JP2018183134A - Methods for producing ascochlorin and ilicicolin a - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide isoprenoid-producing methods capable of a high yield production of ilicicolin A and ascochlorin and derivatives thereof compared to the prior art so as to enable industrial scale production of isoprenoids such as ilicicolin and ascochlorin.SOLUTION: To solve the above problem, the invention provides a method for producing an isoprenoid such as ilicicolin A and ascochlorin, which comprises a step of obtaining isoprenoids such as ilicicolin A and ascochlorin by using a transformant transformed with an ascochlorin-synthesizing gene.SELECTED DRAWING: Figure 1

Description

The present invention relates to a gene for synthesizing iricicholine A and ascochlorin, and a method for producing ascochlorin and iricicholine A using the gene.

In developed and developing countries, including Japan, where there are densely populated areas, infectious diseases caused by viruses and protozoa are often a problem. In addition, lifestyle-related diseases such as type 2 diabetes, hypercholesterolemia, cancer, and complications resulting from these diseases lead to an increase in medical costs and a decrease in labor force, and are particularly serious problems in Japan. ing.

Therefore, development of substances effective for the treatment and prevention of these diseases is desired. As one of such substances, ascochlorin and ascofuranone, which are isoprenoid-based physiologically active substances, are known. Ascochlorin and ascofuranone are promising in the treatment and prevention of African sleeping sickness, which is a protozoal infection caused by protozoan trypanosomes mediated by, for example, the fly, by inhibiting the electron transport system and reducing the intracellular ATP concentration. (For example, refer to Patent Document 1).

When suffering from African sleeping sickness, protozoa grow in the bloodstream early in the infection. At the beginning of the chronic phase, the central nervous system is invaded and symptoms such as mental confusion and generalized convulsions occur, eventually falling into lethargy and dying. The number of deaths in Africa due to African sleeping sickness is more than 10,000 per year, and the population at risk of infection is said to be more than 70 million. At present, there is no vaccine prevention method for African sleeping sickness, and its treatment relies on drug therapy. However, effective treatments for African sleeping sickness have problems such as strong side effects.

Therefore, prevention and treatment of African sleeping sickness is expected by specifically inhibiting the trypanosome electron transport system with ascochlorin and ascofuranone. When protozoa invade the mammalian body, ATP synthesis is mainly carried out by glycolysis within glycosomes, which requires regeneration of NAD ⁺ catalyzed by trypanosome, alternative native oxidase (TAO). Ascofuranone inhibits this TAO function. Since infected mammals do not have the same enzyme as TAO, it is possible to specifically combat trypanosomes. In particular, ascofuranone and its derivatives have been reported to inhibit TAO even at very low concentrations.

In addition, it is known that ascochlorin, ascofuranone and derivatives thereof have antitumor activity, blood glucose lowering action, blood lipid lowering action, glycation inhibiting action, antioxidant action and the like (for example, patent documents) 2). Furthermore, iricicholine A (LL-Z1272α), which is an intermediate in the biosynthetic pathway of ascochlorin and ascofuranone, is also a high antiprotozoal agent (Patent Document 3), immunosuppressant, rheumatic agent, anticancer agent, and rejection treatment. Drugs, antiviral drugs, anti-H. It is expected as an active ingredient of new pharmaceuticals such as pylori drugs and antidiabetic drugs (Patent Document 4). Furthermore, Iricicolin A is known as an intermediate for biosynthesis of not only ascochlorin and ascofuranone but also other isoprenoid compounds, and is also a useful compound as a raw material thereof.

As a method for producing ascofuranone and ascochlorin, culturing Ascochyta genus (Ascochyta) fungi, a method of separating collected ascofuranone accumulated in the mycelia are known (e.g., see Patent Documents 5 and 6) . Incidentally, Ascochyta & Vicia was known as a production strain of ascofuranone (Ascochyta viciae) is correct, it has been reported by Non-Patent Document 1 is a Acremonium sclerotinia Kye Nam (Acremonium sclerotigenum).

JP 09-165332 A JP 2006-213644 A International Publication No. 2012/060387 International Publication No. 2013/180140 Japanese Patent Publication No.56-25310 Japanese Examined Patent Publication No. 45-9832

J Antibiot (Tokyo). 2016 Nov 2. Re-identification of the ascofuranone-producing fungus Ascochyta viciae as Acremonium scrorotigenum.

According to the method using a filamentous fungus known to produce ascochlorin, such as the method described in Patent Document 6, the yield of ascochlorin greatly depends on the filamentous fungus used. However, the content of ascochlorin in the microorganisms known so far is small for industrial production, and the production amount varies greatly with a slight difference in culture conditions. There is a problem that stable production of chlorin cannot be realized. In addition, Iricicholine A has not been known so far for a production method for obtaining it in large quantities.

For example, in order to realize mass production of ascochlorin and its intermediate, iricicholine A, it is necessary to isolate or breed wild strains that stably produce ascochlorin and iricicholine A at high concentrations, It is conceivable to construct a transformant in which a gene involved in biosynthesis of ascochlorin and iricicholine A is inserted. However, few wild strains have been known so far that stably produce ascochlorin and iricicholine A at high concentrations, and there are still many unclear parts regarding the biosynthetic pathways of ascochlorin and iricicholine A.

In addition, there are still many unknown parts of the biosynthetic genes for ascochlorin and iricicholine A.

Accordingly, the problem to be solved by the present invention is that iricicholine A and ascochlorin and their derivatives can be stably produced in high yield as compared with the prior art. It is an object of the present invention to provide a method for producing an isoprenoid that enables the production of an isoprenoid.

As a result of intensive studies to solve the above-mentioned problems, the present inventors have conducted reactions involving the biosynthesis of iricicholine A and ascochlorin in Acremonium sclerotigenum , which is a type of filamentous fungus. We have succeeded in identifying a group of genes that encode enzymes that catalyze.

Then, the present inventors have prepared a DNA construct for over-expressing proteins encoded by the genes, and then the resulting DNA construct as the host organism, Aspergillus is a kind of filamentous fungi (Aspergillus) By introducing and transforming into a genus microorganism, we succeeded in producing a transformed filamentous fungus overexpressing the protein encoded by the above gene group.

The transformed filamentous fungus can be cultured according to the usual method for culturing filamentous fungi, and the growth rate and the like thereof are not particularly different from those of the host organism. From these results, it was found that iricicholine A and ascochlorin can be produced by using the above-mentioned transformed filamentous fungus. In addition, a DNA construct that forcibly expresses the protein encoded by the above gene group was prepared for a strain that originally produced iricicholine A or ascochlorin, such as A. sclerotigenum. Then, it is conceivable to stably mass-produce iricicholine A and ascochlorin by using the transformant in which the enzyme performance of the above gene group is enhanced by introducing the obtained DNA construct. The present invention has been completed based on such successful examples and knowledge.

Therefore, according to one aspect of the present invention, the following gene, transformant and production method are provided.
[1] A gene ascF comprising the nucleotide sequence of any one of the following (1) to (5), which encodes an amino acid sequence of an enzyme having an activity of catalyzing an epoxidation reaction of iricicholine A.
(1) a base sequence shown in SEQ ID NO: 5 in the sequence listing or a base sequence that hybridizes with a base sequence complementary to the base sequence under stringent conditions (2) a gene comprising the base sequence shown in SEQ ID NO: 5 And a nucleotide sequence having an identity of 60% or more (3) a nucleotide sequence encoding an amino acid sequence of an enzyme having an activity of catalyzing an epoxidation reaction of iricicholine A (4) and an amino acid sequence represented by SEQ ID NO: 12 or 33; Base sequence encoding amino acid sequence having% or more identity (5) Encoding amino acid sequence in which one or several amino acids of amino acid sequence shown in SEQ ID NO: 12 or 33 are deleted, substituted and / or added Base sequence [2] A base sequence of any one of the following (1) to (5), wherein the amino acid of the enzyme has an activity of catalyzing the cyclization reaction of iricicholine A epoxide. Comprising a nucleotide sequence encoding an acid sequences, gene ASCG.
(1) a base sequence shown in SEQ ID NO: 6 in the sequence listing or a base sequence that hybridizes with a base sequence complementary to the base sequence under stringent conditions (2) a gene comprising the base sequence shown in SEQ ID NO: 6 And a base sequence having 60% or more identity (3) a base sequence encoding an amino acid sequence of an enzyme having an activity of catalyzing a cyclization reaction of iricicholine A epoxide (4) an amino acid sequence described in SEQ ID NO: 13 or 34; Nucleotide sequence encoding an amino acid sequence having 60% or more identity (5) encoding an amino acid sequence in which one or several amino acids of the amino acid sequence set forth in SEQ ID NO: 13 or 34 have been deleted, substituted and / or added The base sequence [3] is the base sequence of any one of the following (1) to (5), wherein the anti-reaction of Iscicolin A to AscF protein and AscG protein Comprising a nucleotide sequence encoding the amino acid sequence of the enzyme having activity to catalyze a reaction for generating a ascochlorin by dehydrogenation of the compound produced by gene ASCH.
(1) A nucleotide sequence that hybridizes under stringent conditions with a nucleotide sequence set forth in SEQ ID NO: 7 in the sequence listing or a nucleotide sequence complementary to the nucleotide sequence. (2) A gene comprising the nucleotide sequence set forth in SEQ ID NO: 7. (3) an amino acid sequence of an enzyme having an activity of catalyzing a reaction for producing ascochlorin by dehydrogenation of a compound produced by the reaction of AscF protein and AscG protein from iricicholine A. Base sequence encoding (4) Base sequence encoding amino acid sequence having 60% or more identity with the amino acid sequence described in SEQ ID NO: 14 or 35 (5) One or number of amino acid sequences described in SEQ ID NO: 14 or 35 Nucleotide sequence encoding an amino acid sequence in which one amino acid is deleted, substituted and / or added [4] (1) to ( ) It is any of the nucleotide sequences of, comprising a nucleotide sequence encoding the amino acid sequence of the enzyme having activity to catalyze a reaction for generating a Irishikorin A from LL-Z1272β, gene ASCE.
(1) a nucleotide sequence that is hybridized under stringent conditions with a nucleotide sequence shown in SEQ ID NO: 4 of the sequence listing or a nucleotide sequence complementary to the nucleotide sequence; (2) a gene comprising the nucleotide sequence shown in SEQ ID NO: 4 (3) a base sequence encoding an amino acid sequence of an enzyme having an activity of catalyzing a reaction for producing iricicholine A from LL-Z1272β (4) in SEQ ID NO: 11, 32 or 40 A nucleotide sequence encoding an amino acid sequence having 60% or more identity with the described amino acid sequence (5) wherein one or several amino acids of the amino acid sequence of SEQ ID NO: 11, 32 or 40 are deleted, substituted and / or Base sequence encoding added amino acid sequence [5] The base sequence of any one of the following (1) to (5), wherein acetyl CoA to o-orselic acid The resulting reaction comprises a nucleotide sequence encoding the amino acid sequence of the enzyme having activity to catalyze a gene ASCD.
(1) a nucleotide sequence that hybridizes under stringent conditions with a nucleotide sequence set forth in SEQ ID NO: 3 in the sequence listing or a nucleotide sequence complementary to the nucleotide sequence; (2) a gene comprising the nucleotide sequence set forth in SEQ ID NO: 3 (3) a base sequence encoding an amino acid sequence of an enzyme having an activity of catalyzing a reaction for producing o-orselic acid from acetyl CoA (4) SEQ ID NO: 10, 31, or 39 A nucleotide sequence encoding an amino acid sequence having 60% or more identity with the amino acid sequence described in (5), wherein one or several amino acids of the amino acid sequence described in SEQ ID NO: 10, 31 or 39 are deleted, substituted and / or Or a base sequence encoding the added amino acid sequence [6] The base sequence of any one of the following (1) to (5), wherein o-orselinic acid to iricicolic acid B The product reacts comprising a nucleotide sequence encoding the amino acid sequence of the enzyme having activity to catalyze a gene ASCB.
(1) A nucleotide sequence that hybridizes under stringent conditions with a nucleotide sequence set forth in SEQ ID NO: 1 in the sequence listing or a nucleotide sequence complementary to the nucleotide sequence. (2) A gene comprising the nucleotide sequence set forth in SEQ ID NO: 1. (3) a base sequence encoding an amino acid sequence of an enzyme having an activity of catalyzing a reaction for producing iricicoline acid B from o-orceric acid (4) SEQ ID NO: 8, 29, or A nucleotide sequence encoding an amino acid sequence having 60% or more identity with the amino acid sequence described in (37), wherein one or several amino acids of the amino acid sequence described in SEQ ID NO: 8, 29 or 37 are deleted, substituted and / Or base sequence encoding the added amino acid sequence [7] The base sequence of any one of the following (1) to (5), wherein irilicolinic acid B to LL-Z1272β The product reacts comprising a nucleotide sequence encoding the amino acid sequence of the enzyme having activity to catalyze a gene ASCC.
(1) A nucleotide sequence that hybridizes under stringent conditions with a nucleotide sequence set forth in SEQ ID NO: 2 in the sequence listing or a nucleotide sequence complementary to the nucleotide sequence (2) A gene comprising the nucleotide sequence set forth in SEQ ID NO: 2 (3) a base sequence encoding an amino acid sequence of an enzyme having an activity of catalyzing a reaction for producing LL-Z1272β from iricicolic acid B (4) SEQ ID NO: 9, 30 or 38 A nucleotide sequence encoding an amino acid sequence having 60% or more identity with the amino acid sequence described in (5) wherein one or several amino acids of the amino acid sequence described in SEQ ID NO: 9, 30 or 38 are deleted, substituted and / or Or base sequence encoding added amino acid sequence [8] genes ascF, ascG, ascH, ascE, ascD, a described in [1] to [7] above Genes cB and any one gene or a combination thereof ascC is inserted, and expressing the inserted gene, transformants (except for human).
[9] A method for producing iricicholine A, comprising a step of obtaining iricicholine A using the transformant according to [8] above.
[10] A method for producing ascochlorin, comprising the step of obtaining ascochlorin using the transformant according to [8] above.

According to the present invention, high yields of isoprenoids such as iricicholine A and ascochlorin can be produced. As a result, according to the present invention, it can be expected to produce isoprenoids such as iricicholine A and ascochlorin on an industrial scale.

FIG. 1 is a diagram showing an ascochlorin biosynthesis gene cluster predicted by transcriptome analysis. FIG. 2 is a diagram showing the results of HPLC analysis of As-DBCE strain extract and iricicholine A standard product as described in Examples described later. FIG. 3 is a diagram showing HPLC analysis results of respective extracts of As-DBCE strain, As-DBCEF strain, As-DBCEFG strain, and As-DBCEFGH strain as described in Examples described later. . FIG. 4A is a diagram showing LC / MS analysis results of a reaction product when using a wild-type reaction solution and an As-F reaction solution, respectively, as described in Examples described later. FIG. 4B is a diagram showing an LC / MS analysis result of a reaction product when using an As-F reaction solution and an As-FG reaction solution, respectively, as described in Examples described later. FIG. 5 is a diagram showing LC / MS analysis results of a reaction product when using an As-FG reaction solution and an As-FGH reaction solution, respectively, as described in Examples described later. FIG. 6 is a schematic diagram showing the relationship between each enzyme reaction and the reactants in the reaction system of iricicholine A and ascochlorin.

Hereinafter, the details of a gene, a transformant, a knockout organism, and a production method which are one embodiment of the present invention will be described, but the technical scope of the present invention is not limited only to the matters of this item. Can take various forms as long as the object is achieved.

(Isoprenoid)
The “isoprenoid” in the present specification is not particularly limited as long as it is a compound having isoprene as a structural unit as generally known. For example, iricicolic acid B, iricicolic acid A, LL-Z1272β, iricicholine A (LL- Z1272α), iricicholine A epoxide, iricicholine C, ascochlorin, ascofuranone and derivatives thereof. However, in the present specification, ascofuranone, iricicholine A, ascochlorin, and derivatives thereof are sometimes referred to as “isoprenoids”.

(Amino acid sequence of enzymes (1) to (7))
The gene ascB of one embodiment of the present invention is a base sequence that encodes the amino acid sequence of an enzyme that has an activity of catalyzing a reaction for producing iricicolinic acid B from o-orceric acid (hereinafter also referred to as “enzyme (1)”). including.

The gene ascC of one embodiment of the present invention has a nucleotide sequence that encodes an amino acid sequence of an enzyme (hereinafter also referred to as “enzyme (2)”) having an activity of catalyzing a reaction for generating LL-Z1272β from iricicolic acid B. Including.

The gene ascD of one embodiment of the present invention has a base sequence encoding an amino acid sequence of an enzyme having an activity of catalyzing a reaction for generating o-orselic acid from acetyl CoA (hereinafter also referred to as “enzyme (3)”). Including.

The gene ascE of one embodiment of the present invention includes a base sequence encoding an amino acid sequence of an enzyme having an activity of catalyzing a reaction for producing iricicholine A from LL-Z1272β (hereinafter also referred to as “enzyme (4)”). .

The gene ascF of one embodiment of the present invention includes a base sequence encoding an amino acid sequence of an enzyme having an activity of catalyzing an epoxidation reaction of iricicholine A (hereinafter also referred to as “enzyme (5)”).

The gene ascG of one embodiment of the present invention includes a base sequence encoding an amino acid sequence of an enzyme having an activity of catalyzing the cyclization reaction of iricicholine A epoxide (hereinafter also referred to as “enzyme (6)”).

The gene ascH of one embodiment of the present invention is an enzyme having an activity that catalyzes a reaction for producing ascochlorin by dehydrogenation of a compound produced by the reaction of AscF protein and AscG protein from iricicholine A (hereinafter referred to as “enzyme (7)”. Also includes a base sequence encoding the amino acid sequence.

Although the technical scope of the present invention is not limited to any speculation or reasoning, enzyme (1) has a function similar to prenyl transferase; enzyme (2) has a function similar to oxidoreductase. Enzyme (3) has a function similar to polyketide synthase; enzyme (4) has a function similar to halogenase; enzyme (5) has a function similar to p450 / p450 reductase as an epoxidase Enzyme (6) has the same function as terpene cyclase; enzyme (7) has a probability of having the same function as p450 as a dehydrogenase.

The enzymes (1) to (7) are not particularly limited as long as they have the enzyme activity described above.

For example, as an embodiment of the enzyme (1) having the enzyme activity described above, there are amino acid sequences shown in SEQ ID NOs: 8, 29 and 37; as an embodiment of the enzyme (2) having the enzyme activity described above, SEQ ID NOS: 9, 30 and As an embodiment of the enzyme (3) having the above enzyme activity, there are amino acid sequences shown in SEQ ID NOs: 10, 31 and 39; As an embodiment of the enzyme (4) having the above enzyme activity There are amino acid sequences shown in SEQ ID NOs: 11, 32 and 40; as an embodiment of the enzyme (5) having the enzyme activity described above, there are amino acid sequences shown in SEQ ID NOs: 12 and 33; As one embodiment, there are amino acid sequences shown in SEQ ID NOs: 13 and 34; as an embodiment of the enzyme (7) having the enzyme activity described above, amino acids shown in SEQ ID NOs: 14 and 35 There is acid sequence.

The enzymes having the amino acid sequences shown in SEQ ID NOs: 8 to 14 are all derived from Acremonium filamentous fungi, and are named by the present inventors as AscB, AscC, AscD, AscE, AscF, AscG and AscH proteins, respectively. . The base sequences of the genes encoding these enzymes are the base sequences shown in SEQ ID NOs: 1-7.

The enzymes having the amino acid sequences shown in SEQ ID NOs: 29 to 35 are all derived from Neonectoria ditissima , and the present inventors have made AscB (SEQ ID NO: 29), AscC (SEQ ID NO: 30), AscD, respectively. (SEQ ID NO: 31), AscE (SEQ ID NO: 32), AscF (SEQ ID NO: 33), AscG (SEQ ID NO: 34) and AscH (SEQ ID NO: 35) proteins.

The enzymes having the amino acid sequences shown in SEQ ID NOs: 37 to 40 are all derived from Trichoderma reesei , and the present inventors have made AscB (SEQ ID NO: 37), AscC (SEQ ID NO: 38), AscD, respectively. (SEQ ID NO: 39), named AscE (SEQ ID NO: 40).

AscB, AscC, AscD, AscE, AscF, AscG and AscH proteins are those encoded by genes encoding these enzymes present on the chromosomal DNA of the genera Acremonium, Neonectria or Trichoderma. When a gene existing on the chromosomal DNA of such an organism and a protein or enzyme encoded by the gene are referred to as “wild type gene”, “wild type protein” or “wild type enzyme”, respectively, in this specification There is.

As long as the amino acid sequences of the enzymes (1) to (7) have the enzyme activities of the enzymes (1) to (7) described above, the amino acid sequence of the wild-type enzyme lacks one to several amino acids. It may consist of an amino acid sequence having loss, substitution, addition and the like. Here, the range of “1 to several” in “deletion, substitution and addition of 1 to several amino acids” of the amino acid sequence is not particularly limited, but for example, the unit of 100 amino acids in the amino acid sequence is one unit. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 per unit, preferably It means about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, more preferably about 1, 2, 3, 4 or 5. In addition, “amino acid deletion” means deletion or disappearance of an amino acid residue in the sequence, and “amino acid substitution” means that an amino acid residue in the sequence is replaced with another amino acid residue. “Addition of amino acid” means that a new amino acid residue is added to the sequence.

A specific embodiment of “deletion, substitution, addition of 1 to several amino acids” includes an embodiment in which one to several amino acids are replaced with another chemically similar amino acid. For example, a case where a certain hydrophobic amino acid is substituted with another hydrophobic amino acid, a case where a certain polar amino acid is substituted with another polar amino acid having the same charge, and the like can be mentioned. Such chemically similar amino acids are known in the art for each amino acid. Specific examples include non-polar (hydrophobic) amino acids such as alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, and methionine. Examples of polar (neutral) amino acids include glycine, serine, threonine, tyrosine, glutamine, asparagine, and cysteine. Examples of the basic amino acid having a positive charge include arginine, histidine, and lysine. Examples of acidic amino acids having a negative charge include aspartic acid and glutamic acid.

Examples of amino acid sequences having a deletion, substitution, addition, etc. of one to several amino acids in the amino acid sequence possessed by the wild type enzyme include amino acid sequences having a certain sequence identity with the amino acid sequence possessed by the wild type enzyme. For example, 60% or more, preferably 65% or more, preferably 70% or more, preferably 75% or more, preferably 80% or more, preferably 85% or more, more preferably 90% with the amino acid sequence of the wild-type enzyme As mentioned above, the amino acid sequence having the identity of 95% or more is most preferable.

(Gene encoding enzymes (1) to (7))
The genes ascB, ascC, ascD, ascE, ascF, ascG and ascH (hereinafter, these may be collectively referred to as “genes encoding enzymes (1) to (7)”) have the enzyme activity described above. It does not specifically limit if it contains the base sequence which codes the amino acid sequence which the enzyme (1) thru | or (7) which has has. Enzymes (1) to (7) are produced by expressing genes encoding enzymes (1) to (7) in the transformant. As used herein, “gene expression” means that an enzyme encoded by a gene is produced in such a manner as to have an original catalytic activity through transcription or translation. In addition, “gene expression” includes high expression of a gene, that is, insertion of a gene causes production of an enzyme encoded by the gene in excess of the amount originally expressed by the host organism. To do.

The gene encoding the enzymes (1) to (7) may be a gene that can produce the enzymes (1) to (7) via splicing after transcription of the gene when introduced into the host organism. The gene may be any gene that can produce enzymes (1) to (7) without splicing after transcription of the gene.

The gene encoding the enzymes (1) to (7) does not have to be completely identical to the gene originally possessed by the derived organism (that is, the wild type gene), and is a gene encoding the enzyme having the enzyme activity described above. As long as it is a DNA having a base sequence that hybridizes with a base sequence complementary to the base sequence of the wild-type gene under stringent conditions.

The term “base sequence that hybridizes under stringent conditions” in the present specification refers to a colony hybridization method, plaque hybridization method, Southern blot hybridization method using a DNA having a base sequence of a wild-type gene as a probe. It means the base sequence of DNA obtained by using.

The term “stringent conditions” in the present specification is a condition in which a specific hybrid signal is clearly distinguished from a non-specific hybrid signal. The hybridization system used, the type of probe, and the sequence It depends on the length. Such conditions can be determined by changing the hybridization temperature, washing temperature and salt concentration. For example, when a non-specific hybrid signal is strongly detected, the specificity can be increased by raising the hybridization and washing temperature and, if necessary, lowering the washing salt concentration. If no specific hybrid signal is detected, the hybrid can be stabilized by lowering the hybridization and washing temperatures and, if necessary, raising the washing salt concentration.

As a specific example of stringent conditions, for example, a DNA probe is used as a probe, hybridization is 5 × SSC, 1.0% (w / v) blocking reagent for nucleic acid hybridization (manufactured by Boehringer Mannheim), 0.1% (w / v) N-lauroyl sarcosine, 0.02% (w / v) SDS is used overnight (about 8 to 16 hours). Washing is performed using 0.1-0.5 × SSC, 0.1% (w / v) SDS, preferably 0.1 × SSC, 0.1% (w / v) SDS, twice for 15 minutes. Do. The temperature for performing hybridization and washing is 65 ° C or higher, preferably 68 ° C or higher.

Examples of the DNA having a base sequence that hybridizes under stringent conditions include, for example, a DNA having a base sequence of a wild-type gene derived from a colony or plaque or a filter on which a fragment of the DNA is immobilized, as described above. After carrying out hybridization at 40 to 75 ° C. in the presence of DNA obtained by hybridization under a gentle condition or 0.5 to 2.0 M NaCl, preferably 0.7 to 1.0 M After hybridization at 65 ° C. in the presence of NaCl, a 0.1-1 × SSC solution (1 × SSC solution is 150 mM sodium chloride, 15 mM sodium citrate) was used, and the filter was filtered at 65 ° C. Examples thereof include DNA that can be identified by washing. Probe preparation and hybridization methods are described in Molecular Cloning: A laboratory Manual, 2nd-Ed. , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. , 1989, Current Protocols in Molecular Biology, Supplement 1-38, John Wiley & Sons, 1987-1997 (hereinafter, these documents are also referred to as “reference technical documents”) and the like. it can. In addition to the conditions such as the salt concentration and temperature of the buffer, those skilled in the art will consider other conditions such as probe concentration, probe length, reaction time, etc. Conditions for obtaining a DNA having a base sequence that hybridizes under stringent conditions with a complementary base sequence can be appropriately set.

Examples of DNA containing a base sequence that hybridizes under stringent conditions include DNA having a certain sequence identity with a base sequence of a DNA having a base sequence of a wild-type gene used as a probe. 60% or more, preferably 65% or more, preferably 70% or more, preferably 75% or more, preferably 80% or more, preferably 85% or more, more preferably 90% or more, more preferably Examples thereof include DNA having 95% or more sequence identity.

As a base sequence that hybridizes under stringent conditions with a base sequence complementary to the base sequence of the wild-type gene, for example, if the number of bases in the base sequence is 500 units, the base sequence of the wild-type gene 1 to several, preferably 1 to 200, more preferably 1 to 175, more preferably 1 to 150, more preferably 1 to 125, more preferably 1 to 100 per unit, More preferably 1 to 75, more preferably 1 to 50, more preferably 1 to 30, more preferably 1 to 20, still more preferably 1, 2, 3, 4, 5, 6, 7, Includes base sequences with 8, 9 or 10 base deletions, substitutions, additions, etc. Here, “base deletion” means that there is a deletion or disappearance in the base in the sequence, and “base replacement” means that the base in the sequence is replaced with another base, “Addition of a base” means that a new base is added to be inserted.

The enzyme encoded by the base sequence that hybridizes with the base sequence complementary to the base sequence of the wild-type gene under stringent conditions is 1 to several in the amino acid sequence of the enzyme encoded by the base sequence of the wild-type gene. Although it is likely to be an enzyme having an amino acid sequence having deletion, substitution, addition, etc. of individual amino acids, it has the same enzyme activity as the enzyme encoded by the base sequence of the wild-type gene.

In addition, the gene encoding the enzymes (1) to (7) has the same or similar amino acid sequence as the amino acid sequence of the enzyme encoded by the wild type gene using the fact that there are several types of codons corresponding to one amino acid. May include a nucleotide sequence different from that of the wild-type gene. Examples of the base sequence in which codon modification is performed on the base sequence of such a wild type gene include the base sequences described in SEQ ID NOs: 15 to 18 and 22 to 24, and the like. The base sequence subjected to codon modification is preferably, for example, a base sequence subjected to codon modification so as to be easily expressed in a host organism.

(Means for calculating sequence identity)
The method for determining the sequence identity of the base sequence or amino acid sequence is not particularly limited. For example, using a commonly known method, the target is the amino acid sequence of the wild type gene or the enzyme encoded by the wild type gene. It is obtained by aligning the base sequence and amino acid sequence and using a program for calculating the coincidence rate of both sequences.

As a program for calculating the coincidence ratio between two amino acid sequences or base sequences, for example, the algorithm of Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87: 2264-2268, 1990; Proc. Natl. Acad. USA 90: 5873-5877, 1993), and a BLAST program using this algorithm has been developed by Altschul et al. (J. Mol. Biol. 215: 403-410, 1990). Furthermore, Gapped BLAST, which is a program for determining sequence identity with higher sensitivity than BLAST, is also known (Nucleic Acids Res. 25: 3389-3402, 1997). Therefore, a person skilled in the art can search, for example, a sequence showing high sequence identity from a database using a program described above. These are available, for example, at the National Center for Biotechnology Information website (http://blast.ncbi.nlm.nih.gov/Blast.cgi).

Each of the above methods can be generally used to search a sequence showing sequence identity from a database. As a means for determining the sequence identity of individual sequences, Genetyx network version version 12.0. A homology analysis of 1 (Genetics) can also be used. This method is based on the Lipman-Pearson method (Science 227: 1435-1441, 1985). When analyzing the sequence identity of a base sequence, a region encoding a protein (CDS or ORF) is used if possible.

(Origin of gene encoding enzymes (1) to (7))
Genes encoding enzymes (1) to (7) are derived from, for example, biological species capable of producing isoprenoids such as iricicholine A and ascochlorin and biological species in which the expression of enzymes (1) to (7) is observed. To do. Examples of the organism derived from the gene encoding the enzymes (1) to (7) include microorganisms. Among the microorganisms, filamentous fungi are preferable because there are many bacterial species known to have the ability to produce ascochlorin. Examples of filamentous fungi with ascochlorin producing ability, Acremonium filamentous fungus, Trichoderma filamentous fungus, Fusarium (Fusarium) filamentous fungi, cylindroconical Cal Pont genus (Cylindrocarpon) filamentous fungi, Verticillium genus (Verticillium ) Filamentous fungi, Nectria genus ( Nectria ) filamentous fungi, and the like, and more specifically, Acremonium sclerotigenum and the like.

As described above, the organism from which the genes encoding the enzymes (1) to (7) are derived is not particularly limited, but the enzymes (1) to (7) expressed in the transformant are inactivated depending on the growth conditions of the host organism. Without activity. Therefore, the organism derived from the gene encoding the enzymes (1) to (7) is a microorganism whose growth conditions are close to those of the host organism to be transformed by inserting the genes encoding the enzymes (1) to (7). Preferably there is.

(Cloning of genes encoding enzymes (1) to (7) by genetic engineering techniques)
The gene encoding the enzymes (1) to (7) can be inserted into various appropriate known vectors. Furthermore, this vector can be introduced into an appropriate known host organism to produce a transformant into which a recombinant vector (recombinant DNA) containing a gene encoding enzymes (1) to (7) has been introduced. Methods for obtaining genes encoding enzymes (1) to (7), nucleotide sequences of genes encoding enzymes (1) to (7), methods for obtaining amino acid sequence information of enzymes (1) to (7), various methods A method for producing a vector, a method for producing a transformant, and the like can be appropriately selected by those skilled in the art. Moreover, in this specification, a transformation and a transformant include a transduction and a transductant, respectively. An example of the cloning of the gene encoding the enzymes (1) to (7) will be described later without limitation.

In order to clone the gene encoding the enzymes (1) to (7), a generally used gene cloning method can be appropriately used. For example, chromosomal DNA and mRNA can be extracted from microorganisms and various cells capable of producing the enzymes (1) to (7) by conventional methods, for example, methods described in the reference technical literature. CDNA can be synthesized using the extracted mRNA as a template. A chromosomal DNA or cDNA library can be prepared using the chromosomal DNA or cDNA thus obtained.

For example, the gene encoding the enzymes (1) to (7) can be obtained by cloning using a chromosomal DNA or cDNA of a living organism having the gene as a template. The organism from which the genes encoding the enzymes (1) to (7) are derived is as described above, and specific examples include Acremonium sclerotigenum. For example, culturing Acremonium sclerotigenum, removing moisture from the obtained bacterial cells, and physically pulverizing with a mortar etc. while cooling in liquid nitrogen to form fine powdered bacterial cell pieces Then, a chromosomal DNA fraction is extracted from the cell piece by a usual method. A commercially available chromosomal DNA extraction kit such as DNeasy Plant Mini Kit (manufactured by Qiagen) can be used for the chromosomal DNA extraction operation.

Next, using the chromosomal DNA as a template, DNA is amplified by performing a polymerase chain reaction (hereinafter referred to as “PCR”) using a synthetic primer complementary to the 5 ′ end sequence and the 3 ′ end sequence. The primer is not particularly limited as long as a DNA fragment containing the gene can be amplified. As another method, DNA containing the target gene fragment can be amplified by appropriate PCR such as 5'RACE method or 3'RACE method, and these can be ligated to obtain DNA containing the full length target gene.

In addition, the method for obtaining the gene encoding the enzymes (1) to (7) is not particularly limited. For example, the enzymes (1) to (7) can be obtained using a chemical synthesis method without using a genetic engineering technique. It is possible to construct a gene to encode.

Confirmation of the base sequence in the amplification product amplified by PCR or the chemically synthesized gene can be performed, for example, as follows. First, a DNA whose sequence is to be confirmed is inserted into an appropriate vector according to a normal method to produce a recombinant DNA. For cloning into a vector, commercially available kits such as TA Cloning Kit (manufactured by Invitrogen); pUC19 (manufactured by Takara Bio Inc.), pUC18 (manufactured by Takara Bio Inc.), pBR322 (manufactured by Takara Bio Inc.), pBluescript SK + (Stratagene) Commercially available plasmid vector DNA such as pYES2 / CT (manufactured by Invitrogen); commercially available bacteriophage vector DNA such as λEMBL3 (manufactured by Stratagene). The recombinant DNA is used to transform a host organism, for example, E. coli ( Escherichia coli ), preferably E. coli JM109 strain (Takara Bio) or E. coli DH5α strain (Takara Bio). The recombinant DNA contained in the obtained transformant is purified using QIAGEN Plasmid Mini Kit (manufactured by Qiagen).

The base sequence of each gene inserted into the recombinant DNA is determined by the dideoxy method (Methods in Enzymology, 101, 20-78, 1983). The sequence analysis apparatus used for determining the base sequence is not particularly limited. For example, Li-COR MODEL 4200L sequencer (manufactured by Aroka), 370 DNA sequence system (manufactured by PerkinElmer), CEQ2000XL DNA analysis system (manufactured by Beckman) ) And the like. Based on the determined base sequence, the translated protein, that is, the amino acid sequences of the enzymes (1) to (7) can be known.

(Construction of a recombinant vector containing a gene encoding enzymes (1) to (7))
A recombinant vector (recombinant DNA) containing a gene encoding enzymes (1) to (7) comprises a PCR amplification product containing any of the genes encoding enzymes (1) to (7) and various vectors. It can be constructed by binding in a form that allows expression of the genes encoding the enzymes (1) to (7). For example, it can be constructed by excising a DNA fragment containing any of the genes encoding enzymes (1) to (7) with an appropriate restriction enzyme and ligating the DNA fragment with a plasmid cut with an appropriate restriction enzyme. it can. Alternatively, a DNA fragment containing the gene in which a sequence homologous to a plasmid is added to both ends and a plasmid-derived DNA fragment amplified by inverse PCR are commercially available, such as In-Fusion HD Cloning Kit (manufactured by Clontech). It can be obtained by ligation using a recombinant vector preparation kit.

(Method for producing transformant)
The method for producing the transformant is not particularly limited, and examples thereof include a method of inserting into the host organism in such a manner that the gene encoding the enzymes (1) to (7) is expressed according to a conventional method. Specifically, a DNA construct in which any of the genes encoding enzymes (1) to (7) is inserted between the expression-inducing promoter and terminator is prepared, and then the genes encoding enzymes (1) to (7) By transforming a host organism with a DNA construct containing, a transformant overexpressing the gene encoding the enzymes (1) to (7) can be obtained. In the present specification, a DNA fragment comprising a gene-terminator encoding an expression-inducible promoter-enzyme (1) to (7) and a recombinant vector containing the DNA fragment, which are prepared for transforming a host organism, are DNA. Collectively called a construct.

The method of inserting into the host organism in such a manner that the gene encoding the enzymes (1) to (7) is expressed is not particularly limited. For example, the gene is directly inserted into the host organism chromosome by using homologous recombination. Techniques: A technique for introducing into a host organism by ligation onto a plasmid vector.

In the method using homologous recombination, a DNA construct can be ligated and inserted into the genome of the host organism between sequences homologous to the upstream region and downstream region of the recombination site on the chromosome. The high expression promoter is not particularly limited. For example, the promoter region of the translation elongation factor TEF1 gene (tef1), the promoter region of the α-amylase gene (amy), the alkaline protease gene (alp) promoter region, glyceraldehyde-3 -Phosphate dehydrogenase (gpd) promoter region and the like.

In a method using a vector, a DNA construct can be incorporated into a plasmid vector used for transformation of a host organism by a conventional method, and the corresponding host organism can be transformed by a conventional method.

Such a suitable vector-host system is not particularly limited as long as it is a system capable of producing the enzymes (1) to (7) in the host organism. For example, pUC19 and a filamentous fungal system, pSTA14 (Mol Gen. Genet. 218, 99-104, 1989) and filamentous fungal systems.

The DNA construct is preferably used after being introduced into the chromosome of the host organism. However, as another method, the DNA construct is introduced into an autonomously replicating vector (Ozeki et al. Biosci. Biotechnol. Biochem. 59, 1133 (1995)). By incorporating it, it can be used in a form not introduced into the chromosome.

The DNA construct may include a marker gene to allow selection of transformed cells. The marker gene is not particularly limited, and examples thereof include genes that complement the auxotrophy of host organisms such as pyrG, niaD, and adeA; drug resistance genes for drugs such as pyrithiamine, hygromycin B, and oligomycin. The DNA construct also contains a promoter, terminator, and other control sequences (eg, enhancer, polyadenylation sequence, etc.) that allow overexpression of the gene encoding enzymes (1) to (7) in the host organism. It is preferable. The promoter is not particularly limited, and examples thereof include an appropriate expression-inducing promoter and a constitutive promoter. Examples thereof include a tef1 promoter, an alp promoter, an amy promoter, and a gpd promoter. The terminator is also not particularly limited, and examples thereof include an alp terminator, an amy terminator, and a tef1 terminator.

In the DNA construct, the expression control sequence of the gene encoding the enzymes (1) to (7) is such that the DNA fragment containing the gene encoding the inserted enzyme (1) to (7) has an expression control function. It is not always necessary to include sequences. Further, when transformation is performed by the co-transformation method, the DNA construct may not have a marker gene.

The DNA construct can be tagged for purification. For example, a linker sequence is appropriately connected upstream or downstream of the gene encoding enzymes (1) to (7), and a base sequence encoding histidine is connected by 6 codons or more, thereby enabling purification using a nickel column. can do.

The DNA construct may contain homologous sequences necessary for marker recycling. For example, the pyrG marker adds a sequence homologous to the sequence upstream of the insertion site (5 ′ homologous recombination region) downstream of the pyrG marker, or downstream of the insertion site (3 ′ homologous recombination region) upstream of the pyrG marker. By adding a sequence homologous to the sequence, the pyrG marker can be removed on a medium containing 5-fluoroorotic acid (5FOA). The length of the homologous sequence suitable for marker recycling is preferably 0.5 kb or more.

In one embodiment of the DNA construct, for example, a tef1 gene promoter, a gene encoding enzymes (1) to (7), an alp gene terminator, and a pyrG marker gene are linked to In-Fusion Cloning Site at the multi-cloning site of pUC19. DNA construct.

One aspect of the DNA construct when inserting a gene by homologous recombination is as follows: 5 ′ homologous recombination sequence, tef1 gene promoter, gene encoding enzymes (1) to (7), alp gene terminator and pyrG marker gene, 3 ′ homology A DNA construct in which recombination sequences are linked.

In one embodiment of the DNA construct when a gene is inserted by homologous recombination and the marker is recycled, a 5 ′ homologous recombination sequence, a tef1 gene promoter, a gene encoding enzymes (1) to (7), an alp gene terminator, It is a DNA construct in which a homologous sequence for marker recycling, a pyrG marker gene, and a 3 ′ homologous recombination sequence are linked.

When the host organism is a filamentous fungus, a method known to those skilled in the art can be appropriately selected as a method for transformation into the filamentous fungus. For example, after preparing a protoplast of the host organism, polyethylene glycol and calcium chloride are added. The protoplast PEG method to be used (for example, refer to Mol. Gen. Genet. 218, 99-104, 1989, JP 2007-2222055 A, etc.) can be used. As a medium for regenerating the transformant, an appropriate medium is used according to the host organism to be used and the transformation marker gene. For example, when Aspergillus oryzae ( A. oryzae ) or Aspergillus sojae ( A. sojae ) is used as the host organism and the pyrG gene is used as the transformation marker gene, regeneration of the transformant is, for example, 0.5 Czapek-Dox minimal medium (manufactured by Difco) containing 1% agar and 1.2 M sorbitol.

Further, for example, in order to obtain a transformant, the promoter of the gene encoding the enzymes (1) to (7) that the host organism originally has on the chromosome is changed to a high expression promoter such as tef1 using homologous recombination. It may be replaced. Also in this case, it is preferable to insert a transformation marker gene such as pyrG in addition to the high expression promoter. For example, for this purpose, referring to the examples described in JP2011-239681 and FIG. 1, the upstream region of the gene encoding the enzymes (1) to (7) -transformation marker gene-high An expression cassette-transformation cassette comprising all or part of the gene encoding the enzyme (1) to (7) can be used. In this case, the upstream region of the gene encoding enzymes (1) to (7) and the whole or part of the gene encoding enzymes (1) to (7) are used for homologous recombination. As the whole or part of the gene encoding the enzymes (1) to (7), those containing a region in the middle from the start codon can be used. The length of the region suitable for homologous recombination is preferably 0.5 kb or more.

Confirmation that the transformant was produced is obtained by culturing the transformant under conditions where the enzyme activities of the enzymes (1) to (7) are observed, and then the target product in the culture obtained after the culture, for example, It can be carried out by confirming that ascochlorin, iricicholine A or the like is detected, or that the amount of the target product detected is larger than the amount of the target product in the culture of the host organism cultured under the same conditions. it can.

In addition, confirmation that the transformant was produced was performed by extracting chromosomal DNA from the transformant, performing PCR using this as a template, and confirming that a PCR product that can be amplified is produced when transformation occurs. It may be done by doing. In this case, for example, PCR is performed with a combination of a forward primer for the base sequence of the promoter used and a reverse primer for the base sequence of the transformation marker gene to confirm that a product of the expected length is generated.

When transformation is performed by homologous recombination, PCR is performed using a combination of a forward primer located upstream from the upstream homologous region used and a reverse primer located downstream from the used homologous region. It is preferable to confirm that a product of the expected length is produced when recombination occurs.

(Host organism)
The host organism is not particularly limited as long as it is an organism capable of producing enzymes (1) to (7) by transformation with a DNA construct containing a gene encoding enzymes (1) to (7). , including microorganisms and plants. Examples of the microorganisms, Aspergillus microorganism Acremonium microorganism Neonekutoria microorganisms, Fusarium microorganism, Escherichia (Escherichia) microorganism belonging to the genus Saccharomyces (Saccharomyces) microorganisms belonging to the genus Pichia (Pichia) sp microorganism , Schizosaccharomyces (Schizosaccharomyces) a microorganism belonging to the genus, Gigot Saccharomyces Seth (Zygosaccharomyces) microorganism belonging to the genus Trichoderma (Trichoderuma) microorganism belonging to the genus, Penicillium ( Penicillium) microorganism belonging to the genus, Rhizopus (Rhizopus) microorganism belonging to the genus, Neurospora crassa (Neurospora) microorganism belonging to the genus, Mucor (Mucor) a microorganism belonging to the genus, Neosartorya (Neosartorya) microorganism belonging to the genus, Bissokuramisu (Byssochlamys) microorganism belonging to the genus, Talaromyces (Talaromyces) microorganism belonging to the genus, Ajeromisesu ( Ajellomyces) a microorganism belonging to the genus, para cock Sidi Oy death (Paracoccidioides) microorganism belonging to the genus, Anshinokarupusu (Uncinocarpus) a microorganism belonging to the genus, cock Sidi Oy death (Coccidioides) microorganism belonging to the genus, Arufuroderuma (Arthroderma) microorganism belonging to the genus, Trichophyton (Trichophyton) microorganism belonging to the genus, Ekusofira ( xophiala) microorganism belonging to the genus, Kapuronia (Capronia) a microorganism belonging to the genus, Cladosporium Fear Russia follower La (Cladophialophora) microorganism belonging to the genus, Makurohomina (Macrophomina) a microorganism belonging to the genus, Leptosphaeria file area (Leptosphaeria) microorganism belonging to the genus, Bipolaris (Bipolaris) microorganism belonging to the genus, Dochisutoroma (Dothistroma) a microorganism belonging to the genus , Pyrenophora (Pyrenophora) microorganism belonging to the genus, Neofushikokkamu (Neofusicoccum) microorganism belonging to the genus, Setosufaeria (Setosphaeria) microorganism belonging to the genus, Baudoinia (Baudoinia) microorganism belonging to the genus, Gaeumanomisesu (Gaeumannomyces) microorganism belonging to the genus, Marussonina (Marssonina) a microorganism belonging to the genus, Sufae Rina (Sphaerulina) microorganism belonging to the genus, Sclerotinia (Sclerotinia) microorganism belonging to the genus, Magunaporuse (Magnaporthe) a microorganism belonging to the genus, Vel lethal potassium (Verticillium) a microorganism belonging to the genus, pseudoephedrine Serco Supora (Pseudocercospora) microorganism belonging to the genus, Colletotrichum (Colletotrichum) a microorganism belonging to the genus, ophiolite stoma ( Ophiostoma) microorganism, Metaruhijiumu (Metarhizium) microorganisms, sports Ross helix (Sporothrix) microorganism, Sorudaria (Sordaria) microorganisms, such as Arabidopsis (Arabidopsis) plants of the genus, and the like, microorganisms and plants are preferred. Filamentous fungi having the ability to produce isoprenoids such as iricicholine A, ascochlorin, and ascofuranone, and filamentous fungi having genes encoding enzymes (1) to (7) on genomic DNA may be used. However, in any case, humans are excluded from the host organism.

Among filamentous fungi, Aspergillus oryzae, Aspergillus soya, Aspergillus niger ( A. niger), Aspergillus tamari ( A. tamarii ), Aspergillus awamori ( A. awamori), Aspergillus Usami (A.usami), Aspergillus kawachii (A.kawachii), such as Aspergillus microorganisms, such as Aspergillus saitoi (A.saitoi) is preferable.

(Specific examples of genes encoding enzymes (1) to (7))
Examples of genes encoding enzymes (1) to (7) derived from Acremonium sclerotigenum include genes asbB, ascC, ascD, ascE, ascF, ascG each having the base sequence described in SEQ ID NOs: 1-7. And ascH. The amino acid sequences of AscB, AscC, AscD, AscE, AscF, AscG, and AscH proteins are shown as SEQ ID NOs: 8 to 14, respectively.

A method for obtaining a gene encoding the enzymes (1) to (7) from organisms other than Acremonium sclerotigenum and Acremonium sclerotigenum is not particularly limited. For example, genes ascB, ascC, ascD, ascE, ascF Based on the base sequences (SEQ ID NOs: 1 to 7) of ascG and ascH, the genomic DNA of the target organism is subjected to BLAST homology search, and the base sequences and sequences of the genes ascB, ascC, ascD, ascE, ascF, ascG and ascH It can be obtained by specifying a gene having a highly identical base sequence. Further, based on the total protein of the target organism, specify a protein having an amino acid sequence having a high sequence identity with the amino acid sequences of the AscB, AscC, AscD, AscE, AscF, AscG and AscH proteins (SEQ ID NOs: 8 to 14), It can be obtained by specifying the gene encoding the protein. For example, the amino acid sequences having high sequence identity with the amino acid sequences of AscB, AscC, AscD, AscE, AscF, AscG and AscH proteins derived from Acremonium sclerotigenum include the amino acid sequences of SEQ ID NOs: 29 to 35 derived from Neonectoria The amino acid sequences having high sequence identity with the amino acid sequences of AscB, AscC, AscD and AscE proteins derived from Acremonium sclerotigenum include the amino acid sequences of SEQ ID NOs: 37 to 40 derived from Trichoderma.

Aspergillus as a host organism, a gene encoding enzymes (1) to (7) obtained from Acremonium sclerotigenum or a gene encoding an enzyme having sequence identity with enzymes (1) to (7) It can be transformed by introducing it into an arbitrary host cell such as a microorganism or Acremonium microorganism.

(Transformant)
In one aspect of the transformant, filamentous fungi or plants are used as host organisms, and any one of the genes ascD, ascB, ascC, ascE, ascF, ascG and ascH, or a combination thereof is inserted, and A transformant transformed to express the inserted gene (hereinafter also referred to as “transformant (1)”). When the host organism is an organism that can produce ascochlorin and ascofuranone, such as Acremonium and sclerotigenum, the inserted gene is constantly expressed higher than forced or endogenous expression. It is desirable that the condition is expressed in the later stage of the culture after cell growth. Such a transformant can detect iricicholine A or ascochlorin which is not produced in the host organism by the action of expressed AscD, AscB, AscC, AscE, AscF, AscG and / or AscH, or even if it is produced in a trace amount. It is possible to produce more.

Another aspect of the transformant is a wild-type biosynthetic gene cluster gene containing all or part of the genes ascD, ascB, ascC, ascE, ascF, ascG and ascH using filamentous fungi or plants as host organisms (Including a promoter sequence other than the ORF), and a DNA construct designed to highly express or underexpress a transcription factor that controls transcription of the biosynthetic gene cluster, and the insertion A transformant transformed to express the expressed gene (hereinafter also referred to as “transformant (2)”). When the host organism is an organism that can produce ascochlorin and ascofuranone, such as Acremonium and sclerotigenum, the inserted gene is constantly expressed higher than forced or endogenous expression. It is desirable that the condition is expressed in the later stage of the culture after cell growth. Such a transformant is not produced in the host organism or is produced in a trace amount even if it is produced by culturing or growing under the conditions suitable for the host organism or the transformant by the action of the transcription factor whose expression level is changed. Certain iricicholine A or ascochlorin can be produced beyond detection.

(Production method)
The production method of one embodiment of the present invention includes at least a step of obtaining iricicholine A or ascochlorin by culturing the transformant (1) or the transformant (2) under conditions suitable for host cells. Or it is a manufacturing method of ascochlorin.

According to another embodiment of the present invention, a method for producing a transformant (1) or a transformant (2) using an iricholine A or ascochlorin precursor such as LL-Z1272β or iricicholine A (LL-Z1272α). A method for producing iricicholine A or ascochlorin, comprising at least a step of obtaining iricicholine A or ascochlorin. For example, the method of allowing iricicholine A to act on a transformant is not particularly limited as long as it is a method in which iricicholine A and the transformant are brought into contact with each other so that ascochlorin can be produced by the enzyme of the transformant. And a method for producing ascochlorin by culturing the transformant under a culture condition suitable for growth of the transformant using a medium containing A and suitable for the growth of the transformant. It is done. The culture method is not particularly limited. For example, when the host organism is a filamentous fungus, a solid culture method or a liquid culture method performed under aerated or non-aerated conditions can be used.

In another embodiment of the present invention, a method for extracting a precursor of iricicholine A or ascochlorin, such as LL-Z1272β or iricicholine A (LL-Z1272α), from the transformant (1) or the transformant (2). This is a method for producing iricicholine A or ascochlorin, comprising at least a step of obtaining iricicholine A or ascochlorin by causing the enzyme to act.

Below, although the manufacturing method in case a host organism and a wild type organism are filamentous fungi is mainly described, the manufacturing method of each aspect of this invention is not limited to the following description.

The medium is a normal medium for culturing host organisms and wild-type organisms (hereinafter collectively referred to as “host organisms”), that is, carbon sources, nitrogen sources, minerals, and other nutrients in appropriate proportions. Any of a synthetic medium and a natural medium can be used. When the host organism or the like is a microorganism belonging to the genus Aspergillus, a GPY medium as described in the examples described later can be used, but is not particularly limited.

The culture conditions for transformants and knockout organisms (hereinafter collectively referred to as “transformants etc.”) may be culture conditions such as host organisms ordinarily known by those skilled in the art. When the host organism or the like is a filamentous fungus, the initial pH of the medium is adjusted to 5 to 10, the culture temperature is 20 to 40 ° C., the culture time is several hours to several days, preferably 1 to 7 days, more preferably 2 It can be appropriately set such as ˜4 days. The culture means is not particularly limited, and aeration and agitation deep culture, shaking culture, static culture, and the like can be adopted, but culture is preferably performed under conditions that provide sufficient dissolved oxygen. For example, as an example of a culture medium and culture conditions for culturing an Aspergillus microorganism, shaking culture at 30 ° C. and 160 rpm for 3 to 5 days using a GPY medium described in Examples described later can be mentioned.

A method for extracting a target product such as ascochlorin and iricicholine A from the culture after completion of the culture is not particularly limited. For the extraction, the cells recovered from the culture by filtration, centrifugation, or the like may be used as they are, or the cells recovered after drying and further crushed cells may be used. The method for drying the cells is not particularly limited, and examples thereof include freeze drying, sun drying, hot air drying, vacuum drying, aeration drying, and reduced pressure drying.

The extraction solvent is not particularly limited as long as the target product can be dissolved. For example, organic solvents such as methanol, ethanol, isopropanol, and acetone; hydrous organic solvents obtained by mixing these organic solvents and water; water, hot water, and Hot water etc. are mentioned. After adding the solvent, the target product is extracted as appropriate while subjecting the cells to disruption.

Further, instead of the above-mentioned heating treatment, for example, a method of destroying bacterial cells using a disrupting means such as an ultrasonic crusher, a French press, a dynomill, or a mortar; a cell cell wall using a cell wall lytic enzyme such as yatalase Method of dissolving; The cells may be subjected to cell disruption treatment such as a method of dissolving cells using a surfactant such as SDS or Triton X-100. These methods can be used alone or in combination.

The obtained extract is centrifuged, filtered, ultrafiltered, gel filtered, separated by solubility difference, solvent extraction, chromatography (adsorption chromatography, hydrophobic chromatography, cation exchange chromatography, anion exchange chromatography). The target product can be purified by subjecting it to purification treatment such as reverse phase chromatography, crystallization, activated carbon treatment, membrane treatment, etc.

Qualitative analysis or quantitative analysis can be performed by LC-MS, LC-ICP-MS, MS / MS, and the like. Those skilled in the art can appropriately select the conditions for these analyses, and for example, the conditions described in the examples described later can be used.

In the manufacturing method of each aspect of the present invention, as long as the problems of the present invention can be solved, various processes and operations can be added before, after, or in the process described above.

(Use)
Isoprenoids such as ascochlorin and iricicholine A obtained using the gene, transformant, knockout organism and production method of one embodiment of the present invention have antiprotozoal activity, antitumor activity, blood glucose lowering effect, blood lipid lowering It is a functional biological substance that can be expected to have various physiological activities such as action, glycation-inhibiting action, antioxidant action, etc., and to make use of its characteristics to produce pharmaceuticals, quasi drugs, etc. It can be used as a raw material.

Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to these examples, and the present invention can take various modes as long as the problems of the present invention can be solved. .

(Search for Ascochlorin biosynthesis genes)
Using the Ascofuranone producing bacterium Acremonium sclerotigenum ( Acremonium sclerotigenum F-1392 strain; see J. Antibiot. 70: 304-307 (2016)) A culture sample was obtained.

From these samples, 50 to 100 mg of cells were collected, and total RNA was collected using TRIzol Reagent (manufactured by Thermo Fisher Scientific) according to a standard protocol.

From the recovered total RNA, mRNA was isolated using Dynabeads mRNA DIRECT Micro Kit (Thermo Fisher Scientific), and transcriptome library (cDNA library) was obtained from Ion Total RNA-seq Kit v2 (ThermoFisher Scientific). ).

The quality of total RNA, mRNA, and cDNA and the measurement of transcriptome library concentration were confirmed using Agilent RNA 6000 pico kit and Agilent 2100 bioanalyzer system (both manufactured by Agilent).

RNA sequence analysis of the obtained cDNA library was performed as follows using a system of Thermo Fisher Scientific.

The obtained cDNA libraries were each diluted to 20 pmol / L, and the libraries were amplified by emulsion PCR using Ion OneTouch 2. The amplified library was concentrated using Ion OneTouch ES, and RNA sequence analysis was performed using Ion PGM system. Ion PGM Template OT2 200 Kit was used for Ion OneTouch 2, and Ion PGM sequencing 200 Kit v2 was used for Ion PGM.

Ion PGM Ion 316 v2 chip was used for RNA sequencing. The obtained sequence information was mapped to the genome sequence database of Acremonium sclerotigenum, and the difference in gene expression level between the two samples was analyzed.

Based on a value obtained by correcting the number of mapped cDNA reads by the length of each gene (RPKM: reads per kilobase of exon mapped mapped reads), the expression magnification between the high production sample and the low production sample is calculated. Calculated.

As a result of searching for genes that are highly expressed at a magnification of 300 times or more compared to those of a low production sample among the genes of the high production sample, two or more genes are continuous, and the magnification is 300 times or more. We were able to find the only region where the highly expressed genes were concentrated. It was predicted that this was an ascofuranone biosynthetic gene cluster, and genes that were highly expressed at a magnification of 300 times or more were named ascA to H (see FIG. 1).

In addition, as a result of performing a blast search or a domain search by Pfam for the protein encoded by each gene, AscA to H were predicted to have the functions shown in Table 1. Of these, AscA was considered to be a transcription factor, and Ascfuranone biosynthesis involves AscB to H protein (SEQ ID NOs: 8 to 14) encoded by genes ascB to H (SEQ ID NOs: 1 to 7). Expected.

(Preparation of AscD, AscB, AscC and AscE expression transformants)
An expression cassette containing any of the genes asbB, ascC, ascD and ascE of SEQ ID NOs: 15 to 18 having codons modified for the expression of Neisseria gonorrhoeae to a pyrG disrupted strain / ku70 disrupted strain of Aspergillus sojae ( Aspergillus sojae NRRC 4239 strain) Introduced.

Specifically, as an expression cassette for expressing each asc gene, a promoter sequence of translation elongation factor gene tef1 Ptef (upstream 748 bp of tef1 gene, SEQ ID NO: 19) is used as a promoter, and alkaline protease gene alp The terminator sequence Talp (800 bp downstream of the alp gene, SEQ ID NO: 20) was used as the terminator. As a selection marker, a transformation marker gene pyrG (1,838 bp including upstream 407 bp, coding region 896 bp and downstream 5,35 bp, SEQ ID NO: 21) that complements uracil / uridine requirement was used.

For example, Yuen et al. (Appl Microbiol Biotechnol. 2009 Mar; 82 (4): 691-701. Doi: 10.1007 / s00253-008-1815-5. Epub 2008 Dec 24, Construction of Quintetrupt intuitp As reported in protein production in Aspergillus oryzae.), when DNA used for transformation contains a sequence homologous to the sequence upstream or downstream of the gene insertion position (homologous recombination region), The pyrG marker can be removed on a medium containing orotic acid (5FOA), and the pyrG marker should be used many times. Marker recycling) is possible. Therefore, 5 ′ homologous recombination sequence (5′arm), Ptef, asc gene, Talp, homologous sequence for marker recycling (homologous sequence with downstream gene; loop out region), pyrG, 3 ′ homologous recombination sequence (3′arm) ) Was used as transformation DNA to perform pyrG marker recycling, and each ac gene expression cassette was inserted in the order of ascD, ascB, ascC, and ascE onto the chromosome of Aspergillus sojae.

In addition, In-Fusion HD Cloning Kit (Clontech) was used for linking each DNA. For example, when Ptef and gene ascD were ligated, each DNA fragment was amplified by PCR reaction using Ptef using the primers of SEQ ID NOs: 25 and 26 and ascD using the primers of SEQ ID NOs: 27 and 28. At this time, since 15 bp of a sequence homologous to Ptef is added to the 5 ′ end of the forward primer for gene ascD, Ptef and gene ascD can be linked by the Infusion reaction.

5'arm-Pef-ascD-Talp-loop out region-pyrG-3'arm, 5'arm-Ptef-ascB-Talp-loopout region-pyrG-3'arm, 5'arm prepared as described above -Aspergillus soya NRRC 4239 strain using DNA for transformation of Ptef-ascC-Talp-loop out region-pyrG-3'arm, 5'arm-Pef-ascE-Talp-loopout region-pyrG-3'arm An As-D strain, an As-DB strain, in which one copy of each of the expression cassettes containing any of the genes ascD, ascB, ascC, and ascE was introduced in order by transforming the pyrG disrupted strain / ku70 disrupted strain of As-DB C strain and As-DBCE strain were obtained.

Next, GPY medium supplemented with 1% (w / v) NaCl (2% (w / v) glucose, 1%
(W / v) polypeptone, 0.5% (w / v) yeast extract, 0.5% (w / v) monopotassium dihydrogen phosphate, 0.05% (w / v) magnesium sulfate heptahydrate The As-D strain, As-DB strain, As-DBC strain and As-DBCE strain were inoculated into the product and cultured at 30 ° C. for 4 days. After the cultured cells were collected on a filter paper, water was removed by suction filtration.

The collected microbial cells were immersed in acetone overnight and filtered to obtain an acetone extract of As-DBCE strain. The obtained acetone extract was concentrated to dryness, dissolved in methanol, and then subjected to HPLC analysis and MS analysis (negative mode). As a result, a new strain that was not found in the host strain (NBRC4239 strain) was found in the As-D strain. The peak was confirmed at the same elution position as the standard o-orceric acid. In addition, a new peak that was not found in the As-D strain was confirmed in the As-DB strain, and as a result of MS analysis, it was found that the m / z value of the peak was 371 corresponding to iricicolinic acid B. In addition, a new peak that was not found in the As-DB strain was confirmed in the As-DBC strain, and as a result of MS analysis, it was found that the m / z value of the peak was 355 corresponding to L-Z1272β. Furthermore, the As-DBCE strain showed a slight new peak that was not seen in the As-DBC strain, and the peak was found at the same elution position as that of the iricicolin A standard (see FIG. 2). .

2 is a TSKgel ODS-100V (manufactured by TOSOH) having a mobile phase (1 ml / min) of methanol: water: acetic acid (450: 50: 10) and a particle diameter of 3 μm, 4.6 mm × 100 mm. Performed using an ODS column.

Furthermore, in the As-DBCE-multi-copy strain in which the genes ascD, ascB, ascC and ascE were introduced in multiple copies using the pyrG3 gene (SEQ ID NO: 44) described in Japanese Patent Application No. 2006-209178, It was found that a large amount of A was accumulated (see FIG. 2). The pyrG3 gene is a selectable marker gene in which the promoter region in pyrG is modified to reduce the expression level in order to incorporate an arbitrary gene into a chromosome in multiple copies in a filamentous fungus.

Also, the LC / MS analysis shows that the peak compound shows the same m / z value 389 (negative mode) as the iricicholine A standard product, and the LC / MS / MS analysis also shows the same peak pattern as the iricicholine A standard product. As shown, it was found that iricicholine A was biosynthesized by the expressed AscD, AscB, AscC and AscE proteins. In FIG. 2, the peak seen at the elution position of about 7 minutes matches the elution position with the peak confirmed in the As-DBC strain. As a result of MS analysis, m / z of LL-Z1272β It was found to agree with the value.

(Preparation of transformants expressing AscD, AscB, AscC, AscE, AscF, AscG and AscH)
In the same manner as the As-DBCE strain, an AscF expression cassette was introduced by sequentially introducing an expression cassette containing any of the genes ascF, ascG and ascH of SEQ ID NOs: 22 to 24 whose codons were modified for the expression of Aspergillus. -DBCEF strain; As-DBCEFG strain introduced with AscF and AscG expression cassettes; and As-DBCEFGH strain introduced with AscF, AscG and AscH expression cassettes. These strains were cultured as described above and subjected to HPLC analysis.

In HPLC, acetonitrile + 0.1% (v / v) formic acid is liquid A and water + 0.1% (v / v) formic acid is liquid B, and under a gradient condition of liquid A 40 to 100% (50 min). The analysis was performed using an ODS column of L-column 2 ODS (particle size: 3 μm, 2.1 mm × 100 mm; manufactured by Chemical Substance Evaluation Research Corporation) at a flow rate of 0.25 ml / min.

As shown in FIG. 3, a new peak that was not found in the As-DBCE strain was confirmed in the As-DBCEF strain. In addition, a new peak that was not seen in the As-DBCEF strain was confirmed in the As-DBCEFG strain. Furthermore, a new peak that was not observed in the As-DBCEFG strain was confirmed in the As-DBCEFG strain.

From this, it has been found that after Iricicolin A, the AscF, AscG and AscH proteins act sequentially and the reaction proceeds.

(In vitro analysis using crude enzyme solution)
As-F strain, As-F strain in which any of the expression cassettes of genes ascF, ascG and ascH of SEQ ID NOs: 22 to 24 whose codons have been modified for the expression of Aspergillus is introduced into multiple copies of the Aspergillus soja NRRC4239 strain pyrG-disrupted strain -G strain and As-H strain were prepared. In preparing these strains, plasmid DNA in which Ptef-asc gene-Talp-pyrG3 was inserted into pUC19 was used as DNA for transformation.

Aspergillus soja NRRC 4239 strain (wild strain), As-F strain, As-G strain and As-H strain were each cultured in GPY medium for 1 day, and after removing the water of the cultured cells, liquid nitrogen was used. The cells were frozen and the cells were crushed with a multi-bead shocker. By adding 20 mM HEPES-NaOH (pH 7.0) to the pulverized cells, the crude enzyme solutions of the wild strain, As-F strain, As-G strain and As-H strain were extracted.

The following reaction solutions (1) to (4) were prepared using the obtained crude enzyme solution (for 5 to 10 mg of bacterial cells):
(1) Wild strain reaction solution: crude enzyme solution of wild strain, standard product of iricicholine A, mixed solution of 1 mM NADPH, 1 mM NADH, 1 mM ATP and 3 mM MgCl ₂ (2) As-F reaction solution: As-F strain Crude enzyme solution, standard of iricicholine A, mixed solution of 1 mM NADPH, 1 mM NADH, 1 mM ATP and 3 mM MgCl ₂ (3) As-FG reaction solution: crude enzyme solution of As-F strain, crude enzyme of As-G strain Liquid, iricicholine A standard, 1 mM NADPH, 1 mM NADH, 1 mM ATP and 3 mM MgCl ₂ mixed solution (4) As-FGH reaction solution: crude enzyme solution of As-F strain, crude enzyme solution of As-G strain, Crude enzyme solution of As-H strain, standard product of iricicholine A, mixed solution of 1 mM NADPH, 1 mM NADH, 1 mM ATP and 3 mM MgCl ₂

The reaction solutions (1) to (4) were each reacted overnight at room temperature. Next, each reaction solution was extracted with ethyl acetate, and the obtained extract was concentrated to dryness, and then subjected to LC / MS analysis.

LC analysis was performed using a column of L-column 2 ODS (particle size: 3 μm, 2.1 mm × 100 mm; manufactured by Chemical Substance Evaluation and Research Institute), acetonitrile + 0.1% (v / v) formic acid as liquid A, water + 0 .1% (v / v) formic acid was used as solution B, and the solution was analyzed at a flow rate of 0.25 ml / min under gradient conditions of solution A of 40% to 100% (50 min), and MS analysis was performed in negative mode.

As a result, as shown in FIG. 4A, a new peak that was not visible in the wild-type reaction solution was confirmed at the m / z value 423 in the As-F reaction solution. Further, as shown in FIG. 4B, a new peak that was not observed in the As-F reaction solution was confirmed at the m / z value 405 in the As-FG reaction solution. Furthermore, as shown in FIG. 5, a new peak that was not observed in the As-FG reaction solution or the As-FG reaction solution was confirmed at the m / z value 403 in the As-FGH reaction solution. In addition, the peak confirmed in the As-FGH reaction solution coincided with the peak of the ascochlorin standard product, and the m / z value of ascochlorin was 403. It was found that Ascochlorin was biosynthesized by the sequential action of AscG and AscH proteins.

As described above, the gene cluster predicted to be involved in ascofuranone biosynthesis was found to be an ascochlorin biosynthesis gene cluster. A predicted biosynthesis scheme of ascochlorin is shown in FIG. As shown in FIG. 6, the biosynthetic pathway of ascochlorin was found to be completely different from the biosynthetic pathway of ascofuranone, although there was some overlap. From this, it was found that the product produced by the transformant into which the biosynthesis gene cluster of ascochlorin was introduced was ascochlorin and not ascofuranone.

(Analysis of endogenous epoxide hydrolase gene-disrupted strain)
Since the peak of m / z value 423 was confirmed in the As-F reaction solution by the above in vitro analysis, the reaction product of the crude enzyme solution of AscF expressed in the Aspergillus soja NRRC 4239 strain was dihydroxylated Iricicolin A (See FIG. 6). However, according to Hosono et al. (J Antibiot (Tokyo). 2009 Oct; 62 (10): 571-4.), Iricicholine A epoxide (m / z value 405) is accumulated in Acremonium microorganisms. Thus, the original AscF reaction product was considered to be iricicholine A epoxide. That is, in Aspergillus soja NRRC4239 strain, it was considered that iricicholine A epoxide was opened by endogenous epoxide hydrolase and dihydroxylated iricicholine A was generated. Therefore, in the As-DBCEF strain, an As-DBCEF-ΔEH strain in which the epoxide hydrolase gene (SEQ ID NO: 36) having the highest expression level among genes predicted to encode an epoxide hydrolase derived from Aspergillus sojae is deleted. did. When As-DBCEF-ΔEH strain was cultured in the same manner as described above and subjected to HPLC analysis, a new peak that was not found in As-DBCEF strain was confirmed. Moreover, it was found by MS analysis that the peak had an m / z value corresponding to the epoxide compound. Thus, AscF was found to catalyze the epoxidation reaction of Iricicolin A.

(Functional analysis of AscC derived from Trichoderma reesei)
As a result of a Blast search based on the amino acid sequences of AscB to E of SEQ ID NOs: 8 to 11 derived from Acremonium sclerotigenum, AscB to E homologs of SEQ ID NOs: 37 to 40 (sequence) in Trichoderma reesei The identities are 47%, 53%, 52%, and 66%, respectively, and the ascB to E genes encoding them were found to be adjacent on the genome. From this, it was estimated that SEQ ID NOs: 37 to 40 are also biosynthesis enzymes of iricicholine A. Therefore, the ascC gene (Tr-ascC) of SEQ ID NO: 43 was cloned by performing PCR reaction using the genomic DNA of Trichoderma reesei NBRC31329 strain purchased from NITE as a template and using the primers of SEQ ID NOs: 41 and 42. The Tr-ascC gene of SEQ ID NO: 43 has a base sequence containing an intron, but as a result of intron prediction, it was considered to encode the AscC protein of SEQ ID NO: 38.

Cloned Tr-ascC was ligated in the same manner as described above to prepare DNA for transformation of 5'arm-Ptef-Tr-ascC-Talp-pyrG-3'arm. Next, the 5′arm-Ptef-Tr-ascC- of the DNA for transformation was transformed into the strain obtained by recycling the pyrG marker of the As-DB strain into which the ascD gene and the ascB gene derived from acremonium were prepared. By transforming with Talp-pyrG-3′arm, the As-DB-Tr-C strain into which one copy of each of the expression cassettes containing ascD and ascB derived from Acremonium and ascC derived from Trichoderma was introduced. I got it.

Next, GPY medium (2% (w / v) glucose, 1% (w / v) polypeptone, 0.5% (w / v) yeast extract, 0.5% (w / v) dihydrogen phosphate 1 The As-DB-Tr-C strain was inoculated into potassium (0.05% (w / v) magnesium sulfate heptahydrate) and cultured at 30 ° C. for 4 days. After the cultured cells were collected on a filter paper, water was removed by suction filtration.

The collected microbial cells were immersed in acetone overnight and filtered to obtain an acetone extract of As-DB-Tr-C strain. The obtained acetone extract was concentrated to dryness, dissolved in methanol, and then subjected to HPLC analysis. As-DB-Tr-C strain corresponds to L-Z1272β of m / z value of As-DBC strain. A new peak was detected at the same elution position as the 355 peak. From this, it can be seen that the Tr-ascC gene of SEQ ID NO: 43 has the same function as the ascC gene derived from Acremonium, as expected, so that SEQ ID NOs: 37-40 derived from Trichoderma are biosynthesis enzymes of iricicholine A. It was thought that there was. Furthermore, AscB~H of Neonekutoria-Ditishima (Neonecrtria ditissima) from SEQ ID NO: of 29 to 35, the identity of the AscB~H from Acremonium is in all 60% or more, and each of the encoding genes in the genome Since it is adjacent, this group of enzymes was considered to be ascochlorin biosynthetic enzymes.

The sequences listed in the sequence listing are as follows:
[SEQ ID NO: 1] ascB
[SEQ ID NO: 2] ascC
[SEQ ID NO: 3] ascD
[SEQ ID NO: 4] ascE
[SEQ ID NO: 5] ascF
[SEQ ID NO: 6] ascG
[SEQ ID NO: 7] ascH
[SEQ ID NO: 8] AscB protein [SEQ ID NO: 9] AscC protein [SEQ ID NO: 10] AscD protein [SEQ ID NO: 11] AscE protein [SEQ ID NO: 12] AscF protein [SEQ ID NO: 13] AscG protein [SEQ ID NO: 14] AscH protein [ SEQ ID NO: 15] codon modified ascB
[SEQ ID NO: 16] codon-modified ascC
[SEQ ID NO: 17] Codon-modified ascD
[SEQ ID NO: 18] Codon-modified ascE
[SEQ ID NO: 19] Ptef
[SEQ ID NO: 20] Talp
[SEQ ID NO: 21] pyrG
[SEQ ID NO: 22] codon-modified ascF
[SEQ ID NO: 23] codon-modified ascG
[SEQ ID NO: 24] codon-modified ascH
[SEQ ID NO: 25] Ptef-Fw
[SEQ ID NO: 26] Ptef-Rv
[SEQ ID NO: 27] ascD-Fw
[SEQ ID NO: 28] ascD-Rv
[SEQ ID NO: 29] AscB protein [SEQ ID NO: 30] AscC protein [SEQ ID NO: 31] AscD protein [SEQ ID NO: 32] AscE protein [SEQ ID NO: 33] AscF protein [SEQ ID NO: 34] AscG protein [SEQ ID NO: 35] AscH protein [ SEQ ID NO: 36] A. Sojae-derived epoxide hydrolase gene [SEQ ID NO: 37] AscB protein [SEQ ID NO: 38] AscC protein [SEQ ID NO: 39] AscD protein [SEQ ID NO: 40] AscE protein [SEQ ID NO: 41] Tr-ascC-Fw
[SEQ ID NO: 42] Tr-ascC-Rv
[SEQ ID NO: 43] Tr-ascC
[SEQ ID NO: 44] pyrG3

The gene, transformant, knockout organism and production method which are one embodiment of the present invention can be used to produce iricicholine A and ascochlorin in large quantities. Thus, the present invention can be used to produce Iricicolin A and Ascochlorin on an industrial scale.

Claims (10)

The gene ascF comprising any one of the following base sequences (1) to (5), the base sequence encoding an amino acid sequence of an enzyme having an activity of catalyzing an epoxidation reaction of iricicholine A:
(1) a base sequence shown in SEQ ID NO: 5 in the sequence listing or a base sequence that hybridizes with a base sequence complementary to the base sequence under stringent conditions (2) a gene comprising the base sequence shown in SEQ ID NO: 5 And a nucleotide sequence having 90% or more identity (3) a nucleotide sequence encoding an amino acid sequence of an enzyme having an activity of catalyzing an epoxidation reaction of iricicholine A (4) 90% or more with the amino acid sequence of SEQ ID NO: 12 (5) a base sequence encoding an amino acid sequence in which one or several amino acids of the amino acid sequence shown in SEQ ID NO: 12 have been deleted, substituted and / or added A gene ascG comprising any one of the following base sequences (1) to (5), the base sequence encoding an amino acid sequence of an enzyme having an activity of catalyzing a cyclization reaction of iricicholine A epoxide.
(1) a base sequence shown in SEQ ID NO: 6 in the sequence listing or a base sequence that hybridizes with a base sequence complementary to the base sequence under stringent conditions (2) a gene comprising the base sequence shown in SEQ ID NO: 6 And a nucleotide sequence having 90% identity or more (3) a nucleotide sequence encoding an amino acid sequence of an enzyme having an activity of catalyzing a cyclization reaction of iricicholine A epoxide (4) a nucleotide sequence of 90% Base sequence encoding amino acid sequence having the above identity (5) Base sequence encoding amino acid sequence in which one or several amino acids of SEQ ID NO: 13 have been deleted, substituted and / or added The base sequence of any of the following (1) to (5), which has an activity to catalyze a reaction for producing ascochlorin by dehydrogenation of a compound produced by the reaction of AscF protein and AscG protein from iricicholine A Gene ascH, comprising a base sequence encoding the amino acid sequence of
(1) A nucleotide sequence that hybridizes under stringent conditions with a nucleotide sequence set forth in SEQ ID NO: 7 in the sequence listing or a nucleotide sequence complementary to the nucleotide sequence. (2) A gene comprising the nucleotide sequence set forth in SEQ ID NO: 7. (3) An amino acid sequence of an enzyme having an activity of catalyzing a reaction for producing ascochlorin by dehydrogenation of a compound produced by the reaction of AscF protein and AscG protein from iricicholine A with 90% or more identity Encoding base sequence (4) Base sequence encoding amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 14 (5) One or several amino acids of the amino acid sequence shown in SEQ ID NO: 14 Base sequence encoding amino acid sequence deleted, substituted and / or added The gene ascE, comprising any one of the following base sequences (1) to (5), the base sequence encoding an amino acid sequence of an enzyme having an activity of catalyzing a reaction for producing iricicholine A from LL-Z1272β.
(1) a nucleotide sequence that is hybridized under stringent conditions with a nucleotide sequence shown in SEQ ID NO: 4 of the sequence listing or a nucleotide sequence complementary to the nucleotide sequence; (2) a gene comprising the nucleotide sequence shown in SEQ ID NO: 4 (3) a base sequence encoding an amino acid sequence of an enzyme having an activity of catalyzing a reaction for producing iricicholine A from LL-Z1272β (4) an amino acid sequence described in SEQ ID NO: 11 Nucleotide sequence encoding an amino acid sequence having 90% or more identity with (5) encoding an amino acid sequence in which one or several amino acids of the amino acid sequence set forth in SEQ ID NO: 11 have been deleted, substituted and / or added Base sequence A gene ascD comprising any one of the following base sequences (1) to (5), the base sequence encoding an amino acid sequence of an enzyme having an activity of catalyzing a reaction for producing o-orselic acid from acetyl CoA.
(1) a nucleotide sequence that hybridizes under stringent conditions with a nucleotide sequence set forth in SEQ ID NO: 3 in the sequence listing or a nucleotide sequence complementary to the nucleotide sequence; (2) a gene comprising the nucleotide sequence set forth in SEQ ID NO: 3 (3) A base sequence encoding an amino acid sequence of an enzyme having an activity of catalyzing a reaction for producing o-orselic acid from acetyl CoA (4) The amino acid described in SEQ ID NO: 10 A nucleotide sequence encoding an amino acid sequence having 90% or more identity with the sequence (5) encoding an amino acid sequence in which one or several amino acids of the amino acid sequence of SEQ ID NO: 10 have been deleted, substituted and / or added Base sequence The gene ascB comprising any one of the following base sequences (1) to (5), wherein the base sequence encodes an amino acid sequence of an enzyme having an activity of catalyzing a reaction for producing iricicolic acid B from o-orceric acid: .
(1) A nucleotide sequence that hybridizes under stringent conditions with a nucleotide sequence set forth in SEQ ID NO: 1 in the sequence listing or a nucleotide sequence complementary to the nucleotide sequence. (2) A gene comprising the nucleotide sequence set forth in SEQ ID NO: 1. (3) a nucleotide sequence encoding an amino acid sequence of an enzyme having an activity of catalyzing a reaction for producing iricicoline acid B from o-orceric acid (4) described in SEQ ID NO: 8 A base sequence encoding an amino acid sequence having 90% or more identity with the amino acid sequence (5) An amino acid sequence in which one or several amino acids of the amino acid sequence shown in SEQ ID NO: 8 have been deleted, substituted and / or added Base sequence to encode The gene ascC comprising any one of the following base sequences (1) to (5), wherein the base sequence encodes an amino acid sequence of an enzyme having an activity of catalyzing a reaction for producing LL-Z1272β from iricicolinic acid B.
(1) A nucleotide sequence that hybridizes under stringent conditions with a nucleotide sequence set forth in SEQ ID NO: 2 in the sequence listing or a nucleotide sequence complementary to the nucleotide sequence (2) A gene comprising the nucleotide sequence set forth in SEQ ID NO: 2 (3) a base sequence encoding an amino acid sequence of an enzyme having an activity of catalyzing a reaction for producing LL-Z1272β from iricicolinic acid B (4) an amino acid described in SEQ ID NO: 9 A nucleotide sequence encoding an amino acid sequence having 90% or more identity with the sequence (5) encoding an amino acid sequence in which one or several amino acids of the amino acid sequence of SEQ ID NO: 9 have been deleted, substituted and / or added Base sequence A gene of any one of the genes ascF, ascG, ascH, ascE, ascD, ascB and ascC according to claim 1 or a combination thereof is inserted and expresses the inserted gene , Transformants (except for humans). The manufacturing method of iricicholine A including the process of obtaining iricicholine A using the transformant of Claim 8. The manufacturing method of an ascochlorin including the process of obtaining an ascochlorin using the transformant of Claim 8.