JP2012065578A - Production of tricyclic non-natural type alkaloid using vegetable polyketide synthase - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a means to make a complicated alkaloid skeleton efficiently and at once under the clean and mild conditions and under the ordinary temperature and normal pressure.SOLUTION: A compound expressed by formula (I) (the definition of substituents in the formula is given in the specification) or its salt is disclosed. A method of producing the compound expressed by the formula (I) includes a process in which a mutation III type polyketide synthase is used as a catalyst, and thereby the condensation reaction of a 2-carbamoyl benzoyl CoA derivative and a malonyl-CoA derivative is carried out.

Description

  The present invention relates to a plant-derived polyketide synthase mutant enzyme and production of a novel compound using the enzyme.

  Some of the secondary metabolic enzymes that make up the basic skeleton of natural products change their reaction patterns greatly due to subtle differences in the structure of the active site, and this is one of the major factors generating the molecular diversity of natural products. It has become. On the other hand, the substrate specificity of enzymes is generally strict and the degree of freedom is low, but the wide substrate specificity and catalytic ability of type III polyketide synthase (PKS) that constructs the basic skeleton of plant polyphenols is notable. (Non-Patent Documents 1-3). These cannot be said to be “exquisite enzyme systems” in which the stereochemistry of the reaction is strictly controlled, but rather can be regarded as “carbon chain elongation machines” by simple repeated acyl group transfer.

  The inventors have succeeded in cloning a novel type III polyketide synthase (PKS1) from Huperzia serrata and have reported that the enzyme has a wide range of substrate specificities and catalytic ability (non- Patent Document 4).

  Development of pharmaceutical seeds by chemical synthesis requires harsh conditions such as the use of metal catalysts under strong acid or base conditions, and complicated processes such as repeated protection / deprotection or functional group transformation. And The PKS1 cloned by the present inventors can generate a tricyclic alkaloid having an acridone skeleton (Non-patent Document 4) or a tricyclic alkaloid having a 6-5-6 condensed ring structure (FIG. 1B). . On the other hand, it was impossible to produce a tricyclic alkaloid having a more complicated 6-7-6 condensed ring structure using type III PKS.

Abe, I., Morita, H., Nat. Prod. Rep., 27, 809-838 (2010) Abe, I., Topics in Current Chemistry, 2010, 1-22, DOI: 10.1007 / 128_2009_22 Abe, I. et al., J. Am. Chem. Soc. 122, 11242 (2000) Wanibuchi, K. et al., FEBS Journal 274, 1073-1082 (2007)

  The object is to provide a means for efficiently and efficiently constructing a complex alkaloid skeleton at room temperature and normal pressure under clean and mild conditions.

  The present inventors have intensively studied the three-dimensional structure of type III polyketide synthase and created a single amino acid substitution mutant of the wild type enzyme. It has been found that the created mutant enzyme can further expand the wild-type substrate specificity and generate a compound having a more complex alkaloid skeleton, and has completed the present invention.

That is, the present invention is as follows.
[1] Formula (I):

(Where
R ¹ , R ² , R ³ and R ⁴ are the same or different and each represents a hydrogen atom, a halogen atom, a C _1-6 alkyl group or an alkoxy group,
R ⁵ represents a hydrogen atom, a C _1-6 alkyl group, a C _2-6 alkenyl group, a C _3-6 cycloalkyl group, an aryl group, an aralkyl group, a heterocyclic group or an acyl group,
R ⁶ is the same or different and represents a hydrogen atom, a C _1-6 alkyl group or an alkoxy group,
Furthermore, a nitrogen atom may be introduced at the position of the carbon atom to which R ¹ , R ² , R ³ or R ⁴ is bonded. Or a salt thereof.
[2] The compound or a salt thereof according to the above [1], wherein R ¹ , R ² , R ³ and R ⁴ are hydrogen atoms, R ⁵ is a hydrogen atom and R ⁶ is a hydrogen atom.
[3] Using a mutant type III polyketide synthase as a catalyst, the following formula:

(Where
R ¹ , R ² , R ³ and R ⁴ are the same or different and each represents a hydrogen atom, a halogen atom, a C _1-6 alkyl group or an alkoxy group,
R ⁵ represents a hydrogen atom, a C _1-6 alkyl group, a C _2-6 alkenyl group, a C _3-6 cycloalkyl group, an aryl group, an aralkyl group, a heterocyclic group or an acyl group,
Furthermore, the C—R ¹ , C—R ² , C—R ³ or C—R ⁴ bond may be substituted with a nitrogen atom. 2-carbamoylbenzoyl CoA derivative represented by the following formula:

(Wherein R ⁶ represents a hydrogen atom, a C _1-6 alkyl group or an alkoxy group), and a step of subjecting it to a condensation reaction with a malonyl CoA derivative represented by the above [1] or [2] Compound production method.
[4] The production method according to [3], wherein the mutant type III polyketide synthase is a point mutant enzyme from serine residue constituting glycine or alanine residue constituting the active center cavity of wild type III polyketide synthase .
[5] The production method according to [4], wherein the wild type III polyketide synthase is a Huperzia serrata-derived wild type III polyketide synthase.
[6] Mutant type III polyketide synthase comprising the following amino acid sequences (A1) to (A3):
(A1) the amino acid sequence of SEQ ID NO: 2;
(A2) the amino acid sequence of SEQ ID NO: 3; or (A3) 1 to several amino acids are further substituted, deleted or added at positions other than position 348 of the amino acid sequence of any of the above (A1) or (A2) An amino acid sequence having the ability to synthesize a tricyclic alkaloid in which the protein comprising the amino acid sequence after the mutation is introduced has a 6-7-6 condensed ring structure.
[7] A gene encoding the mutant type III polyketide synthase according to any one of [3] to [6].
[8] The gene according to [7] above, comprising the following polynucleotides (B1) to (B3):
(B1) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 5;
(B2) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 6; or (B3) a polynucleotide comprising a nucleotide sequence having 70% or more sequence identity to any of the nucleotide sequences of (B1) or (B2) above. And a polynucleotide encoding an enzyme having the ability to synthesize a tricyclic alkaloid having a 6-7-6 fused ring structure.
[9] A recombinant vector containing the gene according to [7] or [8].
[10] A transformant into which the recombinant vector according to [9] is introduced.

  The mutant type III polyketide synthase of the present invention is a highly versatile enzyme with greatly expanded substrate specificity compared to the wild type enzyme, and various alkaloid skeletons, particularly 6- A tricyclic alkaloid having a 7-6 fused ring structure can be produced. The method for producing a compound using the mutant type III polyketide synthase of the present invention can efficiently produce alkaloids and the like under clean and mild conditions as compared with the organic synthesis method. The novel tricyclic alkaloid having a 6-7-6 fused ring structure produced in this manner is useful as a raw material for pharmaceuticals, cosmetics, and foods (functional foods).

FIG. 1 shows a reaction mechanism using various substrates of type III polyketide synthase (PKS1) derived from Huperzia serrata. (A) Chalcone production reaction by wild-type PKS1. (B) Formation reaction of tricyclic alkaloid having a 6-5-6 condensed ring structure by wild-type PKS1. (C) Formation reaction of tricyclic alkaloid having 6-7-6 condensed ring structure by mutant PKS1. FIG. 2 shows the results of the antimicrobial assay of Example 8. In the figure, Amp represents ampicillin, Compound 1 represents a compound of formula (II), and Compound 2 represents a tricyclic alkaloid having a 6-5-6 condensed ring structure described in FIG. 1B. Biofilm formation inhibition was observed in the wells in the frame.

  Hereinafter, in the present specification, indications using abbreviations such as amino acids, (poly) peptides, (poly) nucleotides, etc. are defined in IUPAC-IUB [IUPAC-IUB Communication on Biological Nomenclature, Eur. J. Biochem., 138: 9 ( 1984)], “Guidelines for the preparation of specifications including base sequences or amino acid sequences” (edited by the Japan Patent Office), and conventional symbols in the field.

1.6-7-6 Tricyclic Alkaloid Having Condensed Ring Structure The present invention provides a compound represented by formula (I) (hereinafter sometimes referred to as “the compound of the present invention”) or a salt thereof. .

In formula (I), R ¹ , R ² , R ³ and R ⁴ are the same or different and each represents a hydrogen atom, a halogen atom, a C _1-6 alkyl group or an alkoxy group.
Examples of the halogen atom in R ¹ , R ² , R ³ and R ⁴ include chlorine, fluorine, bromine, iodine and the like. Of these, chlorine or fluorine is preferred.
_Examples of the C _1-6 alkyl group in R ¹ , R ² , R ³ and R ⁴ include a methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, sec-butyl group, tert- Examples thereof include a butyl group, an n-amyl group, an isoamyl group, a sec-amyl group, a tert-amyl group, and a hexyl group. Of these, a methyl group or an ethyl group is preferable.
_Examples of the C _1-6 alkoxy group for R ¹ , R ² , R ³ and R ⁴ include a methoxy group, an ethoxy group, a propoxy group, an isopropoxy group, a butoxy group, a pentyloxy group, and a hexyloxy group. Of these, a methoxy group and an ethoxy group are preferable.
Preferred combinations of R ^{1, R} 2, ^{R 3} and ^{R 4,} R ^1, R 2, ^{R 3} and ^{R 4} are hydrogen atoms.

In formula (I), R ⁵ represents a hydrogen atom, a C _1-6 alkyl group, a C _2-6 alkenyl group, a C _3-6 cycloalkyl group, an aryl group, an aralkyl group, a heterocyclic group or an acyl group.
The C _1-6 alkyl group for R ⁵ is the same as R ¹ , R ² , R ³ and R ⁴ above.
C _2-6 alkenyl groups include vinyl, 1-propenyl, allyl, isopropenyl, butenyl and isobutenyl.
C _3-6 cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and the like.
Examples of the aryl group include C _6-14 aryl groups such as phenyl, 1-naphthyl, 2-naphthyl, biphenylyl and 2-anthryl.
Examples of the aralkyl group include C _7-15 aralkyl groups such as benzyl, α-methylbenzyl and phenethyl.
The heterocyclic group is a 5- to 14-membered (preferably 5- to 9-membered, more preferably 1 to 4 hetero-atoms selected from a nitrogen atom, an oxygen atom and a sulfur atom in addition to a carbon atom. Is a 5- or 6-membered) non-aromatic heterocycle (eg, pyrrolidinyl, tetrahydrofuryl, tetrahydrothienyl, piperidyl, tetrahydropyranyl, morpholinyl, thiomorpholinyl, piperazinyl) or an aromatic heterocyclic group (eg, furyl, thienyl, pyrrolyl, Oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, imidazolyl, pyrazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,3,4-oxadiazolyl, furazanyl, 1,2,3-thiadiazolyl, 1,2, 4-thiadiazolyl, 1,3,4-thiadiazolyl, 1,2,3 Triazolyl, 1,2,4-triazolyl, tetrazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl) and the like. These “heterocyclic groups” may have 1 to 3 substituents such as a halogen atom, optionally halogenated C _1-6 alkyl, C _1-6 alkoxy, oxo and the like.
Acyl groups include those represented by the formula-(C = O) -R ^a ,-(C = S) -R ^a ,-(C = S) -OR ^a ,-(C = O) -OR ^a , -SO _2-. R ^a , —SO—R ^a , — (C═O) N (R ^b ) (R ^{b ′} ) or — (P═O) (OR ^b ) (OR ^{b ′} ) (where R ^a is a hydrogen atom Represents a hydrocarbon group which may have a substituent or a heterocyclic group which may have a substituent, and R ^b and R ^{b ′} are the same or different and have a hydrogen atom or a substituent. A hydrocarbon group which may be substituted or a heterocyclic group which may have a substituent, and the like. Here, examples of the hydrocarbon group include an aliphatic hydrocarbon group, a monocyclic saturated hydrocarbon group, and an aromatic hydrocarbon group, and those having 1 to 16 carbon atoms are preferable. Specific examples include an alkyl group, an alkenyl group, an alkynyl group, a cycloalkyl group, and an aryl group. Moreover, examples of the heterocyclic group which may have a substituent include the same heterocyclic groups as those described above for R ⁵ .

In formula (I), R ⁶ is the same or different and represents a hydrogen atom, a C _1-6 alkyl group or an alkoxy group. The C _1-6 alkyl group and the alkoxy group in R ⁶ are the same as R ¹ , R ² , R ³ and R ⁴ described above. R ⁶ is preferably a hydrogen atom, a methyl group, an ethyl group, a methoxy group or an ethoxy group, more preferably a hydrogen atom, a methyl group and an ethyl group, and most preferably a hydrogen atom.

In formula (I), a nitrogen atom may be further introduced at the position of the carbon atom to which R ¹ , R ² , R ³ or R ⁴ is bonded. The introduction position of the nitrogen atom is not particularly limited, and the number of nitrogen atoms introduced may be 1, 2 or 3.

In the formula (I), compounds in which R ¹ , R ² , R ³ and R ⁴ are hydrogen atoms, R ⁵ is a hydrogen atom and R ⁶ is a hydrogen atom are preferable.

  Examples of the salt of the compound of the present invention include metal salts, ammonium salts, salts with organic bases, salts with inorganic acids, salts with organic acids, salts with basic or acidic amino acids, and the like. Preferable examples of the metal salt include alkali metal salts such as sodium salt and potassium salt; alkaline earth metal salts such as calcium salt, magnesium salt and barium salt; aluminum salt and the like. Preferable examples of the salt with an organic base include, for example, trimethylamine, triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, N, N′-dibenzylethylenediamine. And salt. Preferable examples of the salt with inorganic acid include salts with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid and the like. Preferable examples of the salt with organic acid include formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, and benzenesulfone. And salts with acid, p-toluenesulfonic acid and the like. Preferable examples of salts with basic amino acids include salts with arginine, lysine, ornithine and the like, and preferable examples of salts with acidic amino acids include salts with aspartic acid and glutamic acid, for example. It is done.

  The compound represented by formula (I) may take the form of a solvate, and such a compound is also included in the compound of the present invention. Preferred solvates include hydrates and ethanolates.

  The compound of the present invention can be synthesized according to a general organic synthesis method, but is preferably produced by the following method using a mutant type III polyketide synthase described later as a catalyst.

2.6-7-6 Method for Producing Tricyclic Alkaloid Having Condensed Ring Structure The production method of the present invention uses a mutant type III polyketide synthase described later as a catalyst and a 2-carbamoylbenzoyl CoA derivative as a starter substrate. And a condensation reaction using a malonyl CoA derivative as an extended chain substrate.

  The starter substrate 2-carbamoylbenzoyl CoA derivative has the following formula:

(Wherein R ¹ , R ² , R ³ , R ⁴ and R ⁵ are as defined in the above formula (I)).

  The 2-carbamoylbenzoyl CoA derivative constitutes the 6-7 ring in the left half of the tricyclic alkaloid having a 6-7-6 condensed ring structure, and has a predetermined structure depending on the structure of the target alkaloid. You can choose what you have.

  The 2-carbamoylbenzoyl CoA derivative can be obtained, for example, as follows. The phthalamic acid derivative was reacted at 4 to 40 ° C. for 1 to 24 hours in the presence of N-hydroxysuccinimide, N-ethyl-N′-3-dimethylaminopropylcarbodiimide (WSCI), etc. A succinimide ester (NHS ester) is used, and the resulting NHS ester solution and Coenzyme A aqueous solution are mixed and reacted at 0 to 40 ° C. for 1 to 48 hours to produce a 2-carbamoylbenzoyl CoA derivative.

  The extended chain substrate malonyl CoA derivative has the following formula:

(Wherein R ⁶ is as defined in the above formula (I)), one type may be used, or two or more types may be used.

As the malonyl CoA derivative, commercially available products such as malonyl CoA (R ⁶ = H), methyl malonyl CoA (R ⁶ = CH ₃ ), ethyl malonyl CoA (R ⁶ = C ₂ H ₅ ) can be used. Other malonyl CoA derivatives can be obtained according to a conventional method using the product as a raw material.

  The conditions for the condensation reaction using the mutant type III polyketide synthase are not particularly limited as long as a tricyclic alkaloid is produced from the starter substrate and the extended chain substrate. Examples are 0.1 to 24 hours at 20 to 50 ° C. in the presence of an enzyme (for example, 0.01 to 0.1 mM) and a substrate having a predetermined concentration (for example, 0.05 to 10 mM). The mixing ratio of the starter substrate and the extended chain substrate is exemplified by 1: 1 to 1: 3, and a mixing ratio of 1: 2 is preferable.

  After completion of the reaction, the produced tricyclic alkaloid having a 6-7-6 fused ring structure is recovered from the reaction solution and purified according to the purpose. Recovery and purification of the tricyclic alkaloid can be performed using means well known in the art, and examples thereof include reverse phase HPLC, silica gel column and the like. In the condensation step, in addition to the tricyclic alkaloid having a 6-7-6 condensed ring structure in which three molecules of a malonyl CoA derivative are condensed with respect to one molecule of a 2-carbamoylbenzoyl CoA derivative, 2-carbamoyl A tricyclic alkaloid having a 6-5-6 condensed ring structure in which two molecules of a malonyl CoA derivative are condensed to one molecule of a benzoyl CoA derivative is also produced. Therefore, depending on the purpose, a product having a 6-5-6 fused ring structure can also be recovered and purified.

  A tricyclic alkaloid (compound of formula (II)) having a 6-7-6 condensed ring structure produced by a mutant type III polyketide synthase using 2-carbamoylbenzoyl CoA as a starter substrate and malonyl CoA as an extended chain substrate The reaction formula is shown below and shown in FIG. 1C.

  As shown in Example 8, the compound represented by the above formula (II) inhibits colony formation of bacteria such as S. aureus. Therefore, the compound represented by the formula (II) and the compound represented by the formula (I) are used as a new component such as an antibacterial agent and a biofilm remover, or the compound is a seed compound of a new component such as an antibacterial agent. It is expected to be used as

Bacteria belonging to Gram-positive bacteria include those that are subject to inhibition of colony formation, antibacterial activity, and biofilm formation:
The genus such as Bacillus, Listeria, Staphylococcus, Streptococcus, Enterococcus, Clostridium, broadly categorized as gram-positive bacteria such as Mycoplasma; and among others, as a typical human pathogen, Staphylococcus spp.), Neisseria gonorrhoeae (Corynebacterium spp., Listeria spp., Bacillus spp., Clostridium spp.); And bacteria belonging to Gram negative bacteria:
Escherichia coli, Salmonella, Enterobacteriaceae, Pseudomonas, Moraxella, Helicobacter, Stenotrophomona, Buderobibrio, acetic acid bacteria, Legionella, Wolbachia and other α-proteobacteria; and cyanobacteria, Spirochaetes, green sulfur bacteria, green Examples include non-sulfur bacteria.

3. Mutant type III polyketide synthase (mutant PKS1)
In the present invention, a mutant type III polyketide synthase (hereinafter referred to as “mutant enzyme of the present invention” or “mutant PKS1”) in which at least one amino acid in the amino acid sequence constituting the active center cavity of the type III polyketide synthase is substituted. Provide).

  In the present invention, a type III polyketide synthase (hereinafter sometimes abbreviated as “PKS1”) is a condensate of 3 molecules of malonyl CoA starting from 4 coumaroyl CoA as a starting substrate to generate chalcone (FIG. 1A). . “Mutant PKS1” is included in “PKS1” when classified from the viewpoint of retaining the activity of generating chalcone. “Wild-type PKS1” is used for the purpose of removing “mutant PKS1” from “PKS1”.

  The active center of PKS1 (SEQ ID NO: 1) consists of three sets of catalytic amino acid residues (Cys-174, His-313 and Asn-346), active site residues (Thr-207, Phe-225, Gly-266, Phe-275 and Ser-348) (see Wanibuchi, K. et al., FEBS Journal 274, 1073-1082 (2007), Fig. 2). In the present invention, based on the homology between the amino acid sequence of the active center of the enzyme found from PKS1 and the amino acid sequence of another PKS, the corresponding amino acid residue present in the active center of other PKS other than PKS1. It is possible to analogize the position.

  The substitution of one amino acid from a serine residue constituting the active center cavity to a glycine or alanine residue is to acquire the ability to synthesize a tricyclic alkaloid having a 6-7-6 fused ring structure not found in wild-type PKS1. Is a replacement. Preferably, in the case of wild-type PKS1 (SEQ ID NO: 1), the substitution at position 348 is a serine residue to a glycine residue or an alanine residue.

  The substitution of one amino acid described above can be introduced into a gene encoding PKS1 using a method known per se such as site-directed mutagenesis.

  The “capability of synthesizing a tricyclic alkaloid having a 6-7-6 condensed ring structure” in “acquiring the ability to synthesize a tricyclic alkaloid having a 6-7-6 condensed ring structure” of the present invention means “2-carbamoylbenzoyl” It refers to the property of synthesizing a tricyclic alkaloid having a 6-7-6 condensed ring structure using a CoA derivative and a malonyl CoA derivative as substrates. Measurement of “ability to synthesize a tricyclic alkaloid having a 6-7-6 condensed ring structure” was carried out by reacting the mutant enzyme of the present invention with 2-carbamoylbenzoyl CoA and malonyl CoA as substrates, and the reaction product by HPLC, LC- It can be performed by detecting the presence or absence of alkaloid synthesis using chromatography such as ESI MS. Details of the measurement method are described in Example 6.

  The wild-type PKS used in the present invention may be PKS derived from any plant or bacteria. Examples include PKS derived from plants such as Huperzia serrata, Selaginella moellendorffii, Medicago sativa, Pisum sativum, Marchantia polymorpha, and Trifolium subterraneum, but are not limited thereto.

  Wild-type PKS is preferably PKS1 derived from Huperzia serrata (DQ979827.1, GeneID: ABI94386.1, SEQ ID NO: 1).

Mutant PKS1 can be easily obtained by introducing the 1 amino acid substitution described above into wild-type PKS1. Preferably,
(A1) The amino acid sequence of SEQ ID NO: 2 or (A2) the amino acid sequence of SEQ ID NO: 3 (preferably consisting of the amino acid sequence).
Mutant PKS1 consisting of the amino acid sequence of SEQ ID NO: 2 or 3 is referred to as S348G or S348A, respectively.

The mutant PKS1 of the present invention is
(A3) An amino acid sequence obtained by introducing substitution, deletion or addition mutations at one to several amino acids at positions other than position 348 of the amino acid sequence of any of (A1) or (A2) above. Thus, the protein comprising the amino acid sequence after introduction of the mutation may contain an amino acid sequence having the ability to synthesize a tricyclic alkaloid having a 6-7-6 condensed ring structure. Here, the number of amino acids to be substituted, deleted or added is not limited as long as the alkaloid synthesis ability of the mutant PKS1 is maintained. For example, about 1 to 30, preferably about 1 to 20, more preferably about 1 to 10, even more preferably about 1 to 5, most preferably 1 or 2. The position where amino acid substitution, deletion or addition is performed is not particularly limited as long as the alkaloid synthesis ability is maintained.

4). Mutant PKS1 gene The present invention provides a gene encoding the mutant PKS1 (sometimes simply referred to as mutant PKS1 gene or mutant PKS1 DNA).
In this specification, “gene” or “DNA” is used to include not only double-stranded DNA but also each single-stranded DNA such as a sense strand and an antisense strand constituting the DNA. The length is not particularly limited. Therefore, in this specification, unless otherwise specified, a gene (DNA) is a double-stranded DNA containing genomic DNA and a single-stranded DNA containing cDNA (positive strand) and a sequence having a sequence complementary to the positive strand. Both double-stranded DNA (complementary strand) and fragments thereof are included. The “gene” or “DNA” includes not only the “gene” or “DNA” represented by a specific base sequence (SEQ ID NOs: 4 to 6), but also the protein and biological function encoded by these. Included are “genes” or “DNA” that encode proteins that are equivalent (eg, homologs (such as homologs and splice variants) and derivatives). The gene or DNA does not ask for distinction of the functional region, and can include, for example, an expression control region, a coding region, an exon, or an intron.
In the present specification, “polynucleotide” indicates a concept when the above “gene” is viewed from the viewpoint of chemical structure, and “poly” is 2 or more (preferably about 10 to 10,000, more preferably 20 to The number of nucleotides is about 5000), and “nucleotide” includes not only DNA but also RNA.

  The gene encoding wild-type PKS1 is preferably PKS1 derived from Huperzia serrata (DQ979827.1, GeneID: ABI94386.1, SEQ ID NO: 4).

  The mutant PKS1 gene of the present invention can be easily obtained by introducing the mutation that enables the substitution of one amino acid as described above into the wild-type PKS1 gene. Examples of the substitution from serine to glycine include mutagenesis of a codon (GGT, GGC, GGA or GGG) encoding glycine with respect to a codon encoding serine (TCT, TCC, TCA or TCG). For substitution of serine to alanine, mutagenesis of an alanine-encoding codon (GCT, GCC, GCA or GCG) to a codon encoding serine (TCT, TCC, TCA or TCG), preferably point mutagenesis Can be given.

The mutagenesis is performed by a known technique such as the Kunkel method or the Gapped duplex method or a method equivalent thereto, for example, using a mutagenesis kit using site-directed mutagenesis, or by using the QuickChange ^™ XL Kit (Stratagene ) And the like.

Preferably, the mutant PKS1 gene includes a polynucleotide having a base sequence as shown below.
(B1) a polynucleotide comprising the base sequence of SEQ ID NO: 5, or (B2) a polynucleotide comprising the base sequence of SEQ ID NO: 6.
Mutant PKS1 genes containing a polynucleotide having the base sequence of SEQ ID NO: 5 or 6 are referred to as S348G gene (DNA) and S348A gene (DNA), respectively.

The mutant PKS1 gene of the present invention is
(B3) Tricyclic which consists of a polynucleotide comprising a base sequence having 70% or more sequence identity with any one of the base sequences (B1) or (B2) and has a 6-7-6 condensed ring structure It may include a polynucleotide encoding an enzyme having alkaloid synthesis ability. Such polynucleotides also include degenerate isomers of mutant PKS1 genes. Degenerate isomers refer to DNAs that differ only in degenerate codons and can encode the same protein. For example, for a DNA consisting of the nucleotide sequence of SEQ ID NO: 5 or 6, the codon corresponding to any of its amino acids, for example, the codon corresponding to Phe (TTC) has been changed to, for example, TTT which is degenerate. In the present invention, these are called degenerate isomers.

  Here, “sequential identity of 70% or more” is not limited as long as the alkaloid synthesis ability of the encoded mutant PKS1 is maintained, but it is about 70% or more, preferably about 80% or more, more preferably between base sequences. Means about 90% or more, more preferably about 95% or more, and most preferably 98% or more. The identity (%) of the base sequence in the present specification can be determined by using a homology search program (for example, BLAST, FASTA, etc.) commonly used in the art in an initial setting. In the present invention, “identity” of base sequences is calculated by aligning two types of base sequences to be compared and dividing the number of base sequences matched by the alignment by the total number of base sequences as a reference. It is a number showing the percentage in%. Note that the gap generated by the alignment is calculated as a mismatch.

5. Recombinant vector The recombinant vector of the present invention is the above-mentioned 4. This gene can be constructed by introducing the gene into an appropriate vector. Here, the vector is preferably selected as appropriate according to the host to be introduced.

  As the plant introduction vector, pBI, pPZP, and pSMA vectors that can introduce a target gene into a plant via Agrobacterium are preferably used. In particular, pBI binary vectors or intermediate vector systems are preferably used, and examples thereof include pBI121, pBI101, pBI101.2, pBI101.3 and the like. A binary vector is a shuttle vector that can replicate in Escherichia coli and Agrobacterium. When a plant is infected with Agrobacterium that holds the binary vector, it is a border sequence consisting of LB and RB sequences on the vector. It is possible to incorporate the enclosed DNA into plant nuclear DNA (EMBO Journal, 10 (3), 697-704 (1991)). On the other hand, a pUC vector can directly introduce a gene into a plant, and examples thereof include pUC18, pUC19, and pUC9. Also, plant virus vectors such as cauliflower mosaic virus (CaMV), kidney bean mosaic virus (BGMV), tobacco mosaic virus (TMV) and the like can be used.

  A baculovirus-derived vector or the like can be used for the insect cell system. In yeast, many vectors containing constitutive promoters such as ADH or LEU2 or inducible promoters such as GAL can be used.

  Examples of vectors for introducing bacteria such as Escherichia coli include the above-described pBI binary vectors and pUC vectors.

  Moreover, those skilled in the art can very easily select appropriate vectors other than these for carrying out the present invention. Depending on the vector used, many suitable transcription and translation elements can be used, such as constitutive or inducible promoters, transcription enhancer elements, transcription terminators, etc., which are known to those skilled in the art. is there.

  The gene of the present invention needs to be incorporated into a vector so that the function of the gene is expressed. Therefore, the vector may contain components such as a promoter, intron, enhancer, translation termination codon, terminator, poly A addition signal, 5′-UTR sequence, selection marker gene, etc. upstream, inside, or downstream of the gene. it can. These can be used in combination with known ones.

  As the promoter, a promoter capable of expressing DNA in a target host may be used. Such promoters are incorporated into various vectors that are commercially available as expression vectors, and can be isolated from the vectors. In addition, components such as introns, enhancers, translation termination codons, terminators, poly A addition signals, 5′-UTR sequences, and selectable marker genes are also incorporated into various vectors that are commercially available as expression vectors. These components can also be isolated from the vector.

  In order to insert a gene into a vector, first, a method in which purified DNA is cleaved with an appropriate restriction enzyme, inserted into a restriction enzyme site or a multicloning site of an appropriate vector DNA, and ligated to the vector is employed. .

6). Transformant “Transformation” means that by introducing DNA that is new (foreign) to a cell, the cell undergoes a permanent genetic change. If the cell is a mammalian cell, permanent genetic changes are achieved by introducing DNA into the cell's genome. A “transformant” can be easily obtained by transforming a target host using the recombinant vector.

  When the host is eukaryotic, DNA can be transfected into eukaryotic cells by the calcium phosphate method, microinjection, electroporation, liposome, or viral vector. Also, a eukaryotic cell is transfected with a DNA molecule encoding the protein of the present invention and a second DNA molecule encoding a selectable trait (for example, a herpes simplex virus thymidine kinase gene). Transformants can be selected. Transformed eukaryotic cells can express the protein of the present invention by culturing under a predetermined condition in a medium suitable for the growth of the cells.

  Examples of host cell strains that can be used as mammalian cells include CHO, VERO, BHK, HeLa, COS, MDCK, Jurkat, HEK-293, WI38, and the like. Examples of host cell strains that can be used as insect cells include Sf9. Examples of host cell strains that can be used as yeast cells include Saccharomyces cerevisiae.

  When the host is a prokaryotic cell (for example, E. coli), competent cells having the ability to take up DNA can be prepared by treating with the calcium chloride method by procedures well known to those skilled in the art. Transformation can also be performed by alternative methods such as electroporation. A prokaryotic cell together with a DNA molecule encoding the protein of the present invention and a second DNA molecule encoding a selectable trait (for example, a gene resistant to antibiotics such as ampicillin and kanamycin). Transfectants can be selected by transfection. The transformed prokaryotic cell can be cultured in a medium suitable for the growth of the cell (preferably in the presence of an antibiotic) under predetermined conditions to express the protein of the present invention.

  The mutant PKS1 of the present invention is preferably obtained by expressing DNA encoding the mutant PKS1 in a prokaryotic organism using the recombinant vector of the present invention. For example, when E. coli is used as a host, mutant PKS1 can be expressed by culturing E. coli on a large scale.

  Regarding the technique of isolation or purification after expressing the mutant PKS1 of the present invention, immunological methods using conventionally known means such as chromatographic separation, ammonium sulfate precipitation, and antigen or antibody (monoclonal antibody, polyclonal antibody) are used. This can be done by various separation methods.

  For example, the purification of the mutant PKS1 of the present invention from microorganisms such as Escherichia coli can be simplified by using nickel chelate chromatography with a label for purification in one step in the protein sequence. For example, a polyhistidine label composed of a plurality of (preferably 5 to 15) histidine residues is incorporated into the amino terminus or carboxyl terminus of the mutant PKS1 of the present invention and labeled with polyhistidine, and nickel chelate chromatography is used. Thus, protein can be efficiently isolated in one step. The mutant PKS1 of the present invention can also be designed to include a suitable enzyme cleavage site for the purpose of improving recovery.

7). Transformed plant body 5. Using the recombinant vector prepared in (1) above, a transformed plant body carrying the mutant PKS1 of the present invention can be prepared by transforming and regenerating cells of the target plant.

  In preparing a transformed plant body, various methods already reported and established can be used as appropriate, and preferable examples thereof include, for example, biological methods such as viruses and Agrobacterium. Examples include methods using Ti plasmids, Ri plasmids and the like as vectors. Physical methods include electroporation, polyethylene glycol, particle gun, microinjection (Plant Genetic Transformation and Gene Expression; a laboratory manual, J. Draper et al. al., Blackwell Scientific Publication (1988)), Silicon Whisker (Euphytica, 85, 75-80 (1995); In Vitro Cell. Dev. Biol., 31, 101-104 (1995); Plant Science, 132, 31 -43 (1998)), liposomes, vacuum infiltration (CR Acad. Sci. Paris, Life Science, 316: 1194 (1993)), floral dip, etc. For example, a method of introducing a child. The introduction method can be appropriately selected and used by those skilled in the art.

  In general, a gene introduced into a plant is integrated into the genome of the host plant, but in this case, a phenomenon called a position effect, in which the expression of the transgene is different due to different positions on the introduced genome, is seen. Confirmation of the transgene is necessary.

  Whether or not a gene has been incorporated into a plant can be confirmed by PCR, Southern hybridization, or the like. For example, DNA is prepared from a transformed plant, PCR is performed by designing DNA-specific primers. After performing PCR, the amplified product is subjected to electrophoresis such as agarose gel electrophoresis, stained with ethidium bromide solution, etc., and the amplified product is detected as a single band to confirm that it has been transformed. Can be confirmed.

  Examples of the plant used for transformation in the present invention include monocotyledonous plants such as rice and taro, dicotyledonous plants such as Arabidopsis thaliana and tobacco, and particularly preferred plant types include Arabidopsis thaliana and tobacco.

  In the present invention, as plant material to be transformed, for example, plant body, growth point, shoot primordia, meristem, leaf piece, stem piece, root piece, tuber piece, petiole piece, protoplast, callus, cocoon, Examples thereof include cells such as pollen, pollen tube, flower pattern piece, flower stem piece, petal, and sepal piece.

  In the case of targeting plant cells, a known tissue culture method may be used to regenerate a transformant from the obtained transformed cells. Such an operation can be easily performed by those skilled in the art by a method generally known as a method for regenerating plant cells from plant cells. For regeneration from plant cells to plant bodies, for example, references such as “Plant Cell Culture Manual” (Yasuyuki Yamada, Kodansha Scientific, 1984) can be referred to.

  The transformed plant of the present invention is not only the “T0 generation”, which is a regenerated generation that has undergone transformation treatment, but also the “T1 generation”, a progeny obtained from seeds of self-propagation or other breeding of the plant, Progeny plants such as the next generation (T2 generation) obtained by self-propagation or other breeding of "T1 generation" plants that have been found to be transgenic by selection or analysis by the Southern method, etc. It also includes generations and individuals that can be obtained by any means of cultivation and breeding based on the T1 generation, such as individuals that have maintained growth, and mutant individuals whose specific traits have changed from the next generation such as the T1 generation.

7). Production of tricyclic alkaloid having 6-7-6 fused ring structure using transformant (7-1) Method using transformed plant Transformed plant is produced under conditions suitable for the plant and alkaloid The target alkaloid is enriched by cultivating, harvesting and recovering the product under conditions suitable for the production of (eg, giving 2-carbamoylbenzoyl CoA derivatives and malonyl CoA derivatives as substrates at appropriate concentrations). It can be obtained in yield.

(7-2) Method of using transformant (excluding plant body) In another embodiment, the transformant of the present invention (excluding the plant body) is cultured in the presence of a substrate to produce 6-7- A tricyclic alkaloid having a 6-fused ring structure can be produced.

  As the transformant to be used, a transformant according to the purpose can be selected from the viewpoint of the growth rate of the transformant, the ease of setting the expression of mutant PKS1 and the enzyme reaction conditions, and the like. A suitable example is an E. coli transformant. Hereinafter, E. coli will be described as an example.

  As the substrate to be used, 2. It is as described in.

  The mutant PKS1 is expressed in the Escherichia coli as a foreign gene product in the transformed Escherichia coli, and the enzyme reaction proceeds using the substrate in the medium by continuing the cultivation. It is expected to accumulate inside or be released outside the cell. Although it does not specifically limit as reaction conditions, In presence of the substrate of predetermined concentration (for example, 1-1000 mM) in the culture solution (for example, LB (Luria-Bertani) culture medium) of pH 6-9, 20- Examples are 24 to 90 hours at 40 ° C. The culture can be scaled up using a bioreactor or the like and can be continued continuously.

  After completion of the reaction, the produced tricyclic alkaloid is recovered from the culture or culture medium and purified according to the purpose. Recovery and purification of the tricyclic alkaloid can be performed using means well known in the art.

  The present invention will be described in more detail with reference to examples below, but the present invention is not limited to these examples.

Example 1 Cloning of polyketide synthase (ABI94386.1) from Huperzia serrata
The target sequence was amplified by PCR using PrimeSTAR (registered trademark) HS DNA Polymerase and the following primers. Electrophoresis using 1.2% agarose gel, excise the 1.2 kbp band of the desired size, purify using QIAquick (registered trademark) Gel Extraction Kit (QIAGEN), ethanol precipitate, and use as insert DNA .
The oligonucleotide primer used was produced by Invitrogen.
5'-CTG CTG GTC GAC ATG ACA ATC AAG GGA-3 '(SalI site is underlined) (SEQ ID NO: 7)
5'-CCG CCG CTG CAG TCA AAT GTT GAT ACT TCT-3 '(PstI site is underlined) (SEQ ID NO: 8)
The purified DNA and pQE81L (QIAGEN) were subjected to restriction enzyme treatment using SalI and PstI, and the resulting restriction enzyme-cleaved fragment was purified by the DNA purification method described above. Using DNA Ligation Kit Ver.2.1 (TAKARA), a plasmid DNA for enzyme expression having a 6x histidine tag added to the N-terminus was prepared and transformed into M15 [pREP4] (QIAGEN).

Example 2 Production of Mutant Enzyme Mutation was performed by PCR using QuickChange ^™ XL Kit (Stratagene) with the following primers using wild type enzyme expression plasmid DNA (pET + insert DNA) as a template. The oligonucleotide primer used was produced by Invitrogen.
S348G Fw 5'-CGGCAACATGGGAAGCGCCTCAGTACTGTTTGTTTTGG-3 '(SEQ ID NO: 9)
S348G Rv 5'-CCAAAACAAACAGTACTGAGGCGCTTCCCATGTTGCCG-3 '(SEQ ID NO: 10)
Plasmid DNA introduced with the S348G mutation was transformed into expression E. coli BL21 (DE3) pLysS (NOVAGEN).

Example 3 Protein Expression in E. coli The E. coli transformed in Examples 1 and 2 was cultured with shaking in 1000 mL of Luria-Bertani medium containing 100 mg of Ampicillin until 37 ° C. and OD 600 = 0.6. Isopropylthio-β-D-galactoside (IPTG) was added to a final concentration of 1 mM and cultured with shaking at 23 ° C. for 12 hours.

Example 4 Protein Extraction and Purification After collecting the enzyme-induced E. coli of Example 3, the cells were suspended in 40 mM potassium phosphate buffer (KPB), pH 7.9 containing 0.1 M NaCl and 5 mM imidazole, and E. coli cells were used using a sonicator. Was crushed. After crushing, centrifugation was performed at 10,000 rpm, 4 ° C. for 30 minutes, and the supernatant was used as a crude protein extract. Subjecting the crude extract to ^{Ni Sepharose TM 6 Fast Flow (GE} Healthcare), washed with 0.5M NaCl and 40 mM imidazole-containing 40mM KPB, 10% (w / v) glycerol and 500 mM imidazole-containing 15 mM KPB, pH 7.5 To obtain a purified enzyme. The purified enzyme solution was desalted and concentrated using an Amicon Ultra-4 (Ultracel-10k) filter using 0.1 mM HEPES, pH 7.0 containing 5% (w / v) glycerol and 0.2M NaCl. went.

Example 5 Synthesis of enzyme reaction substrate (2-carbamoylbenzoyl CoA)
1) Synthesis of activated ester Under argon, 3 g of phthalamic acid, 3.6 g of N-hydroxysuccinimide and 8.0 g of WSCI · H ₂ O were mixed in acetonitrile and reacted at room temperature for 2 hours. After the reaction, the mixture was separated with CH ₂ Cl ₂ and water to recover the organic layer, and the solvent was distilled off under reduced pressure. The obtained liquid was purified with a silica gel column (hexane: ethyl acetate = 10: 1) to obtain 200 mg of N-hydroxysuccinimide ester (NHS ester) of phthalamic acid.
2) Synthesis of CoA ester An acetone solution in which 60 mg of the obtained NHS ester was dissolved was mixed with an aqueous solution in which 60 mg of Coenzyme A and 60 mg of sodium bicarbonate were dissolved, and the mixture was reacted at 4 ° C for 24 hours. Acetone was distilled off under reduced pressure, and the mixture was partitioned between ethyl acetate and water, and the aqueous layer was recovered. The CoA ester contained in the aqueous layer was purified with a C18 Sep-Pack column (Waters).
3) Purification method Ammonium acetate was added to the aqueous layer obtained by liquid separation to prepare a 4% aqueous ammonium acetate solution (sample). After washing the column with methanol and water, an equal volume of 4% aqueous ammonium acetate was eluted from the sample to equilibrate the column. The sample was then eluted, followed by an equal volume of 4% ammonium acetate. Finally, 3 times the amount of water as the sample was eluted and collected. Water was removed by lyophilization to obtain 12 mg of CoA ester.

Example 6 Enzyme Reaction and Compound Analysis Mutant enzyme 20 μg, CoA ester 25 μg, malonyl CoA 50 μg were mixed in 500 μl of 0.1 M phosphate buffer (pH 6.5) and reacted at 30 ° C. for 24 hours. After adding 20 μl of 20% HCl, the enzyme reaction product was extracted with ethyl acetate. The obtained substance was measured by LC-ESIMS using Bruker Daltonics esquire4000. Using a TSK-gel ODS-80TsQA (2.0 × 150 mm) (Tosoh) column with a flow rate of 0.2 mL / min, water containing 1% acetic acid and methanol (MeOH), 0-5 min, 30% MeOH; 5-17 min, 30-60% MeOH; 17-25min, 60% MeOH; 25-27min, 60-70% MeOH; 27-35min, 70% MeOH; 35-40min, 100% MeOH, measured at 280nm detection wavelength It was.

Example 7 Structure Determination of Compound The structure of the obtained product having a molecular weight (MW) of 255 was determined by NMR.
NMR measuring instrument JNM-ECX 500 (JEOL)
¹ H-NMR (500 MHz, DMSO) (ppm): 9.38 (s, 1H, OH), 8.27 (s, 1H, CONH), 7.91 (d, J = 7.0 Hz, 1H, Ar), 7.51 (t, J = 7.0, 7.0 Hz, 2H, Ar), 7.40 (d, J = 7.0 Hz, 1H, Ar), 6.85 (d, J = 2.0 Hz, 1H, Ar), 5.06 (d, J = 2.0 Hz, 1H , Ar)
¹³ C-NMR (125 MHz, DMSO) (ppm): 188.5 (C = O), 172.1 (Ar), 165.1 (CONH), 163.5 (Ar), 159.8 (Ar), 137.0 (Ar), 136.2 (Ar) , 132.8 (Ar), 132.7 (Ar), 121.3 (Ar), 121.3 (Ar), 121.0 (Ar), 95.5 (Ar), 94.6 (Ar), 86.3 (Ar)
The molecular weight was measured by Bruker Daltonics microTOF.
m / z 256.0604 (M + H) ⁺ , C ₁₄ H ₁₀ NO ₄
The structural formula is as follows.

Example 8 Antibacterial Assay A liquid medium containing bacteria was placed in a well together with the compound whose structure was determined in Example 7 and cultured, and whether or not there was antibacterial activity was determined by the presence or absence of colony formation. The bacterium used in this assay was methicillin sensitive S. aureus (MSSA).
Bacteria were cultured in LB liquid medium at 37 ° C. for 16 hours. After culturing, 10 μl of the bacterial solution was mixed with 10 ml of Mueller Hinton medium, and 100 μl of each well was put into a 96-well plate. The column at one end was 198 μl. Compounds 1 and 2 were each dissolved in DMSO to give 2.4 mg / ml. Ampicillin (10 mg / ml) was used as a positive control, and 0.9% physiological saline was used as a negative control.
2 μl of ampicillin, sample, and physiological saline were added to each column at one end and diluted 100 times to obtain the maximum concentration. Thereafter, the mixture was diluted half-fold while transferring half by half to the next well and incubated at 37 ° C. for 16 hours.
As a result, bacterial growth was observed at all concentrations assayed, and bactericidal activity could not be confirmed at the current concentration. However, in the wells to which Compound 1 was added, colony formation was inhibited and biofilm inhibition was observed. Compound 1 is a compound of formula (II), Compound 2 is an enzyme reaction product obtained together with the compound of formula (II) by the enzyme reaction of Example 6, and is a compound represented by the following formula.
Minimum Inhibitory Concentration (MIC) of the compound of formula (II) was 12.5 μg / ml.

Creation of unnatural new alkaloid skeleton using nitrogen-containing artificial substrate PKS1 derived from Huperzia serrata, which was previously cloned by the present inventors, is a novel type III PKS having a relatively large substrate binding site, coumaroyl After sequentially condensing three molecules of malonyl CoA using CoA as the starting substrate, chalcone is generated (FIG. 1A). In this case, when a chemically synthesized CoA thioester of 2-carbamoylbenzoic acid was used as an initiation substrate, two molecules of malonyl CoA (or methylmalonyl CoA) were condensed sequentially, followed by a 6-5-6 condensed ring. It was found that a novel tricyclic unnatural alkaloid having a structure was obtained as a single product in a yield of 80% (FIG. 1B). On the other hand, in contrast to the reaction shown in FIG. 1B described above, the S348G point mutant enzyme with an enlarged active center cavity based on the X-ray crystal structure analysis of PKS1 sequentially condensed three molecules of malonyl CoA. Later, it was found that a novel tricyclic unnatural alkaloid having a 6-7-6 fused ring structure was produced (FIG. 1C).

  The present inventors have developed rational control of enzyme catalytic function based on X-ray crystal structure analysis of type III PKS, which has been considered difficult until now. If an artificial substrate into which a heteroatom such as N is introduced is allowed to act on a type III PKS that catalyzes a simple “carbonyl chemistry” composed of C, H, and O atoms, a new one utilizing the basicity of the N atom It is also possible to form a C—N or C—C bond. Using various artificial substrates and malonyl CoA or derivatives thereof introduced with such heteroatoms, an intramolecular cyclization reaction proceeds through the formation of a Schiff base from a highly reactive β-polyketomethylene intermediate, resulting in a complicated It is possible to create a novel supernatural biocatalyst that efficiently constructs the alkaloid skeleton at once.

  The method for producing alkaloids using the mutant type III polyketide synthase of the present invention can efficiently produce non-natural new alkaloids and the like under clean and mild conditions as compared with organic synthesis methods. The mutant type III polyketide synthase and the production method using the same of the present invention provide various novel alkaloids useful as raw materials for pharmaceuticals, cosmetics, and foods (functional foods) by selecting appropriate substrates. Can do.

Claims (10)

Formula (I):
(Where
R ¹ , R ² , R ³ and R ⁴ are the same or different and each represents a hydrogen atom, a halogen atom, a C _1-6 alkyl group or an alkoxy group,
R ⁵ represents a hydrogen atom, a C _1-6 alkyl group, a C _2-6 alkenyl group, a C _3-6 cycloalkyl group, an aryl group, an aralkyl group, a heterocyclic group or an acyl group,
R ⁶ is the same or different and represents a hydrogen atom, a C _1-6 alkyl group or an alkoxy group,
Furthermore, a nitrogen atom may be introduced at the position of the carbon atom to which R ¹ , R ² , R ³ or R ⁴ is bonded. Or a salt thereof. The compound or a salt thereof according to claim ¹ , wherein R ¹ , R ² , R ³ and R ⁴ are hydrogen atoms, R ⁵ is a hydrogen atom and R ⁶ is a hydrogen atom. Using a mutant type III polyketide synthase as a catalyst, the following formula:
(Where
R ¹ , R ² , R ³ and R ⁴ are the same or different and each represents a hydrogen atom, a halogen atom, a C _1-6 alkyl group or an alkoxy group,
R ⁵ represents a hydrogen atom, a C _1-6 alkyl group, a C _2-6 alkenyl group, a C _3-6 cycloalkyl group, an aryl group, an aralkyl group, a heterocyclic group or an acyl group,
Furthermore, the C—R ¹ , C—R ² , C—R ³ or C—R ⁴ bond may be substituted with a nitrogen atom. 2-carbamoylbenzoyl CoA derivative represented by the following formula:
(In the formula, R ⁶ represents a hydrogen atom, a C _1-6 alkyl group or an alkoxy group.) The production of the compound according to claim 1 or 2 comprising a step of subjecting the malonyl CoA derivative to a condensation reaction. Method.   The production method according to claim 3, wherein the mutant type III polyketide synthase is a point mutant enzyme from serine residue to glycine or alanine residue constituting the active center cavity of wild type III polyketide synthase.   The production method according to claim 4, wherein the wild type III polyketide synthase is a Huperzia serrata-derived wild type III polyketide synthase. Mutant type III polyketide synthase comprising the following amino acid sequences (A1) to (A3):
(A1) the amino acid sequence of SEQ ID NO: 2;
(A2) the amino acid sequence of SEQ ID NO: 3; or (A3) 1 to several amino acids are further substituted, deleted or added at positions other than position 348 of the amino acid sequence of any of the above (A1) or (A2) An amino acid sequence having the ability to synthesize a tricyclic alkaloid in which the protein comprising the amino acid sequence after the mutation is introduced has a 6-7-6 condensed ring structure.   A gene encoding the mutant type III polyketide synthase according to any one of claims 3 to 6. The gene according to claim 7, comprising the following polynucleotides (B1) to (B3):
(B1) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 5;
(B2) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 6; or (B3) a polynucleotide comprising a nucleotide sequence having 70% or more sequence identity to any of the nucleotide sequences of (B1) or (B2) above. And a polynucleotide encoding an enzyme having the ability to synthesize a tricyclic alkaloid having a 6-7-6 fused ring structure.   A recombinant vector containing the gene according to claim 7 or 8.   A transformant into which the recombinant vector according to claim 9 has been introduced.