# Patent

## Title
Method for producing ambrein

## Abstract

Provided is a method for producing ambrein, comprising reacting tetraprenyl-β-curcumene cyclase with 3-deoxyachilleol A to obtain ambrein.

## Background
RELATED APPLICATION DATA
This application is a National Stage Application under 35 U.S.C. 371 of co-pending PCT application number PCT/JP2014/071333 designating the United States and filed Aug. 12, 2014; which claims the benefit of JP application number 2013-184143 and filed Sep. 5, 2013 each of which are hereby incorporated by reference in their entireties.
TECHNICAL FIELD
The present invention relates to a method for producing ambrein.
BACKGROUND ART
Ambergris is a high grade perfume which has been used from around the seventh century, and has been also used as a Chinese medicinal drug. Ambergris is considered to be formed by sperm whales due to lithification of indigestibles of foods (octopuses, squids, or the like) by gastrointestinal secretions and excreted therefrom. The detailed production mechanism thereof, however, is unknown. The principal component of ambergris is ambrein, and it is considered that ambrein is subjected to oxidative decomposition by sunlight and oxygen while the ambergris is floating on the ocean's surface, thereby producing compounds having a variety of fragrances.
Although ambrein, the principal component of ambergris, is used as perfumes or pharmaceuticals, it is impossible to obtain a large amount of ambrein as a natural product. A variety of organic synthesis methods have thus been proposed.
For example, as a method of producing (+)-ambrein easily, efficiently, and inexpensively, Japanese Patent Application Laid-Open (JP-A) No. H10-236996 discloses a method comprising a step of producing a new sulfonic acid derivative from ambrenolide and coupling therewith an optically active γ-cyclogeranyl halide.
Tetrahedron Asymmetry, (2006) Vol. 17, pp. 3037-3045 discloses a method of obtaining ambrein by a convergent synthesis using a Julia coupling reaction between 2-((1R,2R,4αS,8αS)-2-(methoxymethoxy)-2,5,5,8α-tetramethyl decahydronaphthalene-1-yl)acetaldehyde synthesized from (±)(5,5,8α-trimethyloctahydro-1H-spiro[naphthalene-2,2′-oxirane]-1-yl)methanol and 5-((4-((S)-2,2-dimethyl-6-methylenecyclohexyl)butane-2-yl)sulfonyl)-1-phenyl-1H-tetrazole synthesized from (±)methyl 6-hydroxy-2,2-dimethyl cyclohexanecarboxylate.
A method in which 3-deoxyachilleol A which is a monocyclic triterpene is obtained from squalene by using a mutant enzyme (D377C, D377N, Y420H, Y420W, or the like) of a squalene-hopene cyclase is also known (Biosci. Biotechnol. Biochem., (1999) Vol. 63, pp. 2189-2198, Biosci. Biotechnol. Biochem., (2001) Vol. 65, pp. 2233-2242, and Biosci. Biotechnol. Biochem., (2002) Vol. 66, pp. 1660-1670).
It is also reported that tetraprenyl-β-curcumene cyclase is a bifunctional enzyme which is involved in two reactions: a reaction in which a tetracyclic C35 terpenol is produced from tetraprenyl-β-curcumene; and a reaction in which a bicyclic triterpene is produced from squalene (J. Am. Chem. Soc., (2011) Vol. 133, pp. 17540-17543).
SUMMARY OF INVENTION
Technical Problem
Since conventional organic synthesis methods of ambrein involve many synthesis stages, the reaction systems are complex, and therefore commercialization thereof has not been accomplished. In addition, no specific enzyme that is involved in production of ambrein is known.
Accordingly, an object in the invention is to provide a method for producing ambrein in which ambrein can be produced more easily than conventionally known organic synthesis methods.
Solution to Problem
The invention is as follows:
[1] A method for producing ambrein, comprising reacting a tetraprenyl-β-curcumene cyclase with 3-deoxyachilleol A to obtain ambrein.
[2] The method for producing ambrein according to [1], wherein the tetraprenyl-β-curcumene cyclase is derived from a bacterium of the genus Bacillus. 
[3] The method for producing ambrein according to [1] or [2], wherein the tetraprenyl-β-curcumene cyclase is derived from any one of Bacillus megaterium, Bacillus subtilis or Bacillus licheniformis. 
[4] The method for producing ambrein according to any one of [1] to [3], further comprising reacting a mutant squalene-hopene cyclase, which can produce 3-deoxyachilleol A from squalene, with squalene to obtain 3-deoxyachilleol A.
[5] The method for producing ambrein according to [4], wherein the mutant squalene-hopene cyclase has an amino acid substitution at at least one position selected from the group consisting of position 377, position 420, position 607, and position 612 in the amino acid sequence represented by SEQ ID NO:1.
[6] The method for producing ambrein according to [4] or [5], wherein the mutant squalene-hopene cyclase has the amino acid sequence represented by any one of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8.
[7] The method for producing ambrein according to any one of [1] to [6], wherein the tetraprenyl-β-curcumene cyclase has the amino acid sequence represented by any one of SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:18.
Effects of Invention
According to the invention, there is provided a method for producing ambrein in which ambrein can be produced more easily than conventionally known organic synthesis methods.


DESCRIPTION OF EMBODIMENTS
The term “step” herein includes not only independent steps. Even when a step cannot be clearly distinguished from other steps, the step is included in this term as long as the expected purpose of the step can be achieved.
Further, each numerical range represented using “to” herein means the range having the numerical values described before and after the “to” as the minimum value and the maximum value, respectively.
When the composition contains a plurality of substances corresponding to the each component, the amount of the each component in the composition herein means the total amount of the plurality of substances contained in the composition unless otherwise specified.
In the invention, each amino acid residue in an amino acid sequence may be represented by the single-letter code (for example, “G” represents a glycine residue) or three-letter code (for example, “Gly” represents a glycine residue), which are well known in the art.
In the invention, “%” as used in relation to the amino acid sequence of a protein or a polypeptide is based on the number of amino acid residues, unless otherwise specified.
In the following, embodiments in the invention will now be described. These descriptions and Examples are for illustration of the invention and should not limit the scope of the present invention.
The method of producing ambrein of the present invention is a method comprising: reacting a tetraprenyl-β-curcumene cyclase with 3-deoxyachilleol A to obtain ambrein.
In the invention, ambrein can be easily produced since tetraprenyl-β-curcumene cyclase is reacted with 3-deoxyachilleol A to obtain ambrein.
Although tetraprenyl-β-curcumene cyclase has been known to be an enzyme which produces a bicyclic terpenol from squalene which is a C30 linear unsaturated hydrocarbon, it has been found that 3-deoxyachilleol A which comprises a monocycle at one end can be employed as a substrate. It has been also found that, when 3-deoxyachilleol A is utilized as a substrate, a tetraprenyl-β-curcumene cyclase selectively forms a ring on the end of the 3-deoxyachilleol A on which a ring has not been formed to produce a compound which is cyclized at both ends. The invention has been made based on these findings. Due to the above-described activity of tetraprenyl-β-curcumene cyclase, ambrein can be produced easily using 3-deoxyachilleol A comprising a monocycle on one end as a material by using one enzyme.
The method for producing ambrein in the invention comprises: reacting tetraprenyl-β-curcumene cyclase with 3-deoxyachilleol A to obtain ambrein (hereinafter referred to as an “ambrein production step”). The method comprises other steps as needed.
Ambrein is (1R,4αα)-1-[(E)-6-[(S)-2,2-dimethyl-6-methylenecyclohexyl]-4-methyl-3-hexenyl]decahydro-2,5,5,8αβ-tetramethylnaphthalene-2α-al, and a compound which is cyclized at both ends. Ambrein has a composition formula of C30H52O and a molecular weight of 428.745, which is a triterpene alcohol having the following structure (CAS registration number:473-03-0):




In the method for producing ambrein in the invention, 3-deoxyachilleol A is utilized as a substrate of tetraprenyl-β-curcumene cyclase.
3-deoxyachilleol A is (S)-1,1-dimethyl-3-methylene-2-((3E,7E,11E)-3,8,12,16-tetramethyl heptadeca-3,7,11,15-tetraen-1-yl)cyclohexane, and a compound which is cyclized at one end. 3-deoxyachilleol A has a composition formula of C30H50, and the following structure. This compound is used in the invention as a material for producing ambrein. A method of obtaining 3-deoxyachilleol A is not particularly restricted. 3-deoxyachilleol A may be obtained by chemical synthesis, or may be obtained from a known compound by using enzyme reaction.




The production method in the invention preferably further comprises: reacting a mutant squalene-hopene cyclase with squalene to obtain 3-deoxyachilleol A (hereinafter, referred to as a “3-deoxyachilleol A production step”). This makes it possible to efficiently and easily produce ambrein through two enzyme reactions using a mutant squalene-hopene cyclase and a tetraprenyl-β-curcumene cyclase by using inexpensive squalene as a material.
[3-Deoxyachilleol A Production Step]
In a 3-deoxyachilleol A production step, a mutant squalene-hopene cyclase, which can produce 3-deoxyachilleol A from squalene, is reacted with squalene to obtain 3-deoxyachilleol A. The term “mutant squalene-hopene cyclase” herein refers to a mutant squalene-hopene cyclase, which can produce 3-deoxyachilleol A from squalene, unless otherwise specified.
In the invention, a mutant squalene-hopene cyclase is an enzyme that is obtained by modifying wild-type squalene-hopene cyclase and that can produce 3-deoxyachilleol A from squalene. A wild-type squalene-hopene cyclase is known to be an enzyme (ECS. 4. 99. -) which cyclizes squalene to produce a pentacyclic hopene or hopanol, which is widely found in a prokaryote such as the genus Alicyclobacillus, the genus Zymomonas, or the genus Bradyrhizobium. The amino acid sequence of a wild-type squalene-hopene cyclase is known. For example, the amino acid sequence (SEQ ID NO:1) (Table 1) of wild-type squalene-hopene cyclase of Alicyclobacillus acidocaldarius is shown in GenBank Accession No.: AB007002.







TABLE 1





Wild-type squalene-hopene cyclase derived from


Alicyclobacillus acidocaldarius

















MAEQLVEAPA YARTLDRAVE YLLSCQKDEG YWWGPLLSNV



 



TMEAEYVLLC HILDRVDRDR MEKIRRYLLH EQREDGTWAL



 



YPGGPPDLDT TIEAYVALKY IGMSRDEEPM QKALRFIQSQ



 



GGIESSRVFT RMWLALVGEY PWEKVPMVPP EIMFLGKRMP



 



LNIYEFGSWA RATVVALSIV MSRQPVFPLP ERARVPELYE



 



TDVPPRRRGA KGGGGWIFDA LDRALHGYQK LSVHPFRRAA



 



EIRALDWLLE RQAGDGSWGG IQPPWFYALI ALKILDMTQH



 



PAFIKGWEGL ELYGVELDYG GWMFQASISP VWDTGLAVLA



 



LRAAGLPADH DRLVKAGEWL LDRQITVPGD WAVKRPNLKP



 



GGFAFQFDNV YYPDVDDTAV VVWALNTLRL PDERRRRDAM



 



TKGFRWIVGM QSSNGGWGAY DVDNTSDLPN HIPFCDFGEV



 



TDPPSEDVTA HVLECFGSFG YDDAWKVIRR AVEYLKREQK



 



PDGSWFGRWG VNYLYGTGAV VSALKAVGID TREPYIQKAL



 



DWVEQHQNPD GGWGEDCRSY EDPAYAGKGA STPSQTAWAL



 



MALIAGGRAE SEAARRGVQY LVETQRPDGG WDEPYYTGTG



 



FPGDFYLGYT MYRHVFPTLA LGRYKQAIER R










The mutant squalene-hopene cyclase is an enzyme which contains a mutation in the amino acid sequence of a wild-type squalene-hopene cyclase and which has an activity by which 3-deoxyachilleol A having a monocycle can be produced from squalene. It is known that, in a case that a mutation is contained in the amino acid sequence of a wild-type squalene-hopene cyclase, an incomplete cyclization reaction occurs, and that a monocycle compound can be produced when squalene is reacted therewith while a wild-type squalene-hopene cyclase without a mutation produces a pentacyclic compound.
From the viewpoint of production efficiency of 3-deoxyachilleol A, the mutant squalene-hopene cyclase is preferably a mutant squalene-hopene cyclase having an amino acid substitution(s) at least one site selected from the group consisting of position 377, position 420, position 607, and position 612 in the amino acid sequence represented by SEQ ID NO:1, more preferably a mutant squalene-hopene cyclase having a mutation(s) at one or two sites selected from the group consisting of position 377, position 420, position 607, and position 612 in the amino acid sequence represented by SEQ ID NO:1, and still more preferably a mutant squalene-hopene cyclase having a mutation at any one of sites selected from the group consisting of position 377, position 420, position 607, and position 612 in the amino acid sequence represented by SEQ ID NO:1.
The above-described mutation sites in the mutant squalene-hopene cyclase are relative ones. For example, “position 377” is actually position 376 when one amino acid residue on the N terminal side of the position 377 is deleted. When the amino acid sequence of a wild-type squalene-hopene cyclase includes a species-specific variation irrespective of the function of squalene-hopene cyclase itself depending on the species, the above-described mutation sites should be read as sites on which an alignment has been performed in a known method in the art.
Amino acid substitution in a mutant squalene-hopene cyclase is that an amino acid residue of a wild-type squalene-hopene cyclase is substituted with another amino acid residue. The other amino acid residue with which the amino acid residue of the wild-type squalene-hopene cyclase is to be substituted may be any amino acid residue as long as it is an amino acid residue by which a mutant squalene-hopene cyclase after the substitution can produce 3-deoxyachilleol A from squalene.
The mutation site and the substituted amino acid of a mutant squalene-hopene cyclase are preferably the following mutation site in the amino acid sequence represented by SEQ ID NO:1 and substituted amino acid.
(i) The aspartic acid residue (D) at position 377 is substituted with a cysteine residue (C) or an asparagine residue (N).
(ii) The tyrosine residue (Y) at position 420 is substituted with a histidine residue (H) or a tryptophan residue (W).
(iii) The leucine residue (L) at position 607 is substituted with a phenylalanine residue (F) or a tryptophan residue (W).
(iv) The tyrosine residue (Y) at position 612 is substituted with an alanine residue (A).
The mutant squalene-hopene cyclase is preferably an enzyme having at least one substitution selected from the group consisting of the above-described (i) to (iv) in the amino acid sequence represented by SEQ ID NO:1, more preferably an enzyme having one or two substitutions selected from the group consisting of the above-described (i) to (iv) in the amino acid sequence represented by SEQ ID NO:1, and further preferably an enzyme having one substitution selected from the group consisting of the above-described (i) to (iv) in the amino acid sequence represented by SEQ ID NO:1.
The mutant squalene-hopene cyclase may have an amino acid sequence wherein one or more amino acid residues are substituted, deleted, inserted, or added at a site(s) in the amino acid sequence of the wild-type squalene-hopene cyclase other than the above-described mutation sites as long as a function of producing 3-deoxyachilleol A from squalene is maintained. In this case, the number of the one or several amino acid residues which is/are substituted, deleted, inserted, or added varies depending on the positions of the amino acid residue(s) in the spatial structure of the protein and the types of the amino acid residue(s) or the like. Specifically, the number thereof is preferably 1 to 20, more preferably 1 to 10, and further preferably 1 to 5.
The origin of the mutant squalene-hopene cyclase is not particularly limited, and the mutant squalene-hopene cyclase is preferably derived from, for example, a bacterium of the genus Alicyclobacillus, a bacterium of the genus Zymomonas, or a bacterium of the genus Bradyrhizobium. From the viewpoint of enzyme activity, the mutant squalene-hopene cyclase is more preferably a mutant squalene-hopene cyclase derived from a bacterium of the genus Alicyclobacillus, and particularly preferably a mutant squalene-hopene cyclase derived from Alicyclobacillus acidocaldarius among others.
From the viewpoint of enzyme activity, the mutant squalene-hopene cyclase is preferably polypeptides A to G (SEQ ID NOs:2 to 8) listed below. In Table 2, the amino acid residues of the polypeptides are the same as the amino acid residue in the amino acid sequence represented by SEQ ID NO:1 except for the mutations represented by “mutation”.










TABLE 2





Polypeptide





Name
Origin
Mutation
SEQ ID No.







A
Alicyclobacillus acidocaldarius
D377C
2


B
Alicyclobacillus acidocaldarius
D377N
3


C
Alicyclobacillus acidocaldarius
Y420H
4


D
Alicyclobacillus acidocaldarius
Y420W
5


E
Alicyclobacillus acidocaldarius
L607F
6


F
Alicyclobacillus acidocaldarius
L607W
7


G
Alicyclobacillus acidocaldarius
Y612A
8









The polypeptides A to G which are mutant squalene-hopene cyclases respectively encompass polypeptides which have the amino acid sequences represented by SEQ ID NOs:2 to 8 wherein one or several amino acid residues are substituted, deleted, inserted, or added, and in which a function of producing 3-deoxyachilleol A from squalene is maintained. The number of amino acid residues which are substituted, deleted, inserted, or added in each of the amino acid sequences represented by SEQ ID NOs:2 to 8 is, specifically, preferably 1 to 20, more preferably 1 to 10, and further preferably 1 to 5.
The polypeptides A to G which are mutant squalene-hopene cyclases respectively encompass polypeptides which have sequence identity of, for example, 80% or higher, preferably 90% or higher, more preferably 95% or higher, more preferably 97% or higher, more preferably 98% or higher, and particular preferably 99% or higher, to the whole amino acid sequences each represented by SEQ ID NOs:2 to 8, and in which a function of producing 3-deoxyachilleol A from squalene is maintained.
A polynucleotide which can express a mutant squalene-hopene cyclase can be obtained on the basis of information on the sequence of the wild-type mutant squalene-hopene cyclase. Examples of the polynucleotide which can express a mutant squalene-hopene cyclase include polynucleotides A to G having the base sequences represented by SEQ ID NOs:9 to 15 (Table 3). In Table 3, the base sequences are the same as the base sequence (GenBank Accession No.: AB007002) of the wild-type squalene-hopene cyclase gene of Alicyclobacillus acidocaldarius except for the sites listed in “mutation site”.











TABLE 3





Polynucleotide






Name
Mutation
Mutation Site
Base
SEQ ID No.



















A
D377C
1129-1131
gac→tgc
9


B
D377N
1129-1131
gac→aac
10


C
Y420H
1258-1260
tac→cac
11


D
Y420W
1258-1260
tac→tgg
12


E
L607F
1819-1821
ctc→ttc
13


F
L607W
1819-1821
ctc→tgg
14


G
Y612A
1834-1836
tac→gcc
15









The polynucleotides A to G encompass respectively polynucleotides which have the base sequences represented by SEQ ID NOs:9 to 15 wherein one or several bases are substituted, deleted, inserted, or added, and which encode a polypeptide in which a function of producing 3-deoxyachilleol A from squalene is maintained. The number of bases which are substituted, deleted, inserted, or added in each of the base sequences represented by SEQ ID NOs:9 to 15 is, specifically, preferably 1 to 20, more preferably 1 to 10, and further preferably 1 to 5.
The polynucleotides A to G respectively encompass polynucleotides which have sequence identity of, for example, 80% or higher, preferably 90% or higher, more preferably 95% or higher, more preferably 97% or higher, more preferably 98% or higher, and particular preferably 99% or higher, to the whole base sequences each represented by SEQ ID NOs:9 to 15, and which encode polypeptides in which a function of producing 3-deoxyachilleol A from squalene is maintained.
The polynucleotides A to G respectively encompass polynucleotides which hybridize with complementary strands of the base sequences represented by SEQ ID NOs:9 to 15 under stringent conditions, and which encode a polypeptide in which a function of producing 3-deoxyachilleol A from squalene.
Hybridization can be performed according to a known method or a method according to a known method, such as a method described in Molecular Cloning 3rd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 2001). The stringent conditions mean conditions under which a specific hybrid is formed while nonspecific hybrids are not formed. Typical examples of the stringent conditions include conditions under which hybridization is performed with a potassium concentration of about 25 mM to about 50 mM and a magnesium concentration of about 1.0 mM to about 5.0 mM. In the invention, examples of the conditions include conditions under which hybridization is performed in Tris-HCl buffer (pH 8.6), 25 mM KCl and 1.5 mM MgCl2, but the conditions are not limited thereto. Other examples of the stringent conditions include those described in Molecular Cloning 3rd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 2001). Those skilled in the art can easily select stringent conditions by changing the conditions of the hybridization reaction such as the concentrations of salts condition of the hybridization reaction liquid.
A recombinant vector which is used for expressing a polynucleotide which encodes a mutant squalene-hopene cyclase is not particularly restricted, and examples thereof include a vector which can be expressed by Escherichia coli such as pET-3a, a vector which can be expressed by Bacillus subtilis such as pHT01, and a vector which can be expressed by yeast such as pYES2. By introducing a polynucleotide encoding a mutant squalene-hopene cyclase into such a vector, an enzyme expression vector can be obtained. A host bacterium which is a target for introducing an enzyme expression vector can be appropriately selected according to the type of a recombinant vector to be used, and examples thereof include Escherichia coli such as BL21 (DE3), Bacillus subtilis such as strain 168, and yeast such as Saccharomyces cerevisiae. 
The recombinant vector may contain, as needed, a promoter, a splicing signal, a poly(A) addition signal, a selection marker, a ribosome binding sequence (SD sequence), a terminator such as NOS, and/or the like. As the selection marker, a known one such as an antibiotic resistance gene such as a kanamycin resistance gene, an ampicillin resistance gene or a tetracycline resistance gene is used without particular restriction.
The recombinant vector may contain a reporter gene for confirming that an objective gene is introduced. Examples of such a reporter gene include a GUS (β-glucuronidase) gene, a luciferase gene, and a GFP (green fluorescent protein) gene.
The mutant squalene-hopene cyclase is produced by culturing a transformant that is obtained by introducing an enzyme expression vector into a bacterium. A culture medium used for culturing a transformant may be a culture medium which is usually used, and is appropriately selected according to the type of a host. For example, when Escherichia coli are cultured, an LB medium or the like is used. An antibiotic may be added to a culture medium according to the type of a selection marker.
The mutant squalene-hopene cyclase may be obtained by extraction followed by purification from a culture medium which has been obtained by culturing a transformant capable of expressing the enzyme. An extraction liquid containing the enzyme, which has been extracted from a transformant in a culture medium, may be used as it is. As a method of extracting an enzyme from a transformant, a known method may be applied. A step of extracting an enzyme may comprise, for example, crushing a transformant in an extraction solvent and separating cell contents from crushed pieces of the transformant. The obtained cell contents contain a target mutant squalene-hopene cyclase. Cell contents obtained by extracting from a cell and separating from crushed pieces of the cell are herein referred to as a “cell-free extract”.
Regarding a method of crushing a transformant, a method of separating cell contents from crushed pieces of a microorganism, the composition of an extraction solvent, and pH conditions, those identical to the description of the ambrein production step described below are applied as they are.
Mutant squalene-hopene cyclases may be used singly, or in combination of two or more kinds thereof.
The conditions of a reaction between a mutant squalene-hopene cyclase and squalene are not particularly restricted as long as the conditions are such that an enzyme reaction can be proceeded. For example, the reaction temperature and the reaction time may be appropriately selected based on the activity of a mutant squalene-hopene cyclase or the like. From the viewpoint of reaction efficiency, the reaction temperature and the reaction time may be, for example, from 4° C. to 100° C. and from 0.1 hour to 48 hours, and preferably 30° C. to 60° C. and 16 hours to 24 hours. From the viewpoint of reaction efficiency, the pH is, for example, from 3 to 10, and preferably from 6 to 8.
A reaction solvent is not particularly restricted as long as the reaction solvent does not inhibit the enzyme reaction, and a buffer or the like which is usually used can be used. For example, the same solvent as an extraction solvent which is used in a step of extracting the enzyme can be used. An extraction liquid (for example, cell-free extract) containing a mutant squalene-hopene cyclase may be used as it is as an enzyme liquid in the reaction.
From the viewpoint of reaction efficiency, in a production reaction of 3-deoxyachilleol A, the concentration ratio between a mutant squalene-hopene cyclase and squalene which is the substrate thereof in a production reaction of 3-deoxyachilleol A is preferably from 10 to 10000, more preferably from 100 to 5000, still more preferably from 1000 to 3000, and still further preferably from 1000 to 2000 in terms of the molar concentration ratio (substrate/enzyme) of the substrate to the enzyme.
From the viewpoint of reaction efficiency, the concentration of squalene to be used for an enzyme reaction is preferably from 0.000001% by mass to 0.002% by mass, and more preferably from 0.00001% by mass to 0.0002% by mass based on the total mass of the reaction solvent.
3-deoxyachilleol A obtained by a reaction using a mutant squalene-hopene cyclase can be purified by a known method, and can then be subjected to a reaction with a tetraprenyl-β-curcumene cyclase.
The purification method of 3-deoxyachilleol A is not particularly restricted as long as 3-deoxyachilleol A in a reaction liquid can be taken out, and a purification method which is usually used may be appropriately selected. Specific examples of the purification method include solvent extraction, recrystallization, distillation, column chromatography, and high performance liquid chromatography (HPLC).
A step of reaction between a mutant squalene-hopene cyclase and squalene may be repeated a plurality of times. This can increase the yield of 3-deoxyachilleol A. In a case that a plurality of reaction steps are repeated, the purification method may comprise: a step of recharging squalene to be the substrate; a step of recovering and purifying a reaction product in a reaction liquid after deactivating the enzyme by a known method; and the like. In a case that squalene is recharged, a charging point in time and the amount of charging of squalene can be appropriately set according to the concentration of the mutant squalene-hopene cyclase in the reaction liquid, the amount of the substrate remained in the reaction liquid, or the like.
[Ambrein Production Step]
In an ambrein production step, a tetraprenyl-β-curcumene cyclase is reacted with 3-deoxyachilleol A to obtain ambrein.
A tetraprenyl-β-curcumene cyclase, which is classified as belonging to EC 4.2.1.129, is an enzyme capable of catalyzing a reaction which produces baciterpenol A from water and tetraprenyl-β-curcumene or a reaction which produces 8α-hydroxypolypoda-13,17,21-triene from squalene.
A tetraprenyl-β-curcumene cyclase is known as an enzyme which a bacterium such as the genus Bacillus produces. From the viewpoint of reaction efficiency, a tetraprenyl-β-curcumene cyclase is preferably derived from a bacterium of the genus Bacillus. 
The tetraprenyl-β-curcumene cyclase derived from a bacterium of the genus Bacillus is preferably an enzyme derived from Bacillus megaterium, Bacillus subtilis, Bacillus licheniformis, or the like, and from the viewpoint of reaction efficiency, it is more preferably an enzyme derived from Bacillus megaterium or Bacillus subtilis, and particularly preferably an enzyme derived from Bacillus megaterium. 
The amino acid sequence of a tetraprenyl-β-curcumene cyclase of Bacillus bacteria is known.
The amino acid sequence of a tetraprenyl-β-curcumene cyclase derived from Bacillus megaterium is shown in GenBank Accession No.: ADF38987 (SEQ ID NO:16) (Table 4).
The amino acid sequence of a tetraprenyl-β-curcumene cyclase derived from Bacillus subtilis is shown in GenBank Accession No.: AB618206 (SEQ ID NO:17) (Table 5).
The amino acid sequence of a tetraprenyl-β-curcumene cyclase derived from Bacillus licheniformis is shown in GenBank Accession No.: AAU41134 (SEQ ID NO:18) (Table 6).
From the viewpoint of reaction efficiency, the tetraprenyl-β-curcumene cyclase is preferably a tetraprenyl-β-curcumene cyclase having the amino acid sequence represented by SEQ ID NO:16, SEQ ID NO:17, or SEQ ID NO:18, and more preferably a tetraprenyl-β-curcumene cyclase having the amino acid sequence represented by SEQ ID NO:16.







TABLE 4





Tetraprenyl-β-curcumene Cyclase Derived from


Bacillus megaterium

















MIILLKEVQL EIQRRIAYLR PTQKNDGSFR YCFETGVMPD



 



AFLIMLLRTF DLDKEVLIKQ LTERIVSLQN EDGLWTLFDD



 



EEHNLSATIQ AYTALLYSGY YQKNDRILRK AERYIIDSGG



 



ISRAHFLTRW MLSVNGLYEW PKLFYLPLSL LLVPTYVPLN



 



FYELSTYARI HFVPMMVAGN KKFSLTSRHT PSLSHLDVRE



 



QKQESEETTQ ESRASIFLVD HLKQLASLPS YIHKLGYQAA



 



ERYMLERIEK DGTLYSYATS TFFMIYGLLA LGYKKDSFVI



 



QKAIDGICSL LSTCSGHVHV ENSTSTVWDT ALLSYALQEA



 



GVPQQDPMIK GTTRYLKKRQ HTKLGDWQFH NPNTAPGGWG



 



FSDINTNNPD LDDTSAAIRA LSRRAQTDTD YLESWQRGIN



 



WLLSMQNKDG GFAAFEKNTD SILFTYLPLE NAKDAATDPA



 



TADLTGRVLE CLGNFAGMNK SHPSIKAAVK WLFDHQLDNG



 



SWYGRWGVCY IYGTWAAITG LRAVGVSASD PRIIKAINWL



 



KSIQQEDGGF GESCYSASLK KYVPLSFSTP SQTAWALDAL



 



MTICPLKDRS VEKGIKFLLN PNLTEQQTHY PTGIGLPGQF



 



YIQYHSYNDI FPLLALAHYA KKHSS

















TABLE 5





Tetraprenyl-β-curcumene Cyclase Derived from


Bacillus subtilis

















MGTLQEKVRR YQKKTIAELK NRQNADGSWT FCFEGPIMTN



 



SFFILLLTSL DEGENEKELI SALAAGIREK QQPDGTFINY



 



PDETSGNITA TVQGYVGMLA SGCFHRSDPH MRKAEQSIIS



 



HGGLRHVHFM TKWMLAVNGL YPWPVLYLPL SLMALPPTLP



 



VHFYQFSAYA RIHFAPMAVT LNQRFVLKNR NIPSLRHLDP



 



HMTKNPFTWL RSDAFEERDL TSIWSHWNRI FHAPFAFQQL



 



GLQTAKTYML DRIEKDGTLY SYASATIFMV YSLLSLGVSR



 



YSPVIKRAIN GIKSLMTKCN GIPYLENSTS TVWDTALISY



 



ALQKNGVTET DGSITKAAAY LLERQHTKRA DWSVKNPSAA



 



PGGWGFSNIN TNNPDCDDTA AVLKAIPHSY SPSAWERGVS



 



WLLSMQNNDG GFSAFEKNVN HPLIRLLPLE SAEDAAVDPS



 



TADLTGRVLH FLGEKAGFTE KHQHIQRAVN WLFEHQEQNG



 



SWYGRWGVCY IYGTWAALTG MHACEVDRKH PAIQKALRWL



 



KSIQHDDGSW GESCNSAEVK TYVPLHKGTI VQTAWALDAL



 



LTYESSEHPS VVKGMQYLTD SSYHGADSLA YPAGIGLPKQ



 



FYIRYHSYPY VFSLLAVGKY LNSIEKETAN ET

















TABLE 6





Tetraprenyl-β-curcumene Cyclase Derived from


Bacillus licheniformis

















MTDSFFILML TSLGDQDSSL IASLAERIRS RQSEDGAFRN



 



HPDERAGNLT ATVQGYTGML ASGLYDRKAP HMQKAEAFIK



 



DAGGLKGVHF MTKWMLAANG LYPWPRAYIP LSFLLIPSYF



 



PLHFYHFSTY ARIHFVPMAI TFNRRFSLKN NQIGSLRHLD



 



EAMSKNPLEW LNIRAFDERT FYSFNLQWKQ LFQWPAYVHQ



 



LGFEAGKKYM LDRIEEDGTL YSYASATMFM IYSLLAMGIS



 



KNAPVVKKAV SGIKSLISSC GKEGAHLENS TSTVWDTALI



 



SYAMQESGVP EQHSSTSSAA DYLLKRQHVK KADWAVSNPQ



 



AVPGGWGFSH INTNNPDLDD TAAALKAIPF QRRPDAWNRG



 



LAWLLSMQNK DGGFAAFEKD VDHPLIRNLP LESAAEAAVD



 



PSTADLTGRV LHLLGLKGRF TDNHPAVRRA LRWLDHHQKA



 



DGSWYGRWGV CFIYGTWAAL TGMKAVGVSA NQTSVKKAIS



 



WLKSIQREDG SWGESCKSCE AKRFVPLHFG TVVQSSWALE



 



ALLQYERPDD PQIIKGIRFL IDEHESSRER LEYPTGIGLP



 



NQFYIRYHSY PFVFSLLASS AFIKKAEMRE TY










The tetraprenyl-β-curcumene cyclase encompasses polypeptides which have the amino acid sequences represented by SEQ ID NOs:16 to 18 wherein one or several amino acid residues are substituted, deleted, inserted, or added, and in which a function of producing ambrein from 3-deoxyachilleol A is maintained. The number of amino acid residues which are substituted, deleted, inserted, or added in each of the amino acid sequences represented by SEQ ID NOs:16 to 18 is, specifically, preferably 1 to 20, more preferably 1 to 10, and further preferably 1 to 5.
The tetraprenyl-β-curcumene cyclase encompasses polypeptides which have sequence identity of, for example, 80% or higher, preferably 90% or higher, more preferably 95% or higher, more preferably 97% or higher, more preferably 98% or higher, and particular preferably 99% or higher, to the whole amino acid sequences each represented by SEQ ID NOs:16 to 18, and in which a function of producing ambrein from 3-deoxyachilleol A is maintained.
The tetraprenyl-β-curcumene cyclase may be obtained by genetic engineering based on the amino acid sequence of a tetraprenyl-β-curcumene cyclase which a bacterium of the genus Bacillus produces and/or based on the base sequence of a tetraprenyl-β-curcumene cyclase gene present in a bacterium of the genus Bacillus. Examples of the tetraprenyl-β-curcumene cyclase gene, which is used when a tetraprenyl-β-curcumene cyclase is produced by genetic engineering, include a polynucleotide having the base sequence of the wild-type gene present in a bacterium of the genus Bacillus or a synthesized polynucleotide based on the base sequence of the wild-type gene.
The base sequence of a tetraprenyl-β-curcumene cyclase gene present in a bacterium of the genus Bacillus is known.
As for Bacillus megaterium, the polynucleotide ranging from 2130781 to 2132658 in the genomic sequence of GenBank:CP001982.1 (SEQ ID NO:19, the base sequence starting from the 2130781st base in the genomic sequence of GenBank:CP001982.1) is known.
As for Bacillus subtilis, the polynucleotide (SEQ ID NO:20) described in GenBank:AB618206 is known.
As for Bacillus licheniformis, the polynucleotide ranging from 2209539 to 2211428 in the genomic sequence of GenBank:CP000002.3 (SEQ ID NO:21, the base sequence starting from the 2209539th base in the genomic sequence of GenBank:CP000002.3) is known.
The polynucleotide encoding a tetraprenyl-β-curcumene cyclase encompasses polynucleotides which have the base sequences represented by SEQ ID NOs:19 to 21 wherein one or several bases are substituted, deleted, inserted, or added, and which encode a polypeptide in which a function of producing ambrein from 3-deoxyachilleol A is maintained. The number of bases which are substituted, deleted, inserted, or added in each of the base sequences represented by SEQ ID NOs:19 to 21 is, specifically, preferably 1 to 20, more preferably 1 to 10, and further preferably 1 to 5.
The polynucleotide encoding a tetraprenyl-β-curcumene cyclase encompasses polynucleotides encoding polypeptides which have sequence identity of, for example, 80% or higher, preferably 90% or higher, more preferably 95% or higher, more preferably 97% or higher, more preferably 98% or higher, and particular preferably 99% or higher, to the whole base sequences each represented by SEQ ID NOs:19 to 21, and in which a function of producing ambrein from 3-deoxyachilleol A is maintained.
The polynucleotide encoding a tetraprenyl-β-curcumene cyclase encompasses polynucleotides which hybridize with complementary strands of the base sequences represented by SEQ ID NOs:19 to 21 under stringent conditions, and which encode a polypeptide in which a function of producing ambrein from 3-deoxyachilleol A from squalene. The conditions of hybridization and the stringent conditions are the same as the conditions described for a mutant squalene-hopene cyclase.
Examples of the tetraprenyl-β-curcumene cyclase include a polypeptide which is encoded by the base sequence represented by any one of SEQ ID NOs:19 to 21, a polypeptide which is encoded by the base sequence represented by SEQ ID NO:19 or SEQ ID NO:20, and a polypeptide which is encoded by the base sequence represented by SEQ ID NO:19.
A recombinant vector which is used for expressing a polynucleotide which encodes a tetraprenyl-β-curcumene cyclase is not particularly restricted, and examples thereof include a vector which can be expressed by Escherichia coli such as pCold TF, a vector which can be expressed by Bacillus subtilis such as pHT01, and a vector which can be expressed by yeast such as pYES2. By introducing a polynucleotide encoding a tetraprenyl-β-curcumene cyclase into such a vector, an enzyme expression vector can be obtained. A host bacterium which is a target for introducing an enzyme expression vector can be appropriately selected according to the type of a recombinant vector to be used, and examples thereof include Escherichia coli such as BL21 (DE3), Bacillus subtilis such as strain 168, and yeast such as Saccharomyces cerevisiae. 
The recombinant vector may contain, as needed, a promoter, a splicing signal, a poly(A) addition signal, a selection marker, a ribosome binding sequence (SD sequence), a terminator such as NOS, and/or the like. As the selection marker, a known one such as an antibiotic resistance gene such as a kanamycin resistance gene, an ampicillin resistance gene or a tetracycline resistance gene is used without particular restriction.
The recombinant vector may contain a reporter gene for confirming that an objective gene is introduced. Examples of such a reporter gene include a GUS (β-glucuronidase) gene, a luciferase gene, and a GFP (green fluorescent protein) gene.
A tetraprenyl-β-curcumene cyclase may be produced by culturing a transformant obtained by introducing an enzyme expression vector into a bacterium. A culture medium used for culturing a transformant may be a culture medium which is usually used, and is appropriately selected according to the type of a host. For example, when Escherichia coli are cultured, an LB medium or the like is used. An antibiotic may be added to a culture medium according to the type of a selection marker.
A tetraprenyl-β-curcumene cyclase may be obtained by extraction followed by purification from a culture medium which has been obtained by culturing a transformant capable of expressing the enzyme. An extraction liquid containing the enzyme which has been extracted from a transformant in a culture medium may be used as it is. As a method of extracting an enzyme from a transformant, a known method may be applied. A step of extracting an enzyme may comprise, for example, crushing a transformant in an extraction solvent and separating cell contents from crushed pieces of the transformant. The obtained cell contents contain the target tetraprenyl-β-curcumene cyclase.
As the method of crushing a transformant, a known method in which a transformant is crushed and an enzyme liquid can be recovered may be applied, and examples thereof include ultrasonic crushing and glass beads crushing. The conditions of crushing are not particularly restricted as long as the enzyme is not inactivated, such as a condition of not higher than 10° C. and for 15 minutes.
Examples of the method of separating cell contents from crushed pieces of microorganism include sedimentation, centrifugation, filtering separation, and a combination of two or more thereof. Conditions for these separation methods are known to those skilled in the art. The conditions are, for example, from 8,000×g to 15,000×g and from 10 to 20 minutes in the case of centrifugation.
The extraction solvent may be a solvent which is usually used as a solvent for extracting an enzyme, and examples thereof include Tris-HCl buffer and potassium phosphate buffer. The pH of an extraction solvent is, from the viewpoint of enzyme stability, preferably from 3 to 10 and more preferably from 6 to 8.
The extraction solvent may contain a surfactant. Examples of the surfactant include a nonionic surfactant and an ampholytic surfactant. Examples of the nonionic surfactant include: a polyoxyethylene sorbitan fatty acid ester such as poly(oxyethylene)sorbitan monooleate (Tween 80); alkylglucoside such as n-octyl β-D-glucoside; a sucrose fatty acid ester such as sucrose stearate; and a polyglycerol fatty acid ester such as polyglycerol stearate. Examples of the ampholytic surfactant include N,N-dimethyl-N-dodecylglycine betaine which is an alkylbetaine. Besides the above, surfactants generally used in the art such as Triton X-100 (TRITON X-100), polyoxyethylene(20)cetyl ether (BRIJ-58), and nonylphenol ethoxylate (TERGITOL NP-40) can be utilized.
The concentration of a surfactant in an extraction solvent is, from the viewpoint of enzyme stability, preferably from 0.001% by mass to 10% by mass, more preferably from 0.10% by mass to 3.0% by mass, and further preferably from 0.10% by mass to 1.0% by mass.
From the viewpoint of enzyme activity, an extraction solvent preferebly contains a reducing agent such as dithiothreitol or β-mercaptoethanol. The reducing agent is preferably dithiothreitol. The concentration of dithiothreitol in an extraction solvent is preferably from 0.1 mM to 1M and more preferably from 1 mM to 10 mM. In a case that dithiothreitol is present in an extraction solvent, a structure such as a disulfide bond in the enzyme is easily to be retained and enzyme activity is easely to be enhanced.
From the viewpoint of enzyme activity, the extraction solvent preferably contains chelating agent such as ethylenediaminetetraacetic acid (EDTA). The concentration of EDTA in the extraction solvent is preferably from 0.01 mM to 1 M and more preferably from 0.1 mM to 10 mM. In a case that EDTA is present in the extraction solvent, a metal ion which may reduce enzyme activity is chelated, and therefore, enzyme activity is easily to be enhanced.
The extraction solvent may contain, besides the ingredients described above, a known ingredient which can be added to an enzyme extraction solvent.
Tetraprenyl-β-curcumene cyclases may be used singly, or in combination of two or more kinds thereof.
The conditions of a reaction between a tetraprenyl-β-curcumene cyclase and 3-deoxyachilleol A are not particularly restricted as long as the conditions are such that an enzyme reaction can be proceeded. For example, the reaction temperature and the reaction time may be appropriately selected based on the activity of a tetraprenyl-β-curcumene cyclase or the like. From the viewpoint of reaction efficiency, the reaction temperature and the reaction time may be, for example, from 4° C. to 100° C. and from 0.1 hour to 48 hours, and preferably 30° C. to 60° C. and 16 hours to 24 hours. From the viewpoint of reaction efficiency, the pH is, for example, from 3 to 10, and preferably from 6 to 8.
A reaction solvent is not particularly restricted as long as the reaction solvent does not inhibit an enzyme reaction, and a buffer or the like which is usually used can be used. For example, the same solvent as an extraction solvent which is used in a step of extracting an enzyme can be used. An extraction liquid (for example, cell-free extract) containing a tetraprenyl-β-curcumene cyclase may be used as it is as an enzyme liquid in the reaction.
From the viewpoint of reaction efficiency, the concentration ratio between a tetraprenyl-β-curcumene cyclase and 3-deoxyachilleol A which is the substrate thereof in a production reaction of ambrein is preferably from 10 to 10000, more preferably from 100 to 5000, still more preferably from 1000 to 3000, and still further preferably from 1000 to 2000 in terms of the molar concentration ratio (substrate/enzyme) of the substrate to the enzyme.
From the viewpoint of reaction efficiency, the concentration of 3-deoxyachilleol A to be used for an enzyme reaction is preferably from 0.000001% by mass to 0.002% by mass, and more preferably from 0.00001% by mass to 0.0002% by mass with respect to the total mass of the reaction solvent.
A step of reaction between a tetraprenyl-β-curcumene cyclase and 3-deoxyachilleol A may be repeated a plurality of times. This can increase the yield of ambrein. In a case that a plurality of reaction steps are repeated, the purification method may comprise: a step of recharging 3-deoxyachilleol A to be the substrate; a step of recovering and purifying a reaction product in a reaction liquid after inactivating the enzyme by a known method; and the like. In a case that 3-deoxyachilleol A is recharged, a charging point in time and the amount of charging of squalene can be appropriately set according to the concentration of the tetraprenyl-β-curcmene cyclase in the reaction liquid, the amount of the substrate remained in the reaction liquid, or the like.
From the viewpoints of production efficiency of ambrein and simplicity of the production method thereof, the method for producing ambrein in the invention is, in a case that the method comprises a step of producing 3-deoxyachilleol A and an ambrein production step, preferably a method comprising: reacting a tetraprenyl-β-curcumene cyclase derived from a bacterium of the genus Bacillus with 3-deoxyachilleol A obtained from a reaction between a mutant squalene-hopene cyclase derived from a bacterum of the genus Alicyclobacillus and squalene to produce ambrein. The method for producing ambrein in the invention is more preferably a method comprising: reacting a tetraprenyl-β-curcumene cyclase derived from Bacillus megaterium or Bacillus subtilis with 3-deoxyachilleol A obtained from a reaction between a mutant squalene-hopene cyclase derived from Alicyclobacillus acidocaldarius and squalene to produce ambrein.
[Other Steps]
The method for producing ambrein in the invention may further comprise a purification step which purifies produced ambrein. The purification method of ambrein is not particularly restricted as long as ambrein in a reaction liquid can be taken out, and a purification method which is usually used may be appropriately selected. Specific examples of the purification method include solvent extraction, recrystallization, distillation, column chromatography, and HPLC.
The obtained product can be confirmed to be ambrein by a conventional method using a gas chromatography-mass spectrometer (GC-MS) or a nuclear magnetic resonance apparatus (NMR).
EXAMPLES
In the following, the invention will be described in detail by way of Examples. The invention, however, should not be limited thereto in any way.
Example 1
Ambrein was obtained using squalene as a material by two steps: a step of reacting a mutant squalene-hopene cyclase with squalene; and a step of reacting a tetraprenyl-β-curcumene cyclase with 3-deoxyachilleol A. A reaction scheme of the two steps is illustrated below.




(1) Synthesis of 3-deoxyachilleol A
Escherichia coli BL21 (DE3) (Biosci. Biotechnol. Biochem., (1999) Vol. 63, pp. 2189-2198) that is transformed with a recombinant vector containing the polynucleotide (SEQ ID NO:9) encoding the mutant squalene-hopene cyclase (SEQ ID NO:2) was prepared. This transformant was inoculated onto an LB medium (6 L) containing ampicillin (50 mg/L) and cultured at 37° C. for 16 hours while shaking. After culturing, bacterial cells were harvested by centrifugation (6,000×g, 10 minutes). The harvested bacterial cells were washed with 50 mM Tris-HCl buffer (pH 8.0), then suspended in 300 mL buffer A [containing 50 mM Tris-HCl buffer (pH 8.0), 1 v/v % Triton X-100], and ultrasonically crushed (4° C., 15 minutes) using UP2005 sonicator (Hielscher Ultrasonics, Teltow, Germany). The crushed sample was centrifuged (12,000×g, 15 minutes), and a supernatant obtained after the centrifugation was designated as “cell-free extract A”.
Squalene (50 mg) was mixed with Triton X-100 (1 g) to be solubilized, and a buffer A (5 mL) was added thereto to prepare squalene liquid. The whole of the squalene liquid was added to the cell-free extract A to obtain a reaction liquid, followed by incubation at 60° C. for 16 hours. In the reaction liquid, the mole ratio (substrate/enzyme) of squalene (substrate) to the mutant squalene-hopene cyclase (enzyme) was about 1,000.
After the incubation, ethanol solution containing 15% by mass of potassium hydroxide (KOH/MeOH, 450 mL) was added to the reaction liquid to stop an enzyme reaction. Thereafter, n-hexane (750 mL) was added to the reaction liquid, and the reaction product was extracted three times. The obtained extract was subjected to silica gel column chromatography (solvent: n-hexane) to obtain pure 3-deoxyachilleol A (42.2 mg). The structure of 3-deoxyachilleol A was confirmed by gas chromatography-mass spectrometer (GC-MS) and nuclear magnetic resonance apparatus (NMR).
(2) Synthesis of Ambrein
Escherichia coli BL21 (DE3) (J. Am. Chem. Soc., (2011) Vol. 133, pp. 17540-17543) transformed with a recombinant vector containing the polynucleotide (SEQ ID NO:19) encoding the tetraprenyl-β-curcumene cyclase (SEQ ID NO:16) derived from Bacillus megaterium was prepared. This transformant was inoculated onto an LB medium (18 L) and cultured at 37° C. for 3 hours while shaking. After culturing, 0.1M isopropyl-β-thiogalactopyranoside (IPTG) was added thereto and shaken at 15° C. for 24 hours, and the expression of a tetraprenyl-β-curcumene cyclase was induced.
Thereafter, bacterial cells that was harvested by centrifugation (6,000×g, 10 minutes) were washed with 50 mM Tris-HCl buffer (pH 8.0), then suspended in 540 mL buffer B [containing 50 mM Tris-HCl buffer (pH 7.5), 0.1 v/v % Triton X-100, 2.5 mM dithiothreitol, and 1 mM EDTA], and ultrasonically crushed (4° C., 20 minutes) using UP2005 sonicator (Hielscher Ultrasonics, Teltow, Germany). The crushed sample was centrifuged (12,300×g, 20 minutes), and a supernatant obtained after the centrifugation was designated as “cell-free extract B”.
3-deoxyachilleol A (35 mg) obtained in the step (1) was mixed with Triton X-100 (700 mg) to be solubilized, and a buffer B (5 mL) was added thereto to prepare 3-deoxyachilleol A liquid. The whole of the 3-deoxyachilleol A liquid was added to the cell-free extract B (180 mL) to obtain a reaction liquid, followed by incubation at 30° C. for 16 hours. In the reaction liquid, the mole ratio (substrate/enzyme) of 3-deoxyachilleol A (substrate) to a tetraprenyl-β-curcumene cyclase (enzyme) was about 1,000.
After the incubation, ethanol solution containing 15% by mass of potassium hydroxide (KOH/MeOH, 220 mL) was added to the reaction liquid, and a heat treatment was further performed at 70° C. for 30 minutes to stop an enzyme reaction. Thereafter, n-hexane (400 mL) was added to the reaction liquid, and the reaction product was extracted three times. The obtained extract was solubilized by adding Triton X-100 (470 mg) thereto. The solubilized extract was added to buffer B (5 mL), and the buffer was then added to cell-free extract B (180 mL), followed by performing incubation, stopping a reaction, and performing n-hexane extraction in the same manner as above. Subsequently, solubilization of the extract, addition to the cell-free extract B, incubation, stopping of the reaction, and n-hexane extraction were performed once more in the same manner as above.
The obtained extract was subjected to silica gel column chromatography (solvent: n-hexane, n-hexane:ethyl acetate=100:20; volume ratio) to obtain n-hexane:ethyl acetate=100:20 fraction. The obtained fraction was concentrated and subjected to HPLC (solvent: n-hexane:THF=100:20) to obtain pure ambrein (0.4 mg). The structure of ambrein was confirmed by gas chromatography-mass spectrometer (GC-MS) and nuclear magnetic resonance apparatus (NMR). The optical rotation thereof was approximately agreed with the literature value.
Example 2
The step (1) and step (2) were performed in the similar manner as Example 1 except that the tetraprenyl-β-curcumene cyclase was changed from an enzyme derived from Bacillus megaterium to an enzyme derived from Bacillus subtilis to synthesize ambrein. The tetraprenyl-β-curcumene cyclase used in Example 2 is an enzyme encoded by the polynucleotide represented by SEQ ID NO:20 and has the amino acid sequence represented by SEQ ID NO:17.
As a result, in the same manner as Example 1, ambrein was obtainable from squalene via 3-deoxyachilleol A. The yield of the synthesized ambrein was about 10% of the yield in a case (Example 1) in which a tetraprenyl-β-curcumene cyclase derived from Bacillus megaterium was used.
According to the invention, ambrein can be easily produced from 3-deoxyachilleol A by using a tetraprenyl-β-curcumene cyclase.
According to the invention, ambrein can be easily produced from squalene via 3-deoxyachilleol A by using a mutant squalene-hopene cyclase and a tetraprenyl-β-curcumene cyclase.
The disclosure of Japanese Patent Application No. 2013-184143 filed on Sep. 5, 2013 is hereby incorporated by reference in its entirety.
All the references, patent applications and technical standards that are described in the present specification are hereby incorporated by reference to the same extent as if each individual reference, patent application or technical standard is concretely and individually described to be incorporated by reference.

## Claims

1. A method for producing ambrein, comprising reacting a tetraprenyl-β-curcumene cyclase with 3-deoxyachilleol A to obtain ambrein.

2. The method for producing ambrein according to claim 1, wherein the tetraprenyl-β-curcumene cyclase is derived from a bacterium of the genus Bacillus.

3. The method for producing ambrein according to claim 2, wherein the tetraprenyl-β-curcumene cyclase is derived from any one of Bacillus megaterium, Bacillus subtilis or Bacillus licheniformis.

4. The method for producing ambrein according to claim 2, further comprising reacting a mutant squalene-hopene cyclase-with squalene to obtain 3-deoxyachilleol A, wherein the mutant squalene-hopene cyclase has an amino acid substitution at at least one position selected from the group consisting of position 377, position 420, position 607, and position 612 in the amino acid sequence of SEQ ID NO:1.

5. The method for producing ambrein according to claim 4, wherein the mutant squalene-hopene cyclase has an amino acid sequence of any one of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8.

6. The method for producing ambrein according to claim 1, wherein the tetraprenyl-β-curcumene cyclase is derived from any one of Bacillus megaterium, Bacillus subtilis or Bacillus licheniformis.

7. The method for producing ambrein according to claim 6, further comprising reacting a mutant squalene-hopene cyclase-with squalene to obtain 3-deoxyachilleol A, wherein the mutant squalene-hopene cyclase has an amino acid substitution at at least one position selected from the group consisting of position 377, position 420, position 607, and position 612 in the amino acid sequence of SEQ ID NO:1.

8. The method for producing ambrein according to claim 7, wherein the mutant squalene-hopene cyclase has an amino acid sequence of any one of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8.

9. The method for producing ambrein according to claim 1, further comprising reacting a mutant squalene-hopene cyclase-with squalene to obtain 3-deoxyachilleol A, wherein the mutant squalene-hopene cyclase has an amino acid substitution at at least one position selected from the group consisting of position 377, position 420, position 607, and position 612 in the amino acid sequence of SEQ ID NO:1.

10. The method for producing ambrein according to claim 9, wherein the mutant squalene-hopene cyclase has an amino acid sequence of any one of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8.