Method for producing sclareol glycol

Abstract

A purpose of the present invention is to provide: a more efficient and environmentally friendly method for producing a novel ambroxide precursor; and an enzyme therefor. The present invention provides a polypeptide having a cyclization activity and comprising (i) the amino acid sequence represented by SEQ ID NO: 1, (ii) an amino acid sequence having 95% or more identity to the amino acid sequence represented by SEQ ID NO: 1, or (iii) an amino acid sequence derived from the amino acid sequence represented by SEQ ID NO: 1 by substitution, deletion, insertion, or addition of one to several amino acids and having at least one mutation selected from the group consisting of the following (a) to (d): (a) a mutation in which tyrosine at position 319 of the amino acid sequence of SEQ ID NO: 1 or a position corresponding thereto is substituted with phenylalanine; (b) a mutation in which phenylalanine at position 509 of the amino acid sequence of SEQ ID NO: 1 or a position corresponding thereto is substituted with tyrosine; (c) a mutation in which alanine at position 511 of the amino acid sequence of SEQ ID NO: 1 or a position corresponding thereto is substituted with tyrosine or phenylalanine; and (d) a mutation in which phenylalanine at position 518 of the amino acid sequence of SEQ ID NO: 1 or a position corresponding thereto is substituted with alanine, serine, or histidine.

Description

Method for producing sclareol glycol

[0001] The present invention relates to a method for producing an ambroxide precursor (sclareol glycol), a compound that can be used as a starting material in the production method, and an enzyme that can be used in the production method.

[0002] Ambroxide is an economically important chemical substance of extremely high value as a fragrance. Currently, ambroxide is manufactured from naturally derived starting materials, and the supply of these starting materials depends on climatic conditions and socioeconomic factors. Therefore, in order to ensure a stable industrial supply of ambroxide, which is economically important, a more cost-effective and industrializable manufacturing method is needed. A known method for producing ambroxide uses sclareol as a starting material and proceeds via sclareolide, but this method has problems such as using natural products as raw materials and requiring multi-step conversion (Non-Patent Document 1). On the other hand, a method using homofarnesol as a starting material and a cyclase is also known, but this method has the problem of a low conversion rate (Patent Documents 1-3). Sclareol glycol, an ambroxide precursor, is known to be able to be produced from sclareol using microorganisms (Patent Documents 4-6). Numerous reaction conditions have also been reported for the conversion from sclareol glycol to ambroxide (Patent Documents 7, 8, Non-Patent Document 1).

[0003]

[0004] Japanese Patent Publication No. 2009-060799, Japanese Patent Publication No. 2012-528578, Japanese Patent Publication No. 2018-513691, Japanese Patent Publication No. 2007-252365, Japanese Patent Publication No. 2008-029252, Japanese Patent Publication No. 2008-212087, Japanese Patent Publication No. 62-74281, Japanese Patent Publication No. 3-224478

[0005] "Synthetic Fragrance Chemistry and Product Knowledge, Revised and Expanded Edition," Chemical Daily Co., Ltd., 2016, pp. 390-392. ACS Chem. Biol. 2022, 17, 1226-1238.

[0006] Conventional ambroxide manufacturing methods suffer from low efficiency and yield, as well as high production costs. In particular, the complex reaction process makes them unsuitable for commercial-scale production. Furthermore, the production process can generate harmful by-products, posing a significant environmental burden. Additionally, the low utilization efficiency of raw materials makes cost reduction difficult. To address these challenges and stabilize market supply, a novel, more efficient, and environmentally friendly manufacturing method is needed.

[0007] In view of the above issues, the inventors focused on raw materials and enzymes used in order to develop a manufacturing method different from the conventional technology. The inventors have previously discovered and reported a cyclase derived from the marine microorganism Aquimarina spongiae (Aquimarina spongiae drimenol synthase: AsDMS) (Non-Patent Literature 2). AsDMS is mainly involved in the biosynthesis of drimenol (a cyclic sesquiterpene). AsDMS efficiently cyclizes and dephosphorylates farnesyl diphosphate (FPP) to produce drimenol, and its potent enzymatic activity suggests that it may be applicable to the industrial production of terpenes. Therefore, the inventors diligently researched a method for producing ambroxide using this enzyme that differs from the conventional technology. As a result, they succeeded in producing an ambroxide precursor from a homofarnesol derivative in one step using a mutant of AsDMS, thus completing the present invention.

[0008] That is, the present invention provides the following: Item 1. A polypeptide having cyclization activity, comprising an amino acid sequence having at least one mutation selected from the group consisting of (a) to (d) below, in which (i) the amino acid sequence represented by SEQ ID NO: 1, (ii) an amino acid sequence having 95% or more identity with the amino acid sequence represented by SEQ ID NO: 1, or (iii) an amino acid sequence having one to several amino acid substitutions, deletions, insertions or additions in the amino acid sequence represented by SEQ ID NO: 1: (a) A mutation in which tyrosine at position 319 or a corresponding position in the amino acid sequence of SEQ ID NO: 1 is substituted with phenylalanine. (b) A mutation in which phenylalanine at position 509 or a corresponding position in the amino acid sequence of SEQ ID NO: 1 is substituted with tyrosine. (c) A mutation in which alanine at position 511 or a corresponding position in the amino acid sequence of SEQ ID NO: 1 is substituted with tyrosine or phenylalanine. (d) A mutation in which phenylalanine at position 518 or a corresponding position in the amino acid sequence of SEQ ID NO: 1 is substituted with alanine, serine or histidine. Item 2. A polypeptide according to claim 1, wherein at least two mutations are selected from the group consisting of (a) to (d). Claim 3. A polypeptide according to claim 1, wherein at least three mutations are selected from the group consisting of (a) to (d). Claim 4. A polypeptide according to claim 1, having all of the mutations (a) to (d). Claim 5. A polypeptide according to any one of claims 1 to 4, further having (e) a mutation in which arginine at position 513 or equivalent in the amino acid sequence of SEQ ID NO: 1 is replaced with valine, histidine, alanine, threonine, proline, or aspartic acid, and / or (f) a mutation in which serine at position 514 or equivalent in the amino acid sequence of SEQ ID NO: 1 is replaced with threonine, isoleucine, cysteine, or valine. Claim 6. A polypeptide according to any one of claims 1 to 5, having all of the mutations (a) to (f). Claim 7. Furthermore, (g-1) a mutation in which leucine at position 44 or equivalent in the amino acid sequence of SEQ ID NO: 1 is replaced with cysteine, glutamine, or alanine.(g-2) A mutation in which leucine at position 49 or equivalent in the amino acid sequence of SEQ ID NO: 1 is replaced by cysteine, isoleucine, threonine, or serine. (g-3) A mutation in which tryptophan at position 51 or equivalent in the amino acid sequence of SEQ ID NO: 1 is replaced by isoleucine, phenylalanine, or leucine. (g-4) A mutation in which valine at position 70 or equivalent in the amino acid sequence of SEQ ID NO: 1 is replaced by isoleucine, leucine, phenylalanine, or tyrosine. (g-5) A mutation in which leucine at position 107 or equivalent in the amino acid sequence of SEQ ID NO: 1 is replaced by valine. (g-6) A mutation in which isoleucine at position 111 or equivalent in the amino acid sequence of SEQ ID NO: 1 is replaced by phenylalanine, serine, asparagine, leucine, valine, or threonine. (g-7) A mutation in which leucine at position 114 or equivalent in the amino acid sequence of SEQ ID NO: 1 is replaced by valine or phenylalanine. (g-8) A mutation in which valine at position 140 or equivalent in the amino acid sequence of SEQ ID NO: 1 is replaced with glutamine, asparagine, cysteine, or serine. (g-9) A mutation in which serine at position 144 or equivalent in the amino acid sequence of SEQ ID NO: 1 is replaced with valine, leucine, aspartic acid, histidine, or tyrosine. (g-10) A mutation in which leucine at position 148 or equivalent in the amino acid sequence of SEQ ID NO: 1 is replaced with threonine, isoleucine, cysteine, or serine. (g-11) A mutation in which valine at position 264 or equivalent in the amino acid sequence of SEQ ID NO: 1 is replaced with leucine, threonine, cysteine, asparagine, or glutamine. (g-12) A mutation in which cysteine ​​at position 317 or equivalent in the amino acid sequence of SEQ ID NO: 1 is replaced with asparagine or glutamine. (g-13) A mutation in which phenylalanine at position 318 or equivalent in the amino acid sequence of SEQ ID NO: 1 is replaced by tyrosine. (g-14) A mutation in which phenylalanine at position 328 or equivalent in the amino acid sequence of SEQ ID NO: 1 is replaced by tyrosine or histidine.(g-15) A mutation in which proline at position 512 or equivalent in the amino acid sequence of SEQ ID NO: 1 is replaced with tryptophan or aspartic acid. (g-16) A mutation in which threonine at position 516 or equivalent in the amino acid sequence of SEQ ID NO: 1 is replaced with arginine or histidine. Polypeptide according to any one of claims 1 to 6. Claim 8. A polypeptide according to any one of claims 1 to 7, having (a) a mutation in which tyrosine at position 319 or equivalent in the amino acid sequence of SEQ ID NO: 1 is replaced with phenylalanine. (b) A mutation in which phenylalanine at position 509 or equivalent in the amino acid sequence of SEQ ID NO: 1 is replaced with tyrosine. (c) A mutation in which alanine at position 511 or equivalent in the amino acid sequence of SEQ ID NO: 1 is replaced with tyrosine or phenylalanine. (d) A mutation in which phenylalanine at position 518 or equivalent in the amino acid sequence of SEQ ID NO: 1 is replaced with alanine, serine or histidine. Claim 9. The polypeptide according to any one of claims 1 to 8, wherein the mutation in (c) is a substitution of alanine to tyrosine at position 511 or equivalent in the amino acid sequence of SEQ ID NO: 1. Claim 10. The polypeptide according to any one of claims 1 to 9, wherein the mutation in (d) is a substitution of phenylalanine to alanine at position 518 or equivalent in the amino acid sequence of SEQ ID NO: 1. Claim 11. A polypeptide selected from (i) to (iii) below: (i) A polypeptide consisting of the amino acid sequence represented by Sequence ID No. 2; (ii) A polypeptide consisting of an amino acid sequence having 95% or more identity with the amino acid sequence represented by Sequence ID No. 2 and possessing cyclization activity; (iii) A polypeptide consisting of an amino acid sequence having one to several amino acid substitutions, deletions, insertions, or additions in the amino acid sequence represented by Sequence ID No. 2 and possessing cyclization activity, provided that in polypeptides (ii) and (iii), the amino acids at positions 319 or equivalent, 509 or equivalent, 511 or equivalent, 518 or equivalent, and 513 or equivalent in the amino acid sequence of Sequence ID No. 2 are not mutated. Item 12.A method for producing sclareol glycol, characterized by contacting homofarnesyl monophosphate or a salt thereof represented by the following formula (I) or homofarnesyl pyrophosphate or a salt thereof represented by the following formula (II) with a polypeptide described in any of items 1 to 11, a microorganism or cell capable of producing the polypeptide, an extract of the microorganism or cell, and / or a culture medium containing the polypeptide obtained by culturing the microorganism or cell, thereby cyclizing the homofarnesyl monophosphate or a salt thereof or homofarnesyl pyrophosphate or a salt thereof.

[0009]

[0010] Item 13. The method according to item 12, wherein the salt is an ammonium salt. Item 14. The method according to item 13, wherein the ammonium salt is a diammonium salt. Item 15. The method according to item 13, wherein the ammonium salt is an alkylammonium salt. Item 16. The method according to item 13, wherein the ammonium salt is a benzylammonium salt. Item 17. A compound represented by the following formula (I) or a salt thereof.

[0011]

[0012] Item 18. The compound or salt thereof according to Item 17, wherein the salt is an ammonium salt. Item 19. The compound or salt thereof according to Item 18, wherein the ammonium salt is a diammonium salt. Item 20. The compound or salt thereof according to Item 18, wherein the ammonium salt is an alkylammonium salt. Item 21. The compound or salt thereof according to Item 18, wherein the ammonium salt is a benzylammonium salt.

[0013] [1] Polypeptides having cyclization activity, comprising an amino acid sequence having at least one mutation selected from the group consisting of (a) to (f) below, in which (i) the amino acid sequence represented by SEQ ID NO: 1, (ii) an amino acid sequence having 95% or more identity with the amino acid sequence represented by SEQ ID NO: 1, or (iii) an amino acid sequence having one to several amino acid substitutions, deletions, insertions or additions in the amino acid sequence represented by SEQ ID NO: 1: (a) A mutation in which tyrosine at position 319 or equivalent in the amino acid sequence of SEQ ID NO: 1 is substituted with phenylalanine. (b) A mutation in which phenylalanine at position 509 or equivalent in the amino acid sequence of SEQ ID NO: 1 is substituted with tyrosine. (c) A mutation in which alanine at position 511 or equivalent in the amino acid sequence of SEQ ID NO: 1 is substituted with tyrosine or phenylalanine. (d) A mutation in which phenylalanine at position 518 or equivalent in the amino acid sequence of SEQ ID NO: 1 is substituted with alanine, serine or histidine. (e) A mutation in which arginine at position 513 or equivalent in the amino acid sequence of Sequence ID No. 1 is replaced with valine or histidine. (f) A mutation in which serine at position 514 or equivalent in the amino acid sequence of Sequence ID No. 1 is replaced with threonine, provided that in the amino acid sequences of (ii) and (iii), the amino acids at positions 319 or equivalent, 509 or equivalent, 511 or equivalent, 518 or equivalent, 513 or equivalent, and 514 or equivalent in the amino acid sequence of Sequence ID No. 1 are not mutated.[1A] Polypeptides having cyclization activity, and (i) comprising an amino acid sequence in the amino acid sequence represented by SEQ ID NO: 1, having at least one mutation selected from the group consisting of (a) to (f) below: (a) A mutation in which the 319th tyrosine in the amino acid sequence of SEQ ID NO: 1 is replaced by phenylalanine. (b) A mutation in which the 509th phenylalanine in the amino acid sequence of SEQ ID NO: 1 is replaced by tyrosine. (c) A mutation in which the 511th alanine in the amino acid sequence of SEQ ID NO: 1 is replaced by tyrosine or phenylalanine. (d) A mutation in which the 518th phenylalanine in the amino acid sequence of SEQ ID NO: 1 is replaced by alanine, serine or histidine. (e) A mutation in which the 513th arginine in the amino acid sequence of SEQ ID NO: 1 is replaced by valine or histidine. (f) A mutation in which the 514th serine in the amino acid sequence of SEQ ID NO: 1 is replaced by threonine. [2] The polypeptide according to [1], having (a) a mutation in which tyrosine at position 319 or equivalent in the amino acid sequence of SEQ ID NO: 1 is replaced with phenylalanine, (b) a mutation in which phenylalanine at position 509 or equivalent in the amino acid sequence of SEQ ID NO: 1 is replaced with tyrosine, (c) a mutation in which alanine at position 511 or equivalent in the amino acid sequence of SEQ ID NO: 1 is replaced with tyrosine or phenylalanine, and (d) a mutation in which phenylalanine at position 518 or equivalent in the amino acid sequence of SEQ ID NO: 1 is replaced with alanine, serine or histidine. [2A] The polypeptide according to [1A], having (a) a mutation in which the 319th tyrosine in the amino acid sequence of SEQ ID NO: 1 is replaced with phenylalanine, (b) a mutation in which the 509th phenylalanine in the amino acid sequence of SEQ ID NO: 1 is replaced with tyrosine, (c) a mutation in which the 511th alanine in the amino acid sequence of SEQ ID NO: 1 is replaced with tyrosine or phenylalanine, and (d) a mutation in which the 518th phenylalanine in the amino acid sequence of SEQ ID NO: 1 is replaced with alanine, serine or histidine.[3] A polypeptide according to [1] or [2], having (a) a mutation in which tyrosine at position 319 or equivalent in the amino acid sequence of SEQ ID NO: 1 is replaced by phenylalanine, (b) a mutation in which phenylalanine at position 509 or equivalent in the amino acid sequence of SEQ ID NO: 1 is replaced by tyrosine, (c) a mutation in which alanine at position 511 or equivalent in the amino acid sequence of SEQ ID NO: 1 is replaced by tyrosine or phenylalanine, (d) a mutation in which phenylalanine at position 518 or equivalent in the amino acid sequence of SEQ ID NO: 1 is replaced by alanine, serine or histidine, (e) a mutation in which arginine at position 513 or equivalent in the amino acid sequence of SEQ ID NO: 1 is replaced by valine or histidine, and (f) a mutation in which serine at position 514 or equivalent in the amino acid sequence of SEQ ID NO: 1 is replaced by threonine. [3A] A polypeptide according to [1A] or [2A], having (a) a mutation in which the 319th tyrosine in the amino acid sequence of SEQ ID NO: 1 is replaced with phenylalanine, (b) a mutation in which the 509th phenylalanine in the amino acid sequence of SEQ ID NO: 1 is replaced with tyrosine, (c) a mutation in which the 511th alanine in the amino acid sequence of SEQ ID NO: 1 is replaced with tyrosine or phenylalanine, (d) a mutation in which the 518th phenylalanine in the amino acid sequence of SEQ ID NO: 1 is replaced with alanine, serine or histidine, (e) a mutation in which the 513th arginine in the amino acid sequence of SEQ ID NO: 1 is replaced with valine or histidine, and (f) a mutation in which the 514th serine in the amino acid sequence of SEQ ID NO: 1 is replaced with threonine. [4] A polypeptide according to any one of [1] to [3], wherein the mutation in (c) is a substitution of alanine at the 511th position or a corresponding position in the amino acid sequence of SEQ ID NO: 1 with tyrosine. The polypeptide according to any one of [1A], [2A], and [3A], wherein the mutation in [4A](c) is a substitution of alanine at position 511 to tyrosine in the amino acid sequence of SEQ ID NO: 1. The polypeptide according to any one of [1] to [4], wherein the mutation in [5](d) is a substitution of phenylalanine at position 518 or a corresponding position to alanine in the amino acid sequence of SEQ ID NO: 1.The polypeptide according to any one of [1A], [2A], [3A], and [4A], wherein the mutation in [5A](d) is a substitution of phenylalanine at position 518 to alanine in the amino acid sequence of SEQ ID NO: 1. The polypeptide according to any one of [1] to [5], wherein the mutation in [6](e) is a substitution of arginine at position 513 or a corresponding position to valine in the amino acid sequence of SEQ ID NO: 1. The polypeptide according to any one of [1A], [2A], [3A], [4A], and [5A], wherein the mutation in [6A](e) is a substitution of arginine at position 513 to valine in the amino acid sequence of SEQ ID NO: 1. [7] A polypeptide selected from (i') to (iii') below: (i') A polypeptide consisting of the amino acid sequence represented by Sequence ID No. 2 (ii') A polypeptide consisting of an amino acid sequence having 95% or more identity with the amino acid sequence represented by Sequence ID No. 2 and having cyclization activity (iii') A polypeptide consisting of an amino acid sequence having one to several amino acid substitutions, deletions, insertions or additions in the amino acid sequence represented by Sequence ID No. 2 and having cyclization activity, provided that in polypeptides (ii') and (iii'), the amino acid at position 319 or equivalent, position 509 or equivalent, position 511 or equivalent, position 518 or equivalent, and position 513 or equivalent in the amino acid sequence of Sequence ID No. 2 are not mutated. [7A] A polypeptide consisting of the amino acid sequence represented by Sequence ID No. 2. [8] A method for producing sclareol glycol, characterized by contacting homofarnesyl monophosphate or a salt thereof represented by the following formula (I) with a polypeptide according to any of [1], [1A], [2], [2A], [3], [3A], [4], [4A], [5], [5A], [6], [6A], [7] and [7A], a microorganism or cell capable of producing the polypeptide, an extract of the microorganism or cell, and / or a culture medium containing the polypeptide obtained by culturing the microorganism or cell, thereby cyclizing the homofarnesyl monophosphate or a salt thereof.

[0014]

[0015] [9] The method according to [8], wherein the salt is an ammonium salt.

[10] The method according to [9], wherein the ammonium salt is a diammonium salt.

[11] The method according to [9], wherein the ammonium salt is an alkylammonium salt.

[12] A compound represented by the following formula (I) or a salt thereof.

[0016]

[0017]

[13] The compound or salt thereof according to

[12] , wherein the salt is an ammonium salt.

[14] The compound or salt thereof according to

[13] , wherein the ammonium salt is a diammonium salt.

[15] The compound or salt thereof according to

[13] , wherein the ammonium salt is an alkylammonium salt.

[0018] According to the present invention, it is possible to produce an ambroxide precursor from a homofarnesol derivative in a single step. Therefore, it is possible to provide a more cost-effective manufacturing method for ensuring a stable industrial supply of ambroxide, which is economically important.

[0019] This figure shows an example of the reaction scheme of the present invention. This chart shows the results of GC / MS analysis of the reaction product of the polypeptide of the present invention (AsDMS mutant enzyme: Table 1 No. 6). Total ion chromatogram of sclareol glycol (top) and MS fragment (bottom). Homofarnesyl monophosphate diammonium salt was used as the substrate. This chart shows the results of GC / MS analysis of the reaction product of the polypeptide of the present invention (AsDMS mutant enzyme: Table 1 No. 6). Total ion chromatogram of sclareol glycol (top) and MS fragment (bottom). Homofarnesyl pyrophosphate diammonium salt was used as the substrate. This chart shows the results of GC / MS analysis of the reaction products of the polypeptides of the present invention (AsDMS mutant enzymes: Table 1 No. 1-5). Total ion chromatogram of sclareol glycol is shown. Homofarnesyl monophosphate diammonium salt was used as the substrate. This chart shows the results of GC / MS analysis of the reaction products of the polypeptides of the present invention (AsDMS mutant enzymes: Table 1 No. 1-5). The MS fragment of sclareol glycol is shown. Homofarnesyl monophosphate diammonium salt was used as the substrate.

[0020] The present invention will be described below. Unless otherwise specified, terms used herein have the meanings commonly used in the art.

[0021] 1. The polypeptide cyclization activity of the present invention refers to the activity of an enzyme or catalyst that catalyzes a reaction that forms a cyclic structure in a chemical reaction, and in the present invention, it refers to the activity of an enzyme. An enzyme is defined as having cyclization activity when it changes the molecular structure of a substrate and causes it to form a cyclic structure from an open chain structure. The polypeptide of the present invention is a polypeptide having cyclization activity, comprising an amino acid sequence in which (i) the amino acid sequence represented by Sequence ID No. 1 (hereinafter sometimes referred to as amino acid sequence (i)), (ii) an amino acid sequence having 95% or more identity with the amino acid sequence represented by Sequence ID No. 1 (hereinafter sometimes referred to as amino acid sequence (ii)), or (iii) an amino acid sequence having one to several amino acid substitutions, deletions, insertions or additions in the amino acid sequence represented by Sequence ID No. 1 (hereinafter sometimes referred to as amino acid sequence (iii)), and having at least one, preferably two, more preferably three, and even more preferably all four mutations selected from the group consisting of (a) to (d) below (hereinafter also referred to as the polypeptide of the present invention). (a) A mutation in which tyrosine at position 319 or equivalent in the amino acid sequence of SEQ ID NO: 1 is replaced by phenylalanine. (b) A mutation in which phenylalanine at position 509 or equivalent in the amino acid sequence of SEQ ID NO: 1 is replaced by tyrosine. (c) A mutation in which alanine at position 511 or equivalent in the amino acid sequence of SEQ ID NO: 1 is replaced by tyrosine or phenylalanine. (d) A mutation in which phenylalanine at position 518 or equivalent in the amino acid sequence of SEQ ID NO: 1 is replaced by alanine, serine or histidine. The polypeptide of the present invention has cyclization activity and may therefore be referred to as the cyclase of the present invention.

[0022] The polypeptide of the present invention optionally has the following mutations (e) and / or (f). Preferably, it optionally has the mutation (e) or (f), and more preferably, it optionally has the mutation (e). (e) A mutation in which arginine at position 513 or equivalent in the amino acid sequence of SEQ ID NO: 1 is replaced with valine, histidine, alanine, threonine, proline, or aspartic acid. (f) A mutation in which serine at position 514 or equivalent in the amino acid sequence of SEQ ID NO: 1 is replaced with threonine, isoleucine, cysteine, or valine.

[0023] The polypeptide of the present invention optionally has at least one mutation selected from (g-1) to (g-16) below. The number of mutations is not particularly limited, except that the upper limit is 16 as long as the resulting polypeptide has cyclization activity, but it is preferably one. (g-1) A mutation in which leucine at position 44 or equivalent in the amino acid sequence of SEQ ID NO: 1 is replaced by cysteine, glutamine, or alanine. (g-2) A mutation in which leucine at position 49 or equivalent in the amino acid sequence of SEQ ID NO: 1 is replaced by cysteine, isoleucine, threonine, or serine. (g-3) A mutation in which tryptophan at position 51 or equivalent in the amino acid sequence of SEQ ID NO: 1 is replaced by isoleucine, phenylalanine, or leucine. (g-4) A mutation in which valine at position 70 or equivalent in the amino acid sequence of SEQ ID NO: 1 is replaced by isoleucine, leucine, phenylalanine, or tyrosine. (g-5) A mutation in which leucine at position 107 or equivalent in the amino acid sequence of SEQ ID NO: 1 is replaced by valine. (g-6) A mutation in which isoleucine at position 111 or equivalent in the amino acid sequence of SEQ ID NO: 1 is replaced by phenylalanine, serine, asparagine, leucine, valine, or threonine. (g-7) A mutation in which leucine at position 114 or equivalent in the amino acid sequence of SEQ ID NO: 1 is replaced by valine or phenylalanine. (g-8) A mutation in which valine at position 140 or equivalent in the amino acid sequence of SEQ ID NO: 1 is replaced by glutamine, asparagine, cysteine, or serine. (g-9) A mutation in which serine at position 144 or equivalent in the amino acid sequence of SEQ ID NO: 1 is replaced by valine, leucine, aspartic acid, histidine, or tyrosine. (g-10) A mutation in which leucine at position 148 or equivalent in the amino acid sequence of SEQ ID NO: 1 is replaced by threonine, isoleucine, cysteine, or serine. (g-11) A mutation in which valine at position 264 or equivalent in the amino acid sequence of SEQ ID NO: 1 is replaced by leucine, threonine, cysteine, asparagine, or glutamine.(g-12) A mutation in which the cysteine at position 317 or its corresponding position in the amino acid sequence of SEQ ID NO: 1 is substituted with asparagine or glutamine. (g-13) A mutation in which the phenylalanine at position 318 or its corresponding position in the amino acid sequence of SEQ ID NO: 1 is substituted with tyrosine. (g-14) A mutation in which the phenylalanine at position 328 or its corresponding position in the amino acid sequence of SEQ ID NO: 1 is substituted with tyrosine or histidine. (g-15) A mutation in which the proline at position 512 or its corresponding position in the amino acid sequence of SEQ ID NO: 1 is substituted with tryptophan or aspartic acid. (g-16) A mutation in which the threonine at position 516 or its corresponding position in the amino acid sequence of SEQ ID NO: 1 is substituted with arginine or histidine.

[0024] In the present invention, the polypeptide containing the amino acid sequence (i) before the introduction of the mutations (a) to (f) and (g-1) to (g-16) is Accession No. The polypeptide containing the amino acid sequence (ii) and the polypeptide containing the amino acid sequence (iii) can be obtained from the AsDMS enzyme of WP_084549426.1 by introducing desired mutations into the AsDMS enzyme by a method known per se. For example, it can be carried out according to the method described in Non-Patent Document 2.

[0025] Examples of the method for measuring the cyclization activity include a method for quantifying the product, enzyme immunoassay (ELISA), fluorescence probe method, radioisotope labeling method, reverse transcription PCR (RT-PCR) method, etc. For simplicity, it is a method for quantifying the product. For example, to homofarnesyl monophosphate represented by formula (I) or a salt thereof or homofarnesyl pyrophosphate represented by formula (II) or a salt thereof (hereinafter sometimes referred to as the homofarnesol derivative of the present invention or a salt thereof), the cyclase of the present invention to be measured, a microorganism or cell having the ability to produce the enzyme, an extract of the microorganism or cell, and / or the culture solution containing the enzyme obtained by culturing the microorganism or cell are brought into contact, and the amount of squalene glycol converted from the homofarnesol derivative of the present invention or a salt thereof is directly measured and quantified, whereby its cyclization activity can be confirmed. For quantification, HPLC (high performance liquid chromatography), LC-MS (liquid chromatography mass spectrometry), GC-MS (gas chromatography mass spectrometry), etc. can be used.

[0026] In the present invention, the amino acid sequence represented by SEQ ID NO: 1 is a cyclase (Aquimarina spongiae drimenol synthase: AsDMS) derived from the marine microorganism Aquimarina spongiae, specifically, the amino acid sequence derived from the HAD-IA family hydrolase of accession No. WP_084549426.1 (hereinafter, the cyclase is also referred to as the AsDMS enzyme).

[0027]

[0028] The present invention is characterized by using a cyclase obtained by introducing at least one, preferably two, more preferably three, and even more preferably all four, mutations selected from the group consisting of (a) to (d) below into the AsDMS enzyme; a cyclase obtained by further introducing optionally (e) and / or (f), preferably (e) or (f), more preferably (e) mutations; or a cyclase obtained by further introducing optionally at least one, preferably one, mutation selected from the group consisting of (g-1) to (g-16) (hereinafter, such an AsDMS enzyme into which mutations have been introduced will also be referred to as the AsDMS mutant enzyme of the present invention). As long as the AsDMS mutant enzyme can maintain its cyclization activity, the amino acid sequence before mutation introduction may be amino acid sequence (i), amino acid sequence (ii), or amino acid sequence (iii). (a) A mutation in which tyrosine at position 319 or equivalent in the amino acid sequence of SEQ ID NO: 1 is replaced by phenylalanine. (b) A mutation in which phenylalanine at position 509 or equivalent in the amino acid sequence of SEQ ID NO: 1 is replaced by tyrosine. (c) A mutation in which alanine at position 511 or equivalent in the amino acid sequence of SEQ ID NO: 1 is replaced by tyrosine or phenylalanine. (d) A mutation in which phenylalanine at position 518 or equivalent in the amino acid sequence of SEQ ID NO: 1 is replaced by alanine, serine or histidine. (e) A mutation in which arginine at position 513 or equivalent in the amino acid sequence of SEQ ID NO: 1 is replaced by valine or histidine or alanine or threonine or proline or aspartic acid. (f) A mutation in which serine at position 514 or equivalent in the amino acid sequence of SEQ ID NO: 1 is replaced by threonine or isoleucine or cysteine ​​or valine. (g-1) A mutation in which leucine at position 44 or equivalent in the amino acid sequence of SEQ ID NO: 1 is replaced by cysteine, glutamine, or alanine. (g-2) A mutation in which leucine at position 49 or equivalent in the amino acid sequence of SEQ ID NO: 1 is replaced by cysteine, isoleucine, threonine, or serine. (g-3) A mutation in which tryptophan at position 51 or equivalent in the amino acid sequence of SEQ ID NO: 1 is replaced by isoleucine, phenylalanine, or leucine.(g-4) A mutation in which valine at position 70 or equivalent in the amino acid sequence of SEQ ID NO: 1 is replaced by isoleucine, leucine, phenylalanine, or tyrosine. (g-5) A mutation in which leucine at position 107 or equivalent in the amino acid sequence of SEQ ID NO: 1 is replaced by valine. (g-6) A mutation in which isoleucine at position 111 or equivalent in the amino acid sequence of SEQ ID NO: 1 is replaced by phenylalanine, serine, asparagine, leucine, valine, or threonine. (g-7) A mutation in which leucine at position 114 or equivalent in the amino acid sequence of SEQ ID NO: 1 is replaced by valine or phenylalanine. (g-8) A mutation in which valine at position 140 or equivalent in the amino acid sequence of SEQ ID NO: 1 is replaced by glutamine, asparagine, cysteine, or serine. (g-9) A mutation in which serine at position 144 or equivalent in the amino acid sequence of SEQ ID NO: 1 is replaced by valine, leucine, aspartic acid, histidine, or tyrosine. (g-10) A mutation in which leucine at position 148 or equivalent in the amino acid sequence of SEQ ID NO: 1 is replaced with threonine, isoleucine, cysteine, or serine. (g-11) A mutation in which valine at position 264 or equivalent in the amino acid sequence of SEQ ID NO: 1 is replaced with leucine, threonine, cysteine, asparagine, or glutamine. (g-12) A mutation in which cysteine ​​at position 317 or equivalent in the amino acid sequence of SEQ ID NO: 1 is replaced with asparagine or glutamine. (g-13) A mutation in which phenylalanine at position 318 or equivalent in the amino acid sequence of SEQ ID NO: 1 is replaced with tyrosine. (g-14) A mutation in which phenylalanine at position 328 or equivalent in the amino acid sequence of SEQ ID NO: 1 is replaced with tyrosine or histidine. (g-15) A mutation in which proline at position 512 or equivalent in the amino acid sequence of SEQ ID NO: 1 is replaced with tryptophan or aspartic acid. (g-16) A mutation in which the threonine at position 516 or equivalent in the amino acid sequence of Sequence ID No. 1 is replaced with arginine or histidine.The following describes each mutation in turn. In this specification, the identification of the amino acid residue at the "corresponding position" can be performed by comparing the target amino acid sequence (here, amino acid sequence (ii) or amino acid sequence (iii)) with the reference sequence (amino acid sequence (i)) using a known algorithm, and aligning the sequences to give the greatest homology to the conserved amino acid residues present in the target amino acid sequence. By aligning the amino acid sequences of each protein in this manner, it becomes possible to determine the position of the amino acid residue to be mutated in the sequence, regardless of insertions or deletions in the amino acid sequence.

[0029] The mutation in (a) is a mutation in which the tyrosine at position 319 or the equivalent position in the amino acid sequence of SEQ ID NO: 1 is replaced with phenylalanine. This mutation improves the reaction efficiency in the cyclization reaction.

[0030] The mutation in (b) is a mutation in which phenylalanine at position 509 or the equivalent position in the amino acid sequence of SEQ ID NO: 1 is replaced with tyrosine. This mutation improves the reaction efficiency in the cyclization reaction.

[0031] The mutation in (c) is a mutation in which alanine at position 511 or the equivalent position in the amino acid sequence of SEQ ID NO: 1 is replaced with tyrosine or phenylalanine. This mutation improves the reaction efficiency in the cyclization reaction. A substitution with tyrosine is preferred.

[0032] The mutation in (d) is a mutation in which phenylalanine at position 518 or the equivalent position in the amino acid sequence of SEQ ID NO: 1 is replaced with alanine, serine, or histidine. This mutation improves the reaction efficiency in the cyclization reaction. Substitution with alanine is preferred.

[0033] The mutation in (e) is a mutation in which the arginine at position 513 or the equivalent position in the amino acid sequence of SEQ ID NO: 1 is replaced with valine, histidine, alanine, threonine, proline, or aspartic acid. This mutation improves the reaction efficiency in the cyclization reaction. A substitution with valine or histidine is preferred.

[0034] The mutation in (f) is a mutation in which the serine at position 514 or equivalent in the amino acid sequence of SEQ ID NO: 1 is replaced with threonine, isoleucine, cysteine, or valine. This mutation improves the reaction efficiency in the cyclization reaction. A substitution with threonine is preferred.

[0035] Mutations (g-1) to (g-16) are mutations in which an amino acid at each position or a corresponding position in the amino acid sequence of SEQ ID NO: 1 is replaced with a predetermined (as described above) amino acid. This mutation is expected to improve enzyme activity.

[0036] The cyclase of the present invention, i.e., the AsDMS mutant enzyme, can be obtained by introducing at least one, preferably two, more preferably three, and even more preferably all four, mutations selected from the group consisting of (a) to (d) into amino acid sequence (i), amino acid sequence (ii), or amino acid sequence (iii); optionally (e) and / or (f); and optionally at least one, preferably one, mutation selected from the group consisting of (g-1) to (g-16) into amino acid sequence (i), amino acid sequence (ii), or amino acid sequence (iii); and the mutations into amino acids. The introduction of mutations can be carried out by methods known to those skilled in the art, such as site-directed mutagenesis and PCR.

[0037] Preferably, the AsDMS mutant enzyme of the present invention is a polypeptide selected from the following (i') to (iii'): (i') A polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 (ii') A polypeptide consisting of an amino acid sequence having 95% or more identity with the amino acid sequence represented by SEQ ID NO: 2 and having cyclization activity (iii') A polypeptide consisting of an amino acid sequence having one to several amino acid substitutions, deletions, insertions or additions in the amino acid sequence represented by SEQ ID NO: 2 and having cyclization activity. However, in polypeptides (ii') and (iii'), the amino acid at position 319 or equivalent, position 509 or equivalent, position 511 or equivalent, position 518 or equivalent, and position 513 or equivalent in the amino acid sequence of SEQ ID NO: 2 are not mutated. More preferably, the polypeptide consists of the amino acid sequence represented by SEQ ID NO: 2. The amino acid sequence represented by SEQ ID NO: 2 is obtained by introducing mutations in the combinations of (a) to (e) into the amino acid sequence represented by SEQ ID NO: 1.

[0038]

[0039] In this specification, "an amino acid sequence having one to several amino acid substitutions, deletions, insertions, or additions" means a sequence that has been modified by amino acid substitutions, etc. The number of amino acid modifications (i.e., the number of amino acid substitutions, deletions, insertions, or additions) is usually 1 to 20, preferably 1 to 10, more preferably 1 to 5, and particularly preferably 1, 2, 3, or 4 amino acids. In the case of multiple amino acids, they may be consecutive or not. Examples of modified amino acid sequences that can be used in the present invention are preferably amino acid sequences in which the amino acids have one or several (preferably 1, 2, 3, or 4) conservative substitutions.

[0040] In amino acid sequence (ii), an amino acid sequence having 95% or more identity with the amino acid sequence represented by Sequence ID No. 1 is preferably an amino acid sequence having 96% or more, more preferably 97% or more, even more preferably 98% or more, and even more preferably 99% or more sequence identity with the entire amino acid sequence represented by Sequence ID No. 1.

[0041] In polypeptide (ii'), an amino acid sequence having 95% or more identity with the amino acid sequence represented by SEQ ID NO: 2 is preferably an amino acid sequence having 96% or more, more preferably 97% or more, even more preferably 98% or more, and even more preferably 99% or more sequence identity with the entire amino acid sequence represented by SEQ ID NO: 2.

[0042] An amino acid sequence having "substitution, deletion, insertion, or addition of one to several amino acids" or "an amino acid sequence having 95% or more identity" in the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 is a substitution of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. However, this substitution of the amino acid sequence is a conservative substitution that maintains the function of the polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. A "conservative substitution" means a substitution in which the function of the polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 is not lost. Specifically, it means replacing an amino acid residue with another chemically similar amino acid residue. For example, this could be the substitution of one hydrophobic residue with another hydrophobic residue, or the substitution of one polar residue with another polar residue having the same charge. Functionally similar amino acids that can be created by such substitutions are publicly known in the relevant art for each amino acid. Examples of nonpolar (hydrophobic) amino acids include alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, and methionine. Examples of polar (neutral) amino acids include glycine, serine, threonine, tyrosine, glutamine, asparagine, and cysteine. Examples of positively charged (basic) amino acids include arginine, histidine, and lysine. Examples of negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

[0043] In this specification, the homology (also called identity or similarity) of amino acid sequences can be calculated using the homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) under the following conditions, for example (expected value = 10; gaps allowed; matrix = BLOSUM62; filtering = OFF). Other algorithms for determining amino acid sequence homology include, for example, the algorithm described by Karlin et al., Proc. Natl. Acad. Sci. USA, 90: 5873-5877 (1993) [this algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) (Altschul et al., Nucleic Acids Res., 25: 3389-3402 (1997))], the algorithm described by Needleman et al., J. Mol. Biol., 48: 444-453 (1970) [this algorithm is incorporated into the GAP program in the GCG software package], the algorithm described by Myers and Miller, CABIOS, 4: 11-17 (1988) [this algorithm is incorporated into the ALIGN program (version 2.0), which is part of the CGC sequence alignment software package], and Pearson et al., Proc. Examples include the algorithm described in Natl. Acad. Sci. USA, 85: 2444-2448 (1988) [this algorithm is incorporated into the FASTA program in the GCG software package], and these may also be used as preferred methods.

[0044] Furthermore, the cyclase of the present invention, i.e., the AsDMS mutant enzyme, can also be produced by culturing a transformant containing the nucleic acid encoding it, and separating and purifying the cyclase from the resulting culture. The nucleic acid encoding the cyclase of the present invention may be DNA, RNA, or a DNA / RNA chimera. DNA is preferred. The nucleic acid may also be double-stranded or single-stranded. In the case of double-stranded, it may be double-stranded DNA, double-stranded RNA, or a DNA:RNA hybrid. In the case of single-stranded, it may be a sense strand (i.e., coding strand) or an antisense strand (i.e., non-coding strand).

[0045] Examples of DNA encoding the cyclase of the present invention include synthetic DNA. For example, using a total RNA or mRNA fraction prepared from cells or tissues derived from microorganisms (e.g., marine microorganisms such as Aquimarina; filamentous fungi) as a template, the full-length cyclase cDNA is directly amplified by Reverse Scriptase-PCR, and then processed using a known kit, such as Mutan. ＴＭ -super Express Km (TAKARA BIO INC.), Mutan ＴＭ It can be obtained by converting it using -K (TAKARA BIO INC.), etc., according to known methods such as ODA-LA PCR, Gapped duplex, Kunkel, or similar methods. Alternatively, it can be obtained by converting the cDNA cloned from a cDNA library prepared by inserting the above-mentioned total RNA or mRNA fragments into a suitable vector, using colony or plaque hybridization or PCR, according to the above-mentioned method. The vector used for the library may be any bacteriophage, plasmid, cosmid, phagemid, etc.

[0046] The nucleic acids encoding the cyclase of the present invention are not limited as long as they encode the enzyme, but particularly include those having any of the following base sequences (iv) to (vii): (iv) A nucleic acid having the base sequence represented by Sequence ID No. 3; (v) A nucleic acid having a base sequence in which one or more bases are substituted, deleted, inserted and / or added in the base sequence represented by Sequence ID No. 3, and encoding a polypeptide having the cyclase activity of the present invention; (vi) A nucleic acid having a base sequence having 95% or more sequence identity with the base sequence represented by Sequence ID No. 3, and encoding a polypeptide having the cyclase activity of the present invention; (vii) A nucleic acid having a base sequence that hybridizes under stringent conditions with the complementary strand of the base sequence represented by Sequence ID No. 3, and encoding a polypeptide having the cyclase activity of the present invention. The base sequence represented by Sequence ID No. 3 encodes a polypeptide consisting of the amino acid sequence represented by Sequence ID No. 2.

[0047]

[0048] The nucleic acids shown in (v) above include nucleic acids that encode the cyclase of the present invention, which include a base sequence in which one or more bases are deleted, substituted, inserted and / or added in the base sequence represented by Sequence ID No. 3. In the case of substitution, insertion or addition, it is preferable that one or more bases are substituted, inserted or added. Here, "one or more bases" means, for example, 1 to 60 bases, preferably 1 to 30 bases, more preferably 1 to 15 bases, even more preferably 1 to 10 bases, and particularly preferably 1 to 5 bases.

[0049] The nucleic acids shown in (vi) above include nucleic acids having a base sequence that has 95% or more sequence identity with the base sequence represented by Sequence ID No. 3, and that encode the cyclase of the present invention. Preferably, the nucleic acids have a base sequence that has 96% or more, more preferably 97% or more, even more preferably 98% or more, identity with the base sequence represented by Sequence ID No. 3, and that encode the cyclase of the present invention.

[0050] In this specification, the homology (also called identity) of nucleotide sequences can be calculated using the homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) under the following conditions, for example (expected value = 10; gaps allowed; filtering = ON; match score = 1; mismatch score = -3). Other algorithms for determining the homology of nucleotide sequences include the amino acid sequence homology calculation algorithm described above, which is similarly preferred as an example.

[0051] The nucleic acid shown in (vii) above may be a nucleic acid that hybridizes with the complementary strand of the base sequence of Sequence ID No. 3 under stringent conditions, as long as it encodes the cyclase of the present invention. Here, "stringent conditions" can be appropriately set with reference to previously reported conditions (e.g., Current Protocols in Molecular Biology, John Wiley & Sons, 6.3.16.3.6, 1999). Specifically, examples include conditions in which washing is performed once, more preferably 1 to 3 times, at a salt concentration and temperature equivalent to the normal washing conditions for Southern hybridization, such as 60°C, 1 x SSC, 0.1% SDS, preferably 0.1 x SSC, 0.1% SDS, and even more preferably 65°C, 0.1 x SSC, 0.1% SDS or 68°C, 0.1 x SSC, 0.1% SDS (high stringent conditions).

[0052] Those skilled in the art can introduce desired mutations into nucleic acids having the nucleotide sequence represented by Sequence ID No. 3 by appropriately performing substitutions, deletions, insertions, and / or additions using site-directed mutagenesis methods (Nucleic Acids Res. 10, pp. 6487 (1982), Methods in Enzymol. 100, pp. 448 (1983), Molecular Cloning, PCR A Practical Approach IRL Press pp. 200 (1991)), etc. However, no mutations are introduced into the amino acid sequence of the polypeptide encoded by the nucleic acid having the nucleotide sequence represented by Sequence ID No. 3, i.e., the amino acids at positions 319 or equivalent, 509 or equivalent, 511 or equivalent, 518 or equivalent, and 513 or equivalent in the amino acid sequence of Sequence ID No. 2.

[0053] In the production method of the present invention described later, the above-mentioned cyclizing enzyme may be used directly in the reaction with the homofarnesol derivative represented by formula (I) or formula (II) or a salt thereof, which is the substrate. Alternatively, a microorganism or cell capable of producing the enzyme, an extract of the microorganism or cell, and / or a culture medium containing the enzyme obtained by culturing the microorganism or cell may be used.

[0054] The microorganism or cell capable of producing the cyclase of the present invention may be one that originally possesses the ability to produce the cyclase, or it may be one that has been artificially given the ability to produce the cyclase. The microorganism or cell may be alive or dead.

[0055] As means of artificially conferring the aforementioned production capacity, known methods such as genetic engineering (transformation) and mutagenesis can be employed. Examples of transformation methods include introducing the target DNA and modifying expression regulatory sequences such as promoters on the chromosome to enhance the expression of the target DNA.

[0056] Of these, it is preferable to use microorganisms or cells transformed with DNA encoding the polypeptide of the present invention.

[0057] As described above, the nucleic acid (DNA) encoding the polypeptide (cyclase) of the present invention can be cloned by performing PCR using chromosomal DNA derived from Aquimarina spongiae or the like as a template and appropriate primers.

[0058] Furthermore, the nucleic acid (DNA) encoding the polypeptide (cyclase) of the present invention can be cloned by preparing full-length cyclase cDNA directly amplified by RT-PCR using total RNA or mRNA derived from Aquimarina spongiae or the like as a template, and then performing PCR using appropriate primers, as described above.

[0059] For example, a polypeptide gene expression vector of the present invention is provided by inserting the DNA encoding the polypeptide of the present invention, obtained as described above, into a known expression vector in an expressible configuration. Then, a transformant can be obtained by transforming a host cell with the DNA encoding the polypeptide of the present invention. The transformant can also be obtained by incorporating the DNA encoding the polypeptide of the present invention into the host's chromosomal DNA in an expressible manner using methods such as homologous recombination.

[0060] In this specification, "expression vector" refers to a nucleic acid construct used to replicate and express a protein having a desired function in a host by incorporating a polynucleotide encoding the protein having a desired function and introducing it into the host. Examples include, but are not limited to, plasmids, viruses, phages, and cosmids. Preferably, the expression vector is a plasmid.

[0061] In this specification, "transformed organism" means a microorganism or cell into which a target gene has been introduced using the expression vector, etc., and which is capable of expressing a desired trait related to a polypeptide having a desired function.

[0062] Examples of methods for producing transformants include introducing the DNA encoding the polypeptide of the present invention into a plasmid vector, phage vector, or viral vector that is stably present in a host cell, and then introducing the constructed expression vector into the host cell; or directly introducing the DNA into the host genome and transcribing and translating its genetic information. In this case, it is preferable to ligate a suitable promoter upstream of the 5'- side of the DNA in the host, and it is even more preferable to ligate a terminator downstream of the 3'- side. Such promoters and terminators can be known and used without particular limitation.

[0063] The host microorganisms to be transformed to express the cyclase of the present invention are not particularly limited as long as the host itself does not adversely affect the substrate or target product. For example, the following hosts can be listed.

[0064] The preferred host organisms are mainly microorganisms. The host microorganism is not particularly limited as long as it can be introduced with the AsDMS mutant enzyme gene as an exogenous gene and can express the introduced AsDMS mutant enzyme gene. Microorganisms selected from bacteria, cyanobacteria, fungi, and yeasts are preferred, preferably transgenic or recombinant cells. Preferably, the microorganisms are selected from bacteria of the genera Escherichia, Corynebacterium, Ralstonia, Clostridium, Pseudomonas, Bacillus, Zymomonas, Rhodobacter, Streptomyces, Burkholderia, Lactobacillus, and Lactococcus. More preferably, the cells are selected from the bacteria Escherichia coli, Pseudomonas putida, Burkholderia glumae, Streptomyces lividans, Streptomyces coelicolor, and Zymomonas mobilis, with Escherichia coli being particularly preferred as the host microorganism.

[0065] The procedures for producing transformants, constructing recombinant vectors adapted to the host, and culturing the host can be carried out in accordance with techniques commonly used in the fields of molecular biology, biotechnology, and genetic engineering (for example, Green et al., Molecular Cloning: A Laboratory Manual (4) th (The method described in ed.) Cold Spring Harbor Press, Cold Spring Harbor, NY (2012).

[0066] Beyond microorganisms, various host-vector systems have been established in plants and animals. Furthermore, systems using cell-free protein synthesis methods such as E. coli cell-free extracts and wheat germ have been established and are suitable for use.

[0067] Examples of extracts of microorganisms or cells capable of producing the cyclase of the present invention include extracts of the microorganisms or cells with organic solvents such as acetone, dimethyl sulfoxide (DMSO), and toluene, or surfactants; lysates obtained by freeze-drying or physical or enzymatic disruption; and crude or purified enzyme fractions extracted from microorganisms or cells. The extract may be immobilized on a carrier for convenience during contact with a substrate.

[0068] Examples of culture media containing the cyclase enzyme of the present invention, obtained by culturing microorganisms or cells capable of producing the cyclase enzyme, include a suspension of the cells and a liquid culture medium, or, in the case of cells that secrete the enzyme into the culture medium or other extracellular space, the supernatant or concentrate obtained by removing the cells by centrifugation or the like.

[0069] 2. Method for Manufacturing the Present Invention According to the present invention, the cyclase of the present invention is prepared by homofarnesyl monophosphate represented by the following formula (I) or a salt thereof, or homofarnesyl pyrophosphate represented by the following formula (II) or a salt thereof.

[0070]

[0071] Applying it to the following formula

[0072]

[0073] A method for producing sclareol glycol is provided, which includes producing sclareol glycol represented by [formula].

[0074] The salts of homofarnesyl monophosphate or homofarnesyl pyrophosphate are not particularly limited as long as they do not adversely affect the cyclization activity by cyclases. Examples include salts with inorganic bases, salts with organic bases, and salts with amino acids (e.g., basic amino acids).

[0075] Suitable examples of salts with inorganic bases include alkali metal salts such as sodium salts and potassium salts; alkaline earth metal salts such as calcium salts and magnesium salts; and aluminum salts and ammonium salts. Preferably, sodium salts, potassium salts, calcium salts, and magnesium salts are used, more preferably ammonium salts, sodium salts, and potassium salts, and even more preferably ammonium salts.

[0076] Suitable examples of salts with organic bases include salts with trimethylamine, triethylamine, pyridine, picoline, ethanolamine, diethanolamine, triethanolamine, tromethamine [tris(hydroxymethyl)methylamine], tert-butylamine, cyclohexylamine, benzylamine, dicyclohexylamine, and N,N-dibenzylethylenediamine.

[0077] Preferred examples of amino acid salts include salts with basic amino acids. Suitable examples of salts with basic amino acids include salts with arginine, lysine, and ornithine.

[0078] Ammonium salts are preferred. Examples of ammonium salts include monoammonium salts, diammonium salts, triammonium salts, tetraammonium salts, as well as alkylammonium salts such as tetramethylammonium salt and tetrabutylammonium salt, and benzylammonium salts such as trimethylbenzylammonium salt and triethylbenzylammonium salt. Diammonium salt, tetrabutylammonium salt, and triethylbenzylammonium salt are particularly preferred.

[0079] When contacting the cyclase of the present invention with the homofarnesol derivative or salt thereof, sclareol glycol can be produced by contacting purified or crudely purified cyclase of the present invention, a microorganism or cell capable of producing the cyclase of the present invention (for example, a transformant having DNA encoding the polypeptide of the present invention), an extract of said microorganism or cell, and / or a culture medium containing the enzyme obtained by culturing said microorganism or cell with homofarnesyl monophosphate or a salt thereof. Purified or crudely purified cyclase of the present invention, a microorganism or cell capable of producing the cyclase of the present invention (for example, a transformant having DNA encoding the polypeptide of the present invention), an extract of said microorganism or cell, and / or a culture medium containing the enzyme obtained by culturing said microorganism or cell can all be used as cyclases in the production method of the present invention. Therefore, in the production method of the present invention, unless otherwise specified, these series of substances having cyclization activity are included in the cyclase of the present invention.

[0080] The amount of cyclase added to the reaction solution can be appropriately selected depending on its form (e.g., purified or unpurified, microorganisms or cells themselves, or their extracts or culture media). For example, if the cyclase is a microorganism or cell, it is usually added to the reaction solution so that the concentration of the microorganism or cell is about 0.1 w / v% to 2 w / v%, preferably 0.2 w / v% to 1 w / v%, by wet bacterial weight. If an extract or culture media is used, the enzyme expression level is determined by SDS-polyacrylamide electrophoresis, and the enzyme solution is added so that the final concentration is 0.2 to 2 μM, preferably 0.4 to 0.8 μM. Here, w / v% represents weight / volume%.

[0081] The reaction method is not particularly limited; a reaction substrate, such as the homofarnesol derivative of the present invention or a salt thereof, can be added to a liquid containing the cyclase of the present invention, and the reaction can be carried out at an appropriate temperature and pressure (for example, around atmospheric pressure). This allows for the production of sclareol glycol.

[0082] The amount of the homofarnesol derivative or salt thereof of the present invention, which serves as the reaction substrate, can be appropriately adjusted depending on the activity of the cyclase used, but it can usually be used in a substrate concentration of 0.05 mM to 0.6 mM, preferably 0.1 mM to 0.25 mM.

[0083] The reaction substrates may be added all at once at the start of the reaction, but they can also be added continuously or intermittently to reduce the impact of enzyme substrate inhibition and to improve the accumulation concentration of the product.

[0084] The reaction medium can be appropriately selected depending on the form of the cyclase used. For example, an aqueous medium or a mixture of an aqueous medium and an organic solvent can be used. Examples of aqueous media include water or buffer solutions. As organic solvents, methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, tert-butanol, acetone, dimethyl sulfoxide, etc., which have high solubility of the reaction substrate can be used. In addition, as organic solvents, ethyl acetate, butyl acetate, toluene, chloroform, n-hexane, etc., which are effective in removing reaction byproducts, can also be used.

[0085] The reaction temperature, pH during the reaction, and reaction time can be appropriately selected depending on the form of the cyclase used. For example, the reaction temperature is 4°C to 80°C, preferably 10°C to 70°C, more preferably 20°C to 60°C; the pH during the reaction is pH 3 to 11, preferably pH 4 to 8; and the reaction time is approximately 0.5 hours to 72 hours, 1 hour to 48 hours, or 2 hours to 24 hours.

[0086] The sclareol glycol produced by the manufacturing method of the present invention can be purified after the reaction is complete by separating the bacterial cells, proteins, etc., from the reaction solution using separation or purification methods known to those skilled in the art, such as centrifugation or membrane treatment. This purification can then be carried out by appropriately combining extraction with organic solvents, distillation, column chromatography using ion exchange resins or silica gel, crystallization at the isoelectric point, or crystallization with calcium salts. Furthermore, the sclareol glycol obtained by the manufacturing method of the present invention can be converted to ambroxide by conventional methods.

[0087] Of the homofarnesyl derivatives of the present invention that serve as reaction substrates, homofarnesyl monophosphate represented by formula (I) is a novel compound that can be produced by the method described later. On the other hand, homofarnesyl pyrophosphate represented by formula (II) can be prepared and obtained based on information from known literature (Org Lett. 2012 Aug 17;14(16):4038-41.).

[0088] 3. Compound of the present invention (homofarnesyl monophosphate) The homofarnesyl monophosphate used as a substrate in "2. Method for producing the present invention" is a novel compound and can be produced, for example, by the following method.

[0089]

[0090] (Step 1) This step involves tosyling homofarnesol to convert it into homofarnesyltosylate. Tosyling is performed by tosyling the protecting group tosyl group (-SO ２ C ６ H ４ CH ３ This is a step to introduce ), and in this step it is performed for the purpose of protecting the hydroxyl group of homofarnesol. Homofarnesol is converted to homofarnesyl tosylate by tosylation according to a conventional method. In one embodiment, tosylation is carried out by reacting homofarnesol with tosyl chloride in the presence of an amine, for example, triethylamine or N-methylimidazole, or a base, for example, sodium hydroxide. Tosylation may be carried out in aqueous or organic solvent. Examples of organic solvents include dichloromethane, acetonitrile, dimethyl sulfoxide, dimethylacetamide, and dimethylformamide. Homofarnesol is a terpenoid compound known as 3,7,11-trimethyl-2,6,10-dodecatrien-1-ol, and can be obtained by brominating, cyanating, and hydrolyzing nerolidol to obtain homofarnesylic acid, and then reducing it (Patent Document 1).

[0091] (Step 2) This step is to mono-phosphorylate the tosylated compound (homofarnesyl tosylate) to obtain a phosphoric acid compound (homofarnesyl monophosphate). The mono-phosphorylation is to add a phosphate group (-PO ３ H ２This is a reaction that introduces only one tosylated compound. A method for converting a tosylated compound to a monophosphate compound may include the following steps in one embodiment. First, the tosylated compound is suspended or dissolved in a suitable reaction solvent. Dimethylformamide (DMF), acetonitrile, or a mixture thereof is preferably used as the solvent. Next, a phosphorylation reagent is added under basic conditions. Examples of phosphorylation reagents include triethyl phosphate and potassium dihydrogen phosphate. The base can be either an organic or inorganic base, and triethylamine, pyridine, or sodium carbonate are suitable. The reaction mixture is usually stirred at room temperature to 60°C for several hours to several days, preferably several hours to 24 hours. After the reaction is complete, the mixture is neutralized or treated with water or a dilute acid. If necessary, the product is separated by extraction using an organic solvent. The obtained crude product is purified by chromatography, recrystallization, or a combination thereof. This yields a high-purity monophosphate compound. The obtained monophosphate compound can be converted to a salt if necessary. Alternatively, the tosylated compound can be converted to a salt of the monophosphate compound in a single step. For example, if the salt of a monophosphate compound is a tetrabutylammonium salt, monophosphorylation and salt formation can be carried out in one step by dissolving the tosylated compound in a suitable organic solvent and adding tetrabutylammonium phosphate as a phosphate source. Furthermore, by dissolving the tetrabutylammonium salt of the monophosphate compound in a suitable organic solvent and adding aqueous ammonia, a diammonium salt of the monophosphate compound can be obtained. The solvent used in this process is preferably acetonitrile, dimethylformamide, or a similar anhydrous organic solvent. The reaction time can be from one hour to several hours. The reaction temperature is preferably adjusted to a range of room temperature to 80°C, more preferably from 30°C to 80°C. This effectively substitutes the tosyl group and produces a monophosphate salt. The obtained monophosphate salt can be easily recovered by normal separation and purification operations.

[0092] The present invention will be described in detail below with reference to examples, but the present invention is not limited thereto. Furthermore, the reagents and materials used are commercially available unless otherwise specified. Unless otherwise noted, abbreviations used herein are the same as those commonly used in the art. Amino acid mutations (substitutions) are expressed in the following order: "type of amino acid before substitution, position of the substituted amino acid, type of amino acid after substitution." For example, Y319F means that the tyrosine at position 319 is substituted with phenylalanine. The correspondence between the three-letter and one-letter abbreviations for amino acids used herein is as follows. Alanine: Ala: A Arginine: Arg: R Asparagine: Asn: N Aspartic acid: Asp: D Cysteine: Cys: C Glutamine: Gln: Q Glutamic acid: Glu: E Glycine: Gly: G Histidine: His: H Isoleucine: Ile: I Leucine: Leu: L Lysine: Lys: K Methionine: Met: M Phenylalanine: Phe: F Proline: Pro: P Serine: Ser: S Threonine: Thr: T Tryptophan: Trp: W Tyrosine: Tyr: Y Valine: Val: V

[0093] Example 1: Preparation of homofarnesyl monophosphate (hFMP) (1-1) Preparation of tetrabutylammonium salt

[0094]

[0095] To a 6 mL acetonitrile solution of tetrabutylammonium phosphate (4 g, 6 equivalents), a 3 mL acetonitrile solution of homofarnesyl tosylate (0.78 g, 2 mmol) was added, and the mixture was stirred at 30°C until the starting materials disappeared. After concentrating the reaction mixture under reduced pressure, the resulting residue was dissolved in ethyl acetate, washed with water, and the concentrate was subjected to silica gel column chromatography to obtain homofarnesyl monophosphate tetrabutylammonium salt. Physical properties: １ H-NMR (CHCl ３(TMS, ppm) δ 5.18–5.06 (3H, m), 3.84 (2H, dd, J=7.04, 7.52 Hz), 3.32 (8H, brs), 2.33 (2H, dd, J=7.36, 7.20 Hz), 2.09–2.01 (4H, m), 1.99–1.92 (4H, m), 1.72–1.55 (20H, brm), 1.53–1.39 (8H, brm), 1.05–0.93 (12H, brt). Homofarnesyl monophosphate diammonium salt was obtained by adding 28% aqueous ammonia and acetonitrile to the obtained homofarnesyl monophosphate tetrabutylammonium salt and collecting the resulting solid. Physical properties: １ H-NMR (D ２ O / TMS, ppm) δ5.08-4.96 (3H, m), 3.61 (2H, dd, J = 7.32, 7.24Hz), 2.25 (2H, dd, J = 7 .32, 7.24Hz), 1.96-1.83 (8H, m), 1.55 (6H, d, J = 6.88Hz), 1.47 (6H, d, J = 6.88Hz).

[0096] (1-2) Production of triethylbenzylammonium salt

[0097]

[0098] To a 10 mL solution of homofarnesol (1.5 g, 6.2 mmol) in acetonitrile, N-benzyl-N,N-diethylethaneammonium phosphate dihydrogen salt (1.0 g, 60 wt%, 2.1 mmol) and 2,2,2-trichloroacetonitrile (0.45 g, 3.1 mmol) were sequentially added, and the mixture was stirred at 25°C for 168 hours. After the reaction was complete, the reaction solution was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography to obtain homofarnesyl monophosphate N-benzyl-N,N-diethylethaneammonium salt (81 mg, 80 μmol). Physical properties: １ H-NMR (CDCl ３ / TMS): δ 7.58 (dd, 2H), 7.45–7.41 (m, 3H), 5.09 (t, 3H), 4.60 (2, 2H), 3.85 (brs2H), 3.33 (dd, 6H), 2.29 (dd, 2H), 2.09–1.90 (m, 8H), 1.67 (s, 3H), 1.59 (s, 6H), 1.45 (t, 9H).

[0099] Example 2: Production of ambroxide precursor (sclareol glycol) using AsDMS mutant enzyme (1) Chemicals All reagents and solvents were analytical grade and commercially available. The homofarnesyl monophosphate (hFMP) used as the substrate for the enzymatic reaction in Example 2 was a synthetic product prepared in the same manner as in Example 1.

[0100] (2) The bacterial strains and plasmids Escherichia coli (E. coli) DH5α (TaKaRa) and E. coli BL21 Star™ (DE3) (Invitrogen) were used for general DNA manipulation and recombinant protein expression, respectively.

[0101] (Preparation of competent cells of Escherichia coli) Escherichia coli (BL21 (DE3) star) was cultured overnight at 37°C on an LB plate. Colonies were scraped off and cultured with shaking in 100 mL of SOB at 18°C ​​until OD600 = 0.6. The culture medium was cooled on ice for 10 minutes, then centrifuged at 3,000 rpm at 4°C for 10 minutes. After removing the supernatant, the precipitated Escherichia coli was suspended in 30 mL (1 / 3 vol) of TFB cooled to 0°C and left on ice for 10 minutes. It was centrifuged again at 3,000 rpm at 4°C for 10 minutes. After removing the supernatant, the precipitated Escherichia coli was suspended in 10 mL of TFB cooled to 0°C. 0.7 mL of DMSO was slowly added dropwise and left on ice for 10 minutes. The solution was dispensed in 100 μL portions, frozen in liquid nitrogen, and stored at -80°C. Transformation Buffer (TFB): PIPES 3.0 g, CaCl ２・ 2H ２ Dissolve 2.2g of O and 18.6g of KCl in 950mL of water, then adjust the pH to 6.7 with KOH. Add MnCl to the solution. ２ 4H ２ After adding 10.9g of O, the total volume was adjusted to 1L, filtered (0.22μm), and stored at 4°C. SOB: 20g of Bactorypton, 5g of yeast extract, 2mL of 5M NaCl, and 1.25mL of 2M KCl were dissolved in 990mL of water. After autoclaving this solution, 2M Mg ２＋ Solution (1M MgSO4) ４7H ２ O, 1M MgCl ２ 6H ２ 10 mL of (O) was added.

[0102] (Transformation) 100 μL of BL21 (DE3) star competent cells were thawed on ice, and 100 ng of pET28b vector into which the AsDMS gene had been introduced was added and kept on ice for 30 minutes. After heat treatment at 42°C for 30 seconds, the cells were returned to ice and cooled for 1-2 minutes. 0.5 mL of SOC was added, and the cells were cultured with shaking at 37°C for 1 hour. The cells were then plated on an LB plate containing 50 μg / mL kanamycin and cultured overnight at 37°C. SOC: 20 g of Bactorhynchon, 5 g of yeast extract, 2 mL of 5 M NaCl, and 1.25 mL of 2 M KCl were dissolved in 990 mL of water. After autoclaving this solution, 2 M Mg ２＋ Solution (1M MgSO4) ４ 7H ２ O, 1M MgCl ２ 6H ２ 10 mL of (O) and 10 mL of 2 M glucose were added.

[0103] (3) Preparation of site-directed mutagenesis The AsDMS gene was cloned into a pET28b(+) vector (Novagen) between the NdeI and XhoI restriction sites (Reference 1). pET28b(+)-AsDMS was used as a template. An artificial gene synthesis pET28b-AsDMS (Y319F_F509Y_A511Y_F518A_R513V) was created by introducing a mutation into the AsDMS sequence via GenScript outsourcing. (Reference 1) Nhu Ngoc Quynh Vo, Yuhta Nomura, Kiyomi Kinugasa, Hiroshi Takagi, and Shunji Takahashi, Identification and Characterization of Bifunctional Drimenol Synthases of Marine Bacterial Origin. ACS Chem. Biol. 2022, 17: p.1226-1238. Similarly, artificial genes were synthesized by introducing the mutations listed in Table 1 into the AsDMS sequence.

[0104]

[0105] (a) Y319F (b) F509Y (c) A511Y or A511F (d) F518A or F518S or F518H (e) R513V or R513H (f) S514T

[0106] Artificial genes were synthesized by further introducing the mutations listed in Table 2, based on one of the AsDMS mutant enzyme genes created above (Y319F_F509Y_A511Y_R513H_S514T_F518A). In Table 2, "Before Mutation" indicates the type of amino acid at each position in Sequence ID No. 1, and "After Mutation" indicates the type of amino acid after substitution at that position.

[0107]

[0108]

[0109] (4) Enzyme Purification A protein expression strain of Escherichia coli BL21 (DE3) star was prepared by introducing the gene for the AsDMS mutant enzyme [pET28b-AsDMS(Y319F_F509Y_A511Y_F518A_R513V)]. It was cultured in Luria-Bertani (LB) medium (4 mL) containing kanamycin (50 μg / mL) at 37°C and 225 rpm for approximately 16 hours. Terrifico Bloc (TB) medium (100 mL) containing kanamycin (50 μg / mL) was inoculated so that the initial O.D. at 600 nm was 0.04, and the O.D. was purified at 37°C and 220 rpm. Isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to the culture medium to a final concentration of 0.5 mM when the 600 nm wavelength reached 0.9–1.0. The culture was then incubated at 18°C ​​and 220 rpm for 24 hours. The bacterial cells were then collected from the culture medium by centrifugation (9,000 x g, 5 minutes). All enzyme purification procedures were performed in instruments and a chromatography chamber set to 4°C, or under ice cooling. 1 g of bacterial cells were suspended in 20 mL of buffer solution (Buffer A [50 mM Tris-HCl pH 7.5, 500 mM NaCl, 20% glycerol] containing 0.5 mg / mL Lysozyme and 0.4 μL / mL Sm2 Nuclease) and then sonication was performed (QSONICA Sonicator, 3 sets of [10 sec ON / 10 sec OFF x 2 minutes, 4 minutes on ice]). Next, the supernatant was collected by centrifugation (13,000 x g, 30 minutes) and deposited onto a Ni-NTA agarose (2 mL) column. The column was washed with 10 times the column volume of buffer (Buffer A + 30 mM imidazole), and then the target enzyme was eluted with 2.5 times the column volume of elution buffer (Buffer A containing 200 mM imidazole). The enzyme solution was concentrated by ultrafiltration (Amicon® Ultra 30K filter) (4,000x g). The enzyme protein concentration was quantified using Protein Assay Dye Reagent Concentrate (Bio-Rad) with bovine serum albumin as the standard protein. The purified enzyme was rapidly frozen in liquid nitrogen and stored at -80°C.Terrific Broth (TB) medium: Tryptone 12g, yeast extract 24g, glycerol 8mL, potassium (K). ２ HPO ４ 9.4g, KH ２ PO ４ 2.2g (H ２ (Total volume 1 L) In the same manner, mutant proteins were obtained in which the mutations described in Tables 1 and 2 were introduced into the AsDMS sequence. Furthermore, the cyclization activity of each mutant protein was confirmed, and purified AsDMS mutant enzymes were obtained.

[0110] (5) Sclareol glycol production reaction using AsDMS mutant enzyme The AsDMS mutant enzyme used was Y319F_F509Y_A511Y_F518A_R513V (Table 1, No. 6). Reaction solution (17 mM sodium phosphate buffer (pH 7.5), 0.5 mM MgCl ２0.2 ml of 0.1 mM homofarnesyl monophosphate diammonium salt (hFMP) or 0.2 ml of 0.1 mM homofarnesyl pyrophosphate diammonium salt (hFPP) was thoroughly mixed, incubated in a 30°C water bath for 5 minutes, and then AsDMS mutant enzyme was added to a final concentration of 0.4 μM. The mixture was reacted at 35°C for 16 hours. Subsequently, 0.2 mL of ethyl acetate was added to the reaction mixture, and after thorough stirring, the organic layer was recovered. This procedure was repeated twice, and an appropriate amount of anhydrous magnesium sulfate was added to the recovered ethyl acetate solution to dehydrate it. After precipitating the magnesium sulfate by centrifugation (13,000 x g, 3 min), 50 μL of the supernatant was transferred to a GC-MS vial and GC-MS analysis was performed (sclareol glycol RT = 20.5-21.5 min). The results are shown in Figures 2 and 3. The conversion efficiencies to sclareol glycol were over 90% (hFMP) and 5% (hFPP), respectively. Furthermore, sclareol glycol was converted to ambroxide by a known reaction. Similarly, the sclareol glycol production reaction was carried out using the following AsDMS mutant enzymes: Y319F_F509Y_A511F_F518A (Table 1, No. 1), Y319F_F509Y_A511F_F518S (Table 1, No. 2), Y319F_F509Y_A511F_F518H (Table 1, No. 3), Y319F_F509Y_A511Y_F518A (Table 1, No. 4), and Y319F_F509Y_A511Y_F518S (Table 1, No. 5). The results are shown in Figures 4 and 5. In all cases, conversion to sclareol glycol was confirmed.

[0111] (6) GC-MS Analysis Conditions The ethyl acetate extracted sample after the enzymatic reaction was analyzed using GC-MS (5795C, Agilent). The column used was HP-5MS UI (30 m, 0.25 mm, Agilent). High-purity helium was used as the carrier gas. The starting temperature was 70°C, and the temperature was raised to 230°C over 30 minutes. The analysis was then performed using a program that raised the temperature to 300°C over 4.5 minutes.

[0112] According to the present invention, it is possible to produce an ambroxide precursor from a homofarnesol derivative in a single step. Therefore, it is possible to provide a more cost-effective manufacturing method for ensuring a stable industrial supply of ambroxide, which is economically important. This application is based on Japanese Patent Application No. 2024-218536 (filing date: December 13, 2024), the contents of which are fully incorporated herein.

Claims

1. A polypeptide having cyclization activity, comprising an amino acid sequence having at least one mutation selected from the group consisting of (a) to (d) below, in which (i) the amino acid sequence represented by SEQ ID NO: 1, (ii) an amino acid sequence having 95% or more identity with the amino acid sequence represented by SEQ ID NO: 1, or (iii) an amino acid sequence having one to several amino acid substitutions, deletions, insertions or additions in the amino acid sequence represented by SEQ ID NO: 1: (a) A mutation in which tyrosine at position 319 or equivalent in the amino acid sequence of SEQ ID NO: 1 is substituted with phenylalanine. (b) A mutation in which phenylalanine at position 509 or equivalent in the amino acid sequence of SEQ ID NO: 1 is substituted with tyrosine. (c) A mutation in which alanine at position 511 or equivalent in the amino acid sequence of SEQ ID NO: 1 is substituted with tyrosine or phenylalanine. (d) A mutation in which phenylalanine at position 518 or equivalent in the amino acid sequence of SEQ ID NO: 1 is substituted with alanine, serine or histidine.

2. The polypeptide according to claim 1, wherein at least two mutations are selected from the group consisting of (a) to (d).

3. The polypeptide according to claim 1, wherein at least three mutations are selected from the group consisting of (a) to (d).

4. The polypeptide according to claim 1, having all of the mutations (a) to (d).

5. The polypeptide according to claim 4, further comprising: (e) a mutation in which arginine at position 513 or equivalent in the amino acid sequence of SEQ ID NO: 1 is replaced with valine, histidine, alanine, threonine, proline, or aspartic acid; and / or (f) a mutation in which serine at position 514 or equivalent in the amino acid sequence of SEQ ID NO: 1 is replaced with threonine, isoleucine, cysteine, or valine.

6. The polypeptide according to claim 5, having all of the mutations (a) to (f).

7. Furthermore, (g-1) Mutations in which leucine at position 44 or equivalent in the amino acid sequence of SEQ ID NO: 1 is replaced by cysteine, glutamine, or alanine. (g-2) Mutations in which leucine at position 49 or equivalent in the amino acid sequence of SEQ ID NO: 1 is replaced by cysteine, isoleucine, threonine, or serine. (g-3) Mutations in which tryptophan at position 51 or equivalent in the amino acid sequence of SEQ ID NO: 1 is replaced by isoleucine, phenylalanine, or leucine. (g-4) Mutations in which valine at position 70 or equivalent in the amino acid sequence of SEQ ID NO: 1 is replaced by isoleucine, leucine, phenylalanine, or tyrosine. (g-5) Mutations in which leucine at position 107 or equivalent in the amino acid sequence of SEQ ID NO: 1 is replaced by valine. (g-6) Mutations in which isoleucine at position 111 or equivalent in the amino acid sequence of SEQ ID NO: 1 is replaced by phenylalanine, serine, asparagine, leucine, valine, or threonine. (g-7) A mutation in which leucine at position 114 or equivalent in the amino acid sequence of SEQ ID NO: 1 is replaced by valine or phenylalanine. (g-8) A mutation in which valine at position 140 or equivalent in the amino acid sequence of SEQ ID NO: 1 is replaced by glutamine, asparagine, cysteine, or serine. (g-9) A mutation in which serine at position 144 or equivalent in the amino acid sequence of SEQ ID NO: 1 is replaced by valine, leucine, aspartic acid, histidine, or tyrosine. (g-10) A mutation in which leucine at position 148 or equivalent in the amino acid sequence of SEQ ID NO: 1 is replaced by threonine, isoleucine, cysteine, or serine. (g-11) A mutation in which valine at position 264 or equivalent in the amino acid sequence of SEQ ID NO: 1 is replaced by leucine, threonine, cysteine, asparagine, or glutamine. (g-12) A mutation in which cysteine ​​at position 317 or equivalent in the amino acid sequence of SEQ ID NO: 1 is replaced with asparagine or glutamine. (g-13) A mutation in which phenylalanine at position 318 or equivalent in the amino acid sequence of SEQ ID NO: 1 is replaced with tyrosine.The polypeptide according to claim 6, having at least one mutation selected from: (g-14) a mutation in which phenylalanine at position 328 or equivalent in the amino acid sequence of SEQ ID NO: 1 is replaced with tyrosine or histidine; (g-15) a mutation in which proline at position 512 or equivalent in the amino acid sequence of SEQ ID NO: 1 is replaced with tryptophan or aspartic acid; or (g-16) a mutation in which threonine at position 516 or equivalent in the amino acid sequence of SEQ ID NO: 1 is replaced with arginine or histidine.

8. The polypeptide according to claim 1, having (a) a mutation in which tyrosine at position 319 or a corresponding position in the amino acid sequence of SEQ ID NO: 1 is replaced by phenylalanine; (b) a mutation in which phenylalanine at position 509 or a corresponding position in the amino acid sequence of SEQ ID NO: 1 is replaced by tyrosine; (c) a mutation in which alanine at position 511 or a corresponding position in the amino acid sequence of SEQ ID NO: 1 is replaced by tyrosine or phenylalanine; and (d) a mutation in which phenylalanine at position 518 or a corresponding position in the amino acid sequence of SEQ ID NO: 1 is replaced by alanine, serine or histidine.

9. The polypeptide according to claim 1, wherein the mutation in (c) is a substitution of alanine to tyrosine at the 511th position or a corresponding position in the amino acid sequence of SEQ ID NO:

1.

10. The polypeptide according to claim 1, wherein the mutation in (d) is a substitution of phenylalanine to alanine at position 518 or a corresponding position in the amino acid sequence of SEQ ID NO:

1.

11. A polypeptide selected from (i) to (iii) below: (i) A polypeptide consisting of the amino acid sequence represented by Sequence ID No. 2; (ii) A polypeptide consisting of an amino acid sequence having 95% or more identity with the amino acid sequence represented by Sequence ID No. 2 and possessing cyclization activity; (iii) A polypeptide consisting of an amino acid sequence having one to several amino acid substitutions, deletions, insertions or additions in the amino acid sequence represented by Sequence ID No. 2 and possessing cyclization activity, provided that in polypeptides (ii) and (iii), the amino acids at position 319 or equivalent, position 509 or equivalent, position 511 or equivalent, position 518 or equivalent, and position 513 or equivalent in the amino acid sequence of Sequence ID No. 2 are not mutated.

12. A method for producing sclareol glycol, characterized by contacting homofarnesyl monophosphate or a salt thereof represented by the following formula (I) or homofarnesyl pyrophosphate or a salt thereof represented by the following formula (II) with a polypeptide according to any one of claims 1 to 11, a microorganism or cell capable of producing the polypeptide, an extract of the microorganism or cell, and / or a culture medium containing the polypeptide obtained by culturing the microorganism or cell, thereby cyclizing the homofarnesyl monophosphate or a salt thereof or homofarnesyl pyrophosphate or a salt thereof.

13. The method according to claim 12, wherein the salt is an ammonium salt.

14. The method according to claim 13, wherein the ammonium salt is a diammonium salt.

15. The method according to claim 13, wherein the ammonium salt is an alkylammonium salt.

16. The method according to claim 13, wherein the ammonium salt is benzylammonium salt.

17. A compound represented by the following formula (I) or a salt thereof.

18. The compound or salt thereof according to claim 17, wherein the salt is an ammonium salt.

19. The compound or salt thereof according to claim 18, wherein the ammonium salt is a diammonium salt.

20. The compound or salt thereof according to claim 18, wherein the ammonium salt is an alkylammonium salt.

21. The compound or salt thereof according to claim 18, wherein the ammonium salt is benzylammonium salt.

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