なpAAG-Creを使用した。
NRPS-fh-F: TCGACAAGCTTGCGGCCGCCACGTGACTAGTATGGCCAGCGACATCAATACTCATCCAG(配列番号110)
NRPS-fh-R: ACTAGTCACGTGGCGGCCGCGGCGCGCCAAGATCGTCTTGCTGTACG(配列番号111)
NRPS-rh-IF-Fa: GATGCGCTAGCGGCCGCGAAGTGGTCCTTGTCGCTGGTGAC(配列番号112)
NRPS-rh-IF-Ra: TGCCGTTCGCATTCATAGGCATCTCGTC(配列番号113)
NRPS-rh-IF-Fb: TGAATGCGAACGGCAAGGTTGACAG(配列番号114)
NRPS-rh-IF-Rb: CTTGGTTGCTGGCTTCGTCGTTGTC(配列番号115)
NRPS-rh-IF-Fc: AAGCCAGCAACCAAGTCGAAGATTG(配列番号116)
NRPS-rh-IF-Rc: GTCACTAGTGCGGCCGCCTATTTTTGCAAGATCTTGTTCAAAC(配列番号117)
3)クラスター遺伝子導入ベクターの構築(図22)
実施例2で記載したとおり、KK-1生合成遺伝子クラスターに含まれる遺伝子のうち、TRAF01000068000004はKK-1の生合成に必須ではないことが示唆されている。また、TRAF01000068000005はクラスター遺伝子の発現を制御する転写因子であるため、全遺伝子を麹菌用のプロモーターで制御する場合、必要でないことが予想される。上述したNRPS遺伝子及びこれら2種の遺伝子を除く7遺伝子を麹菌に導入するため、図22に示したように、これらの遺伝子を搭載した遺伝子導入ベクターの構築を実施した。
TR02-SpeI-F: GGACTAGTATGACTGAACCCACATGGAAG(配列番号118)
TR02-SpeI-R: GGACTAGTTTAATAATCTACTTCAAGCAC(配列番号119)
TR03-NotI-F: ATAAGAATGCGGCCGCATGGCGTTGCAAGAGCG(配列番号120)
TR03-NotI-R: ATAAGAATGCGGCCGCTCAAGATGGGAAAGCCGCTG(配列番号121)
TR06-NheI-F: CTAGCTAGCATGAGTGCTATCGAGCTGC(配列番号122)
TR06-NheI-R: CTAGCTAGCTCAGCGATTGAGGGCCTGG(配列番号123)
TR07-NotI-F: ATAAGAATGCGGCCGCATGAAGCTCACCGTTTTCAG(配列番号124)
TR07-NotI-R: ATAAGAATGCGGCCGCTCAGAGCCGCGCCAAC(配列番号125)
TR08-SpeI-F: GGACTAGTATGACGAAAAGGGAAAGCAAC(配列番号126)
TR08-SpeI-R: GGACTAGTCTACGCGTTTTCTTTCGAC(配列番号127)
TR09-NheI-F: CTAGCTAGCATGGAGAGCGAAGACAATCC(配列番号128)
TR09-NheI-R: CTAGCTAGCTCAGCAGTATCCCATCGG(配列番号129)
OMT-NotI-F: ATTTGCGGCCGCATGGACCCGAGACAGTCACGGATC(配列番号130)
OMT-NotI-R: ATTTGCGGCCGCTTATGGTGTGGTGGGTTGCCATTC(配列番号131)
4) 麹菌への遺伝子導入方法
上記3)で作製した各種プラスミドを用いた麹菌の形質転換にはプロトプラストPEG法を用いて行った。親株(NS4 ΔadeA株)の分生子1×107個を、200ml容三角フラスコ中の100mlのYPD液体培地(1% yeast extract、2% polypeptone及び2% glucose)に接種し、30℃で20時間、160回転で振盪培養した菌糸をミラクロス(CALIBIOCHEM 社)で濾過、集菌した。滅菌水で洗浄し、乾熱滅菌したスパーテルで菌体をプレスし脱水した。集めた菌糸を50ml容チューブに入れ、0.20μmのフィルターで濾過したプロトプラスト化溶液[10mg/ml Lysing Enzymes (Sigma社)、5mg/ml Cellulase Onozuka (Yakult Pharmaceutical Ind.Co.,Ltd.)、2.5mg/ml Yatalase (TAKARA) in 0.8M NaCl及び10mM Phosphate buffer (pH 6.0)]を25ml加えて懸濁した。30℃、83rpmで3時間振盪し、細胞壁を消化することでプロトプラストを作製した。反応後、滅菌したミラクロスで未消化の菌体を濾過し、濾液を4℃、2,500×gで5分間遠心分離しプロトプラストを回収した。回収したプロトプラストを10mlの0.8M NaClで洗浄し、4℃、2,500×gで5分間再度遠心分離することでプロトプラストを沈殿させ回収した。プロトプラストが2×108個/mlとなるようにSol.I[0.8M NaCl、10mM CaCl2、10mM Tris-HCl (pH 8.0)]を加え、懸濁した後、1/5量のSol.II [40%(w/v) PEG4000、50 mM CaCl2、50 mM Tris-HCl (pH 8.0)]を加え、よく混合した。240μlのプロトプラスト液を15ml容チューブに分注し、DNA溶液を5~20μg分加えよく混合し、氷中で30分放置した。次に、1mlのSol.IIを加え、よく混合した後、室温で20分間放置した。10mlのSol.Iを加え、よく混合した後室温、2,500×gで5分間遠心し、上清を取り除き、300μlのSol.Iを加えた。プロトプラストを均一に懸濁し、0.8M NaClを含むCDE選択寒天培地に散らしてまいた後、55℃に温めておいた同組成の軟寒天培地[0.6%(w/v)Agar]5 mlを周りから注ぎ、プロトプラストを手早く均一に懸濁するように重層した。その後、30℃でコロニーを形成するまで培養した。
5)麹菌におけるマーカーリサイクル
麹菌におけるマーカーリサイクルは、以下の方法に従って行った。すなわち、変異型loxP配列(lox66とlox71)の間にadeA選抜マーカーとCreリコンビナーゼを配置し、Creリコンビナーゼを発現させることでloxP配列間でのループアウトを誘発し、adeA選抜マーカーを抜取る方法である。ここで、CreリコンビナーゼはXylose誘導型プロモーターで発現を誘導できる。すなわち、上記システムを組込んだ菌株を炭素源をXyloseとした培地に植菌することでCreリコンビナーゼが発現し、adeA選抜マーカーが抜け落ちた菌株を取得することができる。adeA選抜マーカーが抜け落ちた菌株では、アデニン要求性が回復するので、再びadeA選抜マーカーを使用して遺伝子組換え体を選抜することが可能となる。
6) 麹菌内でのKK-1 NRPS遺伝子再構築(図23)
Cre-loxPシステムを利用してNRPS遺伝子の前半部分及び後半部分を2段階で導入するスキームを模式的に図23に示した。図23に示すように、麹菌へのベクターの導入にあたり、まずはNRPS遺伝子前半部分を搭載したpAAG-Cre/NRPSfhを導入した。得られた形質転換体について、ベクターの導入をPCRにより確認した。本株に対して後半部分を導入するため、Cre-loxPシステムによるマーカーリサイクルを実施した。ベクター内のCreリコンビナーゼを発現させるため(xynG2プロモーターで制御)、Xylose含有かつアデニン添加CDE培地、すなわちCDEAX培地で菌体を生育させた。生育してきた菌体について、マーカーであるadeA遺伝子欠失の特徴である赤みを帯びたコロニーを選抜した。これらの株について核純化を行い、アデニン無添加の培地では生育できないことを確認した。
7) 麹菌内におけるクラスター内必須遺伝子の高発現
上記3)で作製した、クラスター遺伝子を搭載したプラスミドpATR0203、pATR678及びpATR09OMTを順にマーカーリサイクルを繰返して麹菌へと導入した。しかしながら、全プラスミドを導入した株において、TRAF01000068000009遺伝子の脱落が認められた。また、本系統の株については、NRPS遺伝子についても部分的な脱落が生じていることが明らかになった。さらに別系統の株について、遺伝子脱落の有無を調査したところ、度々遺伝子の脱落が観察された。これらの結果から、マーカーリサイクルあるいは形質転換操作時に近接するプロモーター配列間でループアウトが生じている可能性が考えられた。そこで導入戦略を立て直し、図24に示すように、マーカーリサイクルあるいは形質転換操作を可能な限り減数するため、7遺伝子を搭載した3種のベクターを同時に導入することとした。その結果、全遺伝子の導入が確認できた株が得られた。
8) 転写解析(図25)
次に、以上のようにして作製されたKK-1生合成クラスター遺伝子を導入した形質転換麹菌について、導入遺伝子の発現解析を実施した。菌株をYPM培地(プロモーター誘導基質としてマルトース2%を含む)で24時間振盪培養した後、培養液をミラクロスによりろ過して菌体を回収した。菌体は、液体窒素で瞬間凍結し、速やかに液体窒素下で乳鉢・乳棒で破砕した。破砕菌体を1.5ml容エッペンドルフチューブに移し、RNeasy Plant Mini Kit(QIAGEN) のプロトコルに従いRNAを抽出した。DNAの混入を避けるため、付属のプロトコルに従いオンカラムでDNase処理も行った。最終的に50μlのRNase free waterで2回溶出することでTotal RNAを取得した。
・TRAF01000135000001遺伝子用
NRPS-RT1-F:GACGCCACGAACGCATAGAC(配列番号132)
NRPS-RT1-R:TTCCCAGAGAGGTAGATCGAC(配列番号133)
・TRAF01000135000001遺伝子用
NRPS-RT2-F:GACCGTTACAGCGAGTTCAG(配列番号134)
NRPS-RT2-R:CTGAATTCCTCGCACAGAAC(配列番号135)
・TRAF01000135000001遺伝子用
NRPS-RT3-F:GAAGTTGAGAACGCCATGCT(配列番号136)
NRPS-RT3-R:GATGCGAGATGGGAGCATGT(配列番号137)
・TRAF01000068000002遺伝子用
TR02-RT-F:GCCCTACTAGATCTGACCAC(配列番号138)
TR02-RT-R:GCTGTTACCTTTTCCTCCTC(配列番号139)
・TRAF01000068000003遺伝子用
TR03-RT-F:AGATCTTAGACGAGCTGCTC(配列番号140)
TR03-RT-R:AAACAGTCGCGAAGCGACTG(配列番号141)
・TRAF01000068000006遺伝子用
TR06-RT-F:ACGTCCAGGAAGCTATCGAG(配列番号142)
TR06-RT-R:ATTGAGGGCCTGGGCTTGAC(配列番号143)
・TRAF01000068000007遺伝子用
TR07-RT-F:GTGATGAAGGCGCTGAAGAG(配列番号144)
TR07-RT-R:CTCCGCAATTTCCGTGAGTG(配列番号145)
・TRAF01000068000008遺伝子用
TR08-RT-F:TGACTCTATGGTGGATGGTG(配列番号146)
TR08-RT-R:CCTTGTTCAAGTGCCAGTAG(配列番号147)
・TRAF01000068000009遺伝子用
TR09-RT-F:GATTCCGTCACGAGACACTG(配列番号148)
TR09-RT-R:AGTATCCCATCGGGCAACAG(配列番号149)
・TRAF01000135000002遺伝子用
OMT-RT-F: ACGTTCAAGACCTTCCAG(配列番号150)
OMT-RT-R:GTTCCGGATGATTTGCAG(配列番号151)
定量リアルタイムPCRの結果を図25に示した。なお、図25において、O-MTはTRAF01000135000002遺伝子を意味し、NRPS-1~NRPS-3はTRAF01000135000001遺伝子(実施例1のNRPS遺伝子)を意味し、TR02はTRAF01000068000002遺伝子を意味し、TR03はTRAF01000068000003遺伝子を意味し、TR06はTRAF01000068000006遺伝子を意味し、TR07はTRAF01000068000007遺伝子を意味し、TR08はTRAF01000068000008遺伝子を意味し、TR09はTRAF01000068000009遺伝子を意味している。図25に示すように、導入した全ての遺伝子がヒストンと同程度程の発現レベルを示しており、クラスター遺伝子を導入した麹菌において、KK-1生合成に必要な全ての遺伝子が高発現していることが明らかになった。
9) KK-1生産性評価
また、以上のようにして作製されたKK-1生合成クラスター遺伝子を導入した形質転換麹菌について、KK-1の生産性を評価した。
Claims (16)
- 配列番号1に示すアミノ酸配列又は配列番号1に示すアミノ酸配列に対して70%以上の同一性を有するアミノ酸配列からなる第1のアデニレーションドメインと、配列番号2に示すアミノ酸配列又は配列番号2に示すアミノ酸配列に対して70%以上の同一性を有するアミノ酸配列からなる第1のペプチジルキャリアタンパク質ドメインとをN末端側からこの順で有する第1モジュールと、
配列番号3に示すアミノ酸配列又は配列番号3に示すアミノ酸配列に対して70%以上の同一性を有するアミノ酸配列からなる第1の縮合ドメインと、配列番号4に示すアミノ酸配列又は配列番号4に示すアミノ酸配列に対して70%以上の同一性を有するアミノ酸配列からなる第2のアデニレーションドメインと、配列番号5に示すアミノ酸配列又は配列番号5に示すアミノ酸配列に対して70%以上の同一性を有するアミノ酸配列からなる第2のペプチジルキャリアタンパク質ドメインとをN末端側からこの順で有する第2モジュールと、
配列番号6に示すアミノ酸配列又は配列番号6に示すアミノ酸配列に対して70%以上の同一性を有するアミノ酸配列からなる第2の縮合ドメインと、配列番号7に示すアミノ酸配列又は配列番号7に示すアミノ酸配列に対して70%以上の同一性を有するアミノ酸配列からなる第3のアデニレーションドメインと、配列番号8に示すアミノ酸配列又は配列番号8に示すアミノ酸配列に対して70%以上の同一性を有するアミノ酸配列からなる第1のN-メチルトランスフェラーゼドメインと、配列番号9に示すアミノ酸配列又は配列番号9に示すアミノ酸配列に対して70%以上の同一性を有するアミノ酸配列からなる第3のペプチジルキャリアタンパク質ドメインとをN末端側からこの順で有する第3モジュールと、
配列番号10に示すアミノ酸配列又は配列番号10に示すアミノ酸配列に対して70%以上の同一性を有するアミノ酸配列からなる第3の縮合ドメインと、配列番号11に示すアミノ酸配列又は配列番号11に示すアミノ酸配列に対して70%以上の同一性を有するアミノ酸配列からなる第4のアデニレーションドメインと、配列番号12に示すアミノ酸配列又は配列番号12に示すアミノ酸配列に対して70%以上の同一性を有するアミノ酸配列からなる第4のペプチジルキャリアタンパク質ドメインとをN末端側からこの順で有する第4モジュールと、
配列番号13に示すアミノ酸配列又は配列番号13に示すアミノ酸配列に対して70%以上の同一性を有するアミノ酸配列からなる第4の縮合ドメインと、配列番号14に示すアミノ酸配列又は配列番号14に示すアミノ酸配列に対して70%以上の同一性を有するアミノ酸配列からなる第5のアデニレーションドメインと、配列番号15に示すアミノ酸配列又は配列番号15に示すアミノ酸配列に対して70%以上の同一性を有するアミノ酸配列からなる第2のN-メチルトランスフェラーゼドメインと、配列番号16に示すアミノ酸配列又は配列番号16に示すアミノ酸配列に対して70%以上の同一性を有するアミノ酸配列からなる第5のペプチジルキャリアタンパク質ドメインとをN末端側からこの順で有する第5モジュールと、
配列番号17に示すアミノ酸配列又は配列番号17に示すアミノ酸配列に対して70%以上の同一性を有するアミノ酸配列からなる第5の縮合ドメインと、配列番号18に示すアミノ酸配列又は配列番号18に示すアミノ酸配列に対して70%以上の同一性を有するアミノ酸配列からなる第6のアデニレーションドメインと、配列番号19に示すアミノ酸配列又は配列番号19に示すアミノ酸配列に対して70%以上の同一性を有するアミノ酸配列からなる第3のN-メチルトランスフェラーゼドメインと、配列番号20に示すアミノ酸配列又は配列番号20に示すアミノ酸配列に対して70%以上の同一性を有するアミノ酸配列からなる第6のペプチジルキャリアタンパク質ドメインとをN末端側からこの順で有する第6モジュールと、
配列番号21に示すアミノ酸配列又は配列番号21に示すアミノ酸配列に対して70%以上の同一性を有するアミノ酸配列からなる第6の縮合ドメインと、配列番号22に示すアミノ酸配列又は配列番号22に示すアミノ酸配列に対して70%以上の同一性を有するアミノ酸配列からなる第7のアデニレーションドメインと、配列番号23に示すアミノ酸配列又は配列番号23に示すアミノ酸配列に対して70%以上の同一性を有するアミノ酸配列からなる第4のN-メチルトランスフェラーゼドメインと、配列番号24に示すアミノ酸配列又は配列番号24に示すアミノ酸配列に対して70%以上の同一性を有するアミノ酸配列からなる第7のペプチジルキャリアタンパク質ドメインとをN末端側からこの順で有する第7モジュールと、
配列番号25に示すアミノ酸配列又は配列番号25に示すアミノ酸配列に対して70%以上の同一性を有するアミノ酸配列からなる第7の縮合ドメインと、配列番号26に示すアミノ酸配列又は配列番号26に示すアミノ酸配列に対して70%以上の同一性を有するアミノ酸配列からなる第8のアデニレーションドメインと、配列番号27に示すアミノ酸配列又は配列番号27に示すアミノ酸配列に対して70%以上の同一性を有するアミノ酸配列からなる第8のペプチジルキャリアタンパク質ドメインとをN末端側からこの順で有する第8モジュールと、
配列番号28に示すアミノ酸配列又は配列番号28に示すアミノ酸配列に対して70%以上の同一性を有するアミノ酸配列からなる第8の縮合ドメインと、配列番号29に示すアミノ酸配列又は配列番号29に示すアミノ酸配列に対して70%以上の同一性を有するアミノ酸配列からなる第9のアデニレーションドメインと、配列番号30に示すアミノ酸配列又は配列番号30に示すアミノ酸配列に対して70%以上の同一性を有するアミノ酸配列からなる第5のN-メチルトランスフェラーゼドメインと、配列番号31に示すアミノ酸配列又は配列番号31に示すアミノ酸配列に対して70%以上の同一性を有するアミノ酸配列からなる第9のペプチジルキャリアタンパク質ドメインとをN末端側からこの順で有する第9モジュールと、
配列番号32に示すアミノ酸配列又は配列番号32に示すアミノ酸配列に対して70%以上の同一性を有するアミノ酸配列からなる第9の縮合ドメインと、配列番号33に示すアミノ酸配列又は配列番号33に示すアミノ酸配列に対して70%以上の同一性を有するアミノ酸配列からなる第10のアデニレーションドメインと、配列番号34に示すアミノ酸配列又は配列番号34に示すアミノ酸配列に対して70%以上の同一性を有するアミノ酸配列からなる第10のペプチジルキャリアタンパク質ドメインと、配列番号35に示すアミノ酸配列又は配列番号35に示すアミノ酸配列に対して70%以上の同一性を有するアミノ酸配列からなる第10の縮合ドメインとをN末端側からこの順で有する第10モジュールと、
をN末端側からこの順で有し、Curvularia属糸状菌が生産する環状ペプチド化合物の非リボソーム型ペプチド合成活性を有するタンパク質をコードする環状ペプチド化合物合成関連遺伝子。 - 上記タンパク質は、以下(a)~(c)のいずれかのタンパク質であることを特徴とする請求項1記載の環状ペプチド化合物合成関連遺伝子。
(a)配列番号37に示すアミノ酸配列からなるタンパク質
(b)配列番号37に示すアミノ酸配列に対して70%以上の同一性を有するアミノ酸配列からなり、Curvularia属糸状菌が生産する環状ペプチド化合物の非リボソーム型ペプチド合成活性を有するタンパク質
(c)配列番号36に示す塩基配列の相補鎖に対してストリンジェントな条件下でハイブリダイズするポリヌクレオチドによりコードされ、Curvularia属糸状菌が生産する環状ペプチド化合物の非リボソーム型ペプチド合成活性を有するタンパク質 - Curvularia属糸状菌由来であることを特徴とする請求項1記載の環状ペプチド化合物合成関連遺伝子。
- 上記糸状菌は、Curvularia clavataであることを特徴とする請求項3記載の環状ペプチド化合物合成関連遺伝子。
- 以下(a)~(c)のいずれかのタンパク質をコードする環状ペプチド化合物合成関連遺伝子。
(a)配列番号39に示すアミノ酸配列からなるタンパク質
(b)配列番号39に示すアミノ酸配列に対して70%以上の同一性を有するアミノ酸配列からなり、転写因子活性を有するタンパク質
(c)配列番号38に示す塩基配列の相補鎖に対してストリンジェントな条件下でハイブリダイズするポリヌクレオチドによりコードされ、転写因子活性を有するタンパク質 - Curvularia属糸状菌由来であることを特徴とする請求項5記載の環状ペプチド化合物合成関連遺伝子。
- 上記糸状菌は、Curvularia clavataであることを特徴とする請求項6記載の環状ペプチド化合物合成関連遺伝子。
- 請求項1乃至4いずれか一項記載の環状ペプチド化合物合成関連遺伝子と、Curvularia属糸状菌における環状ペプチド化合物の生産に関与する遺伝子群とを導入した形質転換体を培養する工程と、
培養した形質転換体及び/又は培養液から上記環状ペプチド化合物を回収する工程とを含む、
Curvularia属糸状菌が生産する環状ペプチド化合物の製造方法。 - 上記遺伝子群は以下[1]~[7]の遺伝子を含むことを特徴とする請求項8記載の環状ペプチド化合物の製造方法。
[1]配列番号41に示すアミノ酸配列又は配列番号41に示すアミノ酸配列に対して70%以上の同一性を有するアミノ酸配列からなるタンパク質をコードする遺伝子
[2]配列番号43に示すアミノ酸配列又は配列番号43に示すアミノ酸配列に対して70%以上の同一性を有するアミノ酸配列からなるタンパク質をコードする遺伝子
[3]配列番号45に示すアミノ酸配列又は配列番号45に示すアミノ酸配列に対して70%以上の同一性を有するアミノ酸配列からなるタンパク質をコードする遺伝子
[4]配列番号47に示すアミノ酸配列又は配列番号47に示すアミノ酸配列に対して70%以上の同一性を有するアミノ酸配列からなるタンパク質をコードする遺伝子
[5]配列番号49に示すアミノ酸配列又は配列番号49に示すアミノ酸配列に対して70%以上の同一性を有するアミノ酸配列からなるタンパク質をコードする遺伝子
[6]配列番号51に示すアミノ酸配列又は配列番号51に示すアミノ酸配列に対して70%以上の同一性を有するアミノ酸配列からなるタンパク質をコードする遺伝子
[7]配列番号53に示すアミノ酸配列又は配列番号53に示すアミノ酸配列に対して70%以上の同一性を有するアミノ酸配列からなるタンパク質をコードする遺伝子 - 上記形質転換体は、麹菌(Aspergillus oryzae)を宿主とすることを特徴とする請求項8記載の環状ペプチド化合物の製造方法。
- 請求項1乃至4いずれか一項記載の環状ペプチド化合物合成関連遺伝子と、Curvularia属糸状菌における環状ペプチド化合物の生産に関与する遺伝子群とを導入した形質転換体。
- 上記遺伝子群は以下[1]~[7]の遺伝子を含むことを特徴とする請求項11記載の形質転換体。
[1]配列番号41に示すアミノ酸配列又は配列番号41に示すアミノ酸配列に対して70%以上の同一性を有するアミノ酸配列からなるタンパク質をコードする遺伝子
[2]配列番号43に示すアミノ酸配列又は配列番号43に示すアミノ酸配列に対して70%以上の同一性を有するアミノ酸配列からなるタンパク質をコードする遺伝子
[3]配列番号45に示すアミノ酸配列又は配列番号45に示すアミノ酸配列に対して70%以上の同一性を有するアミノ酸配列からなるタンパク質をコードする遺伝子
[4]配列番号47に示すアミノ酸配列又は配列番号47に示すアミノ酸配列に対して70%以上の同一性を有するアミノ酸配列からなるタンパク質をコードする遺伝子
[5]配列番号49に示すアミノ酸配列又は配列番号49に示すアミノ酸配列に対して70%以上の同一性を有するアミノ酸配列からなるタンパク質をコードする遺伝子
[6]配列番号51に示すアミノ酸配列又は配列番号51に示すアミノ酸配列に対して70%以上の同一性を有するアミノ酸配列からなるタンパク質をコードする遺伝子
[7]配列番号53に示すアミノ酸配列又は配列番号53に示すアミノ酸配列に対して70%以上の同一性を有するアミノ酸配列からなるタンパク質をコードする遺伝子 - 麹菌(Aspergillus oryzae)を宿主とすることを特徴とする請求項11記載の形質転換体。
- 請求項1乃至4いずれか一項記載の環状ペプチド化合物合成関連遺伝子を有するCurvularia属糸状菌。
- Curvularia clavataであることを特徴とする請求項14記載のCurvularia属糸状菌。
- 受託番号NITE BP-02399であることを特徴とする請求項14記載のCurvularia属糸状菌。
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CN109337916A (zh) * | 2018-09-21 | 2019-02-15 | 华南农业大学 | 一种稻瘟菌modip基因及其应用 |
CN109371044A (zh) * | 2018-10-29 | 2019-02-22 | 华南农业大学 | 一种稻瘟菌基因Movan及其应用 |
WO2023032950A1 (ja) | 2021-08-30 | 2023-03-09 | 天野エンザイム株式会社 | 向上したタンパク質生産性を有する微生物及びその利用 |
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CN109337916A (zh) * | 2018-09-21 | 2019-02-15 | 华南农业大学 | 一种稻瘟菌modip基因及其应用 |
CN109371044A (zh) * | 2018-10-29 | 2019-02-22 | 华南农业大学 | 一种稻瘟菌基因Movan及其应用 |
WO2023032950A1 (ja) | 2021-08-30 | 2023-03-09 | 天野エンザイム株式会社 | 向上したタンパク質生産性を有する微生物及びその利用 |
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US20220259634A1 (en) | 2022-08-18 |
EP3567107A4 (en) | 2020-07-22 |
TW201829771A (zh) | 2018-08-16 |
EP3567107A1 (en) | 2019-11-13 |
BR112019013234A2 (pt) | 2020-02-11 |
CN111684066B (zh) | 2024-03-08 |
AR110725A1 (es) | 2019-04-24 |
TWI826363B (zh) | 2023-12-21 |
US11946086B2 (en) | 2024-04-02 |
AU2017391000A1 (en) | 2019-07-18 |
US11230726B2 (en) | 2022-01-25 |
KR20190117511A (ko) | 2019-10-16 |
CA3048176A1 (en) | 2018-07-12 |
JPWO2018128140A1 (ja) | 2019-11-07 |
KR102633026B1 (ko) | 2024-02-01 |
US20190316164A1 (en) | 2019-10-17 |
PH12019501577A1 (en) | 2020-02-24 |
CN111684066A (zh) | 2020-09-18 |
JP7072750B2 (ja) | 2022-05-23 |
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