WO2013031975A1

WO2013031975A1 - Method for producing intermediate for biosynthesis of fusicoccin a, and synthase for use in said method

Info

Publication number: WO2013031975A1
Application number: PCT/JP2012/072221
Authority: WO
Inventors: 徹大利; 修雄加藤; 基義野池; 祐介小野; 雄介樋口
Original assignee: Dairi Tohru; Kato Nobuo; Noike Motoyoshi; Ono Yusuke; Higuchi Yusuke
Priority date: 2011-08-31
Filing date: 2012-08-31
Publication date: 2013-03-07

Abstract

The present invention relates to: a method for producing an intermediate for the biosynthesis of fusicoccin A; an enzyme which can be used in the production method; and a product of the enzyme. According to the present invention, it becomes possible to obtain an enzyme capable of biosynthesizing fusicoccin A and an intermediate for the biosynthesis of fusicoccin A, and it also becomes possible to produce fusicoccin A and an intermediate for the biosynthesis of fusicoccin A using the obtained enzyme. Further, it also becomes possible to produce an intermediate for the biosynthesis of fusicoccin A with high efficiency using a filamentous fungus in which the enzyme is deleted.

Description

Method for producing biosynthetic intermediate of fusicoccin A and its synthase

The present invention relates to a method for producing a biosynthetic intermediate of fusicoccin A, an enzyme that can be used in the method, and a product thereof.

Fusicoccin (hereinafter, also simply referred to as “FC”) A is a diterpene glycoside produced by the phytopathogenic fungus Phomopsis amygdali . FC A exhibits plant hormone-like activity through the activation of cell membrane H ⁺ -ATPase. By analyzing the crystal structure of a ternary complex of a phosphorylated peptide derived from the C terminal of plant 14-3-3 protein, FC A and H ⁺ -ATPase, FC A was found to be of 14-3-3 protein and phosphorylated peptide. The association state is stabilized by the formation of a ternary complex, resulting in continuous activation of H ⁺ -ATPase (Non-patent Document 1). H ⁺ -ATPase plays an important role in plant growth regulation, and the activity of H ⁺ -ATPase is controlled by phosphorylation of threonine at the C-terminal, and when this threonine is phosphorylated, 14 Associates with 3-3 protein and activates H ⁺ -ATPase. When this complex of H ⁺ -ATPase and 14-3-3 protein is stabilized by FCA, it brings about physiological effects such as inducing germination of dormant seeds, opening of pores, and enlargement of cotyledons.

Therefore, the enzyme responsible for the synthesis of FC A and its gene are also important in the intentional growth regulation of plants. In addition, FC A is structurally similar to brassicene (hereinafter, also simply referred to as “BC”) C, etc., and other FC A structural analogs may be produced from the biosynthesis intermediate of FC A. It is considered possible.

Furthermore, the 14-3-3 protein is a protein that exists throughout eukaryotic organisms, and in mammals, it is known to be included in a part of a signal transduction pathway including a cancer-related factor. It is considered that FC A that binds to a 3-3 protein and its structural analogs have high utility value as pharmaceuticals.

It is believed that at least 13 enzymes are required for FCA biosynthesis. Previously, the inventors have described P.P. Genes including a part of genes of enzymes responsible for FCA synthesis from amygdali , genes encoding cyclase (ORF1), dioxygenase (ORF2), cytochrome P450-1 (ORF3) and short chain dehydrogenase / reductase (ORF4) Although the cluster (cluster 1) was identified (Non-patent document 2 and Non-patent document 3), the genes encoding the remaining nine enzymes have not yet been elucidated.

The present invention elucidates the entire biosynthetic pathway of FC A, obtains an enzyme that produces fusicoccin A and its biosynthetic intermediate, and produces fusicoccin A and its biosynthetic intermediate using the obtained enzyme. It aims to provide a way to do.

The “FC A biosynthetic intermediate” is a compound produced in the process of FC A biosynthesis. These compounds correspond to “manufacturing intermediate of FC A” in the manufacturing method of FC A of the present invention. Hereinafter, the term “FC A biosynthetic intermediate” is unified in this specification, but this also means “FC A production intermediate” and is not restricted by biosynthesis.

The present inventors have found that, in order to solve the above problems, was carried out extensive research, P. From the genome of amygdali , a new enzyme group considered to be involved in the biosynthesis pathway of FCA , specifically, four types of cytochrome P450 (hereinafter sometimes simply referred to as “P450”), one type. A gene cluster (cluster 2) was found that contained genes encoding glycosyltransferase, two acetyltransferases, one methyltransferase, and one prenyltransferase, respectively.

Based on this, the present inventors used four enzymes encoded in cluster 1 isolated earlier and nine enzymes identified this time, so that FCA was obtained using geranylgeranyl diphosphate as a starting material. Successfully synthesized. In addition, P. In amygdali , it was found that a compound serving as a substrate for the enzyme can be efficiently biosynthesized in the microbial cells by deleting a gene encoding a part of the enzyme and blocking the enzyme reaction. The present invention has been completed on the basis of these series of findings, and includes the inventions of the embodiments listed below.

(1) Method for producing fusicoccin A (FC A) and its biosynthetic intermediate (1-1). Formula (I) comprising the following steps (a) to (g):

A method for producing fusicoccin H (hereinafter also referred to as “FC H”) represented by:
(A). Cyclase (ORF1) is allowed to act on geranylgeranyl diphosphate (hereinafter also referred to as “GGDP”) to produce a compound represented by the following formula (II) (hereinafter referred to as “fusicocca-2,10 (14) -diene”). The process of obtaining

(B). P450-2 (ORF5) is allowed to act on the compound (II) obtained above in the presence of P450 reductase and nicotinamide adenine dinucleotide (NADH) or nicotinamide adenine dinucleotide phosphate (NADPH). A step of obtaining a compound represented by (III) (hereinafter also referred to as “fusicocca-2,10 (14) -dien-8β-ol”);

(C). The compound (III) obtained above is reacted with P450 reductase and NADH or NADPH, P450-1 (ORF3) to give a compound represented by the following formula (IV) (hereinafter referred to as “fusicocca-2,10 (14 ) -Diene-8β, 16-diol ”).

(D). Compound (IV) obtained above is reacted with dioxygenase (ORF2) in the presence of an iron ion, a diatomic oxygen donor, and ascorbic acid to give a compound represented by the following formula (V) or (VI) ( A step of obtaining the compound (VI) hereinafter also referred to as “8β-hydroxyfusicocca-1,10 (14) -dien-16-al”);

(E). The compound (VI) obtained above is allowed to act on a short-chain dehydrogenase reductase (ORF4) to produce a compound represented by the following formula (VII) (hereinafter referred to as “fusicocca-1,10 (14) -diene-8β, 16- also referred to as “diol”),

(F). The compound (VII) obtained above is allowed to act on P450-3 (ORF7) in the presence of P450 reductase and NADH or NADPH to obtain compound (VIII) (hereinafter also referred to as “FC H aglycon”). Process,

(G). A step of obtaining a compound (FC H) represented by the above formula (I) by allowing a glycosyltransferase (ORF6) to act on the compound (VIII) obtained above in the presence of a glycosyl group donor.

(1-2). In addition to the steps (a) to (g) described in (1-1), the fusicoccin Q represented by the formula (IX) further including the following step (h) (hereinafter also referred to as “FC Q”) .) Manufacturing method:
(H). (G) P450-4 (ORF10) is allowed to act on the compound (I) obtained in the step in the presence of P450 reductase and NADH or NADPH to give the compound (FC Q) represented by the following formula (IX): Obtaining process

.

(1-3). In addition to the steps (a) to (g) described in (1-1) and the step (h) described in (1-2), the method is further represented by the formula (X) including the step (i). Method for producing fusicoccin P (hereinafter also referred to as “FC P”):
(I). (H) A step of obtaining a compound (FCP) represented by the following formula (X) by allowing methyltransferase (ORF8) to act on the compound (IX) obtained in the step in the presence of a methyl group donor.

.

(1-4). In addition to steps (a) to (g) described in (1-1), step (h) described in (1-2), and step (i) described in (1-3), (j ) Process for producing fusicoccin J represented by formula (XI) (hereinafter also referred to as “FC J”):
(J). (I) A step of obtaining a compound (FC J) represented by the following formula (XI) by allowing prenyl transferase (ORF11) to act on the compound (X) obtained in the step in the presence of a prenyl group donor:

.

(1-5). Steps (a) to (g) described in (1-1), step (h) described in (1-2), step (i) described in (1-3), and (1-4) A method for producing dideacetyl fusicoccin A represented by formula (XII) (hereinafter also referred to as “deacetylyl-FC A”), which further comprises step (k) in addition to the step (j) described. :
(K). (J) The compound (XI) obtained in the step is allowed to act on P450-5 (ORF13) in the presence of P450 reductase and NADH or NADPH to give a compound represented by the following formula (XII) (deacetylyl-FC A )

.

(1-6). Steps (a) to (g) described in (1-1), Step (h) described in (1-2), Step (i) described in (1-3), Step (1-4) In addition to step (j) and step (k) described in (1-5), fusicoccine A represented by formula (XIII) (hereinafter referred to as “FC A”) Also known as :) Manufacturing method:
(L). (K) A step of obtaining a compound (FC A) represented by the following formula (XIII) by allowing two kinds of acetyltransferases ORF9 and ORF12 to act on the compound (XII) obtained in the step in the presence of acetyl CoA:

.

(1-7). P450-2 (ORF5) used in step (b) is an enzyme encoded by the base sequence described in any one of (1) to (3), or any one of (4) to (6) A method for producing FCA or a biosynthetic intermediate thereof according to any one of (1-1) to (1-6), which is an enzyme comprising an amino acid sequence:
(1) the base sequence shown in SEQ ID NO: 1,
(2) having a base sequence that hybridizes with a complementary strand of the base sequence shown in SEQ ID NO: 1 under stringent conditions, and similar to the gene product encoded by the base sequence shown in SEQ ID NO: 1, P450 reductase and NADH or A base sequence encoding a protein having an enzyme action that catalyzes the reaction of step (b) in the presence of NADPH;
(3) In the nucleotide sequence shown in SEQ ID NO: 1, P450 has a nucleotide sequence in which one or more bases are substituted, deleted, or added, and is the same as the gene product encoded by the nucleotide sequence shown in SEQ ID NO: 1. A base sequence encoding a protein having an enzyme action that catalyzes the reaction of step (b) in the presence of a reductase and NADH or NADPH, or (4) an amino acid sequence shown in SEQ ID NO: 2,
(5) The reaction of step (b) in the presence of P450 reductase and NADH or NADPH, which is 85% or more identical to the amino acid sequence shown in SEQ ID NO: 2 and consists of the amino acid sequence of SEQ ID NO: 2 An amino acid sequence encoding a protein having an enzyme action that catalyzes or (6) an amino acid sequence represented by SEQ ID NO: 2 having an amino acid sequence in which one or more amino acids are substituted, deleted, or added; The amino acid sequence which codes the protein which has the enzyme effect | action which catalyzes reaction of a process (b) in presence of P450 reductase and NADH or NADPH similarly to the enzyme which consists of an amino acid sequence of 2.

(1-8). The P450-3 (ORF7) used in the step (f) is an enzyme encoded by the base sequence described in any of (7) to (9) or the enzyme described in any of (10) to (12) A method for producing FCA or a biosynthetic intermediate thereof according to any one of (1-1) to (1-6), which is an enzyme comprising an amino acid sequence:
(7) the base sequence shown in SEQ ID NO: 5,
(8) having a base sequence that hybridizes with a complementary strand of the base sequence shown in SEQ ID NO: 5 under stringent conditions, and similar to the gene product encoded by the base sequence shown in SEQ ID NO: 5, P450 reductase and NADH or In the presence of NADPH, a base sequence encoding a protein having an enzyme action that catalyzes the reaction of step (f), or (9) one or more bases substituted or deleted in the base sequence shown in SEQ ID NO: 5, Alternatively, it has an added base sequence and has an enzyme action that catalyzes the reaction of step (f) in the presence of P450 reductase and NADH or NADPH, as in the gene product encoded by the base sequence shown in SEQ ID NO: 5. A base sequence encoding a protein, or (10) an amino acid sequence shown in SEQ ID NO: 6,
(11) In the presence of P450 reductase and NADH or NADPH, which is 85% or more identical to the amino acid sequence shown in SEQ ID NO: 6 and consists of the amino acid sequence shown in SEQ ID NO: 6, in the step (f) An amino acid sequence encoding a protein having an enzyme action that catalyzes a reaction, or (12) an amino acid sequence represented by SEQ ID NO: 6 having an amino acid sequence in which one or more amino acids are substituted, deleted, or added; The amino acid sequence which codes the protein which has the enzyme effect | action which catalyzes reaction of a process (f) in presence of P450 reductase and NADH or NADPH similarly to the enzyme which consists of an amino acid sequence shown by the number 6.

(1-9). The glycosyltransferase (ORF6) used in the step (g) is an enzyme encoded by the base sequence according to any one of (13) to (15), or any one of (16) to (18) A method for producing FCA or a biosynthetic intermediate thereof according to any one of (1-1) to (1-6), which is an enzyme comprising an amino acid sequence:
(13) the base sequence shown in SEQ ID NO: 3,
(14) Presence of a glycosyl group donor having a base sequence that hybridizes with a complementary strand of the base sequence shown in SEQ ID NO: 3 under stringent conditions, as in the gene product encoded by the base sequence shown in SEQ ID NO: 3. Below, in the base sequence encoding a protein having an enzyme action that catalyzes the reaction of step (g), or (15) one or more bases are substituted, deleted, or added in the base sequence shown in SEQ ID NO: 3. A base sequence encoding a protein having a base sequence and having the enzyme action of catalyzing the reaction of step (g) in the presence of a glycosyl group donor, in the same manner as the gene product encoded by the base sequence shown in SEQ ID NO: 3; Or (16) the amino acid sequence shown in SEQ ID NO: 4,
(17) Catalyzing the reaction of step (g) in the presence of a glycosyl group donor having the identity of 85% or more to the amino acid sequence shown in SEQ ID NO: 4 and the enzyme consisting of the amino acid sequence shown in SEQ ID NO: 4 An amino acid sequence encoding a protein having an enzyme action, or (18) an amino acid sequence represented by SEQ ID NO: 4, wherein one or a plurality of amino acids are substituted, deleted, or added; The amino acid sequence which codes the protein which has the enzyme action which catalyzes reaction of a process (g) similarly to the enzyme which consists of an amino acid sequence shown in presence of a glycosyl group donor.

(1-10). P450-4 (ORF10) used in the step (h) is an enzyme encoded by the base sequence described in any one of (19) to (21), or any one of (22) to (24) A method for producing FCA or a biosynthetic intermediate thereof according to any one of (1-1) to (1-6), which is an enzyme comprising the amino acid sequence:
(19) the base sequence shown in SEQ ID NO: 11,
(20) having a base sequence that hybridizes under stringent conditions with a complementary strand of the base sequence shown in SEQ ID NO: 11 and, like the gene product encoded by the base sequence shown in SEQ ID NO: 11, P450 reductase and NADH or In the presence of NADPH, in the base sequence encoding a protein having an enzyme action that catalyzes the reaction of step (h), or (21) one or more bases are substituted, deleted, or deleted in the base sequence shown in SEQ ID NO: 11, or A protein having an added base sequence and having an enzyme action that catalyzes the reaction of step (h) in the presence of P450 reductase and NADH or NADPH, as in the gene product encoded by the base sequence shown in SEQ ID NO: 11 (22) the amino acid sequence shown in SEQ ID NO: 12,
(23) In the presence of P450 reductase and NADH or NADPH, which has 85% or more identity with the amino acid sequence shown in SEQ ID NO: 12 and consists of the amino acid sequence shown in SEQ ID NO: 12, An amino acid sequence encoding a protein having an enzyme action that catalyzes a reaction, or (24) an amino acid sequence represented by SEQ ID NO: 12 having an amino acid sequence in which one or more amino acids are substituted, deleted, or added; The amino acid sequence which codes the protein which has the enzyme effect | action which catalyzes reaction of a process (h) in presence of P450 reductase and NADH or NADPH similarly to the enzyme which consists of an amino acid sequence shown in number 12.

(1-11). The methyltransferase (ORF8) in step (i) is an enzyme encoded by the base sequence described in any one of (25) to (27) or the amino acid sequence described in any one of (28) to (30). A method for producing FCA or a biosynthetic intermediate thereof according to any one of (1-1) to (1-6):
(25) the base sequence shown in SEQ ID NO: 7,
(26) having a base sequence that hybridizes with a complementary strand of the base sequence shown in SEQ ID NO: 7 under stringent conditions, and in the presence of a methyl group donor in the same manner as the gene product encoded by the base sequence of SEQ ID NO: 7 , A base sequence encoding a protein having an enzyme action that catalyzes the reaction of step (i), or (27) a base in which one or more bases are substituted, deleted, or added in the base sequence shown in SEQ ID NO: 7 A nucleotide sequence that encodes a protein having an enzyme action that catalyzes the reaction of step (i) in the presence of a methyl group donor, as in the gene product having the sequence and encoded by the nucleotide sequence shown in SEQ ID NO: 7; (28) the amino acid sequence shown in SEQ ID NO: 8,
(29) Catalyzing the reaction of step (i) in the presence of a methyl donor, having 85% or more identity with the amino acid sequence shown in SEQ ID NO: 8 and the enzyme consisting of the amino acid sequence shown in SEQ ID NO: 8 An amino acid sequence encoding a protein having an enzyme action, or (30) an amino acid sequence represented by SEQ ID NO: 8, wherein one or a plurality of amino acids are substituted, deleted, or added; The amino acid sequence which codes the protein which has the enzyme effect | action which catalyzes reaction of a process (i) similarly to the enzyme which consists of an amino acid sequence shown in presence of a methyl group donor.

(1-12). The prenyl transferase (ORF11) used in the step (j) is an enzyme encoded by the base sequence according to any of (31) to (33), or any of (34) to (36) A method for producing FCA or a biosynthetic intermediate thereof according to any one of (1-1) to (1-6), which is an enzyme comprising an amino acid sequence:
(31) the base sequence shown in SEQ ID NO: 13,
(32) having a base sequence that hybridizes with a complementary strand of the base sequence shown in SEQ ID NO: 13 under stringent conditions, and in the presence of a prenyl group donor in the same manner as the gene product encoded by the base sequence of SEQ ID NO: 13 , A base sequence encoding a protein having an enzyme action that catalyzes the reaction of step (j), or (33) a base in which one or more bases are substituted, deleted, or added in the base sequence shown in SEQ ID NO: 13 A nucleotide sequence encoding a protein having an enzyme action that catalyzes the reaction of the step (j) in the presence of a prenyl group donor, as in the gene product having the sequence and encoded by the nucleotide sequence of SEQ ID NO: 13; 34) the amino acid sequence shown in SEQ ID NO: 14,
(35) Catalyzing the reaction of the step (j) in the presence of a prenyl group donor having the identity of 85% or more to the amino acid sequence shown in SEQ ID NO: 14 and the enzyme consisting of the amino acid sequence shown in SEQ ID NO: 14 An amino acid sequence encoding a protein having an enzyme action, (36) having an amino acid sequence in which one or more amino acids are substituted, deleted, or added in the amino acid sequence shown in SEQ ID NO: 14, and shown in SEQ ID NO: 14 The amino acid sequence which codes the protein which has the enzyme effect | action which catalyzes reaction of a process (j) in presence of a prenyl group donor similarly to the enzyme which consists of an amino acid sequence.

(1-13). The enzyme encoded by the nucleotide sequence according to any of (37) to (39), or the amino acid sequence according to any of (40) to (42), wherein P450-5 (ORF13) in step (k) A method for producing FCA or a biosynthetic intermediate thereof according to any one of (1-1) to (1-6), which is an enzyme comprising:
(37) the base sequence shown in SEQ ID NO: 17,
(38) having a base sequence that hybridizes under stringent conditions with a complementary strand of the base sequence shown in SEQ ID NO: 17, and, like the gene product encoded by the base sequence shown in SEQ ID NO: 17, P450 reductase and NADH or In the presence of NADPH, in the base sequence encoding a protein having an enzyme action that catalyzes the reaction of step (k), or (39) the base sequence shown in SEQ ID NO: 17, one or more bases are substituted, deleted, or A protein having an added base sequence and having an enzyme action that catalyzes the reaction of step (k) in the presence of P450 reductase and NADH or NADPH, in the same manner as the gene product encoded by the base sequence shown in SEQ ID NO: 17. (40) the amino acid sequence shown in SEQ ID NO: 18,
(41) In the presence of P450 reductase and NADH or NADPH in the presence of P450 reductase and NADH or NADPH having the identity of 85% or more to the amino acid sequence shown in SEQ ID NO: 18 and consisting of the amino acid sequence shown in SEQ ID NO: 18 An amino acid sequence encoding a protein having an enzyme action that catalyzes a reaction, or (42) an amino acid sequence represented by SEQ ID NO: 18 having an amino acid sequence in which one or more amino acids are substituted, deleted, or added; The amino acid sequence which codes the protein which has the enzyme action which catalyzes reaction of a process (k) in presence of P450 reductase and NADH or NADPH similarly to the enzyme which consists of an amino acid sequence shown in number 18.

(1-14). From the enzyme encoded by the base sequence described in any of (43) to (45) or the amino acid sequence described in any of (46) to (48), the acetyltransferase (ORF12) in step (l) A method for producing FCA or a biosynthetic intermediate thereof according to any one of (1-1) to (1-6):
(43) the base sequence represented by SEQ ID NO: 15,
(44) having a base sequence that hybridizes with a complementary strand of the base sequence shown in SEQ ID NO: 15 under stringent conditions, and in the presence of an acetyl donor in the same manner as the gene product encoded by the base sequence shown in SEQ ID NO: 15 , A base sequence encoding a protein having an enzyme action that catalyzes acetylation of the 19th hydroxyl group of compound (XII), or (45) one or more bases substituted or deleted in the base sequence shown in SEQ ID NO: 15 Or an enzyme that catalyzes the acetylation of the hydroxyl group at position 19 of compound (XII) in the presence of an acetyl donor in the same manner as the gene product encoded by the nucleotide sequence shown in SEQ ID NO: 15 A base sequence encoding a protein having an action, or (46) an amino acid sequence shown in SEQ ID NO: 16,
(47) Acetyl of the hydroxyl group at position 19 of compound (XII) in the presence of an acetyl donor having the identity of 85% or more to the amino acid sequence shown in SEQ ID NO: 16 and the same as the enzyme encoded by SEQ ID NO: 16 An amino acid sequence encoding a protein having an enzyme action that catalyzes oxidization, or (48) an amino acid sequence represented by SEQ ID NO: 16, wherein one or a plurality of amino acids are substituted, deleted, or added; The amino acid sequence which codes the protein which has the enzyme effect | action which catalyzes the acetylation of the 19th hydroxyl group of compound (XII) in presence of an acetyl donor similarly to the enzyme which consists of an amino acid sequence shown in number 14.

(1-15). The acetyltransferase (ORF9) in step (l) is an enzyme encoded by the base sequence described in any of (49) to (51) or an enzyme consisting of the amino acid sequence described in (52) to (54) A method for producing FC A or a biosynthetic intermediate thereof according to any one of (1-1) to (1-6):
(49) the base sequence represented by SEQ ID NO: 9,
(50) having a base sequence that hybridizes with a complementary strand of the base sequence shown in SEQ ID NO: 9 under stringent conditions, and in the presence of an acetyl donor in the same manner as the gene product encoded by the base sequence shown in SEQ ID NO: 9 Or a base sequence encoding a protein having an enzyme action that catalyzes the acetylation of the 3′-position hydroxyl group of the compound of formula (XII), or (51) one or more bases substituted in the base sequence shown in SEQ ID NO: 9 In the presence of an acetyl donor in the presence of an acetyl donor, the hydroxyl group at the 3 ′ position of the compound of formula (XII), which has a deleted or added base sequence and is encoded by the base sequence shown in SEQ ID NO: 9. A base sequence encoding a protein having an enzyme action that catalyzes acetylation, or (52) an amino acid sequence shown in SEQ ID NO: 10,
(53) 3 of the compound of formula (XII) in the presence of an acetyl donor in the same manner as the enzyme having 85% or more identity with the amino acid sequence shown in SEQ ID NO: 10 and consisting of the amino acid sequence shown in SEQ ID NO: 10 An amino acid sequence encoding a protein having an enzyme action that catalyzes acetylation of the hydroxyl group at the position, or (54) an amino acid in which one or more amino acids are substituted, deleted, or added in the amino acid sequence shown in SEQ ID NO: 10 A protein having an enzyme action that catalyzes the acetylation of the hydroxyl group at the 3 ′ position of the compound of formula (XII) in the presence of an acetyl donor in the same manner as the enzyme having the sequence and the amino acid sequence shown in SEQ ID NO: 10 Amino acid sequence to encode.

(2) Enzymes and their genes used in the production of FC A and its biosynthetic intermediates (2-1). A gene encoding P450-2 (ORF5) comprising the base sequence according to any one of (1) to (3):
(1) the base sequence shown in SEQ ID NO: 1,
(2) having a base sequence that hybridizes with a complementary strand of the base sequence shown in SEQ ID NO: 1 under stringent conditions, and similar to the gene product encoded by the base sequence shown in SEQ ID NO: 1, P450 reductase and NADH or A base sequence encoding a protein having an enzyme action that catalyzes the reaction of step (b) in the presence of NADPH;
(3) In the nucleotide sequence shown in SEQ ID NO: 1, P450 has a nucleotide sequence in which one or more bases are substituted, deleted, or added, and is the same as the gene product encoded by the nucleotide sequence shown in SEQ ID NO: 1. A base sequence encoding a protein having an enzyme action that catalyzes the reaction of step (b) in the presence of a reductase and NADH or NADPH.

(2-2). P450-2 (ORF5) comprising the amino acid sequence according to any one of (4) to (6):
(4) the amino acid sequence shown in SEQ ID NO: 2,
(5) The reaction of step (b) in the presence of P450 reductase and NADH or NADPH, which is 85% or more identical to the amino acid sequence shown in SEQ ID NO: 2 and consists of the amino acid sequence of SEQ ID NO: 2 An amino acid sequence encoding a protein having an enzyme action that catalyzes or (6) an amino acid sequence represented by SEQ ID NO: 2 having an amino acid sequence in which one or more amino acids are substituted, deleted, or added; The amino acid sequence which codes the protein which has the enzyme effect | action which catalyzes reaction of a process (b) in presence of P450 reductase and NADH or NADPH similarly to the enzyme which consists of an amino acid sequence of 2.

(2-3). A gene encoding P450-3 (ORF7), comprising the base sequence according to any one of (7) to (9):
(7) the base sequence shown in SEQ ID NO: 5,
(8) having a base sequence that hybridizes with a complementary strand of the base sequence shown in SEQ ID NO: 5 under stringent conditions, and, like the gene product encoded by the base sequence shown in SEQ ID NO: 5, P450 reductase and NADH or A base sequence encoding a protein having an enzyme action that catalyzes the reaction of step (f) in the presence of NADPH;
(9) In the base sequence shown in SEQ ID NO: 5, the base sequence has one or more bases substituted, deleted, or added, and is the same as the gene product encoded by the base sequence shown in SEQ ID NO: 5, A base sequence encoding a protein having an enzyme action that catalyzes the reaction of step (f) in the presence of P450 reductase and NADH or NADPH.

(2-4). P450-3 (ORF7) comprising the amino acid sequence according to any one of (10) to (12):
(10) the amino acid sequence shown in SEQ ID NO: 6,
(11) In the presence of P450 reductase and NADH or NADPH, which has 85% or more identity with the amino acid sequence shown in SEQ ID NO: 6 and consists of the amino acid sequence shown in SEQ ID NO: 6, An amino acid sequence encoding a protein having an enzymatic action to catalyze the reaction;
(12) A P450 reductase having the same amino acid sequence as that shown in SEQ ID NO: 6, wherein the amino acid sequence shown in SEQ ID NO: 6 has an amino acid sequence in which one or more amino acids are substituted, deleted, or added. And an amino acid sequence encoding a protein having an enzyme action that catalyzes the reaction of step (f) in the presence of NADH or NADPH.

(2-5). A gene encoding a glycosyltransferase (ORF6) comprising the base sequence according to any one of (13) to (15):
(13) the base sequence shown in SEQ ID NO: 3,
(14) Presence of a glycosyl group donor having a base sequence that hybridizes with a complementary strand of the base sequence shown in SEQ ID NO: 3 under stringent conditions, as in the gene product encoded by the base sequence shown in SEQ ID NO: 3. A base sequence encoding a protein having an enzyme action that catalyzes the reaction in the step (g)
(15) In the base sequence shown in SEQ ID NO: 3, a glycosyl group having the base sequence in which one or more bases are substituted, deleted, or added, and the gene product encoded by the base sequence of SEQ ID NO: 3 A base sequence encoding a protein having an enzyme action that catalyzes the reaction of step (g) in the presence of a donor.

(2-6). Glycosyltransferase (ORF6) comprising the amino acid sequence according to any one of (16) to (18):
(16) the amino acid sequence shown in SEQ ID NO: 4,
(17) Catalyzing the reaction of step (g) in the presence of a glycosyl group donor having the identity of 85% or more to the amino acid sequence shown in SEQ ID NO: 4 and the enzyme consisting of the amino acid sequence shown in SEQ ID NO: 4 An amino acid sequence encoding a protein having an enzyme action,
(18) A glycosyl group donor having an amino acid sequence in which one or more amino acids are substituted, deleted, or added in the amino acid sequence shown in SEQ ID NO: 4 and consisting of the amino acid sequence shown in SEQ ID NO: 4 An amino acid sequence encoding a protein having an enzyme action that catalyzes the reaction of step (g) in the presence of.

(2-7). (19) A gene encoding P450-4 (ORF10) comprising the base sequence according to any one of (21):
(19) the base sequence shown in SEQ ID NO: 11,
(20) A P450 reductase and NADH or NADPH having a base sequence that hybridizes with a complementary strand of the base sequence shown in SEQ ID NO: 11 under stringent conditions and the same as the gene product encoded by the base sequence of SEQ ID NO: 11. A base sequence encoding a protein having an enzyme action that catalyzes the reaction of step (h) in the presence of
(21) The base sequence shown in SEQ ID NO: 11 has a base sequence in which one or more bases are substituted, deleted, or added, and is reduced by P450 as in the gene product encoded by the base sequence of SEQ ID NO: 11. A base sequence encoding a protein having an enzyme action that catalyzes the reaction of step (h) in the presence of an enzyme and NADH or NADPH.

(2-8). P450-4 (ORF10) comprising the amino acid sequence according to any one of (22) to (24):
(22) the amino acid sequence shown in SEQ ID NO: 12,
(23) In the presence of P450 reductase and NADH or NADPH, which has 85% or more identity with the amino acid sequence shown in SEQ ID NO: 12 and consists of the amino acid sequence shown in SEQ ID NO: 12, An amino acid sequence encoding a protein having an enzymatic action to catalyze the reaction;
(24) The amino acid sequence shown in SEQ ID NO: 12 has an amino acid sequence in which one or more amino acids are substituted, deleted, or added, and, like the enzyme consisting of the amino acid sequence shown in SEQ ID NO: 12, P450 reductase and An amino acid sequence encoding a protein having an enzyme action that catalyzes the reaction of step (h) in the presence of NADH or NADPH.

(2-9). A gene encoding methyltransferase (ORF8) comprising the base sequence according to any of (25) to (27):
(25) the base sequence shown in SEQ ID NO: 7,
(26) Presence of a methyl group donor having a base sequence that hybridizes with a complementary strand of the base sequence shown in SEQ ID NO: 7 under stringent conditions and the gene product encoded by the base sequence shown in SEQ ID NO: 7 Below, a base sequence encoding a protein having an enzyme action that catalyzes the reaction of step (i),
(27) In the base sequence shown in SEQ ID NO: 7, a base sequence in which one or more bases are substituted, deleted, or added, and methyl as in the gene product encoded by the base sequence shown in SEQ ID NO: 7 A base sequence encoding a protein having an enzyme action that catalyzes the reaction of step (i) in the presence of a group donor.

(2-10). Methyltransferase (ORF8) comprising the amino acid sequence according to any of (28) to (30):
(28) the amino acid sequence shown in SEQ ID NO: 8,
(29) Catalyzing the reaction of step (i) in the presence of a methyl donor, having 85% or more identity with the amino acid sequence shown in SEQ ID NO: 8 and the enzyme consisting of the amino acid sequence shown in SEQ ID NO: 8 An amino acid sequence encoding a protein having an enzyme action,
(30) A methyl group donor having an amino acid sequence in which one or a plurality of amino acids are substituted, deleted, or added in the amino acid sequence shown in SEQ ID NO: 8 and comprising the amino acid sequence shown in SEQ ID NO: 8 An amino acid sequence encoding a protein having an enzyme action that catalyzes the reaction of step (i) in the presence of.

(2-11). A gene encoding prenyltransferase (ORF11) comprising the base sequence according to any of (31) to (33):
(31) the base sequence shown in SEQ ID NO: 13,
(32) having a base sequence that hybridizes with a complementary strand of the base sequence shown in SEQ ID NO: 13 under stringent conditions, and in the presence of a prenyl group donor in the same manner as the gene product encoded by the base sequence in SEQ ID NO: 13 , A base sequence encoding a protein having an enzyme action that catalyzes the reaction of step (j),
(33) In the base sequence shown in SEQ ID NO: 13, a prenyl group has a base sequence in which one or more bases are substituted, deleted, or added, and is the same as the gene product encoded by the base sequence of SEQ ID NO: 13 A base sequence encoding a protein having an enzyme action that catalyzes the reaction of step (j) in the presence of a donor.

(2-12). (34) to (36) prenyltransferase (ORF11) comprising the amino acid sequence according to any one of the above:
(34) the amino acid sequence shown in SEQ ID NO: 14,
(35) Catalyzing the reaction of the step (j) in the presence of a prenyl group donor having the identity of 85% or more to the amino acid sequence shown in SEQ ID NO: 14 and the enzyme consisting of the amino acid sequence shown in SEQ ID NO: 14 An amino acid sequence encoding a protein having an enzyme action,
(36) A prenyl group donor having an amino acid sequence in which one or more amino acids are substituted, deleted, or added in the amino acid sequence shown in SEQ ID NO: 14, and the enzyme comprising the amino acid sequence shown in SEQ ID NO: 14 An amino acid sequence encoding a protein having an enzyme action that catalyzes the reaction of step (j) in the presence of.

(2-13). (450) (ORF13) comprising the base sequence according to any of (37) to (39):
(37) the base sequence shown in SEQ ID NO: 17,
(38) having a base sequence that hybridizes under stringent conditions with a complementary strand of the base sequence shown in SEQ ID NO: 17, and, like the gene product encoded by the base sequence shown in SEQ ID NO: 17, P450 reductase and NADH or A base sequence encoding a protein having an enzyme action that catalyzes the reaction of step (k) in the presence of NADPH;
(39) The base sequence shown in SEQ ID NO: 17 has a base sequence in which one or more bases are substituted, deleted, or added, and is reduced by P450 as in the gene product encoded by the base sequence of SEQ ID NO: 17. A base sequence encoding a protein having an enzyme action that catalyzes the reaction of step (k) in the presence of an enzyme and NADH or NADPH.

(2-14). P450-5 (ORF13) comprising the amino acid sequence according to any one of (40) to (42):
(40) the amino acid sequence shown in SEQ ID NO: 18,
(41) In the presence of P450 reductase and NADH or NADPH in the presence of P450 reductase and NADH or NADPH having 85% or more identity with the amino acid sequence shown in SEQ ID NO: 18, An amino acid sequence encoding a protein having an enzymatic action to catalyze the reaction;
(42) The amino acid sequence shown in SEQ ID NO: 18 has an amino acid sequence in which one or more amino acids are substituted, deleted, or added, and, like the enzyme consisting of the amino acid sequence shown in SEQ ID NO: 18, P450 reductase and An amino acid sequence encoding a protein having an enzyme action that catalyzes the reaction of step (k) in the presence of NADH or NADPH.

(2-15). (43) A gene encoding acetyltransferase (ORF12) comprising the base sequence according to any one of (45):
(43) the base sequence represented by SEQ ID NO: 15,
(44) having a base sequence that hybridizes with a complementary strand of the base sequence shown in SEQ ID NO: 15 under stringent conditions, and in the presence of an acetyl donor in the same manner as the gene product encoded by the base sequence shown in SEQ ID NO: 15 , A base sequence encoding a protein having an enzyme action that catalyzes the acetylation of the 19th hydroxyl group of compound (XII),
(45) In the base sequence shown in SEQ ID NO: 15, one or more bases have a base sequence substituted, deleted, or added, and acetylated like the gene product encoded by the base sequence shown in SEQ ID NO: 15. A base sequence encoding a protein having an enzyme action that catalyzes the acetylation of the 19th hydroxyl group of compound (XII) in the presence of a donor.

(2-16). (46) to (48) acetyltransferase (ORF12) comprising the amino acid sequence according to any one of:
(46) the amino acid sequence shown in SEQ ID NO: 16,
(47) having the identity of 85% or more with the amino acid sequence shown in SEQ ID NO: 16 and, like the enzyme encoded by SEQ ID NO: 16, in the presence of an acetyl group donor, the hydroxyl group at position 19 of compound (XII) An amino acid sequence encoding a protein having an enzymatic action to catalyze acetylation,
(48) An acetyl group donor having the same amino acid sequence as that shown in SEQ ID NO: 14, wherein the amino acid sequence shown in SEQ ID NO: 16 has an amino acid sequence in which one or more amino acids are substituted, deleted, or added. An amino acid sequence encoding a protein having an enzyme action that catalyzes the acetylation of the 19th hydroxyl group of compound (XII) in the presence of

(2-17). (49) A gene encoding acetyltransferase (ORF9) comprising the base sequence according to any one of (51):
(49) the base sequence represented by SEQ ID NO: 9,
(50) having a base sequence that hybridizes with a complementary strand of the base sequence shown in SEQ ID NO: 9 under stringent conditions, and in the presence of an acetyl group donor in the same manner as the gene product encoded by the base sequence of SEQ ID NO: 9 A base sequence encoding a protein having an enzyme action that catalyzes the acetylation of the hydroxyl group at the 3′-position of the compound of formula (XII),
(51) In the base sequence shown in SEQ ID NO: 9, one or more bases have a base sequence substituted, deleted, or added, and the acetyl group is the same as the gene product encoded by the base sequence of SEQ ID NO: 9. A base sequence encoding a protein having an enzyme action that catalyzes the acetylation of the hydroxyl group at the 3'-position of a compound of formula (XII) in the presence of a donor.

(2-18). Acetyltransferase (ORF9) comprising the amino acid sequence according to any of (52) to (54):
(52) the amino acid sequence shown in SEQ ID NO: 10,
(53) In the presence of an acetyl group donor, the compound of the formula (XII) has the identity of 85% or more to the amino acid sequence shown in SEQ ID NO: 10 and consists of the amino acid sequence shown in SEQ ID NO: 10 An amino acid sequence encoding a protein having an enzymatic action to catalyze acetylation of the hydroxyl group at the 3 ′ position,
(54) The amino acid sequence shown in SEQ ID NO: 10 has an amino acid sequence in which one or more amino acids are substituted, deleted, or added, and provides an acetyl group as in the enzyme consisting of the amino acid sequence shown in SEQ ID NO: 10 An amino acid sequence encoding a protein having an enzyme action that catalyzes the acetylation of the 3′-position hydroxyl group of a compound of the formula (XII) in the presence of a body.

(3) fungi used in the preparation of biosynthetic intermediates of FC A (3-1). P450-2 (ORF5), glycosyltransferase (ORF6), P450-3 (ORF7), methyltransferase (ORF8), acetyltransferase (ORF9), P450-4 (ORF10), prenyltransferase (ORF11), acetyltransferase (ORF12) And a filamentous fungus that lacks any one of the enzyme-producing ability selected from the group consisting of P450-5 (ORF13).
(3-2). A filamentous fungus lacking the ability to produce any one of ORF10 or ORF13.
(3-3). The filamentous fungus according to (3-1) or (3-2), wherein the filamentous fungus is derived from Pomopsis amygdali .

According to the present invention, it is possible to obtain an enzyme that produces fusicoccin A and its biosynthetic intermediate, and to produce fusicoccin A and its biosynthetic intermediate using the obtained enzyme. In addition, since FC A is structurally similar to BC C and the like, it is also possible to produce an analog such as BC C from a biosynthetic intermediate of FC A.

In particular, when trying to obtain a useful compound by developing the structure of a compound having no substituent at the 19-position, FC J is more useful than FC A, and the structure of a compound having no hydroxyl group at the 12-position For this, FC H is useful.

In addition, P450-2 (ORF5), glycosyltransferase (ORF6), P450-3 (ORF7), methyltransferase (ORF8), acetyltransferase (ORF9), P450-4 (ORF10), prenyltransferase (ORF11), acetyltransferase ( Filamentous fungi lacking any one of the enzyme-producing ability selected from the group consisting of ORF12) and P450-5 (ORF13) are useful for obtaining a biosynthesis intermediate of FC A. Among them, filamentous fungi lacking the enzyme producing ability of P450-4 (ORF10) or P450-5 (ORF13) are useful because they produce FC H or FC J efficiently.

P450-2 (ORF5), glycosyltransferase (ORF6), P450-3 (ORF7), methyltransferase (ORF8), acetyltransferase (ORF9), P450-4 (ORF10), prenyltransferase (ORF11), acetyltransferase (ORF12) And P450-5 (ORF13) can be expressed in yeast or obtained as a recombinant protein, and can also be used in an in vitro reaction system.

It is the figure which showed the process which manufactures fusicoccin A (FC A) using GGDP as a starting material with the compound obtained by each reaction, and the enzyme which catalyzes each reaction process. P. Fig. 2 shows a fusicoccin A biosynthetic gene cluster isolated from amygdali . (A) Cluster 1 includes four genes: a cyclase gene: orf1, a dioxygenase gene: orf2, a P450-1 gene: orf3, and a short chain dehydrogenase / reductase gene: orf4 (B) Cluster 2 includes P450-2 gene (orf5), glycosyltransferase gene: orf6, P450-3 gene: orf7, methyltransferase gene: orf8, acetyltransferase gene : Orf9, P450-4 gene: o f10, prenyl transferases (prenyltransferase) gene (orf11), acetyltransferase (acetyltransferase) gene: orf12 and P450-5 gene: contains nine genes ORF13. (A) Cyclase [fusicocca-2,10 (14) -diene synthase gene] (orf1), S with P450 reductase gene and P450-2 gene (orf5). cerevisiae transformant pentane extract (upper [+ P450-2]) and cyclase [fusicocca-2,10 (14) -diene synthase gene] (orf1), P450 reductase gene and P450-2 gene (Orf5) without S. The HPLC chromatogram of the pentane extract (bottom [-P450-2]) of the cerevisiae transformant is shown. (B) GC / MS spectrum (top) of a peak (indicated by an arrow in the figure) specifically detected in the culture solution of the transformant [+ P450-2] and fusicocca-2, 10 (14)- The GC / MS spectrum (lower part) of a dien-8β-ol standard is shown. (A) S. A reaction product obtained by reacting a microsomal fraction prepared from a cerevisiae transformant with fusicocca-1,10 (14) -diene-8β, 16-diol in the presence of NADH or NADPH and the P450-3 gene is not present. S. The HPLC chromatogram of the reaction product (lower stage) obtained using the microsome of cerevisiae (control) is shown. (B) LC-MS spectrum (upper) of fusicocca-1,10 (14) -diene-8β, 9,16-triol sample and a peak specifically detected in the reaction product of the transformant (in the figure) , Labeled “product”) (lower column). The results of HPLC analysis of culture solutions of (A) orf10-deficient strain and (B) orf10 non-deficient strain are shown. (C) Disruption of orf10 gene by homologous recombination: Electrophoretic image (C left) confirmed by PCR analysis that the gene was deleted by homologous recombination. to construct a plasmid for Recombination inserting the hygromycin gene between between the upstream and downstream regions of orf10 gene, P. Operation outline | summary figure (C right) which introduce | transduced into amygdali and showed having performed the double crossover homologous recombination. (A) HPLC chromatogram of culture solution of orf13-deficient strain and (B) orf13 non-deficient strain. (C) Disruption of orf13 gene by homologous recombination: Electrophoretic image (C left) confirmed by PCR analysis that the gene was deleted. to construct a plasmid for Recombination inserting the hygromycin gene between between the upstream and downstream regions of orf13 gene, P. Operation outline | summary figure (C right) which introduce | transduced into amygdali and showed having performed the double crossover homologous recombination. (A) Reaction product (upper) synthesized by in vitro glycosyltransferase reaction in which His tag-binding ORF6 recombinant protein is reacted with FC H aglycon and UDP-glucose in vitro and FC H aglycon preparation (lower) as a substrate HPLC chromatogram of (B) LC-MS spectrum (upper) of the product produced by in vitro glycosyltransferase reaction and LC-MS spectrum (lower) of FC H standard. (A) Reaction product synthesized by in vitro methyltransferase reaction in which MBP tag-binding ORF8 recombinant protein is reacted with FC H aglycon and S-adenosyl methionine in vitro (upper) and FC H aglycon preparation as substrate (lower) HPLC chromatogram of (B) LC-MS spectrum (upper) of the product synthesized by in vitro methyltransferase reaction and LC-MS spectrum (lower) of 16- O- methyl-FC H alycon preparation. (A) HPLC chromatogram of a product synthesized by an in vitro prenyltransferase reaction (upper) and a substrate of FC P standard (lower), in which a His tag-binding ORF11 recombinant protein is reacted in vitro with FC P and dimethylallyl diphosphate. (B) LC / MS spectrum of the product synthesized from FC P and dimethylallyl diphosphate (upper) and LC / MS spectrum of FC J standard (lower). (A) HPLC chromatogram of the product synthesized by in vitro prenyl transferase reaction in which His-tag-binding ORF11 recombinant protein is reacted with FC H and dimethylallyl diphosphate in vitro (upper panel) and the FC H standard (lower panel) as a substrate. (B) ¹ H and ¹³ C NMR structural analysis results of 6′- O 2-(1,1-dimethyl) allylated ( t -pentenylated) FC H are shown. (A) LC / MS spectrum of the product synthesized by in vitro prenyl transferase reaction in which His tag-bound ORF11 recombinant protein is reacted with FCP and geranyl diphosphate in vitro; total ion chromatogram (top) and UV detection (bottom) . (B) LC-MS spectrum of 6′- O- linearized-FC P of the product. 1H and ¹³ C NMR structural analysis of a new FC-related compound 6′- O ^1- linarylated FC P produced using FCP and geranyl diphosphate using prenyltransferase (ORF11) as an enzyme. TF (trigger factor) -tagged ORF12 recombinant protein in vitro reacts with diethylacetyl-FC A and acetyl CoA. The reaction product (upper) obtained by in vitro acetyltransferase reaction and the substrate, 3 ′, 19-deacetylyl-FC HPLC chromatogram of A standard (bottom). (B) 3'-deacetyl-FC A standard LC / MS spectrum (upper) and reaction product LC / MS spectrum (lower).

(I) Explanation of Terms First, terms used in this specification will be explained. Production includes in vivo biosynthesis and production of each compound in vitro.

“Stringent conditions” for hybridizing with a complementary strand of a base sequence are, for example, hybridization in a 5 × SSC solution at 65 ° C. (composition of a 1-fold concentration SSC solution is 150 mM sodium chloride, 15 mM sodium citrate). It means the condition of making soybeans and further washing at 65 ° C. with 0.5 × SSC solution containing 0.1% SDS. Each operation of hybridization under stringent conditions is described in “Molecular Cloning (Third Edition)” (J. Sambrook & D. W. Russell, Cold Spring Harbor Press, 2001), etc. It can be performed by a known method. Generally, the higher the temperature and the lower the salt concentration, the higher the stringency.

Under such stringent conditions, a DNA having a base sequence showing a homology of 85% or more, preferably 90% or more, more preferably 95% or more with a specific base sequence described in the sequence listing is described in the sequence listing. It can hybridize with a complementary strand of DNA represented by the base sequence.

The “base sequence in which one or more bases are substituted, deleted, or added” means 1 or 2 to 50, preferably 1 or 2 to 20, more preferably in the specific base sequence described in the sequence listing. Means a base sequence in which 1 or 2 to 10, more preferably about 1 or 2 to 5 bases are substituted, deleted or added. The substitution, deletion, or addition of these base sequences may occur continuously or discontinuously in the base sequences described in the sequence listing.

“85% or more identity with an amino acid” means 85% to 99%, preferably 90% to 99%, more preferably 95% to 99% identity with a specific amino acid sequence described in the sequence listing. An amino acid sequence having Substitution, deletion, or addition in these amino acid sequences may occur continuously or discontinuously in the amino acid sequences described in the sequence listing.

“Amino acid sequence in which one or more amino acids are substituted, deleted or added” means, for example, 1 or 2 to 40, preferably 1 or 2 to 20 in the specific amino acid sequence described in the sequence listing. More preferably, it refers to an amino acid sequence in which 1 or 2 to 12, more preferably 1 or 2 to 9, particularly preferably 1 or 2 to 5 amino acids are substituted, deleted or added. Furthermore, in the substitution of amino acids, substitution with a similar amino acid is preferable because it is predicted that it does not change the phenotype of the polypeptide, that is, it is a conservative amino acid substitution. For example, similar amino acids can be grouped as follows, and specific examples of conservative amino acid substitution are well known in the art (see Science, 247: p1306-1310 (1990)).
Aromatic amino acids: Phe, Trp, Tyr
Aliphatic amino acids: Ala, Leu, Ile, Val
Polar amino acids: Gln, Asn
Basic amino acids: Lys, Arg, His
Acidic amino acids: Glu, Asp
Amino acids having a hydroxyl group: Ser, Thr
Amino acids with small side chains: Gly, Ala, Ser, Thr, Met
“Heterologous expression” refers to P. In cells such as fungi, bacteria or mammals different from Amygdali , P. The expression of a protein derived from amygdali . In contrast, P. P. amygdali as a host. Expression of a protein derived from amygdali is called “homologous expression”. As the host for the heterologous expression system, cultured mammalian cells or cells derived from eukaryotes such as yeast can be used. Saccharomyces cerevisiae is more preferable, and Saccharomyces cerevisiae is more preferable. S. cerevisiae is preferably auxotrophic for amino acids such as lysine and nucleic acids such as adenine, and YPH500 or the like can be used. A cDNA of a protein to be expressed is inserted into a vector containing a promoter sequence transcribed in the host so that the target gene is expressed under the control of the promoter, and the gene is introduced into the host. If necessary, a terminator effective in the host may be included downstream of the target gene cDNA.

The vector is not particularly limited as long as it has a promoter capable of inducing transcription in the host, and is preferably a plasmid. When yeast is used as the host, the promoter is not limited as long as transcription is induced in yeast, but GAL1, GAL10, TEF1, and the like are preferable.

In addition, it is preferable that the vector encodes a gene that can synthesize amino acids and nucleic acids that cannot be synthesized by the host yeast, if necessary. Preferred are the pESC series, pTEF1 series, pTEF1 series, pADH1 series, pTPI1 series, pHXT7, pTDH3, pPGK1, and pPYK1, and more preferred is the pESC series.

“Recombinant protein” refers to a protein that has been expressed in Escherichia coli and purified by transforming a cDNA encoding the target protein according to a conventional method. A promoter that synthesizes DNA containing a base sequence of a tag such as His, Myc, MBP, GST, TF, or FLAG, upstream or downstream of the cDNA to be expressed, and a promoter that is expressed in E. coli such as T3 or T7. Recombinant protein by incorporating the cDNA to be expressed into a vector such as pUC series, pBR322 series, etc., or into a commercially available vector such as pET series, pMAL series, pGEX series, pFLAG series, pCold TF series, etc. An expression vector can be obtained. In addition, by using a plasmid whose protein expression can be controlled by the lac operator, protein expression can be controlled using isopropyl-β-D-galactopyranoside (IPTG).

As the Escherichia coli for transforming the constructed recombinant protein expression vector, JM109 strain, HB101 strain, DH5α strain or BL21 strain can be used. When using a recombinant protein expression vector having a lac operator, it is preferable to use BL21-based E. coli.

Transformation into E. coli, selection with antibiotics, and culture can be performed according to conventional methods. Here, the ordinary method refers to the method described in the above-mentioned “Molecular Cloning (Third Edition)” or a method according to this.

E. coli transformed with the recombinant protein expression vector can be cultured on a large scale, and then the recombinant protein can be purified by a method according to each tag. The lysis of E. coli and the purification of the recombinant protein can be performed according to a protocol attached to a commercially available recombinant protein purification kit.

The “microsome fraction” is a fraction obtained by high-speed centrifugation after disrupting yeast or the like, and includes rough endoplasmic reticulum, smooth endoplasmic reticulum, free ribosome, cell membrane, Golgi apparatus and the like. The microsomal fraction can be prepared according to a known method.
(II) FC A and enzyme available enzymes present invention for the production of biosynthetic intermediates and target, P. 4 new P450s isolated from amygdali, 1 glycosyltransferase, 2 acetyltransferases and 1 methyltransferase and 1 prenyltransferase.

P450 catalyzes the monoatomic oxygenation reaction (monooxygenase reaction or hydroxylation reaction) of a fat-soluble substrate using two electrons and one oxygen molecule in the presence of P450 reductase and NADH or NADPH. An enzyme made of heme protein.

A glycosyltransferase is an enzyme that catalyzes a reaction of transferring a glycosyl group from a donor containing a glycosyl group to a substrate that is an acceptor. As the glycosyl group donor, a glycoside of nucleoside monophosphate or nucleoside diphosphate is used.

Acetyltransferase refers to an enzyme group that transfers an acetyl group to a substrate among acyltransferases that transfer an acyl group from acyl CoA to a substrate.

Methyltransferase refers to an enzyme that transfers a methyl group from a methyl group donor to an amino group, hydroxyl group, or thiol group of a substrate.

Prenyltransferase is a general term for enzymes that transfer a geranylgeranyl group to cysteine at the C-terminal part of a protein. However, the present invention includes an enzyme that transfers a geranylgeranyl group to a sugar.

Hereinafter, each of these enzymes will be specifically described.
1. P450-2 (ORF5)
In the presence of P450 reductase and nicotinamide adenine dinucleotide (NADH) or nicotinamide adenine dinucleotide phosphate (NADPH), the enzyme uses fusicocca-2,10 (14) -diene represented by the formula (II) as a substrate. As described above, it is characterized by having a catalytic action of synthesizing fusicocca-2,10 (14) -dien-8β-ol represented by the formula (III) by hydroxylating the carbon at the 8-position of the compound.

Specific examples of the enzyme (hereinafter also simply referred to as “ORF5”) include (4) a protein comprising the amino acid sequence shown in SEQ ID NO: 2. However, the protein is not limited thereto, and may be a protein comprising any one of the following amino acid sequences (5) or (6) as long as it has the above catalytic action.
(5) an amino acid sequence having 85% or more, preferably 90%, more preferably 95%, and still more preferably 98% or more homology with the amino acid sequence shown in SEQ ID NO: 2;
(6) An amino acid sequence obtained by substituting, deleting, or adding one or more amino acids in the amino acid sequence shown in SEQ ID NO: 2.

The origin of such an enzyme is not particularly limited as long as it has the above catalytic action and falls under any of the above (4) to (6). It may be derived from any organism. It is preferably derived from a fungus, and particularly preferably derived from a filamentous fungus, in particular, a filamentous fungus belonging to the genus Phomopsis , and more preferably from Phomopsis amygdali .

The present invention also provides a gene for the enzyme (the ORF5 gene is also simply referred to as “orf5”).

Specific examples of the ORF5 gene (orf5) include those having (1) the base sequence shown in SEQ ID NO: 1 in the coding region (DNA, cDNA). However, the present invention is not limited to this, and is a gene (DNA, cDNA) having the base sequence of either (2) or (3) below in the coding region, as long as it encodes the protein having the catalytic action described above. May be.
(2) a base sequence that hybridizes with a complementary strand of the base sequence shown in SEQ ID NO: 1 under stringent conditions;
(3) A base sequence in which one or more bases are substituted, deleted, or added in the base sequence shown in SEQ ID NO: 1.

The ORF5 of the present invention can be produced by conventional peptide synthesis based on the amino acid information of the ORF5 described above, particularly the amino acid sequence shown in SEQ ID NO: 2, and can be prepared by the same or different expression system using the orf5. Can be obtained according to. More preferably, budding yeast, particularly S. This is a production method using a heterologous expression system using cerevisiae .

When heterologously expressing ORF5, it is desirable to co-express with cDNA encoding P450 reductase having the amino acid sequence of SEQ ID NO: 20, for example, cDNA having the base sequence of SEQ ID NO: 19.

2. P450-3 (ORF7)
In the presence of P450 reductase and NADH or NADPH, the enzyme uses fusicocca-1,10 (14) -diene-8β, 16-diol represented by the formula (VII) as a substrate, and the 9-position carbon of the compound is It is characterized by having a catalytic action of synthesizing FC H aglycon represented by the formula (VIII) by hydroxylation.

Specific examples of the enzyme (hereinafter also simply referred to as “ORF7”) include (10) a protein consisting of the amino acid sequence shown in SEQ ID NO: 6. However, the protein is not limited thereto, and may be a protein having any one of the following amino acid sequences (11) or (12) as long as it has the above catalytic action.
(11) an amino acid sequence having 85% or more, preferably 90%, more preferably 95%, more preferably 98% or more homology with the amino acid sequence shown in SEQ ID NO: 6,
(12) An amino acid sequence obtained by substituting, deleting, or adding one or more amino acids in the amino acid sequence shown in SEQ ID NO: 6.

The origin of such an enzyme is not particularly limited as long as it has the above catalytic action and falls under any of the above (10) to (12), and is a eukaryotic organism such as a plant, an animal, or a fungus, and an actinomycete. It may be derived from any prokaryotic organism such as a fungus. It is preferably derived from a fungus, and particularly preferably derived from a filamentous fungus, in particular, a filamentous fungus belonging to the genus Phomopsis , and more preferably from Phomopsis amygdali .

The present invention also provides a gene for the enzyme (the ORF7 gene is also simply referred to as “orf7”).

Specific examples of the ORF7 gene (orf7) include those having the base sequence represented by (7) SEQ ID NO: 5 in the coding region (DNA, cDNA). However, the present invention is not limited to this, and is a gene (DNA, cDNA) having any one of the following base sequences (8) or (9) in the coding region, as long as it encodes the protein having the catalytic action described above. May be.
(8) a base sequence that hybridizes with a complementary strand of the base sequence shown in SEQ ID NO: 5 under stringent conditions;
(9) A base sequence in which one or more bases are substituted, deleted, or added in the base sequence shown in SEQ ID NO: 5.

The ORF7 of the present invention can be produced by conventional peptide synthesis based on the amino acid information of ORF7 described above, particularly the amino acid sequence shown in SEQ ID NO: 6, and can be prepared by the same or different expression system using the orf7. Can be obtained according to. More preferably, budding yeast, particularly S. This is a production method using a heterologous expression system using cerevisiae .

When heterologously expressing ORF7, it is desirable to co-express with cDNA encoding a P450 reductase having the amino acid sequence of SEQ ID NO: 20, for example, cDNA having the base sequence of SEQ ID NO: 19.

3. Glycosyltransferase (ORF6)
In the presence of a glycosyl group donor, the enzyme transfers a glycosyl group to the hydroxyl group at the 9-position of the compound using FC H aglycon represented by the formula (VIII) as a substrate, for example, an FC represented by the formula (I) It has a catalytic action for synthesizing glycosides such as H.

Specific examples of the enzyme (hereinafter also simply referred to as “ORF6”) include (16) a protein consisting of the amino acid sequence shown in SEQ ID NO: 4. However, the protein is not limited thereto, and may be a protein comprising any one of the following amino acid sequences (17) or (18) as long as it has the above catalytic action.
(17) Amino acid sequence having 85% or more, preferably 90%, more preferably 95%, more preferably 98% or more homology with the amino acid sequence shown in SEQ ID NO: 4,
(18) An amino acid sequence obtained by substituting, deleting, or adding one or more amino acids in the amino acid sequence shown in SEQ ID NO: 4.

The origin of the enzyme is not particularly limited as long as it has the above catalytic action and falls under any of the above (16) to (18). Eukaryotic organisms such as plants, animals, fungi, and bacteria It may be derived from any prokaryotic organism such as It is preferably derived from a fungus, and particularly preferably derived from a filamentous fungus, in particular, a filamentous fungus belonging to the genus Phomopsis , and more preferably from Phomopsis amygdali .

Glycosyl group donors that can be used with ORF6 of the present invention include, but are not limited to, uridine diphosphate (UDP), guanosine diphosphate (GDP), cytidine monophosphate (CDP), and thymidine diphosphate. Examples include sugar sugar adducts (sugar nucleotides) such as phosphoric acid (TDP). Although not limited, sugar addition products (sugar nucleotides) of uridine diphosphate (UDP) and thymidine diphosphate (TDP) are preferable, and sugar addition products (sugar nucleotides) of UDP are more preferable. Here, the sugar, that is, the glycosyl group transferred from the glycosyl group donor is not particularly limited, and examples thereof include monosaccharides, disaccharides, and trisaccharides. Preferred are monosaccharides or disaccharides, and more preferred are monosaccharides. The sugar may be a deoxy sugar such as fucose, or an amino sugar such as glucosamine or galactosamine. Examples of monosaccharides include tricarbon sugar, tetracarbon sugar, pentose sugar, and hexose sugar. Preferred are pentoses such as xylose and hexoses such as glucose, galactose or mannose, more preferred are hexoses, especially glucose. In particular, ORF6 acts on FC H alycon represented by formula (VIII) in the presence of UDP-glucose or TDP-glucose as a glycosyl group donor to synthesize FC H represented by formula (I). Useful as an enzyme.

The present invention also provides a gene for the enzyme (the ORF6 gene is also simply referred to as “orf6”).

Specific examples of the ORF6 gene (orf6) include those having the base sequence shown in (13) SEQ ID NO: 3 in the coding region (DNA, cDNA). However, the present invention is not limited to this, and is a gene (DNA, cDNA) having any one of the following base sequences (14) or (15) in the coding region, as long as it encodes the protein having the catalytic action described above. May be.
(14) a base sequence that hybridizes with a complementary strand of the base sequence shown in SEQ ID NO: 3 under stringent conditions;
(15) A base sequence in which one or more bases are substituted, deleted, or added in the base sequence shown in SEQ ID NO: 3.

The ORF6 of the present invention can be produced by conventional peptide synthesis based on the amino acid information of the ORF6 described above, particularly the amino acid sequence shown in SEQ ID NO: 2, and can be produced by the same or different expression system using the orf6. Can be obtained according to. More preferably, budding yeast, particularly S. This is a production method using a heterologous expression system using cerevisiae .

ORF6 can be obtained as a recombinant protein. The obtained recombinant protein can be reacted with a glycosyl group donor and FC H aglycon in the range of pH 6.5 to pH 8.5 using, for example, a Tris-HCl buffer or the like. Preferably, it is about pH 7 to pH 8. The glycosyltransferase reaction can be performed, for example, at 25 ° C. to 35 ° C. for about 3 hours to 24 hours, preferably 28 ° C. to 33 ° C. for about 12 hours to 24 hours. By this reaction, sugar can be transferred from the glycosyl group donor to the hydroxyl group at the 9-position of the compound represented by the formula (VIII).

4). P450-4 (ORF10)
In the presence of P450 reductase and NADH or NADPH, the enzyme hydroxylates the carbon at the 12-position of the compound using FCH represented by formula (I) as a substrate to produce FC represented by formula (IX). It has a catalytic action to synthesize Q.

Specific examples of the enzyme (hereinafter also simply referred to as “ORF10”) include (22) a protein consisting of the amino acid sequence shown in SEQ ID NO: 12. However, the protein is not limited thereto, and may be a protein having any one of the following amino acid sequences (23) or (24) as long as it has the above catalytic action.
(23) an amino acid sequence having 85% or more, preferably 90%, more preferably 95%, and still more preferably 98% or more homology with the amino acid sequence shown in SEQ ID NO: 12,
(24) An amino acid sequence obtained by substituting, deleting, or adding one or more amino acids in the amino acid sequence shown in SEQ ID NO: 12.

The origin of such an enzyme is not particularly limited as long as it has the above catalytic action and falls under any of the above (22) to (24), and eukaryotic organisms such as plants, animals, fungi, and the like It may be derived from any prokaryotic organism such as a fungus. It is preferably derived from a fungus, and particularly preferably derived from a filamentous fungus, in particular, a filamentous fungus belonging to the genus Phomopsis , and more preferably from Phomopsis amygdali .

The present invention also provides a gene for the enzyme (the ORF10 gene is also simply referred to as “orf10”).

Specific examples of the ORF10 gene (orf10) include those having the base sequence shown in (19) SEQ ID NO: 11 in the coding region (DNA, cDNA). However, the present invention is not limited to this, and is a gene (DNA, cDNA) having the base sequence of any of (20) or (21) below in the coding region, as long as it encodes the protein having the catalytic action described above. May be.
(20) a base sequence that hybridizes with a complementary strand of the base sequence shown in SEQ ID NO: 11 under stringent conditions;
(21) A base sequence in which one or more bases are substituted, deleted, or added in the base sequence shown in SEQ ID NO: 11.

The ORF10 of the present invention can be produced by conventional peptide synthesis based on the amino acid information of the ORF10 described above, particularly the amino acid sequence shown in SEQ ID NO: 12, and can be produced by the same or different expression system using the above orf10. Can get according to. More preferably, budding yeast, particularly S. This is a production method using a heterologous expression system using cerevisiae .

In the case of heterologous expression of ORF10, it is desirable to coexpress it with a cDNA encoding a P450 reductase having the amino acid sequence of SEQ ID NO: 20, for example, a cDNA having the base sequence of SEQ ID NO: 19.

5). Methyltransferase (ORF8)
The enzyme transfers FCP represented by the formula (X) by transferring the methyl group to the hydroxyl group at the 16-position of the compound using FCQ represented by the formula (IX) as a substrate in the presence of a methyl group donor. It has a catalytic action to synthesize. The enzyme can recognize the three-dimensional structures of FC H aglycon represented by the formula (VIII), FC H represented by the formula (I), and FC Q represented by the formula (IX). It does not react with an epimer formed by epimerization at the 3-position of the compound.

Specific examples of the enzyme (hereinafter also simply referred to as “ORF8”) include (28) a protein comprising the amino acid sequence shown in SEQ ID NO: 8. However, the protein is not limited thereto, and may be a protein comprising any one of the following amino acid sequences (29) or (30) as long as it has the above catalytic action.
(29) an amino acid sequence having 85% or more, preferably 90%, more preferably 95%, and still more preferably 98% or more homology with the amino acid sequence shown in SEQ ID NO: 8;
(30) An amino acid sequence obtained by substituting, deleting, or adding one or more amino acids in the amino acid sequence shown in SEQ ID NO: 8.

The origin of the enzyme is not particularly limited as long as it has the above catalytic action and falls under any of the above (28) to (30). Eukaryotic organisms such as plants, animals, fungi, and bacteria It may be derived from any prokaryotic organism such as It is preferably derived from a fungus, and particularly preferably derived from a filamentous fungus, in particular, a filamentous fungus belonging to the genus Phomopsis , and more preferably from Phomopsis amygdali .

Examples of the methyl group donor that can be used with ORF8 of the present invention include N-methyl compounds such as choline, betaine, 5-methyltetrahydrofolic acid, dimethyltetin, S-adenosyl methionine, and the like. S-adenosyl methionine is more preferable. In particular, ORF8 acts on FC Q represented by formula (IX) in the presence of S-adenosyl methionine as a methyl group donor, and is useful as an enzyme for synthesizing FC P represented by formula (X). is there.

The present invention also provides a gene for the enzyme (the ORF8 gene is also simply referred to as “orf8”).

Specific examples of the ORF8 gene (orf8) include those having a base sequence represented by (25) SEQ ID NO: 7 in the coding region (DNA, cDNA). However, the present invention is not limited to this, and is a gene (DNA, cDNA) having any one of the following base sequences (26) or (27) in the coding region, as long as it encodes the protein having the catalytic action described above. May be.
(26) a base sequence that hybridizes with a complementary strand of the base sequence shown in SEQ ID NO: 7 under stringent conditions;
(27) A base sequence in which one or more bases are substituted, deleted, or added in the base sequence shown in SEQ ID NO: 7.

The ORF8 of the present invention can be produced by conventional peptide synthesis based on the amino acid information of the ORF8 described above, particularly the amino acid sequence shown in SEQ ID NO: 8, and can be prepared by the same or different expression system using the above orf8. Can be obtained according to. More preferably, budding yeast, particularly S. This is a production method using a heterologous expression system using cerevisiae . ORF8 can be obtained as a recombinant protein, and the obtained recombinant protein is in the range of pH 6.5 to pH 8.5 using, for example, a Tris-HCl buffer, preferably at a pH of about 7 to pH 8, and a methyl group donor. Can be reacted with the formula (VIII), (I) or (IX). The methyltransferase reaction can be performed, for example, at 25 ° C. to 35 ° C. for about 3 hours to 24 hours, and preferably at 28 ° C. to 33 ° C. for about 12 hours to 24 hours. By this reaction, a methyl group can be transferred to the hydroxyl group at the 16-position of the compound represented by the formula (VIII), (I) or (IX).

6). Prenyltransferase (ORF11)
The enzyme synthesizes FC J represented by the formula (XI) by transferring the prenyl group to the hydroxyl group of the sugar of the compound using the FCP represented by the formula (X) as a substrate in the presence of a prenyl group donor. It has a catalytic action.

Specific examples of the enzyme (hereinafter also simply referred to as “ORF11”) include (34) a protein consisting of the amino acid sequence shown in SEQ ID NO: 14. However, the protein is not limited thereto, and may be a protein comprising any one of the following amino acid sequences (35) or (36) as long as it has the above catalytic action.
(35) an amino acid sequence having 85% or more, preferably 90%, more preferably 95%, and still more preferably 98% or more homology with the amino acid sequence shown in SEQ ID NO: 14;
(36) An amino acid sequence obtained by substituting, deleting, or adding one or more amino acids in the amino acid sequence shown in SEQ ID NO: 14.

The origin of such an enzyme is not particularly limited as long as it has the above catalytic action and falls under any of the above (34) to (36). It may be derived from any prokaryotic organism such as It is preferably derived from a fungus, and particularly preferably derived from a filamentous fungus, in particular, a filamentous fungus belonging to the genus Phomopsis , and more preferably from Phomopsis amygdali .

Examples of the prenyl group donor that can be used with ORF11 of the present invention include dimethylallyl diphosphate, geranyl diphosphate, farnesyl diphosphate, geranygeryl diphosphate, and the like. Preferred are dimethylallyl diphosphate and geranyl diphosphate, more preferably dimethylallyl diphosphate. In particular, ORF11 is useful as an enzyme that synthesizes FCJ represented by formula (XI) by acting on the 6'-position hydroxyl group of FH P represented by formula (X) using dimethylallyl diphosphate as a prenyl group donor. It is.

The present invention also provides a gene for the enzyme (the ORF11 gene is also simply referred to as “orf11”).

Specific examples of the ORF11 gene (orf11) include those having the base sequence shown in (31) SEQ ID NO: 13 in the coding region (DNA, cDNA). However, the present invention is not limited to this, and is a gene (DNA, cDNA) having the base sequence of any one of (32) and (33) below in the coding region, as long as it encodes the protein having the above catalytic action. May be.
(32) a base sequence that hybridizes with a complementary strand of the base sequence shown in SEQ ID NO: 13 under stringent conditions;
(33) A base sequence in which one or more bases are substituted, deleted, or added in the base sequence shown in SEQ ID NO: 13.

The ORF11 of the present invention can be produced by conventional peptide synthesis based on the amino acid information of the ORF11 described above, particularly the amino acid sequence shown in SEQ ID NO: 14, and can be prepared by the same or different expression system using the orf11. Can be obtained according to. More preferably, budding yeast, particularly S. This is a production method using a heterologous expression system using cerevisiae . ORF11 can be obtained as a recombinant protein, and the obtained recombinant protein is in the range of pH 6.5 to pH 8.5, preferably pH 7.2 to pH 7.8, using, for example, a Tris-HCl buffer or the like. To some extent, the prenyl group donor can be reacted with the compound represented by the formula (I) or (X). The prenyltransferase reaction can be performed, for example, at 25 ° C. to 35 ° C. for about 0.5 hours to 12 hours, and preferably at 28 ° C. to 33 ° C. for about 0.5 hours to 4 hours. be able to. By this reaction, the prenyl group can be transferred to the 6′-position hydroxyl group of the compound represented by the formula (I) or (X).

7). P450-5 (ORF13)
In the presence of P450 reductase and NADH or NADPH, the enzyme is represented by the formula (XII) by hydroxylating the 19th carbon of the compound using FC J represented by the formula (XI) as a substrate. It has a catalytic action for synthesizing dideacetyl-FC A.

Specific examples of the enzyme (hereinafter also simply referred to as “ORF13”) include (40) a protein consisting of the amino acid sequence shown in SEQ ID NO: 18. However, the protein is not limited thereto, and may be a protein comprising any one of the following amino acid sequences (41) or (42) as long as it has the above catalytic action.
(41) an amino acid sequence having 85% or more, preferably 90%, more preferably 95%, and still more preferably 98% or more homology with the amino acid sequence shown in SEQ ID NO: 18;
(42) An amino acid sequence obtained by substituting, deleting, or adding one or more amino acids in the amino acid sequence shown in SEQ ID NO: 18.

The origin of such an enzyme is not particularly limited as long as it has the above catalytic action and falls under any of the above (40) to (42), and eukaryotic organisms such as plants, animals, fungi and actinomycetes. It may be derived from any prokaryotic organism such as a fungus. It is preferably derived from a fungus, and particularly preferably derived from a filamentous fungus, in particular, a filamentous fungus belonging to the genus Phomopsis , and more preferably from Phomopsis amygdali .

The present invention also provides a gene for the enzyme (the ORF13 gene is also simply referred to as “orf13”).

Specific examples of the ORF13 gene (orf13) include those having the base sequence represented by (37) SEQ ID NO: 17 in the coding region (DNA, cDNA). However, the present invention is not limited to this, and is a gene (DNA, cDNA) having the base sequence of any of the following (38) or (39) in the coding region, as long as it encodes the protein having the above catalytic action. May be.
(37) a base sequence that hybridizes with a complementary strand of the base sequence shown in SEQ ID NO: 17 under stringent conditions;
(38) A base sequence in which one or more bases are substituted, deleted, or added in the base sequence shown in SEQ ID NO: 17.

The ORF13 of the present invention can be produced by conventional peptide synthesis based on the amino acid information of the ORF13 described above, particularly the amino acid sequence shown in SEQ ID NO: 18, and can be prepared by the same or different expression system using the orf13. Can be obtained according to. More preferably, budding yeast, particularly S. This is a production method using a heterologous expression system using cerevisiae .
In the case of heterologous expression of ORF13, it is desirable to coexpress it with a cDNA encoding a P450 reductase having the amino acid sequence of SEQ ID NO: 20, for example, a cDNA having the base sequence of SEQ ID NO: 19.

8). Acetyltransferase (ORF12)
The enzyme is characterized by having a catalytic action of transferring an acetyl group to the 19-position hydroxyl group of the compound, using dideacetyl-FC A represented by the formula (XII) as a substrate in the presence of an acetyl group donor.

Specific examples of the enzyme (hereinafter also simply referred to as “ORF12”) include (46) a protein consisting of the amino acid sequence shown in SEQ ID NO: 16. However, the protein is not limited thereto, and may be a protein comprising any one of the following amino acid sequences (47) or (48) as long as it has the above catalytic action.
(47) an amino acid sequence having 85% or more, preferably 90%, more preferably 95%, and still more preferably 98% or more homology with the amino acid sequence shown in SEQ ID NO: 16;
(48) An amino acid sequence obtained by substituting, deleting, or adding one or more amino acids in the amino acid sequence shown in SEQ ID NO: 16.

The origin of the enzyme is not particularly limited as long as it has the above catalytic action and falls under any of the above (46) to (48), and prokaryotic organisms such as plants, animals, fungi, bacteria, etc. From any prokaryotic organism. It is preferably derived from a fungus, and particularly preferably derived from a filamentous fungus, in particular, a filamentous fungus belonging to the genus Phomopsis , and more preferably from Phomopsis amygdali .

Acetyl-coenzyme A is mentioned as an acetyl group donor which can be used with ORF12 of this invention. In particular, ORF12 acts on the hydroxyl group at the 19-position of dideacetyl-FC A represented by the formula (XII) using acyl-coenzyme A as an acetyl group donor, and the compound (3′- It is useful as an enzyme for synthesizing O 2 -deacetyl-FC A).

The present invention also provides a gene for the enzyme (the ORF12 gene is also simply referred to as “orf12”).

Specific examples of the ORF12 gene (orf12) include those having the base sequence represented by (43) SEQ ID NO: 15 in the coding region (DNA, cDNA). However, the present invention is not limited to this, and it is a gene (DNA, cDNA) having the base sequence of either (44) or (45) below in the coding region, as long as it encodes the protein having the above catalytic action. May be.
(44) a base sequence that hybridizes with a complementary strand of the base sequence shown in SEQ ID NO: 15 under stringent conditions;
(45) A base sequence in which one or more bases are substituted, deleted, or added in the base sequence shown in SEQ ID NO: 15.

The ORF12 of the present invention can be produced by conventional peptide synthesis based on the amino acid information of the ORF12 described above, particularly the amino acid sequence shown in SEQ ID NO: 16, and can be prepared by the same or different expression system using the orf12. Can be obtained according to. More preferably, budding yeast, particularly S. This is a production method using a heterologous expression system using cerevisiae . ORF13 can be obtained as a recombinant protein, and the obtained recombinant protein is in the range of pH 6.5 to pH 8.5, preferably about pH 7.2 to pH 7.8 using a phosphate buffer, for example. The acetyl group donor can be reacted with the compound represented by the formula (XII). The acetyltransferase reaction can be performed, for example, at 25 ° C. to 35 ° C. for about 3 hours to 24 hours, and preferably at 28 ° C. to 33 ° C. for about 12 hours to 24 hours. By this reaction, the acetyl group can be transferred to the 19-position hydroxyl group of the compound represented by the formula (XII). As the substrate, a compound represented by the formula (XV) described later can be used instead of the compound represented by the formula (XII).

9. Acetyltransferase (ORF9)
In the presence of an acetyl group donor, the enzyme uses dideacetyl-FC A represented by formula (XII) or a compound represented by formula (XIV) (3′- O- deacetyl FC A) as a substrate. It has a catalytic action of transferring an acetyl group to the hydroxyl group at the 'position.

Specific examples of the enzyme (hereinafter also simply referred to as “ORF9”) include (52) a protein consisting of the amino acid sequence shown in SEQ ID NO: 10. However, the protein is not limited thereto, and may be a protein comprising any one of the following amino acid sequences (53) or (54) as long as it has the above catalytic action.
(53) an amino acid sequence having 85% or more, preferably 90%, more preferably 95%, more preferably 98% or more homology with the amino acid sequence shown in SEQ ID NO: 10;
(54) An amino acid sequence obtained by substituting, deleting, or adding one or more amino acids in the amino acid sequence shown in SEQ ID NO: 10.

The origin of such an enzyme is not particularly limited as long as it has the above catalytic action and falls under any of the above (52) to (54). It may be derived from any prokaryotic organism such as It is preferably derived from a fungus, and particularly preferably derived from a filamentous fungus, in particular, a filamentous fungus belonging to the genus Phomopsis , and more preferably from Phomopsis amygdali .

Examples of the acetyl group donor that can be used with ORF9 of the present invention include acetyl-coenzyme A. In particular, ORF9 acts on the 3′-position hydroxyl group of dideacetyl-FC A represented by the formula (XII) by using acyl-coenzyme A as an acetyl group donor, and the compound (19) represented by the following formula (XV) - or to produce O -deacetyl FC a), also act on the hydroxyl group of the 3'-position of the compound represented by the formula (XIV) (3'- O -deacetyl FC a), represented by the above formula (XIII) It is useful as an enzyme for producing a compound (FCA).

The present invention also provides a gene for the enzyme (the ORF9 gene is also simply referred to as “orf9”).

Specific examples of the ORF9 gene (orf9) include those having the base sequence represented by SEQ ID NO: 9 in the coding region (49) (DNA, cDNA). However, the present invention is not limited to this, and is a gene (DNA, cDNA) having any one of the following base sequences (50) or (51) in the coding region, as long as it encodes a protein having the above catalytic action. May be.
(50) a base sequence that hybridizes with a complementary strand of the base sequence shown in SEQ ID NO: 9 under stringent conditions;
(51) A base sequence in which one or more bases are substituted, deleted, or added in the base sequence shown in SEQ ID NO: 9.

ORF9 of the present invention can be produced by conventional peptide synthesis based on the amino acid information of ORF9 described above, particularly the amino acid sequence shown in SEQ ID NO: 10, and can be prepared by the same or different expression system using orf9. Can be obtained according to. More preferably, budding yeast, particularly S. This is a production method using a heterologous expression system using cerevisiae . ORF13 can be obtained as a recombinant protein, and the obtained recombinant protein is in the range of pH 6.5 to pH 8.5, preferably about pH 7.2 to pH 7.8 using a phosphate buffer, for example. The acetyl group donor can be reacted with the compound represented by the formula (XII). The acetyltransferase reaction can be performed, for example, at 25 ° C. to 35 ° C. for about 3 hours to 24 hours, and preferably at 28 ° C. to 33 ° C. for about 12 hours to 24 hours. By this reaction, the acetyl group can be transferred to the 3′-position hydroxyl group of the compound represented by the formula (XII). As the substrate, the compound represented by the formula (XIV) can be used instead of the compound represented by the formula (XII).

(3) Method for producing FC A and its biosynthetic intermediate The present invention utilizes the above-described enzyme catalytic action to produce a compound represented by formula (XIII) (fusicoccin A: FC A), and its biosynthetic intermediate. A method of manufacturing a body is provided. Here, as a biosynthetic intermediate, a compound represented by formula (I) (fusicoccin H: FCH), a compound represented by formula (IX) (fusicoccin Q: FC Q), represented by formula (X) A compound (fusicoccin P: FC P), a compound represented by formula (XI) (fusicoccin J: FC J), and a compound represented by formula (XII) (dideacetyl fusicoccin A: deacetylacetyl-FC A) Can be mentioned.

Hereinafter, a method for producing these series of compounds will be described according to Scheme I shown in FIG.

(3-1) Method for Producing FC H As shown in FIG. 1, the FC H represented by the formula (I) is prepared from 7 of steps (a) to (g) using geranylgeranyl diphosphate (GGDP) as a starting compound. It can be manufactured through steps. Each step will be described below.

1) Step (a): Production step of compound represented by formula (II) : Cyclase (Orf1: SEQ ID NO: 48) is allowed to act on geranylgeranyl diphosphate (GGDP) to be represented by formula (II) A compound (fusicocca-2,10 (14) -diene) can be obtained. Here, cyclase (ORF1) is a known enzyme described in Non-Patent Document 2, which catalyzes the generation of geranygeranyl diphosphate from Isopentenyl diphosphate and the subsequent cyclization reaction, fusicocca-2, It is a bifunctional enzyme that produces 10 (14) -diene.

In the step (a), the reaction with cyclase is carried out, for example, in the presence of dimethylallyl diphosphate, isopentenyl diphosphate, and magnesium ions at 25 ° C. to 35 ° C., preferably 28 ° C. to 32 ° C. for 0.5 hour to The reaction can be carried out by reacting for about 3 hours, more preferably for about 1 hour.

2) Step (b): Process for producing compound represented by formula (III) Compound (II) (fusicocca-2,10 (14) -diene) obtained in the above step (a) is converted to P450 reductase and NADH. Alternatively, by reacting the aforementioned enzyme P450-2 (ORF5) of the present invention in the presence of NADPH, the compound represented by formula (III) (fusicocca-2,10 (14) -dien-8β-ol) is obtained. Can be manufactured.

In addition, reaction of the said process (a) and (b) can be performed using the enzyme obtained by heterogeneous expression or the enzyme producer which carries out heterogeneous expression, for example. Specifically, first, cDNA encoding ORF5, cDNA encoding P450 reductase, and cDNA encoding cyclase (ORF1) (SEQ ID NO: 48) were obtained from S. cerevisiae. cerevisiae YPH500 (his-, leu-, trp-, ura-, ade-, lys-) is introduced to express these genes. The amino acid sequence of P450 reductase is shown in SEQ ID NO: 20, and the base sequence encoding it is shown in SEQ ID NO: 19.

The transformant produces compound (II) using GGDP in the bacterial body as a substrate by the action of cyclase (ORF1) expressed from the gene-transfected Orf1. Furthermore, compound (III) is produced | generated from compound (II) by the effect | action of the product (ORF5, P450 reductase) expressed from cDNA of ORF5 which introduce | transduced gene, and cDNA of P450 reductase. The produced compound (III) can be extracted and isolated from the cells of the transformant using a solvent such as pentane.

Further, cDNA encoding ORF5 (SEQ ID NO: 1), S was introduced cDNA (SEQ ID NO: 19) encoding the P450 reductase. It is also possible to prepare a microsomal fraction from S. cerevisiae and react the compound (III) with the compound (II) obtained in step (a) in the presence of NADH and NADPH in vitro. Obtainable. The reaction buffer and reaction conditions to be used are not particularly limited as long as P450-2 (ORF5) can exhibit the activity of catalyzing the above reaction.

3) Step (c): Step of producing compound represented by formula (IV) P450-1 (ORF3: SEQ ID NO: 50) is allowed to act on compound (III) obtained in the above step (b) to give the formula A compound represented by (IV) (fusicocca-2,10 (14) -diene-8β, 16-diol) can be obtained. Here, P450-1 (ORF3) is characterized by having a catalytic activity for hydroxylating the methyl group at the 16-position of the compound represented by the formula (III).

This reaction may be carried out by, for example, S. cerevisiae into which cDNA encoding ORF3 (SEQ ID NO: 49) or cDNA encoding P450 reductase was introduced. It is also possible to prepare a microsomal fraction from C. cerevisiae and react the compound (IV) with the compound (III) obtained in step (b) in the presence of NADH and NADPH in vitro. Obtainable. The reaction buffer and reaction conditions to be used are not particularly limited as long as P450-1 (ORF3) can exhibit the activity of catalyzing the above reaction.

Alternatively, BC-ORF7 represented by SEQ ID NO: 56 can be used in place of P450-1. BC-ORF7 is a known enzyme reported by Hashimoto M et al. (Bioorg. Med. Chem. Lett., Vol. 19, p5640-5543, 2009) and acts on the compound represented by the formula (III). As a result, a compound represented by formula (IV) (fusicocca-2,10 (14) -diene-8β, 16-diol) can be obtained.

The cDNAs of cyclase (ORF1), P450-2 (ORF5), P450 reductase represented by SEQ ID NO: 20 and P450-1 (ORF3: SEQ ID NO: 50) or BC-ORF7 (SEQ ID NO: 56) were obtained from S. cerevisiae. By culturing a transformant expressed in C. cerevisiae or the like using an expression vector in an appropriate medium, fusicocca-2,10 (14) -diene-8β, 16- Diol can be stored. The cyclase (ORF1) cDNA is exemplified in SEQ ID NO: 47, and the BC-ORF7 (SEQ ID NO: 56) cDNA is exemplified in SEQ ID NO: 55.

4) Step (d): Process for producing compound represented by formula (V) or formula (VI) Dioxygenase (ORF2: SEQ ID NO: 52) is allowed to act on compound (IV) obtained in the above step (c). Through the compound represented by the formula (V), the compound represented by the formula (VI) (8-hydroxyfusicocca-1,10 (14) -dien-16-al) which is a tautomer thereof is obtained. Obtainable. Here, dioxygenase (ORF2) is a known enzyme described in Non-Patent Document 2 and Non-Patent Document 3, and transfers diatomic oxygen from a diatomic oxygen donor such as α-ketoglutarate to a substrate. It is characterized by that.

ORF2 can be obtained as a recombinant protein using, for example, cDNA containing the base sequence shown in SEQ ID NO: 51. The obtained recombinant protein is used in the presence of divalent iron ions such as glycerol and iron sulfate and ascorbic acid in the range of pH 6 to 8, preferably pH 6.8 to 7.1 using, for example, MOPS buffer or the like. Then, a diatomic oxygen donor such as α-ketoglutaric acid can be reacted with the compound represented by the formula (IV). The reaction can be carried out between 25 ° C. and 35 ° C. for about 4 minutes to 30 minutes, preferably between 28 ° C. and 33 ° C. for less than 5 minutes. By this reaction, the hydroxyl group at the 16-position of the compound represented by the formula (IV) is oxidized with isomerization of the double bond, and converted into the compound represented by the formula (V). The compound is converted to a compound represented by the formula (VI) which is a mutant.

5) Step (e): Production step of compound represented by formula (VII) By reacting short-chain dehydrogenase reductase (ORF4: SEQ ID NO: 54) with compound (VI) obtained in step (d) above, A compound represented by the formula (VII) (fusicocca-1,10 (14) -diene-8β, 16-diol) can be obtained. Here, short-chain dehydrogenase reductase (ORF4) is a known enzyme described in Non-Patent Document 2 and Non-Patent Document 3, and the 16-position aldehyde group of the compound represented by Formula (VI) is reduced to a hydroxyl group. Have catalytic activity.

ORF4 can be obtained as a recombinant protein using, for example, cDNA containing the base sequence shown in SEQ ID NO: 53. The obtained recombinant protein is, for example, a TRIS-HCl buffer at pH 6.5 to 8.0 together with a dioxygenase such as the above recombinant protein of ORF2; glycerol; divalent iron ions such as iron sulfate; and salts such as potassium chloride A diatomic oxygen donor such as α-ketoglutaric acid; ascorbic acid; reaction in the presence of an electron donor such as NADH and NADPH at 25 ° C. to 35 ° C. for about 0.5 to 2 hours, in formula (IV) The represented compound can be converted to a compound represented by the formula (VII). In this reaction, first, dioxygenase converts a compound represented by formula (IV) to a compound represented by formula (VI), and ORF4 converts it to a compound represented by formula (VII). .

6) Step (f): Step of producing compound represented by formula (VIII) In the presence of P450 reductase and NADH or NADPH, the compound (VII) obtained in the above step (e) is subjected to the above-described step of the present invention. By allowing the enzyme P450-3 (ORF7) to act, a compound (FC H aglycone) represented by the formula (VIII) can be produced. This reaction can be performed, for example, in vitro using a microsomal fraction containing P450-3 (ORF7) obtained by heterologous expression. Specifically, first, a cDNA encoding ORF7 is designated as S. cerevisiae YPH500 (his-, leu-, trp-, ura-, ade-, lys-) is introduced to express the ORF7 gene. A microsomal fraction is prepared from the transformant, and the microsomal fraction containing ORF7 and the compound (VII) obtained in step (e) are reacted in vitro in the presence of NADH and NADPH. Compound (VIII) can be obtained. The reaction buffer and reaction conditions to be used are not particularly limited as long as P450-3 (ORF7) can exhibit the activity of catalyzing the above reaction.

7) Step (g): Step for producing compound represented by formula (I) In the presence of a glycosyl donor, compound (VIII) obtained in the above step (f) is subjected to the enzyme glycosyltransferase ( By allowing ORF6) to act, the compound (FCH) represented by the formula (I) can be produced.

This reaction may be performed using ORF6 itself or by using a recombinant protein of ORF6. Specifically, DNA containing a base sequence such as His tag or MBP tag and cDNA encoding ORF6 are combined to transform a host such as Escherichia coli to express a recombinant protein. The tag binding protein thus prepared is purified according to a conventional method using a tag binding protein purification column or the like. The purified ORF6 recombinant protein is reacted with compound (VIII) in the presence of a glucosyl group donor such as UDP-glucose. The reaction buffer used at this time and the pH conditions thereof are not particularly limited as long as the glycosyltransferase (ORF6) can exhibit the activity of catalyzing the above reaction, and is not particularly limited, but is preferably a Tris-HCl buffer and has a pH of 7 It is about ~ 8. The reaction temperature, reaction time, and the like are not particularly limited as long as the glycosyltransferase (ORF6) can exhibit the activity of catalyzing the above reaction, but are preferably 12 hours to 35 ° C. under a temperature condition of 25 ° C. to 35 ° C. A method of reacting for about 24 hours can be exemplified.

In addition, compound (I) is produced from P.I. In amygdali , the gene (orf10) can also be obtained by destroying the gene (orf10) so as to lack the ability to produce ORF10 that catalyzes the reaction from compound (I) to the compound represented by formula (IX). E.g., P obtained by deficient ORF10 gene by a technique such as homo logger Sri combination. As shown in FIG. 1, amygdali cannot block the reaction after step (h) because the reaction at step (h) is blocked. As a result, the compound (I) is accumulated.

ORF10 gene deficient P. In the production of compound (I) by amygdali , first, the deficient body is mass-cultured, and the culture solution obtained by filtering the cells is extracted with an appropriate solvent such as ethyl acetate or methanol-added ethyl acetate. After concentrating the extract to an appropriate amount, saturated aqueous sodium hydrogen carbonate and the like are added and the mixture is separated to obtain an organic phase. The organic phase is washed with saturated saline and the like, concentrated, added again with ethyl acetate and dissolved by heating, and then allowed to stand at room temperature, whereby compound (I) can be obtained as crystals. The method for obtaining and purifying the compound (I) is merely an example, and is not limited to such a method, and can be appropriately changed according to technical common sense in the art.

Compound (I) can also be used as a starting compound for synthesizing other FC A-like compounds such as BC C. In this sense, an efficient method for producing compound (I) (FC H) by the method of the present invention is useful.

(3-2) Method for Producing FC Q As shown in FIG. 1, FC Q represented by the formula (IX) is obtained through the following step (h) in addition to the steps (a) to (g). Can be manufactured.

8) Step (h): Production process of the compound represented by the formula (IX) In the presence of P450 reductase and NADH or NADPH in the presence of P450 reductase and NADH or NADPH, the compound (I) obtained in the above step (g) By allowing the enzyme P450-4 (ORF10) to act, the compound (FC Q) represented by the formula (IX) can be produced.

This reaction can be performed using, for example, a microsomal fraction containing P450-4 (ORF10) obtained by heterologous expression. Specifically, first, cDNA (SEQ ID NO: 11) encoding ORF10 was transformed into S. cerevisiae YPH500 (his-, leu-, trp-, ura-, ade-, lys-) is introduced to express the ORF10 gene (orf10). A microsomal fraction containing ORF10 is prepared from the transformant, and the microsomal fraction and the compound (I) obtained in the above step (g) are combined with each other in the presence of P450 reductase and NADH or NADPH. By reacting in vitro, a compound represented by the formula (IX) can be obtained.

Here, the reaction buffer, pH and salt concentration are not particularly limited as long as P450-4 (ORF10) can exhibit the activity of catalyzing the above reaction. The reaction temperature, reaction time, etc. are not particularly limited as long as P450-4 (ORF10) can exhibit the activity of catalyzing the above reaction.

(3-3) Method for Producing FC P As shown in FIG. 1, FC P represented by the formula (X) is added to the following steps (i) in addition to the steps (a) to (g) and the step (h). ) Can be manufactured.

9) Step (i): Production process of the compound represented by formula (X) In the presence of a methyl group donor, the aforementioned enzyme methyltransferase of the present invention is added to the compound (IX) obtained in the above step (h). By allowing (ORF8) to act, the compound (FCP) represented by the formula (X) can be produced.

This reaction can be performed using ORF8 itself, or can be performed using, for example, a recombinant protein of ORF8. Specifically, DNA containing a base sequence such as a His tag or MBP tag and a cDNA encoding ORF8 are combined, and a host such as E. coli is transformed to express a recombinant protein. The tag binding protein thus prepared is purified according to a conventional method using a tag binding protein purification column or the like. The purified ORF8 recombinant protein is reacted with compound (IX) in the presence of a methyl group donor such as S-adenosyl methionine. The reaction buffer and its pH conditions are not particularly limited as long as methyltransferase (ORF8) can exhibit the activity of catalyzing the above reaction, but it is preferably a Tris-HCl buffer, and the pH is about 7-8. It is. The reaction temperature, reaction time, etc. are not particularly limited as long as methyltransferase (ORF8) can exhibit the activity of catalyzing the above reaction, but it is preferably 25 ° C. to 35 ° C., preferably about 12 hours to 24 hours. It is a reaction.

(3-4) Manufacturing Method of FC J As shown in FIG. 1, FC J represented by the formula (XI) is added to the above steps (a) to (g), step (h), and step (i). And it can manufacture by passing through the following process (j).

10) Step (j): Step of producing compound represented by formula (XI) In the presence of a prenyl group donor, the aforementioned prenyl transferase of the present invention (in the presence of a prenyl group donor) is added to the compound (X) obtained in the above step (i). By allowing ORF11) to act, the compound (FC J) represented by the formula (XI) can be produced.

This reaction can be performed using ORF11 itself, or can be performed using, for example, a recombinant protein of ORF11. Specifically, DNA containing a base sequence such as a His tag or MBP tag and a cDNA encoding ORF11 are combined, and a host such as Escherichia coli is transformed to express a recombinant protein. The tag binding protein thus prepared is purified according to a conventional method using a tag binding protein purification column or the like. The purified ORF11 recombinant protein is reacted with compound (X) in the presence of a prenyl group donor such as dimethylallyl diphosphate.

The reaction buffer and its pH used here are not particularly limited as long as prenyl transferase (ORF11) can exhibit the activity of catalyzing the above reaction, but is preferably a Tris-HCl buffer, and has a pH of 7 It is about ~ 8. The reaction temperature, reaction time, etc. are not particularly limited as long as prenyl transferase (ORF11) can exhibit the activity of catalyzing the above reaction, but is preferably about 25 ° C. to 35 ° C. for 0.5 hour to 4 hours. The reaction is about an hour.

In addition, compound (XI) is a compound of P.sub.1 that produces compound (XI). In Amygdali , the gene (orf13) can also be obtained by destroying the gene (orf13) so as to lack the ability to produce ORF13 that catalyzes the reaction from compound (XI) to the compound represented by formula (XII). P obtained by deficient ORF13 gene by a technique such as homo logger Sri combination. As shown in FIG. 1, the reaction of step (k) is blocked by amygdali , so that the reaction after step (k) cannot be performed. As a result, compound (XI) accumulates in the culture medium.

ORF13 gene deficient P. In the production of compound (XI) by amygdali , first, the deficient body is mass-cultured, and a culture solution obtained by filtering the cells is extracted with an appropriate solvent such as ethyl acetate or methanol-added ethyl acetate. After the extract is concentrated to an appropriate amount, saturated aqueous sodium hydrogen carbonate and the like are added and the mixture is separated to obtain an organic phase. The organic phase is washed with saturated brine and the like, concentrated, and again added with ethyl acetate and dissolved by heating, and then left at room temperature to give compound (XI) as crystals. The method for obtaining and purifying the compound (XI) is an example, and is not limited to such a method, and can be appropriately changed based on the common general technical knowledge in the art.

The compound (XI) can also be used as a starting compound for synthesizing other FC A-like compounds such as BC C. In this sense, an efficient method for producing compound (XI) by the method of the present invention is useful.

(3-5) Method for Producing FC A As shown in FIG. 1, FC A represented by the formula (XIII) is produced by the steps (a) to (g), step (h), step (i) and step ( In addition to j), it can be produced through the following steps (k) and (l).

11) Step (k): Step of producing compound represented by formula (XII) Compound (XI) obtained by the above step (j) is subjected to P450-5 (ORF13) in the presence of P450 reductase and NADH or NADPH. Is allowed to act to produce a compound represented by formula (XII) (dideacetyl-FC A).

This reaction can be performed, for example, using a microsomal fraction containing ORF13 obtained by heterologous expression. Specifically, first, a cDNA encoding ORF13 is obtained from S. cerevisiae . cerevisiae YPH500 (his-, leu-, trp-, ura-, ade-, lys-) is introduced to express the ORF13 gene. A microsomal fraction containing ORF13 is prepared from the transformant, and the microsomal fraction and the compound (XI) obtained in the step (j) are subjected to in vitro in the presence of P450 reductase and NADH or NADPH. To give compound (XII).

Here, the reaction buffer and its pH and salt concentration are not particularly limited as long as P450-5 (ORF13) can exhibit the activity of catalyzing the above reaction. The reaction temperature, reaction time, etc. are not particularly limited as long as P450-5 (ORF13) can exhibit the activity of catalyzing the above reaction.

12) Step (l): Production step of compound represented by formula (XIII): Acetyltransferase ORF9 and ORF12 are allowed to act on compound (XII) obtained in step (k) in the presence of an acetyl group donor. Thus, the compound (FC A) represented by the formula (XIII) can be produced.

This reaction may be performed using

ORFs

9 and 12 themselves, or by using, for example, recombinant proteins of

ORFs

9 and 12. Specifically, a DNA containing a base sequence such as a His tag or an MBP tag and a cDNA encoding ORF9 and a cDNA encoding ORF12 are respectively combined, and a host such as Escherichia coli is transformed with the recombinant protein. To express. Each tag binding protein of

ORF

9 and 12 is purified according to a conventional method using a tag binding protein purification column or the like. The purified ORF9 recombinant protein and ORF12 recombinant protein are reacted with compound (XII) in the presence of an acetyl group donor such as acyl-coenzyme A. The reaction buffer used here is not particularly limited as long as the acetyltransferase (ORF 9 and 12) can exhibit the catalytic activity of the above reaction, but is preferably a phosphate buffer and has a pH of about 7 to 8. is there. The reaction temperature, reaction time, and the like are not particularly limited as long as the acetyltransferase (ORF 9 and 12) can exhibit the activity of catalyzing the above reaction, but it is preferably about 25 ° C. to 35 ° C. for 12 hours to 24 hours. The reaction is about an hour.

(4) Fungi used for the production of FC A biosynthetic intermediate: amygdali
The present invention relates to P450-2 (ORF5), glycosyltransferase (ORF6), P450-3 (ORF7), methyltransferase (ORF8), acetyltransferase (ORF9), P450-4 (ORF10), prenyltransferase (ORF11), acetyl Provided is a filamentous fungus that lacks the ability to produce any one enzyme selected from the group consisting of transferase (ORF12) and P450-13 (ORF13).

Among these enzymes, preferred filamentous fungi are P450-4 (ORF10) that catalyzes the reaction from compound (I) to compound (IX), or P450 that catalyzes the reaction from compound (XI) to compound (XII). -5 (ORF13) is lacking in the ability to produce the enzyme, and the former filamentous fungus can efficiently produce and accumulate compound (I) in the body, and the latter filamentous fungus. Thus, compound (XI) can be efficiently produced and accumulated in the body.

As such a filamentous fungus, a series of compounds shown in FIG. 1 can be biosynthesized, and as such a filamentous fungus, a bacterium belonging to Phomopsis is preferable, and Phomopsis amygdali is more preferable.

P. Amygdali is deposited with the ATCC (American Type Culture Collection: USA), ATCC 42412 strain, ATCC 42610 strain; MAFF numbers 62040, 625041, 625042, 625043, 625044 deposited with the Agricultural Bioresource Genebank (Japan). Etc. can be used.

A filamentous fungus lacking the ability to produce any one of the above-mentioned enzymes does not express the gene encoding any one of the above-mentioned enzymes, or the translation product does not exhibit the desired catalytic activity even if expressed. The gene is disrupted, and such a bacterium is designated as a gene-deficient strain or a gene-deficient P. It is called amygdali . Specifically, lacking genes encoding ORF10 P. In the case of amygdali , ORF10 gene-deficient P. It is called amygdali .

Gene deficient P. Amygdali includes natural mutants, mutants in which gene mutations are induced by mutagenic agents such as ethidium bromide, and mutants artificially produced by gene recombination.

For example, artificially prepared gene-deficient P 2 . Amygdali can be produced by homologous recombination or the like. Specifically, P. A hygromycin resistance gene, a puromycin resistance gene, etc. are ligated downstream of the promoter expressed in Amygdali , and P. coli is further downstream of the drug resistance gene. In Amygdali , a terminator that exhibits a function is bound and inserted into a vector. Here, P. The promoter expressed in amygdali is not particularly limited, but the trpC promoter is preferable. P. The terminator that exhibits the function in amygdali is not particularly limited, but a trpC terminator is preferable.

The vector is not particularly limited, but a plasmid of about 3 to 10 kb is preferable, it can be transformed with E. coli, and preferably has an antibiotic resistance gene such as ampicillin. A gene having a base sequence of 2 to 5 kb upstream of the gene to be deleted is incorporated upstream of the promoter-drug resistance gene-terminator binding DNA, and the gene to be deleted downstream of the promoter-drug resistance gene-terminator binding DNA A DNA having a base sequence of 2 to 5 kb in the downstream region is incorporated. DNA containing the base sequence of the upstream region and the downstream region of the gene to be deleted is P.I. It can be obtained by PCR reaction using amygdali genomic DNA as a template. DNA can be integrated according to a conventional method combining cleavage with a restriction enzyme and ligation. The vector obtained by this operation is called a homologous recombination vector. The obtained homologous recombination vector is transformed into Escherichia coli and cultured in large quantities, and then a large-scale plasmid is prepared according to a conventional method.

The homologous recombination vector thus obtained was designated as P. Introduced into amygdali by the protoplast / polyethylene glycol method or the like. The gene-transferred P.I. Amygdali was cultured in a medium containing a drug suitable for the drug resistance gene contained in the homologous combination vector to delete the target gene. The concentration of the drug contained in the medium is preferably 5 to 100 μg / ml, more preferably 30 to 70 μg / ml. By culturing in a medium containing a drug for about 3 to 7 days, gene-deficient P 2 . amygdali can be obtained.

The gene deficiency may be such that the target gene cannot be expressed or has been modified so that it does not exert the desired enzyme activity even if expressed, and as long as it is expressed, it may occur throughout the target gene. It may happen only to a part.

Hereinafter, the operation and effect of the present invention will be described more clearly using examples. However, this invention is not restrict | limited at all by this Example.
<General operation>
Sequence analysis of PCR fragments was performed by the dideoxy terminator method by Sanger et al. Using an automatic DNA sequencer (Li-Cor, model 4000L). Cell disruption was performed using an Ultrasonic Distortor (TOMY, UD-200).

Analysis of each sample in the protein purification process was performed by SDS-polyacrylamide-gel electrophoresis (SDS-PAGE), and the protein was detected by Coomassie staining. The protein concentration was determined by the Bradford method using bovine serum albumin as a standard.

<Bacterial strain, genomic DNA extraction, RNA extraction, plasmid extraction and gene transfer into E. coli>
As a microbial cell, P.I. Genomic DNA and cDNA were prepared from this using amygdali .

First, the strain was shaken in 100 ml medium containing 5% sucrose, 0.7% soy flour, 0.5% KH ₂ PO ₄ and 0.1% MgSO ₄ .7H ₂ O at 25 ° C. for 4 days. Cultured (120 strokes / min). Thereafter, 6 ml of the preculture solution was inoculated into 100 ml of medium containing 0.5% Yeast Extra (oriental yeast), 5% sucrose, 0.5% KH ₂ PO ₄ and 0.1% MgSO ₄ .7H ₂ O. Then, shaking culture (120 strokes / min) was carried out at 25 ° C. for 6 days.

Genomic DNA was extracted as follows.

First, 10 ml of liquid medium was immediately frozen with liquid nitrogen and ground with a mortar and pestle for 20 minutes. The ground cells were transferred to a 50 ml plastic tube and mixed with 20 ml of lysis buffer (50 mM Tris-HCl pH 8.0, 5 mM EDTA, 1% SDS and 0.1% NaCl). After incubating at 80 ° C. for 10 minutes, the solution was cooled, and the lysate was treated with proteinase K (Sigma Aldrich, 1 mg / ml) and RNase A (Takara Bio Inc., 0.1 mg / ml) at 30 ° C. for 1 hour. Subsequently, the DNA solution was deproteinized by the phenol-chloroform method and then precipitated and collected with isopropanol. The precipitated DNA was washed with 70% ethanol, redissolved in TE buffer and stored at -20 ° C.

Total RNA was prepared as follows.

First, the cells were filtered through a plastic filter, dried on a paper towel, then frozen in liquid nitrogen, and homogenized with SK-mill (Token Inc., Japan). Extraction of Total RNA from the strain was performed using TRIzol (registered trademark) reagent (Invitrogen, US) according to the attached protocol.

A cDNA fragment containing the 5 ′ end or 3 ′ end was obtained using SMART ^™ RACE cDNA Amplification Kit (Clontech, US) and GeneRacer® Kit (Invitrogen).

Plasmids from E. coli were prepared using Qiagen (registered trademark) plasmid kit. All restriction enzymes, T4 ligase, and calf intestinal alkaline phosphatase were from Toyobo (Japan), and were used according to the protocol attached to the product. Introduction of the plasmid into E. coli by electroporation was carried out under standard conditions using a BTX ECM 600 electroporation system (Biotechnologies and Experimental Research).

Example 1
< P. Determination of draft genome sequence of amygdali >
P. The sequence of the base sequence of the Amygdali genome was analyzed by whole genome sequencing using GS FLX (Roche Diagnostics Japan, Tokyo). Actual analysis was performed by Takara Bio Inc. (Shiga, Japan).

First, single-ended analysis was performed (total number of analyzed bases 225,673,483 bp and total number of contigs 35,997). Subsequently, paired-end analysis was performed (final analysis base number 393,803,480 bp and final contig 13,294).

We previously cloned cytochrome P450 gene (BC-Orf1) from cells producing BC structurally similar to FC A, performed characterization, and showed fusicocca-2, 10 (14 ) -Diene has been confirmed to catalyze hydroxylation of the 8β position (Minami A et al., Bioorg. Med. Chem. Lett., Vol. 19, p870-874, 2009; Hashimoto M et al., Bioorg. Med. Chem. Lett., Vol. 19, p5640-5643, 2009). The compound in which the 8β-position is hydroxylated (fusicocca-2,10 (14) -dien-8β-ol) is considered to be an intermediate common to the biosynthesis of BC and FC A. P. Amygdali is also considered to have the ortholog of BC-Orf1. So, we P. This ortholog is searched from the genome of Amygdali . The corresponding specific gene P450-2 (orf5) was isolated from 12716 (length 2.9 kb). This gene product (hereinafter referred to as “P450-2 (ORF5)”) showed 62% identity with the amino acid sequence of the BC-Orf1 gene product.

By repeating such searches, P.I. 9 gene from the genome of amygdali (4 one cytochrome P450 [P450-2 (orf5), P450-3 (orf7), P450-4 (orf10), and P450-5 (orf13)], 2 two acetyltransferase [acetyltransferase ( orf9), and acetyltransferase (orf12)], methyltransferase (orf8), glycosyltransferase (orf6) and prenyltransferase (orf11)) were identified as a gene cluster (21 kb) (FIG. 2 (B)).

The nucleotide sequence of the cytochrome P450-2 gene (P450-2 (orf5)) and the amino acid sequence of the gene product (P450-2 (ORF5)) are shown in SEQ ID NOs: 1 and 2, respectively; glycosyltransferase gene (glycosyltransferase (orf6)) And the amino acid sequence of its gene product (glycosyltransferase (ORF6)) in SEQ ID NOs: 3 and 4, respectively; the nucleotide sequence of the P450-3 gene (P450-3 (orf7)) and its gene product (P450-3 ( The amino acid sequences of ORF7)) are shown in SEQ ID NOs: 5 and 6, respectively. The nucleotide sequence of the methyltransferase gene (methyltransferase (orf8)) and the amino acid sequence of the gene product (methyltransferase (ORF8)) are respectively shown. SEQ ID NOs: 7 and 8; the nucleotide sequence of the acetyltransferase gene (acetyltransferase (orf9)) and the amino acid sequence of the gene product (acetyltransferase (ORF9)) in SEQ ID NOs: 9 and 10, respectively; the P450-4 gene (P450- 4 (orf10)) and the amino acid sequence of its gene product (P450-4 (ORF10)) are shown in SEQ ID NOs: 11 and 12, respectively; the nucleotide sequence of the prenyltransferase gene (prenyltransferase (orf11)) and its gene product ( The amino acid sequence of prenyltransferase (ORF11)) is SEQ ID NO: 13 and 14, respectively; the nucleotide sequence of acetyltransferase (acetyltransferase (orf12)) and the gene product thereof The amino acid sequence of acetyltransferase (ORF12)) is SEQ ID NO: 15 and 16, respectively; the base sequence of the P450-5 gene (P450-5 (orf13)) and the amino acid sequence of its gene product (P450-5 (ORF13)) The

numbers

17 and 18 are shown.

Example 2
<Determining the properties of P450-2 (ORF5)>
According to the method described in the paper of Hashimoto M et al. (Bioorg. Med. Chem. Lett., Vol. 19, p5640-5543, 2009), a heterologous expression experiment (cytochrome P450-2 (ORF5)) was performed. Confirmed the function of.

Coding region of the P450-2 is, P. 5'-CATGGATCCGATGAGCACGCTACAGGGGGCTCTTGG-3 '(SEQ ID NO: 21) and 5'-GGACTCGAGTATAGCTTCCCACCGATAACACGAGATAC-3' (SEQ ID NO: 22) primer set using the cDNA obtained from the genomic DNA of amygdali by the SMART ^™ RACE method as a template A .6 kb full length cDNA fragment was amplified.

The amplified fragment was subcloned into a plasmid, and after confirming the sequence, the BamHI-XhoI digested fragment was inserted into a pESC-URA vector (Agilent Technology) to construct pESC-URA-R450-2.

For functional analysis of cytochrome P450, cytochrome P450 reductase is essential. In order to isolate P450 reductase (SEQ ID NO: 20), P. From the database of amygdali , contig no. The cytochrome P450 reductase in 00872 was searched and the candidate gene was amplified by PCR. About 2.1 kb of full-length cDNA (SEQ ID NO: 19) was obtained by using the cDNA obtained by the SMART ^™ RACE method as a template. ) Was used for amplification.

After the amplified fragment was subcloned into a plasmid and the cytochrome P450 reductase sequence was confirmed, the EcoRI-BglII-digested fragment was inserted into the same enzyme-treated pESC-URA-R450-2. The S. cerevisiae is then combined with the plasmid pESC-TRP-orf8-ADH carrying the fusicocca-2,10 (14) -diene synthase gene (cyclase (orf1)). cerevisiae YPH500 (his ⁻ , leu ⁻ , trp ⁻ , ura ⁻ , ade ⁻ , lys ⁻ ) (Agilent Technology) (Hashimoto M et al., Bioorg. Med. Chem. lett., Vol. 19, p5640- 5643, 2009).

Structural analysis of the compound produced by the transformant was performed by GC / MS analysis (see Non-Patent Document 2 for analysis conditions).

The transformant was cultured, and the pentane extract of the cultured transformant was analyzed by HPLC (FIG. 3A). As a result, as shown in the upper diagram [+ P450-2] of FIG. 3A, one specific peak is detected, and authentic fusicoca-2,10 (14) -dien-8β-ol is generated from the retention time. It was confirmed that (Figure 3 (B)).

Therefore, P450-2 (ORF5) acts on fusicocca-2,10 (14) -diene in FIG. 1 to produce fusicocca-2,10 (14) -dien-8β-ol. In other words, P450-2 (ORF5) generates fusicocca-2,10 (14) -dien-8β-ol from fusicocca-2,10 (14) -diene in the series of reactions shown in FIG. It was confirmed to be useful as an enzyme.

Note that the present inventors have already described P.I. The gene of dioxygenase (ORF2) and short chain dehydrogenase reductase (ORF4) has been isolated and identified from the cyclase (ORF1) gene from the genome of amygdali (Non-patent document 2 and Non-patent document 3).

The nucleotide sequence of the cyclase (ORF1) gene and the amino acid sequence of its gene product are shown in SEQ ID NOs: 47 and 48, respectively. The nucleotide sequence of the gene of P450-1 (ORF3) and the amino acid sequence of its gene product are shown in SEQ ID NOs: 49 and 50, respectively. The nucleotide sequence of the gene for dioxygenase (ORF2) and the amino acid sequence of the gene product are respectively represented by SEQ ID NOs: 51 and 52; the nucleotide sequence of the gene for short-chain dehydrogenase reductase (ORF4) and the amino acid sequence of the gene product are respectively sequenced The numbers 53 and 54 are shown.

As shown in FIG. 1, by using these genes or gene products thereof, fusicocca-1,10 (14) using fusicocca-2,10 (14) -dien-8β-ol obtained above as a substrate. -Diene-8β, 16-diol can be produced. Specifically, fusicocca-2,10 (14) -diene-8β, 16-diol is produced by causing P450-1 (ORF3) to act on fusicocca-2,10 (14) -dien-8β-ol. By reacting this with dioxygenase (ORF2), 8β-hydroxyfusicoccca-1,10 (14) -diene-16-al is produced, and further by reacting with short chain dehydrogenase reductase (ORF4), fusicoca- 1,10 (14) -diene-8β, 16-diol can be obtained.

Example 3
<Isolation of P450-1 (ORF3)>
P. The cDNA obtained from the genome of amygdali in SMART ^TM RACE method fragments with P450-3 gene as a template (P450-1 (orf3)) 5'- GTCGACATGATAGGGGTATGCGGATTCCAGTG-3 '( SEQ ID NO: 57) and 5'-AAGCTTCTATTGTCCCTCACGACTTTTGAAG-3 It was amplified using the primer set of '(SEQ ID NO: 58) and inserted into pGEMT-Easy vector (Promega). After confirming the sequence of P450-1 (orf3), the cDNA of P450-1 was excised by restriction enzyme treatment and ligated to pESC-HIS (Agilent Technology). Thus the pESC-450-3, which was constructed S. cerevisiae YPH500 (his ⁻ , leu ^{− −} , trp ⁻ , ura ⁻ , ade ⁻ and lys ⁻ ) (Agilent Technology).

P450-1 is highly homologous to BC-ORF7 reported by Hashimoto M et al. (Bioorg. Med. Chem. Lett., Vol. 19, p5640-5543, 2009), and therefore has the same activity as BC-ORF7. Presumed to have.

Example 4
<Determining properties of P450-3 (ORF7)>
P. A 1.5 kb fragment containing the P450-3 gene (P450-3 (orf7)) as a template using cDNA obtained from the genome of amygdali by the SMART ^™ RACE method as 5′-ATGCTCTCCCACCATGACACCGTGG-3 ′ (SEQ ID NO: 25) and 5 ′ -TCAACCTGGTACTACTACTACTTCCTCTGC-3 '(SEQ ID NO: 26) was used for amplification and insertion into pGEMT-Easy vector (Promega). After confirming the sequence of P450-3 (orf7), the fragment digested with SpeI-EcoRI was ligated to pESC-HIS (Agilent Technology) digested with the same restriction enzymes. Thus the pESC-450-3, which was constructed S. cerevisiae YPH500 (his ⁻ , leu ^{− −} , trp ⁻ , ura ⁻ , ade ⁻ and lys ⁻ ) (Agilent Technology).

After culturing the transformant in the presence of galactose, the microsomal fraction was prepared and incubated with the substrate fusicocca-1,10 (14) -diene-8β, 16-diol at 30 ° C. for 48 hours ( Microsome fraction extract 87 μl, 100 mM NADH 5 μl, 100 mM NADPH 5 μl, and substrate, total volume 100 μl). Then, the reaction product was analyzed by HPLC under the following conditions (Bioorg. Med. Chem. Lett., Vol. 19, p5640-5643, 2009).
<HPLC conditions>
Column: Mark Mightisil RP-18GP Aqua column (250 × 4.6 mm; Kanto Chemicals, Japan)
Mobile phase: water and acetonitrile gradient: acetonitrile concentration is 40% for 0-5 minutes at the beginning of the run, then 40% to 100% for a linear gradient for 40 minutes;
Flow rate: 1.0 ml / min Detection: 205 nm.

The results of HPLC analysis are shown in FIG. The peak derived from the reaction product shown in the above figure is further subjected to LC / MS analysis, so that the reaction product is fusicocca-1,10 (14) -diene-8β, 9,16-triol (FC H aglycon). (FIG. 4B).

From this, P450-3 (ORF7) acts on fusicocca-1,10 (14) -diene-8β, 16-diol in FIG. 1, and fusicocca-1,10 (14) -diene-8β, 9 , 16-triol (FC-H aglycon), that is, in the series of reactions shown in FIG. 1, P450-3 (ORF7) is fusicocca-1, 10 (14) -diene- It was confirmed that the enzyme is useful as an enzyme for producing FC H aglycon from 8β, 16-diol.

Example 5
<Determination of properties of glycosyltransferase (ORF6)>
P. Using cDNA obtained from the genome of amygdali by SMART ^™ RACE method as a template, 5′-AGTATTTTGAACTCATATTCCCCCCCCTCACAAATCAAACC-3 ′ (SEQ ID NO: 27) and 5′-CTGCAGAGGATCCTCAATTGGTGGAGAGAAATGGTTC-3 ′ (SEQ ID NO: 28) A glycosyltransferase (orf6) cDNA was obtained. After confirming the sequence of glycosyltransferase (orf6), a PCR fragment digested with NdeI-BamHI was inserted into pET15b (Merck) to construct pET15-GLY. Escherichia coli incorporating pET15-GLY and Escherichia coli incorporating pET15b as a control were separately cultured in an LC liquid medium supplemented with 100 μg / ml of ampicillin. Specifically, at 37 ° C., O.D. D. After culturing until ₆₀₀ reached 0.5, 0.5 mM isopropyl β-D-1-thiogalactopyranoside was added, followed by further culturing at 25 ° C. for 18 hours. After culturing, the collected cells are crushed by ultrasonic waves, the crushed material is centrifuged, the supernatant is adsorbed to HisTrap column (GE Healthcare) at 4 ° C., and His-tagged enzyme is used according to a standard protocol to elute and Desalted. After confirming the purity of the recombinant enzyme produced from Escherichia coli incorporating pET15-GLY by SDS-PAGE, the enzyme was used for in vitro analysis using FC H aglycon as a substrate.

Specifically, 100 μl of a reaction solution containing 50 mM Tris-HCl buffer (pH 7.5), FC H aglycon (0.5 mM), UDP-glucose (2.5 mM), and 5 μg / ml of the purified enzyme was added at 30 ° C. The reaction product was analyzed by HPLC under the following conditions. <HPLC analysis conditions>
Column: Mark Mightisil RP-18GP Aqua column (250 × 4.6 mm; Kanto Chemicals, Japan)
Mobile phase: Water and acetonitrile Gradient: The acetonitrile concentration in the water is 30% for 0 to 20 minutes, 30 to 40% for 20 to 30 minutes, 40 to 70% for 30 to 50 minutes, and 50 to 60 minutes for 30 to 50 minutes. Flow rate increased to 70-100%: 1.0 ml / min Detection: 205 nm.

The HPLC result is shown in FIG. By further subjecting the peak derived from the reaction product shown in the above figure to LC / MS analysis, it was confirmed that the reaction product was FC H (FIG. 7B). Note that this enzyme can use not only UDP-glucose but also TDP-glucose as a glucose donor.

From this, the glycosyltransferase (ORF6) is an enzyme having an action of adding FC to FCH aglycon to produce FC-H in FIG. 1, that is, in the series of reactions shown in FIG. It was confirmed that glycosyltransferase (ORF6) is useful as an enzyme that generates FC H from FC H aglycon.

Example 6
<Determining the properties of methyltransferase (ORF8)>
P. Using cDNA obtained from the genome of amygdali by the SMART ^™ RACE method as a template, 5′-ATACTGTGAATTCATGGATGACAGCAAGACAAAAGGCC-3 ′ (SEQ ID NO: 29) and 5′-TGCAGTCGTCGAACTTAGGCGTTTGAAGACTACTTCATC-3 ′ (SEQ ID NO: 30) The cDNA for methyltransferase (orf8) was obtained. After confirming the sequence of methyltransferase (orf8), the PCR fragment digested with EcoRI-SalI was inserted into the EcoRI-SalI site of pMAL-p2X (N-terminal MBP-fused, New England Biolabs). The maltose binding protein fusion enzyme thus prepared was purified by elution and desalting according to a standard protocol. After confirming the purity of the recombinant enzyme by SDS-PAGE, it was used for in vitro analysis using FC-H aglycon as a substrate.

Specifically, 100 μl of a reaction solution containing 50 mM Tris-HCl buffer (pH 7.5), FC H aglycon (0.5 mM), S-adenosyl methionine (5 mM), and 5 μg / ml of the purified enzyme at 30 ° C. After overnight incubation, the reaction products were analyzed by HPLC under the same conditions as described in Example 4.

The HPLC result is shown in FIG. By further subjecting the peak derived from the reaction product shown in the above figure to LC / MS analysis, the reaction product was determined to be 16- O- methyl-FC H aglycon (FIG. 8B). .

Interestingly, when a mixture of FC H and 3-epi-FC H in a ratio of 3.7: 1 was used as the substrate, only FC H was methylated, so methyltransferase (ORF8 ) Was found to specifically recognize the stereochemical structure at position 3 of the substrate.

Therefore, methyltransferase (ORF8) acts on FC H aglycon having a hydroxyl group at position 16 in FIG. 1 or its glycoside, FC H or FC Q, etc., specifically, and the hydroxyl group at position 16 is specifically detected. It was confirmed that the enzyme is a methylating enzyme, that is, methyltransferase (ORF8) is useful as an enzyme that generates FCP from FCQ in the series of reactions shown in FIG.

Example 7
<Determining the properties of prenyltransferase (ORF11)>
P. Using the cDNA obtained from the genome of amygdali by SMART ^™ RACE method as a template, 5′-AGTACTTTGACTGCTAGCATGGGCAACGTGGTTCTGGATGGGTAG-3 ′ (SEQ ID NO: 31) and 5′-TGCAGACTGTCTCGGAGTATATCTGCG-3 The cDNA of prenyltransferase (orf11) was obtained by amplification. After confirming the sequence of prenyltransferase (orf11), this PCR fragment was incorporated into the NheI-XhoI site of pET21b (Merck). Expression and purification of the recombinant enzyme were performed in the same manner as described in Example 4.

After incubating 100 μl of a reaction solution containing 50 mM Tris-HCl buffer (pH 7.5), FC P (0.5 mM), dimethylallyl diphosphate (0.5 mM) and 5 μg / ml of the purified enzyme at 30 ° C. for 2 hours, The reaction product was analyzed by HPLC under the conditions described in Example 4.

The HPLC result is shown in FIG. The peak derived from the reaction product shown in the above figure is further subjected to LC / MS analysis, whereby the reaction product is converted to FC-J (6′- O 2-(1,1-dimethyl) allylated (t-pentenylated). -FCP) was confirmed (Figure 9 (B)).

Interestingly, prenyltransferase (ORF11) can use not only the above-mentioned FCP but also FCH as a prenyl group acceptor, thus 6′- O 2-(1,1-dimethyl) allylated ( t-pentenylated) FC H can be generated (see FIG. 10). In addition, prenyltransferase (ORF11) can use geranyl diphosphate as a prenyl donor, and 6′- O- linylated-FC P was obtained by using FCP as a prenyl group acceptor (FIG. 11). And 12). As far as we know, this enzyme is the first enzyme to prenylate sugars.

From the above, prenyltransferase (ORF11) is an enzyme that acts on glycosides such as FC Q or FCP to prenylate sugar chains in FIG. 1, that is, the series shown in FIG. In this reaction, it was confirmed that prenyltransferase (ORF11) is useful as an enzyme that generates 16- O- demethyl-FC J from FC Q and an enzyme that generates FC J from FC P.

Example 8
<Determining the properties of acetyltransferase-1 (ORF9) and acetyltransferase-2 (ORF12)>
P. The cDNA obtained from the genome of amygdali in SMART ^TM RACE method as a template, 5'-GGCGGTACCGGCATGTCTCAAACAACCGTCCCTGTC-3 '(SEQ ID NO: 33) and 5'-CCGCTGCAGGCCTCAAGGCAGCTTTTGCATCTCC-3' PCR using gene-specific primers (SEQ ID NO: 34) Amplification gave acetyltransferase-2 (orf12) cDNA. The fragment digested with KpnI-PstI was ligated to pCold TF vector (TaKaRa, Japan) digested with the same restriction enzymes to construct pCold-AT-2.

The purified recombinant enzyme was used in an in vitro assay using dideacetyl-FC A as a substrate.

Specifically, 12.4 mM phosphate buffer (pH 7.4), 137 mM NaCl, 2.7 mM KCl, 3 ′, 19-deacetylyl-FC A (50 μM), acetyl-coenzyme A (1 mM) and an appropriate amount of the purification After 100 μl of the reaction solution containing the enzyme was incubated overnight at 30 ° C., the reaction product was analyzed by HPLC under the following conditions.
<HPLC analysis conditions>
Column: Mark Mightisil RP-18GP Aqua column (250 × 4.6 mm) (Kanto Chemicals, Japan)
Mobile phase: Water and acetonitrile Gradient: The acetonitrile concentration in the water is 30% for 0 to 20 minutes, 30 to 40% for 20 to 30 minutes, 40 to 70% for 30 to 50 minutes, and 50 to 60 minutes for 30 to 50 minutes. Flow rate increased to 70-100%: 1.0 ml / min Detection: 205 nm.

The HPLC result is shown in FIG. The peak derived from the reaction product shown in the above figure was further subjected to LC / MS analysis, and it was found that the reaction product was 3′- O- deacetyl-FC A (FIG. 13B). . That is, from this, it was confirmed that acetyltransferase-2 (ORF12) acetylates the hydroxyl group at position 19 of dideacetyl-FC A.

On the other hand, when acetyltransferase-1 (ORF9) purified by the same method was used in an in vitro assay using dideacetyl FC-A as a substrate, it was found that 19- O- deacetyl-FC A was produced. From this, it was confirmed that acetyltransferase-1 (ORF9) acetylates the hydroxyl group at the 3 ′ position of dideacetyl-FC A.

Based on the above, acetyltransferase-1 (ORF9) and acetyltransferase-2 (ORF12) are enzymes that act on dideacetyl-FC A in FIG. 1 to acetylate the hydroxyl groups at positions 3 ′ and 19 respectively. In other words, in the series of reactions shown in FIG. 1, acetyltransferase-1 (ORF9) and acetyltransferase-2 (ORF12) were confirmed to be useful as enzymes that produce FCA from dideacetyl-FCA. .

Example 9
<Production and Properties of P450-4 (orf10) Disrupted Strain and P450-5 (orf13) Disrupted Strain>
(1) Preparation of P450-4 (orf10) -disrupted strain A hygromycin resistance gene expressed under the control of trpC promoter and trpC terminator was used as a template for 5′-ATCAGTATTCTGAGATGCCAGTTGTTCCAGTGAT-3 ′ (SEQ ID NO: 35) and 5 ′. -Two oligonucleotides of TGCAGAGGCTCGAGAATTAAGTCTAGAAAAGAAGG-3 '(SEQ ID NO: 36) were used as primers and amplified by PCR. This fragment was ligated into pGEM-T (Promega) to construct pGEM-Hyg. A fragment having a 3.0 kb SphI site including the upstream region of P450-4 (orf10) and a fragment having a 3.0 kb NotI site including the downstream region of P450-4 (orf10) were obtained by PCR amplification. Primer set used for PCR, respectively, 5'- GCATGC ATGGCGGCACGCAGGAGCCGCTTG-3 '(SEQ ID NO: 37) and 5'- GCATGC GAAGACTGCCGCGCACTATGGTAG-3' (SEQ ID NO: 38); and 5'- GCGGCCGCC TCTGAGTGCCTTCAGATAACAATTTC-3 '(SEQ ID NO: 39) and 5′- GCGGCCCGCG CAAGCTCATCTACCCTCCGGCTAG-3 ′ (SEQ ID NO: 40). These two DNA fragments including the upstream region and the downstream region of P450-4 (orf10) are deleted only in P450-4 (orf10), and the orientation of the upstream region and the downstream region on the chromosome is the same on the plasmid. Inserted into pGEM-Hyg in the direction. The constructed plasmid was transformed into P. p. It was introduced into amygdali by the protoplast / polyethylene glycol method, and colonies showing resistance to 50 μg / ml hygromycin were selected. Confirmation of gene deletion by PCR analysis using chromosomal DNA of the transformant as a template and a primer specific to the upstream region of P450-4 (orf10) and a primer specific to the downstream region of P450-4 (orf10) did. In the target disruption strain, the gene of P450-4 (orf10) was replaced with the hygromycin gene, and a fragment larger than the fragment derived from P450-4 was amplified (FIG. 5C).

(2) Preparation of P450-5 (orf13) -disrupted strain A hygromycin resistance gene expressed under the control of trpC promoter and trpC terminator was used as a template for 5′-ATCAGTATCTCGAGATGCCAGTGTTCCCAGTGAT-3 ′ (SEQ ID NO: 41) and 5 ′. -Two oligonucleotides of TGCAGAGGCTCGAGAATTAAGTCTAGAAAAGAAGG-3 '(SEQ ID NO: 42) were used as primers and amplified by PCR. This fragment was ligated into pGEM-T (Promega) to construct pGEM-Hyg. A fragment having a 2.0 kb NotI site including the upstream region of P450-5 (orf13) and a fragment having a 2.0 kb SphI site including the downstream region of P450-5 (orf13) were obtained by PCR amplification. The primer sets used for PCR were 5′- GCGGCCGCC GATCGACGCACCGTATGTGC-3 ′ (SEQ ID NO: 43) and 5′- GCGGCCGC GTGACCTCCAAATGGTGGCGT- 3CG (SEQ ID NO: 44); and 5′- GCATGCCATGCGCG sequence 45) and 5′- GCATGC AGACAGCCCGTACTGTTACGC-3 ′ (SEQ ID NO: 46). The constructed plasmid was transformed into P. p. Used to introduce into amygdali . The method for selecting the P450-5 (orf13) deletion strain is the same as the construction of the P450-4 (orf10) Orf10 deletion strain.

(3) Identification of P450 that catalyzes hydroxylation at position 12 of fusicoccin by gene disruption experiment <Determination of properties of P450-4 (ORF10) and P450-5 (ORF13)>
By the method described above, P. For amygdali, we succeeded in obtaining several deletions in which P450-4 (orf10) was replaced with a hygromycin resistance gene. The main product of the defect was fusicoccin H (FC H), and FC Q, FC P, FC J, FC A and the like shown in FIG. 1 were not produced. Moreover, the production amount of FCH was almost the same as the sum of a series of FC compounds including FCA produced by the parent strain ( P. amygdali ) (FIG. 5). From this result, it was found that P450-4 (ORF10) is responsible for hydroxylating the 12th position of FC H to generate FC Q.

The function of P450-5 (orf13) was also confirmed by the same method as that for the P450-4 (orf10) deletion. As a result, the main product of the deletion product in which P450-5 (orf13) was replaced with the hygromycin resistance gene was FC J, and dideacetyl-FC A and FC A shown in FIG. 1 were not produced. Moreover, the production amount of FCJ was almost the same as the sum of a series of FC compounds containing FCA produced by the parent strain ( P. amygdali ) (FIG. 6). From this result, it was found that P450-5 (ORF13) has a function of hydroxylating the 19th position of FC J to produce dideacetyl-FC A.

Example 10
<Manufacture of FC H and FC J on a large scale>
(1) P450-4 (orf10) deficient P. Production of FC H by amygdali As seed culture, sucrose 3 g, Pharmamedia 1.5 g, KH ₂ PO ₄ 0.5 g, Na ₂ HPO ₄ · 12H ₂ O 0.3 g, MgSO ₄ · A solution of 0.1 g of 7H ₂ O and 100 ml of purified water was added and sterilized under high pressure (121 ° C., 20 minutes). In this flask, P.I. An amygdali ORF10-deficient strain (growing bacteria on slant, 5 mm square piece) was inoculated and cultured with shaking (150 strokes / min) in the dark at room temperature for 72 hours. Two Sakaguchi flasks were used for seed culture for production culture of the following scale.

Next, as production culture, 5 g of sucrose, 0.7 g of soya flour, 0.75 g of KH ₂ PO ₄ , 0.5 g of Na ₂ HPO ₄ · 12H ₂ O, MgSO ₄ · 7H ₂ O 0 in a Sakaguchi flask (capacity 500 ml). 0.1 g of purified water (100 ml) was added, and after high-pressure sterilization (121 ° C., 20 minutes), a seed culture solution (7 ml) was added, followed by shaking culture (160 strokes / minute) for 144 hours at room temperature in the dark. Since 30 Sakaguchi flasks were used, the total amount of the culture solution was 3 L.

The treatment after shaking culture was divided into two parts. First, half of the culture solution was filtered, and the cell residue was washed with about 1 L of purified water. The mixture was extracted twice with the same amount of ethyl acetate as the filtrate (1% methanol added to about 2 L) and concentrated under reduced pressure. The remaining culture solution was treated in the same manner, and the two treatment solutions were combined and concentrated under reduced pressure until the remaining amount reached 200 ml. This concentrated solution was diluted with ethyl acetate, saturated aqueous sodium hydrogen carbonate (100 ml) was added, and the mixture was separated. The organic phase was washed with saturated brine, and dried over anhydrous magnesium sulfate. After concentrating the solvent under reduced pressure, about 10 times the amount of ethyl acetate as the residue was added, dissolved by heating, and allowed to stand at room temperature. The obtained FC H crystals had sufficient purity as a raw material for semi-synthesis. Yield 1.45 g (0.48 g / L).

(2) P450-5 (orf13) deficient P. aeruginosa Production of FC J by amygdali The same experimental procedure as that for the above orf10-deficient strain was carried out . By culturing an amygdali orf13-deficient strain, 1.86 g (0.62 g / L) of FCJ having sufficient purity as a semi-synthetic raw material was obtained from 3 L of the total culture solution.

Claims

Including the following steps (a) to (g):
Method for producing a compound represented by the formula (I):

(A). A step of allowing a cyclase (ORF1) to act on geranylgeranyl diphosphate to obtain a compound represented by the following formula (II):

(B). P450-2 (ORF5) is allowed to act on the compound (II) obtained above in the presence of P450 reductase and nicotinamide adenine dinucleotide (NADH) or nicotinamide adenine dinucleotide phosphate (NADPH). Obtaining a compound represented by (III),

(C). A step of allowing P450-1 (ORF3) to act on compound (III) obtained above in the presence of P450 reductase and NADH or NADPH to obtain a compound represented by the following formula (IV):

(D). The compound (IV) obtained above is reacted with dioxygenase (ORF2) in the presence of an iron ion, a diatomic oxygen donor and ascorbic acid to obtain a compound represented by the following formula (V) or (VI): Process,

(E). A step of allowing a short-chain dehydrogenase reductase (ORF4) to act on the compound (VI) obtained above to obtain a compound represented by the following formula (VII):

(F). A step of allowing P450-3 (ORF7) to act on compound (VII) obtained above in the presence of P450 reductase and NADH or NADPH to obtain a compound represented by the following formula (VIII):

(G). A step of obtaining a compound represented by the above formula (I) by allowing a glycosyltransferase (ORF6) to act on the compound (VIII) obtained above in the presence of a glycosyl group donor.
In addition to the steps (a) to (g) described in claim 1, the method for producing a compound represented by formula (IX) further comprising the following step (h):
(H). (G) A step of obtaining a compound represented by the following formula (IX) by allowing P450-4 (ORF10) to act on the compound (I) obtained in the step in the presence of P450 reductase and NADH or NADPH.

.
In addition to the steps (a) to (g) described in claim 1 and the step (h) described in claim 2, the method for producing a compound represented by formula (X), further comprising step (i) :
(I). (H) A step of obtaining a compound represented by the following formula (X) by allowing methyltransferase (ORF8) to act on the compound (IX) obtained in the step in the presence of a methyl group donor.

.
In addition to the steps (a) to (g) described in claim 1, the step (h) described in claim 2, and the step (i) described in claim 3, the method further includes a step (j). Method for producing compound represented by (XI):
(J). (I) A step of obtaining a compound represented by the following formula (XI) by allowing prenyl transferase (ORF11) to act on compound (X) obtained in the step in the presence of a prenyl group donor:

.
In addition to the steps (a) to (g) described in claim 1, the step (h) described in claim 2, the step (i) described in claim 3, and the step (j) described in claim 4. And a method for producing a compound represented by formula (XII), further comprising step (k):
(K). (J) A step of obtaining a compound represented by the following formula (XII) by allowing P450-5 (ORF13) to act on the compound (XI) obtained in the step in the presence of P450 reductase and NADH or NADPH:

.
(A) to (g) described in claim 1, (h) process described in claim 2, (i) process described in claim 3, (j) process described in claim 4, and claim In addition to the step (k) described in item 5, the method for producing a compound represented by the formula (XIII) further including the step (l):
(L). (K) A step of obtaining a compound represented by the following formula (XIII) by allowing two kinds of acetyltransferases ORF9 and ORF12 to act on the compound (XII) obtained in the step in the presence of an acetyl group donor:

.
A gene encoding P450-2 (ORF5) comprising the base sequence according to any one of (1) to (3):
(1) the base sequence shown in SEQ ID NO: 1,
(2) having a base sequence that hybridizes with a complementary strand of the base sequence shown in SEQ ID NO: 1 under stringent conditions, and similar to the gene product encoded by the base sequence shown in SEQ ID NO: 1, P450 reductase and NADH or A base sequence encoding a protein having an enzyme action that catalyzes the reaction of step (b) in the presence of NADPH;
(3) In the nucleotide sequence shown in SEQ ID NO: 1, P450 has a nucleotide sequence in which one or more bases are substituted, deleted, or added, and is the same as the gene product encoded by the nucleotide sequence shown in SEQ ID NO: 1. A base sequence encoding a protein having an enzyme action that catalyzes the reaction of step (b) in the presence of a reductase and NADH or NADPH.
P450-2 (ORF5) comprising the amino acid sequence according to any one of (4) to (6):
(4) the amino acid sequence shown in SEQ ID NO: 2,
(5) The reaction of step (b) in the presence of P450 reductase and NADH or NADPH, which is 85% or more identical to the amino acid sequence shown in SEQ ID NO: 2 and consists of the amino acid sequence of SEQ ID NO: 2 An amino acid sequence encoding a protein having an enzyme action that catalyzes or (6) an amino acid sequence represented by SEQ ID NO: 2 having an amino acid sequence in which one or more amino acids are substituted, deleted, or added; The amino acid sequence which codes the protein which has the enzyme effect | action which catalyzes reaction of a process (b) in presence of P450 reductase and NADH or NADPH similarly to the enzyme which consists of an amino acid sequence of 2.
A gene encoding P450-3 (ORF7), comprising the base sequence according to any one of (7) to (9):
(7) the base sequence shown in SEQ ID NO: 5,
(8) having a base sequence that hybridizes with a complementary strand of the base sequence shown in SEQ ID NO: 5 under stringent conditions, and, like the gene product encoded by the base sequence shown in SEQ ID NO: 5, P450 reductase and NADH or A base sequence encoding a protein having an enzyme action that catalyzes the reaction of step (f) in the presence of NADPH;
(9) In the base sequence shown in SEQ ID NO: 5, the base sequence has one or more bases substituted, deleted, or added, and is the same as the gene product encoded by the base sequence shown in SEQ ID NO: 5, A base sequence encoding a protein having an enzyme action that catalyzes the reaction of step (f) in the presence of P450 reductase and NADH or NADPH.
P450-3 (ORF7) comprising the amino acid sequence according to any one of (10) to (12):
(10) the amino acid sequence shown in SEQ ID NO: 6,
(11) In the presence of P450 reductase and NADH or NADPH, which has 85% or more identity with the amino acid sequence shown in SEQ ID NO: 6 and consists of the amino acid sequence shown in SEQ ID NO: 6, An amino acid sequence encoding a protein having an enzymatic action to catalyze the reaction;
(12) A P450 reductase having the same amino acid sequence as that shown in SEQ ID NO: 6, wherein the amino acid sequence shown in SEQ ID NO: 6 has an amino acid sequence in which one or more amino acids are substituted, deleted, or added. And an amino acid sequence encoding a protein having an enzyme action that catalyzes the reaction of step (f) in the presence of NADH or NADPH.
A gene encoding a glycosyltransferase (ORF6) comprising the base sequence according to any one of (13) to (15):
(13) the base sequence shown in SEQ ID NO: 3,
(14) Presence of a glycosyl group donor having a base sequence that hybridizes with a complementary strand of the base sequence shown in SEQ ID NO: 3 under stringent conditions, as in the gene product encoded by the base sequence shown in SEQ ID NO: 3. A base sequence encoding a protein having an enzyme action that catalyzes the reaction in the step (g)
(15) In the base sequence shown in SEQ ID NO: 3, a glycosyl group having the base sequence in which one or more bases are substituted, deleted, or added, and the gene product encoded by the base sequence of SEQ ID NO: 3 A base sequence encoding a protein having an enzyme action that catalyzes the reaction of step (g) in the presence of a donor.
Glycosyltransferase (ORF6) comprising the amino acid sequence according to any one of (16) to (18):
(16) the amino acid sequence shown in SEQ ID NO: 4,
(17) Catalyzing the reaction of step (g) in the presence of a glycosyl group donor having the identity of 85% or more to the amino acid sequence shown in SEQ ID NO: 4 and the enzyme consisting of the amino acid sequence shown in SEQ ID NO: 4 An amino acid sequence encoding a protein having an enzyme action,
(18) A glycosyl group donor having an amino acid sequence in which one or more amino acids are substituted, deleted, or added in the amino acid sequence shown in SEQ ID NO: 4 and consisting of the amino acid sequence shown in SEQ ID NO: 4 An amino acid sequence encoding a protein having an enzyme action that catalyzes the reaction of step (g) in the presence of.
(19) A gene encoding P450-4 (ORF10) comprising the base sequence according to any one of (21):
(19) the base sequence shown in SEQ ID NO: 11,
(20) A P450 reductase and NADH or NADPH having a base sequence that hybridizes with a complementary strand of the base sequence shown in SEQ ID NO: 11 under stringent conditions and the same as the gene product encoded by the base sequence of SEQ ID NO: 11. A base sequence encoding a protein having an enzyme action that catalyzes the reaction of step (h) in the presence of
(21) The base sequence shown in SEQ ID NO: 11 has a base sequence in which one or more bases are substituted, deleted, or added, and is reduced by P450 as in the gene product encoded by the base sequence of SEQ ID NO: 11. A base sequence encoding a protein having an enzyme action that catalyzes the reaction of step (h) in the presence of an enzyme and NADH or NADPH.
P450-4 (ORF10) comprising the amino acid sequence according to any one of (22) to (24):
(22) the amino acid sequence shown in SEQ ID NO: 12,
(23) In the presence of P450 reductase and NADH or NADPH, which has 85% or more identity with the amino acid sequence shown in SEQ ID NO: 12 and consists of the amino acid sequence shown in SEQ ID NO: 12, An amino acid sequence encoding a protein having an enzymatic action to catalyze the reaction;
(24) The amino acid sequence shown in SEQ ID NO: 12 has an amino acid sequence in which one or more amino acids are substituted, deleted, or added, and, like the enzyme consisting of the amino acid sequence shown in SEQ ID NO: 12, P450 reductase and An amino acid sequence encoding a protein having an enzyme action that catalyzes the reaction of step (h) in the presence of NADH or NADPH.
A gene encoding methyltransferase (ORF8) comprising the base sequence according to any of (25) to (27):
(25) the base sequence shown in SEQ ID NO: 7,
(26) Presence of a methyl group donor having a base sequence that hybridizes with a complementary strand of the base sequence shown in SEQ ID NO: 7 under stringent conditions and the gene product encoded by the base sequence shown in SEQ ID NO: 7 Below, a base sequence encoding a protein having an enzyme action that catalyzes the reaction of step (i),
(27) In the base sequence shown in SEQ ID NO: 7, a base sequence in which one or more bases are substituted, deleted, or added, and methyl as in the gene product encoded by the base sequence shown in SEQ ID NO: 7 A base sequence encoding a protein having an enzyme action that catalyzes the reaction of step (i) in the presence of a group donor.
Methyltransferase (ORF8) comprising the amino acid sequence according to any of (28) to (30):
(28) the amino acid sequence shown in SEQ ID NO: 8,
(29) Catalyzing the reaction of step (i) in the presence of a methyl donor, having 85% or more identity with the amino acid sequence shown in SEQ ID NO: 8 and the enzyme consisting of the amino acid sequence shown in SEQ ID NO: 8 An amino acid sequence encoding a protein having an enzyme action,
(30) A methyl group donor having an amino acid sequence in which one or a plurality of amino acids are substituted, deleted, or added in the amino acid sequence shown in SEQ ID NO: 8 and comprising the amino acid sequence shown in SEQ ID NO: 8 An amino acid sequence encoding a protein having an enzyme action that catalyzes the reaction of step (i) in the presence of.
A gene encoding prenyltransferase (ORF11) comprising the base sequence according to any of (31) to (33):
(31) the base sequence shown in SEQ ID NO: 13,
(32) having a base sequence that hybridizes with a complementary strand of the base sequence shown in SEQ ID NO: 13 under stringent conditions, and in the presence of a prenyl group donor in the same manner as the gene product encoded by the base sequence in SEQ ID NO: 13 , A base sequence encoding a protein having an enzyme action that catalyzes the reaction of step (j),
(33) In the base sequence shown in SEQ ID NO: 13, a prenyl group has a base sequence in which one or more bases are substituted, deleted, or added, and is the same as the gene product encoded by the base sequence of SEQ ID NO: 13 A base sequence encoding a protein having an enzyme action that catalyzes the reaction of step (j) in the presence of a donor.
(34) to (36) prenyltransferase (ORF11) comprising the amino acid sequence according to any one of the above:
(34) the amino acid sequence shown in SEQ ID NO: 14,
(35) Catalyzing the reaction of the step (j) in the presence of a prenyl group donor having the identity of 85% or more to the amino acid sequence shown in SEQ ID NO: 14 and the enzyme consisting of the amino acid sequence shown in SEQ ID NO: 14 An amino acid sequence encoding a protein having an enzyme action,
(36) A prenyl group donor having an amino acid sequence in which one or more amino acids are substituted, deleted, or added in the amino acid sequence shown in SEQ ID NO: 14, and the enzyme comprising the amino acid sequence shown in SEQ ID NO: 14 An amino acid sequence encoding a protein having an enzyme action that catalyzes the reaction of step (j) in the presence of.
(450) (ORF13) comprising the base sequence according to any of (37) to (39):
(37) the base sequence shown in SEQ ID NO: 17,
(38) having a base sequence that hybridizes under stringent conditions with a complementary strand of the base sequence shown in SEQ ID NO: 17, and, like the gene product encoded by the base sequence shown in SEQ ID NO: 17, P450 reductase and NADH or A base sequence encoding a protein having an enzyme action that catalyzes the reaction of step (k) in the presence of NADPH;
(39) The base sequence shown in SEQ ID NO: 17 has a base sequence in which one or more bases are substituted, deleted, or added, and is reduced by P450 as in the gene product encoded by the base sequence of SEQ ID NO: 17. A base sequence encoding a protein having an enzyme action that catalyzes the reaction of step (k) in the presence of an enzyme and NADH or NADPH.
P450-5 (ORF13) comprising the amino acid sequence according to any one of (40) to (42):
(40) the amino acid sequence shown in SEQ ID NO: 18,
(41) In the presence of P450 reductase and NADH or NADPH in the presence of P450 reductase and NADH or NADPH having 85% or more identity with the amino acid sequence shown in SEQ ID NO: 18, An amino acid sequence encoding a protein having an enzymatic action to catalyze the reaction;
(42) The amino acid sequence shown in SEQ ID NO: 18 has an amino acid sequence in which one or more amino acids are substituted, deleted, or added, and, like the enzyme consisting of the amino acid sequence shown in SEQ ID NO: 18, P450 reductase and An amino acid sequence encoding a protein having an enzyme action that catalyzes the reaction of step (k) in the presence of NADH or NADPH.
(43) A gene encoding acetyltransferase (ORF12) comprising the base sequence according to any one of (45):
(43) the base sequence represented by SEQ ID NO: 15,
(44) having a base sequence that hybridizes with a complementary strand of the base sequence shown in SEQ ID NO: 15 under stringent conditions, and in the presence of an acetyl donor in the same manner as the gene product encoded by the base sequence shown in SEQ ID NO: 15 , A base sequence encoding a protein having an enzyme action that catalyzes the acetylation of the 19th hydroxyl group of compound (XII),
(45) In the base sequence shown in SEQ ID NO: 15, one or more bases have a base sequence substituted, deleted, or added, and acetylated like the gene product encoded by the base sequence shown in SEQ ID NO: 15. A base sequence encoding a protein having an enzyme action that catalyzes the acetylation of the 19th hydroxyl group of compound (XII) in the presence of a donor.
(46) to (48) acetyltransferase (ORF12) comprising the amino acid sequence according to any one of:
(46) the amino acid sequence shown in SEQ ID NO: 16,
(47) having the identity of 85% or more with the amino acid sequence shown in SEQ ID NO: 16 and, like the enzyme encoded by SEQ ID NO: 16, in the presence of an acetyl group donor, the hydroxyl group at position 19 of compound (XII) An amino acid sequence encoding a protein having an enzymatic action to catalyze acetylation,
(48) An acetyl group donor having the same amino acid sequence as that shown in SEQ ID NO: 14, wherein the amino acid sequence shown in SEQ ID NO: 16 has an amino acid sequence in which one or more amino acids are substituted, deleted, or added. An amino acid sequence encoding a protein having an enzyme action that catalyzes the acetylation of the 19th hydroxyl group of compound (XII) in the presence of
(49) A gene encoding acetyltransferase (ORF9) comprising the base sequence according to any one of (51):
(49) the base sequence represented by SEQ ID NO: 9,
(50) having a base sequence that hybridizes with a complementary strand of the base sequence shown in SEQ ID NO: 9 under stringent conditions, and in the presence of an acetyl group donor in the same manner as the gene product encoded by the base sequence of SEQ ID NO: 9 A base sequence encoding a protein having an enzyme action that catalyzes the acetylation of the hydroxyl group at the 3′-position of the compound of formula (XII),
(51) In the base sequence shown in SEQ ID NO: 9, one or more bases have a base sequence substituted, deleted, or added, and the acetyl group is the same as the gene product encoded by the base sequence of SEQ ID NO: 9. A base sequence encoding a protein having an enzyme action that catalyzes the acetylation of the hydroxyl group at the 3'-position of a compound of formula (XII) in the presence of a donor.
Acetyltransferase (ORF9) comprising the amino acid sequence according to any of (52) to (54):
(52) the amino acid sequence shown in SEQ ID NO: 10,
(53) In the presence of an acetyl group donor, the compound of the formula (XII) has the identity of 85% or more to the amino acid sequence shown in SEQ ID NO: 10 and consists of the amino acid sequence shown in SEQ ID NO: 10 An amino acid sequence encoding a protein having an enzymatic action to catalyze acetylation of the hydroxyl group at the 3 ′ position,
(54) The amino acid sequence shown in SEQ ID NO: 10 has an amino acid sequence in which one or more amino acids are substituted, deleted, or added, and provides an acetyl group as in the enzyme consisting of the amino acid sequence shown in SEQ ID NO: 10 An amino acid sequence encoding a protein having an enzyme action that catalyzes the acetylation of the 3′-position hydroxyl group of a compound of the formula (XII) in the presence of a body.
P450-2 (ORF5), glycosyltransferase (ORF6), P450-3 (ORF7), methyltransferase (ORF8), acetyltransferase (ORF9), P450-4 (ORF10), prenyltransferase (ORF11), acetyltransferase (ORF12) And a filamentous fungus that lacks any one of the enzyme-producing ability selected from the group consisting of P450-5 (ORF13).
The filamentous fungus according to claim 25, wherein the filamentous fungus is derived from Pomopsis amygdali .