WO2021193806A1 - MUTANT TETRAPRENYL-β-CURCUMENE CYCLASE AND METHOD FOR PRODUCING AMBREIN USING SAME - Google Patents

MUTANT TETRAPRENYL-β-CURCUMENE CYCLASE AND METHOD FOR PRODUCING AMBREIN USING SAME Download PDF

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WO2021193806A1
WO2021193806A1 PCT/JP2021/012483 JP2021012483W WO2021193806A1 WO 2021193806 A1 WO2021193806 A1 WO 2021193806A1 JP 2021012483 W JP2021012483 W JP 2021012483W WO 2021193806 A1 WO2021193806 A1 WO 2021193806A1
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amino acid
terminal side
replaced
tyrosine
motif
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佐藤　努
稔彦 竹花
誠治 小池
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国立大学法人新潟大学
株式会社Adeka
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Definitions

  • the present invention relates to a mutant tetraprenyl- ⁇ -curcumen cyclase and a method for producing an umbrella using the same.
  • Ambergris is a high-class fragrance that has been used all over the world since the 7th century, and is also used as a Chinese herbal medicine. Ambergris is thought to have been excreted by sperm whales by calculating indigestible substances of food (octopus, squid, etc.) with gastrointestinal secretions, but the detailed mechanism of production is unknown. The main component of ambergris is ambrein, and it is thought that ambergris undergoes oxidative decomposition by sunlight and oxygen while floating on the sea to produce compounds with various scents.
  • Ambergris the main component of ambergris, is used as a fragrance or a drug, but it is not possible to obtain it in large quantities from the natural world. Therefore, various organic synthesis methods have been proposed.
  • Patent Document 1 as a method for producing (+)-umbrain easily, inexpensively and efficiently, a novel sulfonic acid derivative is produced from umbrella, and an optically active substance of ⁇ -cyclogeranyl halide is added thereto.
  • a method including a step of coupling is disclosed.
  • Non-Patent Document 1 describes 2-((1R) synthesized from ( ⁇ ) (5,5,8a-trimethyloctahydro-1H-spiro [naphthalen-2,2'-oxylan] -1-yl) methanol.
  • the present inventors obtained 3-deoxyachilleol A by reacting squalene with a mutant squalene-hopen cyclase capable of producing 3-deoxyachilleol A from squalene, and further tetraprenyl- ⁇ -curcumen. It has been found that squalene can be produced by reacting with a cyclizing enzyme (Patent Document 2). However, the reaction of converting 3-deoxyacireol A to umbrella, which is the second-stage reaction, has room for improvement in terms of suppression of by-products and ease of scale-up. Furthermore, there was room for improvement in yield.
  • An object of the present invention is to provide a method for producing an umbrella, which can easily and efficiently obtain an umbrella.
  • the present inventor surprisingly, a mutant tetraprenyl- ⁇ -curcumen ring having a slight specific mutation. It has been found that the chemase can efficiently produce umbrellas from bicyclic triterpenes (8 ⁇ -hydroxypolypoda-13,17,21-triene) in high yields. We have found that the mutant tetraprenyl- ⁇ -curcumen cyclase is a second-step bicyclic triterpene (8 ⁇ -hydroxypolypoda-13) in a two-step stepwise method of producing ambrein from squalene.
  • the present invention [1] Asparaginic acid, which is the fourth amino acid residue of the DXDD motif, is replaced with an amino acid other than aspartic acid, and (D / N) G (T / L) (L / F / Y) (Y / F) A mutant tetraprenyl- ⁇ -curcumen cyclase in which the 7th amino acid of the SY motif is replaced with an amino acid other than tyrosine, and (a) the mutant tetraprenyl- ⁇ -curcumen cyclase.
  • the polypeptide constituting the mutant tetraprenyl- ⁇ -curcumen cyclase is (1) the 373rd aspartic acid from the N-terminal side in the amino acid sequence represented by SEQ ID NO: 1 is an amino acid other than aspartic acid.
  • the 373rd aspartic acid is substituted with an amino acid other than aspartic acid, and the 257th tyrosine from the N-terminal side contains an amino acid sequence substituted with an amino acid other than tyrosine, and 8 ⁇ -hydroxypolypoder.
  • the 373rd aspartic acid from the N-terminal side in the amino acid sequence represented by SEQ ID NO: 1 is replaced with an amino acid other than aspartic acid.
  • the acid has been replaced with an amino acid other than aspartic acid
  • the 257th tyrosine from the N-terminal side contains an amino acid sequence having 40% or more identity with an amino acid sequence in which an amino acid other than tyrosine is substituted, and 8 ⁇ -hydroxypolypoda-13,17,21-triene.
  • the 373rd asparagic acid from the N-terminal side in the amino acid sequence represented by SEQ ID NO: 1 is replaced with an amino acid other than aspartic acid
  • the 167th tyrosine from the N-terminal side is an amino acid other than tyrosine.
  • the 167th tyrosine from the N-terminal side is replaced with an amino acid other than tyrosine
  • the 257th tyrosine from the N-terminal side is replaced with an amino acid other than tyrosine.
  • one or several amino acids consist of deleted, substituted, inserted, and / or added amino acid sequences, and umbrella formation using 8 ⁇ -hydroxypolypoda-13,17,21-triene as a substrate.
  • the active polypeptide, (9) the 373rd asparagic acid from the N-terminal side in the amino acid sequence represented by SEQ ID NO: 1 is replaced with an amino acid other than aspartic acid, and the 167th tyrosine from the N-terminal side is tyrosine. It consists of an amino acid sequence that is replaced with an amino acid other than, and the 257th tyrosine from the N-terminal side is replaced with an amino acid other than tyrosine, and has an amino acid sequence of 40% or more identity, and is 8 ⁇ -hydroxy.
  • Polypoda-13,17,21-Triene is a substrate that exhibits umbrella-forming activity.
  • the 373rd aspartic acid from the N-terminal side in the amino acid sequence represented by SEQ ID NO: 1 is an amino acid other than aspartic acid.
  • amino acid sequence in which tyrosine is replaced with an amino acid other than tyrosine and the 257th tyrosine from the N-terminal side is replaced with an amino acid other than tyrosine one or several amino acids are deleted, substituted, inserted, And / or a polypeptide containing an added amino acid sequence and exhibiting umbrella-forming activity using 8 ⁇ -hydroxypolypoda-13,17,21-triene as a substrate, or (12) the amino acid represented by SEQ ID NO: 1.
  • the 373rd asparagic acid from the N-terminal side in the sequence is replaced with an amino acid other than aspartic acid
  • the 167th tyrosine from the N-terminal side is replaced with an amino acid other than tyrosine
  • the 257th position from the N-terminal side Contains an amino acid sequence having 40% or more identity with an amino acid sequence in which tyrosine is replaced with an amino acid other than tyrosine, and has umbrella-forming activity using 8 ⁇ -hydroxypolypoda-13,17,21-triene as a substrate.
  • the fourth amino acid residue of the DXDD motif is replaced with cysteine from asparagic acid, and the (D / N) G (T / L) (L / F / Y) (Y / F)
  • the mutant tetraprenyl- ⁇ -curcumen cyclase according to [1] or [3], wherein the 7th amino acid of the SY motif is replaced with alanine from tyrosine, or the 4th amino acid residue of the DXDD motif.
  • the group is substituted from aspartic acid to cysteine, and the amino acid adjacent to the N-terminal of the (A / S / G) RX (H / N) XPP motif is substituted from tyrosine to alanine.
  • the mutant tetraprenyl- ⁇ -curcumen cyclase has a QXXXGX (W / F) motif at a position separated by 100 amino acid residues or more on the N-terminal side based on the DXDD motif, and a QXXXGX (W / F) motif on the N-terminal side.
  • the (A / S / G) RX (H / N) XXP motif is located 180 to 250 amino acid residues away, and the (D / N) G (T) is located 80 to 140 amino acid residues away from the N-terminal side.
  • a mutant tetraprenyl- ⁇ -curcumen cyclase which exhibits amino acid-forming activity using poda-13,17,21-triene as a substrate.
  • the polypeptide constituting the mutant tetraprenyl- ⁇ -curcumen cyclase is (1) the 373rd aspartic acid from the N-terminal side in the amino acid sequence represented by SEQ ID NO: 1 is an amino acid other than aspartic acid.
  • the 167th tyrosine from the N-terminal side is replaced with an amino acid other than tyrosine
  • the 596th leucine from the N-terminal side is replaced with an amino acid other than leucine, (2).
  • the amino acid sequence represented by SEQ ID NO: 1 the 373rd aspartic acid from the N-terminal side is replaced with an amino acid other than aspartic acid
  • the 167th tyrosine from the N-terminal side is replaced with an amino acid other than tyrosine.
  • one or several amino acids are deleted, substituted, inserted, and / or added.
  • Asparagic acid at the 373rd position from the N-terminal side in the amino acid sequence represented by SEQ ID NO: 1 is aspartic acid.
  • SEQ ID NO: 1 is a polypeptide consisting of an amino acid sequence having an amino acid identity of 40% or more and exhibiting umbrella-forming activity using 8 ⁇ -hydroxypolypoda-13,17,21-triene as a substrate.
  • the 373rd aspartic acid from the N-terminal side in the amino acid sequence is replaced with an amino acid other than aspartic acid
  • the 167th tyrosine from the N-terminal side is replaced with an amino acid other than tyrosine
  • 596 from the N-terminal side is a polypeptide consisting of an amino acid sequence having an amino acid identity of 40% or more and exhibiting umbrella-forming activity using 8 ⁇ -hydroxypolypoda-13,17,21-triene as a substrate.
  • the amino acid sequence represented by 1 the 373rd asparagic acid from the N-terminal side is replaced with an amino acid other than aspartic acid, the 167th tyrosine from the N-terminal side is replaced with an amino acid other than tyrosine, and N.
  • the 596th leucine from the terminal side is replaced with an amino acid other than leucine.
  • the fourth amino acid residue of the DXDD motif is replaced with cysteine from aspartic acid, and the amino acid adjacent to the N-terminal of the (A / S / G) RX (H / N) XXP motif is The mutant tetraprenyl- ⁇ according to [5] or [6], wherein the tyrosine is replaced with alanine and the fourth amino acid of the GXGX (G / A / P) motif is replaced with leucine to alanine.
  • -Kurukumen cyclase [8] A polynucleotide encoding the mutant tetraprenyl- ⁇ -curcumen cyclase according to any one of [1] to [7].
  • a method of manufacturing squalene which is characterized by obtaining [13]
  • a method for producing an umbrella which comprises culturing the microorganism according to [9] or the transformant according to [11].
  • a method for producing an amino acid which comprises a step of reacting curcumen cyclase with 8 ⁇ -hydroxypolypoda-13,17,21-triene to obtain an amino acid, wherein the mutant tetraprenyl- ⁇ -curcumen ring is used.
  • the chemase is the mutant tetraprenyl- ⁇ -curcumen cyclase according to any one of (A) [1] to [7], or (B) aspartic acid, which is the fourth amino acid residue of the DXDD motif.
  • the enzyme has a QXXXGX (W / F) motif at a position separated by 100 amino acid residues or more on the N-terminal side, and the (A / S) at a position 180 to 250 amino acid residues separated on the N-terminal side.
  • / G) RX (H / N) XXP motif said (D / N) G (T / L) (L / F / Y) (Y / F) at a position 80 to 140 amino acid residues away from the N-terminal side.
  • a method for producing an amino acid, which is an active mutant tetraprenyl- ⁇ -curcumen cyclase [15]
  • the polypeptide constituting the mutant tetraprenyl- ⁇ -curcumen cyclase (B) is (1) aspartic acid at the 373rd position from the N-terminal side in the amino acid sequence represented by SEQ ID NO: 1.
  • amino acid sequence in which aspartic acid is replaced with an amino acid other than aspartic acid, and the 167th tyrosine from the N-terminal side is replaced with an amino acid other than tyrosine one or several amino acids are deleted or substituted.
  • the 373rd aspartic acid from the N-terminal side is replaced with an amino acid other than aspartic acid, and the 167th tyrosine from the N-terminal side is replaced with an amino acid other than tyrosine, and 8 ⁇ -hydroxy Polypoda-13,17,21-Triene is a substrate that exhibits umbrella-forming activity.
  • the 373rd aspartic acid from the N-terminal side in the amino acid sequence represented by SEQ ID NO: 1 is an amino acid other than aspartic acid.
  • amino acid in which one or several amino acids are deleted, substituted, inserted, and / or added in an amino acid sequence in which the 167th amino acid from the N-terminal side is replaced with an amino acid other than tyrosine.
  • Asparagic acid is replaced with an amino acid other than asparagic acid
  • the 167th tyrosine from the N-terminal side contains an amino acid sequence having 40% or more identity with an amino acid sequence in which an amino acid other than tyrosine is substituted, and 8 ⁇ -hydroxypolypoda-13, 17, 21 -A polypeptide exhibiting umbrella-forming activity using triene as a substrate
  • the 373rd aspartic acid from the N-terminal side in the amino acid sequence represented by SEQ ID NO: 1 is replaced with an amino acid other than aspartic acid
  • N Polypeptide in which the 596th leucine from the terminal side is replaced with an amino acid other than leucine
  • the 373rd aspartic acid from the N-terminal side in the amino acid sequence represented by SEQ ID NO: 1 is replaced with an amino acid other than aspartic acid.
  • amino acid sequence in which leucine at position 596 from the N-terminal side is replaced with an amino acid other than leucine from the amino acid sequence in which one or several amino acids are deleted, substituted, inserted, and / or added.
  • a polypeptide showing umbrella-forming activity using 8 ⁇ -hydroxypolypoda-13,17,21-triene as a substrate (9) the 373rd asparagine from the N-terminal side in the amino acid sequence represented by SEQ ID NO: 1. It consists of an amino acid sequence in which the acid is replaced with an amino acid other than aspartic acid, and the 596th leucine from the N-terminal side is replaced with an amino acid other than leucine, and the identity is 40% or more.
  • Asparaginic acid at the 373rd position from the N-terminal side in the amino acid sequence represented by SEQ ID NO: 1 is asparagine. It contains an amino acid sequence in which an amino acid other than acid is substituted, and leucine at position 596 from the N-terminal side is substituted with an amino acid other than leucine, and 8 ⁇ -hydroxypolypoda-13,17,21-triene is contained.
  • a polypeptide exhibiting umbrella-forming activity as a substrate (11) the 373rd aspartic acid from the N-terminal side in the amino acid sequence represented by SEQ ID NO: 1 is replaced with an amino acid other than aspartic acid, and from the N-terminal side.
  • the amino acid sequence in which leucine at position 596 is replaced with an amino acid other than leucine one or several amino acids contain the deleted, substituted, inserted, and / or added amino acid sequence, and 8 ⁇ -hydroxypolypoda.
  • Amino acid using -13,17,21-triene as a substrate The 373rd aspartic acid from the N-terminal side in the polypeptide showing the activity of producing an amino acid or (12) the amino acid sequence represented by SEQ ID NO: 1 is replaced with an amino acid other than aspartic acid, and the 596th position from the N-terminal side. Contains an amino acid sequence having 40% or more identity with an amino acid sequence in which leucine is replaced with an amino acid other than leucine, and also produces an umbrella using 8 ⁇ -hydroxypolypoda-13,17,21-triene as a substrate.
  • mutant tetraprenyl- ⁇ -curcumen cyclase (B) the fourth amino acid residue of the DXDD motif is replaced with cysteine from aspartic acid, and the above (A / S / G)
  • the method for producing an amino acid according to [14] or [15], [17] The method for producing an umbrella according to any one of [14] to [16], wherein the steps (1) and (2) are carried out at the same time, and [18] the tetraprenyl- ⁇ -curcumen cyclase.
  • the mutant tetraprenyl- ⁇ -curcumen cyclase of the present invention efficiently produces umbrellas from bicyclic triterpenes (8 ⁇ -hydroxypolypoda-13,17,21-triene) in high yield. Can be done.
  • the mutant tetraprenyl- ⁇ -curcumen cyclase of the present invention is a two-step bicyclic triterpene (8 ⁇ -hydroxypolypoda-13,17, By using it in the step of producing ambrein from 21-trien), ambrein can be efficiently produced in a high yield.
  • Y257A / D373C, where the thyroid was replaced with alanine and aspartic acid at position 373 was replaced with cysteine D373C / L596A, 167 where aspartic acid at position 373 was replaced with cysteine and leucine at position 596 was replaced with alanine.
  • Y167A / Y257A / D373C in which the thyroid of the second was replaced with alanine, the tyrosine of the 257th was replaced with alanine, and the aspartic acid of the 373rd was replaced with cysteine, and the tyrosine of the 167th was replaced with alanine, and the 373rd was It is a figure which showed the amino acid sequence of Y167A / D373C / L596A where aspartic acid was replaced with cysteine, and leucine at position 596 was replaced with alanine.
  • Y257A / D373C, where the thyroid was replaced with alanine and aspartic acid at position 373 was replaced with cysteine D373C / L596A, 167 where aspartic acid at position 373 was replaced with cysteine and leucine at position 596 was replaced with alanine.
  • Y167A / Y257A / D373C in which the thyroid of the second was replaced with alanine, the tyrosine of the 257th was replaced with alanine, and the aspartic acid of the 373rd was replaced with cysteine, and the tyrosine of the 167th was replaced with alanine, and the 373rd was It is a figure which showed the amino acid sequence of Y167A / D373C / L596A where aspartic acid was replaced with cysteine, and leucine at position 596 was replaced with alanine.
  • Tetraprenyl- ⁇ -curcumen cyclase The wild-type tetraprenyl- ⁇ -curcumen cyclase (hereinafter sometimes referred to as TC) can generate an umbrella by using 3-deoxyacireol A having a single ring at one end as a substrate. can. That is, when tetraprenyl- ⁇ -curcumen cyclase utilizes 3-deoxyacireol A as a substrate, the non-cyclized end of 3-deoxyacireol A is selectively cyclized to form both terminal rings. Chemical compounds can be produced.
  • tetraprenyl- ⁇ -curcumen cyclase can produce bicyclic 8 ⁇ -hydroxypolypoda-13,17,21-triene using squalene as a substrate (Non-Patent Document 5).
  • tetraprenyl- ⁇ -curcumen cyclase selectively cyclizes the non-cyclized ends of the bicyclic 8 ⁇ -hydroxypolypoda-13,17,21-triene to cyclize both ends.
  • the compound onoseroids (onoselan oxide and 14 ⁇ -hydroxyonocera-8 (26) -ene) can be produced (Non-Patent Document 6).
  • tetraprenyl- ⁇ -curcumen cyclase is classified into EC4.2.1.129, and is a reaction for producing batyterpenol A from water and tetraprenyl- ⁇ -curcumen, or 8 ⁇ -hydroxypolypoda from squalene. It is an enzyme that can catalyze the reaction that produces 13,17,21-triene.
  • the tetraprenyl- ⁇ -curcumen cyclase is contained in bacteria such as Bacillus, Brevibacillus, Paenibacillus, or Diobacillus. Bacteria of the genus Bacillus include Bacillus subtilis (Bacillus subtilis), Bacillus megaterium, or Bacillus licheniformis.
  • the tetraprenyl- ⁇ -curcumen cyclase is based on the DXDD motif, with the QXXXGX (W / F) motif at a position separated by 100 amino acid residues or more on the N-terminal side and 180 to 250 amino acid residues separated on the N-terminal side.
  • the (A / S / G) RX (H / N) XPP motif is located at the position, and the (D / N) G (T / L) (L / F /) is located at a position 80 to 140 amino acid residues away from the N-terminal side.
  • Squalene-Hopen cyclase is the (A / S / G) RX (H / N) XXP motif, (D / N) G (T / L) (L / F / Y) (Y /) among the above motifs. F) It does not have a SY motif and a GXGX (G / A / P) motif. Further, the squalene-hopen cyclase has a QXXXGXW motif at a position separated by 170 amino acid residues or more on the C-terminal side of the DXDD motif. On the other hand, the tetraprenyl- ⁇ -curcumen cyclase does not have the QXXXGXW motif.
  • the squalene-hopen cyclase has a GXGFP sequence similar to the GXGX (G / A / P) motif on the C-terminal side of the QXXXGXW motif, but the fourth amino acid is phenylalanine (F). It has the characteristic of being.
  • the fourth amino acid is not phenylalanine, but basically leucine (L).
  • mutant tetraprenyl- ⁇ -curcumen cyclase (first aspect) In the mutant tetraprenyl- ⁇ -curcumen cyclase of the first aspect of the present invention, asparagic acid, which is the fourth amino acid residue of the DXDD motif, is replaced with an amino acid other than aspartic acid (D / N). ) G (T / L) (L / F / Y) (Y / F) A mutant tetraprenyl- ⁇ -curcumen cyclase in which the 7th amino acid of the SY motif is replaced with an amino acid other than tyrosine.
  • the mutant tetraprenyl- ⁇ -curcumen cyclase has a QXXXGX (W / F) motif and an N-terminal side at positions separated from the N-terminal side by 100 amino acid residues or more based on the DXDD motif.
  • the (A / S / G) RX (H / N) XXP motif is located 180 to 250 amino acid residues apart, and the (D / N) G (T) is located 80 to 140 amino acid residues separated from the N-terminal side.
  • the mutant tetraprenyl- ⁇ -curcumen cyclase of the first aspect of the present invention the amino acid adjacent to the N-terminal of the (A / S / G) RX (H / N) XXP motif is other than tyrosine. It is replaced with an amino acid.
  • each motif or sequence represents a one-letter abbreviation for an amino acid
  • "X" means an arbitrary amino acid. That is, in the case of the QXXXGX (W / F) motif, glutamine (Q), three arbitrary amino acids (X), glycine (G), any amino acid (X), and further from the N-terminal side to the C-terminal side. Indicates that either tryptophan (W) or phenylalanine (F) is sequenced.
  • amino acid sequence identity is expressed as a percentage by inserting gaps as appropriate so that the amino acid residues of the sequences to be compared match and aligning them, and dividing the number of matched amino acid residues by the total number of amino acid residues. It was done. Identity can be achieved using well-known programs (eg BLAST, FASTA, CLUSTAL W, etc.).
  • a preferred embodiment of the mutant tetraprenyl- ⁇ -curcumen cyclase according to the first aspect of the present invention is the mutant tetraprenyl- ⁇ -curcumen cyclase described in [3] above.
  • the polypeptide constituting the mutant tetraprenyl- ⁇ -curcumen cyclase is SEQ ID NO: 2 or 3.
  • Examples thereof include Bacillus megaterium-derived polypeptides having the amino acid sequence represented by.
  • mutant tetraprenyl- ⁇ -curcumen cyclase aspartic acid, which is the fourth amino acid residue of the DXDD motif, is replaced with cysteine, and the above (D / N) G (T / L) ( The 7th amino acid residue of the L / F / Y) (Y / F) SY motif, tyrosine, is replaced with alanine.
  • mutant tetraprenyl- ⁇ -curcumen cyclase more preferably, the amino acid adjacent to the N-terminal of the (A / S / G) RX (H / N) XXP motif is replaced with alanine from tyrosine.
  • mutant TC substitution of the 4th amino acid residue of the DXDD motif
  • mutant TC substitution of the fourth amino acid residue of the DXDD motif
  • Amino acids other than aspartic acid are not limited as long as the effects of the present invention can be obtained, and are not limited to, alanine, cysteine, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, Although arginine, serine, threonine, valine, tryptophan, or tyrosine can be mentioned, cysteine is preferred.
  • the mutant tetraprenyl- ⁇ -curcumen cyclase of the present invention is 3-from squalene.
  • Deoxyacireol A can be produced, and umbrellas can be produced from 8 ⁇ -hydroxypolypoda-13,17,21-triene.
  • the DXDD motif exists, for example, at the 370th to 373rd positions from the N-terminal side of the amino acid sequence of the tetraprenyl- ⁇ -curcumen cyclase of Bacillus megaterium represented by SEQ ID NO: 1.
  • Amino acids other than tyrosine are not limited as long as the effects of the present invention can be obtained, and are not limited to alanine, cysteine, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, aspartic acid, proline, glutamine, and arginine. , Serine, threonine, valine, tryptophan, or aspartic acid, but also valine, proline, glycine, serine, or phenylalanine. Particularly preferred is alanine or glycine, more preferably alanine.
  • the mutant tetraprenyl- ⁇ -curcumen cyclase of the present invention has an improved function of producing an umbrella from 8 ⁇ -hydroxypolypoda-13,17,21-triene.
  • the (D / N) G (T / L) (L / F / Y) (Y / F) SY motif is, for example, the amino acid of the tetraprenyl- ⁇ -curcumen cyclase of Bacillus megaterium represented by SEQ ID NO: 1. It exists 251 to 257th from the N-terminal side of the sequence. In addition, it is located 256 to 262 from the N-terminal side of the amino acid sequence of the tetraprenyl- ⁇ -curcumen cyclase of Bacillus subtilis represented by SEQ ID NO: 5.
  • the 7th amino acid residue of the (D / N) G (T / L) (L / F / Y) (Y / F) SY motif is extremely conserved and is basically tyrosine in the wild type ( FIG. 8).
  • tetraprenyl- ⁇ -curcumen cyclase can exert umbrella-forming activity using ⁇ -hydroxypolypoda-13,17,21-triene as a substrate. We have found that it can be improved.
  • Amino acids other than tyrosine are not limited as long as the effects of the present invention can be obtained, and are not limited to alanine, cysteine, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, aspartic acid, proline, glutamine, and arginine. , Serine, threonine, valine, tryptophan, or aspartic acid, but preferably hydrophobic amino acids (glycine, alanine, valine, leucine, isoleucine, methionine, proline, phenylalanine, and tryptophan), serine, alanine or cysteine. Can be mentioned.
  • alanine or glycine is particularly preferred, more preferably alanine.
  • alanine or glycine By substituting an amino acid residue adjacent to the N-terminal of the (A / S / G) RX (H / N) XXP motif with an amino acid other than tyrosine (particularly, alanine or glycine), the mutant tetraprenyl of the present invention
  • the - ⁇ -curcumen cyclase has improved functions for producing 3-deoxyacireol A from squalene and for producing umbrellas from 8 ⁇ -hydroxypolypoda-13,17,21-triene.
  • the (A / S / G) RX (H / N) XXP motif is, for example, the 168th to 174th positions from the N-terminal side of the amino acid sequence of the tetraprenyl- ⁇ -curcumen cyclase of Bacillus megaterium represented by SEQ ID NO: 1. Exists in. In addition, it is located 170 to 176th from the N-terminal side of the amino acid sequence of the tetraprenyl- ⁇ -curcumen cyclase of Bacillus subtilis represented by SEQ ID NO: 5.
  • the amino acid residue adjacent to the N-terminal of the (A / S / G) RX (H / N) XXP motif is extremely conserved and is basically tyrosine in the wild type (Fig. 8).
  • the present invention allows the mutant tetraprenyl- ⁇ -curcumen cyclase of the present invention to produce 3-deoxyacireol A from squalene, and Umbranes can be produced from 8 ⁇ -hydroxypolypoda-13,17,21-triene.
  • FIG. 7 shows the wild tetraprenyl- ⁇ -curcumen cyclase of bacillus megaterium, mutant tetraprenyl- ⁇ -curcumen cyclization in which aspartic acid at position 373 was replaced with cysteine and tyrosine at position 257 was replaced with alanine.
  • the amino acid sequence of the enzyme (SEQ ID NO: 3) is shown.
  • the origin of the mutant tetraprenyl- ⁇ -curcumen cyclase of the present invention is not particularly limited, and all tetraprenyl- ⁇ -curcumen cyclases can be used. That is, the tetraprenyl- ⁇ -curcumen cyclase has a QXXXGX (W / F) motif, an (A / S / G) RX (H / N) XXP motif, and (D / N) G (T / L) (L).
  • amino acid sequences of the polypeptides of Bacillus subtilis and Bacillus megaterium are about 50% identical, but due to the characteristics of the present invention, all the enzymes have 8 ⁇ -hydroxypolypoda-13,17, An umbrella can be generated from 21-triene.
  • the amino acid sequence shown in SEQ ID NO: 1 is the amino acid sequence of the wild-type tetraprenyl- ⁇ -curcumen cyclase of Bacillus megaterium
  • amino acid sequence shown in SEQ ID NO: 5 is the wild-type tetraprenyl of Bacillus subtilis. It is an amino acid sequence of - ⁇ -curcumen cyclase.
  • the mutant tetraprenyl- ⁇ -curcumen cyclase (a) the mutant tetraprenyl- ⁇ -curcumen cyclase has 100 amino acid residues on the N-terminal side based on the DXDD motif.
  • the QXXXGX (W / F) motif is located at a distance of 180 to 250 amino acids on the N-terminal side
  • the (A / S / G) RX (H / N) XXP motif is located at a distance of 180 to 250 amino acid residues.
  • the (D / N) G (T / L) (L / F / Y) (Y / F) SY motif is located 140 amino acid residues away, and QXXXX is located 10 to 50 amino acid residues away from the N-terminal side.
  • a preferred embodiment of the mutant tetraprenyl- ⁇ -curcumen cyclase according to the second aspect of the present invention is the mutant tetraprenyl- ⁇ -curcumen cyclase described in [6] above.
  • the polypeptide constituting the mutant tetraprenyl- ⁇ -curcumen cyclase is represented by SEQ ID NO: 4.
  • a polypeptide derived from Bacillus megaterium, which comprises the amino acid sequence to be used, can be mentioned.
  • aspartic acid which is the fourth amino acid residue of the DXDD motif, is replaced with cysteine, and the above (A / S / G) RX (H / N) )
  • the amino acid adjacent to the N-terminal of the XXP motif is substituting tyrosine for alanine
  • the fourth amino acid of the GXGX (G / A / P) motif is substituting leucine for alanine.
  • substitution of the fourth amino acid residue of the DXDD motif and “substitution of the amino acid residue adjacent to the N-terminal of the (A / S / G) RX (H / N) XXP motif” are the same as those of the first aspect. The same is true.
  • Amino acids are not limited as long as the effects of the present invention can be obtained, but are not limited to alanine, cysteine, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, methionine, aspartic acid, proline, glutamine, arginine, serine, and threonine.
  • Valin, tryptophan, tyrosine, or aspartic acid preferably alanine, phenylalanine, valine, methionine, isoleucine, tryptophan.
  • Particularly preferred is alanine or phenylalanine, more preferably alanine.
  • the mutant tetraprenyl- ⁇ -curcumen cyclase of the present invention 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). Has improved ability to generate umbrellas from 8 ⁇ -hydroxypolypoda-13,17,21-triene.
  • the polypeptide of the mutant tetraprenyl- ⁇ -curcumen cyclase of the present invention is derived from an amino acid sequence in which one or several amino acids are deleted, substituted, inserted, and / or added in the amino acid sequence of SEQ ID NO: 1. It may be a polypeptide.
  • the polypeptide of the mutant tetraprenyl- ⁇ -curcumen cyclase of the present invention is a polypeptide exhibiting umbrella-forming activity using 8 ⁇ -hydroxypolypoda-13,17,21-triene as a substrate.
  • polypeptide that uses 8 ⁇ -hydroxypolypoda-13,17,21-triene as a substrate and does not exhibit umbrella-forming activity is included in the polypeptide of the mutant tetraprenyl- ⁇ -curcumen cyclase of the present invention.
  • an amino acid sequence in which one or several amino acids are deleted, substituted, inserted, and / or added means that the amino acid has been modified by 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, which is preferable. Is 1 to 30, more preferably 1 to 10, still 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 acid has one or several (preferably 1, 2, 3 or 4) conservative substitutions. There can be.
  • the polypeptide of the mutant tetraprenyl- ⁇ -curcumen cyclase of the present invention may be a polypeptide consisting of an amino acid sequence having an amino acid sequence identity of 40% or more with respect to the amino acid sequence of SEQ ID NO: 1. good.
  • the polypeptide of the mutant tetraprenyl- ⁇ -curcumen cyclase of the present invention is a polypeptide exhibiting umbrella-forming activity using 8 ⁇ -hydroxypolypoda-13,17,21-triene as a substrate.
  • a polypeptide that uses 8 ⁇ -hydroxypolypoda-13,17,21-triene as a substrate and does not exhibit umbrella-forming activity is included in the polypeptide of the mutant tetraprenyl- ⁇ -curcumen cyclase of the present invention.
  • 17,21-A mutant tetraprenyl- ⁇ -curcumen cyclase comprising or containing a polypeptide exhibiting umbrella-forming activity using triene as a substrate.
  • amino acid sequence of SEQ ID NO: 1 "an amino acid sequence in which one or several amino acids are deleted, substituted, inserted, and / or added” or “an amino acid sequence having 40% or more identity” is shown in SEQ ID NO: 1.
  • the amino acid sequence of the above is substituted, and the substitution of this amino acid sequence is a conservative substitution that maintains the function of the mutant tetraprenyl- ⁇ -curcumen cyclase of the present invention.
  • conservative substitution means a substitution in which the excellent effect of the mutant tetraprenyl- ⁇ -curcumen cyclase of the present invention is not lost.
  • the substitution can improve the umbrella-forming activity by using 8 ⁇ -hydroxypolypoda-13,17,21-triene as a substrate.
  • it means replacing an amino acid residue with another chemically similar amino acid residue.
  • one hydrophobic residue is replaced with another hydrophobic residue
  • one polar residue is replaced with another polar residue having the same charge, and the like.
  • Functionally similar amino acids that can be made by making such substitutions are known in the art for each amino acid.
  • non-polar (hydrophobic) amino acids examples include alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, methionine and the like.
  • polar (neutral) amino acids examples include glycine, serine, threonine, tyrosine, glutamine, asparagine, and cysteine.
  • positively charged (basic) amino acids examples include arginine, histidine, and lysine.
  • negatively charged (acidic) amino acids examples include aspartic acid and glutamic acid.
  • 21-Triene is a positive substitution (mutation) for imparting the activity of producing an umbrella, but the conservative substitution uses 8 ⁇ -hydroxypolypoda-13,17,21-triene as a substrate. It is a substitution for maintaining the activity of producing an amino acid, which can be easily carried out by those skilled in the art.
  • the mutant tetraprenyl- ⁇ -curcumen cyclase of the present invention can be obtained by using a known gene recombination technique or the like.
  • the chromosomal DNA of Bacillus megaterium is obtained, and the tetraprenyl- ⁇ -curcumen cyclase is amplified by PCR or the like using an appropriate primer.
  • the obtained gene is incorporated into an appropriate vector to determine the gene sequence. Then, by introducing the above mutation, a gene encoding the mutant tetraprenyl- ⁇ -curcumen cyclase of the present invention can be obtained.
  • mutant tetraprenyl- ⁇ -curcumen cyclase of the present invention can be obtained.
  • tetraprenyl- ⁇ -curcumen cyclase is known to exist in bacteria such as the genus Bacillus. It is also possible to obtain the derived enzyme (accession number: AAU41134) and the like.
  • the gene encoding the mutant tetraprenyl- ⁇ -curcumen cyclase of the present invention is a method for synthesizing a known synthetic gene, for example, the method of Khorana et al. (Gupta et al., 1968), the method of Narang et al. It is also possible to synthesize by the method of Scarpulla et al., 1982) or Rossi et al. (Rossi et al., 1982). Then, by expressing the synthesized gene, the mutant tetraprenyl- ⁇ -curcumen cyclase of the present invention can be obtained.
  • a pathway with monocyclic 3-deoxyacireol A as an intermediate (hereinafter, may be referred to as a monocyclic pathway) and 8 ⁇ -hydroxypolypoda-13,17,21-triene are intermediates.
  • the umbrella is produced by a route (hereinafter, may be referred to as a bicyclic route).
  • the mutant tetraprenyl- ⁇ -curcumen cyclase of the present invention produces an umbrella, particularly using 8 ⁇ -hydroxypolypoda-13,17,21-triene in the bicyclic pathway as a substrate. Excellent activity.
  • the production of onoseroid which is a by-product, is small.
  • polynucleotide of the present invention is not particularly limited as long as it is a polynucleotide encoding the tetraprenyl- ⁇ -curcumen cyclase of the present invention.
  • polynucleotide encoding the polypeptide represented by SEQ ID NO: 2 SEQ ID NO: 12
  • polynucleotide encoding the polypeptide represented by SEQ ID NO: 3 SEQ ID NO: 13
  • polynucleotide represented by SEQ ID NO: 4 A polynucleotide encoding a peptide (SEQ ID NO: 14) can be mentioned.
  • polynucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 12, 13, or 14 under stringent conditions, and further, it is an umbrella from 8 ⁇ -hydroxypolypoda-13,17,21-triene. Can be mentioned as a polynucleotide having an activity of producing.
  • polynucleotide as used herein includes both DNA and RNA.
  • the polynucleotide of the present invention is preferably changed to the optimum codon base sequence according to the microorganism or host cell into which it is introduced.
  • the microorganism of the present invention is a microorganism having the polynucleotide of the present invention. That is, it is not particularly limited as long as the polynucleotide of the present invention is contained in the cell, and examples thereof include Escherichia coli, Bacillus subtilis, Brevibacillus spp., Radical fungus, baker's yeast, aspergillus, and Neurospora crassa. ..
  • the vector of the present invention is a vector containing a DNA having a polynucleotide encoding the above-mentioned mutant tetraprenyl- ⁇ -curcumen cyclase. That is, the vector of the present invention is not particularly limited as long as it contains the polynucleotide of the present invention.
  • the polynucleotide of the present invention is used in a known expression vector appropriately selected according to the host cell used.
  • the vector obtained by insertion can be mentioned.
  • As the expression vector a vector capable of autonomous replication or integration into a chromosome in a host such as Escherichia coli or baker's yeast and having a high expression efficiency of a foreign protein is preferable.
  • the expression vector for expressing the polynucleotide is preferably a recombinant vector composed of a promoter, a ribosome-binding sequence, the DNA, and a transcription termination sequence while being able to replicate autonomously in a microorganism. It may also contain a gene that controls a promoter.
  • expression vectors for example, pBTrp2, pBTac1, pBTac2 (all commercially available from Hopkins Mannheim), pKK233-2 (manufactured by Pharmacia), pSE280 (manufactured by Invitrogen), pGEMEX-1 (manufactured by Promega). ), PQE-8 (manufactured by QIAGEN), pQE-30 (manufactured by QIAGEN), pKYP10 (Japanese Patent Laid-Open No. 58-110600), pKYP200 [Agricultural Biological Chemistry, 48,669 (1984)], pLSA1 [Agric. Biol. Chem.
  • the promoter may be any promoter as long as it can be expressed in cells such as Escherichia coli and baker's yeast, which are hosts.
  • promoters derived from Escherichia coli, phage and the like SPO1 promoter, SPO2 promoter, penP promoter and the like such as trp promoter (Ptrp), lac promoter (Plac), PL promoter, PR promoter and PSE promoter can be mentioned.
  • an artificially designed and modified promoter such as a promoter in which two Ptprps are connected in series (Pttrpx2), a tac promoter, a letI promoter, and a lacT7 promoter can also be used.
  • a promoter that functions as a strong promoter and is capable of mass production of the target protein is preferable, and an inducible promoter is preferable. More preferred.
  • the inducible promoter include the promoter of the cold shock gene cspA whose expression is induced at low temperature, the T7 promoter induced by the addition of the inducer IPTG, and the like.
  • Constitutive promoters include alcohol dehydrogenase 1 gene (ADH1), translation elongation factor TF-1 ⁇ gene (TEF1), phosphoglycerate kinase gene (PGK1), triosephosphate isomerase gene (TPI1), triosephosphate isomerase gene (TDH3), Examples include the promoter of the pyruvate kinase gene (PYK1).
  • the transformant of the present invention is also not particularly limited as long as it contains the polynucleotide according to the present invention.
  • the transformant according to the present invention is integrated into the chromosome of a host cell. It can be a transformant, or it can be a transformant contained in the form of a vector containing the above-mentioned polynucleotide according to the present invention. Further, it can be a transformant expressing the polypeptide according to the present invention, or it can be a transformant not expressing the polypeptide according to the present invention.
  • the transformant of the present invention can be obtained, for example, by transforming a desired host cell with the vector according to the present invention or with the polynucleotide itself according to the present invention.
  • the host cell is not particularly limited, but a strain that is easy to handle such as Escherichia coli, Bacillus subtilis, Brevibacillus, actinomycete, yeast, aspergillus, and Neurospora crassa is preferable, but insect cells, plant cells, animal cells, etc. may also be used. It is possible. However, in order to produce an enzyme used for production by an enzymatic method (synthesis in vitro by an enzymatic reaction using squalane as a substrate), Escherichia coli, Bacillus subtilis, Brevibacillus spp., And Aspergillus are preferable, and Escherichia coli is most preferable.
  • yeast is most preferable in fermentative production (biosynthesis in vivo by a host using glucose or the like as a carbon source).
  • the most preferable yeast strain is sake yeast.
  • Sake Yeast Association No. 7 or 701 can be mentioned.
  • Association No. 701 yeast is a foamless yeast bred from Association No. 7 and has a feature of not forming high foam, but has the same other properties.
  • the mutant tetraprenyl- ⁇ -curcumen cyclase can be produced by culturing a transformant obtained by introducing an enzyme expression vector into a bacterium or the like.
  • the medium used for culturing the transformant may be a medium usually used, and is appropriately selected according to the type of host. For example, when culturing Escherichia coli, an LB medium or the like is used. Antibiotics may be added to the medium depending on the type of selectable marker.
  • the mutant tetraprenyl- ⁇ -curcumen cyclase may be an enzyme extracted and purified from a culture solution obtained by culturing a transformant capable of expressing the enzyme. Further, it is previously expressed as a fusion protein in which a trigger factor (TF) or His tag is fused to the N-terminal side or C-terminal side of the polypeptide of the mutant tetraprenyl- ⁇ -curcumen cyclase of the present invention, and purified. May be facilitated. Moreover, you may use the extract containing the enzyme extracted from the transformant in the culture solution as it is. As a method for extracting the enzyme from the transformant, a known method may be applied.
  • TF trigger factor
  • the enzyme extraction step may include, for example, disrupting the transformant in an extraction solvent and separating the cell contents from the disrupted fragments of the transformant.
  • the resulting cell content contains the mutant tetraprenyl- ⁇ -curcumen cyclase of interest.
  • a known method capable of crushing the transformant and recovering the enzyme solution may be applied, and examples thereof include ultrasonic crushing and glass bead crushing.
  • the crushing conditions are not particularly limited as long as the enzyme is not inactivated, such as 10 ° C. or lower and 15 minutes.
  • Examples of the method for separating the cell contents and the debris of the microbial body include sedimentation separation, centrifugation, filtration separation, and a combination of two or more separation methods thereof. 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 minutes to 20 minutes.
  • the extraction solvent may be one that is usually used as a solvent for enzyme extraction, and examples thereof include Tris-HCl buffer and potassium phosphate buffer.
  • the pH of the extraction solvent is preferably 3 to 10, more preferably 6 to 8 in terms of enzyme stability.
  • the extraction solvent may contain a surfactant.
  • the surfactant include nonionic surfactants and zwitterionic surfactants.
  • the nonionic surfactant include polyoxyethylene sorbitan fatty acid ester such as poly (oxyethylene) sorbitan monooleic acid ester (Tween80), alkyl glucoside such as n-octyl ⁇ -D-glucoside, and sucrose stearate ester.
  • examples thereof include polyglycerin fatty acid esters such as sucrose fatty acid ester and polyglycerin stearate ester.
  • amphoteric ion surfactant examples include N, N-dimethyl-N-dodecylglycine betaine, which is an alkyl betaine.
  • amphoteric ion surfactant examples include N, N-dimethyl-N-dodecylglycine betaine, which is an alkyl betaine.
  • general in the art such as Triton (registered trademark) X-100 (Triton X-100), polyoxyethylene (20) cetyl ether (Brij-58), nonoxyphenol ethoxylate (Tergitol NP-40), etc.
  • Surfactants used in the art are available.
  • 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 0.10% by mass from the viewpoint of enzyme stability. % To 1.0% by mass is more preferable.
  • the extraction solvent preferably contains a reducing agent such as dithiothreitol or ⁇ -mercaptoethanol.
  • a reducing agent such as dithiothreitol or ⁇ -mercaptoethanol.
  • dithiothreitol is preferable.
  • 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 makes it easier to retain structures such as disulfide bonds in the enzyme, and the enzyme activity tends to increase further.
  • the extraction solvent preferably contains a chelating agent such as ethylenediaminetetraacetic acid (EDTA).
  • EDTA ethylenediaminetetraacetic acid
  • 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 chelate the metal ions that can reduce the enzyme activity, so that the enzyme activity tends to increase further.
  • the extraction solvent may contain known components that can be added to the enzyme extraction solvent.
  • the mutant tetraprenyl- ⁇ -curcumen cyclase may be used alone or in combination of two or more.
  • the conditions for the reaction of the mutant tetraprenyl- ⁇ -curcumen cyclase with squalene or 8 ⁇ -hydroxypolypoda-13,17,21-triene are not particularly limited as long as the enzymatic reaction can proceed.
  • the reaction temperature and reaction time can be appropriately selected based on the activity of the mutant tetraprenyl- ⁇ -curcumen cyclase and the like.
  • the reaction temperature and reaction time are, for example, 4 ° C. to 100 ° C. and 1 hour to 30 days, preferably 30 ° C. to 60 ° C. and 16 hours to 20 days, from the viewpoint of reaction efficiency.
  • the pH condition is, for example, 3 to 10, preferably 6 to 8, from the viewpoint of reaction efficiency.
  • 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. Further, for example, the same extraction solvent as that used in the enzyme extraction step can be used. Further, an extract containing a mutant tetraprenyl- ⁇ -curcumen cyclase (for example, a cell-free extract) may be used as it is in the reaction as an enzyme solution.
  • the concentration ratio of the mutant tetraprenyl- ⁇ -curcumen cyclase and its substrate squalene or 8 ⁇ -hydroxypolypoda-13,17,21-triene in the umbrella formation reaction is an enzyme.
  • the molar concentration ratio (substrate / enzyme) of the substrate to 10000 is preferable, 10 to 5000 is more preferable, 100 to 3000 is more preferable, and 1000 to 2000 is further preferable.
  • the concentration of squalene or 8 ⁇ -hydroxypolypoda-13,17,21-triene used in the enzymatic reaction is preferably 0.000001% by mass to 10% by mass with respect to the total mass of the reaction solvent from the viewpoint of reaction efficiency, and is 0. More preferably, it is .0000001% by mass to 1% by mass.
  • the reaction step in which the mutant tetraprenyl- ⁇ -curcumen cyclase reacts with squalene or 8 ⁇ -hydroxypolypoda-13,17,21-triene may be repeated a plurality of times. This makes it possible to increase the yield of umbrella.
  • the timing and amount of squalene to be added should be appropriately set according to the concentration of the mutant tetraprenyl- ⁇ -curcumen cyclase in the reaction solution, the mass of the group remaining in the reaction solution, and the like. Can be done.
  • Another embodiment of the method for producing an umbrella of the present invention is characterized by culturing the microorganism or transformant of the present invention.
  • An umbrella can be produced by culturing a host cell transformed with the microorganism or an expression vector.
  • yeast may be cultured in a commonly used YPD medium or the like.
  • Yeast gene-introduced by homologous recombination or yeast having an expression vector is pre-cultured, further inoculated into YPD medium or the like, and cultured for 24-240 hours, preferably 72-120 hours.
  • the umbrella secreted in the medium can be used as it is or after being purified by a known method. Specific examples of the purification method include solvent extraction, recrystallization, distillation, column chromatography, HPLC and the like.
  • the second aspect of the method for producing an umbrella of the present invention is (1) a step of reacting tetraprenyl- ⁇ -curcumen cyclase with squalene to obtain 8 ⁇ -hydroxypolypoda-13,17,21-triene (1).
  • the first step it may be referred to as the first step
  • the mutant tetraprenyl- ⁇ -squalene cyclase is reacted with 8 ⁇ -hydroxypolypoda-13,17,21-triene to obtain an umbrella.
  • Including a step hereinafter, may be referred to as a second step).
  • FIG. 3 shows an embodiment in which the wild-type tetraprenyl- ⁇ -curcumen cyclase is used in step (1).
  • the tetraprenyl- ⁇ -curcumen cyclase used in the first step is not particularly limited as long as squalene can be converted to 8 ⁇ -hydroxypolypoda-13,17,21-triene.
  • the mutant tetraprenyl- ⁇ -curcumen cyclase of the present invention may be used, the wild-type tetraprenyl- ⁇ -curcumen cyclase may be used, and the wild-type tetraprenyl- ⁇ -curcumen cyclization.
  • first step mutant TC An enzyme into which a mutation other than the mutant tetraprenyl- ⁇ -curcumen cyclase of the present invention has been introduced (hereinafter, referred to as “first step mutant TC”) may be used.
  • first step mutant TC an enzyme into which a mutation other than the mutant tetraprenyl- ⁇ -curcumen cyclase of the present invention has been introduced.
  • wild-type tetraprenyl- ⁇ -curcumen cyclase include the wild-type tetraprenyl- ⁇ -curcumen cyclase derived from Bacillus megaterium shown in SEQ ID NO: 1 and the wild-type tetraprenyl- ⁇ -curcumen derived from Bacillus subtilis.
  • mutant TC for the first step includes (1) deletion, substitution, insertion, and / of one or several amino acids in the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 5, or SEQ ID NO: 6.
  • mutant TC consisting of an added amino acid sequence and exhibiting 8 ⁇ -hydroxypolypoda-13,17,21-triene-producing activity from squalane, or (2) SEQ ID NO: 1, SEQ ID NO: 5, or SEQ ID NO: 6
  • mutant TCs having an amino acid sequence having an identity of 40% or more with the represented amino acid sequence and exhibiting 8 ⁇ -hydroxypolypoda-13,17,21-triene-producing activity from squalane.
  • mutant tetraprenyl- ⁇ -curcumen cyclase As the mutant tetraprenyl- ⁇ -curcumen cyclase that can be used in the second step, the mutant tetraprenyl- ⁇ -curcumen cyclase used in the first aspect can be used. Further, the following mutant tetraprenyl- ⁇ -curcumen cyclase (hereinafter referred to as mutant TC for the second step) can be used.
  • asparagic acid which is the fourth amino acid residue of the DXDD motif, is replaced with an amino acid other than aspartic acid, and (A / S / G) RX (H / N).
  • a variant tetraprenyl-in which the amino acid adjacent to the N-terminal of the XXP motif is replaced with an amino acid other than tyrosine, or the fourth amino acid of the GXGX (G / A / P) motif is replaced with an amino acid other than leucine.
  • the mutant tetraprenyl- ⁇ -curcumen cyclase is QXXXGX (W) at a position separated by 100 amino acid residues or more on the N-terminal side based on the DXDD motif.
  • a preferred embodiment of the mutant TC for the second step of the present invention is the mutant tetraprenyl- ⁇ -curcumen cyclase described in [14] above.
  • the polypeptide constituting the mutant tetraprenyl- ⁇ -curcumen cyclase comprises the amino acid sequence represented by SEQ ID NO: 8 or 9.
  • examples include polypeptides derived from Bacillus megaterium. That is, in the mutant TC for the second step, aspartic acid, which is the fourth amino acid residue of the DXDD motif, is replaced with cysteine, and the (A / S / G) RX (H / N) XXP motif is used.
  • the amino acid flanking the N-terminal is replaced by alanine from tyrosine, or the fourth amino acid residue of the DXDD motif, aspartic acid, is replaced by cysteine, and the GXGX (G / A / P) motif.
  • the fourth amino acid replaces leucine with alanine.
  • Bacillus megaterium may be abbreviated as "Bme”
  • Example 1 the mutant tetraprenyl- ⁇ -curcumen cyclase was cloned, and the mutant tetraprenyl- ⁇ -curcumen in which tyrosine at position 257 was replaced with alanine and aspartic acid at position 373 was replaced with cysteine.
  • the cyclase gene was constructed. Using the Bacillus megaterium chromosomal DNA as a template, a polynucleotide encoding wild-type tetraprenyl- ⁇ -curcumen cyclase was obtained by PCR, and the amino acid sequence of the wild-type enzyme was determined.
  • the tetraprenyl- ⁇ -curcumen cyclase is a sequence derived from Bacillus megaterium and may be simply referred to as "wild type".
  • the obtained gene is used as a vector pColdI (Takarabio).
  • the expression vector containing the wild-type tetraprenyl- ⁇ -curcumen cyclase gene (SEQ ID NO: 1) was obtained by inserting into the cloning site (restrictor enzyme NdeI / XhoI site) of the company.
  • a transformant of Escherichia coli BL21 (DE3) was prepared from the obtained expression vector containing the mutant tetraprenyl- ⁇ -curcumen cyclase gene.
  • tetraprenyl- ⁇ -curcumen cyclase gene was constructed in which tyrosine at position 257 was replaced with alanine and aspartic acid at position 373 was replaced with cysteine.
  • the 373rd aspartic acid was designed to be replaced with cysteine, and the codon was optimized for the host Escherichia coli and synthesized. bottom.
  • the synthesized gene was inserted into the cloning site (restriction enzyme NdeI / XhoI site) of the vector pColdI (Takara Bio Inc.).
  • a site-specific mutation was introduced by a quick change method using a mutation-introducing primer: OPY257A (SEQ ID NOs: 18 and 19) so that the 257th tyrosine was replaced with alanine, and Y257A /
  • An expression vector containing a mutant tetraprenyl- ⁇ -curcumen cyclase gene (SEQ ID NO: 12) of the D373C mutant was obtained.
  • the pCold vector is a cold shock expression vector that uses the promoter of the cspA gene, which is one of the cold shock genes.
  • the expressed protein is expressed as a fusion protein containing a TEE sequence, a His tag sequence, and a Factor Xa cleavage sequence.
  • the sequence of the start codon (GTG) of the wild-type enzyme inheritance derived from Bacillus megaterium is not changed. Therefore, the N-terminal amino acid of the enzyme protein moiety in the fusion protein is translated into valine.
  • a transformant of Escherichia coli BL21 (DE3) was prepared from the obtained expression vector containing the mutant tetraprenyl- ⁇ -curcumen cyclase gene.
  • Example 2 a mutant tetraprenyl- ⁇ -curcumen cyclase gene in which tyrosine at position 167 was replaced with alanine, tyrosine at position 257 was replaced with alanine, and aspartic acid at position 373 was replaced with cysteine.
  • the mutant tetraprenyl- ⁇ -curcumen cyclase gene is based on the gene sequence of tetraprenyl- ⁇ -curcumen cyclase (wild type), and the 167th tyrosine is alanine and the 373th aspartic acid is cysteine.
  • the synthesized gene is inserted into the cloning site (restriction enzyme NdeI / XhoI site) of the vector pColdI (Takara Bio), and the mutation introduction primer is used so that the 257th tyrosine is replaced with alanine using this vector gene as a template.
  • An expression vector containing the above was obtained.
  • a transformant of Escherichia coli BL21 (DE3) was prepared from the obtained expression vector containing the mutant tetraprenyl- ⁇ -curcumen cyclase gene.
  • ⁇ Reference example 1 ⁇ a mutant tetraprenyl- ⁇ -curcumen cyclase gene in which the 373rd aspartic acid was replaced with cysteine was constructed.
  • the mutant tetraprenyl- ⁇ -curcumen cyclase gene is designed so that aspartic acid at position 373 is replaced with cysteine based on the gene sequence of the wild-type enzyme described in Example 1, and the host Escherichia coli. The codon was optimized and synthesized.
  • the synthesized gene is inserted into the cloning site (restriction enzyme NdeI / XhoI site) of the vector pColdI (Takara Bio Inc.) and contains the mutant tetraprenyl- ⁇ -curcumen cyclase gene (SEQ ID NO: 17) of the D373C variant.
  • An expression vector was obtained.
  • a transformant of Escherichia coli BL21 (DE3) was prepared from the obtained expression vector containing the mutant tetraprenyl- ⁇ -curcumen cyclase gene.
  • a mutant tetraprenyl- ⁇ -curcumen cyclase gene was constructed in which tyrosine at position 167 was replaced with alanine and aspartic acid at position 373 was replaced with cysteine.
  • aspartic acid at position 373 is cysteine based on the gene sequence of the wild-type enzyme described in Example 1 so that tyrosine at position 167 is replaced with alanine. It was designed to be replaced with, and the codon was optimized and synthesized for the host Escherichia coli.
  • the synthesized gene is inserted into the cloning site (restriction enzyme NdeI / XhoI site) of the vector pColdI (Takara Bio Inc.), and the mutant tetraprenyl- ⁇ -curcumen cyclase gene of the Y167A / D373C variant (SEQ ID NO: 15).
  • An expression vector containing the above was obtained.
  • a transformant of Escherichia coli BL21 (DE3) was prepared from the obtained expression vector containing the mutant tetraprenyl- ⁇ -curcumen cyclase gene.
  • a mutant tetraprenyl- ⁇ -curcumen cyclase gene was constructed in which aspartic acid at position 373 was replaced with cysteine and leucine at position 596 was replaced with alanine.
  • aspartic acid at position 373 is replaced with cysteine and leucine at position 596 is replaced with alanine based on the gene sequence of the wild-type enzyme described in Example 1.
  • the codon was optimized and synthesized for the host Escherichia coli.
  • the synthesized gene is inserted into the cloning site (restriction enzyme NdeI / XhoI site) of the vector pColdTF (Takara Bio Inc.), and the mutant tetraprenyl- ⁇ -curcumen cyclase gene of the D373C / L596A variant (SEQ ID NO: 16).
  • An expression vector containing the above was obtained.
  • a transformant of Escherichia coli BL21 (DE3) was prepared from the obtained expression vector containing the mutant tetraprenyl- ⁇ -curcumen cyclase gene.
  • Example 3 the mutant tetraprenyl- ⁇ -curcumen cyclase gene in which tyrosine at position 167 was replaced with alanine, aspartic acid at position 373 was replaced with cysteine, and leucine at position 596 was replaced with alanine.
  • tyrosine at position 167 was replaced with alanine
  • aspartic acid at position 373 is replaced with cysteine
  • leucine at position 596 was designed to be replaced with alanine, and codons were optimized and synthesized for the host Tyrosine.
  • the synthesized gene is inserted into the cloning site (restriction enzyme NdeI / XhoI site) of the vector pColdI (Takara Bio), and the mutant tetraprenyl- ⁇ -curcumen cyclase gene (sequence) of the Y167A / D373C / L596A variant is inserted.
  • An expression vector containing No. 14) was obtained.
  • a transformant of Escherichia coli BL21 (DE3) was prepared from the obtained expression vector containing the mutant tetraprenyl- ⁇ -curcumen cyclase gene.
  • ⁇ Manufacturing method example 1 In this production method example, the mutant tetraprenyl- ⁇ -curcumen cyclase of Examples 1 to 3 and Reference Example 3 is used as a substrate using the bicyclic compound 8 ⁇ -hydroxypolypoda-13,17,21-triene. The enzyme activity was examined. The transformants prepared in Examples 1 to 3 and Reference Example 3 were inoculated into LB medium (1 L) containing ampicillin (50 mg / L), respectively, and cultured with shaking at 37 ° C. for 3 hours. After culturing, 1 mM isopropyl- ⁇ -thiogalactopyranoside (IPTG) was added and shaken at 15 ° C.
  • IPTG isopropyl- ⁇ -thiogalactopyranoside
  • the amount of enzyme protein was 50 ⁇ g, and 8 ⁇ -hydroxypolypoda-13,17,21-triene (substrate) and mutant tetraprenyl- ⁇ -curcumen.
  • the molar ratio (substrate / enzyme) to the cyclizing enzyme (enzyme) was about 170.
  • 15% potassium hydroxide / methanol 1.2 mL was added to the reaction solution to stop the enzymatic reaction.
  • n-hexane 2.0 mL was added to the reaction solution, and the reaction product was extracted three times.
  • the content ratio of the reaction product (umbrain) in the obtained extract (reaction composition) is shown in FIG. 4 (A). Moreover, the content ratio of umbrella and onoseroid excluding 8 ⁇ -hydroxypolypoda-13,17,21-triene in the obtained extract (reaction composition) is shown in FIG. 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 are 8 ⁇ of the bicyclic compound.
  • Umbrain could be produced using -hydroxypolypoda-13,17,21-triene as a substrate.
  • the Y257A / D373C mutant of Example 1 and the Y167A / Y257A / D373C mutant of Example 2 produced a large amount of umbrella when 8 ⁇ -hydroxypolypoda-13,17,21-triene was used as a substrate. There were many.
  • the Y167A / D373C / L596A mutant of Example 3 produced no by-product onoseroid.
  • the Y167A / Y257A / D373C mutant of Example 2 produced umbrellas from 8 ⁇ -hydroxypolypoda-13,17,21-triene at an production rate of 85.5% (production efficiency) and a production rate of 88.6%.
  • the Y257A / D373C mutant of Example 1 had a production rate of 45.7% (production efficiency) and a production rate of 61.3%, and the Y167A / D373C / L596A mutant of Example 3 had a production rate of 9.0. It was possible to produce at a production rate of% (production efficiency) and a production rate of 100%.
  • the Y167A / Y257A / D373C mutant of Example 2 As described above, the Y167A / Y257A / D373C mutant of Example 2, the Y257A / D373C mutant of Example 1, and the Y167A / D373C / L596A mutant of Example 3 are 8 ⁇ -hydroxypolypoda-13,17,21. -The reaction selectivity from triene (matrix) to umbrella is improved, and umbrella can be produced efficiently.
  • Squalene (100 ⁇ g) was mixed with Tween 80 (2 mg) to solubilize it, 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 prepare a reaction solution, and the mixture was incubated at 30 ° C. for 64 hours.
  • the amount of enzyme protein was 50 ⁇ g, and the molar ratio (substrate / enzyme) of squalene (substrate) to mutant tetraprenyl- ⁇ -curcumen cyclase (enzyme) was about 170.
  • the content of the reaction product (Ambrein) in the obtained extract (reaction composition) is shown in FIG. 5 (A).
  • the content ratios of umbrella, monocyclic compound, bicyclic compound and onoseroid in the obtained extract (reaction composition) excluding squalene are shown in FIG. 5 (B).
  • the Y167A / Y257A / D373C mutant of Example 2 produced a larger amount of umbrella than the D373C mutant of Reference Example 1.
  • the Y167A / Y257A / D373C mutant of Example 1 was able to produce umbrella from squalene at a production rate of 13.6% (production efficiency) and a production ratio of 44.0%.
  • the Y167A / Y257A / D373C mutant of Example 2 has high reaction selectivity from squalene (substrate) to umbrella, and can efficiently produce umbrella.
  • ⁇ Manufacturing method example 3 In this production method example, using squalene as a substrate, a wild-type tetraprenyl- ⁇ -curcumen cyclase having different substrate specificity and each mutant tetraprenyl- ⁇ -curcumen cyclase are used in combination. The production of umbrella by the Wise (two-step) reaction method was examined. As the mutant tetraprenyl- ⁇ -curcumen cyclase, the Y167A / Y257A / D373C mutant of Example 2 and the Y167A / D373C mutant of Reference Example 2 were used.
  • the recombinant enzyme solution (protein concentration 100 ⁇ g / mL) was prepared in the same manner as in Production Method Example 1. Squalene (100 ⁇ g) was mixed with Tween 80 (2 mg) to solubilize it, and then added to buffer A (1 mL) to prepare a squalene solution. A wild-type tetraprenyl- ⁇ -curcumen cyclase solution (0.25 mL) was added to this squalene solution (0.5 mL) to prepare a reaction solution, which was incubated at 30 ° C. for 64 hours. After the reaction, the mixture was incubated at 70 ° C. for 1 hour to stop the enzymatic reaction.
  • a mutant tetraprenyl- ⁇ -curcumen cyclase solution (0.25 mL) was added to the reaction solution, and the mixture was incubated at 30 ° C. for 64 hours.
  • the amount of enzyme protein of the wild-type and mutant tetraprenyl- ⁇ -curcumen cyclase was 25 ⁇ g, respectively.
  • the molar ratio (substrate / enzyme) of squalene to mutant tetraprenyl- ⁇ -curcumen cyclase (enzyme) or wild-type tetraprenyl- ⁇ -curcumen cyclase (enzyme) was about 170. rice field.
  • the content of the reaction product (umbrain) in the obtained extract (reaction composition) is shown in FIG. 6 (A).
  • the content ratios of umbrella, monocyclic compound, bicyclic compound and onoseroid in the obtained extract (reaction composition) excluding squalene are shown in FIG. 6 (B).
  • the combination of the wild-type tetraprenyl- ⁇ -squalene cyclase and the Y167A / Y257A / D373C mutant of Example 2 had a production rate (production efficiency) of 59.9% (production efficiency), 65.
  • the combination of the wild-type tetraprenyl- ⁇ -squalene cyclase and the Y167A / D373C mutant of Reference Example 2 had a production rate of 35.6% (production efficiency) of 46.7% at a production rate of 0.6%.
  • At the production ratio we were able to produce umbrellas from squalene.
  • 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 likenformis WT amino acid sequence
  • SEQ ID NO: 7 Alicyclobacillus acidcardarius
  • SEQ ID NO: 8 Bacillus megaterium Y167A / D373C amino acid sequence number 9: Bacillus megaterium D373C / L596A Amino acid sequence No.
  • Bacillus megaterium D373C Amino acid sequence No. 11 Bacillus megaterium WT base sequence SEQ ID NO: 12: Bacillus megaterium Y257A / D373C base sequence No. 13: Bacillus megaterium Y167A / Y257A / D373C Nucleotide Sequence No. 14: Bacillus Megaterium Y167A / D373C / L596A Nucleotide Sequence No. 15: Bacillus Megaterium Y167A / D373C Nucleotide Sequence No. 16: Bacillus Megaterium D373C / L596A Nucleotide Sequence No. 17: Bacillus Megaterium D373C Nucleotide Sequence No. 18: OPY257A_F SEQ ID NO: 19: OPY257A_R
  • the mutant tetraprenyl- ⁇ -curcumen cyclase of the present invention can be used for efficient production of umbrellas by microorganisms.
  • the umbrella obtained by the present invention can be used as a raw material for the production of, for example, pharmaceuticals.

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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

変異型テトラプレニル－β－クルクメン環化酵素及びそれを用いたアンブレインの製造方法Mutant tetraprenyl-β-curcumen cyclase and method for producing umbrella using it
　本発明は、変異型テトラプレニル－β－クルクメン環化酵素及びそれを用いたアンブレインの製造方法に関する。 The present invention relates to a mutant tetraprenyl-β-curcumen cyclase and a method for producing an umbrella using the same.
　龍涎香（ａｍｂｅｒｇｒｉｓ）は、７世紀ごろから世界各地で使用されてきた高級香料であり、漢方薬としても使用されている。龍涎香は、マッコウクジラが食物（タコ、イカ等）の不消化物を消化管分泌物により結石化させ、排泄したものと考えられているが、詳細な生成メカニズムは不明である。龍涎香の主成分はアンブレインであり、龍涎香が海上を浮遊する間に日光と酸素によって酸化分解を受け、各種の香りを持つ化合物を生成すると考えられている。 Ambergris is a high-class fragrance that has been used all over the world since the 7th century, and is also used as a Chinese herbal medicine. Ambergris is thought to have been excreted by sperm whales by calculating indigestible substances of food (octopus, squid, etc.) with gastrointestinal secretions, but the detailed mechanism of production is unknown. The main component of ambergris is ambrein, and it is thought that ambergris undergoes oxidative decomposition by sunlight and oxygen while floating on the sea to produce compounds with various scents.
　龍涎香の主成分アンブレインは、香料又は薬剤として用いられているが、自然界から大量に入手することは不可能である。このため、種々の有機合成法が提案されている。
　例えば、特許文献１には、（＋）－アンブレインを簡便かつ安価に効率よく製造する方法として、アンブレノリドから、新規なスルホン酸誘導体を製造し、これにγ－シクロゲラニルハライドの光学活性体をカップリングさせる工程を含む方法が開示されている。 Ambergris, the main component of ambergris, is used as a fragrance or a drug, but it is not possible to obtain it in large quantities from the natural world. Therefore, various organic synthesis methods have been proposed.
For example, in Patent Document 1, as a method for producing (+)-umbrain easily, inexpensively and efficiently, a novel sulfonic acid derivative is produced from umbrella, and an optically active substance of γ-cyclogeranyl halide is added thereto. A method including a step of coupling is disclosed.
　また、非特許文献１には、（±）（５，５，８ａ－トリメチルオクタヒドロ－１Ｈ－スピロ［ナフタレン－２，２’－オキシラン］－１－イル）メタノールから合成した２－（（１Ｒ，２Ｒ，４ａＳ，８ａＳ）－２－（メトキシメトキシ）－２，５，５，８ａ－テトラメチルデカヒドロナフタレン－１－イル）アセトアルデヒドと、（±）メチル　６－ヒドロキシ－２，２－ジメチルシクロヘキサンカルボキシレートから合成した５－（（４－（（Ｓ）－２，２－ジメチル－６－メチレンシクロヘキシル）ブタン－２－イル）スルホニル）－１－フェニル－１Ｈ－テトラゾールとを、Ｊｕｌｉａカップリング反応によって収束的合成して、アンブレインを得る方法が開示されている。 In addition, Non-Patent Document 1 describes 2-((1R) synthesized from (±) (5,5,8a-trimethyloctahydro-1H-spiro [naphthalen-2,2'-oxylan] -1-yl) methanol. , 2R, 4aS, 8aS) -2- (methoxymethoxy) -2,5,5,8a-tetramethyldecahydronaphthalen-1-yl) acetaldehyde and (±) methyl 6-hydroxy-2,2-dimethylcyclohexane Julia coupling reaction with 5-((4-((S) -2,2-dimethyl-6-methylenecyclohexyl) butane-2-yl) sulfonyl) -1-phenyl-1H-tetrazole synthesized from carboxylate. Disclosed is a method of obtaining an umbrella by convergent synthesis by.
　しかしながら、従来のアンブレインの有機合成法は、多くの合成段階を必要とすることから、反応系が複雑であり、事業化には至っていない。
　一方、スクアレン－ホペン環化酵素の変異型酵素（Ｄ３７７Ｃ、Ｄ３７７Ｎ、Ｙ４２０Ｈ、Ｙ４２０Ｗ等）を用いることによって、スクアレンから、単環性トリテルペンである３－デオキシアキレオールＡを得る方法が知られている（非特許文献２～４）。
　本発明者らは、スクアレンから３－デオキシアキレオールＡを生成可能な変異型スクアレン－ホペン環化酵素をスクアレンに反応させることによって、３－デオキシアキレオールＡを得、更にテトラプレニル－β－クルクメン環化酵素を反応させることによって、アンブレインの製造ができることを見出した（特許文献２）。
　しかしながら、２段階目の反応である３－デオキシアキレオールＡからアンブレインへ変換させる反応は、副生成物の抑制、スケールアップの容易さの点で改善の余地があった。更に、収率についても改善の余地があった。 However, the conventional organic synthesis method of umbrella requires many synthesis steps, so that the reaction system is complicated and has not been commercialized.
On the other hand, a method of obtaining 3-deoxyacireol A, which is a monocyclic triterpene, from squalene by using a mutant enzyme of squalene-hopen cyclase (D377C, D377N, Y420H, Y420W, etc.) is known. (Non-Patent Documents 2 to 4).
The present inventors obtained 3-deoxyachilleol A by reacting squalene with a mutant squalene-hopen cyclase capable of producing 3-deoxyachilleol A from squalene, and further tetraprenyl-β-curcumen. It has been found that squalene can be produced by reacting with a cyclizing enzyme (Patent Document 2).
However, the reaction of converting 3-deoxyacireol A to umbrella, which is the second-stage reaction, has room for improvement in terms of suppression of by-products and ease of scale-up. Furthermore, there was room for improvement in yield.
特開平１０－２３６９９６号公報Japanese Unexamined Patent Publication No. 10-236996 国際公開ＷＯ２０１５／０３３７４６号パンフレットInternational Publication WO2015 / 033746 Pamphlet 国際公開ＷＯ２０１７／１５０６９５号パンフレットInternational Publication WO2017 / 150695 Pamphlet
　そこで、本発明者らは、変異型テトラプレニル－β－クルクメン環化酵素（Ｄ３７３Ｃ等）を用いることによって、スクアレンから、１個の酵素を使用した２段階の反応で、アンブレインを得る方法を見出し、提案している（特許文献３）。
　しかし、この方法では、特許文献２の方法に比べ、収率は大きく改善されたものの、スクアレンからまず３－デオキシアキレオールＡと二環性トリテルペン（８α－ヒドロキシポリポダ－１３，１７，２１－トリエン）が生成し、そこからアンブレインを得るため、副生成物の抑制の点で、依然として改善の余地があった。
　本発明の目的は、簡便に且つ効率的にアンブレインを得ることができるアンブレインの製造方法を提供することにある。 Therefore, the present inventors have described a method for obtaining an umbrella from squalene in a two-step reaction using one enzyme by using a mutant tetraprenyl-β-curcumen cyclase (D373C, etc.). Finding and proposing (Patent Document 3).
However, although the yield of this method was significantly improved as compared with the method of Patent Document 2, squalene was first treated with 3-deoxyacireol A and bicyclic triterpenes (8α-hydroxypolypoda-13,17,21-). There was still room for improvement in terms of suppression of by-products, as triene) was produced and squalene was obtained from it.
An object of the present invention is to provide a method for producing an umbrella, which can easily and efficiently obtain an umbrella.
　本発明者は、簡便且つ効率的にアンブレインを得ることができるアンブレインの製造方法について、鋭意研究した結果、驚くべきことに、わずかな特定の変異を有する変異型テトラプレニル－β－クルクメン環化酵素が２環性トリテルペン（８α－ヒドロキシポリポダ－１３，１７，２１－トリエン）から効率的に、高い収率でアンブレインを生成することができることを見出した。本発明者らは、変異型テトラプレニル－β－クルクメン環化酵素は、スクアレンからアンブレインを産生する２段階のステップワイズ法において、２段階目の２環性トリテルペン（８α－ヒドロキシポリポダ－１３，１７，２１－トリエン）からアンブレインを産生する工程に使用することによって、効率的に高い収率でアンブレインを産生することができることを見出した。
　本発明は、こうした知見に基づくものである。
　従って、本発明は、
［１］ＤＸＤＤモチーフの第４番目のアミノ酸残基であるアスパラギン酸がアスパラギン酸以外のアミノ酸に置換しており、（Ｄ／Ｎ）Ｇ（Ｔ／Ｌ）（Ｌ／Ｆ／Ｙ）（Ｙ／Ｆ）ＳＹモチーフの７番目のアミノ酸がチロシン以外のアミノ酸に置換している、変異型テトラプレニル－β－クルクメン環化酵素であって、（ａ）前記変異型テトラプレニル－β－クルクメン環化酵素は、前記ＤＸＤＤモチーフを基準として、Ｎ末端側に１００アミノ酸残基以上離れた位置にＱＸＸＸＧＸ（Ｗ／Ｆ）モチーフ、Ｎ末端側に１８０～２５０アミノ酸残基離れた位置に（Ａ／Ｓ／Ｇ）ＲＸ（Ｈ／Ｎ）ＸＸＰモチーフ、Ｎ末端側に８０～１４０アミノ酸残基離れた位置に前記（Ｄ／Ｎ）Ｇ（Ｔ／Ｌ）（Ｌ／Ｆ／Ｙ）（Ｙ／Ｆ）ＳＹモチーフ、Ｎ末端側に１０～５０アミノ酸残基離れた位置にＱＸＸＸＸ（Ｇ／Ａ／Ｓ）Ｘ（Ｆ／Ｗ／Ｙ）モチーフ、Ｃ末端側に２０～５０アミノ酸残基離れた位置にＱＸＸＸＧＸ（Ｆ／Ｗ／Ｙ）モチーフ、Ｃ末端側に５０～１２０アミノ酸残基離れた位置にＱＸＸＸＧＸＷモチーフ、Ｃ末端側に１２０～１７０アミノ酸残基離れた位置にＱＸＸＸＧＸ（Ｆ／Ｗ）モチーフ、及びＣ末端側に１８０～２５０アミノ酸残基離れた位置に前記ＧＸＧＸ（Ｇ／Ａ／Ｐ）モチーフを有し、そしてＤＸＤＤモチーフを基準として、Ｃ末端側に１７０アミノ酸残基以上離れた位置にＱＸＸＸＧＸＷモチーフを有さず、（ｂ）配列番号１で表されるアミノ酸配列との同一性が４０％以上であり、そして（ｃ）８α－ヒドロキシポリポダ－１３，１７，２１－トリエンを基質としてアンブレイン生成活性を示す、変異型テトラプレニル－β－クルクメン環化酵素、
［２］更に、（Ａ／Ｓ／Ｇ）ＲＸ（Ｈ／Ｎ）ＸＸＰモチーフのＮ末に隣接するアミノ酸がチロシン以外のアミノ酸に置換している、［１］に記載の変異型テトラプレニル－β－クルクメン環化酵素、
［３］前記変異型テトラプレニル－β－クルクメン環化酵素を構成するポリペプチドが、（１）配列番号１で表されるアミノ酸配列におけるＮ末端側から３７３番目のアスパラギン酸がアスパラギン酸以外のアミノ酸に置換しており、そしてＮ末端側から２５７番目のチロシンがチロシン以外のアミノ酸の置換しているポリペプチド、（２）配列番号１で表されるアミノ酸配列におけるＮ末端側から３７３番目のアスパラギン酸がアスパラギン酸以外のアミノ酸に置換しており、そしてＮ末端側から２５７番目のチロシンがチロシン以外のアミノ酸に置換しているアミノ酸配列において、１又は数個のアミノ酸が欠失、置換、挿入、及び／又は付加されたアミノ酸配列からなり、しかも、８α－ヒドロキシポリポダ－１３，１７，２１－トリエンを基質としてアンブレイン生成活性を示すポリペプチド、（３）配列番号１で表されるアミノ酸配列におけるＮ末端側から３７３番目のアスパラギン酸がアスパラギン酸以外のアミノ酸に置換しており、そしてＮ末端側から２５７番目のチロシンがチロシン以外のアミノ酸に置換しているアミノ酸配列との同一性が４０％以上であるアミノ酸配列からなり、しかも、８α－ヒドロキシポリポダ－１３，１７，２１－トリエンを基質としてアンブレイン生成活性を示すポリペプチド、（４）配列番号１で表されるアミノ酸配列におけるＮ末端側から３７３番目のアスパラギン酸がアスパラギン酸以外のアミノ酸に置換しており、そしてＮ末端側から２５７番目のチロシンがチロシン以外のアミノ酸に置換しているアミノ酸配列を含み、しかも、８α－ヒドロキシポリポダ－１３，１７，２１－トリエンを基質としてアンブレイン生成活性を示すポリペプチド、（５）配列番号１で表されるアミノ酸配列におけるＮ末端側から３７３番目のアスパラギン酸がアスパラギン酸以外のアミノ酸に置換しており、そしてＮ末端側から２５７番目のチロシンがチロシン以外のアミノ酸に置換しているアミノ酸配列において、１又は数個のアミノ酸が欠失、置換、挿入、及び／又は付加されたアミノ酸配列を含み、しかも、８α－ヒドロキシポリポダ－１３，１７，２１－トリエンを基質としてアンブレイン生成活性を示すポリペプチド、又は（６）配列番号１で表されるアミノ酸配列におけるＮ末端側から３７３番目のアスパラギン酸がアスパラギン酸以外のアミノ酸に置換しており、そしてＮ末端側から２５７番目のチロシンがチロシン以外のアミノ酸に置換しているアミノ酸配列との同一性が４０％以上であるアミノ酸配列を含み、しかも、８α－ヒドロキシポリポダ－１３，１７，２１－トリエンを基質としてアンブレイン生成活性を示すポリペプチド、である、［１］に記載の変異型テトラプレニル－β－クルクメン環化酵素、あるいは前記変異型テトラプレニル－β－クルクメン環化酵素を構成するポリペプチドが、（７）配列番号１で表されるアミノ酸配列におけるＮ末端側から３７３番目のアスパラギン酸がアスパラギン酸以外のアミノ酸に置換しており、Ｎ末端側から１６７番目のチロシンがチロシン以外のアミノ酸に置換しており、そしてＮ末端側から２５７番目のチロシンがチロシン以外のアミノ酸の置換しているポリペプチド、（８）配列番号１で表されるアミノ酸配列におけるＮ末端側から３７３番目のアスパラギン酸がアスパラギン酸以外のアミノ酸に置換しており、Ｎ末端側から１６７番目のチロシンがチロシン以外のアミノ酸に置換しており、そしてＮ末端側から２５７番目のチロシンがチロシン以外のアミノ酸に置換しているアミノ酸配列において、１又は数個のアミノ酸が欠失、置換、挿入、及び／又は付加されたアミノ酸配列からなり、しかも、８α－ヒドロキシポリポダ－１３，１７，２１－トリエンを基質としてアンブレイン生成活性を示すポリペプチド、（９）配列番号１で表されるアミノ酸配列におけるＮ末端側から３７３番目のアスパラギン酸がアスパラギン酸以外のアミノ酸に置換しており、Ｎ末端側から１６７番目のチロシンがチロシン以外のアミノ酸に置換しており、そしてＮ末端側から２５７番目のチロシンがチロシン以外のアミノ酸に置換しているアミノ酸配列との同一性が４０％以上であるアミノ酸配列からなり、しかも、８α－ヒドロキシポリポダ－１３，１７，２１－トリエンを基質としてアンブレイン生成活性を示すポリペプチド、（１０）配列番号１で表されるアミノ酸配列におけるＮ末端側から３７３番目のアスパラギン酸がアスパラギン酸以外のアミノ酸に置換しており、Ｎ末端側から１６７番目のチロシンがチロシン以外のアミノ酸に置換しており、そしてＮ末端側から２５７番目のチロシンがチロシン以外のアミノ酸に置換しているアミノ酸配列を含み、しかも、８α－ヒドロキシポリポダ－１３，１７，２１－トリエンを基質としてアンブレイン生成活性を示すポリペプチド、（１１）配列番号１で表されるアミノ酸配列におけるＮ末端側から３７３番目のアスパラギン酸がアスパラギン酸以外のアミノ酸に置換しており、Ｎ末端側から１６７番目のチロシンがチロシン以外のアミノ酸に置換しており、そしてＮ末端側から２５７番目のチロシンがチロシン以外のアミノ酸に置換しているアミノ酸配列において、１又は数個のアミノ酸が欠失、置換、挿入、及び／又は付加されたアミノ酸配列を含み、しかも、８α－ヒドロキシポリポダ－１３，１７，２１－トリエンを基質としてアンブレイン生成活性を示すポリペプチド、又は（１２）配列番号１で表されるアミノ酸配列におけるＮ末端側から３７３番目のアスパラギン酸がアスパラギン酸以外のアミノ酸に置換しており、Ｎ末端側から１６７番目のチロシンがチロシン以外のアミノ酸に置換しており、そしてＮ末端側から２５７番目のチロシンがチロシン以外のアミノ酸に置換しているアミノ酸配列との同一性が４０％以上であるアミノ酸配列を含み、しかも、８α－ヒドロキシポリポダ－１３，１７，２１－トリエンを基質としてアンブレイン生成活性を示すポリペプチド、である、［２］に記載の変異型テトラプレニル－β－クルクメン環化酵素、
［４］前記ＤＸＤＤモチーフの第４番目のアミノ酸残基がアスパラギン酸から、システインに置換しており、前記（Ｄ／Ｎ）Ｇ（Ｔ／Ｌ）（Ｌ／Ｆ／Ｙ）（Ｙ／Ｆ）ＳＹモチーフの７番目のアミノ酸がチロシンからアラニンに置換している、［１］又は［３］に記載の変異型テトラプレニル－β－クルクメン環化酵素、あるいは前記ＤＸＤＤモチーフの第４番目のアミノ酸残基がアスパラギン酸から、システインに置換しており、前記（Ａ／Ｓ／Ｇ）ＲＸ（Ｈ／Ｎ）ＸＸＰモチーフのＮ末に隣接するアミノ酸がチロシンからアラニンに置換しており、前記（Ｄ／Ｎ）Ｇ（Ｔ／Ｌ）（Ｌ／Ｆ／Ｙ）（Ｙ／Ｆ）ＳＹモチーフの７番目のアミノ酸がチロシンからアラニンに置換している、［２］又は［３］に記載の変異型テトラプレニル－β－クルクメン環化酵素、
［５］ＤＸＤＤモチーフの第４番目のアミノ酸残基であるアスパラギン酸がアスパラギン酸以外のアミノ酸に置換しており、（Ａ／Ｓ／Ｇ）ＲＸ（Ｈ／Ｎ）ＸＸＰモチーフのＮ末に隣接するアミノ酸がチロシン以外のアミノ酸に置換しており、ＧＸＧＸ（Ｇ／Ａ／Ｐ）モチーフの４番目のアミノ酸がロイシン以外のアミノ酸に置換している変異型テトラプレニル－β－クルクメン環化酵素であって、（ａ）前記変異型テトラプレニル－β－クルクメン環化酵素は、前記ＤＸＤＤモチーフを基準として、Ｎ末端側に１００アミノ酸残基以上離れた位置にＱＸＸＸＧＸ（Ｗ／Ｆ）モチーフ、Ｎ末端側に１８０～２５０アミノ酸残基離れた位置に前記（Ａ／Ｓ／Ｇ）ＲＸ（Ｈ／Ｎ）ＸＸＰモチーフ、Ｎ末端側に８０～１４０アミノ酸残基離れた位置に前記（Ｄ／Ｎ）Ｇ（Ｔ／Ｌ）（Ｌ／Ｆ／Ｙ）（Ｙ／Ｆ）ＳＹモチーフ、Ｎ末端側に１０～５０アミノ酸残基離れた位置にＱＸＸＸＸ（Ｇ／Ａ／Ｓ）Ｘ（Ｆ／Ｗ／Ｙ）モチーフ、Ｃ末端側に２０～５０アミノ酸残基離れた位置にＱＸＸＸＧＸ（Ｆ／Ｗ／Ｙ）モチーフ、Ｃ末端側に５０～１２０アミノ酸残基離れた位置にＱＸＸＸＧＸＷモチーフ、Ｃ末端側に１２０～１７０アミノ酸残基離れた位置にＱＸＸＸＧＸ（Ｆ／Ｗ）モチーフ、及びＣ末端側に１８０～２５０アミノ酸残基離れた位置に前記ＧＸＧＸ（Ｇ／Ａ／Ｐ）モチーフを有し、そしてＤＸＤＤモチーフを基準として、Ｃ末端側に１７０アミノ酸残基以上離れた位置にＱＸＸＸＧＸＷモチーフを有さず、（ｂ）配列番号１で表されるアミノ酸配列との同一性が４０％以上であり、そして（ｃ）８α－ヒドロキシポリポダ－１３，１７，２１－トリエンを基質としてアンブレイン生成活性を示す、変異型テトラプレニル－β－クルクメン環化酵素、
［６］前記変異型テトラプレニル－β－クルクメン環化酵素を構成するポリペプチドが、（１）配列番号１で表されるアミノ酸配列におけるＮ末端側から３７３番目のアスパラギン酸がアスパラギン酸以外のアミノ酸に置換しており、Ｎ末端側から１６７番目のチロシンがチロシン以外のアミノ酸に置換しており、そしてＮ末端側から５９６番目のロイシンがロイシン以外のアミノ酸に置換しているポリペプチド、（２）配列番号１で表されるアミノ酸配列におけるＮ末端側から３７３番目のアスパラギン酸がアスパラギン酸以外のアミノ酸に置換しており、Ｎ末端側から１６７番目のチロシンがチロシン以外のアミノ酸に置換しており、そしてＮ末端側から５９６番目のロイシンがロイシン以外のアミノ酸に置換しているアミノ酸配列において、１又は数個のアミノ酸が欠失、置換、挿入、及び／又は付加されたアミノ酸配列からなり、しかも、８α－ヒドロキシポリポダ－１３，１７，２１－トリエンを基質としてアンブレイン生成活性を示すポリペプチド、（３）配列番号１で表されるアミノ酸配列におけるＮ末端側から３７３番目のアスパラギン酸がアスパラギン酸以外のアミノ酸に置換しており、Ｎ末端側から１６７番目のチロシンがチロシン以外のアミノ酸に置換しており、そしてＮ末端側から５９６番目のロイシンがロイシン以外のアミノ酸に置換しているアミノ酸配列との同一性が４０％以上であるアミノ酸配列からなり、しかも、８α－ヒドロキシポリポダ－１３，１７，２１－トリエンを基質としてアンブレイン生成活性を示すポリペプチド、（４）配列番号１で表されるアミノ酸配列におけるＮ末端側から３７３番目のアスパラギン酸がアスパラギン酸以外のアミノ酸に置換しており、Ｎ末端側から１６７番目のチロシンがチロシン以外のアミノ酸に置換しており、そしてＮ末端側から５９６番目のロイシンがロイシン以外のアミノ酸に置換しているアミノ酸配列を含み、しかも、８α－ヒドロキシポリポダ－１３，１７，２１－トリエンを基質としてアンブレイン生成活性を示すポリペプチド、（５）配列番号１で表されるアミノ酸配列におけるＮ末端側から３７３番目のアスパラギン酸がアスパラギン酸以外のアミノ酸に置換しており、Ｎ末端側から１６７番目のチロシンがチロシン以外のアミノ酸に置換しており、そしてＮ末端側から５９６番目のロイシンがロイシン以外のアミノ酸に置換しているアミノ酸配列において、１又は数個のアミノ酸が欠失、置換、挿入、及び／又は付加されたアミノ酸配列を含み、しかも、８α－ヒドロキシポリポダ－１３，１７，２１－トリエンを基質としてアンブレイン生成活性を示すポリペプチド、又は（６）配列番号１で表されるアミノ酸配列におけるＮ末端側から３７３番目のアスパラギン酸がアスパラギン酸以外のアミノ酸に置換しており、Ｎ末端側から１６７番目のチロシンがチロシン以外のアミノ酸に置換しており、そしてＮ末端側から５９６番目のロイシンがロイシン以外のアミノ酸に置換しているアミノ酸配列との同一性が４０％以上であるアミノ酸配列を含み、しかも、８α－ヒドロキシポリポダ－１３，１７，２１－トリエンを基質としてアンブレイン生成活性を示すポリペプチドである、［５］に記載の変異型テトラプレニル－β－クルクメン環化酵素、
［７］前記ＤＸＤＤモチーフの第４番目のアミノ酸残基がアスパラギン酸から、システインに置換しており、前記（Ａ／Ｓ／Ｇ）ＲＸ（Ｈ／Ｎ）ＸＸＰモチーフのＮ末に隣接するアミノ酸がチロシンからアラニンに置換しており、そしてＧＸＧＸ（Ｇ／Ａ／Ｐ）モチーフの４番目のアミノ酸がロイシンからアラニンに置換している、［５］又は［６］に記載の変異型テトラプレニル－β－クルクメン環化酵素、
［８］［１］～［７］のいずれかに記載の変異型テトラプレニル－β－クルクメン環化酵素をコードするポリヌクレオチド、
［９］［８］に記載のポリヌクレオチドを有する微生物、
［１０］［８］に記載のポリヌクレオチドを持つＤＮＡを含むベクター、
［１１］［１０］に記載のベクターを有する形質転換体、
［１２］［１］～［７］のいずれかに記載の変異型テトラプレニル－β－クルクメン環化酵素をスクアレン又は８α－ヒドロキシポリポダ－１３，１７，２１－トリエンに反応させて、アンブレインを得ることを特徴とする、アンブレインの製造方法、
［１３］［９］に記載の微生物、又は［１１］に記載の形質転換体を培養することを特徴とする、アンブレインの製造方法、
［１４］（１）テトラプレニル－β－クルクメン環化酵素をスクアレンに反応させて、８α－ヒドロキシポリポダ－１３，１７，２１－トリエンを得る工程、及び（２）変異型テトラプレニル－β－クルクメン環化酵素を８α－ヒドロキシポリポダ－１３，１７，２１－トリエンに反応させて、アンブレインを得る工程、を含むアンブレインの製造方法であって、前記変異型テトラプレニル－β－クルクメン環化酵素が、（Ａ）［１］～［７］のいずれかに記載の変異型テトラプレニル－β－クルクメン環化酵素、又は（Ｂ）ＤＸＤＤモチーフの第４番目のアミノ酸残基であるアスパラギン酸がアスパラギン酸以外のアミノ酸に置換しており、そして（Ａ／Ｓ／Ｇ）ＲＸ（Ｈ／Ｎ）ＸＸＰモチーフのＮ末に隣接するアミノ酸がチロシン以外のアミノ酸に置換しているか、又はＧＸＧＸ（Ｇ／Ａ／Ｐ）モチーフの４番目のアミノ酸がロイシン以外のアミノ酸に置換している変異型テトラプレニル－β－クルクメン環化酵素であって、（ａ）前記変異型テトラプレニル－β－クルクメン環化酵素は、前記ＤＸＤＤモチーフを基準として、Ｎ末端側に１００アミノ酸残基以上離れた位置にＱＸＸＸＧＸ（Ｗ／Ｆ）モチーフ、Ｎ末端側に１８０～２５０アミノ酸残基離れた位置に前記（Ａ／Ｓ／Ｇ）ＲＸ（Ｈ／Ｎ）ＸＸＰモチーフ、Ｎ末端側に８０～１４０アミノ酸残基離れた位置に前記（Ｄ／Ｎ）Ｇ（Ｔ／Ｌ）（Ｌ／Ｆ／Ｙ）（Ｙ／Ｆ）ＳＹモチーフ、Ｎ末端側に１０～５０アミノ酸残基離れた位置にＱＸＸＸＸ（Ｇ／Ａ／Ｓ）Ｘ（Ｆ／Ｗ／Ｙ）モチーフ、Ｃ末端側に２０～５０アミノ酸残基離れた位置にＱＸＸＸＧＸ（Ｆ／Ｗ／Ｙ）モチーフ、Ｃ末端側に５０～１２０アミノ酸残基離れた位置にＱＸＸＸＧＸＷモチーフ、Ｃ末端側に１２０～１７０アミノ酸残基離れた位置にＱＸＸＸＧＸ（Ｆ／Ｗ）モチーフ、及びＣ末端側に１８０～２５０アミノ酸残基離れた位置に前記ＧＸＧＸ（Ｇ／Ａ／Ｐ）モチーフを有し、そしてＤＸＤＤモチーフを基準として、Ｃ末端側に１７０アミノ酸残基以上離れた位置にＱＸＸＸＧＸＷモチーフを有さず、（ｂ）配列番号１で表されるアミノ酸配列との同一性が４０％以上であり、そして（ｃ）８α－ヒドロキシポリポダ－１３，１７，２１－トリエンを基質としてアンブレイン生成活性を示す、変異型テトラプレニル－β－クルクメン環化酵素、であるアンブレインの製造方法、
［１５］前記変異型テトラプレニル－β－クルクメン環化酵素（Ｂ）を構成するポリペプチドが、（１）配列番号１で表されるアミノ酸配列におけるＮ末端側から３７３番目のアスパラギン酸がアスパラギン酸以外のアミノ酸に置換しており、そしてＮ末端側から１６７番目のチロシンがチロシン以外のアミノ酸に置換しているポリペプチド、（２）配列番号１で表されるアミノ酸配列におけるＮ末端側から３７３番目のアスパラギン酸がアスパラギン酸以外のアミノ酸に置換しており、そしてＮ末端側から１６７番目のチロシンがチロシン以外のアミノ酸に置換しているアミノ酸配列において、１又は数個のアミノ酸が欠失、置換、挿入、及び／又は付加されたアミノ酸配列からなり、しかも、８α－ヒドロキシポリポダ－１３，１７，２１－トリエンを基質としてアンブレイン生成活性を示すポリペプチド、（３）配列番号１で表されるアミノ酸配列におけるＮ末端側から３７３番目のアスパラギン酸がアスパラギン酸以外のアミノ酸に置換しており、そしてＮ末端側から１６７番目のチロシンがチロシン以外のアミノ酸に置換しているアミノ酸配列との同一性が４０％以上であるアミノ酸配列からなり、しかも、８α－ヒドロキシポリポダ－１３，１７，２１－トリエンを基質としてアンブレイン生成活性を示すポリペプチド、（４）配列番号１で表されるアミノ酸配列におけるＮ末端側から３７３番目のアスパラギン酸がアスパラギン酸以外のアミノ酸に置換しており、そしてＮ末端側から１６７番目のチロシンがチロシン以外のアミノ酸に置換しているアミノ酸配列を含み、しかも、８α－ヒドロキシポリポダ－１３，１７，２１－トリエンを基質としてアンブレイン生成活性を示すポリペプチド、（５）配列番号１で表されるアミノ酸配列におけるＮ末端側から３７３番目のアスパラギン酸がアスパラギン酸以外のアミノ酸に置換しており、そしてＮ末端側から１６７番目のチロシンがチロシン以外のアミノ酸に置換しているアミノ酸配列において、１又は数個のアミノ酸が欠失、置換、挿入、及び／又は付加されたアミノ酸配列を含み、しかも、８α－ヒドロキシポリポダ－１３，１７，２１－トリエンを基質としてアンブレイン生成活性を示すポリペプチド、（６）配列番号１で表されるアミノ酸配列におけるＮ末端側から３７３番目のアスパラギン酸がアスパラギン酸以外のアミノ酸に置換しており、そしてＮ末端側から１６７番目のチロシンがチロシン以外のアミノ酸に置換しているアミノ酸配列との同一性が４０％以上であるアミノ酸配列を含み、しかも、８α－ヒドロキシポリポダ－１３，１７，２１－トリエンを基質としてアンブレイン生成活性を示すポリペプチド、（７）配列番号１で表されるアミノ酸配列におけるＮ末端側から３７３番目のアスパラギン酸がアスパラギン酸以外のアミノ酸に置換しており、そしてＮ末端側から５９６番目のロイシンがロイシン以外のアミノ酸に置換しているポリペプチド、（８）配列番号１で表されるアミノ酸配列におけるＮ末端側から３７３番目のアスパラギン酸がアスパラギン酸以外のアミノ酸に置換しており、そしてＮ末端側から５９６番目のロイシンがロイシン以外のアミノ酸に置換しているアミノ酸配列において、１又は数個のアミノ酸が欠失、置換、挿入、及び／又は付加されたアミノ酸配列からなり、しかも、８α－ヒドロキシポリポダ－１３，１７，２１－トリエンを基質としてアンブレイン生成活性を示すポリペプチド、（９）配列番号１で表されるアミノ酸配列におけるＮ末端側から３７３番目のアスパラギン酸がアスパラギン酸以外のアミノ酸に置換しており、そしてＮ末端側から５９６番目のロイシンがロイシン以外のアミノ酸に置換しているアミノ酸配列との同一性が４０％以上であるアミノ酸配列からなり、しかも、８α－ヒドロキシポリポダ－１３，１７，２１－トリエンを基質としてアンブレイン生成活性を示すポリペプチド、（１０）配列番号１で表されるアミノ酸配列におけるＮ末端側から３７３番目のアスパラギン酸がアスパラギン酸以外のアミノ酸に置換しており、そしてＮ末端側から５９６番目のロイシンがロイシン以外のアミノ酸に置換しているアミノ酸配列を含み、しかも、８α－ヒドロキシポリポダ－１３，１７，２１－トリエンを基質としてアンブレイン生成活性を示すポリペプチド、（１１）配列番号１で表されるアミノ酸配列におけるＮ末端側から３７３番目のアスパラギン酸がアスパラギン酸以外のアミノ酸に置換しており、そしてＮ末端側から５９６番目のロイシンがロイシン以外のアミノ酸に置換しているアミノ酸配列において、１又は数個のアミノ酸が欠失、置換、挿入、及び／又は付加されたアミノ酸配列を含み、しかも、８α－ヒドロキシポリポダ－１３，１７，２１－トリエンを基質としてアンブレイン生成活性を示すポリペプチド、又は（１２）配列番号１で表されるアミノ酸配列におけるＮ末端側から３７３番目のアスパラギン酸がアスパラギン酸以外のアミノ酸に置換しており、そしてＮ末端側から５９６番目のロイシンがロイシン以外のアミノ酸に置換しているアミノ酸配列との同一性が４０％以上であるアミノ酸配列を含み、しかも、８α－ヒドロキシポリポダ－１３，１７，２１－トリエンを基質としてアンブレイン生成活性を示すポリペプチド、である、［１４］に記載のアンブレインの製造方法、
［１６］前記変異型テトラプレニル－β－クルクメン環化酵素（Ｂ）が、前記ＤＸＤＤモチーフの第４番目のアミノ酸残基がアスパラギン酸から、システインに置換しており、そして前記（Ａ／Ｓ／Ｇ）ＲＸ（Ｈ／Ｎ）ＸＸＰモチーフのＮ末に隣接するアミノ酸がチロシンからアラニンに置換している変異型テトラプレニル－β－クルクメン環化酵素、又は前記ＤＸＤＤモチーフの第４番目のアミノ酸残基がアスパラギン酸から、システインに置換しており、そしてＧＸＧＸ（Ｇ／Ａ／Ｐ）モチーフの４番目のアミノ酸がロイシンからアラニンに置換している変異型テトラプレニル－β－クルクメン環化酵素である、［１４］又は［１５］に記載のアンブレインの製造方法、
［１７］前記工程（１）及び工程（２）を同時に行う、［１４］～［１６］のいずれかに記載のアンブレインの製造方法、及び
［１８］前記テトラプレニル－β－クルクメン環化酵素が野生型テトラプレニル－β－クルクメン環化酵素である、［１４］～［１７］のいずれかに記載のアンブレインの製造方法、
に関する。 As a result of diligent research on a method for producing an enzyme that can easily and efficiently obtain an enzyme, the present inventor surprisingly, a mutant tetraprenyl-β-curcumen ring having a slight specific mutation. It has been found that the chemase can efficiently produce umbrellas from bicyclic triterpenes (8α-hydroxypolypoda-13,17,21-triene) in high yields. We have found that the mutant tetraprenyl-β-curcumen cyclase is a second-step bicyclic triterpene (8α-hydroxypolypoda-13) in a two-step stepwise method of producing ambrein from squalene. , 17, 21-triene), it has been found that ambrein can be efficiently produced in a high yield by using it in the step of producing ambrein.
The present invention is based on these findings.
Therefore, the present invention
[1] Asparaginic acid, which is the fourth amino acid residue of the DXDD motif, is replaced with an amino acid other than aspartic acid, and (D / N) G (T / L) (L / F / Y) (Y / F) A mutant tetraprenyl-β-curcumen cyclase in which the 7th amino acid of the SY motif is replaced with an amino acid other than tyrosine, and (a) the mutant tetraprenyl-β-curcumen cyclase. Is a QXXXGX (W / F) motif at a position 100 amino acid residues or more away from the N-terminal side and 180 to 250 amino acid residues away from the N-terminal side (A / S / G) based on the DXDD motif. ) RX (H / N) XXP motif, the above (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-terminal side. , QXXXXX (G / A / S) X (F / W / Y) motif at a position 10 to 50 amino acid residues away from the N-terminal side, QXXXXX (F) at a position 20 to 50 amino acid residues away from the C-terminal side / W / Y) motif, QXXXGXW motif at a position 50 to 120 amino acid residues away from the C-terminal side, QXXXGX (F / W) motif at a position 120 to 170 amino acid residues away from the C-terminal side, and C-terminal side It has the GXGX (G / A / P) motif at a position 180 to 250 amino acid residues apart, and has a QXXXGXW motif at a position separated by 170 amino acid residues or more on the C-terminal side based on the DXDD motif. However, (b) the identity with the amino acid sequence represented by SEQ ID NO: 1 is 40% or more, and (c) the umbrella-forming activity using 8α-hydroxypolypoda-13,17,21-triene as a substrate. Shown, mutant tetraprenyl-β-curcumen cyclase,
[2] Further, the mutant tetraprenyl-β according to [1], wherein the amino acid adjacent to the N-terminal of the (A / S / G) RX (H / N) XPP motif is replaced with an amino acid other than tyrosine. -Kurukumen cyclase,
[3] The polypeptide constituting the mutant tetraprenyl-β-curcumen cyclase is (1) the 373rd aspartic acid from the N-terminal side in the amino acid sequence represented by SEQ ID NO: 1 is an amino acid other than aspartic acid. Polypeptide in which the 257th tyrosine from the N-terminal side is substituted with an amino acid other than tyrosine, and (2) the 373th aspartic acid from the N-terminal side in the amino acid sequence represented by SEQ ID NO: 1. Is replaced with an amino acid other than aspartic acid, and one or several amino acids are deleted, substituted, inserted, and in the amino acid sequence in which the 257th tyrosine from the N-terminal side is replaced with an amino acid other than tyrosine. / Or a polypeptide consisting of an added amino acid sequence and exhibiting umbrella-forming activity using 8α-hydroxypolypoda-13,17,21-triene as a substrate, (3) in the amino acid sequence represented by SEQ ID NO: 1. 40% or more identity with the amino acid sequence in which the 373rd asparagic acid from the N-terminal side is replaced with an amino acid other than aspartic acid and the 257th tyrosine from the N-terminal side is replaced with an amino acid other than tyrosine. A polypeptide consisting of the amino acid sequence of, which exhibits umbrella-forming activity using 8α-hydroxypolypoda-13,17,21-triene as a substrate, (4) N-terminal side in the amino acid sequence represented by SEQ ID NO: 1. The 373rd aspartic acid is substituted with an amino acid other than aspartic acid, and the 257th tyrosine from the N-terminal side contains an amino acid sequence substituted with an amino acid other than tyrosine, and 8α-hydroxypolypoder. A polypeptide showing umbrella-forming activity using 13,17,21-triene as a substrate, (5) The 373rd aspartic acid from the N-terminal side in the amino acid sequence represented by SEQ ID NO: 1 is replaced with an amino acid other than aspartic acid. Includes 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 257th tyrosine from the N-terminal side is replaced with an amino acid other than tyrosine. Moreover, a polypeptide showing umbrella-forming activity using 8α-hydroxypolypoda-13,17,21-triene as a substrate, or (6) the 373rd asparagine from the N-terminal side in the amino acid sequence represented by SEQ ID NO: 1. The acid has been replaced with an amino acid other than aspartic acid, and The 257th tyrosine from the N-terminal side contains an amino acid sequence having 40% or more identity with an amino acid sequence in which an amino acid other than tyrosine is substituted, and 8α-hydroxypolypoda-13,17,21-triene. The polypeptide that exhibits umbrella-forming activity using the above-mentioned as a substrate, the mutant tetraprenyl-β-curcumen cyclase according to [1], or the poly that constitutes the mutant tetraprenyl-β-curcumen cyclase. In the peptide, (7) the 373rd asparagic acid from the N-terminal side in the amino acid sequence represented by SEQ ID NO: 1 is replaced with an amino acid other than aspartic acid, and the 167th tyrosine from the N-terminal side is an amino acid other than tyrosine. Polypeptide in which the 257th tyrosine from the N-terminal side is substituted with an amino acid other than tyrosine, and (8) the 373th aspartic acid from the N-terminal side in the amino acid sequence represented by SEQ ID NO: 1. Is replaced with an amino acid other than aspartic acid, the 167th tyrosine from the N-terminal side is replaced with an amino acid other than tyrosine, and the 257th tyrosine from the N-terminal side is replaced with an amino acid other than tyrosine. In the amino acid sequence, one or several amino acids consist of deleted, substituted, inserted, and / or added amino acid sequences, and umbrella formation using 8α-hydroxypolypoda-13,17,21-triene as a substrate. The active polypeptide, (9) the 373rd asparagic acid from the N-terminal side in the amino acid sequence represented by SEQ ID NO: 1 is replaced with an amino acid other than aspartic acid, and the 167th tyrosine from the N-terminal side is tyrosine. It consists of an amino acid sequence that is replaced with an amino acid other than, and the 257th tyrosine from the N-terminal side is replaced with an amino acid other than tyrosine, and has an amino acid sequence of 40% or more identity, and is 8α-hydroxy. Polypoda-13,17,21-Triene is a substrate that exhibits umbrella-forming activity. (10) The 373rd aspartic acid from the N-terminal side in the amino acid sequence represented by SEQ ID NO: 1 is an amino acid other than aspartic acid. Contains an amino acid sequence in which the 167th tyrosine from the N-terminal side is replaced with an amino acid other than tyrosine, and the 257th tyrosine from the N-terminal side is replaced with an amino acid other than tyrosine. , 8α-Hydroxypolypoda-13,17,21-triene as substrate The 373rd asparagic acid from the N-terminal side in the amino acid sequence represented by (11) SEQ ID NO: 1 is replaced with an amino acid other than aspartic acid, and is 167th from the N-terminal side. In the amino acid sequence in which tyrosine is replaced with an amino acid other than tyrosine and the 257th tyrosine from the N-terminal side is replaced with an amino acid other than tyrosine, one or several amino acids are deleted, substituted, inserted, And / or a polypeptide containing an added amino acid sequence and exhibiting umbrella-forming activity using 8α-hydroxypolypoda-13,17,21-triene as a substrate, or (12) the amino acid represented by SEQ ID NO: 1. The 373rd asparagic acid from the N-terminal side in the sequence is replaced with an amino acid other than aspartic acid, the 167th tyrosine from the N-terminal side is replaced with an amino acid other than tyrosine, and the 257th position from the N-terminal side. Contains an amino acid sequence having 40% or more identity with an amino acid sequence in which tyrosine is replaced with an amino acid other than tyrosine, and has umbrella-forming activity using 8α-hydroxypolypoda-13,17,21-triene as a substrate. The mutant tetraprenyl-β-curcumen cyclase according to [2], which is a polypeptide showing the above.
[4] The fourth amino acid residue of the DXDD motif is replaced with cysteine from asparagic acid, and the (D / N) G (T / L) (L / F / Y) (Y / F) The mutant tetraprenyl-β-curcumen cyclase according to [1] or [3], wherein the 7th amino acid of the SY motif is replaced with alanine from tyrosine, or the 4th amino acid residue of the DXDD motif. The group is substituted from aspartic acid to cysteine, and the amino acid adjacent to the N-terminal of the (A / S / G) RX (H / N) XPP motif is substituted from tyrosine to alanine. N) The mutant tetra according to [2] or [3], wherein the 7th amino acid of the G (T / L) (L / F / Y) (Y / F) SY motif is substituted from tyrosine to alanine. Plenyl-β-curcumen cyclase,
[5] Asparaginic acid, which is the fourth amino acid residue of the DXDD motif, is replaced with an amino acid other than aspartic acid, and is adjacent to the N-terminal of the (A / S / G) RX (H / N) XPP motif. A mutant tetraprenyl-β-curcumen cyclase in which the amino acid is replaced with an amino acid other than tyrosine and the fourth amino acid of the GXGX (G / A / P) motif is replaced with an amino acid other than leucine. , (A) The mutant tetraprenyl-β-curcumen cyclase has a QXXXGX (W / F) motif at a position separated by 100 amino acid residues or more on the N-terminal side based on the DXDD motif, and a QXXXGX (W / F) motif on the N-terminal side. The (A / S / G) RX (H / N) XXP motif is located 180 to 250 amino acid residues away, and the (D / N) G (T) is located 80 to 140 amino acid residues away from the N-terminal side. / L) (L / F / Y) (Y / F) SY motif, QXXX (G / A / S) X (F / W / Y) motif at a position 10 to 50 amino acid residues away from the N-terminal side, QXXXGX (F / W / Y) motif at a position 20 to 50 amino acid residues away from the C-terminal side, QXXXGXGXW motif at a position 50 to 120 amino acid residues away from the C-terminal side, 120-170 amino acid residue at the C-terminal side It has a QXXXGX (F / W) motif at a position distant from the base and the GXGX (G / A / P) motif at a position 180 to 250 amino acid residues away from the C-terminal side, and C is based on the DXDD motif. It does not have a QXXXGXW motif at a position separated by 170 amino acid residues or more on the terminal side, has (b) 40% or more identity with the amino acid sequence represented by SEQ ID NO: 1, and (c) 8α-hydroxypoly. A mutant tetraprenyl-β-curcumen cyclase, which exhibits amino acid-forming activity using poda-13,17,21-triene as a substrate.
[6] The polypeptide constituting the mutant tetraprenyl-β-curcumen cyclase is (1) the 373rd aspartic acid from the N-terminal side in the amino acid sequence represented by SEQ ID NO: 1 is an amino acid other than aspartic acid. , The 167th tyrosine from the N-terminal side is replaced with an amino acid other than tyrosine, and the 596th leucine from the N-terminal side is replaced with an amino acid other than leucine, (2). In the amino acid sequence represented by SEQ ID NO: 1, the 373rd aspartic acid from the N-terminal side is replaced with an amino acid other than aspartic acid, and the 167th tyrosine from the N-terminal side is replaced with an amino acid other than tyrosine. Then, in the amino acid sequence in which the 596th leucine from the N-terminal side is replaced with an amino acid other than leucine, one or several amino acids are deleted, substituted, inserted, and / or added. Polypeptide showing umbrella-forming activity using 8α-hydroxypolypoda-13,17,21-triene as a substrate, (3) Asparagic acid at the 373rd position from the N-terminal side in the amino acid sequence represented by SEQ ID NO: 1 is aspartic acid. With an amino acid sequence in which the 167th tyrosine from the N-terminal side is replaced with an amino acid other than tyrosine, and the 596th leucine from the N-terminal side is replaced with an amino acid other than leucine. (4) SEQ ID NO: 1 is a polypeptide consisting of an amino acid sequence having an amino acid identity of 40% or more and exhibiting umbrella-forming activity using 8α-hydroxypolypoda-13,17,21-triene as a substrate. The 373rd aspartic acid from the N-terminal side in the amino acid sequence is replaced with an amino acid other than aspartic acid, the 167th tyrosine from the N-terminal side is replaced with an amino acid other than tyrosine, and 596 from the N-terminal side. A polypeptide containing an amino acid sequence in which the second leucine is replaced with an amino acid other than leucine and exhibiting umbrella-forming activity using 8α-hydroxypolypoda-13,17,21-triene as a substrate, (5) SEQ ID NO: In the amino acid sequence represented by 1, the 373rd asparagic acid from the N-terminal side is replaced with an amino acid other than aspartic acid, the 167th tyrosine from the N-terminal side is replaced with an amino acid other than tyrosine, and N. The 596th leucine from the terminal side is replaced with an amino acid other than leucine. Contains an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted, and / or added, and the amino acid sequence contains 8α-hydroxypolypoda-13,17,21-triene as a substrate. The 373rd asparagic acid from the N-terminal side in the polypeptide showing brain-forming activity or (6) the amino acid sequence represented by SEQ ID NO: 1 is replaced with an amino acid other than aspartic acid, and the 167th asparagic acid from the N-terminal side is substituted. It contains an amino acid sequence in which tyrosine is replaced with an amino acid other than tyrosine, and leucine at position 596 from the N-terminal side is 40% or more identical to the amino acid sequence substituted with an amino acid other than leucine, and moreover. The mutant tetraprenyl-β-curcumen cyclase according to [5], which is a polypeptide showing umbrella-forming activity using 8α-hydroxypolypoda-13,17,21-triene as a substrate.
[7] The fourth amino acid residue of the DXDD motif is replaced with cysteine from aspartic acid, and the amino acid adjacent to the N-terminal of the (A / S / G) RX (H / N) XXP motif is The mutant tetraprenyl-β according to [5] or [6], wherein the tyrosine is replaced with alanine and the fourth amino acid of the GXGX (G / A / P) motif is replaced with leucine to alanine. -Kurukumen cyclase,
[8] A polynucleotide encoding the mutant tetraprenyl-β-curcumen cyclase according to any one of [1] to [7].
[9] A microorganism having the polynucleotide according to [8].
[10] A vector containing a DNA having the polynucleotide according to [8].
[11] A transformant having the vector according to [10],
[12] The mutant tetraprenyl-β-curcumen cyclase according to any one of [1] to [7] is reacted with squalene or 8α-hydroxypolypoda-13,17,21-triene to give an umbrella. A method of manufacturing squalene, which is characterized by obtaining
[13] A method for producing an umbrella, which comprises culturing the microorganism according to [9] or the transformant according to [11].
[14] (1) A step of reacting tetraprenyl-β-curcumen cyclase with squalene to obtain 8α-hydroxypolypoda-13,17,21-triene, and (2) mutant tetraprenyl-β-. A method for producing an amino acid, which comprises a step of reacting curcumen cyclase with 8α-hydroxypolypoda-13,17,21-triene to obtain an amino acid, wherein the mutant tetraprenyl-β-curcumen ring is used. The chemase is the mutant tetraprenyl-β-curcumen cyclase according to any one of (A) [1] to [7], or (B) aspartic acid, which is the fourth amino acid residue of the DXDD motif. Is replaced with an amino acid other than aspartic acid, and the amino acid adjacent to the N-terminal of the (A / S / G) RX (H / N) XXP motif is replaced with an amino acid other than tyrosine, or GXGX (G) / A / P) A mutant tetraprenyl-β-curcumen cyclase in which the fourth amino acid of the motif is replaced with an amino acid other than leucine, and (a) the mutant tetraprenyl-β-curcumen cyclization. Based on the DXDD motif, the enzyme has a QXXXGX (W / F) motif at a position separated by 100 amino acid residues or more on the N-terminal side, and the (A / S) at a position 180 to 250 amino acid residues separated on the N-terminal side. / G) RX (H / N) XXP motif, said (D / N) G (T / L) (L / F / Y) (Y / F) at a position 80 to 140 amino acid residues away from the N-terminal side. SY motif, QXXX (G / A / S) X (F / W / Y) motif at a position 10 to 50 amino acid residues away from the N-terminal side, QXXXGX at a position 20 to 50 amino acid residues away from the C-terminal side (F / W / Y) motif, QXXXGXW motif at a position 50 to 120 amino acid residues away from the C-terminal side, QXXXGX (F / W) motif at a position 120 to 170 amino acid residues away from the C-terminal side, and C The GXGX (G / A / P) motif is located 180 to 250 amino acid residues away from the terminal side, and the QXXXGXW motif is located 180 to 250 amino acid residues or more away from the C terminal side based on the DXDD motif. No, (b) identity with the amino acid sequence represented by SEQ ID NO: 1 is 40% or more, and (c) umbrella formation using 8α-hydroxypolypoda-13,17,21-triene as a substrate. A method for producing an amino acid, which is an active mutant tetraprenyl-β-curcumen cyclase,
[15] The polypeptide constituting the mutant tetraprenyl-β-curcumen cyclase (B) is (1) aspartic acid at the 373rd position from the N-terminal side in the amino acid sequence represented by SEQ ID NO: 1. A polypeptide in which an amino acid other than tyrosine is substituted and the 167th tyrosine from the N-terminal side is replaced with an amino acid other than tyrosine, (2) 373rd from the N-terminal side in the amino acid sequence represented by SEQ ID NO: 1. In the amino acid sequence in which aspartic acid is replaced with an amino acid other than aspartic acid, and the 167th tyrosine from the N-terminal side is replaced with an amino acid other than tyrosine, one or several amino acids are deleted or substituted. A polypeptide consisting of an inserted and / or added amino acid sequence and exhibiting umbrella-forming activity using 8α-hydroxypolypoda-13,17,21-triene as a substrate, (3) represented by SEQ ID NO: 1. The identity with the amino acid sequence in which the 373rd asparagic acid from the N-terminal side in the amino acid sequence is replaced with an amino acid other than aspartic acid and the 167th tyrosine from the N-terminal side is replaced with an amino acid other than tyrosine. A polypeptide consisting of an amino acid sequence of 40% or more and exhibiting umbrella-forming activity using 8α-hydroxypolypoda-13,17,21-triene as a substrate, (4) in the amino acid sequence represented by SEQ ID NO: 1. It contains an amino acid sequence in which the 373rd aspartic acid from the N-terminal side is replaced with an amino acid other than aspartic acid, and the 167th tyrosine from the N-terminal side is replaced with an amino acid other than tyrosine, and 8α-hydroxy Polypoda-13,17,21-Triene is a substrate that exhibits umbrella-forming activity. (5) The 373rd aspartic acid from the N-terminal side in the amino acid sequence represented by SEQ ID NO: 1 is an amino acid other than aspartic acid. An amino acid in which one or several amino acids are deleted, substituted, inserted, and / or added in an amino acid sequence in which the 167th amino acid from the N-terminal side is replaced with an amino acid other than tyrosine. A polypeptide containing a sequence and exhibiting umbrella-forming activity using 8α-hydroxypolypoda-13,17,21-triene as a substrate, (6) 373rd from the N-terminal side in the amino acid sequence represented by SEQ ID NO: 1. Asparagic acid is replaced with an amino acid other than asparagic acid, The 167th tyrosine from the N-terminal side contains an amino acid sequence having 40% or more identity with an amino acid sequence in which an amino acid other than tyrosine is substituted, and 8α-hydroxypolypoda-13, 17, 21 -A polypeptide exhibiting umbrella-forming activity using triene as a substrate, (7) The 373rd aspartic acid from the N-terminal side in the amino acid sequence represented by SEQ ID NO: 1 is replaced with an amino acid other than aspartic acid, and N Polypeptide in which the 596th leucine from the terminal side is replaced with an amino acid other than leucine, (8) The 373rd aspartic acid from the N-terminal side in the amino acid sequence represented by SEQ ID NO: 1 is replaced with an amino acid other than aspartic acid. And in the amino acid sequence in which leucine at position 596 from the N-terminal side is replaced with an amino acid other than leucine, from the amino acid sequence in which one or several amino acids are deleted, substituted, inserted, and / or added. Moreover, a polypeptide showing umbrella-forming activity using 8α-hydroxypolypoda-13,17,21-triene as a substrate, (9) the 373rd asparagine from the N-terminal side in the amino acid sequence represented by SEQ ID NO: 1. It consists of an amino acid sequence in which the acid is replaced with an amino acid other than aspartic acid, and the 596th leucine from the N-terminal side is replaced with an amino acid other than leucine, and the identity is 40% or more. , 8α-Hydroxypolypoda-13,17,21-Triene as a substrate, a polypeptide showing umbrella-forming activity, (10) Asparaginic acid at the 373rd position from the N-terminal side in the amino acid sequence represented by SEQ ID NO: 1 is asparagine. It contains an amino acid sequence in which an amino acid other than acid is substituted, and leucine at position 596 from the N-terminal side is substituted with an amino acid other than leucine, and 8α-hydroxypolypoda-13,17,21-triene is contained. A polypeptide exhibiting umbrella-forming activity as a substrate, (11) the 373rd aspartic acid from the N-terminal side in the amino acid sequence represented by SEQ ID NO: 1 is replaced with an amino acid other than aspartic acid, and from the N-terminal side. In the amino acid sequence in which leucine at position 596 is replaced with an amino acid other than leucine, one or several amino acids contain the deleted, substituted, inserted, and / or added amino acid sequence, and 8α-hydroxypolypoda. Amino acid using -13,17,21-triene as a substrate The 373rd aspartic acid from the N-terminal side in the polypeptide showing the activity of producing an amino acid or (12) the amino acid sequence represented by SEQ ID NO: 1 is replaced with an amino acid other than aspartic acid, and the 596th position from the N-terminal side. Contains an amino acid sequence having 40% or more identity with an amino acid sequence in which leucine is replaced with an amino acid other than leucine, and also produces an umbrella using 8α-hydroxypolypoda-13,17,21-triene as a substrate. The method for producing an amino acid according to [14], which is an active polypeptide.
[16] In the mutant tetraprenyl-β-curcumen cyclase (B), the fourth amino acid residue of the DXDD motif is replaced with cysteine from aspartic acid, and the above (A / S / G) A mutant tetraprenyl-β-curcumen cyclase in which the amino acid adjacent to the N-terminal of the RX (H / N) XXP motif is replaced with alanine from tyrosine, or the fourth amino acid residue of the DXDD motif. Is a mutant tetraprenyl-β-curcumen cyclase in which aspartic acid is replaced with cysteine and the fourth amino acid of the GXGX (G / A / P) motif is replaced with leucine to alanine. The method for producing an amino acid according to [14] or [15],
[17] The method for producing an umbrella according to any one of [14] to [16], wherein the steps (1) and (2) are carried out at the same time, and [18] the tetraprenyl-β-curcumen cyclase. The method for producing an umbrella according to any one of [14] to [17], wherein is a wild-type tetraprenyl-β-curcumen cyclase.
Regarding.
　本発明の変異型テトラプレニル－β－クルクメン環化酵素は、２環性トリテルペン（８α－ヒドロキシポリポダ－１３，１７，２１－トリエン）から効率的に、高い収率でアンブレインを生成することができる。
　本発明の変異型テトラプレニル－β－クルクメン環化酵素は、スクアレンからアンブレインを産生する２段階のステップワイズ法において、２段階目の２環性トリテルペン（８α－ヒドロキシポリポダ－１３，１７，２１－トリエン）からアンブレインを産生する工程に使用することによって、効率的に高い収率でアンブレインを産生することができる。 The mutant tetraprenyl-β-curcumen cyclase of the present invention efficiently produces umbrellas from bicyclic triterpenes (8α-hydroxypolypoda-13,17,21-triene) in high yield. Can be done.
The mutant tetraprenyl-β-curcumen cyclase of the present invention is a two-step bicyclic triterpene (8α-hydroxypolypoda-13,17, By using it in the step of producing ambrein from 21-trien), ambrein can be efficiently produced in a high yield.
変異型テトラプレニル－β－クルクメン環化酵素を用いてスクアレンからアンブレインを生成する２つの経路を示した図である。It is a figure which showed two pathways which generate an umbrella from squalene using a mutant tetraprenyl-β-curcumen cyclase. 本発明の変異型テトラプレニル－β－クルクメン環化酵素を用いて、８α－ヒドロキシポリポダ－１３，１７，２１－トリエンからアンブレインを生成する経路を示した図である。It is a figure which showed the pathway which produces the umbrella from 8α-hydroxypolypoda-13,17,21-triene using the mutant tetraprenyl-β-curcumen cyclase of this invention. テトラプレニル－β－クルクメン環化酵素及び本発明の変異型テトラプレニル－β－クルクメン環化酵素を用いて、スクアレンからからアンブレインを生成する本発明の製造方法を示した図である。It is a figure which showed the production method of this invention which produces umbrella from squalene using tetraprenyl-β-curcumen cyclase and the mutant tetraprenyl-β-curcumen cyclase of this invention. 本発明の変異型テトラプレニル－β－クルクメン環化酵素（実施例１～３、製造方法例１）を用いて、８α－ヒドロキシポリポダ－１３，１７，２１－トリエンを基質として得られた反応組成物中のアンブレインの含有量比（Ａ）、及び８α－ヒドロキシポリポダ－１３，１７，２１－トリエンを除いた、アンブレイン及びオノセロイドの含有量比（Ｂ）に示したグラフである。Reaction obtained using 8α-hydroxypolypoda-13,17,21-triene as a substrate using the mutant tetraprenyl-β-curcumen cyclase of the present invention (Examples 1 to 3, Production Method Example 1). It is a graph shown in the content ratio (A) of umbrella in the composition, and the content ratio (B) of umbrella and onoseroid excluding 8α-hydroxypolypoda-13,17,21-triene. 発明の変異型テトラプレニル－β－クルクメン環化酵素（実施例２、製造方法例２）を用いて、スクアレンを基質として得られた反応組成物中のアンブレイン含有量（Ａ）、及びスクアレンを除いた、アンブレイン、単環化合物、２環化合物及びオノセロイドの含有量比（Ｂ）を示したグラフである。Using the mutant tetraprenyl-β-curcumen cyclase of the present invention (Example 2, Production Method Example 2), the umbrella content (A) and squalene in the reaction composition obtained using squalene as a substrate were used. It is the graph which showed the content ratio (B) of the squalene, the monocyclic compound, the bicyclic compound and onoseroid excluding. 野生型テトラプレニル－β－クルクメン環化酵素及び本発明の変異型テトラプレニル－β－クルクメン環化酵素（実施例２、製造方法例３）を用いて、ステップワイズ（二段階）反応法によって得られた反応組成物中のアンブレイン含有量（Ａ）、及びスクアレンを除いた、アンブレイン、単環化合物、２環化合物及びオノセロイドの含有量比（Ｂ）示したグラフである。Obtained by a stepwise (two-step) reaction method using the wild-type tetraprenyl-β-squalene cyclase and the mutant tetraprenyl-β-squalene cyclase of the present invention (Example 2, Production Method Example 3). It is a graph which showed the umbrella content (A) in the obtained reaction composition, and the content ratio (B) of umbrella, monocyclic compound, bicyclic compound and onoseroid excluding squalene. 野生型テトラプレニル－β－クルクメン環化酵素、３７３番目のアスパラギン酸がシステインに置換したＤ３７３Ｃ、１６７番目のチロシンがアラニンに置換し、そして３７３番目のアスパラギン酸がシステインに置換したＹ１６７Ａ／Ｄ３７３Ｃ、２５７番目のチロシンがアラニンに置換し、そして３７３番目のアスパラギン酸がシステインに置換したＹ２５７Ａ／Ｄ３７３Ｃ、３７３番目のアスパラギン酸がシステインに置換し、そして５９６番目のロイシンがアラニンに置換したＤ３７３Ｃ／Ｌ５９６Ａ、１６７番目のチロシンがアラニンに置換し、２５７番目のチロシンがアラニンに置換し、そして３７３番目のアスパラギン酸がシステインに置換したＹ１６７Ａ／Ｙ２５７Ａ／Ｄ３７３Ｃ、及び１６７番目のチロシンがアラニンに置換し、３７３番目のアスパラギン酸がシステインに置換し、そして５９６番目のロイシンがアラニンに置換したＹ１６７Ａ／Ｄ３７３Ｃ／Ｌ５９６Ａのアミノ酸配列を示した図である。Wild-type tetraprenyl-β-curcumen cyclase, D373C in which aspartic acid at position 373 was replaced with cysteine, Y167A / D373C, 257 in which aspartic acid at position 167 was replaced with alanine and aspartic acid at position 373 was replaced with cysteine. Y257A / D373C, where the thyroid was replaced with alanine and aspartic acid at position 373 was replaced with cysteine, D373C / L596A, 167 where aspartic acid at position 373 was replaced with cysteine and leucine at position 596 was replaced with alanine. Y167A / Y257A / D373C, in which the thyroid of the second was replaced with alanine, the tyrosine of the 257th was replaced with alanine, and the aspartic acid of the 373rd was replaced with cysteine, and the tyrosine of the 167th was replaced with alanine, and the 373rd was It is a figure which showed the amino acid sequence of Y167A / D373C / L596A where aspartic acid was replaced with cysteine, and leucine at position 596 was replaced with alanine. 野生型テトラプレニル－β－クルクメン環化酵素、３７３番目のアスパラギン酸がシステインに置換したＤ３７３Ｃ、１６７番目のチロシンがアラニンに置換し、そして３７３番目のアスパラギン酸がシステインに置換したＹ１６７Ａ／Ｄ３７３Ｃ、２５７番目のチロシンがアラニンに置換し、そして３７３番目のアスパラギン酸がシステインに置換したＹ２５７Ａ／Ｄ３７３Ｃ、３７３番目のアスパラギン酸がシステインに置換し、そして５９６番目のロイシンがアラニンに置換したＤ３７３Ｃ／Ｌ５９６Ａ、１６７番目のチロシンがアラニンに置換し、２５７番目のチロシンがアラニンに置換し、そして３７３番目のアスパラギン酸がシステインに置換したＹ１６７Ａ／Ｙ２５７Ａ／Ｄ３７３Ｃ、及び１６７番目のチロシンがアラニンに置換し、３７３番目のアスパラギン酸がシステインに置換し、そして５９６番目のロイシンがアラニンに置換したＹ１６７Ａ／Ｄ３７３Ｃ／Ｌ５９６Ａのアミノ酸配列を示した図である。Wild-type tetraprenyl-β-curcumen cyclase, D373C in which aspartic acid at position 373 was replaced with cysteine, Y167A / D373C, 257 in which aspartic acid at position 167 was replaced with alanine and aspartic acid at position 373 was replaced with cysteine. Y257A / D373C, where the thyroid was replaced with alanine and aspartic acid at position 373 was replaced with cysteine, D373C / L596A, 167 where aspartic acid at position 373 was replaced with cysteine and leucine at position 596 was replaced with alanine. Y167A / Y257A / D373C, in which the thyroid of the second was replaced with alanine, the tyrosine of the 257th was replaced with alanine, and the aspartic acid of the 373rd was replaced with cysteine, and the tyrosine of the 167th was replaced with alanine, and the 373rd was It is a figure which showed the amino acid sequence of Y167A / D373C / L596A where aspartic acid was replaced with cysteine, and leucine at position 596 was replaced with alanine. バチルス・メガテリウム、バチルス・サブチリス、及びバチルス・リケニフォルミスのテトラプレニル－β－クルクメン環化酵素のアミノ酸配列、並びにアリシクロバチルス・アシドカルダリウスのスクアレン－ホペン環化酵素のアミノ酸配列のアラインメントを示した図である。The figure which showed the amino acid sequence of the tetraprenyl-β-curcumen cyclase of Bacillus megaterium, Bacillus subtilis, and Bacillus licheniformis, and the amino acid sequence of the squalene-hopen cyclase of Alicyclobacillus acidocardarius. Is. バチルス・メガテリウム、バチルス・サブチリス、及びバチルス・リケニフォルミスのテトラプレニル－β－クルクメン環化酵素のアミノ酸配列、並びにアリシクロバチルス・アシドカルダリウスのスクアレン－ホペン環化酵素のアミノ酸配列のアラインメントを示した図である。The figure which showed the amino acid sequence of the tetraprenyl-β-curcumen cyclase of Bacillus megaterium, Bacillus subtilis, and Bacillus licheniformis, and the amino acid sequence of the squalene-hopen cyclase of Alicyclobacillus acidocardarius. Is.
（テトラプレニル－β－クルクメン環化酵素）
　野生型のテトラプレニル－β－クルクメン環化酵素（以下、ＴＣと称することがある）は、片末端に単環を有する３－デオキシアキレオールＡを基質として利用し、アンブレインを生成することができる。すなわち、テトラプレニル－β－クルクメン環化酵素は、３－デオキシアキレオールＡを基質として利用すると、３－デオキシアキレオールＡの環状化されていない端を選択的に環化させて、両末端環化化合物を生成することができる。
　また、テトラプレニル－β－クルクメン環化酵素は、スクアレンを基質とし、２環性の８α－ヒドロキシポリポダ－１３，１７，２１－トリエンを生成することができる（非特許文献５）。更に、テトラプレニル－β－クルクメン環化酵素は、２環性の８α－ヒドロキシポリポダ－１３，１７，２１－トリエンの環状化されていない端を選択的に環化させて、両末端環化化合物のオノセロイド（オノセランオキサイド及び１４β－ヒドロキシオノセラ－８（２６）－エン）を生成することができる（非特許文献６）。 (Tetraprenyl-β-curcumen cyclase)
The wild-type tetraprenyl-β-curcumen cyclase (hereinafter sometimes referred to as TC) can generate an umbrella by using 3-deoxyacireol A having a single ring at one end as a substrate. can. That is, when tetraprenyl-β-curcumen cyclase utilizes 3-deoxyacireol A as a substrate, the non-cyclized end of 3-deoxyacireol A is selectively cyclized to form both terminal rings. Chemical compounds can be produced.
In addition, tetraprenyl-β-curcumen cyclase can produce bicyclic 8α-hydroxypolypoda-13,17,21-triene using squalene as a substrate (Non-Patent Document 5). In addition, tetraprenyl-β-curcumen cyclase selectively cyclizes the non-cyclized ends of the bicyclic 8α-hydroxypolypoda-13,17,21-triene to cyclize both ends. The compound onoseroids (onoselan oxide and 14β-hydroxyonocera-8 (26) -ene) can be produced (Non-Patent Document 6).
　すなわち、テトラプレニル－β－クルクメン環化酵素は、ＥＣ４．２．１．１２９に分類され、水とテトラプレニル－β－クルクメンからバチテルペノールＡを生成する反応、又は、スクアレンから８α－ヒドロキシポリポダ－１３，１７，２１－トリエンを生成する反応を触媒し得る酵素である。
　テトラプレニル－β－クルクメン環化酵素は、例えばバチルス属、ブレビバチルス属、パエニバチルス属、又はジオバチルス属などの細菌が有している。バチルス属の細菌としては、枯草菌（バチルス・サブチルス）、バチルス・メガテリウム、又はバチルス・リケニフォルミスを挙げることができる。テトラプレニル－β－クルクメン環化酵素は、ＤＸＤＤモチーフを基準として、Ｎ末端側に１００アミノ酸残基以上離れた位置にＱＸＸＸＧＸ（Ｗ／Ｆ）モチーフ、Ｎ末端側に１８０～２５０アミノ酸残基離れた位置に前記（Ａ／Ｓ／Ｇ）ＲＸ（Ｈ／Ｎ）ＸＸＰモチーフ、Ｎ末端側に８０～１４０アミノ酸残基離れた位置に前記（Ｄ／Ｎ）Ｇ（Ｔ／Ｌ）（Ｌ／Ｆ／Ｙ）（Ｙ／Ｆ）ＳＹモチーフ、Ｎ末端側に１０～５０アミノ酸残基離れた位置にＱＸＸＸＸ（Ｇ／Ａ／Ｓ）Ｘ（Ｆ／Ｗ／Ｙ）モチーフ、Ｃ末端側に２０～５０アミノ酸残基離れた位置にＱＸＸＸＧＸ（Ｆ／Ｗ／Ｙ）モチーフ、Ｃ末端側に５０～１２０アミノ酸残基離れた位置にＱＸＸＸＧＸＷモチーフ、Ｃ末端側に１２０～１７０アミノ酸残基離れた位置にＱＸＸＸＧＸ（Ｆ／Ｗ）モチーフ、及びＣ末端側に１８０～２５０アミノ酸残基離れた位置に前記ＧＸＧＸ（Ｇ／Ａ／Ｐ）モチーフを有している。スクアレン－ホペン環化酵素は、前記のモチーフのうち（Ａ／Ｓ／Ｇ）ＲＸ（Ｈ／Ｎ）ＸＸＰモチーフ、（Ｄ／Ｎ）Ｇ（Ｔ／Ｌ）（Ｌ／Ｆ／Ｙ）（Ｙ／Ｆ）ＳＹモチーフ、及びＧＸＧＸ（Ｇ／Ａ／Ｐ）モチーフを有していない。また、スクアレン－ホペン環化酵素はＤＸＤＤモチーフのＣ末端側に１７０アミノ酸残基以上離れた位置にＱＸＸＸＧＸＷモチーフを有している。一方、テトラプレニル－β－クルクメン環化酵素は、前記ＱＸＸＸＧＸＷモチーフを有していない。更に、スクアレン－ホペン環化酵素は、ＱＸＸＸＧＸＷモチーフのＣ末側に、前記ＧＸＧＸ（Ｇ／Ａ／Ｐ）モチーフに似たＧＸＧＦＰ配列を有しているが、その４番目のアミノ酸がフェニルアラニン（Ｆ）であるという特徴を有する。テトラプレニル－β－クルクメン環化酵素のＧＸＧＸ（Ｇ／Ａ／Ｐ）モチーフは、４番目のアミノ酸がフェニルアラニンではなく、基本的には、ロイシン（Ｌ）である。 That is, tetraprenyl-β-curcumen cyclase is classified into EC4.2.1.129, and is a reaction for producing batyterpenol A from water and tetraprenyl-β-curcumen, or 8α-hydroxypolypoda from squalene. It is an enzyme that can catalyze the reaction that produces 13,17,21-triene.
The tetraprenyl-β-curcumen cyclase is contained in bacteria such as Bacillus, Brevibacillus, Paenibacillus, or Diobacillus. Bacteria of the genus Bacillus include Bacillus subtilis (Bacillus subtilis), Bacillus megaterium, or Bacillus licheniformis. The tetraprenyl-β-curcumen cyclase is based on the DXDD motif, with the QXXXGX (W / F) motif at a position separated by 100 amino acid residues or more on the N-terminal side and 180 to 250 amino acid residues separated on the N-terminal side. The (A / S / G) RX (H / N) XPP motif is located at the position, and the (D / N) G (T / L) (L / F /) is located at a position 80 to 140 amino acid residues away from the N-terminal side. Y) (Y / F) SY motif, QXXX (G / A / S) X (F / W / Y) motif at 10 to 50 amino acid residues on the N-terminal side, 20 to 50 amino acids on the C-terminal side QXXXGX (F / W / Y) motif at the residue-distant position, QXXXGXW motif at the C-terminal 50-120 amino acid residue, and QXXXGX (F / W / Y) at the C-terminal 120-170 amino acid residue. It has the / W) motif and the GXGX (G / A / P) motif at a position 180 to 250 amino acid residues away from the C-terminal side. Squalene-Hopen cyclase is the (A / S / G) RX (H / N) XXP motif, (D / N) G (T / L) (L / F / Y) (Y /) among the above motifs. F) It does not have a SY motif and a GXGX (G / A / P) motif. Further, the squalene-hopen cyclase has a QXXXGXW motif at a position separated by 170 amino acid residues or more on the C-terminal side of the DXDD motif. On the other hand, the tetraprenyl-β-curcumen cyclase does not have the QXXXGXW motif. Furthermore, the squalene-hopen cyclase has a GXGFP sequence similar to the GXGX (G / A / P) motif on the C-terminal side of the QXXXGXW motif, but the fourth amino acid is phenylalanine (F). It has the characteristic of being. In the GXGX (G / A / P) motif of the tetraprenyl-β-curcumen cyclase, the fourth amino acid is not phenylalanine, but basically leucine (L).
［１］変異型テトラプレニル－β－クルクメン環化酵素
（第１態様）
　本発明の第１態様の変異型テトラプレニル－β－クルクメン環化酵素は、ＤＸＤＤモチーフの第４番目のアミノ酸残基であるアスパラギン酸がアスパラギン酸以外のアミノ酸に置換しており、（Ｄ／Ｎ）Ｇ（Ｔ／Ｌ）（Ｌ／Ｆ／Ｙ）（Ｙ／Ｆ）ＳＹモチーフの７番目のアミノ酸がチロシン以外のアミノ酸に置換している、変異型テトラプレニル－β－クルクメン環化酵素であって、（ａ）前記変異型テトラプレニル－β－クルクメン環化酵素は、前記ＤＸＤＤモチーフを基準として、Ｎ末端側に１００アミノ酸残基以上離れた位置にＱＸＸＸＧＸ（Ｗ／Ｆ）モチーフ、Ｎ末端側に１８０～２５０アミノ酸残基離れた位置に（Ａ／Ｓ／Ｇ）ＲＸ（Ｈ／Ｎ）ＸＸＰモチーフ、Ｎ末端側に８０～１４０アミノ酸残基離れた位置に前記（Ｄ／Ｎ）Ｇ（Ｔ／Ｌ）（Ｌ／Ｆ／Ｙ）（Ｙ／Ｆ）ＳＹモチーフ、Ｎ末端側に１０～５０アミノ酸残基離れた位置にＱＸＸＸＸ（Ｇ／Ａ／Ｓ）Ｘ（Ｆ／Ｗ／Ｙ）モチーフ、Ｃ末端側に２０～５０アミノ酸残基離れた位置にＱＸＸＸＧＸ（Ｆ／Ｗ／Ｙ）モチーフ、Ｃ末端側に５０～１２０アミノ酸残基離れた位置にＱＸＸＸＧＸＷモチーフ、Ｃ末端側に１２０～１７０アミノ酸残基離れた位置にＱＸＸＸＧＸ（Ｆ／Ｗ）モチーフ、及びＣ末端側に１８０～２５０アミノ酸残基離れた位置に前記ＧＸＧＸ（Ｇ／Ａ／Ｐ）モチーフを有し、そしてＤＸＤＤモチーフを基準として、Ｃ末端側に１７０アミノ酸残基以上離れた位置にＱＸＸＸＧＸＷモチーフを有さず、（ｂ）配列番号１で表されるアミノ酸配列との同一性が４０％以上であり、そして（ｃ）８α－ヒドロキシポリポダ－１３，１７，２１－トリエンを基質としてアンブレイン生成活性を示す。
　本発明の第１態様の変異型テトラプレニル－β－クルクメン環化酵素の好ましい態様は、更に（Ａ／Ｓ／Ｇ）ＲＸ（Ｈ／Ｎ）ＸＸＰモチーフのＮ末に隣接するアミノ酸がチロシン以外のアミノ酸に置換している。 [1] Mutant tetraprenyl-β-curcumen cyclase (first aspect)
In the mutant tetraprenyl-β-curcumen cyclase of the first aspect of the present invention, asparagic acid, which is the fourth amino acid residue of the DXDD motif, is replaced with an amino acid other than aspartic acid (D / N). ) G (T / L) (L / F / Y) (Y / F) A mutant tetraprenyl-β-curcumen cyclase in which the 7th amino acid of the SY motif is replaced with an amino acid other than tyrosine. (A) The mutant tetraprenyl-β-curcumen cyclase has a QXXXGX (W / F) motif and an N-terminal side at positions separated from the N-terminal side by 100 amino acid residues or more based on the DXDD motif. The (A / S / G) RX (H / N) XXP motif is located 180 to 250 amino acid residues apart, and the (D / N) G (T) is located 80 to 140 amino acid residues separated from the N-terminal side. / L) (L / F / Y) (Y / F) SY motif, QXXX (G / A / S) X (F / W / Y) motif at a position 10 to 50 amino acid residues away from the N-terminal side, QXXXGX (F / W / Y) motif at a position 20 to 50 amino acid residues away from the C-terminal side, QXXXGXGXW motif at a position 50 to 120 amino acid residues away from the C-terminal side, 120-170 amino acid residue at the C-terminal side It has a QXXXGX (F / W) motif at a position distant from the base and the GXGX (G / A / P) motif at a position 180 to 250 amino acid residues away from the C-terminal side, and C is based on the DXDD motif. It does not have a QXXXGXW motif at a position separated by 170 amino acid residues or more on the terminal side, has (b) 40% or more identity with the amino acid sequence represented by SEQ ID NO: 1, and (c) 8α-hydroxypoly. It shows amino acid-forming activity using poda-13,17,21-triene as a substrate.
In a preferred embodiment of the mutant tetraprenyl-β-curcumen cyclase of the first aspect of the present invention, the amino acid adjacent to the N-terminal of the (A / S / G) RX (H / N) XXP motif is other than tyrosine. It is replaced with an amino acid.
　ここで、各モチーフや配列を定義しているアルファベットはアミノ酸の一文字略号を表しており、「Ｘ」は任意のアミノ酸を意味する。すなわち、ＱＸＸＸＧＸ（Ｗ／Ｆ）モチーフの場合、Ｎ末端側からＣ末端側に向かってグルタミン（Ｑ）、任意のアミノ酸（Ｘ）が３つ、グリシン（Ｇ）、任意のアミノ酸（Ｘ）、更にトリプトファン（Ｗ）又はフェニルアラニン（Ｆ）のいずれか、が配列することを示す。また、「ＤＸＤＤモチーフを基準として、Ｎ末端側１００アミノ酸残基以上離れた位置にＱＸＸＸＧＸ（Ｗ／Ｆ）モチーフを有する」とは、ＤＸＤＤモチーフとＱＸＸＸＧＸ（Ｗ／Ｆ）モチーフの間に１００アミノ酸残基以上存在することを意味するものとし、その他モチーフの特定についても同様である。
　また、ＧＸＧＸ（Ｇ／Ａ／Ｐ）モチーフの４番目のアミノ酸とは、Ｎ末端側から数えて４番目であることを示し、他の配列についても同様である。以下、特に記載のない限り同様である。
　なお、アミノ酸配列の同一性は、比較する配列のアミノ酸残基が一致するように適宜ギャップを挿入して整列させ、一致したアミノ酸残基数を全アミノ酸残基数で除したものを百分率で表したものである。同一性は、周知のプログラム（例えばBLAST、FASTA、CLUSTAL W等）を用いて行うことができる。 Here, the alphabet that defines each motif or sequence represents a one-letter abbreviation for an amino acid, and "X" means an arbitrary amino acid. That is, in the case of the QXXXGX (W / F) motif, glutamine (Q), three arbitrary amino acids (X), glycine (G), any amino acid (X), and further from the N-terminal side to the C-terminal side. Indicates that either tryptophan (W) or phenylalanine (F) is sequenced. In addition, "having a QXXXGX (W / F) motif at a position separated by 100 amino acid residues or more on the N-terminal side with respect to the DXDD motif" means that 100 amino acids remain between the DXDD motif and the QXXXGX (W / F) motif. It means that there are more than one group, and the same applies to the identification of other motifs.
Further, the fourth amino acid of the GXGX (G / A / P) motif indicates that it is the fourth amino acid counting from the N-terminal side, and the same applies to other sequences. Hereinafter, the same applies unless otherwise specified.
The amino acid sequence identity is expressed as a percentage by inserting gaps as appropriate so that the amino acid residues of the sequences to be compared match and aligning them, and dividing the number of matched amino acid residues by the total number of amino acid residues. It was done. Identity can be achieved using well-known programs (eg BLAST, FASTA, CLUSTAL W, etc.).
　本発明の第１態様の変異型テトラプレニル－β－クルクメン環化酵素の好ましい態様としては、前記［３］に記載した変異型テトラプレニル－β－クルクメン環化酵素である。 A preferred embodiment of the mutant tetraprenyl-β-curcumen cyclase according to the first aspect of the present invention is the mutant tetraprenyl-β-curcumen cyclase described in [3] above.
　また、本発明の第１態様の変異型テトラプレニル－β－クルクメン環化酵素の最も好ましい態様としては、変異型テトラプレニル－β－クルクメン環化酵素を構成するポリペプチドが、配列番号２又は３で表されるアミノ酸配列からなるBacillus megaterium由来のポリペプチド、を挙げることができる。すなわち、変異型テトラプレニル－β－クルクメン環化酵素は、ＤＸＤＤモチーフの第４番目のアミノ酸残基であるアスパラギン酸がシステインに置換しており、前記（Ｄ／Ｎ）Ｇ（Ｔ／Ｌ）（Ｌ／Ｆ／Ｙ）（Ｙ／Ｆ）ＳＹモチーフの７番目のアミノ酸残基であるチロシンがアラニンに置換している。また、前記変異型テトラプレニル－β－クルクメン環化酵素は、更に好ましくは、（Ａ／Ｓ／Ｇ）ＲＸ（Ｈ／Ｎ）ＸＸＰモチーフのＮ末に隣接するアミノ酸がチロシンからアラニンに置換している。 In addition, as the most preferable embodiment of the mutant tetraprenyl-β-curcumen cyclase of the first aspect of the present invention, the polypeptide constituting the mutant tetraprenyl-β-curcumen cyclase is SEQ ID NO: 2 or 3. Examples thereof include Bacillus megaterium-derived polypeptides having the amino acid sequence represented by. That is, in the mutant tetraprenyl-β-curcumen cyclase, aspartic acid, which is the fourth amino acid residue of the DXDD motif, is replaced with cysteine, and the above (D / N) G (T / L) ( The 7th amino acid residue of the L / F / Y) (Y / F) SY motif, tyrosine, is replaced with alanine. Further, in the mutant tetraprenyl-β-curcumen cyclase, more preferably, the amino acid adjacent to the N-terminal of the (A / S / G) RX (H / N) XXP motif is replaced with alanine from tyrosine. There is.
（ＤＸＤＤモチーフの第４番目のアミノ酸残基の置換）
　本発明の変異型テトラプレニル－β－クルクメン環化酵素（以下、変異型ＴＣと称することがある）においては、ＤＸＤＤモチーフの第４番目のアミノ酸残基がアスパラギン酸以外のアミノ酸に置換している。アスパラギン酸以外のアミノ酸は、本発明の効果が得られる限りにおいて、限定されるものではなく、アラニン、システイン、グルタミン酸、フェニルアラニン、グリシン、ヒスチジン、イソロイシン、リシン、ロイシン、メチオニン、アスパラギン、プロリン、グルタミン、アルギニン、セリン、トレオニン、バリン、トリプトファン、又はチロシンを挙げることができるが、好ましくはシステインである。
　ＤＸＤＤモチーフの第４番目のアミノ酸残基がアスパラギン酸以外のアミノ酸（特には、システイン又はグリシン）に置換することによって、本発明の変異型テトラプレニル－β－クルクメン環化酵素は、スクアレンから３－デオキシアキレオールＡを生成することができ、そして８α－ヒドロキシポリポダ－１３，１７，２１－トリエンからアンブレインを生成することができる。
　ＤＸＤＤモチーフは、例えば配列番号１で表されるバチルス・メガテリウムのテトラプレニル－β－クルクメン環化酵素のアミノ酸配列のＮ末端側から３７０～３７３番目に存在する。また、配列番号５で表されるバチルス・サブチルスのテトラプレニル－β－クルクメン環化酵素のアミノ酸配列のＮ末端側から３７５～３７８番目に位置する。ＤＸＤＤモチーフのアスパラギン酸は極めて保存性が高く、通常Ｎ末端側から４番目のアミノ酸残基はアスパラギン酸である（図８）。 (Substitution of the 4th amino acid residue of the DXDD motif)
In the mutant tetraprenyl-β-curcumen cyclase of the present invention (hereinafter, may be referred to as mutant TC), the fourth amino acid residue of the DXDD motif is replaced with an amino acid other than aspartic acid. .. Amino acids other than aspartic acid are not limited as long as the effects of the present invention can be obtained, and are not limited to, alanine, cysteine, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, Although arginine, serine, threonine, valine, tryptophan, or tyrosine can be mentioned, cysteine is 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-curcumen cyclase of the present invention is 3-from squalene. Deoxyacireol A can be produced, and umbrellas can be produced from 8α-hydroxypolypoda-13,17,21-triene.
The DXDD motif exists, for example, at the 370th to 373rd positions from the N-terminal side of the amino acid sequence of the tetraprenyl-β-curcumen cyclase of Bacillus megaterium represented by SEQ ID NO: 1. In addition, it is located at the 375th to 378th positions from the N-terminal side of the amino acid sequence of the tetraprenyl-β-curcumen cyclase of Bacillus subtilis represented by SEQ ID NO: 5. The DXDD motif aspartic acid is extremely conserved, and the fourth amino acid residue from the N-terminal side is usually aspartic acid (Fig. 8).
（（Ｄ／Ｎ）Ｇ（Ｔ／Ｌ）（Ｌ／Ｆ／Ｙ）（Ｙ／Ｆ）ＳＹモチーフの７番目のアミノ酸残基の置換）
　本発明の変異型ＴＣにおいては、（Ｄ／Ｎ）Ｇ（Ｔ／Ｌ）（Ｌ／Ｆ／Ｙ）（Ｙ／Ｆ）ＳＹモチーフの７番目のアミノ酸残基がチロシン以外のアミノ酸に置換している。チロシン以外のアミノ酸は、本発明の効果が得られる限りにおいて、限定されるものではなく、アラニン、システイン、グルタミン酸、フェニルアラニン、グリシン、ヒスチジン、イソロイシン、リシン、ロイシン、メチオニン、アスパラギン、プロリン、グルタミン、アルギニン、セリン、トレオニン、バリン、トリプトファン、又はアスパラギン酸を挙げることができるが、バリン、プロリン、グリシン、セリン、又はフェニルアラニンを挙げることができる。特に好ましくは、アラニン又はグリシンであり、より好ましくはアラニンである。
　（Ｄ／Ｎ）Ｇ（Ｔ／Ｌ）（Ｌ／Ｆ／Ｙ）（Ｙ／Ｆ）ＳＹモチーフの７番目のアミノ酸残基がチロシン以外のアミノ酸（特には、アラニン又はグリシン）に置換することによって、本発明の変異型テトラプレニル－β－クルクメン環化酵素は、８α－ヒドロキシポリポダ－１３，１７，２１－トリエンからアンブレインを生成する機能が向上している。
　（Ｄ／Ｎ）Ｇ（Ｔ／Ｌ）（Ｌ／Ｆ／Ｙ）（Ｙ／Ｆ）ＳＹモチーフは、例えば配列番号１で表されるバチルス・メガテリウムのテトラプレニル－β－クルクメン環化酵素のアミノ酸配列のＮ末端側から２５１～２５７番目に存在する。また、配列番号５で表されるバチルス・サブチルスのテトラプレニル－β－クルクメン環化酵素のアミノ酸配列のＮ末端側から２５６～２６２番目に位置する。（Ｄ／Ｎ）Ｇ（Ｔ／Ｌ）（Ｌ／Ｆ／Ｙ）（Ｙ／Ｆ）ＳＹモチーフの７番目のアミノ酸残基は極めて保存性が高く、野生型では基本的にはチロシンである（図８）。本発明は、この保存性の高い特定のアミノ酸を変異させることで、テトラプレニル－β－クルクメン環化酵素が、α－ヒドロキシポリポダ－１３，１７，２１－トリエンを基質としてアンブレイン生成活性を向上できることを見出したものである。 (Replacement 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 7th amino acid residue of the (D / N) G (T / L) (L / F / Y) (Y / F) SY motif is replaced with an amino acid other than tyrosine. There is. Amino acids other than tyrosine are not limited as long as the effects of the present invention can be obtained, and are not limited to alanine, cysteine, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, aspartic acid, proline, glutamine, and arginine. , Serine, threonine, valine, tryptophan, or aspartic acid, but also valine, proline, glycine, serine, or phenylalanine. Particularly preferred is alanine or glycine, more preferably alanine.
By substituting the 7th 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-curcumen cyclase of the present invention has an improved function of producing an umbrella from 8α-hydroxypolypoda-13,17,21-triene.
The (D / N) G (T / L) (L / F / Y) (Y / F) SY motif is, for example, the amino acid of the tetraprenyl-β-curcumen cyclase of Bacillus megaterium represented by SEQ ID NO: 1. It exists 251 to 257th from the N-terminal side of the sequence. In addition, it is located 256 to 262 from the N-terminal side of the amino acid sequence of the tetraprenyl-β-curcumen cyclase of Bacillus subtilis represented by SEQ ID NO: 5. The 7th amino acid residue of the (D / N) G (T / L) (L / F / Y) (Y / F) SY motif is extremely conserved and is basically tyrosine in the wild type ( FIG. 8). In the present invention, by mutating this highly conserved specific amino acid, tetraprenyl-β-curcumen cyclase can exert umbrella-forming activity using α-hydroxypolypoda-13,17,21-triene as a substrate. We have found that it can be improved.
（（Ａ／Ｓ／Ｇ）ＲＸ（Ｈ／Ｎ）ＸＸＰモチーフのＮ末に隣接するアミノ酸残基の置換）
　本発明の変異型ＴＣにおいては、（Ａ／Ｓ／Ｇ）ＲＸ（Ｈ／Ｎ）ＸＸＰモチーフのＮ末に隣接するアミノ酸がチロシン以外のアミノ酸に置換している。チロシン以外のアミノ酸は、本発明の効果が得られる限りにおいて、限定されるものではなく、アラニン、システイン、グルタミン酸、フェニルアラニン、グリシン、ヒスチジン、イソロイシン、リシン、ロイシン、メチオニン、アスパラギン、プロリン、グルタミン、アルギニン、セリン、トレオニン、バリン、トリプトファン、又はアスパラギン酸を挙げることができるが、好ましくは疎水性アミノ酸（グリシン、アラニン、バリン、ロイシン、イソロイシン、メチオニン、プロリン、フェニルアラニン、及びトリプトファン）、セリン、トレオニン又はシステインを挙げることができる。特に好ましくは、アラニン又はグリシンであり、より好ましくはアラニンである。
　（Ａ／Ｓ／Ｇ）ＲＸ（Ｈ／Ｎ）ＸＸＰモチーフのＮ末に隣接するアミノ酸残基がチロシン以外のアミノ酸（特には、アラニン又はグリシン）に置換することによって、本発明の変異型テトラプレニル－β－クルクメン環化酵素は、スクアレンから３－デオキシアキレオールＡを生成する機能、そして８α－ヒドロキシポリポダ－１３，１７，２１－トリエンからアンブレインを生成する機能が向上している。
　（Ａ／Ｓ／Ｇ）ＲＸ（Ｈ／Ｎ）ＸＸＰモチーフは、例えば配列番号１で表されるバチルス・メガテリウムのテトラプレニル－β－クルクメン環化酵素のアミノ酸配列のＮ末端側から１６８～１７４番目に存在する。また、配列番号５で表されるバチルス・サブチルスのテトラプレニル－β－クルクメン環化酵素のアミノ酸配列のＮ末端側から１７０～１７６番目に位置する。（Ａ／Ｓ／Ｇ）ＲＸ（Ｈ／Ｎ）ＸＸＰモチーフのＮ末に隣接するアミノ酸残基は極めて保存性が高く、野生型では基本的にはチロシンである（図８）。本発明は、この保存性の高い特定のアミノ酸を変異させることで、本発明の変異型テトラプレニル－β－クルクメン環化酵素は、スクアレンから３－デオキシアキレオールＡを生成することができ、そして８α－ヒドロキシポリポダ－１３，１７，２１－トリエンからアンブレインを生成することができる。 (Substitution of amino acid residue adjacent to N-terminal of (A / S / G) RX (H / N) XXP motif)
In the mutant TC of the present invention, the amino acid adjacent to the N-terminal of the (A / S / G) RX (H / N) XXP motif is replaced with an amino acid other than tyrosine. Amino acids other than tyrosine are not limited as long as the effects of the present invention can be obtained, and are not limited to alanine, cysteine, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, aspartic acid, proline, glutamine, and arginine. , Serine, threonine, valine, tryptophan, or aspartic acid, but preferably hydrophobic amino acids (glycine, alanine, valine, leucine, isoleucine, methionine, proline, phenylalanine, and tryptophan), serine, alanine or cysteine. Can be mentioned. Particularly preferred is alanine or glycine, more preferably alanine.
By substituting an amino acid residue adjacent to the N-terminal of the (A / S / G) RX (H / N) XXP motif with an amino acid other than tyrosine (particularly, alanine or glycine), the mutant tetraprenyl of the present invention The -β-curcumen cyclase has improved functions for producing 3-deoxyacireol A from squalene and for producing umbrellas from 8α-hydroxypolypoda-13,17,21-triene.
The (A / S / G) RX (H / N) XXP motif is, for example, the 168th to 174th positions from the N-terminal side of the amino acid sequence of the tetraprenyl-β-curcumen cyclase of Bacillus megaterium represented by SEQ ID NO: 1. Exists in. In addition, it is located 170 to 176th from the N-terminal side of the amino acid sequence of the tetraprenyl-β-curcumen cyclase of Bacillus subtilis represented by SEQ ID NO: 5. The amino acid residue adjacent to the N-terminal of the (A / S / G) RX (H / N) XXP motif is extremely conserved and is basically tyrosine in the wild type (Fig. 8). By mutating this highly conserved specific amino acid, the present invention allows the mutant tetraprenyl-β-curcumen cyclase of the present invention to produce 3-deoxyacireol A from squalene, and Umbranes can be produced from 8α-hydroxypolypoda-13,17,21-triene.
　図７にバチルス・メガテリウムの野生型テトラプレニル－β－クルクメン環化酵素、３７３番目のアスパラギン酸がシステインに置換し、そして２５７番目のチロシンがアラニンに置換した変異型テトラプレニル－β－クルクメン環化酵素（配列番号２）、及び３７３番目のアスパラギン酸がシステインに置換し、そして２５７番目のチロシンがアラニンに置換し、そして１６７番目のチロシンがアラニンに置換した変異型テトラプレニル－β－クルクメン環化酵素（配列番号３）のアミノ酸配列を示す。 FIG. 7 shows the wild tetraprenyl-β-curcumen cyclase of bacillus megaterium, mutant tetraprenyl-β-curcumen cyclization in which aspartic acid at position 373 was replaced with cysteine and tyrosine at position 257 was replaced with alanine. Mutant tetraprenyl-β-curcumen cyclization in which the enzyme (SEQ ID NO: 2) and aspartic acid at position 373 were replaced with cysteine, and tyrosine at position 257 was replaced with alanine, and tyrosine at position 167 was replaced with alanine. The amino acid sequence of the enzyme (SEQ ID NO: 3) is shown.
　本発明の変異型テトラプレニル－β－クルクメン環化酵素の由来は、特に限定されるものではなく、全てのテトラプレニル－β－クルクメン環化酵素を用いることができる。すなわち、テトラプレニル－β－クルクメン環化酵素は、ＱＸＸＸＧＸ（Ｗ／Ｆ）モチーフ、（Ａ／Ｓ／Ｇ）ＲＸ（Ｈ／Ｎ）ＸＸＰモチーフ、（Ｄ／Ｎ）Ｇ（Ｔ／Ｌ）（Ｌ／Ｆ／Ｙ）（Ｙ／Ｆ）ＳＹモチーフ、ＱＸＸＸＸ（Ｇ／Ａ／Ｓ）Ｘ（Ｆ／Ｗ／Ｙ）モチーフ、ＤＸＤＤモチーフ、ＱＸＸＸＧＸ（Ｆ／Ｗ／Ｙ）モチーフ、ＱＸＸＸＧＸＷモチーフ、ＱＸＸＸＧＸ（Ｆ／Ｗ）モチーフ、及びＧＸＧＸ（Ｇ／Ａ／Ｐ）モチーフの９つのモチーフを有するものであり、このようなテトラプレニル－β－クルクメン環化酵素を限定することなく、利用できる。例えば、バチルス・サブチルスとバチルス・メガテリウムとのポリペプチドのアミノ酸配列の同一性は５０％程度であるが、本発明の特徴を有することにより、いずれの酵素も８α－ヒドロキシポリポダ－１３，１７，２１－トリエンからアンブレインを生成することができる。なお、配列番号１に記載のアミノ酸配列は、バチルス・メガテリウムの野生型テトラプレニル－β－クルクメン環化酵素のアミノ酸配列であり、配列番号５に記載のアミノ酸配列はバチルス・サブチルスの野生型テトラプレニル－β－クルクメン環化酵素のアミノ酸配列である。 The origin of the mutant tetraprenyl-β-curcumen cyclase of the present invention is not particularly limited, and all tetraprenyl-β-curcumen cyclases can be used. That is, the tetraprenyl-β-curcumen cyclase has a QXXXGX (W / F) motif, an (A / S / G) RX (H / N) XXP motif, and (D / N) G (T / L) (L). / F / Y) (Y / F) SY motif, QXXXX (G / A / S) X (F / W / Y) motif, DXDD motif, QXXXGX (F / W / Y) motif, QXXXGXW motif, QXXXGX (F) It has nine motifs, a / W) motif and a GXGX (G / A / P) motif, and such tetraprenyl-β-curcumen cyclase can be used without limitation. For example, the amino acid sequences of the polypeptides of Bacillus subtilis and Bacillus megaterium are about 50% identical, but due to the characteristics of the present invention, all the enzymes have 8α-hydroxypolypoda-13,17, An umbrella can be generated from 21-triene. The amino acid sequence shown in SEQ ID NO: 1 is the amino acid sequence of the wild-type tetraprenyl-β-curcumen cyclase of Bacillus megaterium, and the amino acid sequence shown in SEQ ID NO: 5 is the wild-type tetraprenyl of Bacillus subtilis. It is an amino acid sequence of -β-curcumen cyclase.
（第２態様）
　本発明の第１態様の変異型テトラプレニル－β－クルクメン環化酵素は、ＤＸＤＤモチーフの第４番目のアミノ酸残基であるアスパラギン酸がアスパラギン酸以外のアミノ酸に置換しており、（Ａ／Ｓ／Ｇ）ＲＸ（Ｈ／Ｎ）ＸＸＰモチーフのＮ末に隣接するアミノ酸がチロシン以外のアミノ酸に置換しており、ＧＸＧＸ（Ｇ／Ａ／Ｐ）モチーフの４番目のアミノ酸がロイシン以外のアミノ酸に置換している変異型テトラプレニル－β－クルクメン環化酵素であって、（ａ）前記変異型テトラプレニル－β－クルクメン環化酵素は、前記ＤＸＤＤモチーフを基準として、Ｎ末端側に１００アミノ酸残基以上離れた位置にＱＸＸＸＧＸ（Ｗ／Ｆ）モチーフ、Ｎ末端側に１８０～２５０アミノ酸残基離れた位置に前記（Ａ／Ｓ／Ｇ）ＲＸ（Ｈ／Ｎ）ＸＸＰモチーフ、Ｎ末端側に８０～１４０アミノ酸残基離れた位置に前記（Ｄ／Ｎ）Ｇ（Ｔ／Ｌ）（Ｌ／Ｆ／Ｙ）（Ｙ／Ｆ）ＳＹモチーフ、Ｎ末端側に１０～５０アミノ酸残基離れた位置にＱＸＸＸＸ（Ｇ／Ａ／Ｓ）Ｘ（Ｆ／Ｗ／Ｙ）モチーフ、Ｃ末端側に２０～５０アミノ酸残基離れた位置にＱＸＸＸＧＸ（Ｆ／Ｗ／Ｙ）モチーフ、Ｃ末端側に５０～１２０アミノ酸残基離れた位置にＱＸＸＸＧＸＷモチーフ、Ｃ末端側に１２０～１７０アミノ酸残基離れた位置にＱＸＸＸＧＸ（Ｆ／Ｗ）モチーフ、及びＣ末端側に１８０～２５０アミノ酸残基離れた位置に前記ＧＸＧＸ（Ｇ／Ａ／Ｐ）モチーフを有し、そしてＤＸＤＤモチーフを基準として、Ｃ末端側に１７０アミノ酸残基以上離れた位置にＱＸＸＸＧＸＷモチーフを有さず、（ｂ）配列番号１で表されるアミノ酸配列との同一性が４０％以上であり、そして（ｃ）８α－ヒドロキシポリポダ－１３，１７，２１－トリエンを基質としてアンブレイン生成活性を示す。
　なお、各モチーフ及び配列のアルファベット等の定義は、第１態様と同様である。 (Second aspect)
In the mutant tetraprenyl-β-curcumen cyclase of the first aspect of the present invention, asparagic acid, which is the fourth amino acid residue of the DXDD motif, is replaced with an amino acid other than aspartic acid (A / S). / G) The amino acid adjacent to the N-terminal of the RX (H / N) XXP motif is replaced with an amino acid other than tyrosine, and the fourth amino acid of the GXGX (G / A / P) motif is replaced with an amino acid other than leucine. The mutant tetraprenyl-β-curcumen cyclase (a) the mutant tetraprenyl-β-curcumen cyclase has 100 amino acid residues on the N-terminal side based on the DXDD motif. The QXXXGX (W / F) motif is located at a distance of 180 to 250 amino acids on the N-terminal side, and the (A / S / G) RX (H / N) XXP motif is located at a distance of 180 to 250 amino acid residues. The (D / N) G (T / L) (L / F / Y) (Y / F) SY motif is located 140 amino acid residues away, and QXXXXX is located 10 to 50 amino acid residues away from the N-terminal side. (G / A / S) X (F / W / Y) motif, QXXXGX (F / W / Y) motif at a position 20 to 50 amino acid residues away from the C terminal side, 50 to 120 amino acid residue on the C terminal side The QXXXGXW motif is located at a distance from the base, the QXXXGX (F / W) motif is located at a position separated by 120 to 170 amino acid residues on the C-terminal side, and the GXGX (G /) is located at a position 180 to 250 amino acid residues away from the C-terminal side. It has an A / P) motif, and does not have a QXXXGXW motif at a position separated by 170 amino acid residues or more on the C-terminal side based on the DXDD motif, and (b) with the amino acid sequence represented by SEQ ID NO: 1. The identity is 40% or more, and (c) shows umbrella-forming activity using 8α-hydroxypolypoda-13,17,21-triene as a substrate.
The definitions of the alphabet and the like of each motif and arrangement are the same as those in the first aspect.
　本発明の第２態様の変異型テトラプレニル－β－クルクメン環化酵素の好ましい態様としては、前記［６］に記載した変異型テトラプレニル－β－クルクメン環化酵素である。 A preferred embodiment of the mutant tetraprenyl-β-curcumen cyclase according to the second aspect of the present invention is the mutant tetraprenyl-β-curcumen cyclase described in [6] above.
　また、本発明の第２態様の変異型テトラプレニル－β－クルクメン環化酵素の最も好ましい態様としては、変異型テトラプレニル－β－クルクメン環化酵素を構成するポリペプチドが、配列番号４で表されるアミノ酸配列からなるBacillus megaterium由来のポリペプチド、を挙げることができる。すなわち、変異型テトラプレニル－β－クルクメン環化酵素は、ＤＸＤＤモチーフの第４番目のアミノ酸残基であるアスパラギン酸がシステインに置換しており、前記（Ａ／Ｓ／Ｇ）ＲＸ（Ｈ／Ｎ）ＸＸＰモチーフのＮ末に隣接するアミノ酸がチロシンからアラニンに置換しており、そしてＧＸＧＸ（Ｇ／Ａ／Ｐ）モチーフの４番目のアミノ酸がロイシンからアラニンに置換している。 In addition, as the most preferable embodiment of the mutant tetraprenyl-β-curcumen cyclase of the second aspect of the present invention, the polypeptide constituting the mutant tetraprenyl-β-curcumen cyclase is represented by SEQ ID NO: 4. A polypeptide derived from Bacillus megaterium, which comprises the amino acid sequence to be used, can be mentioned. That is, in the mutant tetraprenyl-β-curcumen cyclase, aspartic acid, which is the fourth amino acid residue of the DXDD motif, is replaced with cysteine, and the above (A / S / G) RX (H / N) ) The amino acid adjacent to the N-terminal of the XXP motif is substituting tyrosine for alanine, and the fourth amino acid of the GXGX (G / A / P) motif is substituting leucine for alanine.
　「ＤＸＤＤモチーフの第４番目のアミノ酸残基の置換」及び「（Ａ／Ｓ／Ｇ）ＲＸ（Ｈ／Ｎ）ＸＸＰモチーフのＮ末に隣接するアミノ酸残基の置換」は、前記第１態様と同様である。 "Substitution of the fourth amino acid residue of the DXDD motif" and "substitution of the amino acid residue adjacent to the N-terminal of the (A / S / G) RX (H / N) XXP motif" are the same as those of the first aspect. The same is true.
（ＧＸＧＸ（Ｇ／Ａ／Ｐ）モチーフの第４番目のアミノ酸残基の置換）
　本発明の第２態様の変異型テトラプレニル－β－クルクメン環化酵素においては、ＧＸＧＸ（Ｇ／Ａ／Ｐ）モチーフの第４番目のアミノ酸がロイシン以外のアミノ酸に置換している場合、ロイシン以外のアミノ酸は、本発明の効果が得られる限りにおいて、限定されるものではなく、アラニン、システイン、グルタミン酸、フェニルアラニン、グリシン、ヒスチジン、イソロイシン、リシン、メチオニン、アスパラギン、プロリン、グルタミン、アルギニン、セリン、トレオニン、バリン、トリプトファン、チロシン、又はアスパラギン酸を挙げることができるが、好ましくはアラニン、フェニルアラニン、バリン、メチオニン、イソロイシン、トリプトファンを挙げることができる。特に好ましくは、アラニン又はフェニルアラニンであり、より好ましくはアラニンである。
　ＧＸＧＸ（Ｇ／Ａ／Ｐ）モチーフの第４番目のアミノ酸残基がロイシン以外のアミノ酸（特には、アラニン又はフェニルアラニン）に置換することによって、本発明の変異型テトラプレニル－β－クルクメン環化酵素は、８α－ヒドロキシポリポダ－１３，１７，２１－トリエンからアンブレインを生成する機能が向上している。 (Substitution of the 4th amino acid residue of the GXGX (G / A / P) motif)
In the mutant tetraprenyl-β-curcumen cyclase of the second aspect of the present invention, when the fourth amino acid of the GXGX (G / A / P) motif is replaced with an amino acid other than leucine, other than leucine. Amino acids are not limited as long as the effects of the present invention can be obtained, but are not limited to alanine, cysteine, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, methionine, aspartic acid, proline, glutamine, arginine, serine, and threonine. , Valin, tryptophan, tyrosine, or aspartic acid, preferably alanine, phenylalanine, valine, methionine, isoleucine, tryptophan. Particularly preferred is alanine or phenylalanine, more preferably alanine.
The mutant tetraprenyl-β-curcumen cyclase of the present invention 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). Has improved ability to generate umbrellas from 8α-hydroxypolypoda-13,17,21-triene.
　図７にバチルス・メガテリウム由来のテトラプレニル－β－クルクメン環化酵素において、３７３番目のアスパラギン酸がシステインに置換し、１６７番目のチロシンがアラニンに置換し、そして５９６番目のロイシンがアラニンに置換した変異型テトラプレニル－β－クルクメン環化酵素のアミノ酸配列を示す（配列番号４）。 In FIG. 7, in the tetraprenyl-β-curcumen cyclase derived from Bacillus megaterium, aspartic acid at position 373 was replaced with cysteine, tyrosine at position 167 was replaced with alanine, and leucine at position 596 was replaced with alanine. The amino acid sequence of the mutant tetraprenyl-β-curcumen cyclase is shown (SEQ ID NO: 4).
（１又は数個のアミノ酸が欠失、置換、挿入、及び／又は付加されたアミノ酸配列）
　本発明の変異型テトラプレニル－β－クルクメン環化酵素のポリペプチドは、配列番号１のアミノ酸配列において、１又は数個のアミノ酸が欠失、置換、挿入、及び／又は付加されたアミノ酸配列からなるポリペプチドであってもよい。本発明の変異型テトラプレニル－β－クルクメン環化酵素のポリペプチドは、８α－ヒドロキシポリポダ－１３，１７，２１－トリエンを基質として、アンブレイン生成活性を示すポリペプチドである。すなわち、８α－ヒドロキシポリポダ－１３，１７，２１－トリエンを基質として、アンブレイン生成活性を示さないポリペプチドは、本発明の変異型テトラプレニル－β－クルクメン環化酵素のポリペプチドに含まれない。本明細書において、「１又は数個のアミノ酸が欠失、置換、挿入、及び／又は付加されたアミノ酸配列」とは、アミノ酸の置換等により改変がなされたことを意味する。アミノ酸の改変の個数は、例えば１～３３０個、１～３００個、１～２５０個、１～２００個、１～１５０個、１～１００個、又は１～５０個であることができ、好ましくは１～３０個、より好ましくは１～１０個、更に好ましくは１～５個、最も好ましくは１～２個である。本発明に用いることのできる変異ペプチドの改変アミノ酸配列の例は、好ましくは、そのアミノ酸が、１又は数個（好ましくは、１、２、３又は４個）の保存的置換を有するアミノ酸配列であることができる。 (Amino acid sequence in which one or several amino acids are deleted, substituted, inserted, and / or added)
The polypeptide of the mutant tetraprenyl-β-curcumen cyclase of the present invention is derived from an amino acid sequence in which one or several amino acids are deleted, substituted, inserted, and / or added in the amino acid sequence of SEQ ID NO: 1. It may be a polypeptide. The polypeptide of the mutant tetraprenyl-β-curcumen cyclase of the present invention is a polypeptide exhibiting umbrella-forming activity using 8α-hydroxypolypoda-13,17,21-triene as a substrate. That is, a polypeptide that uses 8α-hydroxypolypoda-13,17,21-triene as a substrate and does not exhibit umbrella-forming activity is included in the polypeptide of the mutant tetraprenyl-β-curcumen cyclase of the present invention. No. In the present specification, "an amino acid sequence in which one or several amino acids are deleted, substituted, inserted, and / or added" means that the amino acid has been modified by 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, which is preferable. Is 1 to 30, more preferably 1 to 10, still 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 acid has one or several (preferably 1, 2, 3 or 4) conservative substitutions. There can be.
（アミノ酸配列との同一性が４０％以上であるアミノ酸配列）
　本発明の変異型テトラプレニル－β－クルクメン環化酵素のポリペプチドは、配列番号１のアミノ酸配列に対してアミノ酸配列との同一性が４０％以上であるアミノ酸配列からなるポリペプチドであってもよい。本発明の変異型テトラプレニル－β－クルクメン環化酵素のポリペプチドは、８α－ヒドロキシポリポダ－１３，１７，２１－トリエンを基質として、アンブレイン生成活性を示すポリペプチドである。すなわち、８α－ヒドロキシポリポダ－１３，１７，２１－トリエンを基質として、アンブレイン生成活性を示さないポリペプチドは、本発明の変異型テトラプレニル－β－クルクメン環化酵素のポリペプチドに含まれない。より好ましくは該同一性が４５％以上であるアミノ酸配列、より好ましくは５０％以上であるアミノ酸配列、より好ましくは６０％以上であるアミノ酸配列、より好ましくは７０％以上であるアミノ酸配列、より好ましくは８０％以上であるアミノ酸配列、より好ましくは９０％以上であるアミノ酸配列、最も好ましくは該同一性が９５％以上であるアミノ酸配列からなるポリペプチドであって、且つ８α－ヒドロキシポリポダ－１３，１７，２１－トリエンを基質としてアンブレイン生成活性を示すポリペプチドからなる、又は含む、変異型テトラプレニル－β－クルクメン環化酵素である。 (Amino acid sequence having 40% or more identity with the amino acid sequence)
The polypeptide of the mutant tetraprenyl-β-curcumen cyclase of the present invention may be a polypeptide consisting of an amino acid sequence having an amino acid sequence identity of 40% or more with respect to the amino acid sequence of SEQ ID NO: 1. good. The polypeptide of the mutant tetraprenyl-β-curcumen cyclase of the present invention is a polypeptide exhibiting umbrella-forming activity using 8α-hydroxypolypoda-13,17,21-triene as a substrate. That is, a polypeptide that uses 8α-hydroxypolypoda-13,17,21-triene as a substrate and does not exhibit umbrella-forming activity is included in the polypeptide of the mutant tetraprenyl-β-curcumen cyclase of the present invention. No. More preferably, an amino acid sequence having the same identity of 45% or more, more preferably 50% or more, a more preferably 60% or more amino acid sequence, more preferably 70% or more, a more preferable. Is a polypeptide consisting of an amino acid sequence of 80% or more, more preferably an amino acid sequence of 90% or more, most preferably an amino acid sequence having the sameness of 95% or more, and 8α-hydroxypolypoda-13. , 17,21-A mutant tetraprenyl-β-curcumen cyclase comprising or containing a polypeptide exhibiting umbrella-forming activity using triene as a substrate.
　前記配列番号１のアミノ酸配列において「１又は数個のアミノ酸が欠失、置換、挿入、及び／又は付加されたアミノ酸配列」又は「同一性が４０％以上であるアミノ酸配列」は、配列番号１のアミノ酸配列が置換されたものであるが、このアミノ酸配列の置換は、本発明の変異型テトラプレニル－β－クルクメン環化酵素の機能を維持する保存的置換である。換言すると「保存的置換」とは、本発明の変異型テトラプレニル－β－クルクメン環化酵素の優れた効果が失われない置換を意味する。すなわち、前記挿入、置換、又は欠失、若しくは付加された場合であっても、８α－ヒドロキシポリポダ－１３，１７，２１－トリエンを基質としてアンブレイン生成活性を向上できる置換である。具体的には、アミノ酸残基を、別の化学的に類似したアミノ酸残基で置き換えることを意味する。例えば、ある疎水性残基を別の疎水性残基によって置換する場合、ある極性残基を同じ電荷を有する別の極性残基によって置換する場合などが挙げられる。このような置換を行うことでできる機能的に類似のアミノ酸は、アミノ酸毎に当該技術分野において公知である。非極性（疎水性）アミノ酸としては、例えば、アラニン、バリン、イソロイシン、ロイシン、プロリン、トリプトファン、フェニルアラニン、メチオニンなどが挙げられる。極性（中性）アミノ酸としては、例えば、グリシン、セリン、トレオニン、チロシン、グルタミン、アスパラギン、システインなどが挙げられる。正電荷をもつ（塩基性）アミノ酸としては、アルギニン、ヒスチジン、リジンなどが挙げられる。また、負電荷をもつ（酸性）アミノ酸としては、アスパラギン酸、グルタミン酸などが挙げられる。 In the amino acid sequence of SEQ ID NO: 1, "an amino acid sequence in which one or several amino acids are deleted, substituted, inserted, and / or added" or "an amino acid sequence having 40% or more identity" is shown in SEQ ID NO: 1. The amino acid sequence of the above is substituted, and the substitution of this amino acid sequence is a conservative substitution that maintains the function of the mutant tetraprenyl-β-curcumen cyclase of the present invention. In other words, "conservative substitution" means a substitution in which the excellent effect of the mutant tetraprenyl-β-curcumen cyclase of the present invention is not lost. That is, even when the insertion, substitution, deletion, or addition is performed, the substitution can improve the umbrella-forming activity by using 8α-hydroxypolypoda-13,17,21-triene as a substrate. Specifically, it means replacing an amino acid residue with another chemically similar amino acid residue. For example, there is a case where one hydrophobic residue is replaced with another hydrophobic residue, a case where one polar residue is replaced with another polar residue having the same charge, and the like. Functionally similar amino acids that can be made by making such substitutions are known in the art for each amino acid. Examples of non-polar (hydrophobic) amino acids include alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, methionine and the like. 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.
　本発明の変異型テトラプレニル－β－クルクメン環化酵素におけるＤＸＤＤモチーフの第４番目のアミノ酸残基であるアスパラギン酸をアスパラギン酸以外のアミノ酸へ変異（置換）させること、（Ｄ／Ｎ）Ｇ（Ｔ／Ｌ）（Ｌ／Ｆ／Ｙ）（Ｙ／Ｆ）ＳＹモチーフの７番目のアミノ酸をチロシン以外のアミノ酸に置換させること、（Ａ／Ｓ／Ｇ）ＲＸ（Ｈ／Ｎ）ＸＸＰモチーフのＮ末に隣接するアミノ酸がチロシン以外のアミノ酸に置換させること、又はＧＸＧＸ（Ｇ／Ａ／Ｐ）モチーフの４番目のアミノ酸をロイシン以外のアミノ酸に置換させることは、８α－ヒドロキシポリポダ－１３，１７，２１－トリエンを基質としてアンブレインを生成する活性を付与するための、積極的な置換（変異）であるが、前記保存的置換は８α－ヒドロキシポリポダ－１３，１７，２１－トリエンを基質としてアンブレインを生成する活性を維持するための置換であり、当業者であれば容易に実施することのできるものである。 (D / N) G (D / N) G (D / N) G (D / N) G Replacing the 7th amino acid of the T / L) (L / F / Y) (Y / F) SY motif with an amino acid other than tyrosine, N of the (A / S / G) RX (H / N) XPP motif Replacing the amino acid adjacent to the end with an amino acid other than tyrosine, or substituting the fourth amino acid of the GXGX (G / A / P) motif with an amino acid other than leucine is 8α-hydroxypolypoda-13,17. , 21-Triene is a positive substitution (mutation) for imparting the activity of producing an umbrella, but the conservative substitution uses 8α-hydroxypolypoda-13,17,21-triene as a substrate. It is a substitution for maintaining the activity of producing an amino acid, which can be easily carried out by those skilled in the art.
　本発明の変異型テトラプレニル－β－クルクメン環化酵素は、公知の遺伝子組換え技術などを用いて取得することができる。例えば、バチルス・メガテリウムの染色体ＤＮＡを取得し、適当なプライマーを用いて、テトラプレニル－β－クルクメン環化酵素を、ＰＣＲなどによって増幅する。得られた遺伝子を、適当なベクターに組み込み、遺伝子配列を決定する。そして上記の変異を導入することによって、本発明の変異型テトラプレニル－β－クルクメン環化酵素をコードする遺伝子を取得することができる。その遺伝子を、酵母等の宿主に組み込み、発現させることによって、本発明の変異型テトラプレニル－β－クルクメン環化酵素を得ることが可能である。
　またテトラプレニル－β－クルクメン環化酵素は、バチルス・メガテリウムの他に、バチルス属等の細菌に存在することが知られており、バチルス・サブチリス由来酵素（アクセッション番号：ＡＢ６１８２０６）、バチルス・リケニフォルミス由来酵素（アクセッション番号：ＡＡＵ４１１３４）等を取得することも可能である。 The mutant tetraprenyl-β-curcumen cyclase of the present invention can be obtained by using a known gene recombination technique or the like. For example, the chromosomal DNA of Bacillus megaterium is obtained, and the tetraprenyl-β-curcumen cyclase is amplified by PCR or the like using an appropriate primer. The obtained gene is incorporated into an appropriate vector to determine the gene sequence. Then, by introducing the above mutation, a gene encoding the mutant tetraprenyl-β-curcumen cyclase of the present invention can be obtained. By incorporating the gene into a host such as yeast and expressing it, the mutant tetraprenyl-β-curcumen cyclase of the present invention can be obtained.
In addition to Bacillus megaterium, tetraprenyl-β-curcumen cyclase is known to exist in bacteria such as the genus Bacillus. It is also possible to obtain the derived enzyme (accession number: AAU41134) and the like.
　また、本発明の変異型テトラプレニル－β－クルクメン環化酵素をコードする遺伝子は、公知の合成遺伝子の合成方法、例えば、Ｋｈｏｒａｎａらの方法（Ｇｕｐｔａ　ｅｔ　ａｌ．，１９６８）、Ｎａｒａｎｇらの方法（Ｓｃａｒｐｕｌｌａ　ｅｔ　ａｌ．，１９８２）、又はＲｏｓｓｉらの方法（Ｒｏｓｓｉ　ｅｔ　ａｌ．，１９８２）によって合成することも可能である。そして、合成された遺伝子を発現させることにより、本発明の変異型テトラプレニル－β－クルクメン環化酵素を得ることができる。 Further, the gene encoding the mutant tetraprenyl-β-curcumen cyclase of the present invention is a method for synthesizing a known synthetic gene, for example, the method of Khorana et al. (Gupta et al., 1968), the method of Narang et al. It is also possible to synthesize by the method of Scarpulla et al., 1982) or Rossi et al. (Rossi et al., 1982). Then, by expressing the synthesized gene, the mutant tetraprenyl-β-curcumen cyclase of the present invention can be obtained.
（作用）
　従来、スクアレンからアンブレインを生成する場合、スクアレンを変異型スクアレン－ホペン環化酵素（以下、変異型ＳＨＣと称することがある）によって、３－デオキシアキレオールＡに変換し、そして３－デオキシアキレオールＡを野生型のテトラプレニル－β－クルクメン環化酵素によって、アンブレインに変換することによって製造されていた（特許文献２）。本発明者らは、図１に示すように、変異型テトラプレニル－β－クルクメン環化酵素（Ｄ３７３Ｃ）を用いることによって、スクアレンから、１個の酵素を使用した２段階の反応で、アンブレインを得る方法を見出し、提案している（特許文献３）。この場合、単環性の３－デオキシアキレオールＡを中間体とする経路（以下、単環性経路と称することがある）と、８α－ヒドロキシポリポダ－１３，１７，２１－トリエンを中間体とする経路（以下、２環性経路と称することがある）によって、アンブレインが製造される。
　本発明の変異型テトラプレニル－β－クルクメン環化酵素は、図２示すように、特に２環性経路の８α－ヒドロキシポリポダ－１３，１７，２１－トリエンを基質として、アンブレインを産生する活性が優れている。また、副産物であるオノセロイドの生成が少ない。 (Action)
Conventionally, when producing ambrein from squalene, squalene is converted to 3-deoxyachilleol A by a mutant squalene-hopen cyclase (hereinafter sometimes referred to as mutant SHC), and then 3-deoxyachille. It was produced by converting all-A to ambrein with a wild-type tetraprenyl-β-squalene cyclase (Patent Document 2). As shown in FIG. 1, the present inventors used a mutant tetraprenyl-β-curcumen cyclase (D373C) to convert squalene into an umbrella in a two-step reaction using one enzyme. Has been found and proposed (Patent Document 3). In this case, a pathway with monocyclic 3-deoxyacireol A as an intermediate (hereinafter, may be referred to as a monocyclic pathway) and 8α-hydroxypolypoda-13,17,21-triene are intermediates. The umbrella is produced by a route (hereinafter, may be referred to as a bicyclic route).
As shown in FIG. 2, the mutant tetraprenyl-β-curcumen cyclase of the present invention produces an umbrella, particularly using 8α-hydroxypolypoda-13,17,21-triene in the bicyclic pathway as a substrate. Excellent activity. In addition, the production of onoseroid, which is a by-product, is small.
［２］ポリヌクレオチド
　本発明のポリヌクレオチドは、本発明のテトラプレニル－β－クルクメン環化酵素をコードするポリヌクレオチドである限りにおいて、特に限定されるものではない。例えば、配列番号２で表されるポリペプチドをコードするポリヌクレオチド（配列番号１２）、配列番号３で表されるポリペプチドをコードするポリヌクレオチド（配列番号１３）、配列番号４で表されるポリペプチドをコードするポリヌクレオチド（配列番号１４）が挙げられる。
　更には、配列番号１２、１３、又は１４で表される塩基配列からなるポリヌクレオチドとストリンジェントな条件下でハイブリダイズし、しかも、８α－ヒドロキシポリポダ－１３，１７，２１－トリエンからアンブレインを生成する活性を有するポリヌクレオチドを挙げることができる。なお、本明細書における用語「ポリヌクレオチド」には、ＤＮＡ及びＲＮＡの両方が含まれる。
　また、本発明のポリヌクレオチドは、それを導入する微生物又は宿主細胞に合わせて、最適なコドンの塩基配列に変更することが好ましい。 [2] Polynucleotide The polynucleotide of the present invention is not particularly limited as long as it is a polynucleotide encoding the tetraprenyl-β-curcumen cyclase of the present invention. For example, the polynucleotide encoding the polypeptide represented by SEQ ID NO: 2 (SEQ ID NO: 12), the polynucleotide encoding the polypeptide represented by SEQ ID NO: 3 (SEQ ID NO: 13), and the polynucleotide represented by SEQ ID NO: 4. A polynucleotide encoding a peptide (SEQ ID NO: 14) can be mentioned.
Furthermore, it hybridizes with a polynucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 12, 13, or 14 under stringent conditions, and further, it is an umbrella from 8α-hydroxypolypoda-13,17,21-triene. Can be mentioned as a polynucleotide having an activity of producing. The term "polynucleotide" as used herein includes both DNA and RNA.
In addition, the polynucleotide of the present invention is preferably changed to the optimum codon base sequence according to the microorganism or host cell into which it is introduced.
［３］微生物
　本発明の微生物は、本発明のポリヌクレオチドを有する微生物である。すなわち、本発明のポリヌクレオチドを細胞内に含む限りにおいて特に限定されるものではないが、例えば、大腸菌、枯草菌、ブレビバチルス属菌、放線菌、パン酵母、麹菌、又はアカパンカビを挙げることができる。 [3] Microorganism The microorganism of the present invention is a microorganism having the polynucleotide of the present invention. That is, it is not particularly limited as long as the polynucleotide of the present invention is contained in the cell, and examples thereof include Escherichia coli, Bacillus subtilis, Brevibacillus spp., Radical fungus, baker's yeast, aspergillus, and Neurospora crassa. ..
［４］ベクター
　本発明のベクターは、上記変異型テトラプレニル－β－クルクメン環化酵素をコードするポリヌクレオチドを持つＤＮＡを含むベクターである。すなわち、本発明のベクターは、本発明による前記ポリヌクレオチドを含む限り、特に限定されるものではなく、例えば、用いる宿主細胞に応じて適宜選択した公知の発現ベクターに、本発明による前記ポリヌクレオチドを挿入することにより得られるベクターを挙げることができる。
　発現ベクターとしては、宿主である大腸菌、パン酵母等において自立複製可能ないしは染色体中への組込みが可能で、外来タンパク質の発現効率の高いものが好ましい。前記ポリヌクレオチドを発現させるための発現ベクターは、微生物中で自立複製可能であると同時に、プロモーター、リボソーム結合配列、前記ＤＮＡ及び転写終結配列より構成された組換えベクターであることが好ましい。また、プロモーターを制御する遺伝子が含まれていてもよい。 [4] Vector The vector of the present invention is a vector containing a DNA having a polynucleotide encoding the above-mentioned mutant tetraprenyl-β-curcumen cyclase. That is, the vector of the present invention is not particularly limited as long as it contains the polynucleotide of the present invention. For example, the polynucleotide of the present invention is used in a known expression vector appropriately selected according to the host cell used. The vector obtained by insertion can be mentioned.
As the expression vector, a vector capable of autonomous replication or integration into a chromosome in a host such as Escherichia coli or baker's yeast and having a high expression efficiency of a foreign protein is preferable. The expression vector for expressing the polynucleotide is preferably a recombinant vector composed of a promoter, a ribosome-binding sequence, the DNA, and a transcription termination sequence while being able to replicate autonomously in a microorganism. It may also contain a gene that controls a promoter.
　より具体的には、発現ベクターとして、例えば、ｐＢＴｒｐ２、ｐＢＴａｃ１、ｐＢＴａｃ２（いずれもベーリンガーマンハイム社より市販）、ｐＫＫ２３３－２（Ｐｈａｒｍａｃｉａ社製）、ｐＳＥ２８０（Ｉｎｖｉｔｒｏｇｅｎ社製）、ｐＧＥＭＥＸ－１（Ｐｒｏｍｅｇａ社製）、ｐＱＥ－８（ＱＩＡＧＥＮ社製）、ｐＱＥ－３０（ＱＩＡＧＥＮ社製）、ｐＫＹＰ１０（特開昭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）〕、ｐＢｌｕｅｓｃｒｉｐｔＩＩ　ＳＫ＋、ｐＢｌｕｅｓｃｒｉｐｔＩＩ　ＳＫ（－）（Ｓｔｒａｔａｇｅｎｅ社製）、ｐＴｒＳ３０（ＦＥＲＭＢＰ－５４０７）、ｐＴｒＳ３２（ＦＥＲＭ　ＢＰ－５４０８）、ｐＧＥＸ（Ｐｈａｒｍａｃｉａ社製）、ｐＥＴ－３（Ｎｏｖａｇｅｎ社製）、ｐＴｅｒｍ２（ＵＳ４６８６１９１、ＵＳ４９３９０９４、ＵＳ５１６０７３５）、ｐＳｕｐｅｘ、ｐＵＢ１１０、ｐＴＰ５、ｐＣ１９４、ｐＵＣ１８〔ｇｅｎｅ，３３，１０３（１９８５）〕、ｐＵＣ１９〔Ｇｅｎｅ，３３，１０３（１９８５）〕、ｐＳＴＶ２８（宝酒造社製）、ｐＳＴＶ２９（宝酒造社製）、ｐＵＣ１１８（宝酒造社製）、ｐＰＡ１（特開昭６３－２３３７９８号公報）、ｐＥＧ４００〔Ｊ．Ｂａｃｔｅｒｉｏｌ．，１７２，２３９２（１９９０）〕、ｐＣｏｌｄＩ、ｐＣｏｌｄII、ｐＣｏｌｄIII、ｐＣｏｌｄIV、ｐＮＩＤＮＡ、ｐＮＩ－ＨｉｓＤＮＡ（タカラバイオ社製）等を例示することができる。 More specifically, as expression vectors, for example, pBTrp2, pBTac1, pBTac2 (all commercially available from Berliner Mannheim), pKK233-2 (manufactured by Pharmacia), pSE280 (manufactured by Invitrogen), pGEMEX-1 (manufactured by Promega). ), PQE-8 (manufactured by QIAGEN), pQE-30 (manufactured by QIAGEN), pKYP10 (Japanese Patent Laid-Open No. 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)], pBluescriptII SK +, pBluescriptII SK (-) (manufactured by Stratagene), pTrS30 (FERMBP-5407), pTrS32 (FERM BP-5408), pGEX (Pha) Novagen), pTerm2 (US4686191, US4939094, US5160735), pSupex, pUB110, pTP5, pC194, pUC18 [gene, 33,103 (1985)], pUC19 [gene, 33,103 (1985)], pSTV28 , PSTV29 (manufactured by Takara Shuzo Co., Ltd.), pUC118 (manufactured by Takara Shuzo Co., Ltd.), pPA1 (Japanese Patent Laid-Open No. 63-233798), pEG400 [J. Bacteriol. , 172, 2392 (1990)], pColdI, pColdII, pColdIII, pColdIV, pNIDNA, pNI-HisDNA (manufactured by Takara Bio Inc.) and the like can be exemplified.
　プロモーターとしては、宿主である大腸菌、パン酵母等の細胞中で発現することができるものであればいかなるものでもよい。例えば、ｔｒｐプロモーター（Ｐｔｒｐ）、ｌａｃプロモーター（Ｐｌａｃ）、ＰＬプロモーター、ＰＲプロモーター、ＰＳＥプロモーター等の、大腸菌やファージ等に由来するプロモーター、ＳＰＯ１プロモーター、ＳＰＯ２プロモーター、ｐｅｎＰプロモーター等を挙げることができる。またＰｔｒｐを２つ直列させたプロモーター（Ｐｔｒｐｘ２）、ｔａｃプロモーター、ｌｅｔＩプロモーター、ｌａｃＴ７プロモーターのように人為的に設計改変されたプロモーター等も用いることができる。酵素法による生産（スクアレンを基質とした酵素反応による生体外での合成）に用いる酵素を生産するには、強いプロモーターとして機能し、目的タンパク質の大量生産が可能なプロモーターが好ましく、誘導型プロモーターがより好ましい。誘導型プロモーターとしては、例えば低温で発現誘導されるコールドショック遺伝子ｃｓｐＡのプロモーター、誘導剤であるＩＰＴＧの添加により誘導されるＴ７プロモーター等を挙げることができる。また発酵生産（グルコースなどを炭素源とした宿主による生体内での生合成）においては、上記プロモーターの中でも組織を問わず常時目的遺伝子を発現させるプロモーター、すなわち構成的プロモーターであることがより好ましい。構成的プロモーターとしては、アルコールデヒドロゲナーゼ１遺伝子（ＡＤＨ１）、翻訳伸長因子ＴＦ－１α遺伝子（ＴＥＦ１）、ホスホグリセリン酸キナーゼ遺伝子（ＰＧＫ１）、トリオースリン酸イソメラーゼ遺伝子（ＴＰＩ１）、トリオースリン酸デヒドロゲナーゼ遺伝子（ＴＤＨ３）、ピルビン酸キナーゼ遺伝子（ＰＹＫ１）のプロモーターが挙げられる。 The promoter may be any promoter as long as it can be expressed in cells such as Escherichia coli and baker's yeast, which are hosts. For example, promoters derived from Escherichia coli, phage and the like, SPO1 promoter, SPO2 promoter, penP promoter and the like such as trp promoter (Ptrp), lac promoter (Plac), PL promoter, PR promoter and PSE promoter can be mentioned. Further, an artificially designed and modified promoter such as a promoter in which two Ptprps are connected in series (Pttrpx2), a tac promoter, a letI promoter, and a lacT7 promoter can also be used. In order to produce an enzyme used for production by an enzymatic method (synthesis in vitro by an enzymatic reaction using squalane as a substrate), a promoter that functions as a strong promoter and is capable of mass production of the target protein is preferable, and an inducible promoter is preferable. More preferred. Examples of the inducible promoter include the promoter of the cold shock gene cspA whose expression is induced at low temperature, the T7 promoter induced by the addition of the inducer IPTG, and the like. Further, in fermentative production (biosynthesis in vivo by a host using glucose or the like as a carbon source), among the above promoters, a promoter that constantly expresses the target gene regardless of the tissue, that is, a constitutive promoter is more preferable. Constitutive promoters include alcohol dehydrogenase 1 gene (ADH1), translation elongation factor TF-1α gene (TEF1), phosphoglycerate kinase gene (PGK1), triosephosphate isomerase gene (TPI1), triosephosphate isomerase gene (TDH3), Examples include the promoter of the pyruvate kinase gene (PYK1).
［５］形質転換体
　本発明の形質転換体も、本発明による前記ポリヌクレオチドを含む限り、特に限定されるものではなく、例えば、本発明による前記ポリヌクレオチドが、宿主細胞の染色体に組み込まれた形質転換体であることもできるし、あるいは、本発明による前記ポリヌクレオチドを含むベクターの形で含有する形質転換体であることもできる。また、本発明によるポリペプチドを発現している形質転換体であることもできるし、あるいは、本発明によるポリペプチドを発現していない形質転換体であることもできる。本発明の形質転換体は、例えば、本発明による前記ベクターにより、あるいは、本発明による前記ポリヌクレオチドそれ自体により、所望の宿主細胞を形質転換することにより得ることができる。 [5] Transformant The transformant of the present invention is also not particularly limited as long as it contains the polynucleotide according to the present invention. For example, the transformant according to the present invention is integrated into the chromosome of a host cell. It can be a transformant, or it can be a transformant contained in the form of a vector containing the above-mentioned polynucleotide according to the present invention. Further, it can be a transformant expressing the polypeptide according to the present invention, or it can be a transformant not expressing the polypeptide according to the present invention. The transformant of the present invention can be obtained, for example, by transforming a desired host cell with the vector according to the present invention or with the polynucleotide itself according to the present invention.
　宿主細胞は、特に限定されないが、大腸菌、枯草菌、ブレビバチルス属菌、放線菌、酵母、麹菌、アカパンカビ等取り扱いが容易な菌株が好ましいが、昆虫細胞、植物細胞、動物細胞などを用いることも可能である。しかしながら、酵素法による生産（スクアレンを基質とした酵素反応による生体外での合成）に用いる酵素を生産するには、大腸菌、枯草菌、ブレビバチルス属菌、麹菌が好ましく、大腸菌が最も好ましい。また発酵生産（グルコースなどを炭素源とした宿主による生体内での生合成）で最も好ましくは酵母である。最も好ましい酵母株としては、清酒酵母が挙げられる。特に好ましくは、清酒酵母協会７号又は７０１号が挙げられる。協会７０１号酵母は、協会７号から育種された泡なし酵母で高泡を形成しない特長があるが、他の性質は同じである。 The host cell is not particularly limited, but a strain that is easy to handle such as Escherichia coli, Bacillus subtilis, Brevibacillus, actinomycete, yeast, aspergillus, and Neurospora crassa is preferable, but insect cells, plant cells, animal cells, etc. may also be used. It is possible. However, in order to produce an enzyme used for production by an enzymatic method (synthesis in vitro by an enzymatic reaction using squalane as a substrate), Escherichia coli, Bacillus subtilis, Brevibacillus spp., And Aspergillus are preferable, and Escherichia coli is most preferable. Further, yeast is most preferable in fermentative production (biosynthesis in vivo by a host using glucose or the like as a carbon source). The most preferable yeast strain is sake yeast. Particularly preferably, Sake Yeast Association No. 7 or 701 can be mentioned. Association No. 701 yeast is a foamless yeast bred from Association No. 7 and has a feature of not forming high foam, but has the same other properties.
［６］アンブレインの製造方法
《第１態様》
　本発明のアンブレインの製造方法は、前記変異型テトラプレニル－β－クルクメン環化酵素をスクアレン又は８α－ヒドロキシポリポダ－１３，１７，２１－トリエンに反応させて、アンブレインを得る。 [6] Method for producing umbrella << first aspect >>
In the method for producing an umbrella of the present invention, the mutant tetraprenyl-β-curcumen cyclase is reacted with squalene or 8α-hydroxypolypoda-13,17,21-triene to obtain an umbrella.
　変異型テトラプレニル－β－クルクメン環化酵素は、酵素発現用ベクターを細菌等に導入することによって得られた形質転換体を培養することにより生成できる。形質転換体の培養に用いられる培地は、通常用いられる培地でよく、宿主の種類に応じて適宜選択される。例えば、大腸菌を培養する場合には、ＬＢ培地等が用いられる。培地には、選択マーカーの種類に応じた抗生物質が添加されていてもよい。 The mutant tetraprenyl-β-curcumen cyclase can be produced by culturing a transformant obtained by introducing an enzyme expression vector into a bacterium or the like. The medium used for culturing the transformant may be a medium usually used, and is appropriately selected according to the type of host. For example, when culturing Escherichia coli, an LB medium or the like is used. Antibiotics may be added to the medium depending on the type of selectable marker.
　変異型テトラプレニル－β－クルクメン環化酵素は、前記酵素を発現可能な形質転換体を培養することにより得られた培養液から酵素を抽出し精製したものであってもよい。また、あらかじめ本発明の変異型テトラプレニル－β－クルクメン環化酵素のポリペプチドのＮ末側又はＣ末側にトリガーファクター（ＴＦ）又はＨｉｓタグなどを融合させた融合タンパク質として発現させ、精製などを容易にしてもよい。また、培養液中の形質転換体から抽出された酵素を含む抽出液をそのまま用いてもよい。形質転換体からの酵素の抽出方法は、公知の方法を適用してよい。酵素の抽出工程は、例えば、形質転換体を抽出溶媒中で破砕し、細胞内容物を形質転換体の破砕片と分離することを含んでよい。得られた細胞内容物には、目的とする変異型テトラプレニル－β－クルクメン環化酵素が含まれている。 The mutant tetraprenyl-β-curcumen cyclase may be an enzyme extracted and purified from a culture solution obtained by culturing a transformant capable of expressing the enzyme. Further, it is previously expressed as a fusion protein in which a trigger factor (TF) or His tag is fused to the N-terminal side or C-terminal side of the polypeptide of the mutant tetraprenyl-β-curcumen cyclase of the present invention, and purified. May be facilitated. Moreover, you may use the extract containing the enzyme extracted from the transformant in the culture solution as it is. As a method for extracting the enzyme from the transformant, a known method may be applied. The enzyme extraction step may include, for example, disrupting the transformant in an extraction solvent and separating the cell contents from the disrupted fragments of the transformant. The resulting cell content contains the mutant tetraprenyl-β-curcumen cyclase of interest.
　形質転換体の破砕方法としては、形質転換体を破砕して、酵素液を回収可能な公知の方法を適用してよく、例えば、超音波破砕、ガラスビーズ破砕等が挙げられる。破砕の条件は、特に制限はなく、１０℃以下及び１５分間などの、酵素が失活しない条件であればよい。
　細胞内容物と微生物体の破砕片との分離方法としては、沈降分離、遠心分離、濾過分離及びこれらの２つ以上の分離方法の組み合わせ等が挙げられる。これらの方法を用いた分離条件は当業者には公知であり、遠心分離の場合には例えば、８，０００×ｇ～１５，０００×ｇ及び１０分間～２０分間である。 As a method for crushing the transformant, a known method capable of crushing the transformant and recovering the enzyme solution may be applied, and examples thereof include ultrasonic crushing and glass bead crushing. The crushing conditions are not particularly limited as long as the enzyme is not inactivated, such as 10 ° C. or lower and 15 minutes.
Examples of the method for separating the cell contents and the debris of the microbial body include sedimentation separation, centrifugation, filtration separation, and a combination of two or more separation methods thereof. 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 minutes to 20 minutes.
　抽出溶媒としては、酵素抽出の溶媒として通常用いられるものでよく、例えば、Ｔｒｉｓ－ＨＣｌ緩衝液、リン酸カリウム緩衝液等が挙げられる。抽出溶媒のｐＨは、酵素の安定性の点で、３～１０が好ましく、６～８がより好ましい。 The extraction solvent may be one that is usually used as a solvent for enzyme extraction, and examples thereof include Tris-HCl buffer and potassium phosphate buffer. The pH of the extraction solvent is preferably 3 to 10, more preferably 6 to 8 in terms of enzyme stability.
　抽出溶媒には、界面活性剤が含まれていてもよい。界面活性剤としては、非イオン界面活性剤、両性イオン界面活性等が挙げられる。非イオン界面活性剤としては、ポリ（オキシエチレン）ソルビタンモノオレイン酸エステル（Ｔｗｅｅｎ８０）等のポリオキシエチレンソルビタン脂肪酸エステル、ｎ－オクチルβ－Ｄ－グルコシド等のアルキルグルコシド、ショ糖ステアリン酸エステル等のショ糖脂肪酸エステル、ポリグリセリンステアリン酸エステル等のポリグリセリン脂肪酸エステル等が挙げられる。両性イオン界面活性剤としては、アルキルベタインであるＮ，Ｎ－ジメチル－Ｎ－ドデシルグリシンベタイン等が挙げられる。これら以外にも、トライトン（登録商標）Ｘ－１００（Triton X-100）、ポリオキシエチレン（２０）セチルエーテル（Brij-58）、ノニルフェノールエトキシレート（Tergitol NP-40）等の当技術分野で一般的に用いられる界面活性剤が利用可能である。
　抽出溶媒中の界面活性剤の濃度は、酵素の安定性の観点から、０．００１質量％～１０質量％が好ましく、０．１０質量％～３．０質量％がより好ましく、０．１０質量％～１．０質量％が更に好ましい。 The extraction solvent may contain a surfactant. Examples of the surfactant include nonionic surfactants and zwitterionic surfactants. Examples of the nonionic surfactant include polyoxyethylene sorbitan fatty acid ester such as poly (oxyethylene) sorbitan monooleic acid ester (Tween80), alkyl glucoside such as n-octyl β-D-glucoside, and sucrose stearate ester. Examples thereof include polyglycerin fatty acid esters such as sucrose fatty acid ester and polyglycerin stearate ester. Examples of the amphoteric ion surfactant include N, N-dimethyl-N-dodecylglycine betaine, which is an alkyl betaine. In addition to these, general in the art such as Triton (registered trademark) X-100 (Triton X-100), polyoxyethylene (20) cetyl ether (Brij-58), nonoxyphenol ethoxylate (Tergitol NP-40), etc. Surfactants used in the art are available.
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 0.10% by mass from the viewpoint of enzyme stability. % To 1.0% by mass is more preferable.
　抽出溶媒には、酵素活性の観点から、ジチオスレイトール、β－メルカプトエタノール等の還元剤が含まれていることが好ましい。還元剤としては、ジチオスレイトールが好ましい。抽出溶媒中のジチオスレイトールの濃度は、０．１ｍＭ～１Ｍが好ましく、１ｍＭ～１０ｍＭがより好ましい。抽出溶媒中にジチオスレイトールが存在することによって、酵素におけるジスルフィド結合等の構造が保持されやすくなり、酵素活性がより上昇する傾向がある。 From the viewpoint of enzyme activity, the extraction solvent preferably contains a reducing agent such as dithiothreitol or β-mercaptoethanol. As the reducing agent, dithiothreitol is preferable. 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 makes it easier to retain structures such as disulfide bonds in the enzyme, and the enzyme activity tends to increase further.
　抽出溶媒には、酵素活性の観点から、エチレンジアミン四酢酸（ＥＤＴＡ）等のキレート剤が含まれていることが好ましい。抽出溶媒中のＥＤＴＡの濃度は、０．０１ｍＭ～１Ｍが好ましく、０．１ｍＭ～１０ｍＭがより好ましい。抽出溶媒中にＥＤＴＡが存在することによって、酵素活性を低下させ得る金属イオンがキレートされるため、酵素活性がより上昇する傾向がある。 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 chelate the metal ions that can reduce the enzyme activity, so that the enzyme activity tends to increase further.
　抽出溶媒には、上記の成分以外に、酵素抽出溶媒に添加可能な公知の成分が含まれていてよい。 In addition to the above components, the extraction solvent may contain known components that can be added to the enzyme extraction solvent.
　変異型テトラプレニル－β－クルクメン環化酵素は、１種単独で用いてもよく、２種以上を組み合わせて用いてもよい。
　変異型テトラプレニル－β－クルクメン環化酵素とスクアレン又は８α－ヒドロキシポリポダ－１３，１７，２１－トリエンとの反応の条件は、酵素反応が進行可能な条件であれば特に制限はない。例えば、反応温度及び反応時間は、変異型テトラプレニル－β－クルクメン環化酵素の活性等に基づいて適宜選択することができる。反応温度及び反応時間は、反応効率の観点から、例えば４℃～１００℃及び１時間～３０日であり、３０℃～６０℃及び１６時間～２０日が好ましい。ｐＨ条件は、反応効率の観点から、例えば３～１０であり、６～８が好ましい。 The mutant tetraprenyl-β-curcumen cyclase may be used alone or in combination of two or more.
The conditions for the reaction of the mutant tetraprenyl-β-curcumen cyclase with squalene or 8α-hydroxypolypoda-13,17,21-triene are not particularly limited as long as the enzymatic reaction can proceed. For example, the reaction temperature and reaction time can be appropriately selected based on the activity of the mutant tetraprenyl-β-curcumen cyclase and the like. The reaction temperature and reaction time are, for example, 4 ° C. to 100 ° C. and 1 hour to 30 days, preferably 30 ° C. to 60 ° C. and 16 hours to 20 days, from the viewpoint of reaction efficiency. The pH condition is, for example, 3 to 10, preferably 6 to 8, from the viewpoint of reaction efficiency.
　反応溶媒は、酵素反応を阻害しないものであれば特に制限はなく、通常用いられる緩衝液等を用いることができる。また、例えば、酵素の抽出工程で使用する抽出溶媒と同一のものを用いることができる。また、変異型テトラプレニル－β－クルクメン環化酵素を含む抽出液（例えば、無細胞抽出液）を酵素液としてそのまま反応に用いてもよい。 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. Further, for example, the same extraction solvent as that used in the enzyme extraction step can be used. Further, an extract containing a mutant tetraprenyl-β-curcumen cyclase (for example, a cell-free extract) may be used as it is in the reaction as an enzyme solution.
　アンブレイン生成反応における変異型テトラプレニル－β－クルクメン環化酵素と、その基質であるスクアレン又は８α－ヒドロキシポリポダ－１３，１７，２１－トリエンとの濃度比は、反応効率の観点から、酵素に対する基質のモル濃度比（基質／酵素）として、１～１００００が好ましく、１０～５０００がより好ましく、１００～３０００がより好ましく、１０００～２０００が更に好ましい。
　酵素反応に用いるスクアレン又は８α－ヒドロキシポリポダ－１３，１７，２１－トリエンの濃度は、反応効率の観点から、反応溶媒の全質量に対して０．０００００１質量％～１０質量％が好ましく、０．００００１質量％～１質量％がより好ましい。 From the viewpoint of reaction efficiency, the concentration ratio of the mutant tetraprenyl-β-curcumen cyclase and its substrate squalene or 8α-hydroxypolypoda-13,17,21-triene in the umbrella formation reaction is an enzyme. The molar concentration ratio (substrate / enzyme) of the substrate to 10000 is preferable, 10 to 5000 is more preferable, 100 to 3000 is more preferable, and 1000 to 2000 is further preferable.
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 with respect to the total mass of the reaction solvent from the viewpoint of reaction efficiency, and is 0. More preferably, it is .0000001% by mass to 1% by mass.
　変異型テトラプレニル－β－クルクメン環化酵素とスクアレン又は８α－ヒドロキシポリポダ－１３，１７，２１－トリエンとが反応する反応工程は、複数回繰り返してもよい。これにより、アンブレインの収率を高めることができる。反応工程を複数回繰り返す場合には、基質となるスクアレン又は８α－ヒドロキシポリポダ－１３，１７，２１－トリエンを反応系に再投入する工程、公知の方法により酵素を失活させた後、反応液中の反応生成物を回収及び精製する工程等を含むものであってもよい。スクアレンの再投入を行う場合には、反応液中の変異型テトラプレニル－β－クルクメン環化酵素の濃度、反応液中に残存する基質量等によって、投入する時期、投入量を適宜設定することができる。 The reaction step in which the mutant tetraprenyl-β-curcumen cyclase reacts with squalene or 8α-hydroxypolypoda-13,17,21-triene may be repeated a plurality of times. This makes it possible to increase the yield of umbrella. When the reaction step is repeated a plurality of times, a step of re-injecting squalene or 8α-hydroxypolypoda-13,17,21-triene as a substrate into the reaction system, inactivating the enzyme by a known method, and then reacting. It may include a step of recovering and purifying the reaction product in the liquid. When re-adding squalene, the timing and amount of squalene to be added should be appropriately set according to the concentration of the mutant tetraprenyl-β-curcumen cyclase in the reaction solution, the mass of the group remaining in the reaction solution, and the like. Can be done.
　本発明のアンブレインの製造方法の別の実施態様においては、本発明の微生物又は形質転換体を培養することを特徴とする。
　前記微生物又は発現ベクターで形質転換された宿主細胞を培養することによって、アンブレインを製造することができる。例えば、酵母について記載すると、酵母は通常用いられるＹＰＤ培地などで培養すればよい。相同組換えにより遺伝子導入された酵母、又は発現ベクターを有する酵母を前培養し、更にＹＰＤ培地などに植菌し、そして２４～２４０時間、好ましくは７２～１２０時間程度培養する。培地中に分泌されたアンブレインは、そのまま、又は公知の方法により精製して用いることができる。精製方法としては、具体的には、溶媒抽出、再結晶、蒸留、カラムクロマトグラフィー、及びＨＰＬＣ等が挙げられる。 Another embodiment of the method for producing an umbrella of the present invention is characterized by culturing the microorganism or transformant of the present invention.
An umbrella can be produced by culturing a host cell transformed with the microorganism or an expression vector. For example, when describing yeast, yeast may be cultured in a commonly used YPD medium or the like. Yeast gene-introduced by homologous recombination or yeast having an expression vector is pre-cultured, further inoculated into YPD medium or the like, and cultured for 24-240 hours, preferably 72-120 hours. The umbrella secreted in the medium can be used as it is or after being purified by a known method. Specific examples of the purification method include solvent extraction, recrystallization, distillation, column chromatography, HPLC and the like.
《第２態様》
　本発明のアンブレインの製造方法の第２態様は、（１）テトラプレニル－β－クルクメン環化酵素をスクアレンに反応させて、８α－ヒドロキシポリポダ－１３，１７，２１－トリエンを得る工程（以下、第１工程と称することがある）、及び（２）変異型テトラプレニル－β－クルクメン環化酵素を８α－ヒドロキシポリポダ－１３，１７，２１－トリエンに反応させて、アンブレインを得る工程（以下、第２工程と称することがある）を含む。図３に、工程（１）で野生型テトラプレニル－β－クルクメン環化酵素を用いる実施態様を示す。 << Second aspect >>
The second aspect of the method for producing an umbrella of the present invention is (1) a step of reacting tetraprenyl-β-curcumen cyclase with squalene to obtain 8α-hydroxypolypoda-13,17,21-triene (1). Hereinafter, it may be referred to as the first step), and (2) the mutant tetraprenyl-β-squalene cyclase is reacted with 8α-hydroxypolypoda-13,17,21-triene to obtain an umbrella. Including a step (hereinafter, may be referred to as a second step). FIG. 3 shows an embodiment in which the wild-type tetraprenyl-β-curcumen cyclase is used in step (1).
（第１工程）
　本発明のアンブレインの製造方法の第２態様の第１工程においては、テトラプレニル－β－クルクメン環化酵素をスクアレンに反応させて、８α－ヒドロキシポリポダ－１３，１７，２１－トリエンを得る。第１工程は、変異型テトラプレニル－β－クルクメン環化酵素に代えて、テトラプレニル－β－クルクメン環化酵素を用いることを除いては、前記第１態様における、前記「変異型テトラプレニル－β－クルクメン環化酵素をスクアレンに反応させること」と同様に実施することができる。 (First step)
In the first step of the second aspect of the method for producing an umbrella of the present invention, tetraprenyl-β-curcumen cyclase is reacted with squalene to obtain 8α-hydroxypolypoda-13,17,21-triene. .. In the first step, the above-mentioned "variant tetraprenyl-" in the first aspect, except that tetraprenyl-β-squalene cyclase is used instead of the mutant tetraprenyl-β-squalene cyclase. It can be carried out in the same manner as "reacting β-curcumen cyclase with squalene".
　前記第１工程で使用されるテトラプレニル－β－クルクメン環化酵素は、スクアレンを８α－ヒドロキシポリポダ－１３，１７，２１－トリエンに変換できる限りにおいて、特に限定されるものではない。例えば、本発明の変異型テトラプレニル－β－クルクメン環化酵素を用いてもよく、野生型のテトラプレニル－β－クルクメン環化酵素を用いてよく、野生型のテトラプレニル－β－クルクメン環化酵素に、本発明の変異型テトラプレニル－β－クルクメン環化酵素以外の変異が導入されたもの（以下、「第１工程用変異型ＴＣ」と称する）を用いてもよい。
　野生型テトラプレニル－β－クルクメン環化酵素としては、配列番号１に記載のバチルス・メガテリウム由来の野生型テトラプレニル－β－クルクメン環化酵素、バチルス・サブチリス由来の野生型テトラプレニル－β－クルクメン環化酵素（アクセッション番号：ＡＢ６１８２０６）、バチルス・リケニフォルミス由来の野生型テトラプレニル－β－クルクメン環化酵素（アクセッション番号：ＡＡＵ４１１３４）が挙げられる。
　前記第１工程用変異型ＴＣとしては、（１）配列番号１、配列番号５、又は配列番号６で表されるアミノ酸配列において、１又は数個のアミノ酸が欠失、置換、挿入、及び／又は付加されたアミノ酸配列からなり、しかもスクアレンから８α－ヒドロキシポリポダ－１３，１７，２１－トリエン生成活性を示す変異型ＴＣ、又は（２）配列番号１、配列番号５、又は配列番号６で表されるアミノ酸配列との同一性が４０％以上であるアミノ酸配列からなり、しかもスクアレンから８α－ヒドロキシポリポダ－１３，１７，２１－トリエン生成活性を示す変異型ＴＣが挙げられる。 The tetraprenyl-β-curcumen cyclase used in the first step is not particularly limited as long as squalene can be converted to 8α-hydroxypolypoda-13,17,21-triene. For example, the mutant tetraprenyl-β-curcumen cyclase of the present invention may be used, the wild-type tetraprenyl-β-curcumen cyclase may be used, and the wild-type tetraprenyl-β-curcumen cyclization. An enzyme into which a mutation other than the mutant tetraprenyl-β-curcumen cyclase of the present invention has been introduced (hereinafter, referred to as “first step mutant TC”) may be used.
Examples of the wild-type tetraprenyl-β-curcumen cyclase include the wild-type tetraprenyl-β-curcumen cyclase derived from Bacillus megaterium shown in SEQ ID NO: 1 and the wild-type tetraprenyl-β-curcumen derived from Bacillus subtilis. Examples thereof include a cyclization enzyme (accession number: AB618206) and a wild-type tetraprenyl-β-curcumen cyclase derived from Bacillus licheniformis (accession number: AAU41134).
The mutant TC for the first step includes (1) deletion, substitution, insertion, and / of one or several amino acids in the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 5, or SEQ ID NO: 6. Alternatively, a mutant TC consisting of an added amino acid sequence and exhibiting 8α-hydroxypolypoda-13,17,21-triene-producing activity from squalane, or (2) SEQ ID NO: 1, SEQ ID NO: 5, or SEQ ID NO: 6 Examples thereof include mutant TCs having an amino acid sequence having an identity of 40% or more with the represented amino acid sequence and exhibiting 8α-hydroxypolypoda-13,17,21-triene-producing activity from squalane.
（第２工程）
　本発明のアンブレインの製造方法の第２態様の第２工程においては、変異型テトラプレニル－β－クルクメン環化酵素を８α－ヒドロキシポリポダ－１３，１７，２１－トリエンに反応させて、アンブレインを得る。具体的には、第１工程で得られた８α－ヒドロキシポリポダ－１３，１７，２１－トリエンを基質として、前記第１態様における、前記「変異型テトラプレニル－β－クルクメン環化酵素を８α－ヒドロキシポリポダ－１３，１７，２１－トリエンに反応させること」と同様に実施することができる。第２工程において使用できる変異型テトラプレニル－β－クルクメン環化酵素としては、前記第１態様で使用される変異型テトラプレニル－β－クルクメン環化酵素を用いてことができる。更に、下記の変異型テトラプレニル－β－クルクメン環化酵素（以下、第２工程用変異型ＴＣと称する）を用いることができる。 (Second step)
In the second step of the second aspect of the method for producing an umbrella of the present invention, the mutant tetraprenyl-β-curcumen cyclase is reacted with 8α-hydroxypolypoda-13,17,21-triene to obtain an ann. Get Brain. Specifically, using the 8α-hydroxypolypoda-13,17,21-triene obtained in the first step as a substrate, the above-mentioned "mutant tetraprenyl-β-curcumen cyclase 8α" in the first aspect is used. -Reacting with hydroxypolypoda-13,17,21-triene "can be carried out in the same manner. As the mutant tetraprenyl-β-curcumen cyclase that can be used in the second step, the mutant tetraprenyl-β-curcumen cyclase used in the first aspect can be used. Further, the following mutant tetraprenyl-β-curcumen cyclase (hereinafter referred to as mutant TC for the second step) can be used.
　前記第２工程用変異型ＴＣは、ＤＸＤＤモチーフの第４番目のアミノ酸残基であるアスパラギン酸がアスパラギン酸以外のアミノ酸に置換しており、そして（Ａ／Ｓ／Ｇ）ＲＸ（Ｈ／Ｎ）ＸＸＰモチーフのＮ末に隣接するアミノ酸がチロシン以外のアミノ酸に置換しているか、又はＧＸＧＸ（Ｇ／Ａ／Ｐ）モチーフの４番目のアミノ酸がロイシン以外のアミノ酸に置換している変異型テトラプレニル－β－クルクメン環化酵素であって、（ａ）前記変異型テトラプレニル－β－クルクメン環化酵素は、前記ＤＸＤＤモチーフを基準として、Ｎ末端側に１００アミノ酸残基以上離れた位置にＱＸＸＸＧＸ（Ｗ／Ｆ）モチーフ、Ｎ末端側に１８０～２５０アミノ酸残基離れた位置に前記（Ａ／Ｓ／Ｇ）ＲＸ（Ｈ／Ｎ）ＸＸＰモチーフ、Ｎ末端側に８０～１４０アミノ酸残基離れた位置に前記（Ｄ／Ｎ）Ｇ（Ｔ／Ｌ）（Ｌ／Ｆ／Ｙ）（Ｙ／Ｆ）ＳＹモチーフ、Ｎ末端側に１０～５０アミノ酸残基離れた位置にＱＸＸＸＸ（Ｇ／Ａ／Ｓ）Ｘ（Ｆ／Ｗ／Ｙ）モチーフ、Ｃ末端側に２０～５０アミノ酸残基離れた位置にＱＸＸＸＧＸ（Ｆ／Ｗ／Ｙ）モチーフ、Ｃ末端側に５０～１２０アミノ酸残基離れた位置にＱＸＸＸＧＸＷモチーフ、Ｃ末端側に１２０～１７０アミノ酸残基離れた位置にＱＸＸＸＧＸ（Ｆ／Ｗ）モチーフ、及びＣ末端側に１８０～２５０アミノ酸残基離れた位置に前記ＧＸＧＸ（Ｇ／Ａ／Ｐ）モチーフを有し、そしてＤＸＤＤモチーフを基準として、Ｃ末端側に１７０アミノ酸残基以上離れた位置にＱＸＸＸＧＸＷモチーフを有さず、（ｂ）配列番号１で表されるアミノ酸配列との同一性が４０％以上であり、そして（ｃ）８α－ヒドロキシポリポダ－１３，１７，２１－トリエンを基質としてアンブレイン生成活性を示す、変異型テトラプレニル－β－クルクメン環化酵素である。 In the mutant TC for the second step, asparagic acid, which is the fourth amino acid residue of the DXDD motif, is replaced with an amino acid other than aspartic acid, and (A / S / G) RX (H / N). A variant tetraprenyl-in which the amino acid adjacent to the N-terminal of the XXP motif is replaced with an amino acid other than tyrosine, or the fourth amino acid of the GXGX (G / A / P) motif is replaced with an amino acid other than leucine. The β-curcumen cyclase (a) The mutant tetraprenyl-β-curcumen cyclase is QXXXGX (W) at a position separated by 100 amino acid residues or more on the N-terminal side based on the DXDD motif. / F) Motif, 180-250 amino acid residues away from the N-terminal side The (A / S / G) RX (H / N) XPP motif, 80-140 amino acid residues away from the N-terminal side The (D / N) G (T / L) (L / F / Y) (Y / F) SY motif, QXXX (G / A / S) X at a position 10 to 50 amino acid residues away from the N-terminal side. (F / W / Y) motif, QXXXGX (F / W / Y) motif at a position 20 to 50 amino acid residues away from the C-terminal side, QXXXGXGXW motif at a position 50 to 120 amino acid residues away from the C-terminal side, It has a QXXXGX (F / W) motif at a position 120 to 170 amino acid residues away from the C-terminal side, and the GXGX (G / A / P) motif at a position 180 to 250 amino acid residues away from the C-terminal side. And, with reference to the DXDD motif, there is no QXXXGXW motif at a position separated by 170 amino acid residues or more on the C-terminal side, and (b) the identity with the amino acid sequence represented by SEQ ID NO: 1 is 40% or more. , And (c) a mutant tetraprenyl-β-curcumen cyclase that exhibits umbrella-forming activity using 8α-hydroxypolypoda-13,17,21-triene as a substrate.
　本発明の第２工程用変異型ＴＣの好ましい態様としては、前記［１４］に記載した変異型テトラプレニル－β－クルクメン環化酵素である。 A preferred embodiment of the mutant TC for the second step of the present invention is the mutant tetraprenyl-β-curcumen cyclase described in [14] above.
　また、本発明の第２工程用変異型ＴＣの最も好ましい態様としては、変異型テトラプレニル－β－クルクメン環化酵素を構成するポリペプチドが、配列番号８又は９で表されるアミノ酸配列からなるBacillus megaterium由来のポリペプチド、を挙げることができる。すなわち、第２工程用変異型ＴＣは、ＤＸＤＤモチーフの第４番目のアミノ酸残基であるアスパラギン酸がシステインに置換しており、そして（Ａ／Ｓ／Ｇ）ＲＸ（Ｈ／Ｎ）ＸＸＰモチーフのＮ末に隣接するアミノ酸がチロシンからアラニンに置換しているか、又はＤＸＤＤモチーフの第４番目のアミノ酸残基であるアスパラギン酸がシステインに置換しており、そしてＧＸＧＸ（Ｇ／Ａ／Ｐ）モチーフの４番目のアミノ酸がロイシンからアラニンに置換している。 Further, in the most preferable embodiment of the mutant TC for the second step of the present invention, the polypeptide constituting the mutant tetraprenyl-β-curcumen cyclase comprises the amino acid sequence represented by SEQ ID NO: 8 or 9. Examples include polypeptides derived from Bacillus megaterium. That is, in the mutant TC for the second step, aspartic acid, which is the fourth amino acid residue of the DXDD motif, is replaced with cysteine, and the (A / S / G) RX (H / N) XXP motif is used. The amino acid flanking the N-terminal is replaced by alanine from tyrosine, or the fourth amino acid residue of the DXDD motif, aspartic acid, is replaced by cysteine, and the GXGX (G / A / P) motif. The fourth amino acid replaces leucine with alanine.
　図７にバチルス・メガテリウム由来のテトラプレニル－β－クルクメン環化酵素において、３７３番目のアスパラギン酸がシステインに置換し、そして１６７番目のチロシンがアラニンに置換した変異型テトラプレニル－β－クルクメン環化酵素のアミノ酸配列（配列番号８）、及び３７３番目のアスパラギン酸がシステインに置換し、そして５９６番目のロイシンがアラニンに置換した変異型テトラプレニル－β－クルクメン環化酵素のアミノ酸配列（配列番号９）を示す。 In FIG. 7, in the tetraprenyl-β-curcumen cyclase derived from Bacillus megaterium, mutant tetraprenyl-β-curcumen cyclization in which asparagic acid at position 373 was replaced with cysteine and tyrosine at position 167 was replaced with alanine. The amino acid sequence of the enzyme (SEQ ID NO: 8) and the amino acid sequence of the mutant tetraprenyl-β-curcumen cyclase (SEQ ID NO: 9) in which asparagic acid at position 373 was replaced with cysteine and leucine at position 596 was replaced with alanine. ) Is shown.
　以下、実施例によって本発明を具体的に説明するが、これらは本発明の範囲を限定するものではない。本文中又は図表において、バチルス・メガテリウムを「Ｂｍｅ」と略す場合があり、バチルス・メガテリウム由来のテトラプレニル－β－クルクメン環化酵素を「ＢｍｅＴＣ」と略す場合がある。 Hereinafter, the present invention will be specifically described with reference to Examples, but these do not limit the scope of the present invention. In the text or in figures and tables, Bacillus megaterium may be abbreviated as "Bme", and tetraprenyl-β-curcumen cyclase derived from Bacillus megaterium may be abbreviated as "BmeTC".
《実施例１》
　本実施例では、変異型テトラプレニル－β－クルクメン環化酵素のクローニング、及び２５７番目のチロシンがアラニンに置換され、そして３７３番目のアスパラギン酸がシステインに置換された変異型テトラプレニル－β－クルクメン環化酵素遺伝子の構築を行った。
　バチルス・メガテリウム染色体ＤＮＡを鋳型として用いて、ＰＣＲにより野生型テトラプレニル－β－クルクメン環化酵素をコードするポリヌクレオチドを取得し、当該野生型酵素のアミノ酸配列を決定した。（以下、特に記載のない限り、テトラプレニル－β－クルクメン環化酵素はバチルス・メガテリウム由来の配列であり、単に「野生型」ということもある。）得られた遺伝子を、ベクターｐＣｏｌｄＩ（タカラバイオ社）のクローニングサイト（制限酵素ＮｄｅＩ／ＸｈｏＩサイト）に挿入し、野生型テトラプレニル－β－クルクメン環化酵素遺伝子（配列番号１）を含む発現ベクターを得た。得られた変異型テトラプレニル－β－クルクメン環化酵素遺伝子を含む発現ベクターにより、大腸菌ＢＬ２１（ＤＥ３）の形質転換体を作製した。
　続いて、２５７番目のチロシンがアラニンに置換され、そして３７３番目のアスパラギン酸がシステインに置換された変異型テトラプレニル－β－クルクメン環化酵素遺伝子の構築を行った。先ず、テトラプレニル－β－クルクメン環化酵素（野生型）の遺伝子配列をもとに、３７３番目のアスパラギン酸がシステインに置換されるように設計し、宿主である大腸菌にコドンを最適化して合成した。合成した遺伝子は、ベクターｐＣｏｌｄＩ（タカラバイオ社）のクローニングサイト（制限酵素ＮｄｅＩ／ＸｈｏＩサイト）に挿入した。このベクター遺伝子を鋳型として、２５７番目のチロシンがアラニンに置換されるように、変異導入プライマー：ＯＰＹ２５７Ａ（配列番号１８及び１９）を使用したクイックチェンジ法により部位特異的変異を導入して、Ｙ２５７Ａ／Ｄ３７３Ｃ変異体の変異型テトラプレニル－β－クルクメン環化酵素遺伝子（配列番号１２）を含む発現ベクターを得た。
　ｐＣｏｌｄベクターは、コールドショック遺伝子の一つであるｃｓｐＡ遺伝子のプロモーターを利用したコールドショック発現ベクターで、ｃｓｐＡプロモーターの下流に、translation enhancing element（TEE）、Ｈｉｓタグ配列、Ｆａｃｔｏｒ　Ｘａ切断配列などが配置している。このため発現タンパク質は、ＴＥＥ配列、Ｈｉｓタグ配列、Ｆａｃｔｏｒ　Ｘａ切断配列を含む融合タンパク質として発現される。本発明では、バチルス・メガテリウム由来の当該野生型酵素遺伝の開始コドン（ＧＴＧ）の配列に変更は加えていない。そのため、融合タンパク質における酵素タンパク質部位のＮ末端アミノ酸は、バリンに翻訳される。
　続いて、得られた変異型テトラプレニル－β－クルクメン環化酵素遺伝子を含む発現ベクターにより、大腸菌ＢＬ２１（ＤＥ３）の形質転換体を作製した。 << Example 1 >>
In this example, the mutant tetraprenyl-β-curcumen cyclase was cloned, and the mutant tetraprenyl-β-curcumen in which tyrosine at position 257 was replaced with alanine and aspartic acid at position 373 was replaced with cysteine. The cyclase gene was constructed.
Using the Bacillus megaterium chromosomal DNA as a template, a polynucleotide encoding wild-type tetraprenyl-β-curcumen cyclase was obtained by PCR, and the amino acid sequence of the wild-type enzyme was determined. (Unless otherwise specified, the tetraprenyl-β-curcumen cyclase is a sequence derived from Bacillus megaterium and may be simply referred to as "wild type".) The obtained gene is used as a vector pColdI (Takarabio). The expression vector containing the wild-type tetraprenyl-β-curcumen cyclase gene (SEQ ID NO: 1) was obtained by inserting into the cloning site (restrictor enzyme NdeI / XhoI site) of the company. A transformant of Escherichia coli BL21 (DE3) was prepared from the obtained expression vector containing the mutant tetraprenyl-β-curcumen cyclase gene.
Subsequently, a mutant tetraprenyl-β-curcumen cyclase gene was constructed 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-curcumen cyclase (wild type), the 373rd aspartic acid was designed to be replaced with cysteine, and the codon was optimized for the host Escherichia coli and synthesized. bottom. The synthesized gene was inserted into the cloning site (restriction enzyme NdeI / XhoI site) of the vector pColdI (Takara Bio Inc.). Using this vector gene as a template, a site-specific mutation was introduced by a quick change method using a mutation-introducing primer: OPY257A (SEQ ID NOs: 18 and 19) so that the 257th tyrosine was replaced with alanine, and Y257A / An expression vector containing a mutant tetraprenyl-β-curcumen cyclase gene (SEQ ID NO: 12) of the D373C mutant was obtained.
The pCold vector is a cold shock expression vector that uses the promoter of the cspA gene, which is one of the cold shock genes. Translation enhancing element (TEE), His tag sequence, Factor Xa cleavage sequence, etc. are arranged downstream of the cspA promoter. ing. Therefore, the expressed protein is expressed as a fusion protein containing a TEE sequence, a His tag sequence, and a Factor Xa cleavage sequence. In the present invention, the sequence of the start codon (GTG) of the wild-type enzyme inheritance derived from Bacillus megaterium is not changed. Therefore, the N-terminal amino acid of the enzyme protein moiety in the fusion protein is translated into valine.
Subsequently, a transformant of Escherichia coli BL21 (DE3) was prepared from the obtained expression vector containing the mutant tetraprenyl-β-curcumen cyclase gene.
《実施例２》
　本実施例では、１６７番目のチロシンがアラニンに置換され、２５７番目のチロシンがアラニンに置換され、そして３７３番目のアスパラギン酸がシステインに置換された変異型テトラプレニル－β－クルクメン環化酵素遺伝子の構築を行った。
　変異型テトラプレニル－β－クルクメン環化酵素遺伝子は、テトラプレニル－β－クルクメン環化酵素（野生型）の遺伝子配列をもとに、１６７番目のチロシンがアラニンに、３７３番目のアスパラギン酸がシステインに置換されるように設計し、宿主である大腸菌にコドンを最適化して合成した。合成した遺伝子は、ベクターｐＣｏｌｄＩ（タカラバイオ社）のクローニングサイト（制限酵素ＮｄｅＩ／ＸｈｏＩサイト）に挿入し、このベクター遺伝子を鋳型として、２５７番目のチロシンがアラニンに置換されるように、変異導入プライマー：ＯＰＹ２５７Ａ（配列番号１８及び１９）を使用したクイックチェンジ法により部位特異的変異を導入して、Ｙ１６７Ａ／Ｙ２５７Ａ／Ｄ３７３Ｃ変異体の変異型テトラプレニル－β－クルクメン環化酵素遺伝子（配列番号１３）を含む発現ベクターを得た。
　続いて、得られた変異型テトラプレニル－β－クルクメン環化酵素遺伝子を含む発現ベクターにより、大腸菌ＢＬ２１（ＤＥ３）の形質転換体を作製した。 << Example 2 >>
In this example, a mutant tetraprenyl-β-curcumen cyclase gene in which tyrosine at position 167 was replaced with alanine, tyrosine at position 257 was replaced with alanine, and aspartic acid at position 373 was replaced with cysteine. I built it.
The mutant tetraprenyl-β-curcumen cyclase gene is based on the gene sequence of tetraprenyl-β-curcumen cyclase (wild type), and the 167th tyrosine is alanine and the 373th aspartic acid is cysteine. It was designed to be replaced with, and the codon was optimized and synthesized for the host Escherichia coli. The synthesized gene is inserted into the cloning site (restriction enzyme NdeI / XhoI site) of the vector pColdI (Takara Bio), and the mutation introduction primer is used so that the 257th tyrosine is replaced with alanine using this vector gene as a template. : A mutant tetraprenyl-β-curcumen cyclase gene (SEQ ID NO: 13) of a Y167A / Y257A / D373C variant by introducing a site-specific mutation by a quick change method using OPY257A (SEQ ID NOs: 18 and 19). An expression vector containing the above was obtained.
Subsequently, a transformant of Escherichia coli BL21 (DE3) was prepared from the obtained expression vector containing the mutant tetraprenyl-β-curcumen cyclase gene.
≪参考例１≫
　本参考例では、３７３番目のアスパラギン酸がシステインに置換された変異型テトラプレニル－β－クルクメン環化酵素遺伝子の構築を行った。
　変異型テトラプレニル－β－クルクメン環化酵素遺伝子は、実施例１記載の野生型酵素の遺伝子配列をもとに、３７３番目のアスパラギン酸がシステインに置換されるように設計し、宿主である大腸菌にコドンを最適化して合成した。合成した遺伝子は、ベクターｐＣｏｌｄＩ（タカラバイオ社）のクローニングサイト（制限酵素ＮｄｅＩ／ＸｈｏＩサイト）に挿入し、Ｄ３７３Ｃ変異体の変異型テトラプレニル－β－クルクメン環化酵素遺伝子（配列番号１７）を含む発現ベクターを得た。
　続いて、得られた変異型テトラプレニル－β－クルクメン環化酵素遺伝子を含む発現ベクターにより、大腸菌ＢＬ２１（ＤＥ３）の形質転換体を作製した。 ≪Reference example 1≫
In this reference example, a mutant tetraprenyl-β-curcumen cyclase gene in which the 373rd aspartic acid was replaced with cysteine was constructed.
The mutant tetraprenyl-β-curcumen cyclase gene is designed so that aspartic acid at position 373 is replaced with cysteine based on the gene sequence of the wild-type enzyme described in Example 1, and the host Escherichia coli. The codon was optimized and synthesized. The synthesized gene is inserted into the cloning site (restriction enzyme NdeI / XhoI site) of the vector pColdI (Takara Bio Inc.) and contains the mutant tetraprenyl-β-curcumen cyclase gene (SEQ ID NO: 17) of the D373C variant. An expression vector was obtained.
Subsequently, a transformant of Escherichia coli BL21 (DE3) was prepared from the obtained expression vector containing the mutant tetraprenyl-β-curcumen cyclase gene.
≪参考例２≫
　本参考例では、１６７番目のチロシンがアラニンに置換され、そして３７３番目のアスパラギン酸がシステインに置換された変異型テトラプレニル－β－クルクメン環化酵素遺伝子の構築を行った。変異型テトラプレニル－β－クルクメン環化酵素遺伝子は、実施例１記載の野生型酵素の遺伝子配列をもとに、１６７番目のチロシンがアラニンに置換されるように、３７３番目のアスパラギン酸がシステインに置換されるように設計し、宿主である大腸菌にコドンを最適化して合成した。合成した遺伝子は、ベクターｐＣｏｌｄＩ（タカラバイオ社）のクローニングサイト（制限酵素ＮｄｅＩ／ＸｈｏＩサイト）に挿入し、Ｙ１６７Ａ／Ｄ３７３Ｃ変異体の変異型テトラプレニル－β－クルクメン環化酵素遺伝子（配列番号１５）を含む発現ベクターを得た。
　続いて、得られた変異型テトラプレニル－β－クルクメン環化酵素遺伝子を含む発現ベクターにより、大腸菌ＢＬ２１（ＤＥ３）の形質転換体を作製した。 ≪Reference example 2≫
In this reference example, a mutant tetraprenyl-β-curcumen cyclase gene was constructed in which tyrosine at position 167 was replaced with alanine and aspartic acid at position 373 was replaced with cysteine. In the mutant tetraprenyl-β-curcumen cyclase gene, aspartic acid at position 373 is cysteine based on the gene sequence of the wild-type enzyme described in Example 1 so that tyrosine at position 167 is replaced with alanine. It was designed to be replaced with, and the codon was optimized and synthesized for the host Escherichia coli. The synthesized gene is inserted into the cloning site (restriction enzyme NdeI / XhoI site) of the vector pColdI (Takara Bio Inc.), and the mutant tetraprenyl-β-curcumen cyclase gene of the Y167A / D373C variant (SEQ ID NO: 15). An expression vector containing the above was obtained.
Subsequently, a transformant of Escherichia coli BL21 (DE3) was prepared from the obtained expression vector containing the mutant tetraprenyl-β-curcumen cyclase gene.
≪参考例３≫
　本参考例では、３７３番目のアスパラギン酸がシステインに置換され、そして５９６番目のロイシンがアラニンに置換された変異型テトラプレニル－β－クルクメン環化酵素遺伝子の構築を行った。変異型テトラプレニル－β－クルクメン環化酵素遺伝子は、実施例１記載の野生型酵素の遺伝子配列をもとに、３７３番目のアスパラギン酸がシステインに置換され、そして５９６番目のロイシンがアラニンに置換されるように設計し、宿主である大腸菌にコドンを最適化して合成した。合成した遺伝子は、ベクターｐＣｏｌｄＴＦ（タカラバイオ社）のクローニングサイト（制限酵素ＮｄｅＩ／ＸｈｏＩサイト）に挿入し、Ｄ３７３Ｃ／Ｌ５９６Ａ変異体の変異型テトラプレニル－β－クルクメン環化酵素遺伝子（配列番号１６）を含む発現ベクターを得た。
　続いて、得られた変異型テトラプレニル－β－クルクメン環化酵素遺伝子を含む発現ベクターにより、大腸菌ＢＬ２１（ＤＥ３）の形質転換体を作製した。 ≪Reference example 3≫
In this reference example, a mutant tetraprenyl-β-curcumen cyclase gene was constructed in which aspartic acid at position 373 was replaced with cysteine and leucine at position 596 was replaced with alanine. In the mutant tetraprenyl-β-curcumen cyclase gene, aspartic acid at position 373 is replaced with cysteine and leucine at position 596 is replaced with alanine based on the gene sequence of the wild-type enzyme described in Example 1. The codon was optimized and synthesized for the host Escherichia coli. The synthesized gene is inserted into the cloning site (restriction enzyme NdeI / XhoI site) of the vector pColdTF (Takara Bio Inc.), and the mutant tetraprenyl-β-curcumen cyclase gene of the D373C / L596A variant (SEQ ID NO: 16). An expression vector containing the above was obtained.
Subsequently, a transformant of Escherichia coli BL21 (DE3) was prepared from the obtained expression vector containing the mutant tetraprenyl-β-curcumen cyclase gene.
≪実施例３≫
　本実施例では、１６７番目のチロシンがアラニンに置換され、３７３番目のアスパラギン酸がシステインに置換され、そして５９６番目のロイシンがアラニンに置換された変異型テトラプレニル－β－クルクメン環化酵素遺伝子の構築を行った。
　先ず、テトラプレニル－β－クルクメン環化酵素（野生型）の遺伝子配列をもとに、１６７番目のチロシンがアラニンに置換され、３７３番目のアスパラギン酸がシステインに置換され、そして５９６番目のロイシンがアラニンに置換されるように設計し、宿主である大腸菌にコドンを最適化して合成した。合成した遺伝子は、ベクターｐＣｏｌｄＩ（タカラバイオ社）のクローニングサイト（制限酵素ＮｄｅＩ／ＸｈｏＩサイト）に挿入して、Ｙ１６７Ａ／Ｄ３７３Ｃ／Ｌ５９６Ａ変異体の変異型テトラプレニル－β－クルクメン環化酵素遺伝子（配列番号１４）を含む発現ベクターを得た。
　続いて、得られた変異型テトラプレニル－β－クルクメン環化酵素遺伝子を含む発現ベクターにより、大腸菌ＢＬ２１（ＤＥ３）の形質転換体を作製した。 << Example 3 >>
In this example, the mutant tetraprenyl-β-curcumen cyclase gene in which tyrosine at position 167 was replaced with alanine, aspartic acid at position 373 was replaced with cysteine, and leucine at position 596 was replaced with alanine. I built it.
First, based on the gene sequence of tetraprenyl-β-curcumen cyclase (wild type), tyrosine at position 167 is replaced with alanine, aspartic acid at position 373 is replaced with cysteine, and leucine at position 596. It was designed to be replaced with alanine, and codons were optimized and synthesized for the host Tyrosine. The synthesized gene is inserted into the cloning site (restriction enzyme NdeI / XhoI site) of the vector pColdI (Takara Bio), and the mutant tetraprenyl-β-curcumen cyclase gene (sequence) of the Y167A / D373C / L596A variant is inserted. An expression vector containing No. 14) was obtained.
Subsequently, a transformant of Escherichia coli BL21 (DE3) was prepared from the obtained expression vector containing the mutant tetraprenyl-β-curcumen cyclase gene.
《製造方法例１》
　本製造方法例では、２環化合物である８α－ヒドロキシポリポダ－１３，１７，２１－トリエンを基質として、実施例１～３及び参考例３の変異型テトラプレニル－β－クルクメン環化酵素の酵素活性を検討した。
　実施例１～３及び参考例３で作製した形質転換体をそれぞれアンピシリン（５０ｍｇ／Ｌ）含有ＬＢ培地（１Ｌ）に植菌し、３７℃で３時間振とう培養した。培養後、１ｍＭのイソプロピル－β－チオガラクトピラノシド（ＩＰＴＧ）を添加し、１５℃で２４時間振とうを行い、変異型テトラプレニル－β－クルクメン環化酵素の発現を誘導した。
　その後、遠心（６，０００×ｇ、１０分間）によって集菌した菌体を、５０ｍＭのＴｒｉｓ－ＨＣｌ緩衝液（ｐＨ７．５）で洗浄した後、５ｇの菌体に対して１５ｍＬの緩衝液Ａ［５０ｍＭのＴｒｉｓ－ＨＣｌ緩衝液（ｐＨ７．５）、０．１ｖ／ｖ％のＴｗｅｅｎ８０、０．１ｗ／ｖ％のアスコルビン酸ナトリウム、２．５ｍＭのジチオスレイトール、１ｍＭのＥＤＴＡを含有。］で懸濁し、ＵＰ２００５　ｓｏｎｉｃａｔｏｒ（Hielscher Ultrasonics, Teltow, Germany）を用いて超音波破砕（４℃、２０分間）した。破砕処理後の試料を遠心（１２，３００×ｇ、２０分間）し、粗酵素液を得た。この粗抽出液からＨｉｓタグを利用して目的タンパク質のアフィニティー精製を行った。精製は、ニッケルカラム（Ni-NTA Agarose、QIAGEN社）を使用し、組み換え酵素液（タンパク質濃度１００μｇ／ｍＬ）を調製した。 << Manufacturing method example 1 >>
In this production method example, the mutant tetraprenyl-β-curcumen cyclase of Examples 1 to 3 and Reference Example 3 is used as a substrate using the bicyclic compound 8α-hydroxypolypoda-13,17,21-triene. The enzyme activity was examined.
The transformants prepared in Examples 1 to 3 and Reference Example 3 were inoculated into LB medium (1 L) containing ampicillin (50 mg / L), respectively, and cultured with shaking at 37 ° C. for 3 hours. After culturing, 1 mM isopropyl-β-thiogalactopyranoside (IPTG) was added and shaken at 15 ° C. for 24 hours to induce the expression of mutant tetraprenyl-β-curcumen cyclase.
Then, the cells collected by centrifugation (6,000 × g, 10 minutes) were washed with 50 mM Tris-HCl buffer (pH 7.5), and then 15 mL of buffer A was added to 5 g of the cells. [Contains 50 mM Tris-HCl buffer (pH 7.5), 0.1 v / v% Tween 80, 0.1 w / v% sodium ascorbate, 2.5 mM dithioslateol, 1 mM EDTA. ], And sonicated (4 ° C., 20 minutes) using UP2005 sonicator (Hielscher Ultrasonics, Teltow, Germany). The crushed sample was centrifuged (12,300 × g, 20 minutes) to obtain a crude enzyme solution. Affinity purification of the target protein was performed from this crude extract using a His tag. For purification, a nickel column (Ni-NTA Agarose, QIAGEN) was used to prepare a recombinant enzyme solution (protein concentration 100 μg / mL).
　８α－ヒドロキシポリポダ－１３，１７，２１－トリエン（１００μｇ）をＴｗｅｅｎ８０（２ｍｇ）に混合して可溶化した後に緩衝液Ａ（１ｍＬ）に添加して８α－ヒドロキシポリポダ－１３，１７，２１－トリエン８α－ヒドロキシポリポダ－１３，１７，２１－トリエン液を調製した。この液（０．５ｍＬ）を酵素溶液（０．５ｍＬ）に加えて反応液とし、３０℃で６４時間インキュベートした。８α－ヒドロキシポリポダ－１３，１７，２１－トリエン反応液において、酵素タンパク質量は５０μｇであり、８α－ヒドロキシポリポダ－１３，１７，２１－トリエン（基質）と変異型テトラプレニル－β－クルクメン環化酵素（酵素）とのモル比（基質／酵素）は、約１７０であった。
　インキュベート後に、１５％水酸化カリウム・メタノール（１．２ｍＬ）を反応液へ添加し、酵素反応を停止させた。その後、反応液へｎ－ヘキサン（２．０ｍＬ）を添加して、反応生成物の抽出を３回行った。 8α-Hydroxypolypoda-13,17,21-triene (100 μg) was mixed with Tween80 (2 mg) to solubilize and then added to buffer A (1 mL) to add 8α-hydroxypolypoda-13,17,21. -Triene 8α-hydroxypolypoda-13, 17, 21-triene solution was prepared. This solution (0.5 mL) was added to the enzyme solution (0.5 mL) to prepare a reaction solution, which was incubated at 30 ° C. for 64 hours. In the 8α-hydroxypolypoda-13,17,21-triene reaction solution, the amount of enzyme protein was 50 μg, and 8α-hydroxypolypoda-13,17,21-triene (substrate) and mutant tetraprenyl-β-curcumen. The molar ratio (substrate / enzyme) to the cyclizing enzyme (enzyme) was about 170.
After incubation, 15% potassium hydroxide / methanol (1.2 mL) was added to the reaction solution to stop the enzymatic reaction. Then, n-hexane (2.0 mL) was added to the reaction solution, and the reaction product was extracted three times.
　得られた抽出物（反応組成物）中の、アンブレイン、２環化合物（８α－ヒドロキシポリポダ－１３，１７，２１－トリエン）、オノセロイドの含有量をガスクロマトグラフィーによって、以下の装置及び条件で測定した。
装置：ガスクロマトグラフ「ＧＣ－２０１４」（SHIMADZU製）、Integrator「Chromatopac CR-8A」（SHIMADZU製）
カラム：DB-1, capillary（Length 30 m, I.D.0.32 mm, Film Thickness 0.25 μm）（J&D製）
インジェクション温度：３００℃
カラム温度：２２０－３００℃（昇温３℃／分）
カラム流量：１．０ｍＬ／分 The contents of umbrella, bicyclic compound (8α-hydroxypolypoda-13,17,21-triene) and onoseroid in the obtained extract (reaction composition) were determined by gas chromatography with the following equipment and conditions. Measured at.
Equipment: Gas chromatograph "GC-2014" (manufactured by SHIMADZU), Integrator "Chromatopac CR-8A" (manufactured by SHIMADZU)
Column: DB-1, capillary (Length 30 m, ID 0.32 mm, Film Thickness 0.25 μm) (manufactured by J & D)
Injection temperature: 300 ° C
Column temperature: 220-300 ° C (temperature rise 3 ° C / min)
Column flow rate: 1.0 mL / min
　得られた抽出物（反応組成物）中の反応生成物（アンブレイン）の含有量比を図４（Ａ）に示す。また、得られた抽出物（反応組成物）中の、８α－ヒドロキシポリポダ－１３，１７，２１－トリエンを除いた、アンブレイン、オノセロイドの含有量比を図４（Ｂ）に示した。実施例１のＹ２５７Ａ／Ｄ３７３Ｃ変異体、実施例２のＹ１６７Ａ／Ｙ２５７Ａ／Ｄ３７３Ｃ変異体、実施例３のＹ１６７Ａ／Ｄ３７３Ｃ／Ｌ５９６Ａ変異体、参考例３のＤ３７３Ｃ／Ｌ５９６Ａ変異体は、２環化合物の８α－ヒドロキシポリポダ－１３，１７，２１－トリエンを基質として、アンブレインを産生できた。特に、実施例１のＹ２５７Ａ／Ｄ３７３Ｃ変異体、実施例２のＹ１６７Ａ／Ｙ２５７Ａ／Ｄ３７３Ｃ変異体は８α－ヒドロキシポリポダ－１３，１７，２１－トリエンを基質としたときの、アンブレインの産生量が多かった。実施例３のＹ１６７Ａ／Ｄ３７３Ｃ／Ｌ５９６Ａ変異体は、副産物のオノセロイド産生が全くなかった。実施例２のＹ１６７Ａ／Ｙ２５７Ａ／Ｄ３７３Ｃ変異体は、８α－ヒドロキシポリポダ－１３，１７，２１－トリエンからアンブレインを、８５．５％の産生率（生成効率）、８８．６％の生成比率で、実施例１のＹ２５７Ａ／Ｄ３７３Ｃ変異体は、４５．７％の産生率（生成効率）、６１．３％の生成比率で、実施例３のＹ１６７Ａ／Ｄ３７３Ｃ／Ｌ５９６Ａ変異体は、９．０％の産生率（生成効率）、１００％の生成比率で生産できていた。このように、実施例２のＹ１６７Ａ／Ｙ２５７Ａ／Ｄ３７３Ｃ変異体、実施例１のＹ２５７Ａ／Ｄ３７３Ｃ変異体、実施例３のＹ１６７Ａ／Ｄ３７３Ｃ／Ｌ５９６Ａ変異体は、８α－ヒドロキシポリポダ－１３，１７，２１－トリエン（基質）からアンブレインへの反応選択性が向上しており、アンブレインを効率的に生産できる。 The content ratio of the reaction product (umbrain) in the obtained extract (reaction composition) is shown in FIG. 4 (A). Moreover, the content ratio of umbrella and onoseroid excluding 8α-hydroxypolypoda-13,17,21-triene in the obtained extract (reaction composition) is shown in FIG. 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 are 8α of the bicyclic compound. Umbrain could be produced using -hydroxypolypoda-13,17,21-triene as a substrate. In particular, the Y257A / D373C mutant of Example 1 and the Y167A / Y257A / D373C mutant of Example 2 produced a large amount of umbrella when 8α-hydroxypolypoda-13,17,21-triene was used as a substrate. There were many. The Y167A / D373C / L596A mutant of Example 3 produced no by-product onoseroid. The Y167A / Y257A / D373C mutant of Example 2 produced umbrellas from 8α-hydroxypolypoda-13,17,21-triene at an production rate of 85.5% (production efficiency) and a production rate of 88.6%. The Y257A / D373C mutant of Example 1 had a production rate of 45.7% (production efficiency) and a production rate of 61.3%, and the Y167A / D373C / L596A mutant of Example 3 had a production rate of 9.0. It was possible to produce at a production rate of% (production efficiency) and a production rate of 100%. As described above, the Y167A / Y257A / D373C mutant of Example 2, the Y257A / D373C mutant of Example 1, and the Y167A / D373C / L596A mutant of Example 3 are 8α-hydroxypolypoda-13,17,21. -The reaction selectivity from triene (matrix) to umbrella is improved, and umbrella can be produced efficiently.
《製造方法例２》
　本製造方法例では、スクアレンを基質として、実施例２及び参考例１の変異型テトラプレニル－β－クルクメン環化酵素の酵素活性を検討した。
　組み換え酵素液（タンパク質濃度１００μｇ／ｍＬ）は、製造方法例１と同様に調製した。 << Manufacturing method example 2 >>
In this production method example, the enzymatic activity of the mutant tetraprenyl-β-curcumen cyclase 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.
　スクアレン（１００μｇ）をＴｗｅｅｎ８０（２ｍｇ）に混合して可溶化した後に緩衝液Ａ（１ｍＬ）に添加してスクアレン液を調製した。このスクアレン液（０．５ｍＬ）を酵素溶液（０．５ｍＬ）に加えて反応液とし、３０℃で６４時間インキュベートした。反応液において、酵素タンパク質量は５０μｇであり、スクアレン（基質）と変異型テトラプレニル－β－クルクメン環化酵素（酵素）とのモル比（基質／酵素）は、約１７０であった。
　インキュベート後に、１５％水酸化カリウム・メタノール（１．２ｍＬ）を反応液へ添加し、酵素反応を停止させた。その後、反応液へｎ－ヘキサン（２．０ｍＬ）を添加して、反応生成物の抽出を３回行った。 Squalene (100 μg) was mixed with Tween 80 (2 mg) to solubilize it, 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 prepare a reaction solution, and the mixture was incubated at 30 ° C. for 64 hours. In the reaction solution, the amount of enzyme protein was 50 μg, and the molar ratio (substrate / enzyme) of squalene (substrate) to mutant tetraprenyl-β-curcumen cyclase (enzyme) was about 170.
After incubation, 15% potassium hydroxide / methanol (1.2 mL) was added to the reaction solution to stop the enzymatic reaction. Then, n-hexane (2.0 mL) was added to the reaction solution, and the reaction product was extracted three times.
　得られた抽出物（反応組成物）中のスクアレン、アンブレイン、単環化合物、２環化合物（８α－ヒドロキシポリポダ－１３，１７，２１－トリエン）、オノセロイドの含有量をガスクロマトグラフィーによって、製造方法例１と同様に測定した。 The contents of squalene, umbrella, monocyclic compound, bicyclic compound (8α-hydroxypolypoda-13,17,21-triene) and onoseroid in the obtained extract (reaction composition) were determined by gas chromatography. The measurement was carried out in the same manner as in Production Method Example 1.
　得られた抽出物（反応組成物）中の反応生成物（アンブレイン、Ambrein）含有量を図５（Ａ）に示す。また、得られた抽出物（反応組成物）中の、スクアレンを除いた、アンブレイン、単環化合物、２環化合物及びオノセロイドの含有量比を図５（Ｂ）に示した。実施例２のＹ１６７Ａ／Ｙ２５７Ａ／Ｄ３７３Ｃ変異体は、参考例１のＤ３７３Ｃ変異体と比べて、アンブレインの産生量が多かった。実施例１のＹ１６７Ａ／Ｙ２５７Ａ／Ｄ３７３Ｃ変異体は、スクアレンからアンブレインを、１３．６％の産生率（生成効率）、４４．０％の生成比率で生産できていた。このように、実施例２のＹ１６７Ａ／Ｙ２５７Ａ／Ｄ３７３Ｃ変異体は、スクアレン（基質）からアンブレインへの反応選択性が高く、アンブレインを効率的に生産できる。 The content of the reaction product (Ambrein) in the obtained extract (reaction composition) is shown in FIG. 5 (A). In addition, the content ratios of umbrella, monocyclic compound, bicyclic compound and onoseroid in the obtained extract (reaction composition) excluding squalene are shown in FIG. 5 (B). The Y167A / Y257A / D373C mutant of Example 2 produced a larger amount of umbrella than the D373C mutant of Reference Example 1. The Y167A / Y257A / D373C mutant of Example 1 was able to produce umbrella from squalene at a production rate of 13.6% (production efficiency) and a production ratio of 44.0%. As described above, the Y167A / Y257A / D373C mutant of Example 2 has high reaction selectivity from squalene (substrate) to umbrella, and can efficiently produce umbrella.
《製造方法例３》
　本製造方法例では、スクアレンを基質として、基質特異性の異なる、野生型テトラプレニル－β－クルクメン環化酵素と、各変異型テトラプレニル－β－クルクメン環化酵素とを組み合わせて使用した、ステップワイズ（二段階）反応法によるアンブレインの製造を検討した。変異型テトラプレニル－β－クルクメン環化酵素としては、実施例２のＹ１６７Ａ／Ｙ２５７Ａ／Ｄ３７３Ｃ変異体、参考例２のＹ１６７Ａ／Ｄ３７３Ｃ変異体を使用した。
　組み換え酵素液（タンパク質濃度１００μｇ／ｍＬ）は、製造方法例１と同様に調製した。スクアレン（１００μｇ）をＴｗｅｅｎ８０（２ｍｇ）に混合して可溶化した後に緩衝液Ａ（１ｍＬ）に添加してスクアレン液を調製した。このスクアレン液（０．５ｍＬ）に、野生型テトラプレニル－β－クルクメン環化酵素溶液（０．２５ｍＬ）を加えて反応液とし、３０℃で６４時間インキュベートした。反応後、７０℃で１時間インキュベートし、酵素反応を停止した。次に、この反応液に変異型テトラプレニル－β－クルクメン環化酵素溶液（０．２５ｍＬ）を加え、３０℃で６４時間インキュベートした。スクアレン反応液において、野生型及び変異型テトラプレニル－β－クルクメン環化酵素の酵素タンパク質量はそれぞれ２５μｇであった。スクアレン（基質）と、変異型テトラプレニル－β－クルクメン環化酵素（酵素）又は野生型テトラプレニル－β－クルクメン環化酵素（酵素）とのモル比（基質／酵素）は、約１７０であった。また、参考例２（Ｙ１６７Ａ／Ｄ３７３Ｃ変異体）では、一種類の酵素溶液を（０．５ｍＬ）加えて反応液とし、同様にスクアレンと酵素を反応させた。
　インキュベート後に、１５％水酸化カリウム・メタノール（１．２ｍＬ）を反応液へ添加し、酵素反応を停止させた。その後、反応液へｎ－ヘキサン（２．０ｍＬ）を添加して、反応生成物の抽出を３回行った。 << Manufacturing method example 3 >>
In this production method example, using squalene as a substrate, a wild-type tetraprenyl-β-curcumen cyclase having different substrate specificity and each mutant tetraprenyl-β-curcumen cyclase are used in combination. The production of umbrella by the Wise (two-step) reaction method was examined. As the mutant tetraprenyl-β-curcumen cyclase, the Y167A / Y257A / D373C mutant of Example 2 and the Y167A / D373C mutant of Reference Example 2 were used.
The recombinant enzyme solution (protein concentration 100 μg / mL) was prepared in the same manner as in Production Method Example 1. Squalene (100 μg) was mixed with Tween 80 (2 mg) to solubilize it, and then added to buffer A (1 mL) to prepare a squalene solution. A wild-type tetraprenyl-β-curcumen cyclase solution (0.25 mL) was added to this squalene solution (0.5 mL) to prepare a reaction solution, which was incubated at 30 ° C. for 64 hours. After the reaction, the mixture was incubated at 70 ° C. for 1 hour to stop the enzymatic reaction. Next, a mutant tetraprenyl-β-curcumen cyclase solution (0.25 mL) was added to the reaction solution, and the mixture was incubated at 30 ° C. for 64 hours. In the squalene reaction solution, the amount of enzyme protein of the wild-type and mutant tetraprenyl-β-curcumen cyclase was 25 μg, respectively. The molar ratio (substrate / enzyme) of squalene to mutant tetraprenyl-β-curcumen cyclase (enzyme) or wild-type tetraprenyl-β-curcumen cyclase (enzyme) was about 170. rice field. Further, in Reference Example 2 (Y167A / D373C mutant), one kind of enzyme solution (0.5 mL) was added to prepare a reaction solution, and squalene and the enzyme were reacted in the same manner.
After incubation, 15% potassium hydroxide / methanol (1.2 mL) was added to the reaction solution to stop the enzymatic reaction. Then, n-hexane (2.0 mL) was added to the reaction solution, and the reaction product was extracted three times.
　得られた抽出物（反応組成物）中のスクアレン、アンブレイン、単環化合物、２環化合物、オノセロイドの含有量をガスクロマトグラフィーによって、製造方法例１と同様に測定した。 The contents of squalene, umbrella, monocyclic compound, bicyclic compound, and onoseroid in the obtained extract (reaction composition) were measured by gas chromatography in the same manner as in Production Method Example 1.
　得られた抽出物（反応組成物）中の、反応生成物（アンブレイン）含有量を図６（Ａ）に示す。また、得られた抽出物（反応組成物）中の、スクアレンを除いた、アンブレイン、単環化合物、２環化合物及びオノセロイドの含有量比を図６（Ｂ）に示した。野生株テトラプレニル－β－クルクメン環化酵素及び実施例２のＹ１６７Ａ／Ｙ２５７Ａ／Ｄ３７３Ｃ変異体の組み合わせ、野生株テトラプレニル－β－クルクメン環化酵素及び参考例２のＹ１６７Ａ／Ｄ３７３Ｃ変異体の組み合わせは、参考例２のＹ１６７Ａ／Ｄ３７３Ｃ変異体のみを使用した場合と比べて、アンブレインの産生量が多かった。このように、ステップワイズ反応法により、野生株テトラプレニル－β－クルクメン環化酵素及び実施例２のＹ１６７Ａ／Ｙ２５７Ａ／Ｄ３７３Ｃ変異体の組み合わせは、５９．９％の産生率（生成効率）、６５．６％の生成比率で、野生株テトラプレニル－β－クルクメン環化酵素及び参考例２のＹ１６７Ａ／Ｄ３７３Ｃ変異体の組み合わせは、３５．６％の産生率（生成効率）、４６．７％の生成比率で、スクアレンからアンブレインを生産できた。 The content of the reaction product (umbrain) in the obtained extract (reaction composition) is shown in FIG. 6 (A). In addition, the content ratios of umbrella, monocyclic compound, bicyclic compound and onoseroid in the obtained extract (reaction composition) excluding squalene are shown in FIG. 6 (B). The combination of the wild strain tetraprenyl-β-curcumen cyclase and the Y167A / Y257A / D373C mutant of Example 2, the combination of the wild strain tetraprenyl-β-curcumen cyclase and the Y167A / D373C mutant of Reference Example 2 As compared with the case where only the Y167A / D373C mutant of Reference Example 2 was used, the amount of umbrella produced was larger. Thus, by the stepwise reaction method, the combination of the wild-type tetraprenyl-β-squalene cyclase and the Y167A / Y257A / D373C mutant of Example 2 had a production rate (production efficiency) of 59.9% (production efficiency), 65. The combination of the wild-type tetraprenyl-β-squalene cyclase and the Y167A / D373C mutant of Reference Example 2 had a production rate of 35.6% (production efficiency) of 46.7% at a production rate of 0.6%. At the production ratio, we were able to produce umbrellas from squalene.
配列番号１：バチルス・メガテリウムWTアミノ酸配列
配列番号２：バチルス・メガテリウムY257A/D373Cアミノ酸配列
配列番号３：バチルス・メガテリウムY167A/Y257A/D373Cアミノ酸配列
配列番号４：バチルス・メガテリウムY167A/D373C/L596Aアミノ酸配列
配列番号５：バチルス・サブチルスWTアミノ酸配列
配列番号６：バチルス・リケンフォルミスWTアミノ酸配列
配列番号７：アリシクロバチルス・アシドカルダリウスSHCアミノ酸配列
配列番号８：バチルス・メガテリウムY167A/D373Cアミノ酸配列
配列番号９：バチルス・メガテリウムD373C/L596Aアミノ酸配列
配列番号１０：バチルス・メガテリウムD373Cアミノ酸配列
配列番号１１：バチルス・メガテリウムWT塩基配列
配列番号１２：バチルス・メガテリウムY257A/D373C塩基配列
配列番号１３：バチルス・メガテリウムY167A/Y257A/D373C塩基配列
配列番号１４：バチルス・メガテリウムY167A/D373C/L596A塩基配列
配列番号１５：バチルス・メガテリウムY167A/D373C塩基配列
配列番号１６：バチルス・メガテリウムD373C/L596A塩基配列
配列番号１７：バチルス・メガテリウムD373C塩基配列
配列番号１８：OPY257A_F
配列番号１９：OPY257A_R 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 likenformis WT amino acid sequence SEQ ID NO: 7: Alicyclobacillus acidcardarius SHC amino acid sequence SEQ ID NO: 8: Bacillus megaterium Y167A / D373C amino acid sequence number 9: Bacillus megaterium D373C / L596A Amino acid sequence No. 10: Bacillus megaterium D373C Amino acid sequence No. 11: Bacillus megaterium WT base sequence SEQ ID NO: 12: Bacillus megaterium Y257A / D373C base sequence No. 13: Bacillus megaterium Y167A / Y257A / D373C Nucleotide Sequence No. 14: Bacillus Megaterium Y167A / D373C / L596A Nucleotide Sequence No. 15: Bacillus Megaterium Y167A / D373C Nucleotide Sequence No. 16: Bacillus Megaterium D373C / L596A Nucleotide Sequence No. 17: Bacillus Megaterium D373C Nucleotide Sequence No. 18: OPY257A_F
SEQ ID NO: 19: OPY257A_R
　本発明の変異型テトラプレニル－β－クルクメン環化酵素は、微生物による効率的なアンブレインの生産に用いることができる。本発明によって得られるアンブレインは、例えば医薬品などの生産の原料として用いることができる。 The mutant tetraprenyl-β-curcumen cyclase of the present invention can be used for efficient production of umbrellas by microorganisms. The umbrella obtained by the present invention can be used as a raw material for the production of, for example, pharmaceuticals.

Claims (18)

  1. 　ＤＸＤＤモチーフの第４番目のアミノ酸残基であるアスパラギン酸がアスパラギン酸以外のアミノ酸に置換しており、（Ｄ／Ｎ）Ｇ（Ｔ／Ｌ）（Ｌ／Ｆ／Ｙ）（Ｙ／Ｆ）ＳＹモチーフの７番目のアミノ酸がチロシン以外のアミノ酸に置換している、変異型テトラプレニル－β－クルクメン環化酵素であって、
    （ａ）前記変異型テトラプレニル－β－クルクメン環化酵素は、前記ＤＸＤＤモチーフを基準として、Ｎ末端側に１００アミノ酸残基以上離れた位置にＱＸＸＸＧＸ（Ｗ／Ｆ）モチーフ、Ｎ末端側に１８０～２５０アミノ酸残基離れた位置に（Ａ／Ｓ／Ｇ）ＲＸ（Ｈ／Ｎ）ＸＸＰモチーフ、Ｎ末端側に８０～１４０アミノ酸残基離れた位置に前記（Ｄ／Ｎ）Ｇ（Ｔ／Ｌ）（Ｌ／Ｆ／Ｙ）（Ｙ／Ｆ）ＳＹモチーフ、Ｎ末端側に１０～５０アミノ酸残基離れた位置にＱＸＸＸＸ（Ｇ／Ａ／Ｓ）Ｘ（Ｆ／Ｗ／Ｙ）モチーフ、Ｃ末端側に２０～５０アミノ酸残基離れた位置にＱＸＸＸＧＸ（Ｆ／Ｗ／Ｙ）モチーフ、Ｃ末端側に５０～１２０アミノ酸残基離れた位置にＱＸＸＸＧＸＷモチーフ、Ｃ末端側に１２０～１７０アミノ酸残基離れた位置にＱＸＸＸＧＸ（Ｆ／Ｗ）モチーフ、及びＣ末端側に１８０～２５０アミノ酸残基離れた位置に前記ＧＸＧＸ（Ｇ／Ａ／Ｐ）モチーフを有し、そして
    ＤＸＤＤモチーフを基準として、Ｃ末端側に１７０アミノ酸残基以上離れた位置にＱＸＸＸＧＸＷモチーフを有さず、
    （ｂ）配列番号１で表されるアミノ酸配列との同一性が４０％以上であり、そして
    （ｃ）８α－ヒドロキシポリポダ－１３，１７，２１－トリエンを基質としてアンブレイン生成活性を示す、
    変異型テトラプレニル－β－クルクメン環化酵素。     Aspartic acid, which is the fourth amino acid residue of the DXDD motif, is replaced with an amino acid other than aspartic acid, and (D / N) G (T / L) (L / F / Y) (Y / F) SY A mutant tetraprenyl-β-curcumen cyclase in which the 7th amino acid of the motif is replaced with an amino acid other than tyrosine.
    (A) The mutant tetraprenyl-β-curcumen cyclase has a QXXXGX (W / F) motif at a position separated by 100 amino acid residues or more on the N-terminal side and 180 on the N-terminal side based on the DXDD motif. (A / S / G) RX (H / N) XXP motif at a position separated by ~ 250 amino acid residues, and the above (D / N) G (T / L) at a position separated by 80 to 140 amino acid residues at the N-terminal side. ) (L / F / Y) (Y / F) SY motif, QXXXXX (G / A / S) X (F / W / Y) motif at a position 10 to 50 amino acid residues away from the N-terminal side, C-terminal QXXXGX (F / W / Y) motif at a position 20 to 50 amino acid residues away from the side, QXXXGXW motif at a position 50 to 120 amino acid residues away from the C-terminal side, 120 to 170 amino acid residues away from the C-terminal side It has a QXXXGX (F / W) motif at a position and the GXGX (G / A / P) motif at a position 180 to 250 amino acid residues away from the C-terminal side, and the C-terminal side with reference to the DXDD motif. Does not have a QXXXGXW motif at a position separated by 170 amino acid residues or more.
    (B) It has an identity of 40% or more with the amino acid sequence represented by SEQ ID NO: 1, and (c) exhibits umbrella-forming activity using 8α-hydroxypolypoda-13,17,21-triene as a substrate.
    Mutant tetraprenyl-β-curcumen cyclase.
  2. 　更に、（Ａ／Ｓ／Ｇ）ＲＸ（Ｈ／Ｎ）ＸＸＰモチーフのＮ末に隣接するアミノ酸がチロシン以外のアミノ酸に置換している、請求項１に記載の変異型テトラプレニル－β－クルクメン環化酵素。 Further, the mutant tetraprenyl-β-curcumen ring according to claim 1, wherein the amino acid adjacent to the N-terminal of the (A / S / G) RX (H / N) XXP motif is replaced with an amino acid other than tyrosine. Chemical enzyme.
  3. 　前記変異型テトラプレニル－β－クルクメン環化酵素を構成するポリペプチドが、
    （１）配列番号１で表されるアミノ酸配列におけるＮ末端側から３７３番目のアスパラギン酸がアスパラギン酸以外のアミノ酸に置換しており、そしてＮ末端側から２５７番目のチロシンがチロシン以外のアミノ酸の置換しているポリペプチド、
    （２）配列番号１で表されるアミノ酸配列におけるＮ末端側から３７３番目のアスパラギン酸がアスパラギン酸以外のアミノ酸に置換しており、そしてＮ末端側から２５７番目のチロシンがチロシン以外のアミノ酸に置換しているアミノ酸配列において、１又は数個のアミノ酸が欠失、置換、挿入、及び／又は付加されたアミノ酸配列からなり、しかも、８α－ヒドロキシポリポダ－１３，１７，２１－トリエンを基質としてアンブレイン生成活性を示すポリペプチド、
    （３）配列番号１で表されるアミノ酸配列におけるＮ末端側から３７３番目のアスパラギン酸がアスパラギン酸以外のアミノ酸に置換しており、そしてＮ末端側から２５７番目のチロシンがチロシン以外のアミノ酸に置換しているアミノ酸配列との同一性が４０％以上であるアミノ酸配列からなり、しかも、８α－ヒドロキシポリポダ－１３，１７，２１－トリエンを基質としてアンブレイン生成活性を示すポリペプチド、
    （４）配列番号１で表されるアミノ酸配列におけるＮ末端側から３７３番目のアスパラギン酸がアスパラギン酸以外のアミノ酸に置換しており、そしてＮ末端側から２５７番目のチロシンがチロシン以外のアミノ酸に置換しているアミノ酸配列を含み、しかも、８α－ヒドロキシポリポダ－１３，１７，２１－トリエンを基質としてアンブレイン生成活性を示すポリペプチド、
    （５）配列番号１で表されるアミノ酸配列におけるＮ末端側から３７３番目のアスパラギン酸がアスパラギン酸以外のアミノ酸に置換しており、そしてＮ末端側から２５７番目のチロシンがチロシン以外のアミノ酸に置換しているアミノ酸配列において、１又は数個のアミノ酸が欠失、置換、挿入、及び／又は付加されたアミノ酸配列を含み、しかも、８α－ヒドロキシポリポダ－１３，１７，２１－トリエンを基質としてアンブレイン生成活性を示すポリペプチド、又は
    （６）配列番号１で表されるアミノ酸配列におけるＮ末端側から３７３番目のアスパラギン酸がアスパラギン酸以外のアミノ酸に置換しており、そしてＮ末端側から２５７番目のチロシンがチロシン以外のアミノ酸に置換しているアミノ酸配列との同一性が４０％以上であるアミノ酸配列を含み、しかも、８α－ヒドロキシポリポダ－１３，１７，２１－トリエンを基質としてアンブレイン生成活性を示すポリペプチド、
    である、請求項１に記載の変異型テトラプレニル－β－クルクメン環化酵素、あるいは
    前記変異型テトラプレニル－β－クルクメン環化酵素を構成するポリペプチドが、
    （７）配列番号１で表されるアミノ酸配列におけるＮ末端側から３７３番目のアスパラギン酸がアスパラギン酸以外のアミノ酸に置換しており、Ｎ末端側から１６７番目のチロシンがチロシン以外のアミノ酸に置換しており、そしてＮ末端側から２５７番目のチロシンがチロシン以外のアミノ酸の置換しているポリペプチド、
    （８）配列番号１で表されるアミノ酸配列におけるＮ末端側から３７３番目のアスパラギン酸がアスパラギン酸以外のアミノ酸に置換しており、Ｎ末端側から１６７番目のチロシンがチロシン以外のアミノ酸に置換しており、そしてＮ末端側から２５７番目のチロシンがチロシン以外のアミノ酸に置換しているアミノ酸配列において、１又は数個のアミノ酸が欠失、置換、挿入、及び／又は付加されたアミノ酸配列からなり、しかも、８α－ヒドロキシポリポダ－１３，１７，２１－トリエンを基質としてアンブレイン生成活性を示すポリペプチド、
    （９）配列番号１で表されるアミノ酸配列におけるＮ末端側から３７３番目のアスパラギン酸がアスパラギン酸以外のアミノ酸に置換しており、Ｎ末端側から１６７番目のチロシンがチロシン以外のアミノ酸に置換しており、そしてＮ末端側から２５７番目のチロシンがチロシン以外のアミノ酸に置換しているアミノ酸配列との同一性が４０％以上であるアミノ酸配列からなり、しかも、８α－ヒドロキシポリポダ－１３，１７，２１－トリエンを基質としてアンブレイン生成活性を示すポリペプチド、
    （１０）配列番号１で表されるアミノ酸配列におけるＮ末端側から３７３番目のアスパラギン酸がアスパラギン酸以外のアミノ酸に置換しており、Ｎ末端側から１６７番目のチロシンがチロシン以外のアミノ酸に置換しており、そしてＮ末端側から２５７番目のチロシンがチロシン以外のアミノ酸に置換しているアミノ酸配列を含み、しかも、８α－ヒドロキシポリポダ－１３，１７，２１－トリエンを基質としてアンブレイン生成活性を示すポリペプチド、
    （１１）配列番号１で表されるアミノ酸配列におけるＮ末端側から３７３番目のアスパラギン酸がアスパラギン酸以外のアミノ酸に置換しており、Ｎ末端側から１６７番目のチロシンがチロシン以外のアミノ酸に置換しており、そしてＮ末端側から２５７番目のチロシンがチロシン以外のアミノ酸に置換しているアミノ酸配列において、１又は数個のアミノ酸が欠失、置換、挿入、及び／又は付加されたアミノ酸配列を含み、しかも、８α－ヒドロキシポリポダ－１３，１７，２１－トリエンを基質としてアンブレイン生成活性を示すポリペプチド、又は
    （１２）配列番号１で表されるアミノ酸配列におけるＮ末端側から３７３番目のアスパラギン酸がアスパラギン酸以外のアミノ酸に置換しており、Ｎ末端側から１６７番目のチロシンがチロシン以外のアミノ酸に置換しており、そしてＮ末端側から２５７番目のチロシンがチロシン以外のアミノ酸に置換しているアミノ酸配列との同一性が４０％以上であるアミノ酸配列を含み、しかも、８α－ヒドロキシポリポダ－１３，１７，２１－トリエンを基質としてアンブレイン生成活性を示すポリペプチド、
    である、請求項２に記載の変異型テトラプレニル－β－クルクメン環化酵素。     The polypeptide constituting the mutant tetraprenyl-β-curcumen cyclase is
    (1) The 373rd aspartic acid from the N-terminal side in the amino acid sequence represented by SEQ ID NO: 1 is replaced with an amino acid other than aspartic acid, and the 257th tyrosine from the N-terminal side is replaced with an amino acid other than tyrosine. Polypeptide,
    (2) The 373rd aspartic acid from the N-terminal side in the amino acid sequence represented by SEQ ID NO: 1 is replaced with an amino acid other than aspartic acid, and the 257th tyrosine from the N-terminal side is replaced with an amino acid other than tyrosine. One or several amino acids are deleted, substituted, inserted, and / or added in the amino acid sequence, and 8α-hydroxypolypoda-13,17,21-triene is used as a substrate. Polypeptides exhibiting amino acid-forming activity,
    (3) The 373rd aspartic acid from the N-terminal side in the amino acid sequence represented by SEQ ID NO: 1 is replaced with an amino acid other than aspartic acid, and the 257th tyrosine from the N-terminal side is replaced with an amino acid other than tyrosine. A polypeptide consisting of an amino acid sequence having an amino acid sequence of 40% or more identity with that of the amino acid sequence, and exhibiting umbrella-forming activity using 8α-hydroxypolypoda-13,17,21-triene as a substrate.
    (4) The 373rd aspartic acid from the N-terminal side in the amino acid sequence represented by SEQ ID NO: 1 is replaced with an amino acid other than aspartic acid, and the 257th tyrosine from the N-terminal side is replaced with an amino acid other than tyrosine. A polypeptide containing the amino acid sequence of tyrosine and exhibiting umbrella-forming activity using 8α-hydroxypolypoda-13,17,21-triene as a substrate.
    (5) The 373rd asparagic acid from the N-terminal side in the amino acid sequence represented by SEQ ID NO: 1 is replaced with an amino acid other than aspartic acid, and the 257th tyrosine from the N-terminal side is replaced with an amino acid other than tyrosine. In the amino acid sequence, one or several amino acids contain deleted, substituted, inserted, and / or added amino acid sequences, and 8α-hydroxypolypoda-13,17,21-triene is used as a substrate. The polypeptide exhibiting umbrella-forming activity, or (6) the 373th aspartic acid from the N-terminal side in the amino acid sequence represented by SEQ ID NO: 1, is replaced with an amino acid other than aspartic acid, and 257 from the N-terminal side. The amino acid sequence in which the second tyrosine is replaced with an amino acid other than tyrosine is 40% or more identical, and the umbrella is composed of 8α-hydroxypolypoda-13,17,21-triene as a substrate. Polypeptide showing production activity,
    The mutant tetraprenyl-β-curcumen cyclase according to claim 1, or the polypeptide constituting the mutant tetraprenyl-β-curcumen cyclase.
    (7) In the amino acid sequence represented by SEQ ID NO: 1, the 373rd aspartic acid from the N-terminal side is replaced with an amino acid other than aspartic acid, and the 167th tyrosine from the N-terminal side is replaced with an amino acid other than tyrosine. And the 257th tyrosine from the N-terminal side is a polypeptide substituted with an amino acid other than tyrosine,
    (8) In the amino acid sequence represented by SEQ ID NO: 1, the 373rd asparagic acid from the N-terminal side is replaced with an amino acid other than aspartic acid, and the 167th tyrosine from the N-terminal side is replaced with an amino acid other than tyrosine. Consists of 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 257th tyrosine from the N-terminal side is replaced with an amino acid other than tyrosine. Moreover, a polypeptide exhibiting umbrella-forming activity using 8α-hydroxypolypoda-13,17,21-triene as a substrate,
    (9) In the amino acid sequence represented by SEQ ID NO: 1, the 373rd aspartic acid from the N-terminal side is replaced with an amino acid other than aspartic acid, and the 167th tyrosine from the N-terminal side is replaced with an amino acid other than tyrosine. It consists of an amino acid sequence having 40% or more identity with an amino acid sequence in which the 257th tyrosine from the N-terminal side is replaced with an amino acid other than tyrosine, and 8α-hydroxypolypoda-13,17. , A polypeptide showing amino acid-forming activity using 21-triene as a substrate,
    (10) In the amino acid sequence represented by SEQ ID NO: 1, the 373rd aspartic acid from the N-terminal side is replaced with an amino acid other than aspartic acid, and the 167th tyrosine from the N-terminal side is replaced with an amino acid other than tyrosine. It contains an amino acid sequence in which the 257th tyrosine from the N-terminal side is replaced with an amino acid other than tyrosine, and further, it has an umbrella-forming activity using 8α-hydroxypolypoda-13,17,21-triene as a substrate. Polypeptide shown,
    (11) In the amino acid sequence represented by SEQ ID NO: 1, the 373rd asparagic acid from the N-terminal side is replaced with an amino acid other than aspartic acid, and the 167th tyrosine from the N-terminal side is replaced with an amino acid other than tyrosine. Includes an amino acid sequence in which one or several amino acids are deleted, substituted, inserted, and / or added in an amino acid sequence in which the 257th tyrosine from the N-terminal side is replaced with an amino acid other than tyrosine. Moreover, a polypeptide showing umbrella-forming activity using 8α-hydroxypolypoda-13,17,21-triene as a substrate, or (12) the 373rd asparagine from the N-terminal side in the amino acid sequence represented by SEQ ID NO: 1. The acid is replaced with an amino acid other than aspartic acid, the 167th tyrosine from the N-terminal side is replaced with an amino acid other than tyrosine, and the 257th tyrosine from the N-terminal side is replaced with an amino acid other than tyrosine. A polypeptide containing an amino acid sequence having 40% or more identity with the amino acid sequence, and exhibiting umbrella-forming activity using 8α-hydroxypolypoda-13,17,21-triene as a substrate.
    The mutant tetraprenyl-β-curcumen cyclase according to claim 2.
  4. 　前記ＤＸＤＤモチーフの第４番目のアミノ酸残基がアスパラギン酸から、システインに置換しており、前記（Ｄ／Ｎ）Ｇ（Ｔ／Ｌ）（Ｌ／Ｆ／Ｙ）（Ｙ／Ｆ）ＳＹモチーフの７番目のアミノ酸がチロシンからアラニンに置換している、請求項１又は３に記載の変異型テトラプレニル－β－クルクメン環化酵素、あるいは
    前記ＤＸＤＤモチーフの第４番目のアミノ酸残基がアスパラギン酸から、システインに置換しており、前記（Ａ／Ｓ／Ｇ）ＲＸ（Ｈ／Ｎ）ＸＸＰモチーフのＮ末に隣接するアミノ酸がチロシンからアラニンに置換しており、前記（Ｄ／Ｎ）Ｇ（Ｔ／Ｌ）（Ｌ／Ｆ／Ｙ）（Ｙ／Ｆ）ＳＹモチーフの７番目のアミノ酸がチロシンからアラニンに置換している、請求項２又は３に記載の変異型テトラプレニル－β－クルクメン環化酵素。     The fourth amino acid residue of the DXDD motif is replaced with cysteine from aspartic acid, and the (D / N) G (T / L) (L / F / Y) (Y / F) SY motif The mutant tetraprenyl-β-curcumen cyclase according to claim 1 or 3, wherein the 7th amino acid is substituted from tyrosine to alanine, or the 4th amino acid residue of the DXDD motif is from aspartic acid. , Cysteine, and the amino acid adjacent to the N-terminal of the (A / S / G) RX (H / N) XXP motif is replaced from tyrosine to alanine, and the (D / N) G (T) / L) (L / F / Y) (Y / F) The mutant tetraprenyl-β-curcumen cyclization according to claim 2 or 3, wherein the 7th amino acid of the SY motif is substituted from tyrosine to alanine. enzyme.
  5. 　ＤＸＤＤモチーフの第４番目のアミノ酸残基であるアスパラギン酸がアスパラギン酸以外のアミノ酸に置換しており、（Ａ／Ｓ／Ｇ）ＲＸ（Ｈ／Ｎ）ＸＸＰモチーフのＮ末に隣接するアミノ酸がチロシン以外のアミノ酸に置換しており、ＧＸＧＸ（Ｇ／Ａ／Ｐ）モチーフの４番目のアミノ酸がロイシン以外のアミノ酸に置換している変異型テトラプレニル－β－クルクメン環化酵素であって、
    （ａ）前記変異型テトラプレニル－β－クルクメン環化酵素は、前記ＤＸＤＤモチーフを基準として、Ｎ末端側に１００アミノ酸残基以上離れた位置にＱＸＸＸＧＸ（Ｗ／Ｆ）モチーフ、Ｎ末端側に１８０～２５０アミノ酸残基離れた位置に前記（Ａ／Ｓ／Ｇ）ＲＸ（Ｈ／Ｎ）ＸＸＰモチーフ、Ｎ末端側に８０～１４０アミノ酸残基離れた位置に前記（Ｄ／Ｎ）Ｇ（Ｔ／Ｌ）（Ｌ／Ｆ／Ｙ）（Ｙ／Ｆ）ＳＹモチーフ、Ｎ末端側に１０～５０アミノ酸残基離れた位置にＱＸＸＸＸ（Ｇ／Ａ／Ｓ）Ｘ（Ｆ／Ｗ／Ｙ）モチーフ、Ｃ末端側に２０～５０アミノ酸残基離れた位置にＱＸＸＸＧＸ（Ｆ／Ｗ／Ｙ）モチーフ、Ｃ末端側に５０～１２０アミノ酸残基離れた位置にＱＸＸＸＧＸＷモチーフ、Ｃ末端側に１２０～１７０アミノ酸残基離れた位置にＱＸＸＸＧＸ（Ｆ／Ｗ）モチーフ、及びＣ末端側に１８０～２５０アミノ酸残基離れた位置に前記ＧＸＧＸ（Ｇ／Ａ／Ｐ）モチーフを有し、そして
    ＤＸＤＤモチーフを基準として、Ｃ末端側に１７０アミノ酸残基以上離れた位置にＱＸＸＸＧＸＷモチーフを有さず、
    （ｂ）配列番号１で表されるアミノ酸配列との同一性が４０％以上であり、そして
    （ｃ）８α－ヒドロキシポリポダ－１３，１７，２１－トリエンを基質としてアンブレイン生成活性を示す、
    変異型テトラプレニル－β－クルクメン環化酵素。     Aspartic acid, which is the fourth amino acid residue of the DXDD motif, is replaced with an amino acid other than aspartic acid, and the amino acid adjacent to the N-terminal of the (A / S / G) RX (H / N) XPP motif is tyrosine. A mutant tetraprenyl-β-curcumen cyclase in which the fourth amino acid of the GXGX (G / A / P) motif is replaced with an amino acid other than leucine.
    (A) The mutant tetraprenyl-β-curcumen cyclase has a QXXXGX (W / F) motif at a position separated by 100 amino acid residues or more on the N-terminal side and 180 on the N-terminal side based on the DXDD motif. The (A / S / G) RX (H / N) XXP motif is located at a position separated by ~ 250 amino acid residues, and the (D / N) G (T / /) is located at a position separated by 80 to 140 amino acid residues at the N-terminal side. L) (L / F / Y) (Y / F) SY motif, QXXX (G / A / S) X (F / W / Y) motif at a position 10 to 50 amino acid residues away from the N-terminal side, C QXXXGX (F / W / Y) motif at a position 20 to 50 amino acid residues away from the terminal side, QXXXGXW motif at a position 50 to 120 amino acid residues away from the C-terminal side, 120 to 170 amino acid residues at the C-terminal side It has a QXXXGX (F / W) motif at a distant position and the GXGX (G / A / P) motif at a position 180 to 250 amino acid residues distant from the C-terminal side, and the C-terminal is based on the DXDD motif. There is no QXXXGXW motif at a position separated by 170 amino acid residues or more on the side,
    (B) It has an identity of 40% or more with the amino acid sequence represented by SEQ ID NO: 1, and (c) exhibits umbrella-forming activity using 8α-hydroxypolypoda-13,17,21-triene as a substrate.
    Mutant tetraprenyl-β-curcumen cyclase.
  6. 　前記変異型テトラプレニル－β－クルクメン環化酵素を構成するポリペプチドが、
    （１）配列番号１で表されるアミノ酸配列におけるＮ末端側から３７３番目のアスパラギン酸がアスパラギン酸以外のアミノ酸に置換しており、Ｎ末端側から１６７番目のチロシンがチロシン以外のアミノ酸に置換しており、そしてＮ末端側から５９６番目のロイシンがロイシン以外のアミノ酸に置換しているポリペプチド、
    （２）配列番号１で表されるアミノ酸配列におけるＮ末端側から３７３番目のアスパラギン酸がアスパラギン酸以外のアミノ酸に置換しており、Ｎ末端側から１６７番目のチロシンがチロシン以外のアミノ酸に置換しており、そしてＮ末端側から５９６番目のロイシンがロイシン以外のアミノ酸に置換しているアミノ酸配列において、１又は数個のアミノ酸が欠失、置換、挿入、及び／又は付加されたアミノ酸配列からなり、しかも、８α－ヒドロキシポリポダ－１３，１７，２１－トリエンを基質としてアンブレイン生成活性を示すポリペプチド、
    （３）配列番号１で表されるアミノ酸配列におけるＮ末端側から３７３番目のアスパラギン酸がアスパラギン酸以外のアミノ酸に置換しており、Ｎ末端側から１６７番目のチロシンがチロシン以外のアミノ酸に置換しており、そしてＮ末端側から５９６番目のロイシンがロイシン以外のアミノ酸に置換しているアミノ酸配列との同一性が４０％以上であるアミノ酸配列からなり、しかも、８α－ヒドロキシポリポダ－１３，１７，２１－トリエンを基質としてアンブレイン生成活性を示すポリペプチド、
    （４）配列番号１で表されるアミノ酸配列におけるＮ末端側から３７３番目のアスパラギン酸がアスパラギン酸以外のアミノ酸に置換しており、Ｎ末端側から１６７番目のチロシンがチロシン以外のアミノ酸に置換しており、そしてＮ末端側から５９６番目のロイシンがロイシン以外のアミノ酸に置換しているアミノ酸配列を含み、しかも、８α－ヒドロキシポリポダ－１３，１７，２１－トリエンを基質としてアンブレイン生成活性を示すポリペプチド、
    （５）配列番号１で表されるアミノ酸配列におけるＮ末端側から３７３番目のアスパラギン酸がアスパラギン酸以外のアミノ酸に置換しており、Ｎ末端側から１６７番目のチロシンがチロシン以外のアミノ酸に置換しており、そしてＮ末端側から５９６番目のロイシンがロイシン以外のアミノ酸に置換しているアミノ酸配列において、１又は数個のアミノ酸が欠失、置換、挿入、及び／又は付加されたアミノ酸配列を含み、しかも、８α－ヒドロキシポリポダ－１３，１７，２１－トリエンを基質としてアンブレイン生成活性を示すポリペプチド、又は
    （６）配列番号１で表されるアミノ酸配列におけるＮ末端側から３７３番目のアスパラギン酸がアスパラギン酸以外のアミノ酸に置換しており、Ｎ末端側から１６７番目のチロシンがチロシン以外のアミノ酸に置換しており、そしてＮ末端側から５９６番目のロイシンがロイシン以外のアミノ酸に置換しているアミノ酸配列との同一性が４０％以上であるアミノ酸配列を含み、しかも、８α－ヒドロキシポリポダ－１３，１７，２１－トリエンを基質としてアンブレイン生成活性を示すポリペプチド
    である、請求項５に記載の変異型テトラプレニル－β－クルクメン環化酵素。     The polypeptide constituting the mutant tetraprenyl-β-curcumen cyclase is
    (1) In the amino acid sequence represented by SEQ ID NO: 1, the 373rd aspartic acid from the N-terminal side is replaced with an amino acid other than aspartic acid, and the 167th tyrosine from the N-terminal side is replaced with an amino acid other than tyrosine. And a polypeptide in which leucine at position 596 from the N-terminal side is replaced with an amino acid other than leucine,
    (2) In the amino acid sequence represented by SEQ ID NO: 1, the 373rd asparagic acid from the N-terminal side is replaced with an amino acid other than aspartic acid, and the 167th tyrosine from the N-terminal side is replaced with an amino acid other than tyrosine. Consists of 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 596th leucine from the N-terminal side is replaced with an amino acid other than leucine. Moreover, a polypeptide exhibiting umbrella-forming activity using 8α-hydroxypolypoda-13,17,21-triene as a substrate,
    (3) In the amino acid sequence represented by SEQ ID NO: 1, the 373rd aspartic acid from the N-terminal side is replaced with an amino acid other than aspartic acid, and the 167th tyrosine from the N-terminal side is replaced with an amino acid other than tyrosine. It consists of an amino acid sequence having 40% or more identity with the amino acid sequence in which leucine at the 596th position from the N-terminal side is replaced with an amino acid other than leucine, and 8α-hydroxypolypoda-13,17. , A polypeptide showing amino acid-forming activity using 21-triene as a substrate,
    (4) In the amino acid sequence represented by SEQ ID NO: 1, the 373rd aspartic acid from the N-terminal side is replaced with an amino acid other than aspartic acid, and the 167th tyrosine from the N-terminal side is replaced with an amino acid other than tyrosine. It contains an amino acid sequence in which leucine at the 596th position from the N-terminal side is replaced with an amino acid other than leucine, and further, it exhibits umbrella-forming activity using 8α-hydroxypolypoda-13,17,21-triene as a substrate. Polypeptide shown,
    (5) In the amino acid sequence represented by SEQ ID NO: 1, the 373rd asparagic acid from the N-terminal side is replaced with an amino acid other than aspartic acid, and the 167th tyrosine from the N-terminal side is replaced with an amino acid other than tyrosine. Includes 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 leucine at position 596 from the N-terminal side is replaced with an amino acid other than leucine. Moreover, a polypeptide showing umbrella-forming activity using 8α-hydroxypolypoda-13,17,21-triene as a substrate, or (6) the 373rd asparagine from the N-terminal side in the amino acid sequence represented by SEQ ID NO: 1. The acid is replaced with an amino acid other than aspartic acid, the 167th tyrosine from the N-terminal side is replaced with an amino acid other than tyrosine, and the 596th leucine from the N-terminal side is replaced with an amino acid other than leucine. 5. A polypeptide containing an amino acid sequence having 40% or more identity with the amino acid sequence, and exhibiting umbrella-forming activity using 8α-hydroxypolypoda-13,17,21-triene as a substrate. The mutant tetraprenyl-β-curcumen cyclase according to.
  7. 　前記ＤＸＤＤモチーフの第４番目のアミノ酸残基がアスパラギン酸から、システインに置換しており、前記（Ａ／Ｓ／Ｇ）ＲＸ（Ｈ／Ｎ）ＸＸＰモチーフのＮ末に隣接するアミノ酸がチロシンからアラニンに置換しており、そしてＧＸＧＸ（Ｇ／Ａ／Ｐ）モチーフの４番目のアミノ酸がロイシンからアラニンに置換している、請求項５又は６に記載の変異型テトラプレニル－β－クルクメン環化酵素。 The fourth amino acid residue of the DXDD motif is replaced with cysteine from aspartic acid, and the amino acid adjacent to the N-terminal of the (A / S / G) RX (H / N) XPP motif is tyrosine to alanine. The mutant tyrosine-β-curcumen cyclase according to claim 5 or 6, wherein the fourth amino acid of the GXGX (G / A / P) motif is substituted from leucine to alanine. ..
  8. 　請求項１～７のいずれか一項に記載の変異型テトラプレニル－β－クルクメン環化酵素をコードするポリヌクレオチド。 A polynucleotide encoding the mutant tetraprenyl-β-curcumen cyclase according to any one of claims 1 to 7.
  9. 　請求項８に記載のポリヌクレオチドを有する微生物。 A microorganism having the polynucleotide according to claim 8.
  10. 　請求項８に記載のポリヌクレオチドを持つＤＮＡを含むベクター。 A vector containing a DNA having the polynucleotide according to claim 8.
  11. 　請求項１０に記載のベクターを有する形質転換体。 A transformant having the vector according to claim 10.
  12. 　請求項１～７のいずれか一項に記載の変異型テトラプレニル－β－クルクメン環化酵素をスクアレン又は８α－ヒドロキシポリポダ－１３，１７，２１－トリエンに反応させて、アンブレインを得ることを特徴とする、アンブレインの製造方法。 The mutant tetraprenyl-β-curcumen cyclase according to any one of claims 1 to 7 is reacted with squalene or 8α-hydroxypolypoda-13,17,21-triene to obtain an umbrella. A method for manufacturing an umbrella, which is characterized by.
  13. 請求項９に記載の微生物、又は請求項１１に記載の形質転換体を培養することを特徴とする、アンブレインの製造方法。 A method for producing an umbrella, which comprises culturing the microorganism according to claim 9 or the transformant according to claim 11.
  14. 　（１）テトラプレニル－β－クルクメン環化酵素をスクアレンに反応させて、８α－ヒドロキシポリポダ－１３，１７，２１－トリエンを得る工程、及び
    （２）変異型テトラプレニル－β－クルクメン環化酵素を８α－ヒドロキシポリポダ－１３，１７，２１－トリエンに反応させて、アンブレインを得る工程、
    を含むアンブレインの製造方法であって、
    前記変異型テトラプレニル－β－クルクメン環化酵素が、
    （Ａ）請求項１～７のいずれか一項に記載の変異型テトラプレニル－β－クルクメン環化酵素、又は
    （Ｂ）ＤＸＤＤモチーフの第４番目のアミノ酸残基であるアスパラギン酸がアスパラギン酸以外のアミノ酸に置換しており、そして（Ａ／Ｓ／Ｇ）ＲＸ（Ｈ／Ｎ）ＸＸＰモチーフのＮ末に隣接するアミノ酸がチロシン以外のアミノ酸に置換しているか、又はＧＸＧＸ（Ｇ／Ａ／Ｐ）モチーフの４番目のアミノ酸がロイシン以外のアミノ酸に置換している変異型テトラプレニル－β－クルクメン環化酵素であって、
    （ａ）前記変異型テトラプレニル－β－クルクメン環化酵素は、前記ＤＸＤＤモチーフを基準として、Ｎ末端側に１００アミノ酸残基以上離れた位置にＱＸＸＸＧＸ（Ｗ／Ｆ）モチーフ、Ｎ末端側に１８０～２５０アミノ酸残基離れた位置に前記（Ａ／Ｓ／Ｇ）ＲＸ（Ｈ／Ｎ）ＸＸＰモチーフ、Ｎ末端側に８０～１４０アミノ酸残基離れた位置に前記（Ｄ／Ｎ）Ｇ（Ｔ／Ｌ）（Ｌ／Ｆ／Ｙ）（Ｙ／Ｆ）ＳＹモチーフ、Ｎ末端側に１０～５０アミノ酸残基離れた位置にＱＸＸＸＸ（Ｇ／Ａ／Ｓ）Ｘ（Ｆ／Ｗ／Ｙ）モチーフ、Ｃ末端側に２０～５０アミノ酸残基離れた位置にＱＸＸＸＧＸ（Ｆ／Ｗ／Ｙ）モチーフ、Ｃ末端側に５０～１２０アミノ酸残基離れた位置にＱＸＸＸＧＸＷモチーフ、Ｃ末端側に１２０～１７０アミノ酸残基離れた位置にＱＸＸＸＧＸ（Ｆ／Ｗ）モチーフ、及びＣ末端側に１８０～２５０アミノ酸残基離れた位置に前記ＧＸＧＸ（Ｇ／Ａ／Ｐ）モチーフを有し、そして
    ＤＸＤＤモチーフを基準として、Ｃ末端側に１７０アミノ酸残基以上離れた位置にＱＸＸＸＧＸＷモチーフを有さず、
    （ｂ）配列番号１で表されるアミノ酸配列との同一性が４０％以上であり、そして
    （ｃ）８α－ヒドロキシポリポダ－１３，１７，２１－トリエンを基質としてアンブレイン生成活性を示す、変異型テトラプレニル－β－クルクメン環化酵素、
    であるアンブレインの製造方法。     (1) A step of reacting tetraprenyl-β-curcumen cyclase with squalene to obtain 8α-hydroxypolypoda-13,17,21-triene, and (2) mutant tetraprenyl-β-curcumen cyclization. A step of reacting an enzyme with 8α-hydroxypolypoda-13,17,21-triene to obtain squalene,
    It is a manufacturing method of umbrella including
    The mutant tetraprenyl-β-curcumen cyclase
    (A) The mutant tetraprenyl-β-curcumen cyclase according to any one of claims 1 to 7, or (B) aspartic acid, which is the fourth amino acid residue of the DXDD motif, is other than aspartic acid. And the amino acid adjacent to the N-terminal of the (A / S / G) RX (H / N) XXP motif is replaced with an amino acid other than tyrosine, or GXGX (G / A / P) ) A mutant tetraprenyl-β-curcumen cyclase in which the fourth amino acid of the motif is replaced with an amino acid other than leucine.
    (A) The mutant tetraprenyl-β-curcumen cyclase has a QXXXGX (W / F) motif at a position separated by 100 amino acid residues or more on the N-terminal side and 180 on the N-terminal side based on the DXDD motif. The (A / S / G) RX (H / N) XXP motif is located at a position separated by ~ 250 amino acid residues, and the (D / N) G (T / /) is located at a position separated by 80 to 140 amino acid residues at the N-terminal side. L) (L / F / Y) (Y / F) SY motif, QXXX (G / A / S) X (F / W / Y) motif at a position 10 to 50 amino acid residues away from the N-terminal side, C QXXXGX (F / W / Y) motif at a position 20 to 50 amino acid residues away from the terminal side, QXXXGXW motif at a position 50 to 120 amino acid residues away from the C-terminal side, 120 to 170 amino acid residues at the C-terminal side It has a QXXXGX (F / W) motif at a distant position and the GXGX (G / A / P) motif at a position 180 to 250 amino acid residues distant from the C-terminal side, and the C-terminal is based on the DXDD motif. There is no QXXXGXW motif at a position separated by 170 amino acid residues or more on the side,
    (B) It has an identity of 40% or more with the amino acid sequence represented by SEQ ID NO: 1, and (c) exhibits umbrella-forming activity using 8α-hydroxypolypoda-13,17,21-triene as a substrate. Variant tetraprenyl-β-curcumen cyclase,
    How to make an umbrella.
  15. 　前記変異型テトラプレニル－β－クルクメン環化酵素（Ｂ）を構成するポリペプチドが、
    （１）配列番号１で表されるアミノ酸配列におけるＮ末端側から３７３番目のアスパラギン酸がアスパラギン酸以外のアミノ酸に置換しており、そしてＮ末端側から１６７番目のチロシンがチロシン以外のアミノ酸に置換しているポリペプチド、
    （２）配列番号１で表されるアミノ酸配列におけるＮ末端側から３７３番目のアスパラギン酸がアスパラギン酸以外のアミノ酸に置換しており、そしてＮ末端側から１６７番目のチロシンがチロシン以外のアミノ酸に置換しているアミノ酸配列において、１又は数個のアミノ酸が欠失、置換、挿入、及び／又は付加されたアミノ酸配列からなり、しかも、８α－ヒドロキシポリポダ－１３，１７，２１－トリエンを基質としてアンブレイン生成活性を示すポリペプチド、
    （３）配列番号１で表されるアミノ酸配列におけるＮ末端側から３７３番目のアスパラギン酸がアスパラギン酸以外のアミノ酸に置換しており、そしてＮ末端側から１６７番目のチロシンがチロシン以外のアミノ酸に置換しているアミノ酸配列との同一性が４０％以上であるアミノ酸配列からなり、しかも、８α－ヒドロキシポリポダ－１３，１７，２１－トリエンを基質としてアンブレイン生成活性を示すポリペプチド、
    （４）配列番号１で表されるアミノ酸配列におけるＮ末端側から３７３番目のアスパラギン酸がアスパラギン酸以外のアミノ酸に置換しており、そしてＮ末端側から１６７番目のチロシンがチロシン以外のアミノ酸に置換しているアミノ酸配列を含み、しかも、８α－ヒドロキシポリポダ－１３，１７，２１－トリエンを基質としてアンブレイン生成活性を示すポリペプチド、
    （５）配列番号１で表されるアミノ酸配列におけるＮ末端側から３７３番目のアスパラギン酸がアスパラギン酸以外のアミノ酸に置換しており、そしてＮ末端側から１６７番目のチロシンがチロシン以外のアミノ酸に置換しているアミノ酸配列において、１又は数個のアミノ酸が欠失、置換、挿入、及び／又は付加されたアミノ酸配列を含み、しかも、８α－ヒドロキシポリポダ－１３，１７，２１－トリエンを基質としてアンブレイン生成活性を示すポリペプチド、
    （６）配列番号１で表されるアミノ酸配列におけるＮ末端側から３７３番目のアスパラギン酸がアスパラギン酸以外のアミノ酸に置換しており、そしてＮ末端側から１６７番目のチロシンがチロシン以外のアミノ酸に置換しているアミノ酸配列との同一性が４０％以上であるアミノ酸配列を含み、しかも、８α－ヒドロキシポリポダ－１３，１７，２１－トリエンを基質としてアンブレイン生成活性を示すポリペプチド、
    （７）配列番号１で表されるアミノ酸配列におけるＮ末端側から３７３番目のアスパラギン酸がアスパラギン酸以外のアミノ酸に置換しており、そしてＮ末端側から５９６番目のロイシンがロイシン以外のアミノ酸に置換しているポリペプチド、
    （８）配列番号１で表されるアミノ酸配列におけるＮ末端側から３７３番目のアスパラギン酸がアスパラギン酸以外のアミノ酸に置換しており、そしてＮ末端側から５９６番目のロイシンがロイシン以外のアミノ酸に置換しているアミノ酸配列において、１又は数個のアミノ酸が欠失、置換、挿入、及び／又は付加されたアミノ酸配列からなり、しかも、８α－ヒドロキシポリポダ－１３，１７，２１－トリエンを基質としてアンブレイン生成活性を示すポリペプチド、
    （９）配列番号１で表されるアミノ酸配列におけるＮ末端側から３７３番目のアスパラギン酸がアスパラギン酸以外のアミノ酸に置換しており、そしてＮ末端側から５９６番目のロイシンがロイシン以外のアミノ酸に置換しているアミノ酸配列との同一性が４０％以上であるアミノ酸配列からなり、しかも、８α－ヒドロキシポリポダ－１３，１７，２１－トリエンを基質としてアンブレイン生成活性を示すポリペプチド、
    （１０）配列番号１で表されるアミノ酸配列におけるＮ末端側から３７３番目のアスパラギン酸がアスパラギン酸以外のアミノ酸に置換しており、そしてＮ末端側から５９６番目のロイシンがロイシン以外のアミノ酸に置換しているアミノ酸配列を含み、しかも、８α－ヒドロキシポリポダ－１３，１７，２１－トリエンを基質としてアンブレイン生成活性を示すポリペプチド、
    （１１）配列番号１で表されるアミノ酸配列におけるＮ末端側から３７３番目のアスパラギン酸がアスパラギン酸以外のアミノ酸に置換しており、そしてＮ末端側から５９６番目のロイシンがロイシン以外のアミノ酸に置換しているアミノ酸配列において、１又は数個のアミノ酸が欠失、置換、挿入、及び／又は付加されたアミノ酸配列を含み、しかも、８α－ヒドロキシポリポダ－１３，１７，２１－トリエンを基質としてアンブレイン生成活性を示すポリペプチド、又は
    （１２）配列番号１で表されるアミノ酸配列におけるＮ末端側から３７３番目のアスパラギン酸がアスパラギン酸以外のアミノ酸に置換しており、そしてＮ末端側から５９６番目のロイシンがロイシン以外のアミノ酸に置換しているアミノ酸配列との同一性が４０％以上であるアミノ酸配列を含み、しかも、８α－ヒドロキシポリポダ－１３，１７，２１－トリエンを基質としてアンブレイン生成活性を示すポリペプチド、
    である、請求項１４に記載のアンブレインの製造方法。     The polypeptide constituting the mutant tetraprenyl-β-curcumen cyclase (B) is
    (1) The 373rd aspartic acid from the N-terminal side in the amino acid sequence represented by SEQ ID NO: 1 is replaced with an amino acid other than aspartic acid, and the 167th tyrosine from the N-terminal side is replaced with an amino acid other than tyrosine. Polypeptide,
    (2) The 373rd aspartic acid from the N-terminal side in the amino acid sequence represented by SEQ ID NO: 1 is replaced with an amino acid other than aspartic acid, and the 167th tyrosine from the N-terminal side is replaced with an amino acid other than tyrosine. One or several amino acids are deleted, substituted, inserted, and / or added in the amino acid sequence, and 8α-hydroxypolypoda-13,17,21-triene is used as a substrate. Polypeptides exhibiting amino acid-forming activity,
    (3) The 373rd aspartic acid from the N-terminal side in the amino acid sequence represented by SEQ ID NO: 1 is replaced with an amino acid other than aspartic acid, and the 167th tyrosine from the N-terminal side is replaced with an amino acid other than tyrosine. A polypeptide consisting of an amino acid sequence having an amino acid sequence of 40% or more identity with that of the amino acid sequence, and exhibiting umbrella-forming activity using 8α-hydroxypolypoda-13,17,21-triene as a substrate.
    (4) The 373rd aspartic acid from the N-terminal side in the amino acid sequence represented by SEQ ID NO: 1 is replaced with an amino acid other than aspartic acid, and the 167th tyrosine from the N-terminal side is replaced with an amino acid other than tyrosine. A polypeptide containing the amino acid sequence of tyrosine and exhibiting umbrella-forming activity using 8α-hydroxypolypoda-13,17,21-triene as a substrate.
    (5) The 373rd aspartic acid from the N-terminal side in the amino acid sequence represented by SEQ ID NO: 1 is replaced with an amino acid other than aspartic acid, and the 167th tyrosine from the N-terminal side is replaced with an amino acid other than tyrosine. In the amino acid sequence, one or several amino acids contain deleted, substituted, inserted, and / or added amino acid sequences, and 8α-hydroxypolypoda-13,17,21-triene is used as a substrate. Polypeptides exhibiting amino acid-forming activity,
    (6) The 373rd aspartic acid from the N-terminal side in the amino acid sequence represented by SEQ ID NO: 1 is replaced with an amino acid other than aspartic acid, and the 167th tyrosine from the N-terminal side is replaced with an amino acid other than tyrosine. A polypeptide containing an amino acid sequence having an amino acid sequence of 40% or more identity with that of the amino acid sequence, and exhibiting umbrella-forming activity using 8α-hydroxypolypoda-13,17,21-triene as a substrate.
    (7) The 373rd aspartic acid from the N-terminal side in the amino acid sequence represented by SEQ ID NO: 1 is replaced with an amino acid other than aspartic acid, and the 596th leucine from the N-terminal side is replaced with an amino acid other than leucine. Polypeptide,
    (8) The 373rd aspartic acid from the N-terminal side in the amino acid sequence represented by SEQ ID NO: 1 is replaced with an amino acid other than aspartic acid, and the 596th leucine from the N-terminal side is replaced with an amino acid other than leucine. One or several amino acids are deleted, substituted, inserted, and / or added in the amino acid sequence, and 8α-hydroxypolypoda-13,17,21-triene is used as a substrate. Polypeptides exhibiting amino acid-forming activity,
    (9) Aspartic acid at position 373 from the N-terminal side in the amino acid sequence represented by SEQ ID NO: 1 is replaced with an amino acid other than aspartic acid, and leucine at position 596 from the N-terminal side is replaced with an amino acid other than leucine. A polypeptide consisting of an amino acid sequence having an amino acid sequence of 40% or more identity with that of the amino acid sequence, and exhibiting umbrella-forming activity using 8α-hydroxypolypoda-13,17,21-triene as a substrate.
    (10) Aspartic acid at position 373 from the N-terminal side in the amino acid sequence represented by SEQ ID NO: 1 is replaced with an amino acid other than aspartic acid, and leucine at position 596 from the N-terminal side is replaced with an amino acid other than leucine. A polypeptide containing the amino acid sequence of 8α-hydroxypolypoda-13,17,21-triene and exhibiting umbrella-forming activity using 8α-hydroxypolypoda-13,17,21-triene as a substrate.
    (11) The 373rd asparagic acid from the N-terminal side in the amino acid sequence represented by SEQ ID NO: 1 is replaced with an amino acid other than aspartic acid, and the 596th leucine from the N-terminal side is replaced with an amino acid other than leucine. In the amino acid sequence, one or several amino acids contain deleted, substituted, inserted, and / or added amino acid sequences, and 8α-hydroxypolypoda-13,17,21-triene is used as a substrate. The 373th asparagic acid from the N-terminal side in the polypeptide exhibiting umbrella-forming activity or (12) the amino acid sequence represented by SEQ ID NO: 1 is replaced with an amino acid other than aspartic acid, and 596 from the N-terminal side. The second leucine contains an amino acid sequence having 40% or more identity with an amino acid sequence in which an amino acid other than leucine is substituted, and an amino acid using 8α-hydroxypolypoda-13,17,21-triene as a substrate is used as an umbrella. Polypeptide showing production activity,
    The method for producing an umbrella according to claim 14.
  16. 　前記変異型テトラプレニル－β－クルクメン環化酵素（Ｂ）が、
    前記ＤＸＤＤモチーフの第４番目のアミノ酸残基がアスパラギン酸から、システインに置換しており、そして前記（Ａ／Ｓ／Ｇ）ＲＸ（Ｈ／Ｎ）ＸＸＰモチーフのＮ末に隣接するアミノ酸がチロシンからアラニンに置換している変異型テトラプレニル－β－クルクメン環化酵素、又は
    前記ＤＸＤＤモチーフの第４番目のアミノ酸残基がアスパラギン酸から、システインに置換しており、そしてＧＸＧＸ（Ｇ／Ａ／Ｐ）モチーフの４番目のアミノ酸がロイシンからアラニンに置換している変異型テトラプレニル－β－クルクメン環化酵素
    である、請求項１４又は１５に記載のアンブレインの製造方法。     The mutant tetraprenyl-β-curcumen cyclase (B)
    The fourth amino acid residue of the DXDD motif is replaced with cysteine from aspartic acid, and the amino acid adjacent to the N-terminal of the (A / S / G) RX (H / N) XPP motif is from tyrosine. The mutant tetraprenyl-β-curcumen cyclase substituting alanine, or the fourth amino acid residue of the DXDD motif, is substituting cysteine from aspartic acid, and GXGX (G / A / P). ) The method for producing an umbrella according to claim 14 or 15, wherein the fourth amino acid of the motif is a mutant tetraprenyl-β-curcumen cyclase in which leucine is replaced with alanine.
  17. 　前記工程（１）及び工程（２）を同時に行う、請求項１４～１６のいずれか一項に記載のアンブレインの製造方法。 The method for producing an umbrella according to any one of claims 14 to 16, wherein the steps (1) and (2) are performed at the same time.
  18. 　前記テトラプレニル－β－クルクメン環化酵素が野生型テトラプレニル－β－クルクメン環化酵素である、請求項１４～１７のいずれか一項に記載のアンブレインの製造方法。 The method for producing an umbrella according to any one of claims 14 to 17, wherein the tetraprenyl-β-curcumen cyclase is a wild-type tetraprenyl-β-curcumen cyclase.
PCT/JP2021/012483 2020-03-25 2021-03-25 MUTANT TETRAPRENYL-β-CURCUMENE CYCLASE AND METHOD FOR PRODUCING AMBREIN USING SAME WO2021193806A1 (en)

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WO2015033746A1 (en) * 2013-09-05 2015-03-12 国立大学法人新潟大学 Method for producing ambrein
WO2017150695A1 (en) * 2016-03-04 2017-09-08 国立大学法人新潟大学 VARIANT TYPE TETRAPRENYL-β-CURCUMENE CYCLASE AND METHOD FOR PRODUCING AMBREIN
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WO2017150695A1 (en) * 2016-03-04 2017-09-08 国立大学法人新潟大学 VARIANT TYPE TETRAPRENYL-β-CURCUMENE CYCLASE AND METHOD FOR PRODUCING AMBREIN
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