Mutant tetraprenyl-β-curcumene cyclase and method for producing ambrein using the same

Abstract

The purpose of the present invention is to provide a method for producing ambrein by which ambrein can be easily and efficiently obtained.　This problem can be solved by a mutant tetraprenyl-β-curcumene cyclase in which aspartic acid that is the fourth amino acid residue of a DXDD motif is substituted by an amino acid other than aspartic acid and the seventh amino acid of a (D / N)G(T / L)(L / F / Y)(Y / F)SY motif is substituted by an amino acid other than tyrosine, said mutant tetraprenyl-β-curcumene cyclase: (a) having nine specific motifs; (b) having 40% or higher sequence identity with the amino acid sequence represented by SEQ ID NO: 1; and (c) showing an ambrein synthesis activity using 8α-hydroxypolypoda-13,17,21-triene as a substrate.

Description

[Technical Field]

[0001] The present invention relates to a mutant tetraprenyl-β-curcumene cyclase and a method for producing ambrein using the same. [Background technology]

[0002] Ambergris (ambergris) is a luxury fragrance that has been used around the world since around the 7th century, and is also used as a traditional Chinese medicine. It is thought that ambergris is formed when sperm whales turn indigestible food (octopus, squid, etc.) into stones using digestive secretions and excrete them, but the exact mechanism of its production is unknown. The main component of ambergris is ambergris, which is thought to be oxidized and decomposed by sunlight and oxygen as it floats on the ocean, producing various fragrant compounds.

[0003] Amberine, the main component of ambergris, is used as a fragrance and medicine, but it is not possible to obtain it in large quantities from nature. For this reason, various organic synthesis methods have been proposed. For example, Patent Document 1 discloses a method for producing (+)-ambreneine simply, inexpensively, and efficiently, which includes the steps of producing a novel sulfonic acid derivative from ambrenolide and coupling this with an optically active γ-cyclogeranyl halide.

[0004] Furthermore, Non-Patent Document 1 discloses a method for obtaining ambrein by convergent synthesis, by Julia coupling reaction, of 2-((1R,2R,4aS,8aS)-2-(methoxymethoxy)-2,5,5,8a-tetramethyldecahydronaphthalen-1-yl)acetaldehyde, synthesized from (±)(5,5,8a-trimethyloctahydro-1H-spiro[naphthalene-2,2'-oxiran]-1-yl)methanol, and 5-((4-((S)-2,2-dimethyl-6-methylenecyclohexyl)butan-2-yl)sulfonyl)-1-phenyl-1H-tetrazole, synthesized from (±)methyl 6-hydroxy-2,2-dimethylcyclohexanecarboxylate.

[0005] However, conventional organic synthesis methods for Ambrein require many synthesis steps, resulting in a complex reaction system, and have not yet been commercialized. On the other hand, a method is known for obtaining 3-deoxyachilleol A, a monocyclic triterpene, from squalene by using mutant squalene-hopene cyclase enzymes (D377C, D377N, Y420H, Y420W, etc.) (Non-Patent Documents 2 to 4). The present inventors discovered that 3-deoxyachilleol A can be obtained by reacting squalene with a mutant squalene-hopene cyclase capable of producing 3-deoxyachilleol A from squalene, and that ambrein can be produced by further reacting this with tetraprenyl-β-curcumene cyclase (Patent Document 2). However, the second step, which converts 3-deoxyachilleol A to ambrein, leaves room for improvement in terms of suppressing by-products, ease of scaling up, and yield. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Japanese Patent Application Publication No. 10-236996 [Patent Document 2] International Publication No. WO2015 / 033746 Pamphlet [Patent Document 3] International Publication No. WO2017 / 150695 Brochure [Non-patent literature]

[0007] [Non-Patent Document 1] Tetrahedron Asymmetry, (2006) Vol.17, pp.3037-3045 [Non-patent document 2] Biosci. Biotechnol. Biochem., (1999) Vol.63, pp.2189-2198 [Non-patent document 3] Biosci. Biotechnol. Biochem., (2001) Vol.65, pp.2233-2242 [Non-patent document 4] Biosci. Biotechnol. Biochem., (2002) Vol.66, pp.1660-1670 [Non-patent document 5] J. Am. Chem. Soc., (2011) Vol.133, pp.17540-17543 [Non-patent document 6] J. Am. Chem. Soc., (2013) Vol.135, pp.18335-18338 Summary of the Invention [Problem to be solved by the invention]

[0008] Therefore, the present inventors have discovered and proposed a method for obtaining ambrein from squalene through a two-step reaction using a single enzyme by using a mutant tetraprenyl-β-curcumene cyclase (D373C, etc.) (Patent Document 3). However, although this method significantly improved the yield compared to the method of Patent Document 2, there was still room for improvement in terms of suppressing by-products, since 3-deoxyachilleol A and a bicyclic triterpene (8α-hydroxypolypoda-13,17,21-triene) were first produced from squalene, and then ambrein was obtained from them. An object of the present invention is to provide a method for producing ambrein, which can easily and efficiently produce ambrein. [Means for solving the problem]

[0009] The present inventors conducted extensive research into a method for producing ambrein that would enable ambrein to be obtained simply and efficiently, and as a result, they surprisingly found that a mutant tetraprenyl-β-curcumene cyclase with a slight specific mutation can efficiently produce ambrein in high yield from a bicyclic triterpene (8α-hydroxypolypoda-13,17,21-triene).The present inventors found that by using the mutant tetraprenyl-β-curcumene cyclase in the second step of a two-step stepwise method for producing ambrein from squalene, where ambrein is produced from a bicyclic triterpene (8α-hydroxypolypoda-13,17,21-triene), ambrein can be produced efficiently and in high yield. The present invention is based on this finding. Therefore, the present invention provides [1] A mutant tetraprenyl-β-curcumene cyclase in which the fourth amino acid residue, aspartic acid, of the DXDD motif is substituted with an amino acid other than aspartic acid, and the seventh amino acid of the (D / N)G(T / L)(L / F / Y)(Y / F)SY motif is substituted with an amino acid other than tyrosine, (a) the mutant tetraprenyl-β-curcumene cyclase has, based on the DXDD motif, a QXXXGX(W / F) motif located 100 or more amino acid residues toward the N-terminus, an (A / S / G)RX(H / N)XXP motif located 180 to 250 amino acid residues toward the N-terminus, the (D / N)G(T / L)(L / F / Y)(Y / F)SY motif located 80 to 140 amino acid residues toward the N-terminus, and a QX motif located 10 to 50 amino acid residues toward the N-terminus. a mutant tetraprenyl-β-curcumene cyclase having a XXX(G / A / S)X(F / W / Y) motif, a QXXXGX(F / W / Y) motif at a position 20 to 50 amino acid residues away from the C-terminus, a QXXXGXW motif at a position 50 to 120 amino acid residues away from the C-terminus, a QXXXGX(F / W) motif at a position 120 to 170 amino acid residues away from the C-terminus, and the GXGX(G / A / P) motif at a position 180 to 250 amino acid residues away from the C-terminus, but not having a QXXXGXW motif at a position 170 or more amino acid residues away from the DXDD motif, (b) having an identity of 40% or more with the amino acid sequence represented by SEQ ID NO: 1, and (c) exhibiting ambuline-forming activity using 8α-hydroxypolypoda-13,17,21-triene as a substrate; [2] The mutant tetraprenyl-β-curcumene cyclase according to [1], wherein the amino acid adjacent to the N-terminus of the (A / S / G)RX(H / N)XXP motif is substituted with an amino acid other than tyrosine. [3] The polypeptide constituting the mutant tetraprenyl-β-curcumene cyclase is (1) a polypeptide in which the aspartic acid at the 373rd position from the N-terminus in the amino acid sequence represented by SEQ ID NO: 1 is substituted with an amino acid other than aspartic acid and the tyrosine at the 257th position from the N-terminus is substituted with an amino acid other than tyrosine, (2) an amino acid sequence in which the aspartic acid at the 373rd position from the N-terminus in the amino acid sequence represented by SEQ ID NO: 1 is substituted with an amino acid other than aspartic acid and the tyrosine at the 257th position from the N-terminus is substituted with an amino acid other than tyrosine, in which one or several amino acids have been deleted, substituted, inserted, and / or added, and which exhibits ambrein-forming activity using 8α-hydroxypolypoda-13,17,21-triene as a substrate, or (3) a polypeptide in which the aspartic acid at the 373rd position from the N-terminus in the amino acid sequence represented by SEQ ID NO: 1 is substituted with an amino acid other than aspartic acid and the tyrosine at the 257th position from the N-terminus is substituted with an amino acid other than tyrosine. (4) a polypeptide comprising an amino acid sequence having an identity of 40% or more with an amino acid sequence in which aspartic acid at position 373 from the N-terminus is substituted with an amino acid other than aspartic acid and tyrosine at position 257 from the N-terminus is substituted with an amino acid other than tyrosine, and which exhibits ambuline-forming activity using 8α-hydroxypolypoda-13,17,21-triene as a substrate; (5) a polypeptide comprising an amino acid sequence in which aspartic acid at position 373 from the N-terminus is substituted with an amino acid other than aspartic acid and tyrosine at position 257 from the N-terminus is substituted with an amino acid other than tyrosine, and which exhibits ambuline-forming activity using 8α-hydroxypolypoda-13,17,21-triene as a substrate, in which one or more amino acids have been deleted, substituted, inserted, and / or added in an amino acid sequence in which aspartic acid at position 373 from the N-terminus is substituted with an amino acid other than aspartic acid and tyrosine at position 257 from the N-terminus is substituted with an amino acid other than tyrosine, and which exhibits ambuline-forming activity using 8α-hydroxypolypoda-13,17,21-triene as a substrate;The mutant tetraprenyl-β-curcumene cyclase according to [1] is (6) a polypeptide that exhibits ambrane-forming activity using 8α-hydroxypolypoda-13,17,21-triene as a substrate, or (7) a polypeptide that comprises an amino acid sequence having 40% or more identity with an amino acid sequence in which the aspartic acid at position 373 from the N-terminus in the amino acid sequence represented by SEQ ID NO: 1 is substituted with an amino acid other than aspartic acid, and the tyrosine at position 257 from the N-terminus is substituted with an amino acid other than tyrosine, and that exhibits ambrane-forming activity using 8α-hydroxypolypoda-13,17,21-triene as a substrate. (8) a polypeptide comprising an amino acid sequence in which one or more amino acids are deleted, substituted, inserted, and / or added in the amino acid sequence represented by SEQ ID NO: 1, in which the aspartic acid at position 373 from the N-terminus is substituted with an amino acid other than aspartic acid, the tyrosine at position 167 from the N-terminus is substituted with an amino acid other than tyrosine, and the tyrosine at position 257 from the N-terminus is substituted with an amino acid other than tyrosine, and which exhibits ambrein-forming activity using 8α-hydroxypolypoda-13,17,21-triene as a substrate; (9) a polypeptide comprising an amino acid sequence having an identity of 40% or more with an amino acid sequence in which the aspartic acid at position 373 from the N-terminus is substituted with an amino acid other than aspartic acid, the tyrosine at position 167 from the N-terminus is substituted with an amino acid other than tyrosine, and the tyrosine at position 257 from the N-terminus is substituted with an amino acid other than tyrosine, and which exhibits ambrein-forming activity using 8α-hydroxypolypoda-13,17,21-triene as a substrate, in which the aspartic acid at position 373 from the N-terminus is substituted with an amino acid other than aspartic acid, the tyrosine at position 167 from the N-terminus is substituted with an amino acid other than tyrosine, and the tyrosine at position 257 from the N-terminus is substituted with an amino acid other than tyrosine, and which has an identity of 40% or more with an amino acid sequence in which the aspartic acid at position 373 from the N-terminus is substituted with an amino acid other than aspartic acid, the tyrosine at position 167 from the N-terminus is substituted with(10) A polypeptide that exhibits ambuline-forming activity using 8α-hydroxypolypoda-13,17,21-triene as a substrate; (11) A polypeptide that contains an amino acid sequence represented by SEQ ID NO: 1 in which the aspartic acid at position 373 from the N-terminus is substituted with an amino acid other than aspartic acid, the tyrosine at position 167 from the N-terminus is substituted with an amino acid other than tyrosine, and the tyrosine at position 257 from the N-terminus is substituted with an amino acid other than tyrosine; and that exhibits ambuline-forming activity using 8α-hydroxypolypoda-13,17,21-triene as a substrate; (12) A polypeptide that contains an amino acid sequence represented by SEQ ID NO: 1 in which the aspartic acid at position 373 from the N-terminus is substituted with an amino acid other than aspartic acid, the tyrosine at position 167 from the N-terminus is substituted with an amino acid other than tyrosine, and the tyrosine at position 257 from the N-terminus is substituted with an amino acid other than tyrosine. (12) a polypeptide comprising an amino acid sequence having 40% or more identity to an amino acid sequence in which the aspartic acid at the 373rd position from the N-terminus in the amino acid sequence represented by SEQ ID NO: 1 is substituted with an amino acid other than aspartic acid, the tyrosine at the 167th position from the N-terminus is substituted with an amino acid other than tyrosine, and the tyrosine at the 257th position from the N-terminus is substituted with an amino acid other than tyrosine, and the mutant tetraprenyl-β-curcumene cyclase according to [2] is a polypeptide having ambulein-forming activity using 8α-hydroxypolypoda-13,17,21-triene as a substrate; [4] A mutant tetraprenyl-β-curcumene cyclase according to [1] or [3], in which the fourth amino acid residue in the DXDD motif is substituted from aspartic acid to cysteine, and the seventh amino acid in the (D / N)G(T / L)(L / F / Y)(Y / F)SY motif is substituted from tyrosine to alanine; or a mutant tetraprenyl-β-curcumene cyclase according to [2] or [3], in which the fourth amino acid residue in the DXDD motif is substituted from aspartic acid to cysteine, the amino acid adjacent to the N-terminus of the (A / S / G)RX(H / N)XXP motif is substituted from tyrosine to alanine, and the seventh amino acid in the (D / N)G(T / L)(L / F / Y)(Y / F)SY motif is substituted from tyrosine to alanine. [5] A mutant tetraprenyl-β-curcumene cyclase in which the fourth amino acid residue, aspartic acid, of the DXDD motif is substituted with an amino acid other than aspartic acid, the amino acid adjacent to the N-terminus of the (A / S / G)RX(H / N)XXP motif is substituted with an amino acid other than tyrosine, and the fourth amino acid of the GXGX(G / A / P) motif is substituted with an amino acid other than leucine, (a) the mutant tetraprenyl-β-curcumene cyclase has, based on the DXDD motif, a QXXXGX(W / F) motif located 100 amino acid residues or more away from the N-terminus, the (A / S / G)RX(H / N)XXP motif located 180 to 250 amino acid residues away from the N-terminus, the (D / N)G(T / L)(L / F / Y)(Y / F)SY motif located 80 to 140 amino acid residues away from the N-terminus, and a mutant tetraprenyl-β-curcumene cyclase having a QXXXX(G / A / S)X(F / W / Y) motif at a position 10 to 50 amino acid residues away from the C-terminus, a QXXXGX(F / W / Y) motif at a position 20 to 50 amino acid residues away from the C-terminus, a QXXXGXW motif at a position 50 to 120 amino acid residues away from the C-terminus, a QXXXGX(F / W) motif at a position 120 to 170 amino acid residues away from the C-terminus, and the GXGX(G / A / P) motif at a position 180 to 250 amino acid residues away from the C-terminus, and not having a QXXXGXW motif at a position 170 or more amino acid residues away from the DXDD motif; (b) having an identity of 40% or more with the amino acid sequence represented by SEQ ID NO: 1; and (c) exhibiting ambuline-forming activity using 8α-hydroxypolypoda-13,17,21-triene as a substrate. [6] The polypeptide constituting the mutant tetraprenyl-β-curcumene cyclase is (1) a polypeptide in which the aspartic acid at position 373 from the N-terminus is substituted with an amino acid other than aspartic acid, the tyrosine at position 167 from the N-terminus is substituted with an amino acid other than tyrosine, and the leucine at position 596 from the N-terminus is substituted with an amino acid other than leucine in the amino acid sequence represented by SEQ ID NO: 1, or (2) an amino acid sequence in which one or several amino acids are deleted, substituted, inserted, and / or added in the amino acid sequence in which the aspartic acid at position 373 from the N-terminus is substituted with an amino acid other than aspartic acid, the tyrosine at position 167 from the N-terminus is substituted with an amino acid other than tyrosine, and the leucine at position 596 from the N-terminus is substituted with an amino acid other than leucine in the amino acid sequence represented by SEQ ID NO: 1, and further, which is capable of cyclizing 8α-hydroxypolypoda-13,17,21-triene as a substrate. (3) a polypeptide exhibiting ambrain-forming activity, (4) an amino acid sequence having 40% or more identity with an amino acid sequence in which the aspartic acid at position 373 from the N-terminus is substituted with an amino acid other than aspartic acid, the tyrosine at position 167 from the N-terminus is substituted with an amino acid other than tyrosine, and the leucine at position 596 from the N-terminus is substituted with an amino acid other than leucine in the amino acid sequence represented by SEQ ID NO: 1, and which exhibits ambrain-forming activity using 8α-hydroxypolypoda-13,17,21-triene as a substrate; (5) an amino acid sequence in which the aspartic acid at position 373 from the N-terminus is substituted with an amino acid other than aspartic acid, the tyrosine at position 167 from the N-terminus is substituted with an amino acid other than tyrosine, and the leucine at position 596 from the N-terminus is substituted with an amino acid other than leucine in the amino acid sequence represented by SEQ ID NO: 1, and which exhibits ambrain-forming activity using 8α-hydroxypolypoda-13,17,21-triene as a substrate;(5) A polypeptide that exhibits ambrane-forming activity using 8α-hydroxypolypoda-13,17,21-triene as a substrate, comprising an amino acid sequence in which one or more amino acids have been deleted, substituted, inserted, and / or added in the amino acid sequence represented by SEQ ID NO: 1, in which the aspartic acid at position 373 from the N-terminus has been replaced with an amino acid other than aspartic acid, the tyrosine at position 167 from the N-terminus has been replaced with an amino acid other than tyrosine, and the leucine at position 596 from the N-terminus has been replaced with an amino acid other than leucine, and which further exhibits ambrane-forming activity using 8α-hydroxypolypoda-13,17,21-triene as a substrate. or (6) the mutant tetraprenyl-β-curcumene cyclase according to [5], which is a polypeptide comprising an amino acid sequence having 40% or more identity with an amino acid sequence in which the aspartic acid at position 373 from the N-terminus in the amino acid sequence represented by SEQ ID NO: 1 is substituted with an amino acid other than aspartic acid, the tyrosine at position 167 from the N-terminus is substituted with an amino acid other than tyrosine, and the leucine at position 596 from the N-terminus is substituted with an amino acid other than leucine, and which exhibits ambuline-forming activity using 8α-hydroxypolypoda-13,17,21-triene as a substrate. [7] The mutant tetraprenyl-β-curcumene cyclase according to [5] or [6], wherein the fourth amino acid residue of the DXDD motif is substituted from aspartic acid to cysteine, the amino acid adjacent to the N-terminus of the (A / S / G)RX(H / N)XXP motif is substituted from tyrosine to alanine, and the fourth amino acid of the GXGX(G / A / P) motif is substituted from leucine to alanine. [8] A polynucleotide encoding the mutant tetraprenyl-β-curcumene cyclase according to any one of [1] to [7]. [9] A microorganism having the polynucleotide according to [8].

[10] A vector containing DNA having the polynucleotide according to [8].

[11] A transformant having the vector according to

[10] .

[12] A method for producing ambrein, comprising reacting the mutant tetraprenyl-β-curcumene cyclase according to any one of [1] to [7] with squalene or 8α-hydroxypolypoda-13,17,21-triene to obtain ambrein.

[13] A method for producing ambrein, comprising culturing the microorganism according to [9] or the transformant according to

[11] .

[14] A method for producing ambrein, comprising: (1) a step of reacting squalene with a tetraprenyl-β-curcumene cyclase to obtain 8α-hydroxypolypoda-13,17,21-triene; and (2) a step of reacting a mutant tetraprenyl-β-curcumene cyclase with 8α-hydroxypolypoda-13,17,21-triene to obtain ambrein, wherein the mutant tetraprenyl-β-curcumene cyclase is (A) the mutant tetraprenyl-β-curcumene cyclase according to any one of [1] to [7], or (B) the fourth position of the DXDD motif. a mutant tetraprenyl-β-curcumene cyclase in which the first amino acid residue, aspartic acid, is substituted with an amino acid other than aspartic acid, and the amino acid adjacent to the N-terminus of the (A / S / G)RX(H / N)XXP motif is substituted with an amino acid other than tyrosine, or the fourth amino acid of the GXGX(G / A / P) motif is substituted with an amino acid other than leucine, (a) the mutant tetraprenyl-β-curcumene cyclase has a QXXXGX(W / F) amino acid residue at a position 100 amino acid residues or more away from the DXDD motif on the N-terminal side; motif, the (A / S / G)RX(H / N)XXP motif located 180 to 250 amino acid residues away from the N-terminus, the (D / N)G(T / L)(L / F / Y)(Y / F)SY motif located 80 to 140 amino acid residues away from the N-terminus, a QXXXX(G / A / S)X(F / W / Y) motif located 10 to 50 amino acid residues away from the N-terminus, a QXXXGX(F / W / Y) motif located 20 to 50 amino acid residues away from the C-terminus, a QXXXGXW motif located 50 to 120 amino acid residues away from the C-terminus, and 120 to 170 amino acid residues away from the C-terminus. a method for producing ambrein, which is a mutant tetraprenyl-β-curcumene cyclase having a QXXXGX(F / W) motif at a position distant from the DXDD motif and the GXGX(G / A / P) motif at a position 180 to 250 amino acid residues away from the DXDD motif on the C-terminal side, and not having a QXXXGXW motif at a position 170 amino acid residues or more away from the DXDD motif on the C-terminal side, (b) having an identity of 40% or more with the amino acid sequence represented by SEQ ID NO: 1, and (c) exhibiting ambrein-forming activity using 8α-hydroxypolypoda-13,17,21-triene as a substrate;

[15] The polypeptide constituting the mutant tetraprenyl-β-curcumene cyclase (B) is (1) a polypeptide in which the aspartic acid at the 373rd position from the N-terminus in the amino acid sequence represented by SEQ ID NO: 1 is substituted with an amino acid other than aspartic acid and the tyrosine at the 167th position from the N-terminus is substituted with an amino acid other than tyrosine, (2) an amino acid sequence in which the aspartic acid at the 373rd position from the N-terminus in the amino acid sequence represented by SEQ ID NO: 1 is substituted with an amino acid other than aspartic acid and the tyrosine at the 167th position from the N-terminus is substituted with an amino acid other than tyrosine, in which one or several amino acids have been deleted, substituted, inserted, and / or added, and which exhibits ambrein-forming activity using 8α-hydroxypolypoda-13,17,21-triene as a substrate, or (3) a polypeptide in which the aspartic acid at the 373rd position from the N-terminus in the amino acid sequence represented by SEQ ID NO: 1 is substituted with an amino acid other than aspartic acid and the tyrosine at the 167th position from the N-terminus is substituted with an amino acid other than tyrosine. (4) a polypeptide comprising an amino acid sequence having an identity of 40% or more with an amino acid sequence in which the aspartic acid at position 373 from the N-terminus is substituted with an amino acid other than aspartic acid and the tyrosine at position 167 from the N-terminus is substituted with an amino acid other than tyrosine, and which exhibits ambuline-forming activity using 8α-hydroxypolypoda-13,17,21-triene as a substrate; (5) a polypeptide comprising an amino acid sequence in which the aspartic acid at position 373 from the N-terminus is substituted with an amino acid other than aspartic acid and the tyrosine at position 167 from the N-terminus is substituted with an amino acid other than tyrosine, and which exhibits ambuline-forming activity using 8α-hydroxypolypoda-13,17,21-triene as a substrate, in which one or more amino acids have been deleted, substituted, inserted, and / or added, and which is not a polypeptide of the present invention;(6) a polypeptide that exhibits ambrane-forming activity using 8α-hydroxypolypoda-13,17,21-triene as a substrate; (7) a polypeptide that exhibits ambrane-forming activity using 8α-hydroxypolypoda-13,17,21-triene as a substrate, and that contains an amino acid sequence that has 40% or more identity with an amino acid sequence in which the aspartic acid at position 373 from the N-terminus in the amino acid sequence represented by SEQ ID NO: 1 has been substituted with an amino acid other than aspartic acid and the tyrosine at position 167 from the N-terminus has been substituted with an amino acid other than tyrosine; (8) a polypeptide that exhibits ambrane-forming activity using 8α-hydroxypolypoda-13,17,21-triene as a substrate, and that has an amino acid sequence represented by SEQ ID NO: 1 in which the aspartic acid at position 373 from the N-terminus has been substituted with an amino acid other than aspartic acid and the leucine at position 596 from the N-terminus has been substituted with an amino acid other than leucine; (9) A polypeptide comprising an amino acid sequence in which one or more amino acids have been deleted, substituted, inserted, and / or added, and which exhibits ambuline-synthesizing activity using 8α-hydroxypolypoda-13,17,21-triene as a substrate; (10) A polypeptide comprising an amino acid sequence in which the aspartic acid at position 373 from the N-terminus of the amino acid sequence represented by SEQ ID NO: 1 has been substituted with an amino acid other than aspartic acid and the leucine at position 596 from the N-terminus has been substituted with an amino acid other than leucine, and which exhibits ambuline-synthesizing activity using 8α-hydroxypolypoda-13,17,21-triene as a substrate; (11) A polypeptide comprising an amino acid sequence in which the aspartic acid at position 373 from the N-terminus of the amino acid sequence represented by SEQ ID NO: 1 has been substituted with an amino acid other than aspartic acid and the leucine at position 596 from the N-terminus has been substituted with an amino acid other than leucine, and which exhibits ambuline-synthesizing activity using 8α-hydroxypolypoda-13,17,21-triene as a substrate;(11) a polypeptide that exhibits ambrane-synthesizing activity using 8α-hydroxypolypoda-13,17,21-triene as a substrate; (12) a polypeptide that exhibits ambrane-synthesizing activity using 8α-hydroxypolypoda-13,17,21-triene as a substrate, comprising an amino acid sequence in which one or more amino acids have been deleted, substituted, inserted, and / or added in the amino acid sequence represented by SEQ ID NO: 1, in which the aspartic acid at position 373 from the N-terminus has been substituted with an amino acid other than aspartic acid and the leucine at position 596 from the N-terminus has been substituted with an amino acid other than leucine; or (13) a polypeptide that exhibits ambrane-synthesizing activity using 8α-hydroxypolypoda-13,17,21-triene as a substrate, comprising an amino acid sequence that has an identity of 40% or more with the amino acid sequence represented by SEQ ID NO: 1, in which the aspartic acid at position 373 from the N-terminus has been substituted with an amino acid other than aspartic acid and the leucine at position 596 from the N-terminus has been substituted with an amino acid other than leucine.

[16] The method for producing ambrane according to

[14] or

[15] , wherein the mutant tetraprenyl-β-curcumene cyclase (B) is a mutant tetraprenyl-β-curcumene cyclase in which the fourth amino acid residue of the DXDD motif is substituted from aspartic acid to cysteine ​​and the amino acid adjacent to the N-terminus of the (A / S / G)RX(H / N)XXP motif is substituted from tyrosine to alanine, or a mutant tetraprenyl-β-curcumene cyclase in which the fourth amino acid residue of the DXDD motif is substituted from aspartic acid to cysteine ​​and the fourth amino acid of the GXGX(G / A / P) motif is substituted from leucine to alanine.

[17] The method for producing ambrane according to any one of

[14] to

[16] , in which the steps (1) and (2) are carried out simultaneously; and

[18] The method for producing ambrane according to any one of

[14] to

[17] , wherein the tetraprenyl-β-curcumene cyclase is a wild-type tetraprenyl-β-curcumene cyclase. Regarding. [Effects of the Invention]

[0010] The mutant tetraprenyl-β-curcumene cyclase of the present invention can efficiently produce ambrein from a bicyclic triterpene (8α-hydroxypolypoda-13,17,21-triene) in high yield. The mutant tetraprenyl-β-curcumene cyclase of the present invention can be used in the second step of a two-step stepwise method for producing ambrein from squalene, where ambrein is produced from a bicyclic triterpene (8α-hydroxypolypoda-13,17,21-triene), to efficiently produce ambrein in high yield. [Brief explanation of the drawings]

[0011] [Figure 1] FIG. 1 shows two pathways for producing ambreine from squalene using mutant tetraprenyl-β-curcumene cyclase. [Figure 2] FIG. 1 shows a pathway for producing ambrein from 8α-hydroxypolypoda-13,17,21-triene using the mutant tetraprenyl-β-curcumene cyclase of the present invention. [Figure 3] FIG. 1 shows a production method of the present invention for producing ambrein from squalene using a tetraprenyl-β-curcumene cyclase and a mutant tetraprenyl-β-curcumene cyclase of the present invention. [Figure 4] This is a graph showing the content ratio of ambrein (A) in a reaction composition obtained using the mutant tetraprenyl-β-curcumene cyclase of the present invention (Examples 1 to 3, Production Method Example 1) and 8α-hydroxypolypoda-13,17,21-triene as a substrate, and the content ratio of ambrein and onoceroid (B) excluding 8α-hydroxypolypoda-13,17,21-triene. [Figure 5]1 is a graph showing the ambrein content (A) in a reaction composition obtained using the mutant tetraprenyl-β-curcumene cyclase of the invention (Example 2, Production Method Example 2) and squalene as a substrate, and the content ratios of ambrein, monocyclic compounds, bicyclic compounds, and onoceroid (B) excluding squalene. [Figure 6] 1 is a graph showing the ambrein content (A) in a reaction composition obtained by a stepwise (two-stage) reaction method using wild-type tetraprenyl-β-curcumene cyclase and the mutant tetraprenyl-β-curcumene cyclase of the present invention (Example 2, Production Method Example 3), and the content ratio of ambrein, monocyclic compounds, bicyclic compounds, and onoceroid (B), excluding squalene. [Figure 7-1] Wild-type tetraprenyl-β-curcumene cyclase, D373C in which aspartic acid at position 373 is replaced by cysteine, Y167A / D373C in which tyrosine at position 167 is replaced by alanine and aspartic acid at position 373 is replaced by cysteine, Y257A / D373C in which tyrosine at position 257 is replaced by alanine and aspartic acid at position 373 is replaced by cysteine, and Y257A / D373C in which aspartic acid at position 373 is replaced by cysteine ​​and aspartic acid at position 373 is replaced by leucine at position 596. FIG. 1 shows the amino acid sequences of D373C / L596A in which tyrosine at position 167 is substituted with alanine, tyrosine at position 257 is substituted with alanine, and aspartic acid at position 373 is substituted with cysteine; and Y167A / D373C / L596A in which tyrosine at position 167 is substituted with alanine, aspartic acid at position 373 is substituted with cysteine, and leucine at position 596 is substituted with alanine. [Figure 7-2]Wild-type tetraprenyl-β-curcumene cyclase, D373C in which aspartic acid at position 373 is replaced by cysteine, Y167A / D373C in which tyrosine at position 167 is replaced by alanine and aspartic acid at position 373 is replaced by cysteine, Y257A / D373C in which tyrosine at position 257 is replaced by alanine and aspartic acid at position 373 is replaced by cysteine, and Y257A / D373C in which aspartic acid at position 373 is replaced by cysteine ​​and aspartic acid at position 373 is replaced by leucine at position 596. FIG. 1 shows the amino acid sequences of D373C / L596A in which tyrosine at position 167 is substituted with alanine, tyrosine at position 257 is substituted with alanine, and aspartic acid at position 373 is substituted with cysteine; and Y167A / D373C / L596A in which tyrosine at position 167 is substituted with alanine, aspartic acid at position 373 is substituted with cysteine, and leucine at position 596 is substituted with alanine. [Figure 8-1] FIG. 1 shows an alignment of the amino acid sequences of the tetraprenyl-β-curcumene cyclases from Bacillus megaterium, Bacillus subtilis, and Bacillus licheniformis, and the squalene-hopene cyclase from Alicyclobacillus acidocaldarius. [Figure 8-2] FIG. 1 shows an alignment of the amino acid sequences of the tetraprenyl-β-curcumene cyclases from Bacillus megaterium, Bacillus subtilis, and Bacillus licheniformis, and the squalene-hopene cyclase from Alicyclobacillus acidocaldarius. DETAILED DESCRIPTION OF THE INVENTION

[0012] (tetraprenyl-β-curcumene cyclase) Wild-type tetraprenyl-β-curcumene cyclase (hereinafter sometimes referred to as TC) can use 3-deoxyachilleol A, which has a single ring at one end, as a substrate to produce ambrein. That is, when 3-deoxyachilleol A is used as a substrate, tetraprenyl-β-curcumene cyclase can selectively cyclize the uncyclized end of 3-deoxyachilleol A to produce a compound cyclized at both ends. Tetraprenyl-β-curcumene cyclase can also use squalene as a substrate to produce bicyclic 8α-hydroxypolypoda-13,17,21-triene (Non-Patent Document 5). Furthermore, tetraprenyl-β-curcumene cyclase can selectively cyclize the uncyclized end of bicyclic 8α-hydroxypolypoda-13,17,21-triene to produce onoceroids (onocerane oxide and 14β-hydroxyonocera-8(26)-ene), which are both-terminally cyclized compounds (Non-Patent Document 6).

[0013] That is, tetraprenyl-β-curcumene cyclase is classified as EC 4.2.1.129 and is an enzyme that can catalyze the reaction of producing batiterpenol A from water and tetraprenyl-β-curcumene, or the reaction of producing 8α-hydroxypolypoda-13,17,21-triene from squalene. Tetraprenyl-β-curcumene cyclases are found in bacteria of the genus Bacillus, Brevibacillus, Paenibacillus, or Geobacillus. Examples of bacteria of the genus Bacillus include Bacillus subtilis, Bacillus megaterium, and Bacillus licheniformis. Tetraprenyl-β-curcumene cyclases have a QXXXGX(W / F) motif at a position 100 or more amino acid residues away from the N-terminus of a DXDD motif, the (A / S / G)RX(H / N)XXP motif at a position 180 to 250 amino acid residues away from the N-terminus, the (D / N)G(T / L)(L / F / Y)(Y / F)SY motif at a position 80 to 140 amino acid residues away from the N-terminus, and the (D / N)G(T / L)(L / F / Y)(Y / F)SY motif at a position 10 to 50 amino acid residues away from the N-terminus of a DXDD motif. The squalene-hopene cyclase has a QXXXX(G / A / S)X(F / W / Y) motif at a position 20 to 50 amino acid residues away from the C-terminus, a QXXXGX(F / W / Y) motif at a position 50 to 120 amino acid residues away from the C-terminus, a QXXXGXW motif at a position 120 to 170 amino acid residues away from the C-terminus, and the GXGX(G / A / P) motif at a position 180 to 250 amino acid residues away from the C-terminus. Among the motifs mentioned above, the squalene-hopene cyclase does not have the (A / S / G)RX(H / N)XXP motif, the (D / N)G(T / L)(L / F / Y)(Y / F)SY motif, or the GXGX(G / A / P) motif. Furthermore, the squalene-hopene cyclase has a QXXXGXW motif at a position 170 amino acid residues or more away from the DXDD motif on the C-terminus. On the other hand, tetraprenyl-β-curcumene cyclase does not have the QXXXGXW motif. Furthermore, squalene-hopene cyclase has a GXGFP sequence similar to the GXGX(G / A / P) motif at the C-terminus of the QXXXGXW motif, but is characterized by the fact that the fourth amino acid is phenylalanine (F). In the GXGX(G / A / P) motif of tetraprenyl-β-curcumene cyclase, the fourth amino acid is basically leucine (L) rather than phenylalanine.

[0014] [1] Mutant tetraprenyl-β-curcumene cyclase (First aspect) A mutant tetraprenyl-β-curcumene cyclase according to a first aspect of the present invention is a mutant tetraprenyl-β-curcumene cyclase in which the fourth amino acid residue, aspartic acid, of the DXDD motif is substituted with an amino acid other than aspartic acid, and the seventh amino acid of the (D / N)G(T / L)(L / F / Y)(Y / F)SY motif is substituted with an amino acid other than tyrosine, wherein: (a) the mutant tetraprenyl-β-curcumene cyclase has, with respect to the DXDD motif, a QXXXGX(W / F) motif at a position 100 or more amino acid residues away from the N-terminus, an (A / S / G)RX(H / N)XXP motif at a position 180 to 250 amino acid residues away from the N-terminus, and the (D / N)G(T / L)(L / F / Y)(Y / F)SY motif at a position 80 to 140 amino acid residues away from the N-terminus. (b) has a QXXXX(G / A / S)X(F / W / Y) motif located 10 to 50 amino acid residues away from the N-terminus, a QXXXGX(F / W / Y) motif located 20 to 50 amino acid residues away from the C-terminus, a QXXXGXW motif located 50 to 120 amino acid residues away from the C-terminus, a QXXXGX(F / W) motif located 120 to 170 amino acid residues away from the C-terminus, and the GXGX(G / A / P) motif located 180 to 250 amino acid residues away from the C-terminus, and does not have a QXXXGXW motif located 170 or more amino acid residues away from the DXDD motif toward the C-terminus; (b) has an identity of 40% or more with the amino acid sequence represented by SEQ ID NO: 1; and (c) exhibits ambuline-forming activity using 8α-hydroxypolypoda-13,17,21-triene as a substrate. In a preferred embodiment of the mutant tetraprenyl-β-curcumene cyclase according to the first aspect of the present invention, the amino acid adjacent to the N-terminus of the (A / S / G)RX(H / N)XXP motif is further substituted with an amino acid other than tyrosine.

[0015] Here, the letters defining each motif and sequence represent the single-letter abbreviation for an amino acid, with "X" representing any amino acid. In other words, the QXXXGX(W / F) motif indicates that, from the N-terminus to the C-terminus, it consists of glutamine (Q), three of any amino acids (X), glycine (G), another of any amino acid (X), and either tryptophan (W) or phenylalanine (F). Furthermore, "having a QXXXGX(W / F) motif at a position 100 or more amino acid residues away from the N-terminus of the DXDD motif" means that there are 100 or more amino acid residues between the DXDD motif and the QXXXGX(W / F) motif. The same applies to the identification of other motifs. The fourth amino acid in the GXGX (G / A / P) motif refers to the fourth amino acid counting from the N-terminus, and the same applies to other sequences. Hereinafter, the same applies unless otherwise specified. The identity of amino acid sequences is determined by aligning the sequences to be compared by inserting appropriate gaps so that the amino acid residues match, dividing the number of matching amino acid residues by the total number of amino acid residues, and expressing the result as a percentage. Identity can be determined using well-known programs (e.g., BLAST, FASTA, CLUSTAL W, etc.).

[0016] A preferred embodiment of the mutant tetraprenyl-β-curcumene cyclase according to the first aspect of the present invention is the mutant tetraprenyl-β-curcumene cyclase described in [3] above.

[0017] Furthermore, in a most preferred embodiment of the mutant tetraprenyl-β-curcumene cyclase of the first aspect of the present invention, the polypeptide constituting the mutant tetraprenyl-β-curcumene cyclase is a polypeptide derived from Bacillus megaterium and consisting of the amino acid sequence set forth in SEQ ID NO: 2 or 3. Specifically, in the mutant tetraprenyl-β-curcumene cyclase, the fourth amino acid residue, aspartic acid, of the DXDD motif is substituted with cysteine, and the seventh amino acid residue, tyrosine, of the (D / N)G(T / L)(L / F / Y)(Y / F)SY motif is substituted with alanine. Furthermore, more preferably, the mutant tetraprenyl-β-curcumene cyclase has the amino acid adjacent to the N-terminus of the (A / S / G)RX(H / N)XXP motif substituted for tyrosine with alanine.

[0018] (Substitution of the fourth amino acid residue in the DXDD motif) In the mutant tetraprenyl-β-curcumene cyclase of the present invention (hereinafter sometimes referred to as mutant TC), the fourth amino acid residue of the DXDD motif is substituted with an amino acid other than aspartic acid. The amino acid other than aspartic acid is not limited as long as the effects of the present invention are obtained, and examples thereof include alanine, cysteine, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, valine, tryptophan, and tyrosine, with cysteine ​​being preferred. By substituting the fourth amino acid residue of the DXDD motif with an amino acid other than aspartic acid (particularly, cysteine ​​or glycine), the mutant tetraprenyl-β-curcumene cyclase of the present invention can produce 3-deoxyachilleol A from squalene and ambrein from 8α-hydroxypolypoda-13,17,21-triene. The DXDD motif is located at positions 370-373 from the N-terminus in the amino acid sequence of the tetraprenyl-β-curcumene cyclase from Bacillus megaterium (SEQ ID NO: 1). It is also located at positions 375-378 from the N-terminus in the amino acid sequence of the tetraprenyl-β-curcumene cyclase from Bacillus subtilis (SEQ ID NO: 5). The aspartic acid in the DXDD motif is highly conserved, and the fourth amino acid residue from the N-terminus is usually aspartic acid (Figure 8).

[0019] (Substitution of the 7th amino acid residue of the (D / N)G(T / L)(L / F / Y)(Y / F)SY motif) In the mutant TC of the present invention, the seventh amino acid residue of the (D / N)G(T / L)(L / F / Y)(Y / F)SY motif is substituted with an amino acid other than tyrosine. The amino acid other than tyrosine is not limited as long as the effects of the present invention are obtained, and examples thereof include alanine, cysteine, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, valine, tryptophan, and aspartic acid, but also includes valine, proline, glycine, serine, and phenylalanine. Alanine or glycine is particularly preferred, and alanine is more preferred. By substituting the seventh amino acid residue of the (D / N)G(T / L)(L / F / Y)(Y / F)SY motif with an amino acid other than tyrosine (particularly alanine or glycine), the mutant tetraprenyl-β-curcumene cyclase of the present invention has improved ability to produce ambrein from 8α-hydroxypolypoda-13,17,21-triene. The (D / N)G(T / L)(L / F / Y)(Y / F)SY motif is present at positions 251 to 257 from the N-terminus of the amino acid sequence of the tetraprenyl-β-curcumene cyclase from Bacillus megaterium (SEQ ID NO: 1). It is also located at positions 256 to 262 from the N-terminus of the amino acid sequence of the tetraprenyl-β-curcumene cyclase from Bacillus subtilis (SEQ ID NO: 5). The seventh amino acid residue of the (D / N)G(T / L)(L / F / Y)(Y / F)SY motif is highly conserved and is essentially tyrosine in wild-type tetraprenyl-β-curcumene cyclases (Figure 8). The present inventors discovered that mutation of this specific, highly conserved amino acid can improve the ambuline-producing activity of tetraprenyl-β-curcumene cyclases using α-hydroxypolypoda-13,17,21-triene as a substrate.

[0020] (Substitution of amino acid residues adjacent to the N-terminus of the (A / S / G)RX(H / N)XXP motif) In the mutant TC of the present invention, the amino acid adjacent to the N-terminus of the (A / S / G)RX(H / N)XXP motif is substituted with an amino acid other than tyrosine. The amino acid other than tyrosine is not limited as long as the effects of the present invention are obtained, and examples thereof include alanine, cysteine, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, valine, tryptophan, and aspartic acid. Hydrophobic amino acids (glycine, alanine, valine, leucine, isoleucine, methionine, proline, phenylalanine, and tryptophan), serine, threonine, or cysteine ​​are preferred. Alanine or glycine is particularly preferred, and alanine is even more preferred. By substituting an amino acid residue adjacent to the N-terminus of the (A / S / G)RX(H / N)XXP motif with an amino acid other than tyrosine (particularly alanine or glycine), the mutant tetraprenyl-β-curcumene cyclase of the present invention has improved functions for producing 3-deoxyachleol A from squalene and for producing ambrein from 8α-hydroxypolypoda-13,17,21-triene. The (A / S / G)RX(H / N)XXP motif is present at positions 168 to 174 from the N-terminus of the amino acid sequence of the tetraprenyl-β-curcumene cyclase of Bacillus megaterium (SEQ ID NO: 1). It is also present at positions 170 to 176 from the N-terminus of the tetraprenyl-β-curcumene cyclase of Bacillus subtilis (SEQ ID NO: 5). The amino acid residue adjacent to the N-terminus of the (A / S / G)RX(H / N)XXP motif is highly conserved and is essentially tyrosine in wild-type tetraprenyl-β-curcumene cyclases (Figure 8). By mutating this specific, highly conserved amino acid, the mutant tetraprenyl-β-curcumene cyclase of the present invention can produce 3-deoxyachilleol A from squalene and ambrein from 8α-hydroxypolypoda-13,17,21-triene.

[0021] Figure 7 shows the amino acid sequences of the wild-type tetraprenyl-β-curcumene cyclase from Bacillus megaterium, the mutant tetraprenyl-β-curcumene cyclase (SEQ ID NO: 2) in which the aspartic acid at position 373 is replaced with cysteine ​​and the tyrosine at position 257 is replaced with alanine, and the mutant tetraprenyl-β-curcumene cyclase (SEQ ID NO: 3) in which the aspartic acid at position 373 is replaced with cysteine, the tyrosine at position 257 is replaced with alanine, and the tyrosine at position 167 is replaced with alanine.

[0022] The origin of the mutant tetraprenyl-β-curcumene cyclase of the present invention is not particularly limited, and all tetraprenyl-β-curcumene cyclases can be used. That is, tetraprenyl-β-curcumene cyclases have nine motifs: QXXXGX(W / F), (A / S / G)RX(H / N)XXP, (D / N)G(T / L)(L / F / Y)(Y / F)SY, QXXXX(G / A / S)X(F / W / Y), DXDD, QXXXGX(F / W / Y), QXXXGXW, QXXXGX(F / W), and GXGX(G / A / P). Such tetraprenyl-β-curcumene cyclases can be used without any limitations. For example, although the amino acid sequence identity of the polypeptides of Bacillus subtilis and Bacillus megaterium is about 50%, both enzymes can produce ambulein from 8α-hydroxypolypoda-13,17,21-triene due to the characteristics of the present invention. The amino acid sequence of SEQ ID NO: 1 is the amino acid sequence of wild-type tetraprenyl-β-curcumene cyclase of Bacillus megaterium, and the amino acid sequence of SEQ ID NO: 5 is the amino acid sequence of wild-type tetraprenyl-β-curcumene cyclase of Bacillus subtilis.

[0023] (Second aspect) A mutant tetraprenyl-β-curcumene cyclase according to a first aspect of the present invention is a mutant tetraprenyl-β-curcumene cyclase in which the fourth amino acid residue, aspartic acid, of the DXDD motif is substituted with an amino acid other than aspartic acid, the amino acid adjacent to the N-terminus of the (A / S / G)RX(H / N)XXP motif is substituted with an amino acid other than tyrosine, and the fourth amino acid of the GXGX(G / A / P) motif is substituted with an amino acid other than leucine, and (a) the mutant tetraprenyl-β-curcumene cyclase has, based on the DXDD motif, a QXXXGX(W / F) motif located 100 amino acid residues or more toward the N-terminus, the (A / S / G)RX(H / N)XXP motif located 180 to 250 amino acid residues toward the N-terminus, and the (D / N)G( (b) the amino acid sequence has a QXXXX(G / A / S)X(F / W / Y) motif located 10 to 50 amino acid residues away from the N-terminus, a QXXXGX(F / W / Y) motif located 20 to 50 amino acid residues away from the C-terminus, a QXXXGXW motif located 50 to 120 amino acid residues away from the C-terminus, a QXXXGX(F / W) motif located 120 to 170 amino acid residues away from the C-terminus, and the GXGX(G / A / P) motif located 180 to 250 amino acid residues away from the C-terminus, and does not have a QXXXGXW motif located 170 or more amino acid residues away from the DXDD motif; (b) it has 40% or more identity with the amino acid sequence represented by SEQ ID NO: 1; and (c) it exhibits ambuline-forming activity using 8α-hydroxypolypoda-13,17,21-triene as a substrate. The definitions of the alphabets of the motifs and sequences are the same as those in the first embodiment.

[0024] A preferred embodiment of the mutant tetraprenyl-β-curcumene cyclase according to the second aspect of the present invention is the mutant tetraprenyl-β-curcumene cyclase described in [6] above.

[0025] Furthermore, the most preferred embodiment of the mutant tetraprenyl-β-curcumene cyclase according to the second aspect of the present invention is a polypeptide derived from Bacillus megaterium, in which the polypeptide constituting the mutant tetraprenyl-β-curcumene cyclase consists of the amino acid sequence set forth in SEQ ID NO: 4. Specifically, in the mutant tetraprenyl-β-curcumene cyclase, the fourth amino acid residue, aspartic acid, of the DXDD motif is substituted with cysteine, the amino acid adjacent to the N-terminus of the (A / S / G)RX(H / N)XXP motif is substituted from tyrosine to alanine, and the fourth amino acid of the GXGX(G / A / P) motif is substituted from leucine to alanine.

[0026] The "substitution of the fourth amino acid residue in the DXDD motif" and the "substitution of the amino acid residue adjacent to the N-terminus of the (A / S / G)RX(H / N)XXP motif" are the same as those in the first embodiment.

[0027] (Substitution of the fourth amino acid residue in the GXGX(G / A / P) motif) In the mutant tetraprenyl-β-curcumene cyclase of the second aspect of the present invention, when the fourth amino acid of the GXGX (G / A / P) motif is substituted with an amino acid other than leucine, the amino acid other than leucine is not limited as long as the effects of the present invention are obtained, and examples thereof include alanine, cysteine, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, valine, tryptophan, tyrosine, and aspartic acid, with alanine, phenylalanine, valine, methionine, isoleucine, and tryptophan being preferred. Alanine or phenylalanine are particularly preferred, and alanine is more preferred. By substituting the fourth amino acid residue of the GXGX (G / A / P) motif with an amino acid other than leucine (particularly alanine or phenylalanine), the mutant tetraprenyl-β-curcumene cyclase of the present invention has improved function in producing ambrein from 8α-hydroxypolypoda-13,17,21-triene.

[0028] Figure 7 shows the amino acid sequence of a mutant tetraprenyl-β-curcumene cyclase derived from Bacillus megaterium, in which aspartic acid at position 373 is replaced by cysteine, tyrosine at position 167 is replaced by alanine, and leucine at position 596 is replaced by alanine (sequence number 4).

[0029] (An amino acid sequence in which one or several amino acids are deleted, substituted, inserted, and / or added) The mutant tetraprenyl-β-curcumene cyclase polypeptide of the present invention may be a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 in which one or several amino acids have been deleted, substituted, inserted, and / or added. The mutant tetraprenyl-β-curcumene cyclase polypeptide of the present invention is a polypeptide that exhibits ambuline-forming activity using 8α-hydroxypolypoda-13,17,21-triene as a substrate. In other words, polypeptides that do not exhibit ambuline-forming activity using 8α-hydroxypolypoda-13,17,21-triene as a substrate are not included in the mutant tetraprenyl-β-curcumene cyclase polypeptide of the present invention. As used herein, the phrase "an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted, and / or added" means that the polypeptide has been modified by amino acid substitution or the like. The number of amino acid modifications can be, for example, 1 to 330, 1 to 300, 1 to 250, 1 to 200, 1 to 150, 1 to 100, or 1 to 50, preferably 1 to 30, more preferably 1 to 10, even more preferably 1 to 5, and most preferably 1 to 2. An example of a modified amino acid sequence of a mutant peptide that can be used in the present invention is preferably an amino acid sequence in which the amino acids have one or several (preferably 1, 2, 3, or 4) conservative substitutions.

[0030] (Amino acid sequence with 40% or more identity to the amino acid sequence) The mutant tetraprenyl-β-curcumene cyclase polypeptide of the present invention may be a polypeptide consisting of an amino acid sequence that has 40% or more identity to the amino acid sequence of SEQ ID NO: 1. The mutant tetraprenyl-β-curcumene cyclase polypeptide of the present invention is a polypeptide that exhibits ambuline-forming activity using 8α-hydroxypolypoda-13,17,21-triene as a substrate. In other words, polypeptides that do not exhibit ambuline-forming activity using 8α-hydroxypolypoda-13,17,21-triene as a substrate are not included in the mutant tetraprenyl-β-curcumene cyclase polypeptide of the present invention. More preferably, the mutant tetraprenyl-β-curcumene cyclase is a polypeptide consisting of an amino acid sequence with an identity of 45% or more, more preferably an amino acid sequence with an identity of 50% or more, more preferably an amino acid sequence with an identity of 60% or more, more preferably an amino acid sequence with an identity of 70% or more, more preferably an amino acid sequence with an identity of 80% or more, more preferably an amino acid sequence with an identity of 90% or more, and most preferably an amino acid sequence with an identity of 95% or more, and is a mutant tetraprenyl-β-curcumene cyclase consisting of or including a polypeptide that exhibits ambuline-producing activity using 8α-hydroxypolypoda-13,17,21-triene as a substrate.

[0031] The "amino acid sequence of SEQ ID NO: 1 in which one or more amino acids have been deleted, substituted, inserted, and / or added" or the "amino acid sequence with 40% or greater identity" refers to a substitution of the amino acid sequence of SEQ ID NO: 1, but this substitution is a conservative substitution that maintains the function of the mutant tetraprenyl-β-curcumene cyclase of the present invention. In other words, a "conservative substitution" refers to a substitution that does not impair the excellent effects of the mutant tetraprenyl-β-curcumene cyclase of the present invention. That is, even when the insertion, substitution, deletion, or addition occurs, the substitution is capable of improving ambuline-producing activity using 8α-hydroxypolypoda-13,17,21-triene as a substrate. Specifically, this refers to replacing an amino acid residue with another chemically similar amino acid residue. Examples include replacing a hydrophobic residue with another hydrophobic residue, or replacing a polar residue with another polar residue having the same charge. Functionally similar amino acids that can be obtained by such substitutions are known for each amino acid in the art. Examples of nonpolar (hydrophobic) amino acids include alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, and methionine. Examples of polar (neutral) amino acids include glycine, serine, threonine, tyrosine, glutamine, asparagine, and cysteine. Examples of positively charged (basic) amino acids include arginine, histidine, and lysine. Examples of negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

[0032] In the mutant tetraprenyl-β-curcumene cyclase of the present invention, mutation (substitution) of the fourth amino acid residue, aspartic acid, in the DXDD motif with an amino acid other than aspartic acid, substitution of the seventh amino acid in the (D / N)G(T / L)(L / F / Y)(Y / F)SY motif with an amino acid other than tyrosine, substitution of the amino acid adjacent to the N-terminus of the (A / S / G)RX(H / N)XXP motif with an amino acid other than tyrosine, or substitution of the fourth amino acid in the GXGX(G / A / P) motif with an amino acid other than leucine are positive substitutions (mutations) intended to confer the activity of synthesizing ambreon using 8α-hydroxypolypoda-13,17,21-triene as a substrate. However, the conservative substitutions are intended to maintain the activity of synthesizing ambreon using 8α-hydroxypolypoda-13,17,21-triene as a substrate and can be easily performed by those skilled in the art.

[0033] The mutant tetraprenyl-β-curcumene cyclase of the present invention can be obtained using known genetic recombination techniques. For example, chromosomal DNA from Bacillus megaterium is obtained, and the tetraprenyl-β-curcumene cyclase is amplified by PCR or other techniques using appropriate primers. The resulting gene is inserted into an appropriate vector, and the gene sequence is determined. The above-described mutations are then introduced to obtain a gene encoding the mutant tetraprenyl-β-curcumene cyclase of the present invention. The mutant tetraprenyl-β-curcumene cyclase of the present invention can be obtained by inserting the gene into a host such as yeast and expressing it. In addition to Bacillus megaterium, tetraprenyl-β-curcumene cyclization enzymes are known to exist in bacteria of the genus Bacillus, such as Bacillus subtilis (accession number: AB618206) and Bacillus licheniformis (accession number: AAU41134).

[0034] Furthermore, the gene encoding the mutant tetraprenyl-β-curcumene cyclase of the present invention can also be synthesized by known synthetic gene synthesis methods, such as the method of Khorana et al. (Gupta et al., 1968), the method of Narang et al. (Scarpulla et al., 1982), or the method of Rossi et al. (Rossi et al., 1982). The mutant tetraprenyl-β-curcumene cyclase of the present invention can then be obtained by expressing the synthesized gene.

[0035] (action) Conventionally, ambrein has been produced from squalene by converting squalene to 3-deoxyachilleol A using a mutant squalene-hopene cyclase (hereinafter sometimes referred to as mutant SHC), and then converting 3-deoxyachilleol A to ambrein using a wild-type tetraprenyl-β-curcumene cyclase (Patent Document 2). The present inventors have discovered and proposed a method for obtaining ambrein from squalene in a two-step reaction using a single enzyme by using a mutant tetraprenyl-β-curcumene cyclase (D373C), as shown in Figure 1 (Patent Document 3). In this method, ambrein is produced via a pathway using monocyclic 3-deoxyachilleol A as an intermediate (hereinafter sometimes referred to as the monocyclic pathway) and a pathway using 8α-hydroxypolypoda-13,17,21-triene as an intermediate (hereinafter sometimes referred to as the bicyclic pathway). As shown in Figure 2, the mutant tetraprenyl-β-curcumene cyclase of the present invention has excellent activity in producing ambrein, particularly from 8α-hydroxypolypoda-13,17,21-triene, which is a substrate of the bicyclic pathway. Furthermore, the production of the by-product onoceroid is low.

[0036] [2] Polynucleotides The polynucleotide of the present invention is not particularly limited as long as it encodes the tetraprenyl-β-curcumene cyclase of the present invention. Examples include a polynucleotide encoding the polypeptide represented by SEQ ID NO: 2 (SEQ ID NO: 12), a polynucleotide encoding the polypeptide represented by SEQ ID NO: 3 (SEQ ID NO: 13), and a polynucleotide encoding the polypeptide represented by SEQ ID NO: 4 (SEQ ID NO: 14). Further examples include polynucleotides that hybridize under stringent conditions to a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 12, 13, or 14 and have the activity of producing ambrein from 8α-hydroxypolypoda-13,17,21-triene. As used herein, the term "polynucleotide" includes both DNA and RNA. Furthermore, it is preferable that the polynucleotide of the present invention be modified to have an optimal codon sequence suited to the microorganism or host cell into which it is introduced.

[0037] [3] Microorganisms The microorganism of the present invention is a microorganism having the polynucleotide of the present invention. That is, the microorganism is not particularly limited as long as it contains the polynucleotide of the present invention in its cells, and examples thereof include Escherichia coli, Bacillus subtilis, Brevibacillus sp., actinomycetes, baker's yeast, Aspergillus oryzae, and Neurospora crassa.

[0038] [4] Vector The vector of the present invention is a vector comprising DNA having a polynucleotide encoding the mutant tetraprenyl-β-curcumene cyclase. That is, the vector of the present invention is not particularly limited as long as it contains the polynucleotide of the present invention, and examples thereof include vectors obtained by inserting the polynucleotide of the present invention into a known expression vector appropriately selected depending on the host cell to be used. The expression vector is preferably one that can replicate autonomously or be integrated into a chromosome in a host such as Escherichia coli or baker's yeast, and that can efficiently express a foreign protein. The expression vector for expressing the polynucleotide is preferably a recombinant vector that can replicate autonomously in a microorganism and is composed of a promoter, a ribosome binding sequence, the DNA, and a transcription termination sequence. It may also contain a gene that controls the promoter.

[0039] More specifically, examples of expression vectors include pBTrp2, pBTac1, pBTac2 (all commercially available from Boehringer Mannheim), pKK233-2 (Pharmacia), pSE280 (Invitrogen), pGEMEX-1 (Promega), pQE-8 (QIAGEN), pQE-30 (QIAGEN), pKYP10 (JP 58-110600), pKYP200 [Agricultural Biological Chemistry, 48, 669 (1984)], pLSA1 [Agric. Biol. Chem., 53, 277 (1989)], pGEL1 [Proc. Natl. Acad. Sci. USA, 82, 4306 (1985)], pBluescript II SK+, pBluescript II SK(-) (Stratagene), pTrS30 (FERM BP-5407), pTrS32 (FERM BP-5408), pGEX (Pharmacia), pET-3 (Novagen), pTerm2 (US4686191, US4939094, US5160735), pSupex, pUB110, pTP5, pC194, pUC18 [gene, 33, 103 (1985)], pUC19 [Gene, 33, 103 (1985)], pSTV28 ( Examples include pColdI, pColdII, pColdIII, pColdIV, pNIDNA, and pNI-HisDNA (manufactured by Takara Bio Inc.).

[0040] Any promoter may be used as long as it can be expressed in host cells such as E. coli or baker's yeast. Examples include promoters derived from E. coli or phages, such as the trp promoter (Ptrp), lac promoter (Plac), PL promoter, PR promoter, and PSE promoter, as well as the SPO1 promoter, SPO2 promoter, and penP promoter. Artificially engineered promoters, such as the Ptrp promoter (Ptrpx2), which combines two Ptrp promoters in tandem, the tac promoter, the letI promoter, and the lacT7 promoter, can also be used. For producing enzymes used in enzymatic production (in vitro synthesis via an enzymatic reaction using squalene as a substrate), a promoter that functions as a strong promoter and enables mass production of the target protein is preferred, with inducible promoters being more preferred. Examples of inducible promoters include the promoter of the cold shock gene cspA, whose expression is induced at low temperatures, and the T7 promoter, which is induced by the addition of the inducer IPTG. Furthermore, in fermentation production (in vivo biosynthesis by a host using glucose or other carbon sources), among the above promoters, promoters that constantly express the target gene regardless of tissue, i.e., constitutive promoters, are more preferred. Examples of constitutive promoters include the promoters of the alcohol dehydrogenase 1 gene (ADH1), translation elongation factor TF-1α gene (TEF1), phosphoglycerate kinase gene (PGK1), triosephosphate isomerase gene (TPI1), triosephosphate dehydrogenase gene (TDH3), and pyruvate kinase gene (PYK1).

[0041] [5] Transformants The transformant of the present invention is not particularly limited as long as it contains the polynucleotide of the present invention. For example, it can be a transformant in which the polynucleotide of the present invention has been integrated into the chromosome of a host cell, or a transformant containing the polynucleotide of the present invention in the form of a vector. Furthermore, the transformant can be one that expresses a polypeptide of the present invention, or one that does not express a polypeptide of the present invention. The transformant of the present invention can be obtained, for example, by transforming a desired host cell with the vector of the present invention or with the polynucleotide of the present invention itself.

[0042] The host cells are not particularly limited, but strains that are easy to handle, such as Escherichia coli, Bacillus subtilis, Brevibacillus sp., actinomycetes, yeast, Aspergillus oryzae, and Neurospora crassa, are preferred. However, insect cells, plant cells, and animal cells can also be used. However, for producing enzymes used in enzymatic production (ex vivo synthesis by enzymatic reaction using squalene as a substrate), Escherichia coli, Bacillus subtilis, Brevibacillus sp., and Aspergillus oryzae are preferred, with Escherichia coli being the most preferred. Furthermore, for fermentative production (in vivo biosynthesis by a host using glucose or the like as a carbon source), yeast is most preferred. The most preferred yeast strain is sake yeast. Sake yeast Kyokai No. 7 or 701 are particularly preferred. Kyokai No. 701 yeast is a non-foaming yeast bred from Kyokai No. 7 and is characterized by not producing a high foam, but other properties are the same.

[0043] [6] Manufacturing method of Ambrein First Aspect The method for producing ambrein of the present invention involves reacting the mutant tetraprenyl-β-curcumene cyclase with squalene or 8α-hydroxypolypoda-13,17,21-triene to obtain ambrein.

[0044] The mutant tetraprenyl-β-curcumene cyclase can be produced by culturing a transformant obtained by introducing an enzyme expression vector into bacteria or the like. The medium used to culture the transformant may be any commonly used medium, and is appropriately selected depending on the type of host. For example, when culturing Escherichia coli, LB medium or the like is used. The medium may contain an antibiotic depending on the type of selection marker.

[0045] The mutant tetraprenyl-β-curcumene cyclase may be obtained by extracting and purifying the enzyme from a culture medium obtained by culturing a transformant capable of expressing the enzyme. Alternatively, the mutant tetraprenyl-β-curcumene cyclase of the present invention may be expressed as a fusion protein by fusing a trigger factor (TF) or a His tag to the N-terminus or C-terminus of the polypeptide, thereby facilitating purification. Alternatively, an extract containing the enzyme extracted from the transformant in the culture medium may be used as is. A known method may be used to extract the enzyme from the transformant. The enzyme extraction step may include, for example, disrupting the transformant in an extraction solvent and separating the cell contents from the transformant debris. The obtained cell contents contain the desired mutant tetraprenyl-β-curcumene cyclase.

[0046] The transformant may be disrupted by any known method capable of disrupting the transformant and recovering the enzyme solution, such as ultrasonic disruption, glass bead disruption, etc. The disruption conditions are not particularly limited, and may be any conditions that do not inactivate the enzyme, such as at 10°C or below for 15 minutes. Methods for separating the intracellular material from the microbial debris include sedimentation, centrifugation, filtration, and combinations of two or more of these separation methods. Separation conditions using these methods are known to those skilled in the art, and in the case of centrifugation, for example, 8,000×g to 15,000×g and 10 to 20 minutes.

[0047] The extraction solvent may be any solvent commonly used for enzyme extraction, such as Tris-HCl buffer, potassium phosphate buffer, etc. The pH of the extraction solvent is preferably 3 to 10, more preferably 6 to 8, from the viewpoint of enzyme stability.

[0048] The extraction solvent may contain a surfactant. Examples of surfactants include nonionic surfactants and zwitterionic surfactants. Nonionic surfactants include polyoxyethylene sorbitan fatty acid esters such as poly(oxyethylene) sorbitan monooleate (Tween 80), alkyl glucosides such as n-octyl β-D-glucoside, sucrose fatty acid esters such as sucrose stearate, and polyglycerin fatty acid esters such as polyglycerin stearate. Zwitterionic surfactants include alkyl betaines such as N,N-dimethyl-N-dodecylglycine betaine. In addition to these, surfactants commonly used in the art, such as Triton® X-100, polyoxyethylene (20) cetyl ether (Brij-58), and nonylphenol ethoxylate (Tergitol NP-40), can also be used. From the viewpoint of enzyme stability, the concentration of the surfactant in the extraction solvent is preferably 0.001% by mass to 10% by mass, more preferably 0.10% by mass to 3.0% by mass, and even more preferably 0.10% by mass to 1.0% by mass.

[0049] From the viewpoint of enzyme activity, the extraction solvent preferably contains a reducing agent such as dithiothreitol or β-mercaptoethanol. Dithiothreitol is preferred as the reducing agent. The concentration of dithiothreitol in the extraction solvent is preferably 0.1 mM to 1 M, more preferably 1 mM to 10 mM. The presence of dithiothreitol in the extraction solvent tends to facilitate the maintenance of structures such as disulfide bonds in the enzyme, thereby further increasing enzyme activity.

[0050] From the viewpoint of enzyme activity, the extraction solvent preferably contains a chelating agent such as ethylenediaminetetraacetic acid (EDTA). The concentration of EDTA in the extraction solvent is preferably 0.01 mM to 1 M, more preferably 0.1 mM to 10 mM. The presence of EDTA in the extraction solvent chelates metal ions that may reduce enzyme activity, which tends to further increase enzyme activity.

[0051] In addition to the above components, the extraction solvent may contain known components that can be added to enzyme extraction solvents.

[0052] The mutant tetraprenyl-β-curcumene cyclase may be used alone or in combination of two or more. The conditions for the reaction of the mutant tetraprenyl-β-curcumene cyclase with squalene or 8α-hydroxypolypoda-13,17,21-triene are not particularly limited as long as they allow the enzymatic reaction to proceed. For example, the reaction temperature and reaction time can be appropriately selected based on the activity of the mutant tetraprenyl-β-curcumene cyclase. From the viewpoint of reaction efficiency, the reaction temperature and reaction time are, for example, 4°C to 100°C and 1 hour to 30 days, with 30°C to 60°C and 16 hours to 20 days being preferred. From the viewpoint of reaction efficiency, the pH condition is, for example, 3 to 10, with 6 to 8 being preferred.

[0053] The reaction solvent is not particularly limited as long as it does not inhibit the enzymatic reaction, and a commonly used buffer solution or the like can be used. For example, the same solvent as the extraction solvent used in the enzyme extraction step can be used. Alternatively, an extract containing a mutant tetraprenyl-β-curcumene cyclase (e.g., a cell-free extract) can be used as an enzyme solution for the reaction.

[0054] From the viewpoint of reaction efficiency, the concentration ratio of the mutant tetraprenyl-β-curcumene cyclase to its substrate, squalene or 8α-hydroxypolypoda-13,17,21-triene, in the ambrein production reaction is preferably 1 to 10,000, more preferably 10 to 5,000, more preferably 100 to 3,000, and even more preferably 1,000 to 2,000, as the molar concentration ratio of substrate to enzyme (substrate / enzyme). From the viewpoint of reaction efficiency, the concentration of squalene or 8α-hydroxypolypoda-13,17,21-triene used in the enzymatic reaction is preferably 0.000001% by mass to 10% by mass, and more preferably 0.00001% by mass to 1% by mass, based on the total mass of the reaction solvent.

[0055] The reaction step in which the mutant tetraprenyl-β-curcumene cyclase reacts with squalene or 8α-hydroxypolypoda-13,17,21-triene may be repeated multiple times. This can increase the yield of ambrein. When the reaction step is repeated multiple times, it may include a step of re-introducing the substrate squalene or 8α-hydroxypolypoda-13,17,21-triene into the reaction system, a step of inactivating the enzyme by a known method, and then recovering and purifying the reaction product in the reaction solution. When squalene is re-introduced, the timing and amount of addition can be appropriately determined depending on the concentration of the mutant tetraprenyl-β-curcumene cyclase in the reaction solution, the amount of substrate remaining in the reaction solution, etc.

[0056] Another embodiment of the method for producing ambulin of the present invention is characterized by culturing the microorganism or transformant of the present invention. Ambrein can be produced by culturing the microorganism or a host cell transformed with the expression vector. For example, in the case of yeast, yeast can be cultured in a commonly used medium such as YPD medium. Yeast into which a gene has been introduced by homologous recombination or yeast carrying an expression vector is pre-cultured, then inoculated into YPD medium or the like and cultured for 24 to 240 hours, preferably 72 to 120 hours. Ambrein secreted into the medium can be used as is, or after purification by known methods. Specific purification methods include solvent extraction, recrystallization, distillation, column chromatography, and HPLC.

[0057] Second Mode A second embodiment of the method for producing ambrein of the present invention includes (1) a step of reacting squalene with tetraprenyl-β-curcumene cyclase to obtain 8α-hydroxypolypoda-13,17,21-triene (hereinafter, sometimes referred to as step 1), and (2) a step of reacting 8α-hydroxypolypoda-13,17,21-triene with mutant tetraprenyl-β-curcumene cyclase to obtain ambrein (hereinafter, sometimes referred to as step 2). Figure 3 shows an embodiment in which wild-type tetraprenyl-β-curcumene cyclase is used in step (1).

[0058] (1st step) In the first step of the second embodiment of the method for producing ambrane of the present invention, squalene is reacted with a tetraprenyl-β-curcumene cyclase to obtain 8α-hydroxypolypoda-13,17,21-triene. Step 1 can be carried out in the same manner as in the "reacting squalene with a mutant tetraprenyl-β-curcumene cyclase" of the first embodiment, except that a tetraprenyl-β-curcumene cyclase is used instead of the mutant tetraprenyl-β-curcumene cyclase.

[0059] The tetraprenyl-β-curcumene cyclase used in the first step is not particularly limited as long as it can convert squalene to 8α-hydroxypolypoda-13,17,21-triene. For example, the mutant tetraprenyl-β-curcumene cyclase of the present invention may be used, or a wild-type tetraprenyl-β-curcumene cyclase may be used, or a wild-type tetraprenyl-β-curcumene cyclase into which a mutation other than that of the mutant tetraprenyl-β-curcumene cyclase of the present invention has been introduced (hereinafter referred to as a "mutant TC for the first step") may be used. Examples of wild-type tetraprenyl-β-curcumene cyclases include the wild-type tetraprenyl-β-curcumene cyclase derived from Bacillus megaterium set forth in SEQ ID NO: 1, the wild-type tetraprenyl-β-curcumene cyclase derived from Bacillus subtilis (accession number: AB618206), and the wild-type tetraprenyl-β-curcumene cyclase derived from Bacillus licheniformis (accession number: AAU41134). The mutant TC for the first step includes (1) a mutant TC consisting of an amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 5, or SEQ ID NO: 6 in which one or more amino acids have been deleted, substituted, inserted, and / or added, and which exhibits the activity of producing 8α-hydroxypolypoda-13,17,21-triene from squalene, or (2) a mutant TC consisting of an amino acid sequence that is 40% or more identical to the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 5, or SEQ ID NO: 6, and which exhibits the activity of producing 8α-hydroxypolypoda-13,17,21-triene from squalene.

[0060] (2nd process) In the second step of the second embodiment of the method for producing ambrein of the present invention, ambrein is obtained by reacting 8α-hydroxypolypoda-13,17,21-triene with a mutant tetraprenyl-β-curcumene cyclase. Specifically, the 8α-hydroxypolypoda-13,17,21-triene obtained in the first step is used as a substrate, and the process can be carried out in the same manner as in the first embodiment, where "8α-hydroxypolypoda-13,17,21-triene is reacted with a mutant tetraprenyl-β-curcumene cyclase." The mutant tetraprenyl-β-curcumene cyclase used in the first embodiment can be used in the second step. Furthermore, the following mutant tetraprenyl-β-curcumene cyclase (hereinafter referred to as mutant TC for the second step) can be used.

[0061] The mutant TC for the second step is a mutant tetraprenyl-β-curcumene cyclase in which the fourth amino acid residue, aspartic acid, of the DXDD motif is substituted with an amino acid other than aspartic acid, and the amino acid adjacent to the N-terminus of the (A / S / G)RX(H / N)XXP motif is substituted with an amino acid other than tyrosine, or the fourth amino acid of the GXGX(G / A / P) motif is substituted with an amino acid other than leucine, and (a) the mutant tetraprenyl-β-curcumene cyclase has, based on the DXDD motif, a QXXXGX(W / F) motif at a position 100 or more amino acid residues away from the N-terminus, the (A / S / G)RX(H / N)XXP motif at a position 180 to 250 amino acid residues away from the N-terminus, and the (D / N)G(T / L)(L / F / Y)(Y / F)SY motif at a position 80 to 140 amino acid residues away from the N-terminus. a QXXXX(G / A / S)X(F / W / Y) motif located 10 to 50 amino acid residues away from the N-terminus, a QXXXGX(F / W / Y) motif located 20 to 50 amino acid residues away from the C-terminus, a QXXXGXW motif located 50 to 120 amino acid residues away from the C-terminus, a QXXXGX(F / W) motif located 120 to 170 amino acid residues away from the C-terminus, and the GXGX(G / A / P) motif located 180 to 250 amino acid residues away from the C-terminus, and does not have a QXXXGXW motif located 170 or more amino acid residues away from the DXDD motif; (b) has an identity of 40% or more with the amino acid sequence represented by SEQ ID NO: 1; and (c) exhibits ambuline-forming activity using 8α-hydroxypolypoda-13,17,21-triene as a substrate.

[0062] A preferred embodiment of the mutant TC for the second step of the present invention is the mutant tetraprenyl-β-curcumene cyclase described in

[14] above.

[0063] Furthermore, the most preferred embodiment of the mutant TC for the second step of the present invention is a polypeptide derived from Bacillus megaterium, in which the polypeptide constituting the mutant tetraprenyl-β-curcumene cyclase consists of the amino acid sequence set forth in SEQ ID NO: 8 or 9. That is, in the mutant TC for the second step, the fourth amino acid residue, aspartic acid, of the DXDD motif is substituted with cysteine, and the amino acid adjacent to the N-terminus of the (A / S / G)RX(H / N)XXP motif is substituted from tyrosine to alanine, or the fourth amino acid residue, aspartic acid, of the DXDD motif is substituted with cysteine, and the fourth amino acid of the GXGX(G / A / P) motif is substituted from leucine to alanine.

[0064] Figure 7 shows the amino acid sequence of a mutant tetraprenyl-β-curcumene cyclase derived from Bacillus megaterium, in which the aspartic acid at position 373 is replaced with cysteine ​​and the tyrosine at position 167 is replaced with alanine (sequence number 8), and the amino acid sequence of a mutant tetraprenyl-β-curcumene cyclase derived from Bacillus megaterium, in which the aspartic acid at position 373 is replaced with cysteine ​​and the leucine at position 596 is replaced with alanine (sequence number 9). [Example]

[0065] The present invention will be described in more detail below with reference to examples, but these examples are not intended to limit the scope of the present invention. In the text and figures, Bacillus megaterium may be abbreviated as "Bme," and tetraprenyl-β-curcumene cyclase derived from Bacillus megaterium may be abbreviated as "BmeTC."

[0066] Example 1 In this example, we cloned a mutant tetraprenyl-β-curcumene cyclase and constructed a mutant tetraprenyl-β-curcumene cyclase gene in which tyrosine at position 257 was replaced with alanine and aspartic acid at position 373 was replaced with cysteine. A polynucleotide encoding wild-type tetraprenyl-β-curcumene cyclase was isolated by PCR using Bacillus megaterium chromosomal DNA as a template, and the amino acid sequence of the wild-type enzyme was determined. (Unless otherwise specified, the tetraprenyl-β-curcumene cyclase is derived from Bacillus megaterium and is sometimes simply referred to as "wild-type.") The resulting gene was inserted into the cloning site (restriction enzyme NdeI / XhoI sites) of the vector pColdI (Takara Bio Inc.) to obtain an expression vector containing the wild-type tetraprenyl-β-curcumene cyclase gene (SEQ ID NO: 1). The resulting expression vector containing the mutant tetraprenyl-β-curcumene cyclase gene was used to generate transformants of Escherichia coli BL21(DE3). Next, we constructed a mutant tetraprenyl-β-curcumene cyclase gene in which tyrosine at position 257 was replaced with alanine and aspartic acid at position 373 was replaced with cysteine. First, based on the gene sequence of tetraprenyl-β-curcumene cyclase (wild-type), the gene was designed so that aspartic acid at position 373 was replaced with cysteine, and the gene was synthesized by optimizing the codons for the host, Escherichia coli. The synthesized gene was inserted into the cloning site (restriction enzyme NdeI / XhoI sites) of the vector pColdI (Takara Bio Inc.). Using this vector gene as a template, site-specific mutagenesis was performed by the QuickChange method using the mutagenesis primer OPY257A (SEQ ID NOs: 18 and 19) to replace tyrosine at position 257 with alanine, yielding an expression vector containing the mutant tetraprenyl-β-curcumene cyclase gene of the Y257A / D373C mutant (SEQ ID NO: 12). The pCold vector is a cold shock expression vector that utilizes the promoter of the cspA gene, a cold shock gene. A translation enhancing element (TEE), His tag sequence, and Factor Xa cleavage sequence are located downstream of the cspA promoter. Therefore, the expressed protein is expressed as a fusion protein containing the TEE sequence, His tag sequence, and Factor Xa cleavage sequence. In the present invention, the start codon (GTG) sequence of the wild-type enzyme gene derived from Bacillus megaterium is unchanged. Therefore, the N-terminal amino acid of the enzyme protein portion in the fusion protein is translated into valine. Subsequently, an Escherichia coli BL21(DE3) transformant was prepared using an expression vector containing the mutant tetraprenyl-β-curcumene cyclase gene.

[0067] Example 2 In this example, a mutant tetraprenyl-β-curcumene cyclase gene was constructed in which tyrosine at position 167 was substituted with alanine, tyrosine at position 257 was substituted with alanine, and aspartic acid at position 373 was substituted with cysteine. The mutant tetraprenyl-β-curcumene cyclase gene was designed based on the gene sequence of tetraprenyl-β-curcumene cyclase (wild-type) so that tyrosine at position 167 was replaced with alanine and aspartic acid at position 373 was replaced with cysteine, and the codons were optimized for the host Escherichia coli. The synthesized gene was inserted into the cloning site (restriction enzyme NdeI / XhoI sites) of the vector pColdI (Takara Bio Inc.). Using this vector gene as a template, site-specific mutations were introduced by the quick-change method using the mutagenesis primer OPY257A (SEQ ID NOs: 18 and 19) to replace tyrosine at position 257 with alanine, yielding an expression vector containing the mutant tetraprenyl-β-curcumene cyclase gene of the Y167A / Y257A / D373C mutant (SEQ ID NO: 13). Subsequently, an Escherichia coli BL21(DE3) transformant was prepared using an expression vector containing the mutant tetraprenyl-β-curcumene cyclase gene.

[0068] ≪Reference example 1≫ In this Reference Example, a mutant tetraprenyl-β-curcumene cyclase gene was constructed in which aspartic acid at position 373 was substituted with cysteine. The mutant tetraprenyl-β-curcumene cyclase gene was designed to replace aspartic acid at position 373 with cysteine, and was synthesized by optimizing the codons for the host Escherichia coli, based on the gene sequence of the wild-type enzyme described in Example 1. The synthesized gene was inserted into the cloning site (restriction enzyme NdeI / XhoI sites) of the vector pColdI (Takara Bio Inc.), to obtain an expression vector containing the mutant tetraprenyl-β-curcumene cyclase gene of the D373C mutant (SEQ ID NO: 17). Subsequently, an Escherichia coli BL21(DE3) transformant was prepared using an expression vector containing the mutant tetraprenyl-β-curcumene cyclase gene.

[0069] ≪Reference example 2≫ In this Reference Example, a mutant tetraprenyl-β-curcumene cyclase gene was constructed in which tyrosine at position 167 was replaced with alanine and aspartic acid at position 373 was replaced with cysteine. The mutant tetraprenyl-β-curcumene cyclase gene was designed based on the gene sequence of the wild-type enzyme described in Example 1, so that tyrosine at position 167 was replaced with alanine and aspartic acid at position 373 was replaced with cysteine, and the gene was synthesized by optimizing the codons for the host Escherichia coli. The synthesized gene was inserted into the cloning site (restriction enzyme NdeI / XhoI sites) of the vector pColdI (Takara Bio Inc.), yielding an expression vector containing the mutant tetraprenyl-β-curcumene cyclase gene of the Y167A / D373C mutant (SEQ ID NO: 15). Subsequently, an Escherichia coli BL21(DE3) transformant was prepared using an expression vector containing the mutant tetraprenyl-β-curcumene cyclase gene.

[0070] ≪Reference example 3≫ In this Reference Example, a mutant tetraprenyl-β-curcumene cyclase gene was constructed in which the aspartic acid at position 373 was replaced with cysteine ​​and the leucine at position 596 was replaced with alanine. The mutant tetraprenyl-β-curcumene cyclase gene was designed based on the gene sequence of the wild-type enzyme described in Example 1, with the aspartic acid at position 373 replaced with cysteine ​​and the leucine at position 596 replaced with alanine, and was synthesized by optimizing the codons for the host Escherichia coli. The synthesized gene was inserted into the cloning site (restriction enzyme NdeI / XhoI sites) of the vector pColdTF (Takara Bio Inc.), yielding an expression vector containing the mutant tetraprenyl-β-curcumene cyclase gene of the D373C / L596A mutant (SEQ ID NO: 16). Subsequently, an Escherichia coli BL21(DE3) transformant was prepared using an expression vector containing the mutant tetraprenyl-β-curcumene cyclase gene.

[0071] Example 3 In this example, a mutant tetraprenyl-β-curcumene cyclase gene was constructed in which tyrosine at position 167 was substituted with alanine, aspartic acid at position 373 was substituted with cysteine, and leucine at position 596 was substituted with alanine. First, based on the gene sequence of wild-type tetraprenyl-β-curcumene cyclase, a gene was designed to replace tyrosine at position 167 with alanine, aspartic acid at position 373 with cysteine, and leucine at position 596 with alanine, and the gene was synthesized by optimizing the codons for the host Escherichia coli. The synthesized gene was inserted into the cloning site (restriction enzyme NdeI / XhoI sites) of the vector pColdI (Takara Bio Inc.) to obtain an expression vector containing the mutant tetraprenyl-β-curcumene cyclase gene of the Y167A / D373C / L596A mutant (SEQ ID NO: 14). Subsequently, an Escherichia coli BL21(DE3) transformant was prepared using an expression vector containing the mutant tetraprenyl-β-curcumene cyclase gene.

[0072] 《Manufacturing method example 1》 In this example of the production method, the enzyme activity of the mutant tetraprenyl-β-curcumene cyclases of Examples 1 to 3 and Reference Example 3 was examined using the bicyclic compound 8α-hydroxypolypoda-13,17,21-triene as a substrate. The transformants prepared in Examples 1 to 3 and Reference Example 3 were each inoculated into LB medium (1 L) containing ampicillin (50 mg / L) and cultured with shaking at 37° C. for 3 hours. After culture, 1 mM isopropyl-β-thiogalactopyranoside (IPTG) was added, and the culture was shaken at 15° C. for 24 hours to induce expression of the mutant tetraprenyl-β-curcumene cyclase. The cells were then collected by centrifugation (6,000 × g, 10 min). The cells were washed with 50 mM Tris-HCl buffer (pH 7.5) and then suspended in 15 mL of buffer A (50 mM Tris-HCl buffer (pH 7.5), 0.1% Tween 80 (v / v), 0.1% sodium ascorbate (v / v), 2.5 mM dithiothreitol, and 1 mM EDTA) per 5 g of cells. The cells were then sonicated (4°C, 20 min) using a UP2005 sonicator (Hielscher Ultrasonics, Teltow, Germany). The disrupted sample was centrifuged (12,300 × g, 20 min) to obtain a crude enzyme solution. The target protein was affinity purified from this crude extract using the His tag. Purification was carried out using a nickel column (Ni-NTA Agarose, QIAGEN) to prepare a recombinant enzyme solution (protein concentration: 100 μg / mL).

[0073] 8α-Hydroxypolypoda-13,17,21-triene (100 μg) was solubilized in Tween 80 (2 mg) and then added to Buffer A (1 mL) to prepare an 8α-hydroxypolypoda-13,17,21-triene solution. 0.5 mL of this solution was added to 0.5 mL of enzyme solution to form a reaction mixture, which was then incubated at 30°C for 64 hours. The enzyme protein content in the 8α-hydroxypolypoda-13,17,21-triene reaction mixture was 50 μg, and the molar ratio (substrate / enzyme) of 8α-hydroxypolypoda-13,17,21-triene (substrate) to mutant tetraprenyl-β-curcumene cyclase (enzyme) was approximately 170. After incubation, 1.2 mL of 15% potassium hydroxide in methanol was added to the reaction mixture to stop the enzyme reaction, and n-hexane (2.0 mL) was then added to the reaction mixture to extract the reaction product three times.

[0074] The contents of ambrein, two-ring compound (8α-hydroxypolypoda-13,17,21-triene), and onoceroid in the obtained extract (reaction composition) were measured by gas chromatography using the following equipment and conditions. Equipment: Gas chromatograph "GC-2014" (Shimadzu), Integrator "Chromatopac CR-8A" (Shimadzu) Column: DB-1, capillary (length 30 m, ID 0.32 mm, film thickness 0.25 μm) (J&D) Injection temperature: 300℃ Column temperature: 220-300°C (heating rate: 3°C / min) Column flow rate: 1.0 mL / min

[0075] The content ratio of the reaction product (ambrane) in the obtained extract (reaction composition) is shown in Figure 4(A). The content ratio of ambrein and onoceroid in the obtained extract (reaction composition), excluding 8α-hydroxypolypoda-13,17,21-triene, is also shown in Figure 4(B). The Y257A / D373C mutant of Example 1, the Y167A / Y257A / D373C mutant of Example 2, the Y167A / D373C / L596A mutant of Example 3, and the D373C / L596A mutant of Reference Example 3 were able to produce ambrein using the bicyclic compound 8α-hydroxypolypoda-13,17,21-triene as a substrate. In particular, the Y257A / D373C mutant in Example 1 and the Y167A / Y257A / D373C mutant in Example 2 produced large amounts of ambrein when 8α-hydroxypolypoda-13,17,21-triene was used as a substrate. The Y167A / D373C / L596A mutant in Example 3 did not produce the by-product onoceroide at all. The Y167A / Y257A / D373C mutant of Example 2 was able to produce ambrein from 8α-hydroxypolypoda-13,17,21-triene at a production rate (production efficiency) of 85.5% and a production ratio of 88.6%, the Y257A / D373C mutant of Example 1 was able to produce ambrein at a production rate (production efficiency) of 45.7% and a production ratio of 61.3%, and the Y167A / D373C / L596A mutant of Example 3 was able to produce ambrein at a production rate (production efficiency) of 9.0% and a production ratio of 100%. Thus, the Y167A / Y257A / D373C mutant of Example 2, the Y257A / D373C mutant of Example 1, and the Y167A / D373C / L596A mutant of Example 3 have improved reaction selectivity from 8α-hydroxypolypoda-13,17,21-triene (substrate) to ambrein, enabling efficient production of ambrein.

[0076] 《Manufacturing method example 2》 In this example of the production method, the enzyme activity of the mutant tetraprenyl-β-curcumene cyclases of Example 2 and Reference Example 1 was examined using squalene as a substrate. The recombinant enzyme solution (protein concentration: 100 μg / mL) was prepared in the same manner as in Production Method Example 1.

[0077] Squalene (100 μg) was solubilized in Tween 80 (2 mg) and then added to Buffer A (1 mL) to prepare a squalene solution. This squalene solution (0.5 mL) was added to the enzyme solution (0.5 mL) to form a reaction mixture, which was then incubated at 30°C for 64 hours. The reaction mixture contained 50 μg of enzyme protein, and the molar ratio (substrate / enzyme) of squalene (substrate) to the mutant tetraprenyl-β-curcumene cyclase (enzyme) was approximately 170. After incubation, 1.2 mL of 15% potassium hydroxide in methanol was added to the reaction mixture to stop the enzyme reaction, and n-hexane (2.0 mL) was then added to the reaction mixture to extract the reaction product three times.

[0078] The contents of squalene, ambrein, monocyclic compounds, bicyclic compounds (8α-hydroxypolypoda-13,17,21-triene), and onoceroid in the obtained extract (reaction composition) were measured by gas chromatography in the same manner as in Production Method Example 1.

[0079] The content of the reaction product (ambrane) in the resulting extract (reaction composition) is shown in Figure 5(A). The content ratios of ambrein, monocyclic compounds, bicyclic compounds, and onoceroid, excluding squalene, in the resulting extract (reaction composition) are shown in Figure 5(B). The Y167A / Y257A / D373C mutant of Example 2 produced a higher amount of ambrein than the D373C mutant of Reference Example 1. The Y167A / Y257A / D373C mutant of Example 1 was able to produce ambrein from squalene with a production rate (production efficiency) of 13.6% and a production ratio of 44.0%. Thus, the Y167A / Y257A / D373C mutant of Example 2 exhibited high reaction selectivity from squalene (substrate) to ambrein, enabling efficient production of ambrein.

[0080] 《Manufacturing method example 3》 In this example of the production method, we investigated the production of ambrein using a stepwise (two-step) reaction method with squalene as a substrate, in which wild-type tetraprenyl-β-curcumene cyclase and each mutant tetraprenyl-β-curcumene cyclase, which have different substrate specificities, were combined. The mutant tetraprenyl-β-curcumene cyclases used were the Y167A / Y257A / D373C mutant of Example 2 and the Y167A / D373C mutant of Reference Example 2. A recombinant enzyme solution (protein concentration 100 μg / mL) was prepared in the same manner as in Production Method Example 1. Squalene (100 μg) was solubilized in Tween 80 (2 mg) and then added to Buffer A (1 mL) to prepare a squalene solution. A wild-type tetraprenyl-β-curcumene cyclase solution (0.25 mL) was added to this squalene solution (0.5 mL) to form a reaction solution, which was then incubated at 30°C for 64 hours. The reaction was then terminated by incubation at 70°C for 1 hour. Next, a mutant tetraprenyl-β-curcumene cyclase solution (0.25 mL) was added to this reaction solution, which was then incubated at 30°C for 64 hours. The amount of enzyme protein in the squalene reaction solution was 25 μg for both the wild-type and mutant tetraprenyl-β-curcumene cyclases. The molar ratio (substrate / enzyme) of squalene (substrate) to the mutant tetraprenyl-β-curcumene cyclase (enzyme) or wild-type tetraprenyl-β-curcumene cyclase (enzyme) was approximately 170. In Reference Example 2 (Y167A / D373C mutant), one type of enzyme solution (0.5 mL) was added to form a reaction solution, and squalene and the enzyme were reacted in the same manner. After incubation, 1.2 mL of 15% potassium hydroxide in methanol was added to the reaction mixture to stop the enzyme reaction, and n-hexane (2.0 mL) was then added to the reaction mixture to extract the reaction product three times.

[0081] The contents of squalene, ambrein, monocyclic compounds, bicyclic compounds, and onoceroid in the obtained extract (reaction composition) were measured by gas chromatography in the same manner as in Production Method Example 1.

[0082] The content of the reaction product (ambrain) in the resulting extract (reaction composition) is shown in Figure 6(A). The content ratios of ambrein, monocyclic compounds, bicyclic compounds, and onoceroid, excluding squalene, in the resulting extract (reaction composition) are shown in Figure 6(B). The combination of wild-type tetraprenyl-β-curcumene cyclase and the Y167A / Y257A / D373C mutant of Example 2, and the combination of wild-type tetraprenyl-β-curcumene cyclase and the Y167A / D373C mutant of Reference Example 2, produced greater amounts of ambrein than the use of the Y167A / D373C mutant of Reference Example 2 alone. Thus, by the stepwise reaction method, the combination of the wild-type tetraprenyl-β-curcumene cyclase and the Y167A / Y257A / D373C mutant of Example 2 was able to produce ambrein from squalene at a production rate (production efficiency) of 59.9% and a production ratio of 65.6%, while the combination of the wild-type tetraprenyl-β-curcumene cyclase and the Y167A / D373C mutant of Reference Example 2 was able to produce ambrein from squalene at a production rate (production efficiency) of 35.6% and a production ratio of 46.7%.

[0083] SEQ ID NO: 1: Bacillus megaterium WT amino acid sequence SEQ ID NO: 2: Bacillus megaterium Y257A / D373C amino acid sequence SEQ ID NO: 3: Bacillus megaterium Y167A / Y257A / D373C amino acid sequence SEQ ID NO: 4: Bacillus megaterium Y167A / D373C / L596A amino acid sequence SEQ ID NO: 5: Bacillus subtilis WT amino acid sequence SEQ ID NO: 6: Bacillus lichenformis WT amino acid sequence SEQ ID NO: 7: Alicyclobacillus acidocaldarius SHC amino acid sequence SEQ ID NO: 8: Bacillus megaterium Y167A / D373C amino acid sequence SEQ ID NO: 9: Bacillus megaterium D373C / L596A amino acid sequence SEQ ID NO: 10: Bacillus megaterium D373C amino acid sequence SEQ ID NO: 11: Bacillus megaterium WT base sequence SEQ ID NO: 12: Bacillus megaterium Y257A / D373C base sequence SEQ ID NO: 13: Bacillus megaterium Y167A / Y257A / D373C base sequence SEQ ID NO: 14: Bacillus megaterium Y167A / D373C / L596A base sequence SEQ ID NO: 15: Bacillus megaterium Y167A / D373C base sequence SEQ ID NO: 16: Bacillus megaterium D373C / L596A base sequence SEQ ID NO: 17: Bacillus megaterium D373C base sequence SEQ ID NO: 18: OPY257A_F SEQ ID NO: 19: OPY257A_R [Industrial Applicability]

[0084] The mutant tetraprenyl-β-curcumene cyclase of the present invention can be used for efficient microbial production of ambrein. Ambrein obtained by the present invention can be used, for example, as a raw material for the production of pharmaceuticals and the like.

Claims

1. A mutant tetraprenyl-β-curcumene cyclase, comprising a polypeptide constituting the mutant tetraprenyl-β-curcumene cyclase: (1) A polypeptide in which the aspartic acid at the 373rd position from the N-terminus in the amino acid sequence represented by SEQ ID NO: 1 is substituted with cysteine ​​and the tyrosine at the 257th position from the N-terminus is substituted with alanine; (2) A polypeptide consisting of an amino acid sequence in which the aspartic acid at position 373 from the N-terminus in the amino acid sequence represented by SEQ ID NO: 1 is substituted with cysteine ​​and the tyrosine at position 257 from the N-terminus is substituted with alanine, and which has an identity of 90% or more with the amino acid sequence represented by SEQ ID NO:

1. (3) A polypeptide comprising an amino acid sequence represented by SEQ ID NO: 1 in which the aspartic acid at the 373rd position from the N-terminus is substituted with cysteine ​​and the tyrosine at the 257th position from the N-terminus is substituted with alanine. (4) A polypeptide comprising an amino acid sequence in which the aspartic acid at position 373 from the N-terminus in the amino acid sequence represented by SEQ ID NO: 1 is substituted with cysteine ​​and the tyrosine at position 257 from the N-terminus is substituted with alanine, and which has an identity of 90% or more with the amino acid sequence represented by SEQ ID NO:

1. (5) A polypeptide in which the aspartic acid at the 373rd position from the N-terminus in the amino acid sequence represented by SEQ ID NO: 1 is substituted with cysteine, the tyrosine at the 167th position from the N-terminus is substituted with alanine, and the tyrosine at the 257th position from the N-terminus is substituted with alanine. (6) A polypeptide consisting of an amino acid sequence in which the aspartic acid at position 373 from the N-terminus in the amino acid sequence represented by SEQ ID NO: 1 is substituted with cysteine, the tyrosine at position 167 from the N-terminus is substituted with alanine, and the tyrosine at position 257 from the N-terminus is substituted with alanine, and which has an identity of 90% or more with the amino acid sequence represented by SEQ ID NO:

1. (7) A polypeptide comprising an amino acid sequence represented by SEQ ID NO: 1 in which the aspartic acid at the 373rd position from the N-terminus is substituted with cysteine, the tyrosine at the 167th position from the N-terminus is substituted with alanine, and the tyrosine at the 257th position from the N-terminus is substituted with alanine; or (8) A polypeptide comprising an amino acid sequence having an identity of 90% or more with the amino acid sequence represented by SEQ ID NO: 1, in which the aspartic acid at position 373 from the N-terminus is substituted with cysteine, the tyrosine at position 167 from the N-terminus is substituted with alanine, and the tyrosine at position 257 from the N-terminus is substituted with alanine, and It exhibits ambrein-forming activity using 8α-hydroxypolypoda-13,17,21-triene as a substrate. Mutant tetraprenyl-β-curcumene cyclase.

2. A mutant tetraprenyl-β-curcumene cyclase, comprising a polypeptide constituting the mutant tetraprenyl-β-curcumene cyclase: (1) A polypeptide in which the aspartic acid at the 373rd position from the N-terminus in the amino acid sequence represented by SEQ ID NO: 1 is substituted with cysteine, the tyrosine at the 167th position from the N-terminus is substituted with alanine, and the leucine at the 596th position from the N-terminus is substituted with alanine; (2) A polypeptide consisting of an amino acid sequence in which the aspartic acid at position 373 from the N-terminus in the amino acid sequence represented by SEQ ID NO: 1 is substituted with cysteine, the tyrosine at position 167 from the N-terminus is substituted with alanine, and the leucine at position 596 from the N-terminus is substituted with alanine, and which has an identity of 90% or more with the amino acid sequence represented by SEQ ID NO:

1. (3) A polypeptide comprising an amino acid sequence represented by SEQ ID NO: 1 in which the aspartic acid at the 373rd position from the N-terminus is substituted with cysteine, the tyrosine at the 167th position from the N-terminus is substituted with alanine, and the leucine at the 596th position from the N-terminus is substituted with alanine; or (4) A polypeptide comprising an amino acid sequence having an identity of 90% or more with the amino acid sequence represented by SEQ ID NO: 1, in which the aspartic acid at position 373 from the N-terminus is substituted with cysteine, the tyrosine at position 167 from the N-terminus is substituted with alanine, and the leucine at position 596 from the N-terminus is substituted with alanine, and It exhibits ambrein-forming activity using 8α-hydroxypolypoda-13,17,21-triene as a substrate. Mutant tetraprenyl-β-curcumene cyclase.

3. A polynucleotide encoding the mutant tetraprenyl-β-curcumene cyclase according to claim 1 or 2.

4. A microorganism having the polynucleotide of claim 3.

5. A vector comprising DNA having the polynucleotide of claim 3.

6. A transformant having the vector according to claim 5.

7. A method for producing ambrein, comprising reacting the mutant tetraprenyl-β-curcumene cyclase according to claim 1 or 2 with squalene or 8α-hydroxypolypoda-13,17,21-triene to obtain ambrein.

8. A method for producing ambrein, comprising culturing the microorganism according to claim 4 or the transformant according to claim 6 in a medium containing squalene or 8α-hydroxypolypoda-13,17,21-triene.

9. (1) reacting tetraprenyl-β-curcumene cyclase with squalene to obtain 8α-hydroxypolypoda-13,17,21-triene; and (2) reacting a mutant tetraprenyl-β-curcumene cyclase with 8α-hydroxypolypoda-13,17,21-triene to obtain ambrein; A method for producing ambraine, comprising: The mutant tetraprenyl-β-curcumene cyclase is (A) The mutant tetraprenyl-β-curcumene cyclase according to claim 1 or 2 This is the manufacturing method of Ambrain.

10. The method for producing ambrane according to claim 9, wherein the steps (1) and (2) are carried out simultaneously.

11. The method for producing ambrene according to claim 9 or 10, wherein the tetraprenyl-β-curcumene cyclase is a wild-type tetraprenyl-β-curcumene cyclase.

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