Ferulic acid decarboxylase and method for producing unsaturated hydrocarbon compound using same

Abstract

By combining (a) an FDC having tryptophan at the 397 position, methionine at the 398 position, tyrosine at the 440 position, and methionine at the 189 position, and / or (b) an FDC having tryptophan at the 397 position, methionine at the 398 position, tyrosine at the 440 position, and tryptophan at the 189 position, and (c) an FDC having histidine at the 397 position, and glutamic acid at the 398 position, compared with the case of using the FDC set forth in (c) alone, the FDC set forth in (c) can be used in combination with the FDC at the 397 position, methionine at the 398 position, tyrosine at the 440 position, and methionine at the 189 position. The catalytic activity related to the production of unsaturated hydrocarbon compounds is significantly improved, and unsaturated hydrocarbon compounds such as butadiene can be produced with high productivity.

Description

Technical Field

[0001] The present invention relates to ferulic acid decarboxylase and a method for producing an unsaturated hydrocarbon compound using the enzyme. More specifically, the present invention relates to ferulic acid decarboxylase having catalytic activity for producing an unsaturated hydrocarbon compound or a combination of the ferulic acid decarboxylases, DNA encoding the ferulic acid decarboxylase or the combination, a vector into which the DNA is inserted, and a host cell into which the DNA or the vector is introduced. The present invention also relates to a method for producing an unsaturated hydrocarbon compound using the combination or the host cell. Further, the present invention also relates to a preparation for promoting the production of an unsaturated hydrocarbon compound, which contains the decarboxylase, the DNA or the vector. Background Art

[0002] Since butadiene (1,3-butadiene) is used as a raw material for various high molecular compounds such as various synthetic rubbers such as butadiene rubber and polymer resins such as ABS resin, it can be said to be an extremely important organic compound in the chemical industry. In addition, these high molecular compounds using butadiene as a raw material are widely used not only for industrial products such as automobile tires but also for daily necessities such as clothing. Therefore, the demand for butadiene has been increasing year by year, with an annual demand of 13 million tons, and the market size has also reached 15 billion US dollars.

[0003] Hitherto, butadiene has mainly been produced by purifying the C4 fraction by-produced when ethylene and propylene are produced from petroleum. However, due to environmental problems such as the depletion of fossil fuels such as petroleum and global warming caused by greenhouse gas emissions, in order to meet the increasing demand for butadiene, the necessity of realizing sustainable butadiene production has increased. Further, as a countermeasure, the development of a method for producing butadiene using an enzyme from a biomass resource as a renewable resource has been actively carried out.

[0004] On the other hand, the present inventors have found that the production of 4-vinylguaiacol (4VG) by the decarboxylation reaction of ferulic acid participated by ferulic acid decarboxylase (FDC) (see Non-Patent Document 1 and the following formula) can be applied to the production of unsaturated hydrocarbon compounds such as butadiene (Patent Document 3).

[0005]

[0006] That is, the present inventors have found that by introducing a mutation into the amino acid of FDC, the substrate specificity of the enzyme can be changed from the original one for ferulic acid to one for muconic acid or the like, and thus butadiene or the like can be produced through the decarboxylation reaction shown by the following formula (Patent Documents 1 and 2).

[0007]

[0008] For example, the inventors have found that in the wild-type FDC (amino acid sequence set forth in SEQ ID NO: 2) derived from Saccharomyces cerevisiae, the catalytic activities of the FDC mutants in which isoleucine at position 398 is replaced with glutamic acid or methionine are increased to 9.3-fold and 16.4-fold, respectively, compared to the wild-type. Furthermore, the inventors have also found that when phenylalanine at position 397 is replaced with histidine or methionine in the FDC mutant in which isoleucine at position 398 is replaced with glutamic acid, the catalytic activities are increased to 75.1-fold and 33.8-fold, respectively, compared to the wild-type. In addition, the inventors have also found that when phenylalanine at position 397 is replaced with histidine in the FDC mutant in which isoleucine at position 398 is replaced with methionine, the catalytic activity is increased to 37.3-fold compared to the wild-type (Patent Document 2).

[0009] Prior Art Documents

[0010] Patent Documents

[0011] Patent Document 1: International Publication No. 2019 / 022083

[0012] Patent Document 2: International Publication No. 2021 / 054441 Summary of the Invention

[0013] Problems to be Solved by the Invention

[0014] An object of the present invention is to provide an enzyme capable of producing unsaturated hydrocarbon compounds such as butadiene with high productivity.

[0015] Means for Solving the Problems

[0016] As described above, the inventors have found that by replacing isoleucine at position 398 with glutamic acid and phenylalanine at position 397 with histidine (F397H / I398Q mutant) in the wild-type FDC (amino acid sequence set forth in SEQ ID NO: 2) derived from Saccharomyces cerevisiae, the catalytic activity is increased up to 75.1-fold compared to the wild-type (Patent Document 2).

[0017] Therefore, this time, in order to achieve the above object, the inventors have tried to further improve the productivity of unsaturated hydrocarbon compounds such as butadiene using the FDC mutant, and have repeatedly conducted in-depth studies. Specifically, first, single mutants or double mutants of FDC were prepared, and the above catalytic activities when these mutants were used in combination with the F397H / I398Q mutant were evaluated.

[0018] As a result, it was found that when combined with the FDC double mutant (F397Y / I398M or F mutant) in which phenylalanine at position 397 was replaced with tyrosine and isoleucine at position 398 was replaced with methionine or phenylalanine, the above-mentioned catalytic activity was increased to 5.0 times compared with the case of using only the F397H / I398Q mutant. In addition, it was also found that when combined with the FDC double mutant (F397W / I398M or F mutant) in which phenylalanine at position 397 was replaced with tryptophan and isoleucine at position 398 was replaced with methionine or phenylalanine, the above-mentioned catalytic activity was increased to 3.4 times compared with the case of using only the F397H / I398Q mutant.

[0019] Furthermore, based on the F397Y / I398M or F mutant and the F397W / I398M or F mutant, further amino acid substitutions were introduced to prepare various FDC triple mutants. Then, the combination with the F397H / I398Q mutant was evaluated.

[0020] As a result, it was found that when phenylalanine at position 440 was replaced with tyrosine in the F397Y / I398M mutant, or when methionine at position 286 was replaced with leucine in the F397Y / I398F mutant, the above-mentioned catalytic activity was further increased (5.9 times or 6.2 times) by more than 5 times.

[0021] In addition, it was also found that when phenylalanine at position 440 was replaced with tyrosine in the F397W / I398M mutant (F397W / I398M / F440Y mutant), or when methionine at position 286 was replaced with leucine in the F397W / I398F mutant (F397W / I398F / M286L mutant), the above-mentioned catalytic activity was further increased (4.6 times or 4.5 times) by more than 3.4 times.

[0022] Furthermore, based on these FDC triple mutants, further amino acid substitutions were introduced to prepare various FDC quadruple mutants. Then, the combination with the F397H / I398Q mutant was evaluated.

[0023] As a result, even if further amino acid substitutions were introduced into the FDC triple mutant of F397Y, it did not exceed 5.9 times or 6.2 times.

[0024] On the other hand, regarding the FDC triple mutant of F397W, when isoleucine at position 189 is replaced with methionine in the F397W / I398F / M286L mutant, or when methionine at position 286 is replaced with leucine in the F397W / I398M / F440Y mutant, the above-mentioned catalytic activity is further increased by more than 4.6 times or 4.5 times (5.6 times or 5.9 times).

[0025] Furthermore, it was also found that when isoleucine at position 189 is replaced with tryptophan or methionine in the F397W / I398M / F440Y mutant, the above-mentioned catalytic activity is also increased to more than about 7 times (7.3 times or 8.3 times) compared with the case of using only the F397H / I398Q mutant. That is, it was found that since even when the F397H / I398Q mutant is used alone, it is 75.1 times that of the wild type, the catalytic activity in the case of being used in combination with the F397W / I398M / F440Y / I189W mutant is increased to about 548 times that of the wild type, and the catalytic activity in the case of being used in combination with the F397W / I398M / F440Y / I189M mutant is increased to about 623 times that of the wild type.

[0026] In this way, by combining with the F397H / I398Q mutant, a further FDC mutant with a catalytic activity for generating unsaturated hydrocarbon compounds such as butadiene that is increased by more than about 500 times compared with the wild type was successfully prepared, thus completing the present invention.

[0027] That is, the present invention provides the following solutions.

[0028] [1] A combination of at least one ferulic acid decarboxylase selected from the following (a) and (b) and the ferulic acid decarboxylase described in the following (c),

[0029] (a) A ferulic acid decarboxylase in which the amino acid at position 397 of the amino acid sequence set forth in SEQ ID NO: 2 or the corresponding amino acid at this position is tryptophan, the amino acid at position 398 or the corresponding amino acid at this position is methionine, the amino acid at position 440 or the corresponding amino acid at this position is tyrosine, the amino acid at position 189 or the corresponding amino acid at this position is methionine, and which has a catalytic activity for generating an unsaturated hydrocarbon compound represented by the following formula (2) or (3) or a geometric isomer thereof,

[0030] (b) An enzyme of ferulic acid decarboxylase, wherein the amino acid at position 397 of the amino acid sequence set forth in SEQ ID NO: 2 or the amino acid corresponding to this position is tryptophan, the amino acid at position 398 or the amino acid corresponding to this position is methionine, the amino acid at position 440 or the amino acid corresponding to this position is tyrosine, the amino acid at position 189 or the amino acid corresponding to this position is tryptophan, and which has catalytic activity for producing an unsaturated hydrocarbon compound represented by the following formula (2) or (3) or a geometric isomer thereof,

[0031] (c) An enzyme of ferulic acid decarboxylase, wherein the amino acid at position 397 of the amino acid sequence set forth in SEQ ID NO: 2 or the amino acid corresponding to this position is histidine, the amino acid at position 398 or the amino acid corresponding to this position is glutamic acid, and which has catalytic activity for producing an unsaturated hydrocarbon compound represented by the following formula (2) or (3) or a geometric isomer thereof,

[0032]

[0033] [In formulas (2) and (3), "R 1 ", "R 2 ", "R 3 ", and "R 4 " each independently represent a hydrogen atom, a linear or branched alkyl group having 1 to 5 carbon atoms, a linear or branched alkoxy group having 1 to 5 carbon atoms, or a hydroxyl group. "A" represents an optionally substituted linear hydrocarbon group having 0 to 5 carbon atoms, and when the number of carbon atoms is 2 to 5, a double bond may be formed between adjacent carbon atoms].

[0034] [2] A DNA encoding the combination of the ferulic acid decarboxylase described in [1].

[0035] [3] A vector containing the DNA encoding the combination of the ferulic acid decarboxylase described in [1].

[0036] [4] A combination of a host cell into which a DNA encoding at least one ferulic acid decarboxylase selected from (a) and (b) described in [1] or a vector containing the DNA has been introduced, and a host cell into which a DNA encoding the ferulic acid decarboxylase described in (c) of [1] or a vector containing the DNA has been introduced.

[0037] [5] A host cell into which a DNA encoding the combination of the ferulic acid decarboxylase described in [1] or a vector containing the DNA has been introduced.

[0038] [6] A method for producing an unsaturated hydrocarbon compound represented by the following formula (3) or a geometric isomer thereof, comprising the step of decarboxylating an unsaturated hydrocarbon dicarboxylic acid compound represented by the following formula (1) or a geometric isomer thereof in the presence of the combination of the ferulic acid decarboxylase described in [1],

[0039]

[0040] [In formulas (1) to (3), "R 1 ", "R 2 ", "R 3 ", and "R 4 " each independently represent a hydrogen atom, a linear or branched alkyl group having 1 to 5 carbon atoms, a linear or branched alkoxy group having 1 to 5 carbon atoms, or a hydroxyl group. "A" represents a linear hydrocarbon group having 0 to 5 carbon atoms that may be substituted, and when the number of carbon atoms is 2 to 5, a double bond may be formed between adjacent carbon atoms].

[0041] [7] A method for producing an unsaturated hydrocarbon compound, comprising: culturing the host cell according to [4], and collecting the unsaturated hydrocarbon compound represented by the following formula (3) or a geometric isomer thereof generated in the host cell and / or its culture,

[0042]

[0043] [In formula (3), "R 1 ", "R 2 ", "R 3 ", and "R 4 " each independently represent a hydrogen atom, a linear or branched alkyl group having 1 to 5 carbon atoms, a linear or branched alkoxy group having 1 to 5 carbon atoms, or a hydroxyl group. "A" represents a linear hydrocarbon group having 0 to 5 carbon atoms that may be substituted, and when the number of carbon atoms is 2 to 5, a double bond may be formed between adjacent carbon atoms].

[0044] [8] A method for producing an unsaturated hydrocarbon compound, comprising: culturing the combination of the host cells according to [5], and collecting the unsaturated hydrocarbon compound represented by the formula (3) or a geometric isomer thereof generated in the combination of the host cells and / or its culture.

[0045] [9] A preparation for decarboxylating an unsaturated hydrocarbon dicarboxylic acid compound represented by the following formula (1) or a geometric isomer thereof and promoting the generation of an unsaturated hydrocarbon compound represented by the following formula (3) or a geometric isomer thereof, comprising the combination of the ferulic acid decarboxylase according to [1], DNA encoding the combination, or a vector containing the DNA,

[0046]

[0047] [In formulas (1) to (3), "R 1 ", "R 2 ", "R 3 ", and "R 4”Each independently represents a hydrogen atom, a linear or branched alkyl group having 1 to 5 carbon atoms, a linear or branched alkoxy group having 1 to 5 carbon atoms, or a hydroxyl group. “A” represents a linear hydrocarbon group having 0 to 5 carbon atoms that may be substituted, and when the number of carbon atoms is 2 to 5, a double bond may be formed between adjacent carbon atoms.”

[0048]

[10] At least one ferulic acid decarboxylase selected from the following (a) and (b),

[0049] (a) A ferulic acid decarboxylase in which the 397th position of the amino acid sequence set forth in SEQ ID NO: 2 or the amino acid corresponding to this position is tryptophan, the 398th position or the amino acid corresponding to this position is methionine, the 440th position or the amino acid corresponding to this position is tyrosine, the 189th position or the amino acid corresponding to this position is methionine, and which has catalytic activity for generating an unsaturated hydrocarbon compound represented by the following formula (2) or (3) or their geometric isomers,

[0050] (b) A ferulic acid decarboxylase in which the 397th position of the amino acid sequence set forth in SEQ ID NO: 2 or the amino acid corresponding to this position is tryptophan, the 398th position or the amino acid corresponding to this position is methionine, the 440th position or the amino acid corresponding to this position is tyrosine, the 189th position or the amino acid corresponding to this position is tryptophan, and which has catalytic activity for generating an unsaturated hydrocarbon compound represented by the following formula (2) or (3) or their geometric isomers.

[0051]

[0052] [In formulas (2) and (3), “R 1 ”, “R 2 ”, “R 3 ” and “R 4 ” each independently represents a hydrogen atom, a linear or branched alkyl group having 1 to 5 carbon atoms, a linear or branched alkoxy group having 1 to 5 carbon atoms, or a hydroxyl group. “A” represents a linear hydrocarbon group having 0 to 5 carbon atoms that may be substituted, and when the number of carbon atoms is 2 to 5, a double bond may be formed between adjacent carbon atoms.]

[0053]

[11] A DNA encoding the ferulic acid decarboxylase described in

[10] .

[0054]

[12] A vector containing the DNA encoding the ferulic acid decarboxylase described in

[10] .

[0055]

[13] A host cell into which the DNA encoding the ferulic acid decarboxylase described in

[10] or a vector containing this DNA has been introduced.

[0056]

[14] A preparation for decarboxylating an unsaturated hydrocarbon dicarboxylic acid compound represented by the following formula (1) or its geometric isomer and promoting the production of an unsaturated hydrocarbon compound represented by the following formula (3) or its geometric isomer, which comprises the ferulic acid decarboxylase described in

[10] , the DNA described in

[11] , or the vector described in

[12] .

[0057] It is characterized by being used in combination with a ferulic acid decarboxylase in which the amino acid at position 397 of the amino acid sequence set forth in SEQ ID NO: 2 or the amino acid corresponding to this position is histidine, the amino acid at position 398 or the amino acid corresponding to this position is glutamic acid, and which has the above-mentioned catalytic activity, a DNA encoding the ferulic acid decarboxylase, or a vector containing the DNA.

[0058]

[0059] [In formulas (1) to (3), "R 1 ", "R 2 ", "R 3 ", and "R 4 " each independently represent a hydrogen atom, a linear or branched alkyl group having 1 to 5 carbon atoms, a linear or branched alkoxy group having 1 to 5 carbon atoms, or a hydroxyl group. "A" represents an optionally substituted linear hydrocarbon group having 0 to 5 carbon atoms, and when the number of carbon atoms is 2 to 5, a double bond can be formed between adjacent carbon atoms].

[0060] Effects of the Invention

[0061] According to the present invention, it is possible to provide an enzyme capable of producing an unsaturated hydrocarbon compound such as butadiene with high productivity, and a method for producing an unsaturated hydrocarbon compound using the enzyme. Detailed Description of the Invention

[0062] <Ferulic Acid Decarboxylase>

[0063] As shown in the examples described later, it was found that by combining (a) a ferulic acid decarboxylase in which the amino acid at position 397 is tryptophan, the amino acid at position 398 is methionine, the amino acid at position 440 is tyrosine, and the amino acid at position 189 is methionine, or (b) a ferulic acid decarboxylase in which the amino acid at position 397 is tryptophan, the amino acid at position 398 is methionine, the amino acid at position 440 is tyrosine, and the amino acid at position 189 is tryptophan, with (c) a ferulic acid decarboxylase in which the amino acid at position 397 is histidine and the amino acid at position 398 is glutamic acid, the catalytic activity of promoting the following reaction for producing an unsaturated hydrocarbon compound represented by the following formula (3) or its geometric isomers (hereinafter also simply referred to as "catalytic activity for producing an unsaturated hydrocarbon compound") is higher than when the ferulic acid decarboxylase described in (c) is used alone.

[0064]

[0065] Accordingly, the present invention relates to at least one ferulic acid decarboxylase selected from the following (a) and (b), and a combination with the ferulic acid decarboxylase described in the following (c).

[0066] (a) A ferulic acid decarboxylase in which the amino acid at position 397 of the amino acid sequence set forth in SEQ ID NO: 2 or the corresponding position thereto is tryptophan, the amino acid at position 398 or the corresponding position thereto is methionine, the amino acid at position 440 or the corresponding position thereto is tyrosine, the amino acid at position 189 or the corresponding position thereto is methionine, and which has catalytic activity for generating the unsaturated hydrocarbon compound represented by the formula (2) or (3) or their geometric isomers.

[0067] (b) A ferulic acid decarboxylase in which the amino acid at position 397 of the amino acid sequence set forth in SEQ ID NO: 2 or the corresponding position thereto is tryptophan, the amino acid at position 398 or the corresponding position thereto is methionine, the amino acid at position 440 or the corresponding position thereto is tyrosine, the amino acid at position 189 or the corresponding position thereto is tryptophan, and which has catalytic activity for generating the unsaturated hydrocarbon compound represented by the formula (2) or (3) or their geometric isomers.

[0068] (c) A ferulic acid decarboxylase in which the amino acid at position 397 of the amino acid sequence set forth in SEQ ID NO: 2 or the corresponding position thereto is histidine, the amino acid at position 398 or the corresponding position thereto is glutamic acid, and which has catalytic activity for generating the unsaturated hydrocarbon compound represented by the formula (2) or (3) or their geometric isomers.

[0069] In addition, in the present invention, the so-called "corresponding position" refers to a position equivalent to isoleucine at position 398 in the amino acid sequence set forth in SEQ ID NO: 2 when aligned with the amino acid sequence set forth in SEQ ID NO: 2 using nucleotide and amino acid sequence analysis software (such as GENETYX-MAC, Sequencher, etc.) or BLAST (http: / / blast.ncbi.nlm.nih.gov / Blast.cgi).

[0070] The so-called "ferulic acid decarboxylase (FDC)" is usually an enzyme registered under the EC number: 4.1.1.102, and refers to an enzyme that catalyzes the following reaction of decarboxylating ferulic acid to produce 4-vinylguaiacol (4VG).

[0071]

[0072] However, in the present invention, as shown in the following embodiments, the FDC has catalytic activity for generating the unsaturated hydrocarbon compounds represented by the above formula (2) or (3) or their geometric isomers because the 397th position, 398th position, etc. are specific amino acids, respectively. In addition, regarding the catalytic activity for generating the unsaturated hydrocarbon compounds, for example, as shown in the following embodiments, it can be evaluated by directly measuring the amount of the unsaturated hydrocarbon compounds using gas chromatography-mass spectrometry (GC-MS).

[0073] As described above, the FDC of the present invention relates to a combination of at least one FDC selected from the above (a) and (b) and the FDC described in the above (c), and exhibits catalytic activity for generating unsaturated hydrocarbon compounds that is higher than that of the FDC described in the above (c). Here, the term "higher" preferably means 3 times or more, more preferably 4 times or more, further preferably 5 times or more, more preferably 6 times or more, further preferably 7 times or more, and more preferably 8 times or more, compared with the FDC described in the above (c). In addition, compared with the catalytic activity of the wild-type FDC (for example, the protein composed of the amino acid sequence described in SEQ ID NO: 2 below), the activity of the FDC of the present invention is preferably 200 times or more, more preferably 300 times or more, further preferably 400 times or more, more preferably 500 times or more, and further preferably 600 times or more.

[0074] The FDC of the present invention that also has this catalytic activity includes not only the ferulic acid decarboxylase in which the amino acids at the 397th position, etc. are artificially replaced with tryptophan, histidine, etc. as shown in the following embodiments (hereinafter also simply referred to as "ferulic acid decarboxylase mutant" or "modified ferulic acid decarboxylase"), but also the naturally occurring ferulic acid decarboxylase in which the amino acids at the 397th position, etc. are tryptophan, histidine, etc. (hereinafter also referred to as "ferulic acid decarboxylase homolog" or "naturally occurring mutant of ferulic acid decarboxylase").

[0075] The FDC of the present invention is not particularly limited with respect to its origin (e.g., Saccharomyces), and preferably comprises a protein having an amino acid sequence having 80% or more (e.g., 81% or more, 82% or more, 83% or more, 84% or more) identity to the FDC from Saccharomyces cerevisiae (the amino acid sequence set forth in SEQ ID NO: 2), more preferably a protein having an amino acid sequence having 85% or more (e.g., 86% or more, 87% or more, 88% or more, 89% or more) identity, still more preferably a protein having an amino acid sequence having 90% or more (e.g., 91% or more, 92% or more, 93% or more, 94% or more) identity, and even more preferably a protein having an amino acid sequence having 95% or more (e.g., 96% or more, 97% or more, 98% or more, 99% or more) identity. In addition, in the present invention, the so-called "identity" refers to the ratio (%) of the number of amino acids identical to the amino acid sequence set forth in SEQ ID NO: 2 in the FDC to the total number of amino acids in the FDC from the Saccharomyces origin.

[0076] The FDC of the present invention may also be a protein having an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted at positions other than positions 397 and 398 (for (a) and (b), further positions 440 and 189) of the amino acid sequence set forth in SEQ ID NO: 2. Here, the so-called "plurality" is not particularly limited, and is usually 2 to 100, preferably 2 to 50, more preferably 2 to 40, still more preferably 2 to 30, even more preferably 2 to 20, and still more preferably 2 to 10 (e.g., 2 to 8, 2 to 4, 2).

[0077] The FDC of the present invention can directly or indirectly add other compounds. As such addition, there is no particular limitation, and it can be addition at the gene level or chemical addition. Additionally, regarding the added site, there is no particular limitation, and it can be either the amino terminus (hereinafter also referred to as the "N-terminus") or the carboxyl terminus (hereinafter also referred to as the "C-terminus") of the FDC of the present invention, or both. Addition at the gene level is achieved by using DNA obtained by adding DNA encoding other proteins in-frame to the DNA encoding the FDC of the present invention. As the "other proteins" added by such operation, there is no particular limitation. When for the purpose of facilitating the purification of the FDC of the present invention, purification tag proteins such as polyhistidine (His-) tag protein, FLAG-tag protein (registered trademark, Sigma-Aldrich), glutathione-S-transferase (GST), etc. are suitable. Additionally, when for the purpose of facilitating the detection of the FDC of the present invention, detection tag proteins such as fluorescent proteins like GFP, chemiluminescent proteins like luciferase, etc. are suitable. Chemical addition can be a covalent bond or a non-covalent bond. As the "covalent bond", there is no particular limitation, and examples include amide bond between amino group and carboxyl group, alkylamine bond between amino group and haloalkyl group, disulfide bond between thiols, and thioether bond between thiol group and maleimide group or haloalkyl group. As the "non-covalent bond", examples include biotin-avidin bond. Additionally, as the "other compounds" chemically added by such operation, when for the purpose of facilitating the detection of the FDC of the present invention, fluorescent dyes such as Cy3, rhodamine, etc. are suitable.

[0078] In addition, the FDC of the present invention can be used in mixture with other components. As the other components, there is no particular limitation, and examples include sterilized water, physiological saline, vegetable oil, surfactant, lipid, cosolvent, buffer, protease inhibitor, preservative.

[0079] <DNA encoding FDC, and vector having the DNA>

[0080] The DNA encoding the FDC of the present invention, etc. will be described. By introducing such DNA, host cells can be transformed, and the FDC of the present invention can be produced in the cells, and further unsaturated hydrocarbon compounds can be produced.

[0081] The DNA of the present invention, as long as it encodes the FDC of the present invention described above, can be either a DNA into which a mutation has been introduced in natural DNA or a DNA composed of a nucleotide sequence designed artificially. Further, there is no particular limitation on its form, and in addition to cDNA, it also includes genomic DNA and chemically synthesized DNA. The preparation of these DNAs can be carried out by those skilled in the art using conventional means. For example, genomic DNA can be prepared as follows: Genomic DNA is extracted from the genus Saccharomyces, a genomic library is made (as a vector, plasmids, phages, cosmids, BACs, PACs, etc. can be used), it is spread out, and colony hybridization or plaque hybridization is carried out using a probe prepared based on the nucleotide sequence of the FDC gene (for example, the nucleotide sequence described in SEQ ID NO: 1) to prepare it. In addition, it can also be prepared by making primers specific to the FDC gene and performing PCR using these primers. In addition, cDNA can be prepared, for example, by synthesizing cDNA based on mRNA extracted from the genus Saccharomyces, inserting it into a vector such as λZAP to make a cDNA library, spreading it out, and performing colony hybridization or plaque hybridization in the same manner as above, and also by performing PCR. And regarding introducing a mutation such as substituting tryptophan or histidine for the 397th position of the amino acid sequence described in SEQ ID NO: 2 or an amino acid corresponding to this site in the DNA thus prepared, those skilled in the art can carry it out by using a known site-directed mutagenesis method. As the site-directed mutagenesis method, for example, the Kunkel method (Kunkel, T.A., Proc Natl Acad Sci USA, 1985, 82, No. 2, 488 - 492) and the SOE (splicing-by-overlap-extention)-PCR method (Ho, S.N., Hunt, H.D., Horton, R.M., Pullen, J.K., and Pease, L.R., Gene, 1989, 77, 51 - 59) can be mentioned. In addition, those skilled in the art can artificially design a nucleotide sequence encoding a protein in which the 397th position of FDC or an amino acid corresponding to this site has been substituted with tryptophan or histidine, and based on this sequence information, chemically synthesize the DNA of the present invention using an automatic nucleic acid synthesizer.

[0082] Furthermore, from the viewpoint of further improving the expression efficiency of the encoded FDC in a host cell, the DNA of the present invention can adopt a scheme of using a DNA encoding the FDC of the present invention with optimized codons according to the type of the host cell.

[0083] In addition, in the present invention, in order to replicate the above DNA in a host cell, a vector into which the DNA is inserted may also be used. In the present invention, the "vector" can exist as an autonomously replicating vector, i.e., an extrachromosomal entity, and can be constructed based on, for example, a plasmid whose replication is independent of chromosomal replication. In addition, the vector can also be a vector that is integrated into the genome of the host cell when introduced into the host cell and is replicated together with the chromosome into which the vector is integrated.

[0084] Examples of such vectors include, for example, plasmids and phage DNAs. In addition, as plasmids, plasmids derived from Escherichia coli (pET22, pBR322, pBR325, pUC118, pUC119, pUC18, pUC19, etc.), plasmids derived from yeast (YEp13, YEp24, YCp50, etc.), and plasmids derived from Bacillus subtilis (pUB110, pTP5, etc.) can be mentioned. As phage DNAs, λ phages (Charon4A, Charon21A, EMBL3, EMBL4, λgt10, λgt11, λZAP, etc.) can be mentioned. Further, if the host cell is derived from an insect, an insect virus vector such as a baculovirus can also be used as the vector of the present invention, if it is derived from a plant, T-DNA, etc. can also be used as the vector of the present invention, and if it is derived from an animal, an animal virus vector such as a retrovirus or an adenovirus vector can also be used as the vector of the present invention. In addition, the steps and methods for constructing the vector of the present invention can use the steps and methods commonly used in the field of genetic engineering. For example, when inserting the DNA of the present invention into a vector, a method of first cutting the purified DNA with an appropriate restriction enzyme and inserting it into the restriction enzyme site or multiple cloning site of an appropriate vector to ligate with the vector is adopted.

[0085] The vector of the present invention may be in the form of an expression vector that contains the FDC encoded by the said DNA in a state capable of being expressed in a host cell. The "expression vector" of the present invention, in order to express the FDC of the present invention by introducing it into a host cell, is expected to contain, in addition to the above DNA, a DNA sequence that controls its expression, a gene marker for selecting the transformed host cell, etc. As the DNA sequence that controls expression, a promoter, an enhancer, a splicing signal, a poly-A addition signal, a ribosome-binding sequence (SD sequence), a terminator, etc. are included therein. The promoter is not particularly limited as long as it shows transcriptional activity in the host cell, and it can be obtained as a DNA sequence that controls the expression of a gene encoding any protein that is the same as or different from the host cell. In addition, in addition to the above DNA sequence that controls expression, a DNA sequence that induces expression may also be included. As such a DNA sequence that induces expression, in the case where the host cell is a bacterium, the lactose operon that can induce the expression of a gene arranged downstream by the addition of isopropyl-β-D-thiogalactopyranoside (IPTG) can be cited. The gene marker in the present invention can be appropriately selected according to the selection method of the transformed host cell. For example, a gene encoding drug resistance, a gene that complements auxotrophy can be used.

[0086] In addition, in the present invention, as described above, a plurality of FDCs are used in combination, but these FDCs can be encoded by 1 vector respectively, and also a plurality of FDCs can be encoded by 1 vector. In the case where 1 vector encodes a plurality of FDCs, for example, by using a DNA encoding an IRES, a 2A peptide sequence, etc., these plurality of FDCs can be expressed polycistronically.

[0087] In addition, the DNA or vector of the present invention can be used in admixture with other components. As the other components, there is no particular limitation, and examples thereof include sterilized water, physiological saline, vegetable oil, surfactant, lipid, solubilizer, buffer, DNase (deoxyribonuclease) inhibitor, preservative.

[0088] <Host cell into which the DNA etc. of the present invention is introduced>

[0089] The host cell into which the DNA or vector of the present invention is introduced will be described. If a host cell transformed by the introduction of the above DNA or vector is used, the FDC of the present invention can be produced, and furthermore, the unsaturated hydrocarbon compound represented by the above formula (3) or its geometric isomers can also be produced.

[0090] The host cell into which the DNA or vector of the present invention is introduced is not particularly limited, and examples thereof include microorganisms (Escherichia coli, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Bacillus subtilis, Actinomycetes, filamentous fungi, etc.), plant cells, insect cells, and animal cells. From the viewpoint of showing high proliferation in a relatively inexpensive medium in a short time and further contributing to the high-productivity production of the unsaturated hydrocarbon compound represented by the above formula (3) or its geometric isomers, it is preferable to use a microorganism as the host cell, and Escherichia coli is more preferable.

[0091] In addition, from the viewpoint of inducing the prenylation of flavin mononucleotide (FMN) and generating prFMN or its isomers that contribute to the improvement of the productivity of the unsaturated hydrocarbon compound represented by the above formula (3) or its geometric isomers, the host cell into which the DNA or vector of the present invention is introduced is preferably a cell retaining flavin prenyltransferase.

[0092] In addition, from the viewpoint of easily generating muconic acid, which is a substrate for the FDC of the present invention, from glucose in the production of butadiene, the host cell into which the DNA or vector of the present invention is introduced is preferably a cell in which the pathway for biosynthesizing muconic acid from glucose via 3-dehydroshikimic acid and catechol is activated. Examples of such cells include cells in which the activities of enzymes of the phosphotransferase system and pyruvate kinase are inhibited and which have enzymes capable of synthesizing aromatic compounds from chorismic acid or iso-chorismic acid (for example, the microorganisms described in International Publication No. 2017 / 033965), Escherichia coli, Pseudomonas putida, or Saccharomyces cerevisiae described in Kruyer NS et al., Curr Opin Biotechnol. 2017, Jun; 45: 136-143.

[0093] The introduction of the DNA or vector of the present invention can also be carried out according to the methods commonly used in the art. For example, as methods for introducing into microorganisms such as Escherichia coli, there are heat shock method, electroporation method, spheroplast method, lithium acetate method; as methods for introducing into plant cells, there are methods using Agrobacterium, particle gun method; as methods for introducing into insect cells, there are methods using baculovirus, electroporation method; as methods for introducing into animal cells, there are calcium phosphate method, lipofection method, electroporation method.

[0094] The DNA etc. thus introduced into the host cell can be maintained by random insertion into its genomic DNA in the host cell, or can be maintained by homologous recombination. In addition, if it is a vector, it can be replicated and maintained as an independent entity outside its genomic DNA.

[0095] <Manufacturing method 1 of unsaturated hydrocarbon compound>

[0096] As described above, the FDC of the present invention has high catalytic activity for generating unsaturated hydrocarbon compounds. Therefore, there is provided a method for producing an unsaturated hydrocarbon compound represented by the following formula (3) or a geometric isomer thereof, including a step of decarboxylating an unsaturated hydrocarbon dicarboxylic acid compound represented by the following formula (1) or a geometric isomer thereof in the presence of the FDC of the present invention.

[0097]

[0098] In the present invention, the "unsaturated hydrocarbon compound or its geometric isomer" generated by the above reaction refers to a hydrocarbon compound having at least one carbon-carbon double bond as shown in the above formula (3), and may be a compound into which a linear or branched alkyl group having 1 to 5 carbon atoms, a linear or branched alkoxy group having 1 to 5 carbon atoms, or a hydroxyl group is introduced. Examples of such compounds include butadiene (1,3-butadiene), 2,4-pentadienoic acid, isocrotonic acid, 3-methylisocrotonic acid, 3-pentenoic acid, and 10-undecenoic acid.

[0099] In the present invention, the "unsaturated hydrocarbon dicarboxylic acid compound or its geometric isomer" that is a raw material for generating an unsaturated hydrocarbon compound refers to a hydrocarbon compound having at least one carbon-carbon double bond and at least two carboxyl groups as shown in the above formula (1), and may be a compound into which a linear or branched alkyl group having 1 to 5 carbon atoms, a linear or branched alkoxy group having 1 to 5 carbon atoms, or a hydroxyl group is introduced. Examples of such compounds include cis,cis-muconic acid, cis,trans-muconic acid, trans,trans-muconic acid, glutaconic acid, 2-methylglutaconic acid, 3-methylglutaconic acid, and traumatic acid.

[0100] The compounds represented by the above formula (1) and their geometric isomers can be purchased as commercially available products as shown in the following examples. In addition, those skilled in the art can appropriately refer to known synthesis methods (for example, the method described in Kiyoshi Kudo et al., Journal of the Japan Petroleum Institute, published on July 13, 1994, Vol. 38, No. 1, pp. 48-51) for synthesis.

[0101] In the compounds represented by the above formulas (1) to (3) and their geometric isomers, "R 1 ", "R 2 ", "R 3 ", and "R 4 " each independently represent a hydrogen atom, a linear or branched alkyl group having 1 to 5 carbon atoms, a linear or branched alkoxy group having 1 to 5 carbon atoms, or a hydroxyl group.

[0102] Examples of the "linear or branched alkyl group having 1 to 5 carbon atoms" include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, and isopentyl.

[0103] Examples of the "linear or branched alkoxy group having 1 to 5 carbon atoms" include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, n-pentyloxy, isopentyloxy, n-pentyloxy, and 1,2-dimethylpropoxy.

[0104] In addition, in the compounds represented by the above formulas (1) to (3) and their geometric isomers, "A" represents a linear hydrocarbon group having 0 to 5 carbon atoms which may be substituted. Further, the "linear hydrocarbon group having 0 carbon atoms which may be substituted" means that the carbon atoms bonded via "A" in the compounds represented by the above formulas (1) to (3) and their geometric isomers are directly bonded to each other without passing through "A". Furthermore, when the number of carbon atoms of the linear hydrocarbon group which may be substituted is 2 to 5, at least one double bond may be formed between adjacent carbon atoms. Examples of the substituents that the hydrocarbon group in "A" may have include linear or branched alkyl groups having 1 to 5 carbon atoms, linear or branched alkoxy groups having 1 to 5 carbon atoms, hydroxyl groups, halogen atoms (e.g., fluorine, chlorine, bromine, iodine), nitro groups, cyano groups, amino groups, carboxyl groups, and formyl groups.

[0105] Regarding the conditions for decarboxylating the unsaturated hydrocarbon dicarboxylic acid compound in the presence of the FDC of the present invention, any conditions under which the decarboxylation is promoted and an unsaturated hydrocarbon compound can be produced are acceptable, and those skilled in the art can appropriately adjust the composition of the reaction solution, the pH of the reaction solution, the reaction temperature, the reaction time, etc. to set the conditions.

[0106] For example, as the reaction solution containing the FDC of the present invention and the unsaturated hydrocarbon dicarboxylic acid compound as its substrate, there is no particular limitation as long as the above reaction is not hindered. Preferably, a buffer solution with a pH of 6 to 8 is selected, and more preferably, a buffer solution containing potassium chloride and sodium phosphate with a pH of 6 to 7 is selected. Further, from the viewpoint of more easily promoting the above reaction, it is preferred to contain prenylated flavin mononucleotide (prFMN) or its isomers (prFMN ketimine , prFMN iminiu , and for these prFMN and its isomers, refer to Non-Patent Document 1).

[0107] The ratio of the FDC used in the production method of the present invention is not particularly limited, but relative to the FDC described in the above (c), in terms of molar ratio, the FDC described in the above (a) and / or the FDC described in the above (b) is usually 0.1 to 10 mol / mol, preferably 0.5 to 5 mol / mol, and more preferably 1 to 2 mol / mol.

[0108] As the reaction temperature, as long as the above reaction is not hindered, there is no particular limitation, and it is usually 20 to 40 °C, preferably 25 to 37 °C. Further, as the reaction time, as long as it is a time capable of generating the above unsaturated hydrocarbon compound, there is no particular limitation, but it is usually 30 minutes to 7 days, preferably 12 hours to 2 days.

[0109] In addition, since the above unsaturated hydrocarbon compound generated under such conditions is generally easy to vaporize, it can be collected by known recovery and purification methods for volatile gases. Examples of such collection methods include stripping, fractional distillation, adsorption, desorption, pervaporation, desorption of isoprene adsorbed on a solid phase from the solid phase using heat or vacuum, extraction using a solvent, or chromatography (e.g., gas chromatography). In addition, even when the generated olefin compound is a liquid, it can be appropriately collected using known recovery and purification methods (distillation, chromatography, etc.). Further, these methods can be carried out alone, and in addition, they can be appropriately combined and carried out in multiple stages.

[0110] <Manufacturing method 2 of unsaturated hydrocarbon compound>

[0111] As described above, by culturing a host cell transformed in a manner expressing the FDC of the present invention, an unsaturated hydrocarbon compound can be produced highly productively. Therefore, in the present invention, there is also provided a method for producing an unsaturated hydrocarbon compound, including culturing a host cell into which a DNA or vector encoding the FDC of the present invention has been introduced, and collecting the unsaturated hydrocarbon compound represented by the above formula (3) or its geometric isomers generated in the host cell and / or its culture.

[0112] Regarding "host cells into which DNA or a vector encoding the FDC of the present invention is introduced", as described above, for example, as shown in the examples described later, host cells into which DNA encoding at least one FDC selected from the above (a) and (b) or a vector containing the DNA is introduced, and a combination with host cells into which DNA encoding the FDC described in the above (c) or a vector containing the DNA is introduced can be cited. In this combination (co-culture), as the ratio of the number of these host cells, when the number of host cells into which DNA encoding the FDC described in the above (c) or a vector containing the DNA is introduced is taken as 1, the number of host cells into which DNA encoding at least one FDC selected from the above (a) and (b) or a vector containing the DNA is introduced is usually 0.1 to 10, preferably 0.5 to 5, more preferably 1 to 2.

[0113] In addition, the cultivation of the host cells in the production method of the present invention is not limited to the above co-culture, and can also be the separate cultivation of host cells into which DNA encoding at least one FDC selected from the above (a) and (b) and DNA encoding the FDC described in the above (c) is introduced. In this case, there is no particular limitation on the ratio of the DNA (or vector) introduced into the host cell. Relative to the DNA encoding the FDC described in the above (c), in terms of molar ratio, the DNA encoding the FD described in the above (a) and / or the DNA encoding the FDC described in the above (b) is usually 0.1 to 10 mol / mol, preferably 0.5 to 5 mol / mol, more preferably 1 to 2 mol / mol.

[0114] In addition, regarding the cultivation conditions of the cells, as described later, it is preferable to add the unsaturated hydrocarbon dicarboxylic acid compound represented by the above formula (1) or their geometric isomers, which are the substrates of the decarboxylase of the present invention, to the medium. The cultivation temperature can be appropriately designed and changed according to the type of host cell used, but is usually 20 to 40 °C, preferably 25 to 37 °C.

[0115] In the present invention, the so-called "cultured product" is a medium obtained by culturing host cells in a medium, containing proliferated host cells, secreted products of the host cells, and metabolites of the host cells, etc., including their dilutions and concentrates.

[0116] Regarding the collection of the unsaturated hydrocarbon compound from such host cells and / or cultured products, there is no particular limitation, and the above-mentioned known recovery and purification methods can be used. In addition, as the collection time, it is appropriately adjusted according to the type of host cell used, as long as it is a time when the unsaturated hydrocarbon compound can be generated, but is usually 30 minutes to 7 days, preferably 12 hours to 2 days.

[0117] <Preparation for Promoting the Formation of Unsaturated Hydrocarbon Compounds>

[0118] As described above, by using the FDC of the present invention, the DNA encoding the FDC, or the vector into which the DNA is inserted, it is possible to decarboxylate the unsaturated hydrocarbon dicarboxylic acid compound represented by the above formula (1) or its geometric isomers, and promote the formation of the unsaturated hydrocarbon compound represented by the above formula (3) or its geometric isomers.

[0119] Therefore, the present invention provides a preparation for decarboxylating the unsaturated hydrocarbon dicarboxylic acid compound represented by the above formula (1) or its geometric isomers and promoting the formation of the unsaturated hydrocarbon compound represented by the above formula (3) or its geometric isomers, comprising: the FDC of the present invention, the DNA encoding the FDC, or the vector into which the DNA is inserted.

[0120] As such a preparation, it is sufficient to contain the FDC of the present invention, etc., and it can also be used in combination with other components. Such other components are not particularly limited, and examples thereof include sterilized water, physiological saline, vegetable oil, surfactant, lipid, cosolvent, buffer, protease inhibitor, DNase inhibitor, preservative.

[0121] In addition, the present invention can also provide a kit containing such a preparation. In the kit of the present invention, the above preparation can be included in the form of the above host cell transformed by introducing the DNA of the present invention, etc. Furthermore, in addition to such a preparation, the compound represented by the above formula (1) or its geometric isomers, the host cell for introducing the DNA of the present invention, etc., the culture medium for culturing the host cell, and their instruction manuals, etc. can also be included in the kit of the present invention. In addition, such an instruction manual is a manual for using the preparation of the present invention, etc. in the above method for manufacturing unsaturated hydrocarbon compounds. The manual can include, for example, the experimental methods, experimental conditions of the manufacturing method of the present invention, and information related to the preparation of the present invention, etc. (such as information such as the vector map showing the nucleotide sequence of the vector, the sequence information of the FDC of the present invention, the source and properties of the host cell, and the culture conditions of the host cell).

[0122] <Method for Producing the FDC of the Present Invention>

[0123] As shown in the examples described later, by culturing the host cell into which the DNA encoding the FDC of the present invention, etc. is introduced, it is possible to produce the FDC in the host cell.

[0124] Therefore, the present invention can also provide a method for producing the FDC of the present invention, comprising: culturing the host cell into which the DNA encoding the FDC of the present invention or the vector containing the DNA is introduced, and collecting the protein expressed in the host cell.

[0125] In the present invention, the conditions for "culturing host cells" are only required to be conditions under which the above host cells can produce the FDC of the present invention. Those skilled in the art can appropriately adjust the temperature, the presence or absence of air addition, the concentration of oxygen, the concentration of carbon dioxide, the pH of the medium, the culture temperature, the culture time, the humidity, etc. according to the type of host cells, the medium used, etc. and make settings.

[0126] As such a medium, it is only required to contain substances that can be assimilated by the host cells. Examples of the contained substances include carbon sources, nitrogen sources, sulfur sources, inorganic salts, metals, peptone, yeast extract, meat extract, casein hydrolysate, serum, etc. In addition, for example, IPTG for inducing the expression of the DNA encoding the FDC of the present invention, an antibiotic corresponding to the drug resistance gene that can be encoded by the vector of the present invention (for example, ampicillin), and a nutrient corresponding to the gene complementary to auxotrophy that can be encoded by the vector of the present invention (for example, arginine, histidine) can be added to such a medium.

[0127] Furthermore, as a method for "collecting the protein expressed in the cell" from the host cells cultured by such an operation, for example, the host cells can be recovered from the medium by filtration, centrifugation, etc., the recovered host cells can be treated by cell lysis, grinding treatment, pressure crushing, etc., and further, the protein expressed in the host cells can be purified and concentrated by ultrafiltration treatment, salting out, solvent precipitation such as ammonium sulfate precipitation, chromatography (for example, gel chromatography, ion exchange chromatography, affinity chromatography), etc. In addition, when the above purification tag protein is added to the FDC of the present invention, purification can also be carried out using a substrate adsorbed with the tag protein for collection. Further, these purification and concentration methods can be carried out alone, and in addition, they can be appropriately combined and implemented in multiple stages.

[0128] In addition, the FDC of the present invention is not limited to the above biological synthesis, and can also be produced using the DNA of the present invention, etc. and a cell-free protein synthesis system. As such a cell-free protein synthesis system, there is no particular limitation, and examples include synthesis systems derived from wheat germ, Escherichia coli, rabbit reticulocytes, and insect cells. Further, those skilled in the art can also chemically synthesize the FDC of the present invention using a commercially available peptide synthesizer, etc.

[0129] As described above, suitable embodiments of the present invention have been described, but the present invention is not limited to the above-described embodiments. For example, as shown in the examples described later, it has also been found that an FDC mutant other than the FDCs described in (a) and (b) above, which has a catalytic activity for generating an unsaturated hydrocarbon compound that can be increased by more than 3 times when combined with the FDC described in (c) above. Thus, in the present invention, not only (a) and (b) above, but also (d) to (m) shown below can be used, and at least one FDC selected from among these can also be used.

[0130] (d) Ferulic acid decarboxylase in which the amino acid at position 397 of the amino acid sequence set forth in SEQ ID NO: 2 or the corresponding amino acid at that position is tyrosine, the amino acid at position 398 or the corresponding amino acid at that position is methionine, and which has a catalytic activity for generating the unsaturated hydrocarbon compound represented by the above formula (2) or (3) or a geometric isomer thereof.

[0131] (e) Ferulic acid decarboxylase in which the amino acid at position 397 of the amino acid sequence set forth in SEQ ID NO: 2 or the corresponding amino acid at that position is tyrosine, the amino acid at position 398 or the corresponding amino acid at that position is methionine, the amino acid at position 440 or the corresponding amino acid at that position is tyrosine, and which has a catalytic activity for generating the unsaturated hydrocarbon compound represented by the above formula (2) or (3) or a geometric isomer thereof.

[0132] (f) Ferulic acid decarboxylase in which the amino acid at position 397 of the amino acid sequence set forth in SEQ ID NO: 2 or the corresponding amino acid at that position is tyrosine, the amino acid at position 398 or the corresponding amino acid at that position is phenylalanine, and which has a catalytic activity for generating the unsaturated hydrocarbon compound represented by the above formula (2) or (3) or a geometric isomer thereof.

[0133] (g) Ferulic acid decarboxylase in which the amino acid at position 397 of the amino acid sequence set forth in SEQ ID NO: 2 or the corresponding amino acid at that position is tyrosine, the amino acid at position 398 or the corresponding amino acid at that position is phenylalanine, the amino acid at position 286 or the corresponding amino acid at that position is leucine, and which has a catalytic activity for generating the unsaturated hydrocarbon compound represented by the above formula (2) or (3) or a geometric isomer thereof.

[0134] (h) Ferulic acid decarboxylase in which the amino acid at position 397 of the amino acid sequence set forth in SEQ ID NO: 2 or the corresponding amino acid at that position is tryptophan, the amino acid at position 398 or the corresponding amino acid at that position is methionine, and which has a catalytic activity for generating the unsaturated hydrocarbon compound represented by the above formula (2) or (3) or a geometric isomer thereof.

[0135] (i) An ferulic acid decarboxylase in which the amino acid at position 397 of the amino acid sequence set forth in SEQ ID NO: 2 or the corresponding amino acid at this position is tryptophan, the amino acid at position 398 or the corresponding amino acid at this position is methionine, the amino acid at position 440 or the corresponding amino acid at this position is tyrosine, and which has catalytic activity for producing the unsaturated hydrocarbon compound represented by the above formula (2) or (3) or their geometric isomers.

[0136] (j) An ferulic acid decarboxylase in which the amino acid at position 397 of the amino acid sequence set forth in SEQ ID NO: 2 or the corresponding amino acid at this position is tryptophan, the amino acid at position 398 or the corresponding amino acid at this position is methionine, the amino acid at position 440 or the corresponding amino acid at this position is phenylalanine, the amino acid at position 286 or the corresponding amino acid at this position is leucine, and which has catalytic activity for producing the unsaturated hydrocarbon compound represented by the above formula (2) or (3) or their geometric isomers.

[0137] (k) An ferulic acid decarboxylase in which the amino acid at position 397 of the amino acid sequence set forth in SEQ ID NO: 2 or the corresponding amino acid at this position is tryptophan, the amino acid at position 398 or the corresponding amino acid at this position is phenylalanine, and which has catalytic activity for producing the unsaturated hydrocarbon compound represented by the above formula (2) or (3) or their geometric isomers.

[0138] (l) An ferulic acid decarboxylase in which the amino acid at position 397 of the amino acid sequence set forth in SEQ ID NO: 2 or the corresponding amino acid at this position is tryptophan, the amino acid at position 398 or the corresponding amino acid at this position is phenylalanine, the amino acid at position 286 or the corresponding amino acid at this position is leucine, and which has catalytic activity for producing the unsaturated hydrocarbon compound represented by the above formula (2) or (3) or their geometric isomers.

[0139] (m) An ferulic acid decarboxylase in which the amino acid at position 397 of the amino acid sequence set forth in SEQ ID NO: 2 or the corresponding amino acid at this position is tryptophan, the amino acid at position 398 or the corresponding amino acid at this position is phenylalanine, the amino acid at position 286 or the corresponding amino acid at this position is leucine, the amino acid at position 189 or the corresponding amino acid at this position is methionine, and which has catalytic activity for producing the unsaturated hydrocarbon compound represented by the above formula (2) or (3) or their geometric isomers.

[0140] Examples

[0141] Hereinafter, the present invention will be described more specifically based on examples, but the present invention is not limited to the following examples.

[0142] As shown in Patent Documents 1 and 2, the inventors found that by introducing mutations accompanied by amino acid substitutions into ferulic acid decarboxylase (FDC), the catalytic activity related to the production of butadiene using muconic acid as a substrate was increased. In particular, the inventors clarified that in the wild-type FDC derived from Saccharomyces cerevisiae (the amino acid sequence described in SEQ ID NO: 2), by substituting isoleucine at position 398 with glutamic acid and phenylalanine at position 397 with histidine (F397H / I398Q mutant), the catalytic activity was increased by 75.1 times compared to the wild-type.

[0143] Then, this time, the inventors attempted to further improve the productivity of unsaturated hydrocarbon compounds such as butadiene using the FDC mutant. As shown below, various amino acid substitution mutants of FDC were prepared, and the catalytic activity when these mutants were used in combination with the F397H / I398Q mutant was evaluated.

[0144] <Preparation of plasmid vector>

[0145] First, in order to efficiently express the wild-type FDC derived from Saccharomyces cerevisiae in Escherichia coli, a form in which a polyhistidine tag was fused to the C-terminus of the wild-type nucleotide sequence encoding the above FDC was used, and it was changed considering the codon usage frequency in Escherichia coli (the changed nucleotide sequence is shown in SEQ ID NO: 3).

[0146] Next, DNA containing such a changed nucleotide sequence was chemically synthesized by a conventional method. Furthermore, the DNA prepared by such an operation was ligated to the pET22b(+) vector (manufactured by Novagen) by the Gibson Assembly method (using the kit NEBuilder HiFi DNA Assembly Master Mix (registered trademark) of New England Biolabs) to prepare a plasmid vector (wild-type FDC vector) capable of expressing the wild-type FDC in Escherichia coli.

[0147] Furthermore, using the wild-type FDC vector thus obtained as a template, a vector encoding FDC into which various amino acid substitutions were introduced was prepared. Specifically, primers encoding the amino acid sequences into which each mutation was introduced were designed and synthesized. Then, using the wild-type FDC vector as a template and the primers, according to the protocol of the Gibson Assembly method, a plasmid vector (FDC mutant vector) capable of expressing the FDC into which each mutation was introduced in a form in which a polyhistidine tag was fused to its C-terminus in Escherichia coli was prepared.

[0148] Similarly, the DNA obtained by amplifying the gene encoding flavoisoprenyltransferase (hereinafter also referred to as "UbiX") (SEQ ID NO: 5) from Escherichia coli (K-12) strain by the polymerase chain reaction method was ligated to the pColADuet vector (manufactured by Novagen) by the Gibson Assembly method to prepare a plasmid vector (UbiX vector) capable of expressing the wild-type UbiX in Escherichia coli.

[0149] <Preparation of enzyme reaction solution and measurement of enzyme activity>

[0150] The vectors prepared as described above (5 μg of F397H / I398Q mutant vector or 5 μg of other FDC mutant vectors, and 5 μg of UbiX vector) were introduced into Escherichia coli C41(DE3) strain (manufactured by Lucigen Corporation, 100 μL) by the heat shock method to prepare a transformant co-expressing the F397H / I398Q mutant and UbiX (hereinafter also referred to as F397H / I398Q mutant-expressing Escherichia coli), and a transformant co-expressing other FDC mutants and UbiX (hereinafter also referred to as other FDC mutant-expressing Escherichia coli).

[0151] Then, these transformants were each cultured in LB medium supplemented with ampicillin and kanamycin for 6 hours. In addition, through such 6-hour culture (pre-culture), the proliferation of these transformants reached the limit. Therefore, the cell mass at the start time of the enzyme reaction described below became uniform among these transformants.

[0152] In addition, lactose was added to a final concentration of 20 g / L in TB medium (112 g / L tryptone, 24 g / L yeast extract, 10 g / L glycerol, 9.4 g / L dipotassium hydrogen phosphate, 2.2 g / L potassium dihydrogen phosphate, 100 mg / L ampicillin, and 50 mg / L kanamycin), and cis,cis-muconic acid (manufactured by Sigma-Aldrich) as a substrate was further added to a final concentration of 5 g / L to prepare a culture medium for enzyme reaction.

[0153] Then, in a 10 mL bottle for a headspace gas chromatography-mass spectrometer (HS / GSMS), 100 μL of the culture solution of the Escherichia coli expressing the F397H / I398Q mutant that had been cultured for 6 hours above, or 50 μL of the culture solution of the Escherichia coli expressing the F397H / I398Q mutant and 50 μL of the culture solution of the Escherichia coli expressing other FDC mutants were added to 1 mL of the above enzyme reaction medium. Immediately after the addition, the lid of the bottle was closed, and further culture was carried out at 37 °C with an oscillation speed of 180 rpm. The peak area was measured by GC-MS (product name: GCMS-QP Ultra, manufactured by Shimadzu Corporation), and this peak area represents the amount of butadiene (1,3-butadiene) generated in the headspace of the bottle 18 hours after the start of this culture. Then, based on the obtained measurement values, the amount of butadiene generated in the co-culture of the Escherichia coli expressing the F397H / I398Q mutant and the Escherichia coli expressing other FDC mutants was compared with the amount of butadiene generated in the single culture of the Escherichia coli expressing the F397H / I398Q mutant.

[0154] (Example 1) Combination with single mutants or double mutants of FDC

[0155] First, various single mutants or double mutants of FDC were prepared, and the amount of butadiene generated when these mutants were combined with the F397H / I398Q mutant (co-cultured as described above) was evaluated. The obtained results are shown in Table 1. In addition, the values in the table represent the ratio of the amount of butadiene generated in each co-culture to the amount of butadiene generated in the single culture of the Escherichia coli expressing the F397H / I398Q mutant (the same applies to Tables 2 and 3 below).

[0156] Table 1

[0157]

[0158] As a result, it was found that when combined with the FDC double mutants (F397Y / I398M or F) in which phenylalanine at position 397 was replaced with tyrosine and isoleucine at position 398 was replaced with methionine or phenylalanine, the amount of butadiene generated increased to 5.0 times compared to the case of using only the F397H / I398Q mutant. It was also found that when combined with the FDC double mutants (F397W / I398M or F) in which phenylalanine at position 397 was replaced with tryptophan and isoleucine at position 398 was replaced with methionine or phenylalanine, the amount of butadiene generated increased to 3.4 times compared to the case of using only the F397H / I398Q mutant.

[0159] (Example 2) Combination with triple mutants of FDC

[0160] Furthermore, based on the F397Y / I398M or F mutants and the F397W / I398M or F mutants, further amino acid substitutions were introduced to prepare various FDC triple mutants. Then, co-culture was carried out as described above, and the combination with the F397H / I398Q mutant was evaluated. The results obtained are shown in Table 2.

[0161] Table 2

[0162]

[0163] As a result, it was also found that when phenylalanine at position 440 was replaced with tyrosine in the F397Y / I398M mutant, or when methionine at position 286 was replaced with leucine in the F397Y / I398F mutant, the butadiene production amount exceeded 5 times that shown in Example 1 and was further increased (5.9 times or 6.2 times).

[0164] In addition, it was also found that when phenylalanine at position 440 was replaced with tyrosine in the F397W / I398M mutant (F397W / I398M / F440Y mutant), or when methionine at position 286 was replaced with leucine in the F397W / I398F mutant (F397W / I398F / M286L mutant), the butadiene production amount exceeded 3.4 times that shown in Example 1 and was further increased (4.6 times or 4.5 times).

[0165] (Example 3) Combination with the quadruple mutant of FDC

[0166] Furthermore, based on these FDC triple mutants, further amino acid substitutions were introduced to prepare various FDC quadruple mutants. Then, co-culture was carried out as described above, and the combination with the F397H / I398Q mutant was evaluated. The results obtained are shown in Table 3.

[0167] Table 3

[0168]

[0169] As a result, even when further amino acid substitutions were introduced into the FDC triple mutant of F397Y, it did not exceed 5.9 times or 6.2 times shown in Example 2.

[0170] On the other hand, for the FDC triple mutant of F397W, when isoleucine at position 189 is replaced with methionine in the F397W / I398F / M286L mutant, or when methionine at position 286 is replaced with leucine in the F397W / I398M / F440Y mutant, the amount of butadiene produced exceeds 4.6 times or 4.5 times as shown in Example 2 and is further increased (5.6 times or 5.9 times).

[0171] Furthermore, as a result of replacing isoleucine at position 189 with tryptophan or methionine in the F397W / I398M / F440Y mutant, the amount of butadiene produced is increased to as high as about 7 times or more (7.3 times or 8.3 times) compared with the case of using only the F397H / I398Q mutant. That is, it was found that since even the F397H / I398Q mutant alone is 75.1 times that of the wild type, the amount of butadiene produced in the case of combination with the F397W / I398M / F440Y / I189W mutant is increased to about 548 times that of the wild type, and the amount of butadiene produced in the case of combination with the F397W / I398M / F440Y / I189M mutant is increased to about 623 times that of the wild type.

[0172] Thus, by combining with the F397H / I398Q mutant, a further FDC mutant with a catalytic activity for producing unsaturated hydrocarbon compounds such as butadiene increased by about 500 times or more compared with the wild type was successfully obtained.

[0173] Industrial Applicability

[0174] As described above, according to the present invention, an enzyme capable of producing unsaturated hydrocarbon compounds such as butadiene with high productivity, and a method for producing an unsaturated hydrocarbon compound using the enzyme can be provided. In addition, according to the present invention, unsaturated hydrocarbon compounds can be produced by biosynthesis without relying on chemical synthesis, and thus the environmental load is small. Therefore, the present invention is extremely useful in the production of raw materials for various synthetic polymers such as synthetic rubber like butadiene.

Claims

1. A combination of at least one ferulic acid decarboxylase selected from the following (a) and (b) and the ferulic acid decarboxylase described in the following (c), (a) The 397th amino acid of the amino acid sequence set forth in SEQ ID NO: 2 or the amino acid corresponding to this position is tryptophan, the 398th amino acid or the amino acid corresponding to this position is methionine, the 440th amino acid or the amino acid corresponding to this position is tyrosine, the 189th amino acid or the amino acid corresponding to this position is methionine, and having the catalytic activity of generating an unsaturated hydrocarbon compound represented by the following formula (2) or (3) or their geometric isomers, (b) The 397th amino acid of the amino acid sequence set forth in SEQ ID NO: 2 or the amino acid corresponding to this position is tryptophan, the 398th amino acid or the amino acid corresponding to this position is methionine, the 440th amino acid or the amino acid corresponding to this position is tyrosine, the 189th amino acid or the amino acid corresponding to this position is tryptophan, and having the catalytic activity of generating an unsaturated hydrocarbon compound represented by the following formula (2) or (3) or their geometric isomers, (c) The 397th amino acid of the amino acid sequence set forth in SEQ ID NO: 2 or the amino acid corresponding to this position is histidine, the 398th amino acid or the amino acid corresponding to this position is glutamic acid, and having the catalytic activity of generating an unsaturated hydrocarbon compound represented by the following formula (2) or (3) or their geometric isomers, In Formulas (2) and (3), "R 1 ", "R 2 ", "R 3 ", and "R 4 " each independently represent a hydrogen atom, a linear or branched alkyl group having 1 to 5 carbon atoms, a linear or branched alkoxy group having 1 to 5 carbon atoms, or a hydroxyl group; "A" represents an optionally substituted linear hydrocarbon group having 0 to 5 carbon atoms, and when the number of carbon atoms is 2 to 5, a double bond may be formed between adjacent carbon atoms.

2. A DNA encoding the combination of ferulic acid decarboxylases described in claim 1.

3. A vector containing the DNA encoding the combination of ferulic acid decarboxylases described in claim 1.

4. A host cell into which a DNA encoding at least one ferulic acid decarboxylase selected from (a) and (b) described in claim 1 or a vector containing the DNA is introduced, and A combination of host cells into which a DNA encoding the ferulic acid decarboxylase described in (c) of claim 1 or a vector containing the DNA is introduced.

5. A host cell into which a DNA encoding the combination of ferulic acid decarboxylases described in claim 1 or a vector containing the DNA is introduced.

6. A method for producing an unsaturated hydrocarbon compound represented by the following formula (3) or its geometric isomers, Comprising: A step of decarboxylating an unsaturated hydrocarbon dicarboxylic acid compound represented by the following formula (1) or its geometric isomers in the presence of the combination of ferulic acid decarboxylases described in claim 1, In formulas (1) to (3), "R 1 ", "R 2 ", "R 3 " and "R 4 " each independently represent a hydrogen atom, a linear or branched alkyl group having 1 to 5 carbon atoms, a linear or branched alkoxy group having 1 to 5 carbon atoms, or a hydroxyl group; "A" represents a linear hydrocarbon group having 0 to 5 carbon atoms which may be substituted, and when the number of carbon atoms is 2 to 5, a double bond may be formed between adjacent carbon atoms.

7. A method for producing an unsaturated hydrocarbon compound, Comprising: Culturing the combination of host cells described in claim 4 or the host cell described in claim 5, and collecting the unsaturated hydrocarbon compound represented by the following formula (3) or their geometric isomers generated in the host cell and / or its culture, In formula (3), "R 1 ", "R 2 ", "R 3 ", and "R 4 " each independently represent a hydrogen atom, a linear or branched alkyl group having 1 to 5 carbon atoms, a linear or branched alkoxy group having 1 to 5 carbon atoms, or a hydroxyl group; "A" represents an optionally substituted linear hydrocarbon group having 0 to 5 carbon atoms, and when the number of carbon atoms is 2 to 5, a double bond may be formed between adjacent carbon atoms.

8. A preparation for decarboxylating an unsaturated hydrocarbon dicarboxylic acid compound represented by the following formula (1) or its geometric isomers and promoting the generation of an unsaturated hydrocarbon compound represented by the following formula (3) or its geometric isomers, comprising the combination of ferulic acid decarboxylases described in claim 1, a DNA encoding the combination, or a vector containing the DNA, In formulas (1) to (3), "R 1 ", "R 2 ", "R 3 " and "R 4 " each independently represent a hydrogen atom, a linear or branched alkyl group having 1 to 5 carbon atoms, a linear or branched alkoxy group having 1 to 5 carbon atoms, or a hydroxyl group; "A" represents an optionally substituted linear hydrocarbon group having 0 to 5 carbon atoms, and when the number of carbon atoms is 2 to 5, a double bond may be formed between adjacent carbon atoms.

9. At least one ferulic acid decarboxylase selected from the following (a) and (b), (a) An abscisic acid decarboxylase in which the amino acid at position 397 of the amino acid sequence set forth in SEQ ID NO: 2 or the corresponding amino acid at this position is tryptophan, the amino acid at position 398 or the corresponding amino acid at this position is methionine, the amino acid at position 440 or the corresponding amino acid at this position is tyrosine, the amino acid at position 189 or the corresponding amino acid at this position is methionine, and which has catalytic activity for generating an unsaturated hydrocarbon compound represented by the following formula (2) or (3) or a geometric isomer thereof, (b) An abscisic acid decarboxylase in which the amino acid at position 397 of the amino acid sequence set forth in SEQ ID NO: 2 or the corresponding amino acid at this position is tryptophan, the amino acid at position 398 or the corresponding amino acid at this position is methionine, the amino acid at position 440 or the corresponding amino acid at this position is tyrosine, the amino acid at position 189 or the corresponding amino acid at this position is tryptophan, and which has catalytic activity for generating an unsaturated hydrocarbon compound represented by the following formula (2) or (3) or a geometric isomer thereof, In Formulas (2) and (3), "R 1 ", "R 2 ", "R 3 ", and "R 4 " each independently represent a hydrogen atom, a linear or branched alkyl group having 1 to 5 carbon atoms, a linear or branched alkoxy group having 1 to 5 carbon atoms, or a hydroxyl group; "A" represents a linear hydrocarbon group having 0 to 5 carbon atoms which may be substituted, and when the number of carbon atoms is 2 to 5, a double bond may be formed between adjacent carbon atoms.

10. A DNA encoding the abscisic acid decarboxylase according to claim 9.

11. A vector comprising the DNA encoding the abscisic acid decarboxylase according to claim 9.

12. A host cell into which the DNA encoding the abscisic acid decarboxylase according to claim 9 or the vector containing the DNA has been introduced.

13. A preparation for decarboxylating an unsaturated hydrocarbon dicarboxylic acid compound represented by the following formula (1) or a geometric isomer thereof and promoting the generation of an unsaturated hydrocarbon compound represented by the following formula (3) or a geometric isomer thereof, which comprises the abscisic acid decarboxylase according to claim 9, the DNA according to claim 10, or the vector according to claim 11, characterized in that, it is used in combination with an abscisic acid decarboxylase in which the amino acid at position 397 of the amino acid sequence set forth in SEQ ID NO: 2 or the corresponding amino acid at this position is histidine, the amino acid at position 398 or the corresponding amino acid at this position is glutamic acid, and which has the said catalytic activity, the DNA encoding this abscisic acid decarboxylase, or a vector containing the DNA, In formulas (1) to (3), "R 1 ", "R 2 ", "R 3 ", and "R 4 " each independently represent a hydrogen atom, a linear or branched alkyl group having 1 to 5 carbon atoms, a linear or branched alkoxy group having 1 to 5 carbon atoms, or a hydroxyl group; "A" represents a linear hydrocarbon group having 0 to 5 carbon atoms which may be substituted, and when the number of carbon atoms is 2 to 5, a double bond may be formed between adjacent carbon atoms.

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